Ph.D. Thesis

Tolerogenic Dendritic Cells: Immunomodulation of Monocyte-Derived Dendritic Cells with

n-Butyrate, NFκB Inhibitor PDTC and JAK3 Inhibitor WHI-P-154

Peter Gyorgy Kelemen, M.D.

Semmelweis University, School of Ph.D-Studies Doctoral School: 7. Molecular Medicine

Doctoral Program: 7/5. Basic and Clinical Immunology Consultant: Prof. Peter Gergely, M.D.

Reviewers: Eva Pocsik, Ph.D. and Zoltan Prohaszka, M.D., Ph.D.

Chairman of the Final Examination Committee: Prof. Bela Fekete, M.D. Members: Miklos Benczur, M.D., Ph.D. and Gabriella Sarmay, Ph.D.

2005

TABLE OF CONTENTS

TABLE OF CONTENTS ................................................................................................. 1

TABLE OF FIGURES ..................................................................................................... 3

ABBREVIATIONS........................................................................................................... 4

INTRODUCTION ............................................................................................................ 5

Dendritic Cells.............................................................................................................. 5

Tolerogenic Dendritic Cells and Allograft Transplantation.........................................12

AIMS OF THE STUDY ...................................................................................................16

MATERIALS AND METH0DS ......................................................................................19

RESULTS ..........................................................................................................................23

A.) „Bacterial Metabolite Interference with Maturation of Human Monocyte-Derived Dendritic Cells”.......................................................................23

1.) Phenotypical and functional impairment of DC maturation by n-butyrate.............23

B.) „Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor-Treated Dendritic Cells: Resistance to Calcineurin Inhibition” ........................................27

1.) Arrest in DC maturation by treatment with the NFκB inhibitor PDTC..................27

2.) PDTC-modulated DC exhibit defective stimulatory capacity for allogeneic

T-cell responses ......................................................................................................30

3.) Induction of allogeneic hyporesponsiveness by PDTC-modulated DC..................31

4.) CsA does not counteract the suppres sive state induced by PDTC-modulated DC 32

5.) T-cell hyporesponsiveness with PDTC-modulated DC in T cells from patients

with renal allografts under calcineurin inhibitor-based immunosuppression.........34

1

C.) „Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” .....................................36

1.) Targeting JAK3 disrupts CD40-triggered DC maturation ......................................36

2.) JAK3 inhibition does not interfere with the activation-associated

down-regulation of the DC-antigen uptake machinery ..........................................36

3.) Prevention of T-cell stimulatory capacity and induction of antigen-specific

hyporeactivity by JAK3 inhibited DC....................................................................39

4.) Effect of JAK3 Inhibitor Treatment on Homotypic T-Cell Aggregation................41

SUMMARY OF THE RESULTS....................................................................................42

DISCUSSION....................................................................................................................45

CONCLUSIONS ...............................................................................................................50

ACKNOWLEDGEMENTS .............................................................................................51

OWN PUBLICATIONS RELATED TO THIS THESIS..............................................52

REFERENCES .................................................................................................................53

2

TABLE OF FIGURES

Figure 1: Hematopoietic development of human dendritic cells. ................................. 7

Table 1: Approaches to therapy of allograft rejection by targeting donor or

recipient DC...................................................................................................15

Figure 2: n-Butyrate prevents DC maturation...............................................................24

Figure 3: Effect of n-butyrate on maturation-associated clustering of DC...................25

Figure 4: Reduced T-cell stimulatory capacity of DC matured in the presence of

n-butyrate.......................................................................................................26

Figure 5: Analysis of the DC antigen-uptake machinery of DC differentiated in

the presence of n-butyrate. ............................................................................26

Figure 6: Treatment with the NFκB inhibitor pyrrolidine dithiocarbamate

(PDTC) prevents the phenotypic changes associated with dendritic cell

(DC) maturation.............................................................................................28

Figure 7: PDTC blocks DC activation to various maturation stimuli...........................29

Figure 8: PDTC-modulated DC fail to support full allogeneic T-cell activation. ........30

Figure 9: Induction of allogeneic T-cell hyporesponsiveness by PDTC-

modulated DC................................................................................................33

Figure 10: Effect of Cyclosporine A (CsA) treatment on allogeneic

hyporesponsiveness induced by PDTC-modulated DC.................................34

Figure 11: Feasibility of modulating alloresponsiveness in T cells from patients

with renal allografts by altered DC................................................................35

Figure 12: JAK3 targeting prevents the phenotypic alterations induced by CD40

ligation. ..........................................................................................................37

Figure 13: Dendritic cells activated by CD40 ligation down-regulate their antigen

uptake machinery in the presence of Janus kinase 3 (JAK3) inhibition........38

Figure 14: Reduced stimulatory activity of dendritic cells (DC) matured in the

pesence of Janus kinase 3 (JAK3) inhibition and induction of a state

of T-cell hyporeactivity. ................................................................................40

Figure 15: Reversal of T-cell hyporesponsiveness by exogenous IL-2. .........................40

Figure 16: JAK3 targeting blocks phenotypical hallmarks of T-cell activation. ............41

3

ABBREVIATIONS

Ab antibody Ag antigen APC antigen-presenting cells CCR chemokine receptor CD cluster of differentiation CsA Cyclosporine A CTL cytotoxic T lymphocytes DC1 myeloid-related dendritic cells DC2 lymphoid-related dendritic cells DC dendritic cells DC-LAMP DC-lysosome-associated

membrane glycoprotein DDC dermal dendritic cells DEX dextran DNA deoxyribonucleic acid ELISA enzyme linked

immunosorbent assay FACS fluorescein-activated cell sorter FCS fetal calf serum FITC fluorescein isothiocyanate FL c-fms-like tyrosine kinase

ligand FLT-3 ligand c-fms-like tyrosine kinase

ligand FLT-3 c-fms-like tyrosine kinase 3 GCDC germinal center dendritic cells G-CSF granulocyte colony

stimulating factor GM-CSF granulocyte-macrophage

colony stimulating factor GVHD graft versus host disease HPC hematopoietic progenitor

cells iDC immature dendritic cells IDC interstitial dendritic cells IFN interferon Ig immunoglobulin IL interleukin IL-R interleukin receptor IPC interferon producing cells JAK3 Janus Kinase 3 KL c-kit-ligand LC Langerhans cells LPS lipopolysaccharide LY lucifer yellow

mAb monoclonal antibody M-CSF macrophage colony

stimulating factor mDC mature DC MFI median fluorescence

intensity MHC major histocompatibility

complex MLC mixed lymphocyte culture moDC monocyte-derived

dendritic cells MR mannose receptor Mr molecular weight OKT3 monoclonal antibody

to CD3 PAMP pathogen-associated

molecular pattern PRR pattern recognition

receptor PBMC peripheral blood

mononuclear cells PBS phosphate-buffered saline pDC2 DC2 precursors PDTC-DC PDTC-modulated

dendritic cells PDTC pyrrolidine dithiocarbamate PE phycoerythrin PGE2 prostaglandin E2 Pg peptidoglycan PMA phorbol myristate acetate pMLC primary mixed lymphocyte

culture PTK protein tyrosine kinase rh recombinant human SCFA short-chain fatty acid SCF stem cell factor SCID severe combined

immunodeficiency SD standard deviation STAT signal transducers and

activators of transcription T regs regulatory T cells TCR T-cell receptor TGF-β transforming growth

factor-β Th1 T helper 1 Th2 T helper 2 TLR Toll-like-receptor TNF-α tumor necrosis factor-α

4

INTRODUCTION

Dendritic Cells

The onset of an immune response requires not only antigen (Ag) and

lymphocytes, but also a third-party cell to present antigens to the lymphocytes. Long

recognized as a requirement for so-called accessory or feeder cells in vitro, the exact cell

type remained elusive until dendritic cells (DC) were first purified and distinguished

from other antigen-presenting cells (APC) in mice, such as B lymphocytes and

macrophages 1,2-4. DC were originally discovered in mice a little more than a quarter

century ago 1,3-4, but only in the past decade has it become feasible to generate either

animal or human DC in sufficiently large numbers and purity for large scale

experimental and clinical investigation.

DC are uniquely specialized among all leukocytes to couple the presentation of

antigen, bound to major histocompatibility complex (MHC) molecules, with all the

adhesive and costimulatory signals, collectively termed accessory molecules, required

to initiate cellular immune responses 5. Unlike B cells that recognize soluble or native

antigen and transform into antibody (Ab)-secreting plasma cells, T-cell receptors

(TCRs) can only recognize peptide fragments of antigen bound to MHC molecules on

APC. MHC molecules are of two types, class I MHC and class II MHC. For the most

part, class I MHC molecules bind intracellular or endogenous antigens that have been

cut into peptides in the cytosol. Class II MHC molecules bind extracellular or

exogenous antigens that have entered the endocytic pathway of the APC. Class I and II

MHC-peptide complexes stimulate CD8+ cytotoxic T lymphocytes (CTL) and CD4+

helper T cells, respectively. DC also have a unique capacity to capture exogenous

antigens from dying cells, apoptotic or necrotic tumor cells, virus-infected cells and

immune complexes, which then access the class I MHC pathway for Ag presentation 6-8. This pathway, called cross-presentation, permits DC to elicit CD8+ as well as CD4+

T-cell responses to exogenous antigens. Cross-presentation is linked to specific DC

antigen uptake receptors, which may be targeted in strategies to load exogenous

antigens onto both MHC I and II. DC also interact directly with B cells and

5

lymphocytes of the innate immune system. Activated myeloid DC can directly induce

B-cell proliferation, immunoglobulin isotype switching, and plasma cell differentiation.

Activated lymphoid DC can induce the differentiation of CD40-activated B cells into

plasma cells. DC can also activate and induce the expansion of resting natural killer

cells. Activated natural killer cells can kill immature, but not mature DC and can

stimulate DC to induce protective CD8+ T-cell responses. Lipid and glycolipid antigens

expressed on pathogens or self tissues are presented by DC to T cells on CD1 molecules

(CD1a-d), which are structurally similar to MHC I but specialized to bind lipids instead

of peptides. Processing of lipid antigens onto CD1 molecules is carried out in

specialized intracellular compartments, much like antigen processing onto MHC II.

CD1 molecules present lipid antigens to a variety of lymphocytes, including T cells with

substantial T-cell receptor diversity as well as relatively invariant natural killer T cells.

While DC express abundant MHC molecules of both types, they also constitutively

express large numbers of accessory molecules. The inducible increase in expression of

these adhesive and costimulatory molecules coordinates with maturation and activation

stimuli in culture. This mimics the in vivo capture of Ags by DC in the periphery,

followed by their maturation and migration to secondary lymphoid organs, where DC

complete their activation while stimulating T cells 9-13. It is this pairing of Ag/MHC

presentation with potent accessory function that enables DC to stimulate T cell

immunity without additional adjuvants. DC have therefore often been termed “nature’s

adjuvants” or “professional APC.”

DC could be regarded as a multilineage system of leukocytes with variable

function rather than as a homogenous cell type with predetermined functional properties.

Because the DC systems a whole can present Ags in an immunogenic or tolerogenic

fashion, it is possible that the outcome of an immune response initiated by DC depends

on a combination of several factors, including Ag presentation by a specific type of DC,

the nature of the DC-activating signal, and the stage of DC maturation. Successful

cytokine-driven growth and differentiation of DC from defined precursors has revealed

that DC comprise at least two types, myeloid and plasmacytoid or lymphoid, of which

the myeloid DC comprise at least two subsets (Figure 1) 14. There is also a spectrum of

differentiation within these divisions, from marrow-derived clonogenic progenitors,

to circulating precursors in blood and lymphatics, to immature DC resident in peripheral

6

Figure 1: Hematopoietic development of human dendritic cells.

All human DC develop originally from CD34+ hematopoietic progenitor cells (HPC) from either bone marrow, cord blood, or cytokine elicited peripheral blood stem cells. Expansion is supported in vitro by factors like FLT-3-ligand (FL) and c-kit-ligand or stem cell factor (KL/SCF), with additional contributions from either granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3). GM-CSF supports myeloid DC expansion and differentiation, whereas IL-3 supports that of lymphoid-related DC. Tumor necrosis factor-alpha (TNF) exerts pleiotropic effects, recruiting transferrin receptor positive CD34+ HPC into cell cycle, at least partially suppressing granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) receptors early in CD34+ HPC differentiation, and later supporting terminal DC maturation. Additional cytokines like interleukin-4 (IL-4) and transforming growth factor-beta 1 (TGF) suppress CD14+ macrophage differentiation. TGF supports the differentiation of Langerhans cells (LC). DC precursors as represented here are no longer in cell cycle. Activated LC continue to express Langerin, but none of the myeloid DC express this antigen. CD1a is expressed by all myeloid DC types 14.

7

tissues, to mature or maturing forms in the thymus and secondary lymphoid organs 15-16.

The two types of myeloid DC are Langerhans cells in epithelial surfaces and dermal or

interstitial DC in the dermis of skin or interstitium of solid organs. C-fms-like tyrosine

kinase ligand (FLT-3 ligand) is a hemopoietic cytokine whose receptor, FLT3, is

expressed on pluripotent stem and progenitor cells. Proceeding from original studies in

mice, FLT-3L also mobilizes both myeloid and lymphoid DC in vivo in humans 17-18.

FLT-3L can increase circulating human myeloid DC by a mean 44-fold and lymphoid

DC by a mean 12-fold, with little toxicity 18. Physical methods can then enrich the DC

from leukopheresis products 19-22. Evidence best supports the direct differentiation of LC

from CD34+ hematopoietic progenitor cells (HPC) 23-27, whereas interstitial or dermal

DC develop from CD34+ HPC via a CD14+ bipotential intermediate 28-31. This CD14+

intermediate is not clonogenic and is termed bipotential because it can alternatively

develop into macrophages instead of DC 29-30. The latter DC type is usually considered

the homologue of the peripheral blood monocyte-derived DC (moDC). CD14+ blood

monocyte cultures require IL-4 in addition to GM-CSF in order to suppress macrophage

differentiation 32 and develop into moDC. LC move into the T cell-rich parafollicular

areas where they are especially likely to encounter naive T cells. In these sites they are

termed interdigitating DC. Although LC have usually been thought of as immature DC,

a new specific marker, Langerin 33-34, still distinguishes some of the mature, activated LC

from the other myeloid interstitial or dermal dendritic cells. They have unique

intracellular organelles called Birbeck granules. Interstitial or dermal DC migrate into

germinal centers where they are termed germinal center DC (GCDC). GCDC present

processed antigen to memory T cells and additionally participate in humoral immunity

by direct interaction with germinal center B cells and indirectly by stimulation of CD4+

T cells that provide cognate help for B cell differentiation 31,35.

In human blood DC are traditionally divided into two populations by staining

with antibodies to CD11c and CD123 (interleukin 3 receptor α [IL-3Rα]). D11c+CD123lo

DC have a monocytoid appearance and are called myeloid DC, whereas CD11c-CD123hi

DC have morphologic features similar to plasma cells and are thus called plasmacytoid

or lymphoid DC. Recently, the terms DC1 and DC2 have been used to distinguish

myeloid DC (DC1) from lymphoid DC (DC2), based on the propensity of each type

to stimulate T helper 1 (Th1) versus T helper 2 (Th2) responses, respectively 36.

8

The myeloid DC1 refers to the moDC or its counterpart in the CD34+-derived system,

the dermal or interstitial DC. The term DC1 was not intended to encompass LC, although

LC are also myeloid and meet many of the criteria for DC1. Most investigators are

focusing on the myeloid DC and/or DC1 to stimulate acquired antiviral and antitumoral

immunity, as these DC stimulate Th1 responses that support CTL generation. DC2

precursors (pDC2) are critical effectors of the innate immune system as they rapidly

produce large amounts of interferon-α (IFN-α, type I IFN) in response to enveloped

viruses, bacteria, and tumor cells, and upon terminal differentiation become professional

antigen-presenting type 2 DC for stimulation of Th2 responses 37-39. The tolerizing role

that DC2 may play, either directly or indirectly, is of considerable interest, but is

more pertinent to transplantation and autoimmunity than to the generation of CTL

against viruses and tumors 40-42.

DC are most likely to encounter and capture Ags in the periphery, leading to

their maturation and migration via afferent lymphatics into draining lymphoid organs.

This culminates in the final activation of DC as they stimulate incoming clones of

Ag-specific naive or resting memory T cells, which then exit to function as producers of

helper cytokines or as CTL in the periphery. Antigen capture and processing versus

antigen presentation and T cell stimulation are processes that are dynamically related

to the maturation state of DC 43-44. Mature DC are more stimulatory of Ag-specific

T cells than are immature DC. Chemokine receptors are also regulated in relation to

maturation and support trafficking to secondary lymphoid organs 45-48. Chemokines are

a group of structurally related polypeptides that have been recognized recently to have

critical roles in the selective recruitment of leukocyte subsets to secondary lymphoid

organs and to sites of inflammation. Upon exposure to maturation signals DC undergo

a chemokine receptor switch: DC down-regulate inflammatory chemokine receptors

(CCR1, CCR2, and CCR5) followed by induction of CCR7. Toll-like receptors (TLRs),

which are type I integral membrane glycoproteins, are highly conserved microbial

pattern recognition receptors. TLRs recognize bacteria, viruses and parasites and trigger

DC maturation and secretion of numerous chemokines and cytokines. Cytokine

production by DC might be crucial to induce T-cell immunity. It is well known that

fully mature DC produce large amounts of the proinflammatory cytokines IL-12p40 and

the bioactive form p70, TNF-α, IL-1β and IL-6 and nitric oxide. The production of IL-2

9

by DC is required to induce T-cell priming and might represent a switch from tolerance

to immunity. Signals that induce cytokine production by DC and full maturation are

associated with microbial recognition as represented by the recognition of evolutionarily

conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition

receptors (PRRs). Such receptors are represented by the recently discovered Toll-like

receptors (TLRs) on DC or indirect recognition through complement and/or omplement

receptors, or antibody and/or Fc-receptor. IL-12-inducing LPS is recognized by TLR4

and might be a prototype for full DC activation. In humans, myeloid DC express TLRs

1 through 5 and, depending on the subset, TLR 7 and/or 8. Human lymphoid DC

express TLRs 1, 7, and 9. Some TLRs act at the cell surface, whereas others such as

TLRs 3, 7, 8, and 9 are found within endosomes and are presumably activated following

capture and internalization of pathogens or their products. Additional stimuli in vitro

ensure that differentiated DC maintain phenotype and function rather than revert to

immature forms or follow alternative differentiation pathways upon cytokine

withdrawal. These stimuli can be in the form of bacterial or viral components

{such as lipopolysaccharide (LPS), unmethylated cytosine poly-guanine (CpG) motifs,

doublestranded RNA products} and pro-inflammatory cytokines {e.g., IL-1β, IL-6,

TNFα, prostaglandin E2 (PGE2), type I IFN α/β} or interactions with molecules of

the TNF receptor family {i.e. CD40, receptor activator of nuclear factor-κB (NF-κB),

TNF-receptor} on the DC surface with their ligands during cognate T cell–DC

interaction 49,50-53. Cytokines secreted by activated macrophages can also support

terminal and irreversible differentiation of monocyte-derived DC 54. However,

standardization between donors can be difficult, leading some investigators to prefer a

recombinant cytokine cocktail that reproduces the effects of macrophage-conditioned

medium 53. CD40-ligand (CD40L or CD154) is also a powerful stimulus for DC, as

evidenced by an activated phenotype, increased T cell stimulatory capacity, and

secretion of IL-12 9-10,55. The importance of CD40 in T cell activation is suggested by its

localization in the immunological synapse where it is found along with MHC II.

CD40 is different than other costimulatory molecules in that it acts bidirectionally.

CD40 on the DC stimulates T cells through CD40 ligand and, conversely, CD40L on

the T cell provides a maturation signal through CD40 on the DC. Soluble CD40L

binding to CD40, which is upregulated on mature DC, mimics the full activation of DC

10

accomplished by their initial interactions and crosstalk with CD4+ T cells at the onset of

an immune response 11-13. Although, CD40 triggering alone is unable to induce

IL-12p70 production in vivo. Therefore, only the combination of microbial plus CD40

signals might be optimal inducers for full DC maturation, at least for Th1 responses.

Upon successful maturation of myeloid DC, there are important properties that are

always useful to distinguish them from their less mature precursors and from

other leukocytes. The circumferential cytoplasmic veils become more prominent with

maturation in vitro. Mature myeloid DC lack any significant expression of epitopes

specific to macrophages (CD14, CD115) or lymphocytes (CD3, CD4, CD8, CD19,

CD20, CD16, CD56). Mature DC increase expression of CD83 56, which is still the best

available marker of maturation. CD83, a cell surface molecule involved in CD4+ T-cell

development and cell-cell interactions, can also be detected intracellularly as evidence

of commitment to the DC lineage. Mature, myeloid DC also express p55/fascin, which

is an actin bundling protein 57-58, abundant class II MHC and DC-lysosome-associated

membrane glycoprotein (DC-LAMP). In addition, they upregulate surface T-cell

costimulatory molecules CD40, B7-2/CD86 more so than B7-1/CD80, OX40 ligand,

inducible costimulator (ICOS) ligand 59-60 and those intercellular adhesion molecules

(CD54 and CD58) required for both physical interaction with T cells and assembly of

immunological synapse. CD45RO, nominally a marker of memory T cells, is also a

marker of activated LC and moDC. Interleukin-2 receptor (IL-2R/CD25) is also

expressed by certain activated DC. All of these mature DC are potent stimulators of

T cells. Comparable T cell proliferation in vitro, used as a measure of the stimulatory

activity of an APC population, requires at least 10- to 100-fold more APC such as

B cells, macrophages, or bulk peripheral blood mononuclear cells, than is required of

mature DC stimulators 43-44.

Investigators now have the tools to generate DC for the control and manipulation

of immune responses against human disease. Initial clinical trials of human DC vaccines

are generating encouraging preliminary results both in patients with cancer and in normal

volunteers 22,61-63,64. Important considerations in the design of such human trials include

antigen selection, methods for introducing the antigen into MHC class I and II rocessing

pathways, methods for isolating and activating dendritic cells, and route of

administration.

11

Tolerogenic Dendritic Cells and Allograft Transplantation

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC),

which are the most potent inducers of primary T-cell responses 65. Beyond this

immunostimulatory function, they play critical roles in central tolerance and in the

maintenance of peripheral tolerance in the normal steady state. Different models have

been proposed to explain the mechanism(s) by which DC may induce/maintain

peripheral T-cell tolerance. It is proposed that under steady-state (normal) conditions,

the uptake of Ags by immature DC expressing low cell-surface levels of MHC and

T-cell costimulatory molecules may induce tolerance to those peptides presented to

Ag-specific T cells. This prediction is based on the fact that binding of the T-cell

receptor (TCR) on naive T cells to MHC–peptide complexes on the APC surface in the

absence of or with low levels of T-cell costimulation leads to anergy/apoptosis of the

Ag-specific T cells or to generation of T cells with regulatory function (regulatory

T cells). In this model of peripheral tolerance, immature DC transport self-antigens

continuously from peripheral tissues to lymph nodes and spleen. The concept of

migratory immature DC as the keepers of peripheral T-cell tolerance disagrees with the

experimental observation that lymph-borne DC (also known as veiled cells), obtained

by cannulation of lymphatic vessels in the steady state, exhibit signs of maturation in all

animal models investigated so far. Lutz and Schuler have coined the term semi-mature

DC for these steady-state migrating DC 66. Semi-mature DC that migrate spontaneously

from the periphery may exhibit certain levels of expression of surface MHC, T-cell

costimulatory and adhesion molecules, low levels of pro-inflammatory cytokines, IL-10

production, and absence of bioactive IL-12p70 synthesis. The detailed mechanism(s) by

which immature or semimature DC induce specific T-cell tolerance to self- or nonself

antigens is not entirely known. The inherent tolerogenicity of DC offers considerable

potential for therapy for allograft rejection. Certain DC subsets have been shown to

induce T-cell tolerance in vitro and in vivo 67. Thus, CD8a+ DEC205+ DC have been

shown to induce antigen-specific T-cell apoptosis 68, intestinal DC to promote Th2 cells 69 or thymic lymphoid DC to induce T-cell anergy in vivo 70. Furthermore, it has been

reported thatimmature DC rather than eliciting a weak T-cell response are able to confer

a state of immunotolerance by the induction of regulatory T cells (T regs) in vitro

12

and in vivo 71-73. The concept has recently emerged that dialogue between T reg cells

and DC is crucial for the regulation of alloimmune responses. Using a human

in vitro model system, it has been shown that immature DC exposed to T reg cells can

increase the expression of inhibitory molecules needed for the tolerogenic activity of

DC, and that tolerogenic DC, in turn, can induce anergy in alloreactive CD4+ T cells,

thus establishing an inhibitory feedback loop. Strikingly, subcutaneous administration

of autologous immature DC induced antigen-specific T-cell tolerance in human

volunteers 74. Likewise, myeloid immature DC have been shown to prolong allograft

survival in vivo 75. These data suggest that the maturation state of DC is critical for the

outcome of an immune response and indicate that administration of immature DC could

be a useful approach for the therapy of several immune-mediated diseases 66,72,76-79.

Allograft rejection is a key problem in the field of organ transplantation. The

principal mechanism underlying the acute rejection of allogeneic tissue/organ grafts is

the vigorous adaptive immune response mounted by recipient T lymphocytes against

donor MHC antigens and donor MHC-derived peptides presented by self-MHC

molecules. Tolerance can be defined as the inability of a host to respond to antigens

without the need for immunosuppressive drugs. In particular, peripheral tolerance,

the ultimate goal in organ transplantation, can be achieved by altering immune

reactivity through Th2-skewing, T-cell deletion or T-cell anergy, and the induction of

regulator T-cell responses. The role of DC in organ transplantation is multifaceted,

because of the coexistence of graft-derived DC from the donor and DC from the

recipient. The role of donor hematopoietic cell microchimerism in the outcome of

organ transplantation has been the subject of intense interest and debate since the

observations that microchimerism could be detected in lymphoid and non-lymphoid

tissues of successful human organ allograft recipients up to many years after

transplantation. These findings prompted the hypothesis that microchimerism provided

an essential basis for organ transplant tolerance. The inflammation triggered by

transplant surgery and necrosis due to ischemia/reperfusion injury is enough to initiate

maturation and migration of graft-resident and graft-infiltrating DC, which migrate as

passenger leukocytes out of the graft into the draining lymphoid tissues. Once in the

lymph nodes or spleen of the recipient, these passenger DC activate alloreactive naive

T cells and initiate acute graft rejection. Stimulation of DC through CD40 also may play

13

a role in DC mobilization after transplantation. CD40–CD40L interaction regulates DC

migration from peripheral tissue indirectly through induction of TNF-a secretion by DC.

In the transplantation setting, graft-infiltrating platelets, activated T cells, and mast cells

resident in the transplanted tissue may be possible sources of CD40L.

Following cell or organ transplantation, DC present antigen to T cells via the

direct or indirect pathways of allorecognition. The direct recognition pathway is mainly

involved in acute rejection, while the indirect pathway is closely related to chronic

rejection. However, more data showed that indirect recognition might play a more

important role in whole allograft rejection. In the direct pathway of allogeneic antigen

recognition, donor dendritic cells leave the graft and migrate towards recipient

lymphoid organs here they present allogeneic (non-self) MHC molecules to recipient

T cells. The vigorous T-cell proliferation that occurs in primary allogeneic mixed

lymphocyte culture (MLC) is caused mainly by direct allorecognition and is due to the

high comparative frequency of allospecific responder T cells (approx. 1 per 200). In the

indirect pathway, recipient DC internalize and process donor antigens (soluble MHC

molecules, fragments/blebs derived from donor apoptotic or necrotic cells, vesicles

exchanged between living cells). These are then presented to recipient T cells as a

restricted repertoire of immunodominant allogeneic peptides bound to self-MHC

molecules on the dendritic cell surface. Most allogeneic peptides derive from

hypervariable regions of donor MHC class II molecules, and to a lesser extent MHC

class I or minor MHC antigens. The frequency of recipient T cells specific for

allopeptides bound to self-MHC molecules is similar to the frequency of T cells

responding to nominal antigen peptides (<1 out of 10 000), and significantly lower than

that of T cells engaged in direct recognition.

There are many experimental models with different grafts and approaches to

therapy of allograft rejection by targeting donor or recipient DC (Table 1.). Various

attempts have been made to convert DC into tolerogenic APC such as treatment of DC

with interleukin-10 (IL-10) 80, transforming growth factor-β (TGF-β) or with low doses

of granulocyte–macrophage-colony-stimulating factor (GM-CSF) 81-83. These treatment

modalities have in common the ability to interfere with the proper maturation of DC.

Several studies indicate that the maturational state of DC is primarily controlled

by NFκB, which regulates a multitude of immunomodulatory genes (e.g., MHC class II,

14

DC SOURCE

APPROACH

TYPE OF DC

TRANSPLANT MODEL

Intravenous administration of DC generated in vitro from bone marrow precursors under specific culture conditions

Immature myeloid DC (expressing low levels of

Heart (mouse) Skin (mouse)

Donor (i.e. low GM-CSF, LPS, etc.) in rodents costimulatory molecules) Heart (rat) Pancreatic islet

(mouse) Intravenous administration of DC generated in vitro from

bone marrow precursors by pharmacological treatment (i.e. vitamin D3)

Immature myeloid DC and/or DC unable to produce IL-12p70

Skin (mouse) Pancreatic islets (mouse)

Intravenous, intraperitoneal, or intraportal administration Of DC generated from bone marrow precursors (mice) or peripheral monocytes (humans) and genetically engineered

Myeloid DC incubated with oligodeoxyribonucleotides (ODNs) or encoding a

Heart (mouse) Kidney (mouse) Human skin

(i.e. FasL, CTLA4-Ig, IL-10, and NFkB decoy ODN) in vitro

transgenic protein (SCID mouse)

Intravenous administration of specific DC subpopulation CD8α+(lymphoid-related) DC isolated from spleen of Flt3L-treated mice

Heart (mouse)

Total DC (CD8α- andCD8α+) isolated from spleen of Flt3L-treated mice

Aorta (mouse)

Plasmacytoid DC from blood of patients treated with G-CSF or isolated from spleen of Flt3L-treated mice

GVHD(human) Heart (mouse)

Intrathymic or intravenous injection of DC pulsed with donor MHC I peptide

Thymic or myeloid DC

Heart (rat) Pancreatic islets (rat)

Recipient

Pharmacologic manipulation (in vivo administration of deoxyspergualin; intravenous injection of recipient-type DC

Lymph node DC (primates) or myeloid DC

Kidney (nonhuman primates)

generated in vitro under IL-10, TGFβ, LPS) GVHD (mouse) Intravenous injection of DC generated in vitro from

recipient-type transgenic mice encoding donor MHC I allele Myeloid DC

Heart (mouse)

or infected with a rAd encoding a donor MHC I allele Intravenous administration of donor MHC+ apoptotic cells

Splenic DC (CD8α- and CD8α+)

Bone marrow(mouse) Heart (mouse)

Table 1: Approaches to therapy of allograft rejection by targeting donor or recipient DC

CD80, CD86and CD40) 84-85. The inhibition of NFκB activation not only hampers

the expression of costimulatory, maturational and major histocompatibility complex

(MHC) molecules 85-86, but is also involved in the induction of T-cell hyporeactivity.

Thus, DC treated with vitamin-D3, corticosteroids or proteasome inhibitors, which are

known to prevent nuclear translocation of NFκB, have been reported to induce a state of

T-cell tolerance both in vitro and in vivo 87-89. Likewise, significant prolongation of

organ allograft survival was achieved when allogeneic DC harboring NFκB decoy

oligonucleotides were administered 90-92. The inhibition of NFκB activity might then

disable also full maturation of DC, as alloreactive T cells are not activated by such DC,

which leads to an arrest of DC at an immature stage with low costimulatory molecule

expression, down-regulated stimulatory capacity and suppressed cytokine production

because of a lack of helper signal provision by T cells 77.

15

A major step towards the realization of allograft tolerance may represent the generation

of defined allogeneic APC facilitating graft acceptance and maintenance of T-cell

hyporeactivity. Several studies indicate that tolerizing APC share particularfeatures such

as low/absent IL-12 production, defective stimulatory capacity and high endocytosis

rate 93. Considering the in vivo application of designer DC defined and reproducible

conditions for their generation are required. Furthermore, administered DC should not

mature upon activating stimuli and the establishment of tolerance should not be

abrogated by conventional immunosuppressive protocols thus enabling their

incorporation into current therapies used to treat allograft rejection.

AIMS OF THE STUDY

Our aim was to generate tolerogenic monocyte-derived dendritic cells by

activating immature DC in the presence of different inhibitory substances in order to

obtain DC that exhibit phenotypical features and cytokine production of immature DC,

as well as defective stimulatory capacity for allogeneic T-cell responses. We aimed to

characterize these in vitro modulated-DC and to analyze the allogeneic tolerance

induced by these APC.

In this study three independent attempts had been made to convert DC into

tolerogenic APC such as treatment of DC with n-butyrate, NFκB inhibitor pyrrolidine

dithiocarbamate (PDTC) and Janus Kinase 3 inhibitor WHI-P-154. These treatments

have in common the ability to interfere with the proper maturation of DC. At first we

planned to use the bacterial metabolite n-butyrate, which occurs physiologically in high

concentrations in the gastrointestinal tract and has well-known anti-inflammatory

effects. Several studies indicate that the maturational state of DC is primarily controlled

by NFκB, which regulates a multitude of immunomodulatory genes. n-Butyrate

profoundly inhibits translocation of NFκB to the nucleus in LPS-stimulated DC,

suggesting that the impairment of nuclear translocation of this transcription factor by

16

n-butyrate may account for most features of the altered DC phenotype. To mimic this

physiologically occurring immunomodulatory phenomenon by n-butyrate we chose the

NFκB inhibitor pyrrolidine dithiocarbamate (PDTC) for the next experimental setting.

These two substances have similar features with regard to their immunomodulatory

capacity of DC via similar mechanism interfering the translocation of NFκB. Our aim

was to study the established T-cell tolerance and the influence of calcineurin inhibition

with Cyclosporine A (CsA) in the allogeneic hyporesponsiveness induced by PDTC-

modulated DC. For further investigation of this T-cell hyporesponsiveness we

established an ex vivo model in T cells from patients with renal allografts under CsA-

based immunosuppression in order to assess the ability of in vitro modulated-DC to

incorporate into a conventional immunosuppressive protocol without abrogation of the

tolerance by the clinical therapy. The third inhibitory substance, which was used in our

experiments is engaged in the CD40 signaling pathway in addition to its involvement in

common-gamma chain (cγ) signaling of cytokine receptors. Our next aim was to assess

the consequences of Janus Kinase 3 inhibition with WHI-P-154 during CD40-induced

maturation of monocyte-derived DC and to test the impact thereof on the induction of

T-cell hyporesponsiveness. At the end we aimed to test the influence of this inhibitory

agent on T cells stimulated by TCR triggering with CD3 and CD28 mAbs in the

presence of the Janus Kinase 3 inhibitor WHI-P-154.

In the current work we aimed to study the followings:

A.) „Bacterial Metabolite Interference with Maturation of Human Monocyte-Derived Dendritic Cells” 94

1.) To investigate how monocyte-derived dendritic cells respond to the bacterial

stimulus (lipopolysaccharide) when applied in the presence of n-butyrate with

regard to phenotypical and morphological changes, T-cell stimulatory capacity

and analysis of the DC antigen-uptake machinery.

17

B.) “Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor-Treated Dendritic Cells: Resistance to Calcineurin Inhibition” 95

1.) To generate tolerogenic monocyte-derived dendritic cells by activating

immature DC in the presence of NFκB inhibitor pyrrolidine dithiocarbamate

(PDTC) in order to obtain DC that exhibit phenotypical features and cytokine

production of immature DC.

2.) To characterize allostimulatory potential of PDTC-modulated dendritic cells in

primary mixed lymphocyte culture, as well as the expression of activation

markers and cytokine production of allogeneic T-cells.

3.) To induce allogeneic hyporesponsiveness by PDTC-modulated dendritic cells

and to analyze this state of allogeneic tolerance after restimulation in secondary

mixed lymphocyte culture.

4.) To study the influence of calcineurin inhibition with Cyclosporine A (CsA) in

functional T-cell responses elicited by different DC pretreatment protocols with

PDTC in primary and secondary mixed lymphocyte culture.

5.) To establish T-cell hyporesponsiveness with PDTC-modulated dendritic cells

in T cells from patients with renal allografts under Cyclosporine A (CsA)-based

immunosuppression.

C.) “Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” 96-98

1.) To explore the impact of Janus Kinase 3 (JAK3) inhibition with WHI-P-154

on the phenotype of monocyte-derived dendritic cells activated through CD40

engagement.

2.) To evaluate receptor-driven endocytosis, macropinocytosis and mannose

receptor expression of JAK3 inhibitor-treated dendritic cells.

3.) To investigate T-cell stimulatory potential of JAK3 inhibitor-treated dendritic

cells (DC) in primary mixed lymphocyte culture, to induce hyporesponsiveness

after restimulation in secondary mixed lymphocyte culture and to assess the

specificity and reversibility of this hyporesponsive state.

4.) To evaluate homotypical cell clustering of T cells activated with CD3 and CD28

mAbs in the presence of Janus Kinase 3 inhibitor WHI-P-154.

18

MATERIALS AND METHODS

Media and reagents

RPMI-1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 2 mM

L-glutamine, 100 µg/mL streptomycin, 100 U/mL penicillin and 10% fetal calf serum

(FCS; Hyclone, Logan, UT, USA) was used as culture medium. The sodium salt of

n-butyric acid, LPS (Escherichia coli 0111:B4), phorbol myristate acetate (PMA),

peptidoglycan and anti-Flag M2 mAbs (IgG1) were purchased from Sigma Chemie

GmbH Co. (Deisenhofen, Germany). Pyrrolidine dithiocarbamate (PDTC) and

recombinant human CD40L fused to a flag-tag were from Alexis (Alexis Co, San

Diego, CA and Lausen, Switzerland). Tumor necrosis factor- α (TNF-α) was purchased

from R and D Systems (Minneapolis, MN, USA). Monoclonal antibody (mAb) to CD3

(OKT3) was from Ortho pharmaceutical Corp. (Raritan, NJ, USA). CD28mAb (Leu-28)

was from Becton Dickinson (San Jose, CA, USA). Recombinant human GM-CSF

(rh-GM-CSF) was from Schering-Plough (Kenilworth,NJ, USA) and rh-IL-4 was from

Strathmann Biotech Gmbh (Hannover, Germany). Human recombinant IL-2 (rh-IL-2)

was from R and D Systems. Cyclosporine A was kindly provided by Novartis AG.

WHI-P-154 [4-(3'-Bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline], a

rationally designed inhibitor of JAK3, was obtained from Calbiochem (San Diego, CA).

Patients

Patients (n = 3) included in the present study had received a cadaveric kidney transplant

at least 2 years prior to evaluation of T-cell function. All patients had stable long-term

allograft function and were on Cyclosporine A-based triple immunosuppression

including low-dose prednisone and azathioprine/mycophenolate mofetil. Serum trough

Cyclosporine A (CsA) levels were between 80 and 120 ng/mL. The study was approved

by the local ethics committee at the medical faculty of the University of Vienna.

Cell isolation

Heparinized blood was obtained from adult healthy volunteers. Buffy coats of blood

donors were obtained through the courtesy of the Austrian Red Cross. Peripheral blood

19

mononuclear cells (PBMC) were isolated by density gradient centrifugation over

Lymphoprep (Nycomed Pharma AS, Oslo, Norway). For monocyte enrichment,

PBMC were depleted of T cells by sheep erythrocyte-rosetting and contained >85%

CD14-positive cells. Resting T cells were isolated by magnetic selection. Briefly,

freshly isolated PBMC were incubated with mAb to CD14 (RMO52), CD11b (Bear1),

CD20 (HRC20) and CD16 (3G8) at 4°C for 1 h (all antibodies were from Immunotech

S.A, Marseille, France). Each antibody was used at a final concentration of 1 µg/ml.

After extensive washing, cells with surface-bound antibodies were removed using

human anti-mouse immunoglobulin G (IgG)-coated magnetic beads (Dynal, Oslo,

Norway). The resulting population contained >98% CD3+ cells. For DC generation

from cadaveric kidney donors, spleens were aseptically removed. Then the spleen cells

were teased out and isolated by density gradient centrifugation. The mononuclear

population was then used for DC differentiation.

DC cultures

Monocytes from healthy donor or mononuclear cells from cadaveric spleens were

cultured in 24-well plates (Costar, Cambridge, MA, USA) at a cell density of

5x105 cells/mL in RPMI-1640/10% FCS medium at 37°C in a humidified CO2-

containing atmosphere. For induction of differentiation, cells were cultured for 5 d with

50 ng/mL rh-GM-CSF and 10 ng/mL rh-IL-4. To induce final maturation LPS (100

ng/mL), CD40L (200 ng/mL) followed by cross-linking with 4.4 µg/mL anti-FLAG

mAbs (Sigma), peptidoglycan (10 µg/mL) or TNF-α (20 ng/mL) was then added for

48 h. Dendritic cell stimulation was performed in the presence or absence of different

concentrations of n-butyrate, PDTC and WHI-P-154 before activation with the

respective DC maturation stimuli.

Flow cytometry and cytokine production

For evaluation of marker expression, cells were incubated with the respective FITC- or

PE-conjugated mAb for 30 min. For control, nonbinding isotypematched FITC- and

PE-conjugated mice IgGs were employed. Cells were analyzed on a FACScalibur flow

cytometer (Becton Dickinson, San Jose, CA). FITC-labeled anti-CD40 (IgG1, clone

LOB7/6) and anti-HLA-ABC (IgG2a, clone W6/32) were from Immunotech S.A

20

(Marseille, France). FITC-conjugated anti-CD25 (IgG1a, clone 2A3), anti-CD69

(FN50), anti-HLA-DR (IgG2a, L243), anti-CD83 (IgG1, clone HB15e) and anti-CD14

(IgG2b, clone MOP9) were from Becton Dickinson. FITC-labeled anti-CD32 (IgG2b,

clone IV.3) was obtained from Medarex (Annandale, NJ). The following PE-labeled

mAbs (obtained from Becton Dickinson) were used: anti-CD80 (IgG1, L307.4),

anti-CD86 (IgG2b, clone IT2.2) and anti-mannose receptor (IgG1, clone 19).

For measurement of cytokines, supernatants were harvested at the indicated time-points.

IL-2, IFN-γ, TNF-α and IL-12p40 were measured by sandwich ELISA using matched

pair antibodies. Capture as well as detection antibodies were obtained from R and D

Systems. Antibodies to human TNF-α were from PharMingen (San Diego, CA, USA).

Standards consisted of human recombinant material from R and D Systems.

Endocytosis assay

To determine mannose receptor (MR)-mediated endocytosis, 1x106 cells/ml were

incubated in medium with FITC-labeled dextran [molecular weight (Mr) 40,000; Sigma

Chemie GmbH Co.] at a concentration of 1 mg/ml. After an incubation period of 60 min

at 37°C or on ice as a control, cells were washed extensively with ice-cold phosphate-

buffered saline (PBS) and analyzed on a FACScalibur. Fluid-phase endocytosis was

measured via cellular uptake of lucifer yellow (LY; Sigma Chemie GmbH Co.) and was

analyzed by flow cytometry.

Allogeneic mixed lymphocyte culture (MLC) and tolerance assays

Stimulator cells as indicated were irradiated (3000 rad, 137Cs source) and added

at the indicated cell numbers to 1x105 allogeneic T cells in 96-well culture plates in

RPMI-1640 medium supplemented with 10% FCS (total volume 200 µL/well). After 4

days, cells were pulsed with 1 µCi [3H]-thymidine (ICN Pharmaceuticals, Irvine, CA).

After another 18 h, the cells were harvested on glass-fiber filters (Packard, Topcount,

Meriden, CI, USA) and DNA-associated radioactivity was determined using a

microplate scintillation counter (Packard). DNA synthesis was expressed as mean cpm

of triplicate cultures. To assess cytokine production in MLC, DC were cocultured with

1x106 purified T cells in 24-well plates. For secondary MLC, first 1x106 purified T cells

were mixed with 1x105 DC subjected to the various treatment protocols (culture volume

21

1 mL in 24-well plates). After washing at day 7, cells (5x104/well) were added to

irradiated DC (5x103/well) from the original donor or unrelated third party donors as a

third-party control in round-bottom 96-well tissue culture plates. Dendritic cells for

restimulation cultures were generated from cryopreserved monocytes and matured by

100 ng/mL LPS for 48 h. For assessment of potential suppressor cells, restimulation

cultures using T cells initially primed with mDC were cocultured with activated DC

from the same donor in the presence/absence of T cells exposed to PDTC-treated DC

stimulated with LPS. For comparison, coculture experiments were performed with

T cells that were incubated with immature DC, immature DC-treated with PDTC or

LPS-treated DC. To evaluate the inhibitory capacity of PDTC-treated DC, PMLC with

T cells and mDC at different ratios were set up in the presence or absence of one of the

respective APC populations. For secondary MLC, 1x106 purified T cells or PBMC

(from renal allografts recipients) were mixed with 1x105 DC subjected to the various

treatment protocols (culture volume 1 mL). After washing at day 7, the cells

(5x104/well) were re-plated in 200 µL culture medium with irradiated cells (5x103/well)

from the original donor or unrelated third party donors as a third party control. DC for

restimulation cultures were generated from monocytes that were cryopreserved

immediately after monocyte isolation. Examination of IL-2 responsiveness of

hyporesponsive T cells was performed in two ways. First, in the respective MLC, 20

U/mL rhIL-2 was added simultaneously with the donor APC to secondary cell cultures.

Second, cells were washed 7 days after initiation of primary MLC, exposed to 20 U/mL

rhIL-2 for 3 days, washed again and then restimulated with the respective APC. DNA

synthesis in secondary MLC was assessed after 3 days. Global T-cell reactivity in

secondary MLC was assayed by stimulation with 10-7 M PMA plus 1 µg/mL CD3 mAb.

Morphological cell analysis

Immature DC were stimulated with LPS in the presence or absence of n-butyrate.

After 4 h, the cells were analyzed by light microscopy on a Leitz Aristoplan microscope

(Wetzlar, Germany). To perform scanning electron microscopy, cells were fixed onto 24

multiwell plates using 2 ml 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for

90 min at 4°C. After rinsing in cacodylate buffer, cells were postfixed with 1% OsO4 for

15 min. The samples were dried in a gradual series of ethanol, transferred to tertiary

22

butanol (Merck, Darmstadt, Germany) for 60 min, and freeze-dried. The samples were

examined in a Stereoscan S90 scanning electron microscope (Cambridge Instruments,

Cambridge, UK). After CD3 and CD28 stimulation (8h) in RPMI 1640 (supplemented

with penicillin (100U/ml), streptomycin (100 µg/ml), glutamine (2mM), and 10% fetal

calf serum) purified T cells were analyzed by light microscopy on a Leitz Aristoplan

microscope (Wetzlar, Germany).

Statistics

Comparisons were performed using the Student’s t-test. A p-value < 0.05 was

considered statistically significant.

RESULTS

A.) “Bacterial Metabolite Interference with Maturation of Human Monocyte Derived Dendritic Cells” 94

1.) Phenotypical and functional impairment of DC maturation by n-butyrate

Because terminal DC maturation critically determines the outcome of

antimicrobial immune responses, we evaluated the impact of n-butyrate on this stage of

the DC life cycle. We first investigated the phenotypical changes in immature DC

exposed to LPS under the influence of this bacterial metabolite. As shown in Figure 2,

addition of LPS to immature DC resulted in the neoexpression of the maturation

markers CD83 and CD25, the α-subunit of the IL-2 receptor, and the upregulation of

major histocompatibility complex (MHC) class I and II and costimulatory molecules.

However, concomitant treatment with n-butyrate yielded DC with a markedly

altered phenotype (Figures 2 and 3). Expression of CD25 and CD83 and of critical

23

costimulatory molecules, i.e., CD40, CD80, and CD86, was reduced substantially.

Furthermore, a clear suppression of the up-regulation of MHC class I and II antigens by

n-butyrate was observed.

Figure 2: n-Butyrate prevents DC maturation.

Monocytes were cultured for 7 days with GM-CSF (50 ng/ml) plus IL-4 (10 ng/ml). Subsequently, these immature DC (5x105/ml) were activated with LPS (100 ng/ml) with or without 1 mM n-butyrate for 48 h. Open profiles (fine line) in the upper panel represent a staining pattern with an isotype control, and open profiles (bold line) represent staining with mAb of the indicated specificity in immature DC before LPS activation. In the lower panel, surface expression results from cultures containing LPS (open profiles with bold line) or LPS plus 1 mM n-butyrate (solid profiles) are depicted. Open profiles (fine line) represent the staining pattern with an isotope control. Data are representative of three independent experiments.

A striking feature of activated DC is the occurrence of large cell clusters arising a few

hours after addition of LPS (Figure 3). In comparison, little or no cell clustering occurs

in cultures activated in the presence of n-butyrate (Figure 3). Moreover, the majority of

cells in the n-butyrate-treated cultures imposed with widespread cytoplasmic

projections. In addition to the reduced expression of DC maturation markers, we found

a significant dose-dependent reduction of the allostimulatory capacity of immature DC

treated with LPS and n-butyrate (Figure 4). A series of experiments confirmed that n-

butyrate does not impede the viability of DC; i.e., we found identical numbers of cells in

cultures treated with or without n-butyrate for 48 h. More importantly, FACS analysis

measuring propidium-iodide uptake confirmed the same number of viable cells in

n-butyrate and nontreated cultures (unpublished results). Thus, the effects of this

24

compound resulted from specific action rather than from unspecific effects, such as cell

death.

Figure 3: Effect of n-butyrate on maturation-associated clustering of DC.

Immature DC were stimulated with LPS (100 ng/ml) in the absence (A, C) or presence (B, D) of 1 mM n-butyrate. After 4 h of cultivation, cells were analyzed by photomicrographs using light microscopy (A, B) or scanning electron microscopy (C, D). The insert in photomicrograph D shows a bulb-like ending of a cytoplasmic projection in n-butyrate-treated cells. Similar results were obtained in four different experiments.

n-Butyrate inhibited the expression of the antigen-uptake molecules CD32 and mannose

receptor (MR). Assessment of macropinocytosis, a special type of actin-dependent

fluid-phase uptake, revealed impaired internalization of lucifer yellow (LY) but

a less pronounced inhibition of receptor-mediated endocytosis of dextran molecules

(Figure 5).

25

Figure 4: Reduced T-cell stimulatory capacity of DC matured in the presence of n-butyrate.

Immature DC were stimulated with LPS (100 ng/ml) with or without n-butyrate at the indicated concentrations. After 48 h, the cells were washed extensively, irradiated, and cocultured with 1x105 allogeneic T cells at the indicated ratios. DNA synthesis was assessed at day 5. Shown are the means ± SE of six to nine experiments. *, P < 0.01.

Figure 5: Analysis of the DC antigen-uptake machinery of DC differentiated in the presence of n-butyrate. Monocytes were cultured for 7 days with GM-CSF (50 ng/ml) plus IL-4 (10 ng/ml) in the absence (Ctrl-DC) or presence (nB-DC) of 1 mM n-butyrate. For assessment of CD32 or MR expression, cells were stained with the respective antibodies and analyzed by flow cytometry. For functional endocytosis assays, cells were pulsed with 1 mg/ml FITC-dextran (DEX) or 1 mg/ml LY for 60 min. Open profiles (fine line) show the background uptake on ice; open profiles with bold line (Ctrl-DC) and solid profiles (nB-DC) indicate antigen uptake of cells at 37°C.

26

B.) “Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor- Treated Dendritic Cells: Resistance to Calcineurin Inhibition” 95

1.) Arrest in DC maturation by treatment with the NFκB inhibitor PDTC

Immature DC and maturation-resistant DC have recently been shown to be

effective inducers of T-cell tolerance in vitro and in vivo 74,83,99. In order to obtain

potentially tolerogenic DC, we aimed to generate DC that exhibit phenotypical features

of immature DC and that are resistant to further activation stimuli. Since triggering

of the DC maturation program represents an irreversible step of DC development,

we employed immature DC that were treated with LPS in the presence of an effective

agent interfering with this activation signal. Because transition of immature DC to the

mature stage is primarily controlled by activation of the transcription factor NFκB 84-

85,100-101 we have chosen the dithiocarbamate NFκB inhibitor PDTC. Dithiocarbamates

such as PDTC have been shown to block NFκB-dependent cytokine production in

human myeloid cells such as monocytes 102-103. As seen in Figure 6A, activation of

immature DC by LPS leads to neoexpression of the DC maturation marker CD83,

upregulation of major histocompatibility complex (MHC) class II antigens and

costimulatory molecules. In contrast, concomitant treatment with PDTC yielded DC

with a markedly altered phenotype. Thus expression of CD83 and of critical

costimulatory molecules such as CD40, CD80 and CD86 was reduced substantially.

Likewise, up-regulation of MHC class II antigens was found to be clearly suppressed by

PDTC. Decreased survival as assessed by propidium-iodide staining was seen only at

doses ≥50 µM (data not shown). Analyzing cytokine production during DC maturation,

we observed a clear inhibition of the production of the immunostimulatory cytokines

IL-12 and TNF-α by PDTC (Figure 6B), which demonstrates the ability of

dithiocarbamate based NFκB inhibition to effectively interfere with the LPS-triggered

process of DC maturation. We further analyzed the ability of PDTC to block other

activation signals. As seen in Figure 7A, PDTC was also effectively abrogating

phenotypical DC maturation initiated by exposure to cross-linked CD40L,

peptidoglycan and TNF-α. Furthermore, also upon other activation stimuli involving

CD40, toll-like-receptor-2 (TLR)-2 and pro-inflammatory cytokine signaling,

27

Figure 6: Treatment with the NFκB inhibitor pyrrolidine dithiocarbamate (PDTC) prevents the phenotypic changes associated with dendritic cell (DC) maturation.

(A) Monocytes were cultured for 7 d with granulocyte–macrophage-colony-stimulating factor (GM-CSF) (50 ng/mL) and interleukin-4 (IL-4) (10 ng/mL). Subsequently, these immature DC (iDC, 5x105/mL) were activated with LPS (100 ng/mL) for 48 h after preincubation with or without 10 µM PDTC for 2 h. Open profiles (fine line) in the upper panel represent the staining pattern with an isotype control and open profiles (bold line) represent staining with mAb of the indicated specificity in immature DC before LPS stimulation. In the lower panel, surface expression results from cultures containing LPS (open profile with bold line) or LPS plus 10 µM PDTC (solid profiles) are depicted. Open profiles (fine line) represent the staining pattern with an isotype control. Data are representative of three independent experiments. (B) DC were cultured as described above and cell-free supernatants were collected 48 h after endotoxin stimulation and analyzed by ELISA. Mean cytokine levels ±SEM obtained from three independent experiments are depicted.*level of significance, p < 0.05 for comparison to cultures in the absence of PDTC.

28

we observed a significant inhibition of cytokine production (Figure 7B). These findings

indicate maturation resistance of such modulated DC, which prompted us to further

evaluate their tolerogenic properties.

Figure 7: PDTC blocks DC activation to various maturation stimuli.

(A) After differentiation to iDC, cells were activated with LPS (100 ng/mL), peptidoglycan (Pg, 10 µg/mL), tumor necrosis factor-α (TNF-α) (20 ng/mL) or CD40L (200 ng/mL) followed by antiflag crosslinking (4.4 µg/mL), for 48 h after pre-incubation with or without 10 µM PDTC for 2 h. Open profiles (fine line) represent the staining pattern with an isotype control and open profiles (bold line) represent staining with mAb of the indicated specificity. Data are representative of three independent experiments. (B) DC were cultured as described above and cell-free supernatants were collected 48 h after respective stimulation and analyzed by ELISA. Mean cytokine levels + SEM obtained from three to five independent experiments are depicted. *level of significance, p<0.05 for comparison to cultures in the absence of PDTC; NA: not applicable because of exogenous TNF-α addition.

29

2.) PDTC-modulated DC exhibit defective stimulatory capacity for allogeneic T-cell responses

To get a first hint towards the immunomodulating properties of PDTC-

modulated DC, we tested their allostimulatory capacity in MLC. As depicted

in Figure 8A, pretreatment of DC with PDTC induced a DC population with a low

stimulatory capacity. Analyzing the production of distinct T cell-derived cytokines, we

found a vigorous production of IL-2 and IFN-γ from T cells stimulated with mature DC.

In contrast, T cells challenged with modulated DC exhibited a dose-dependent

suppression of IL-2 and IFN-γ production (Figure 8B). To get further insight into the

Figure 8: PDTC-modulated DC fail to support full allogeneic T-cell activation.

Immature DC (iDC), LPS-treated DC (mDC) and PDTCmodulated DC stimulated with LPS (PDTC-DC) were generated as described in Materials and Methods and after 48 h the cells were washed and harvested, irradiated, and cocultured with allogeneic T cells (5x105) at the indicated ratios. (A) T-cell proliferation was assessed by DNA synthesis at day 5 and (B) T-cell cytokines were harvested on day 3 (IFN-γ) and 4 (IL-2) after initiation of the MLC and analyzed by ELISA. Results are representative of at least three independent experiments. (C) Analysis of activation marker expression on allogeneic T cells was determined by flow cytometry 48 h after initiation of the MLC. Data are represented as dot blots and percentages in the upper right quadrants indicate percent positivity of the respective markers in CD3-positive lymphocytes. Similar results were obtained in three other independent MLC.

30

activation profile of the responding allogeneic T-cell population, we analyzed surface

molecules indicative of proper T-cell activation such as CD69 and CD25. As shown in

Figure 8C, T-cells challenged with modified DC did not express CD69, similarly

expression of CD25 was profoundly impaired in the respective cell cultures.

To investigate whether PDTC-modulated DC induce T-cell apoptosis, we stained

allogeneic T cells with Annexin V and propidium iodide. After incubation of allogeneic

T cells for 3 and 6 d after initiation of the MLC no increase in cell death as compared

with control MLC was observed (data not shown).

3.) Induction of allogeneic hyporesponsiveness by PDTC-modulated DC

To further determine the impact of the observed lack of allogeneic T-cell

proliferation in primary MLC with PDTC-modulated DC, we analyzed the functional

outcome of T cells challenged with this subset of DC compared with T cells stimulated

with mature and immature DC after restimulation. Therefore, allogeneic T cells were

cocultured with the respective DC for 7 d, washed and incubated with medium alone for

another 24–48 h before they were restimulated. While allogeneic T cells pre-cultured

with immature or mature DC readily responded to antigenspecific restimulation

(Figure 9A), T cells exposed to PDTC-modulated DC did not proliferate upon challenge

with immature or even fully mature DC from both original and third party donor DC

(Figure 9A). In contrast, APC-independent T-cell stimulation with PMA plus OKT-3

was unaffected (Figure 9A). To assess the possibility to revert the hyporesponsive state

exerted by PDTC-modulated DC, rIL-2 was added to secondary MLC. As depicted in

Figure 9B, rIL-2 partially restored proliferative responses towards allogeneic DC in

MLC containing PDTC-modulated DC while it induced an unspecific increase in

secondary proliferative responses. In the absence of T-cell receptor (TCR)-mediated

activation, rIL-2 alone induced a low grade of proliferation in tolerant T cells.

In order to demonstrate specificity for maturation-resistant DC to induce T-cell

hyporesponsiveness, we exposed immature DC to PDTC and then used these cells to

stimulate allogeneic T cells. Interestingly, while such cells induced a T-cell response

comparable to immature DC or maturation-resistant DC in primary MLC (Figure 9D),

secondary stimulation revealed an absence of hyporesponsiveness induction by

31

PDTC-treated immature DC (Figure 9C). To evaluate the possibility of suppressor cells

mediating the observed hyporesponsiveness exerted by PDTC-modulated DC, the

impact of T cells recovered from hyporesponsive T-cell cultures on MLC with the

specific donor cells was analyzed. As shown in Figure 9C, hyporesponsive T cells were

unable to significantly suppress allogeneic activation of syngeneic T cells and allowed

T-cell responsiveness comparable to T cells previously exposed to immature DC or

immature DC treated with PDTC. For the assessment of the inhibitory capacity of

PDTC-treated DC, allogeneic stimulation of T cells with mature DC was performed in

the presence or absence of PDTC-modulated DC. As depicted in Figure 9D such cells

were unable to suppress alloresponsiveness to mature DC from the same donor. In this

respect they behaved similar to immature DC or immature DC exposed to PDTC.

As expected, addition of mature DC resulted in increased stimulation in cultures

set up at a stimulator/responder ratio of 1:40.

4.) CsA does not counteract the suppressive state induced by PDTC-modulated DC

While CsA is the current immunosuppressant for the prevention of renal

allograft rejection, many studies have demonstrated that calcineurin inhibition prevents

the induction of stable tolerance in several experimental models of organ transplantation 104-106. The finding of a profound state of T-cell hyporeactivity by maturation-resistant

DC prompted us to study whether calcineurin inhibition with CsA influences the

functional T-cell responses elicited by different DC pretreatment protocols. Addition of

CsA to primary MLC led to an inhibition of the allogeneic T-cell proliferation

regardless of the mode of APC pretreatment (Figure 10A). However, as shown in Figure

10B,C, the presence of CsA had no influence on the proliferative response of T cells in

secondary cultures when they were stimulated with immature or mature DC in primary

MLC. Interestingly, secondary MLC revealed that the presence of CsA during

alloantigenic priming did not influence the induction of the hyporesponsive state by

PDTC-modulated DC.

32

Figure 9: Induction of allogeneic T-cell hyporesponsiveness by PDTC-modulated DC. Primary MLCs were set up by incubating immature DC (iDC), immature DC plus PDTC (iDC + PDTC), LPS-treated DC (mDC) and PDTC-modulated DC stimulated with LPS (PDTC-DC) with allogeneic T cells as indicated. Seven days after initiation of the primary MLC the cells were washed and restimulated (5x104/well) with irradiated cells (5x103/mL) from the original donor or unrelated third party donors or phorbol myristate acetate (PMA) (1 µM) plus anti-CD3 mAb OKT-3(1 µg/mL). T-cell proliferation was measured after 3d by [3H]-thymidine incorporation for the last 18 h of the culture period. Results are representative of at least three independent experiments. Restimulation cultures were performed in the absence (A) or presence (B) of rIL-2 (20 U/mL). (C) To test for the presence of potential T regs, restimulation cultures using T cells initially primed with mDC (5x104/mL) were cocultured with mDC (5x103/mL) from the same donor in the presence or absence of T cells (5x104/mL) preincubated with one of the four different APC populations. (D) To evaluate the inhibitory capacity of PDTC-treated DC pMLC with T cells (5x104/mL) and mDC were set up in the presence or absence of one of the four different APC populations (5x103/mL).

33

Figure 10: Effect of Cyclosporine A (CsA) treatment on allogeneic hyporesponsiveness induced by PDTC-modulated DC.

Immature DC (iDC), LPS-treated DC (mDC) and PDTC-modulated DC stimulated with LPS (PDTC-DC) were generated as described in Materials and Methods and after 48 h the cells were washed and harvested, irradiated, and cocultured with 5x105/mL allogeneic T cells at an APC/T cell ratio of 1:10 in the absence or presence of 100 ng/mL CsA. Subsequently secondary MLC were set up as described in Figure 8. T-cell proliferation was assessed by DNA synthesis on day 5 in primary MLC and on day 3 in secondary MLC. Results of pMLC (A) and restimulation cultures using cells from pMLC in the absence (B) or presence (C) of CsA are shown. Results are representative of at least three independent experiments.

5.) T-cell hyporesponsiveness with PDTC-modulated DC in T cells from patients with renal allografts under calcineurin inhibitor-based immunosuppression

To further examine the possibility that a state of T-cell hyporesponsiveness can

be established with PDTC-modulated DC in T cells from patients with renal allografts

under CsA-based immunosuppression, freshly isolated T cells were challenged with

various DC subgroups generated from frozen spleen cells of the respective kidney

donors. While the primary MLC response was profoundly reduced using PDTC-

modulated DC (data not shown), a conspicuous state of T-cell hyporesponsiveness was

observed in restimulation cultures supplemented with allogeneic stimulator cells

34

(Figure 11). In contrast, T-cell responsiveness was unimpaired when various polyclonal

stimuli were employed (Figure 11). Similar results were obtained when T cells from

patients with FK506 instead of CsA-based immunosuppression were exposed to in vitro

modulated DC (data not shown). Hence, it is concluded that induction of T-cell

hyporeactivity towards alloantigens by maturation-resistant DC is not dependent on

calcineurin activation and furthermore is feasible in T cells exposed to CsA in vivo.

Figure 11: Feasibility of modulating alloresponsiveness in T cells from patients with renal allografts by altered DC.

Secondary cultures were set up as described in Figure 8. For the generation of DC from kidney donors, spleen cells frozen at the time of organ harvesting were differentiated into the respective DC populations as described in Materials and Methods before they were cocultured with allogeneic T cells. T-cell proliferation was assessed by DNA synthesis 3 d after initiation of the secondary culture. Data represent mean ±SD from triplicate wells. Depicted is one representative experiment out of four different experiments performed: mDC, LPS-treated DC; PDTC-DC, PDTC-modulated DC stimulated with LPS.

35

C.) “Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” 96-98

1.) Targeting JAK3 disrupts CD40-triggered DC maturation

We explored the impact of JAK3 inhibition on the phenotype of DC activated

through CD40 engagement. Such activated DC demonstrated a strong up-regulation of

the costimulatory molecules CD80, CD86 and CD40, the MHC class I and II molecules

as well as of the specific DC maturation marker CD83, when compared with immature

DC. In contrast, DC activated through CD40 in the presence of the JAK3 inhibitor

maintained an immature phenotype (Figure 12A,B). Importantly, JAK3 inhibitor-treated

DC did not revert to a monocyte/macrophage stage as demonstrated by the lack of

expression of CD14 (data not shown).

2.) JAK3 inhibition does not interfere with the activation-associated down-regulation of the DC-antigen uptake machinery

Immature DC are known to effectively internalize antigens through

macropinocytosis and receptor-mediated endocytosis 107-108. When DC mature they

down-regulate their antigen-capturing capability and simultaneously increase their

antigen-presenting and costimulatory efficiency. In the next set of experiments we

analyzed whether JAK3 targeting might affect down-regulation of the DC-antigen

capture machinery upon CD40-ligation. As shown in Figure 13A receptor-driven

endocytosis and macropinocytosis, which were evaluated by incorporation of FITC-

labeled dextran or of lucifer yellow (LY), respectively, were more pronounced in

immature DC than in CD40-activated DC. Interestingly, endocytosis and macro-

pinocytosis was similar in JAK3 inhibitor-treated DC and mature DC (Figure 13B).

Furthermore, CD40 ligation led to a reduction in mannose receptor expression in DC

(Figure 13B). As observed for antigen uptake, JAK3 inhibitor-treated DC behaved

similar to mature DC with regard to mannose receptor expression (Figure 13B).

36

(A)

(B)

Figure 12: JAK3 targeting prevents the phenotypic alterations induced by CD40 ligation.

(A) Monocytes were cultured for 7 days with granulocytemonocyte colony stimulating factor (GM-CSF) (50 ng/mL) plus IL-4 (10 ng/mL). Subsequently these immature DC (5x105/mL) were activated with CD40L (200 ng/mL) with or without the JAK3 inhibitor (10 µg/mL) for 48 h. Open profiles (fine line) in the upper panel represent staining pattern with an isotype control, and open profiles with bold line staining with mAbs of the indicated specificity in immature DC before CD40 activation. In the lower panel surface expression in cultures containing CD40L (open profiles with bold line) or CD40L plus 10 µg/mL of JAK3 inhibitor (black profiles) is depicted. Data are representative of six independent experiments. (B) Percent responses ± SEM calculated from median fluorescence intensities (MFI) from six experiments are shown. Expression of the respective surface markers in mature and JAK3 inhibitor-treated DC was related to expression in immature DC. Median fluorescence intensities of immature DC in the absence of the JAK3 inhibitor were as follows: isotype control-FITC: 5.9 ± 1.8, CD40-FITC: 202 ± 63, CD83-FITC: 12 ± 3, MHCI-FITC: 457 ± 85, MHC II-FITC: 808 ± 272, isotype control-PE: 4.4 ± 1.2, CD86-PE: 79 ± 20, CD80-PE: 92 ± 23. Open bars, immature DC; hatched bars, DC activated by CD40 ligation in the absence of the JAK3 inhibitor; solid bars, DC activated by CD40 ligation in the presence of the JAK3 inhibitor.

37

Figure 13: Dendritic cells (DC) activated by CD40 ligation down-regulate their antigenuptake machinery in the presence of Janus kinase 3 (JAK3) inhibition.

(A) Immature DC or DC activated via CD40 in the absence or presence of the JAK3 inhibitor (10 µg/mL) were pulsed with 1 mg/mL of FITC-dextran (DEX) or 1 mg/mL of Lucifer yellow (LY) for 60 min. Open pro-files with fine line show the background uptake on ice and open profiles with bold line indicate antigen-uptake at 37 °C. Data are representative of four independent experiments. (B) Summary of four experiments ± SD indicating the behavior of immature DC or of DC stimulated via CD40 in the presence or absence of 10 µg/mL of JAK3 inhibitor. MR, mannose receptor expression.

38

3.) Prevention of T-cell stimulatory capacity and induction of antigen-specific hyporeactivity by JAK3 inhibited DC

Mature DC are known to be the strongest stimulators of MHC-mismatched

peripheral blood T-lymphocytes. The finding that the response of DC to CD40

ligation was profoundly modified in the presence of JAK3 inhibition prompted us to

investigate the T-cell stimulatory potential of these cells. CD40 triggering of DC

induced a vigorous T-cell stimulating capacity compared with immature DC (Figure

14A). Treatment of DC with the JAK3-inhibitor during CD40 triggering resulted in an

allostimulatory capacity similar to immature DC (Figure 14A). Regarding cytokine

production lower levels of IL-2 and IFN-γ were detected in cultures stimulated with

JAK3 inhibitor-treated DC than in cultures stimulated with mature DC (data not

shown). To address the functional outcome of defective immunostimulation by JAK3-

inhibited DC, T cells initially primed with allogeneic immature DC or DC that had been

activated by CD40-ligation in the presence or absence of JAK3 inhibition were

restimulated with LPS-matured DC from the original donor and from unrelated third-

party donors. Figure 14B shows that preculturing allogeneic T cells with CD40-

triggered DC led to a strong secondary T-cell alloresponse. In contrast, preculture with

CD40-triggered DC exposed to the JAK3 inhibitor resulted in a dramatically reduced

responsiveness of allogeneic T cells upon restimulation. Priming of allogeneic T cells

with immature DC resulted in a comparably low extent of secondary T-cell reactivity

(Figure 14B). Restimulation with allogeneic mature DC from unrelated third-party

donors was also associated with defective T-cell proliferation in JAK3-treated DC or

immature DC-primed T cells, indicating nonspecific T-cell hyporesponsiveness induced

by both APC populations (Figure 14C). For the assessment of the reversibility of the

hyporesponsive state of allogeneic T cells challenged with JAK3 inhibitor-pretreated

DC, rIL-2 was added to secondary MLC. As shown in Figure 15, addition of IL-2 led to

a complete restoration of the defective T-cell proliferation of T cells coincubated with

JAK3 inhibitor-pretreated DC in primary MLC.

39

Figure 14: Reduced stimulatory activity of dendritic cells (DC) matured in the presence of Janus kinase 3 (JAK3) inhibition and induction of a state of T-cell hyporeactivity. For primary MLC, (A) immature DC (5x105/mL) were activated with rh-CD40L and anti-FLAG mAbs in the presence or absence of the JAK3 inhibitor (10 µg/mL). After 48 h, the cells were washed extensively, irradiated, and cocultured with 1x105 purified allogeneic T cells at the indicated ratios. DNA synthesis was assessed at day 5. Shown are the means ± SEM of six to nine independent experiments. *p < 0.01 for comparison of alloresponses induced by DC activated by CD40L in the presence or absence of the JAK3 inhibitor. For restimulation cultures, purified T cells precultured with immature DC, activated DC or JAK3 inhibitor-treated DC were harvested after 5 days. Then the T cells were restimulated with mature DC from (B) the original donor or (C) third-party donors as described in Materials and Methods. DNA synthesis was assessed at day 3. Data are expressed as mean of five different experiments ± SEM. Open bars, immature DC; hatched bars, DC activated by CD40 ligation in the absenceof the JAK3 inhibitor; solid bars, DC activated by CD40 ligation in the presence of the JAK3 inhibitor.

Figure 15: Reversal of T-cell hypo-responsiveness by exogenous IL-2. T cells were challenged with immature dendritic cells (DC) (white bars), CD40-triggered DC (hatched bars), or DC activated by CD40 ligation in the presence of the Janus kinase 3 (JAK3) inhibitor (black bars) and restimulated with monocytes from the original donor with IL-2 (20 U/mL) or without.

40

4.) Effect of JAK3 Inhibitor Treatment on Homotypic T-Cell Aggregation

We evaluated homotypical cell clustering, which is a typical hallmark of

activated T cells and depends on TCR-induced induction of integrin clustering and

integrin avidity changes 109. Although T cells activated with CD3 and CD28 mAb

exhibited prominent cell clusters 8 hr after initiation of the culture, JAK3 inhibitor-

supplemented cultures were completely devoid of any T-cell clusters (Figure 16).

Figure 16: JAK3 targeting blocks phenotypical hallmarks of T-cell activation.

Effect of JAK3 targeting on the activation-associated homoaggregation of human T cells. Purified T cells were left unstimulated in the absence (upperleft) or presence of 10 µg/mL WHI-P-154 (upper right) or stimulated with CD3 (1 µg/mL) and CD28 (2.5 µg/mL) mAb in the absence (lower left) or presence of 10 µg/mL WHI-154 (lower right) for 8 hr. Data are representative of four different donors.

41

SUMMARY OF THE RESULTS

A.) “Bacterial Metabolite Interference with Maturation of Human

Monocyte-Derived Dendritic Cells” 94

1.) Phenotypical and functional impairment of DC maturation by n-butyrate Expression of CD25 and CD83 and of critical costimulatory molecules, i.e.,

CD40, CD80, and CD86, was reduced substantially. A clear suppression of the

up-regulation of MHC class I and II antigens was observed. Little or no cell

clustering occured in cultures, moreover, the majority of cells imposed with

widespread cytoplasmic projections. We found a significant dose-dependent

reduction of the allostimulatory capacity. The expression of the antigen-uptake

molecules CD32 and mannose receptor was inhibited. Assessment of

macropinocytosis revealed impaired internalization of lucifer yellow but a less

pronounced inhibition of receptor-mediated endocytosis of dextran molecules.

B.) “Hyporesponsiveness in Alloreactive T-Cells by NFκB Inhibitor-Treated Dendritic Cells: Resistance to Calcineurin Inhibition” 95

1.) Arrest in DC maturation by treatment with the NFκB inhibitor PDTC Expression of CD83 and of critical costimulatory molecules such as CD40,

CD80 and CD86 was reduced substantially. Up-regulation of MHC class II

antigens was found to be clearly suppressed. We observed a clear inhibition of

the production of the immunostimulatory cytokines IL-12 and TNF-α.

2.) PDTC-modulated DC exhibit defective stimulatory capacity for allogeneic T-cell responses

Pretreatment with PDTC induced a DC population with a low stimulatory

capacity. T-cells challenged with modified DC did not express CD69, similarly

expression of CD25 was profoundly impaired. We found a dose-dependent

suppression of IL-2 and IFN-γ production.

42

3.) Induction of allogeneic hyporesponsiveness by PDTC-modulated DC

T cells exposed to PDTC-modulated DC did not proliferate upon challenge with

immature or even fully mature DC from both original and third party donor DC.

In contrast, APC-independent T-cell stimulation with PMA plus OKT-3 was

unaffected. Recombinans human IL-2 partially restored proliferative responses

towards allogeneic DC while it induced an unspecific increase in secondary

proliferative responses. In the absence of T-cell receptor (TCR)-mediated

activation, IL-2 alone induced a low grade of proliferation in tolerant T cells.

Hyporesponsive T cells were unable to significantly suppress allogeneic

activation of syngeneic T cells and allowed T-cell responsiveness comparable to

T cells previously exposed to immature DC or immature DC treated with

PDTC. PDTC-modulated DC were unable to suppress alloresponsiveness to

mature DC from the same donor.

4.) CsA does not counteract the suppressive state induced by PDTC-treated DC Addition of CsA to primary MLC led to an inhibition of the allogeneic T-cell

proliferation regardless of the mode of APC pretreatment. The presence of CsA

had no influence on the proliferative response of T cells in secondary cultures

when they were stimulated with immature or mature DC in primary MLC.

Interestingly, secondary MLC revealed that the presence of CsA during

alloantigenic priming did not influence the induction of the hyporesponsive

state by PDTC-modulated DC.

5.) T-cell hyporesponsiveness with PDTC-modulated DC in T cells from patients with renal allografts under calcineurin inhibitor-based immunosuppression While the primary MLC response was profoundly reduced using PDTC-

modulated DC, a conspicuous state of T-cell hyporesponsiveness was observed

in restimulation cultures supplemented with allogeneic stimulator cells. In

contrast, T-cell responsiveness was unimpaired when various polyclonal stimuli

were employed. It is concluded that induction of T-cell hyporeactivity towards

alloantigens by maturation-resistant DC is not dependent on calcineurin

activation and furthermore is feasible in T cells exposed to CsA in vivo.

43

C.) “Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3” 96-98

1.) Targeting JAK3 disrupts CD40-triggered DC maturation

Up-regulation of the costimulatory molecules CD80, CD86 and CD40,

the MHC class I and II molecules as well as of the specific DC maturation

marker CD83 was inhibited. JAK3 inhibitor-treated DC did not revert to a

monocyte/ macrophage stage.

2.) JAK3 inhibition does not interfere with the activation-associated down-regulation of the DC-antigen uptake machinery

Endocytosis and macropinocytosis, which were evaluated by incorporation of

FITC-labeled dextran or of lucifer yellow (LY), respectively, were more

pronounced in immature DC than in CD40-activated DC. Interestingly,

endocytosis and macropinocytosis was similar in JAK3 inhibitor-treated DC and

mature DC. Furthermore, CD40 ligation led to a reduction in mannose receptor

expression in DC. As observed for antigen uptake, JAK3 inhibitor-treated DC

behaved similar to mature DC with regard to mannose receptor expression.

3.) Prevention of T-cell stimulatory capacity and induction of antigen-specific hyporeactivity by JAK3 inhibited DC

CD40 triggering of DC induced a vigorous T-cell stimulating capacity compared

with immature DC. Treatment of DC with the JAK3-inhibitor during CD40

triggering resulted in an allostimulatory capacity similar to immature DC.

Preculturing allogeneic T cells with CD40-triggered DC led to a strong

secondary T-cell alloresponse. In contrast, preculture with CD40-triggered DC

exposed to the JAK3 inhibitor resulted in a dramatically reduced responsiveness

of allogeneic T cells upon restimulation. Restimulation with allogeneic mature

DC from unrelated third-party donors was also associated with defective T-cell

proliferation in JAK3-treated DC or immature DC-primed T cells, indicating

nonspecific T-cell hyporesponsiveness induced by both APC populations.

Addition of IL-2 led to a complete restoration of the defective T-cell

44

proliferation of T cells coincubated with JAK3 inhibitor-pretreated DC in

primary MLC.

4.) Effect of JAK3 Inhibitor Treatment on Homotypic T-Cell Aggregation

T cells activated with CD3 and CD28 mAbs exhibited prominent cell clusters

8 hr after initiation of the culture, JAK3 inhibitor-supplemented cultures were

completely devoid of any T-cell clusters.

DISCUSSION

Several lines of evidence indicate that bacteria and viruses have evolved

strategies to evade immune surveillance and in particular to block DC function 110-119. It

has been shown that n-butyrate, a physiologically occurring short-chain fatty acid

(SCFA) derived from breakdown of carbohydrates by the intestinal microflora, exerts

profound anti-inflammatory effects in vitro 120-128. It was demonstrated that this

bacterial metabolite severely hampers interferon-γ (IFN-γ) production as a result of

defective IL-12 production and IL-12-receptor β1/2-chain expression 121. Mounting

evidence suggests that apart from its physiological function as the essential energy

source for colonocytes, n-butyrate exerts strong anti-inflammatory activity in several

states of mucosal inflammation 129-132. The intestinal lumen has been shown to contain

n-butyrate concentrations from 0.1 mM to 16 mM in the small intestine and 40 mM

in the colon depending on quality and quantity of daily food intake 133. Because

n-butyrate occurs in the portal blood at 0.04 mM 133, it is conceivable that the

concentrations used in our study actually occur in the mucosa, where contact between

butyrate and DC can be expected. Finally, in vitro and in vivo experiments have

demonstrated that n-butyrate is able to induce a state of T-cell anergy 134-136. We

investigated how DC respond to the bacterial DC stimulus lipopolysaccharide (LPS)

45

when applied in the presence of n-butyrate, both of which occur at high concentrations

in the mucosal immune system. We described a novel way of bacterial interference

with DC maturation and provided phenotypical, morphological, and functional

evidence that n-butyrate profoundly suppresses the development of monocyte-derived

DC in vitro. The bacterial metabolite n-butyrate inhibited the development of mature

DC, resulting in impaired DC function and defective cytokine production. n-Butyrate

did not promote a classical differentiation program toward macrophages as has been

demonstrated for corticosteroids, IL-10 and IL-6 80,137-138. Furthermore, n-butyrate

substantially impaired the upregulation of MHC antigens, costimulatory molecules, and

DC-specific maturation markers, correlating with reduced antigen-specific T-cell

proliferation. Inhibition of costimulatory molecule expression on DC by n-butyrate was

also striking given their critical role in T-cell stimulation 139-141. DC exposed to n-

butyrate during maturation by LPS induced a state of hyporeactivity in alloreactive

T cells in secondary cultures (unpublished data not shown). In particular, n-butyrate

prevented homotypic DC clustering, inhibited IL-12 while sparing IL-10 production,

and at the molecular level, blocked NFκB translocation 95,142. It is tempting to speculate

that the intestinal microflora in the gut uses this mechanism to escape immune

surveillance. Mice deficient in functional NFκB have no mature DC and present with

impaired cellular immunity 143-145. Likewise, selective inhibition of NFκB activity has

also been shown to impair DC maturation 146-149. Impairment of nuclear translocation of

this transcription factor by n-butyrate may account for most features of the altered DC

phenotype 150 as well as for the sensitive inhibition of cytokine production by n-

butyrate, because the human IL-12, but not the IL-10 promoter, contains crucial NFκB

binding sites within its promoter 151-152. Endotoxin-stimulated PBMC also showed

inhibited NFκB activity in the presence of n-butyrate, which caused a decreased

production of proinflammatory cytokines 122. Finally, in animal models of colitis

mucosal, NFκB-DNA binding activity was reduced markedly when high intestinal n-

butyrate concentrations were achieved by SCFA enemas or dietary means 122,129-130.

We described conditions for the establishment of a novel form of tolerogenic

dendritic cells. This population of DC was generated from peripheral blood monocytes

applying a pharmacological NFκB-inhibitor (PDTC) in combination with a well-known

maturation stimulus. PDTC-pretreatment of DC leads to an arrest in maturation as

46

reflected by down-regulated major histocompatibility complex (MHC) antigens and

costimulatory molecules, suppressed immunostimulatory cytokines and an impaired

capability to support allogeneic T-cell activation. Tolerization was accompanied by

blunted cytokine production and a failure to express distinct activation molecules such

as CD69 and CD25 in primary MLC. The inhibition of NF-κB activation is also

involved in the induction of a deep state of T-cell hyporeactivity in secondary MLC that

cannot even be reverted by mature allogeneic DC. Allogeneic T cells challenged with

PDTC-modulated DC are refractory upon restimulation with alloantigens but not to

polyclonal stimuli. While DC-based tolerance protocols may be an attractive therapeutic

modality if reproducible in the clinical environment, it is presently not clear whether the

immunotolerizing properties of maturation-resistant DC are affected by the presence of

immunosuppressive drugs currently employed in clinical transplantation. Calcineurin

inhibitors such as CsA or FK506 are presently the basic standard immunosuppressive

agents in allogeneic transplantation. They inhibit the transactivation of the transcription

factor NF-AT thus preventing the induction of a variety of genes such as cytokine

genes. There are conflicting results from studies employing diverse strategies to induce

alloantigen-specific tolerance in combination with CsA. Thus CsA prevents the

induction of anergy when the TCR is engaged in the absence of costimulation in vitro.

Likewise, CsA has been shown to abrogate permanent allograft acceptance induced by

B7 plus CD154 blockade 104-105. Conversely, administration of CsA did not prevent the

establishment of regulatory cells induced by perioperative CD4 mAb therapy 153 or

tolerance employing a protocol with mixed chimerism using post-transplant total

lymphoid irradiation 154. The functional consequences of calcineurin inhibition with

Cyclosporine A were studied in T cells exposed to PDTC-modulated DC. For further

investigation of this T-cell hyporesponsiveness we established an ex vivo model in

T cells from patients with renal allografts under CsA-based immunosuppression in order

to assess the ability of in vitro modulated-DC to incorporate into a conventional

immunosuppressive protocol without abrogation of the tolerance by the clinical therapy.

Interestingly, the successful establishment of alloantigenic hyporesponsiveness is not

prevented by concomitant calcineurin inhibition in vitro as well as in T cells from

patients under CsA-based immunosuppression ex vivo. Our results convincingly

demonstrate that CsA, while synergistically suppressing allogeneic T-cell

47

responsiveness in primary MLC, does not prevent allogeneic T-cell hyporeactivity

induced by PDTC-modulated DC. Importantly, similar results were obtained in T cells

from renal allograft recipients indicating that the effects of DC-based regulation of

allogeneic immune responsiveness is conceivable, when modulated DC are incorporated

into a conventional clinical immunosuppressive protocol. These data may have

important implications for the design of clinical regimens for the establishment of

antidonor hyporeactivity in organ transplantation using in vitro modulated DC.

CD40 ligation by CD154 on activated T cells enables DC for sustained

stimulation of T cells leading to clonal T-cell expansion 155. Impaired DC maturation

may be a pivotal mechanism for the success of the CD154–CD40 blockade 156-162.

Premature decline of antigen-specific T-cell responses was observed in conjunction with

decreased DC persistence when CD40–CD154 interactions were absent 163. Similarly,

the efficacy of the CD40–CD154 blockade as immune intervention strategy for the

treatment of allogeneic rejection is well established 164-167. It has also been demonstrated

that engraftment of immature donor-derived DC in conjunction with anti-CD154 mAb

therapy promotes the induction of transplant tolerance 156-157,168. Recently,

pharmacological JAK3 inhibition has successfully been used to suppress T-cell

reactivity in experimental acute GVHD as well as in allogeneic transplantation 169-173.

Therapeutic targeting of this protein tyrosine kinase (PTK), which is a member of the

Janus family PTK, might be of particular advantage because of its association with the

common-gamma chain (cγ) of cytokine receptors that serves for multiple T-cell

cytokines such as IL-2, IL-4, IL-7, IL-9 and IL-15 174-175. Upon ligand-binding,

receptor associated JAKs are recruited, leading to their phosphorylation and subsequent

activation of STAT (signal transducers and activators of transcription) proteins that

dimerize and transactivate the transcription of specific target genes 176-177. Janus kinase

3 deficiency disables phosphorylation of the transcription factors STAT5a/b 178 and

manifests as severe combined immunodeficiency (SCID) 179-180. This disease is

associated with a profound defect in the immune system with significant alterations in

lymphocyte development, a functional incompetence and increased susceptibility for

apoptosis in peripheral T cells 181. Recent evidence suggests that in addition to its

involvement in common-gamma chain (cγ) signaling of cytokine receptors 182-183, JAK3

is also engaged in the CD40 signaling pathway of peripheral blood monocytes 184-185. In

48

this study, we assessed the consequences of JAK3 inhibition during CD40-induced

maturation of myeloid dendritic cells (DC), and tested the impact thereof on the

induction of T-cell alloreactivity. CD40-triggering of monocyte-derived DC in the

presence of selective JAK3 inhibitors resulted in the generation of APC with low

costimulatory molecule expression, defective cytokine production and impaired

allostimulatory capacity, which even confered a state of hyporeactivity to allogeneic

T cells that was reversible upon exogenous IL-2 supplementation to secondary cultures.

These results suggest that immunosuppressive therapies targeting the tyrosine kinase

JAK3 may also affect the function of myeloid cells. This property of JAK3 inhibitors

therefore represents a further level of interference, which together with the well-

established suppression of common-gamma chain signaling could be responsible for

their clinical efficacy. Current data indicate a critical role for JAK3 in signaling linked

to the T-cell antigen receptor 186-188, therefore, we investigated whether targeting JAK3

with a rationally designed inhibitor affects early T-cell activation events. T-cells were

stimulated by CD3 and CD28 cross-linking. We found that JAK3 inhibitor treatment

dramatically impaired T-cell–receptor (TCR)-induced homotypic T-cell aggregation.

Our finding of a complete block of homotypic cell aggregation by pharmacologic JAK3

inhibition indicates impaired signaling between the TCR and integrins such as LFA-1,

which is required for cell clustering, T-cell proliferation, and stabilization of the

interaction between T cells and antigen presenting cells 189. Various JAK3 inhibitors are

currently evaluated as immunomodulatory agents to treat allogeneic rejection and

GVHD. The systemic administration of glucocorticoids brings about metabolic and

endocrine side effects, and thus administration of JAK3 inhibitors could be

advantageous and is expected to have fewer side effects, because of the restriction of the

JAK3 signaling pathway to lymphoid and myeloid cells/organs 169,171,178,190-194. Drugs

aimed at targeting this particular tyrosine kinase may represent promising candidates for

the development of selective immunosuppressive protocols.

49

CONCLUSIONS

In conclusion, given the therapeutical potential of dendritic cells to induce

tolerance in general, these findings suggest that immunization strategies using in vitro

modulated dendritic cells that are characterized by a maturation blockade might be

potentially useful in the control of allograft rejection.

We provide phenotypical, morphological, and functional evidence that the

physiologically occurring bacterial metabolite n-butyrate profoundly suppresses the

development and maturation of monocyte-derived DC in vitro resulting in impaired DC

function. These findings not only provide a novel interpretation of its potent anti-

inflammatory properties but also suggest a possible in vivo immunomodulatory role of

n-butyrate via interference with the function of DC. Using the cell culture model of

monocyte-derived DC, we generated and described a novel form of tolerogenic DC by

activating immature DC in the presence of NFκB inhibition with PDTC. Importantly,

the successful establishment of alloantigenic hyporesponsiveness is not prevented by

concomitant calcineurin inhibition in vitro as well as in T cells from patients under

Cyclosporine A-based immunosuppression ex vivo. Data obtained from the present

study indicate that the potential benefit of DC-based protocols as a therapeutic strategy

will not be lost if this treatment modality is incorporated into conventional clinical

immunosuppressive treatment. The JAK3 inhibition with the rationally designed JAK3

inhibitor WHI-P-154 arrested the CD40-triggered DC at an immature level. These

results suggest that immunosuppressive therapies targeting the tyrosine kinase JAK3

may also affect the function of myeloid cells. This property of JAK3 inhibitors therefore

represents a further level of interference, which together with the well-established

suppression of common-gamma chain signaling could be responsible for their clinical

efficacy. We demonstrate that T-cell stimulation by TCR triggering in the presence of a

pharmacologic JAK3 inhibitor is characterized by defective early signaling events

leading to a profound impairment of regular T-cell activation. Therefore the selective

prevention of JAK3 activity in peripheral T cells may have important implications for

future immunosuppressive therapy.

50

ACKNOWLEDGEMENTS

I am grateful for the proficiency and support of all collaborators of these studies,

especially on the parts of Dr. Marcus D. Säemann and Professor Gerhard J. Zlabinger,

who provided excellent professional care and research facilities. Special thanks to my

long-term mentor, Professor Peter Gergely for guiding me, as well as Professor Jozsef

Furesz for his help.

51

OWN PUBLICATIONS RELATED TO THIS THESIS

Säemann MD*, Kelemen Peter*, Böhmig GA, Hörl WH, Zlabinger GJ.

Hyporesponsiveness in alloreactive T-cells by NF-κB inhibitor-treated dendritic cells:

resistance to calcineurin inhibition.

American Journal of Transplantation 2004; 4: 1448–1458.

(*Co-first authors: Errata. American Journal of Transplantation 2004; 4: 1928–1929)

Säemann MD, Kelemen Peter, Zeyda M, Böhmig G, Staffler G, Zlabinger G.J.

CD40 triggered human monocyte-derived dendritic cells convert to tolerogenic

dendritic cells when JAK3 activity is inhibited.

Transplantation Proceedings 2002; 34: 1407-1408.

Säemann MD, Diakos C, Kelemen Peter, Kriehuber E, Zeyda M, Böhmig GA, Hörl

WH, Baumruker T, Zlabinger GJ.

Prevention of CD40-triggered dendritic cell maturation and induction of T-cell

hyporeactivity by targeting of Janus Kinase 3.

American Journal of Transplantation 2003; 3: 1341–1349.

Säemann MD, Parolini O, Böhmig GA, Kelemen Peter, Krieger PM, Neumüller J,

Knarr K, Kammlander W, Hörl WH, Diakos C, Stuhlmeier K, Zlabinger GJ.

Bacterial metabolite interference with maturation of human monocyte-derived

dendritic cells.

Journal of Leukocyte Biology 2002; 71: 238-246.

Säemann MD, Zeyda M, Diakos C, Szekeres A, Böhmig GA, Kelemen Peter, Parolini

O, Stockinger H, Prieschl EE, Stulnig TM, Baumruker T, Zlabinger GJ.

Suppression of early T-cell–receptor-triggered cellular activation by the Janus Kinase 3

inhibitor WHI-P-154.

Transplantation 2003; 75: 1864-1872.

52

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