Regulatory T Cells and HIV Infection

ผศ. ศักด์ิชยั เดชตรยัรตัน์ดร. พานทอง สงิหบุ์ตรา

หอ้งปฏิบติัการเอชไอวี แขนงวชิาภมูคิุ้มกัน วทิยาคลินิก

คณะเทคนิคการแพทย์ มหาวยิาลัยเชยีงใหม่

Immune Responses

Adaptive Immune Response

How this is achieved?

Self-ToleranceThe immune system discriminates between self and non-selfEstablishing and maintaining unresponsiveness to self

Central Tolerance (clonal deletion)Occurs within central lymphoid organsBefore lymphocytes mature

Peripheral ToleranceOccurs in peripheral lymphoid organsAfter lymphocyte mature

Proposed Mechanisms of Peripheral Tolerance

Rendered anergic or further deleted if encounter self-antigen in the peripheryFail to be activated because of low avidities to self-antigensLack of co-stimulation from APCsSecluded from target self-antigensRegulatory T cells actively down-regulate the activation and expansion of self-reactive lymphocytes

Regulatory T Cells (Tregs)

“Suppressor cells” (Gershon et al. 1972) CD4+ T cells that express the IL-2 receptor α chain control autoreactive T cells in vivo (Sakaguchi et al. 1995) Antigen-specific T-cell clones suppressed the proliferation of CD4+ T cells in response to antigen and prevented colitis in a severe combined immunodeficiency mouse model (Groux et al., 1997)

Regulatory T Cells (Tregs)

Identification of several types of Tregs“Regulatory T cells” is a preferred term

Types of Tregs

Levi G., et al. Seminars in Immunology. 2011. in press.

CD4+CD25+Foxp3+ Regulatory T Cells

Fundamental in controlling various immune responses Discovered by Sakaguchi et al. 5-10% of peripheral CD4+ T cells Expressed high level of IL-2Rα (CD25), Foxp3 Developed in thymus and present in healthy individuals from birth

γδ Regulatory T Cells

Comprise 5% of total T cells in peripheral lymphoid tissues Enriched in skin, intestine and genito-urinary tract

Natural Killer T Cells (NKT)

Produce large amount of Th1 (IFN-γ, TNF-α) and Th2 (IL-4, IL-10 and IL-13) cytokines Play roles in tumor rejection, resistance to pathogenic infection, autoimmune diseases and allograft acceptance

Regulatory T Cells Type 1 (Tr1)

Arise in the periphery following activation of naïve T cells with Ag in the presence of IL-10 Produce

high IL-10, TGF-β and IL-5 low IL-2 and IFN-γ no IL-4

Do not express high levels of either CD25 or Foxp3

T Helper 3 (Th3)

Induced in gut environment with high TGF-β, Th2 cytokines, subsets of DC and oral antigens Produces TGF-β Induces tolerance to nonpathogenic resident bacteria and food antigens

CD8+ Regulatory T Cells

Arise either from thymus or in response to foreign or self antigens CD8+CD25+ share similar phenotypes and functions with CD4+CD25+ T cells Express increased mRNA levels of Foxp3, GITR, CCR8, TNFR-2 and CTLA-4 Following activation, express TGF-β1 and CTLA-4, do not produce cytokines

Doble Negative T Cells (DN)

CD4-CD8-CD3+ comprised 1-2% of total CD3+ T cells in blood and lymph nodes Express a unique set of cell surface markers

TCRαβ, CD25, LFA-1, CD69, CD45, CD30, CD62L and CTLA-4

Produce High IFN-γ Low IL-10 and IL-4No IL-2

Functions of Tregs

HIV Infection

Loss of CD4+ T cells Chronic immune activation Progressive immune disfunction Impaired immune responses

Possible Roles of Treg in HIV Infection

T cell

Quiescent

resistant to infection

T cell

Activated

establish infection

Possible Roles of Treg in HIV Infection

Loss of Treg

destruction of T cellsdeterioration of immune function

T cell

Hyperactivation

Expand of TregExcessive Treg activity

No protective immune responsesEstablish carrier state of infection

T cell

Suppressed

Changes in Treg Numbers in HIV DiseaseDisease

State Treg Phenotype Change Organ

No ART CD4+CD25+ - Peripheral blood

CD4+Foxp3+ Increase Lymph node

CD4+CD25+Foxp3+ - Peripheral blood

Increase Peripheral blood

Decrease Peripheral blood

Increase Mucosa

Increase Tonsils

Increase Lymph node

CD4+CD25hiCD62Lhi Decrease Peripheral blood

CD4+CD25+CD127lo Increase Peripheral blood

CD4+CD25+CD127lo45RO+ -/ Peripheral blood

Changes in Treg Numbers in HIV Disease

Disease State Treg Phenotype Change Organ

On ART CD4+CD25+ Increase Peripheral blood- Peripheral blood

CD4+Foxp3+ Increase Lymph nodeSpleen

CD4+CD25+Foxp3+ - Mucosa- Mucosa

- Peripheral bloodTonsils

CD4+CD25+CD127lo Increase Peripheral blood

Treg Function in HIV Infection

How to Identify Tregs?

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What are these markers?

A single chain molecule on T-helper cellBinds to 2 domain of MHC-IIIncrease the sinsitivity of T cell to Ag

CD4

Interleukin-2 receptor α-chain (IL-2Rα) Receptor for IL-2, T-cell growth factor

CD25

25

What are these markers?

Forkhead ⁄ winged-helix transcription factor box P3 Transcriptional repressor Most specific Treg marker currently Key factor in controlling Treg development

Foxp3

26

What are these markers?

Cytotoxic T lymphocyte associated protein-4 Binds B7.1 (CD80)/B7.2 (CD86) Major negative regulator of T-cell responses

CTLA-4 (CD152)

27

What are these markers?

Glucocorticoid-induced tumor necrosis factor receptor Required for the induction of apoptosis Expressed on various lymphocytes at different levels High surface expression of GITR is only confined to resting nTreg cells in the periphery and thymus

GIRT

28

What are these markers?

Specific receptor chain for IL-7 IL-7 control thymopoiesis and homeostasis of peripheral T lymphocytes

IL-7Rα (CD127)

%Cell Expressing Treg Markers

Detection of nTregs

1. PBMC Isolation (Ficoll gradient centrifugation)

2. Cell surface staining (CD4 and/or CD25)

3. Intracellular staining (Foxp3)

4. Flow cytometric detection and analysis

FACS profile of CD4+CD25+ Cells

FACS profile of CD4+CD25+ Cells

CD4

Foxp

3Foxp

3

CD4

Isotypes control

FACS profile of CD4+Foxp3+ Cells

FACS profile of CD25+Foxp3+ Cells

Challenges in Identification of Tregs

Current markers are not truely Treg-specific All T cells express CD25 upon activation CTLA-4 is upregulated on all CD4+ and CD8+ T cells, 2–3 days following activation GITR is induced in T cells upon activation CD127 is downregulated in most CD4+ T cells upon activation Most human CD4+ and CD8+ T cells transiently express Foxp3 upon activation

Isolation of Treg using Foxp3 is limited to only phenotypic study