[Advances in Immunology] Volume 124 || A Transendocytosis Perspective on the CD28/CTLA-4 Pathway

CHAPTER FOUR

A Transendocytosis Perspectiveon the CD28/CTLA-4 PathwayBlagoje Soskic*, Omar S. Qureshi†, Tiezheng Hou{,David M. Sansom{,1

*School of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom†Cellular Sciences, UCB, Berkshire, United Kingdom{UCL Institute of Immunity and Transplantation, Royal Free Campus, London, United Kingdom1Corresponding author: e-mail address: [email protected]

Contents

1. Introduction 961.1 The problem of immune self-tolerance 96

2. The CD28 Pathway 972.1 CD28 in the thymus 982.2 CD28 and Treg homeostasis 992.3 CD28 signals and T cell activation 1012.4 CD28 and T cell differentiation 1022.5 CD28 and memory responses 1032.6 CD28 and anergy 1042.7 CD28 and metabolism 105

3. CD80 and CD86: The Ligands for CD28 and CTLA-4 1064. CTLA-4 110

4.1 Cell biology of CTLA-4 1114.2 CTLA-4 function 113

5. Transendocytosis as a Model of CTLA-4 Function 1165.1 CTLA-4-expressing cells can reduce the levels of ligand on APC 1185.2 Transendocytosis exploits the biophysical ligand-binding characteristics

of CTLA-4 1195.3 Transendocytosis explains the requirement for ligand sharing by CD28

and CTLA-4 1195.4 Transendocytosis is a cell-extrinsic, ligand-dependent, CD28-dependent

mechanism 1205.5 Transendocytosis exploits the complex trafficking behavior in CTLA-4 1215.6 Suppression by transendocytosis is easily overridden 121

6. An Integrated Perspective on CD28 and CTLA-4 122References 123

Advances in Immunology, Volume 124 # 2014 Elsevier Inc.ISSN 0065-2776 All rights reserved.http://dx.doi.org/10.1016/B978-0-12-800147-9.00004-2

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http://dx.doi.org/10.1016/B978-0-12-800147-9.00004-2

Abstract

T cell activation is a key event in the adaptive immune response and vital to the gen-eration of both cellular and humoral immunity. Activation is required not only for effec-tive CD4 T cell responses but also to provide help for B cells and the generation ofcytotoxic T cell responses. Unsurprisingly, impaired T cell activation results in infectiouspathology, whereas dysregulated activation can result in autoimmunity. The decision toactivate is therefore tightly regulated and the CD28/CTLA-4 pathway represents this api-cal decision point at the molecular level. In particular, CTLA-4 (CD152) is an essentialcheckpoint control for autoimmunity; however, the molecular mechanism(s) by whichCTLA-4 achieves its regulatory function are not well understood, especially how it func-tionally intersects with the CD28 pathway. In this chapter, we review the establishedmolecular and cellular concepts relating to CD28 and CTLA-4 biology, and attemptto integrate these by discussing the transendocytosis of ligands as a new model ofCTLA-4 function.

1. INTRODUCTION

1.1. The problem of immune self-toleranceThe central problem confronting the adaptive immune system (T and

B cells) is how to generate lymphocytes with a broad enough receptor rep-

ertoire in order to recognize all conceivable foreign antigens. The solution

to this problem—the random rearrangement of gene segments encoding the

T cell receptor and antibodies—generates huge diversity, but initiates a sec-

ond problem: how to prevent these receptors from recognizing our own tis-

sues and causing autoimmunity? For T cells, this problem is addressed in part

during their development in the thymus where highly self-reactive T cells

are removed by negative selection (Xing &Hogquist, 2012). Here, a variety

of self-antigens are also ectopically expressed on thymic epithelial- and

antigen-presenting cells facilitating deletion of overtly self-reactive

T cells. In parallel, this process can also result in the generation of a special-

ized population of regulatory T cells (Treg), which are also essential to self-

tolerance (Cowan et al., 2013). The importance of such ectopic antigen

expression can be observed in genetic deficiency of the AIRE protein where

both mice and humans develop specific autoimmunity (Metzger &

Anderson, 2011). However, despite these processes, it is apparent that the

T cell repertoire is not entirely purged of self-specificities. Interestingly,

recent studies suggest that the frequency of self-reactive specificities within

the peripheral T cell repertoire in humans is ultimately rather similar to that

96 Blagoje Soskic et al.

for other antigens (Su, Kidd, Han, Kotzin, & Davis, 2013). Indeed, it can be

argued that given the likely degree of cross-reactivity required for effective

antigen coverage, the removal of all self-specificities is unfeasible (Sewell,

2012). Accordingly, self-tolerance by deletion does not appear to be the only

solution and the peripheral T cell repertoire is therefore established in the

face of inevitable self-reactivity. The degree of self-reactivity present is spec-

tacularly revealed by a variety of immune defects that result in T cell-

dependent autoimmunity. For example, deficits in the TGFβ (Rubtsov &

Rudensky, 2007), Foxp3 (Sakaguchi, 2005), IL-2 (Malek & Bayer, 2004),

IL-10 (Moore, de Waal Malefyt, Coffman, & O’Garra, 2001), cytotoxic

T lymphocyte antigen-4 (CTLA-4) (Walker & Sansom, 2011) as well as

other pathways can all lead to profound and often fatal autoimmunity

highlighting the lifelong need for immune regulation in the periphery

(Kim, Rasmussen, & Rudensky, 2007). Thus, rather than completely purg-

ing us of self-reactive T cells, the thymus appears to act to select T cells with

constrained self-reactivity which can subsequently be controlled by other

mechanisms (Palmer & Naeher, 2009). The selection of a repertoire that

responds weakly to self-antigens provides an opportunity to use additional

“costimulatory” signals as a mechanism for controlling peripheral T cell acti-

vation. Based on this concept, the CD28/CTLA-4 pathway appears to act as

a molecular checkpoint ideally placed at the decision point between immu-

nity to potential pathogens and peripheral self-tolerance. Here, we explore

this concept in the light of a novel mechanism of CTLA-4 function,

transendocytosis.

2. THE CD28 PATHWAY

CD28 is a 44 kDa, type I transmembrane protein expressed on the sur-

face of the majority of naı̈ve CD4 and CD8 of T cells which consists of a

single extracellular Ig-V-like domain assembled as a homodimer. CD28 is

well established as a major costimulatory molecule in T cell activation

important in the initiation and augmentation of T cell mediated immunity

via its interactions with two ligands CD80 and CD86, found predominantly

on APC (Fig. 4.1; Keir & Sharpe, 2005; Linsley & Ledbetter, 1993; Sansom,

2000). Consequently, mice deficient in CD28 show an array of immune

defects including impaired T cell activation, a lack of T cell help for

B cells and poor memory T cell responses, all highlighting the importance

of CD28 costimulation in the generation of effective T cell responses.

97CTLA-4 Transendocytosis

2.1. CD28 in the thymusAs well as its involvement in the activation of conventional CD4+ and CD8+

Tcells in the periphery,CD28 is expressed on the surfaceof thymocytes during

T cell selection. It is notable that the expression of CD28 varies during

this process with a relatively high level of CD28 expressed on CD4+CD8+

thymocytes that reduces substantially once T cells are selected into single

positive CD4 or CD8 lineages (Liang et al., 2013). Nonetheless, the role of

CD28 in thymic selection is not immediately obvious since CD28-deficient

mice generate relatively normal CD4 and CD8 T cell compartments. How-

ever, the size of positively selected compartment appears to be increased

with loss of CD28 (Vacchio, Williams, & Hodes, 2005). Thismay be related

to the fact that the development and maintenance of the thymic medulla itself

(a1 major site of negative selection) appears to be dependent on self-reactive

TCR engagement and CD28 costimulation (Irla et al., 2012). A further

Figure 4.1 B7-family receptor–ligand interactions. In addition to T cell receptor (TCR)recognition of peptide antigens in the context of major histocompatibility complex II(MHC II), CD4+ T cell activation is controlled by interactions between CD80 or CD86ligands and their receptors CD28 and CTLA-4. CD28 is a costimulatory receptor consti-tutively expressed by T cells which upon interaction with CD80 and CD86 generates sig-nals that promote T cell activation and generation of effector T cell responses. CTLA-4 isalso a receptor for CD80 and CD86 but its expression is limited to activated T cells andregulatory T cells (Treg). CTLA-4 possesses higher affinity for both ligands and has aninhibitory function that downregulates T cell activation. Additional evidence suggeststhat human CD28 and CTLA-4 also interact with ICOS ligand (ICOS-L) and that CD80 spe-cifically interacts with PD-1 ligand (PD-L1); however, the significance of these interac-tions is only just emerging.


possibility is that the shape of the TCR repertoire is altered in CD28-deficient

settings, such that TCRs whose avidity would normally result in deletionmay

then be tolerated in the absence of CD28 signals. Accordingly, several studies

support a role for CD28 in negative selection (Amsen & Kruisbeek, 1996;

Buhlmann, Elkin, & Sharpe, 2003; Noel, Alegre, Reiner, & Thompson,

1998). There is also evidence that alterations in expression of CD28 ligands

(CD80 and CD86) (Williams et al., 2014) and changes in CTLA-4 expression

(Verhagen et al., 2013) can impact on the quality of the selected T cell reper-

toire. The most likely explanation for these findings is that by affecting CD28

costimulation during selection, the shape of the receptor repertoire that is ulti-

mately selected is altered.

2.2. CD28 and Treg homeostasisPerhaps, the most obvious thymic event affected by the CD28 pathway is

the generation and selection of natural CD4+ CD25+ Foxp3+ Treg. Stud-

ies of CD28-deficient mice have revealed a marked lack of Treg (Salomon

et al., 2000), which appear to require specific CD28 signaling motifs for

selection and maintenance independently from the production of IL-2

(Tai, Cowan, Feigenbaum, & Singer, 2005). A number of lines of evidence

further suggest that CD28 signals are important to Treg homeostasis

(Huynh, Zhang, & Turka, 2014; Sansom & Walker, 2006). In addition

to the studies specifically indicating a role in thymic selection, it is clear that

where CD28 signals are prevented, lower numbers of Treg exist in the

periphery (Lohr, Knoechel, Jiang, Sharpe, & Abbas, 2003; Salomon et al.,

2000; Tang et al., 2003). In order to examine the role of CD28 in thymic

selection and peripheral maintenance more precisely, recent studies have

used an inducible CD28-deletion strategy. These studies have confirmed

and extended earlier work with blocking reagents, demonstrating that Treg

are highly CD28 sensitive in their peripheral maintenance in addition to a

role in thymic selection (Gogishvilli et al., 2013; Sansom & Walker, 2013).

Moreover, this effect was found to be Treg intrinsic rather than simply an

effect on IL-2 production. In addition, further refined studies have more

precisely detailed the role of CD28 in Treg by conditional deletion in Foxp3

expressing cells only (Zhang et al., 2013). This revealed a slightly different

picture with a clear, but perhaps surprisingly modest, decrease in the number

of Treg generated in the thymus and generally little impact on peripheral

Treg numbers. Nonetheless, Treg in this system were highly disadvantaged

when in competition with WT Treg indicating a further significant impact


of CD28 on their homeostasis. Moreover, this specific deletion of CD28

in Treg led to the gradual development of a Scurfy-like autoimmune disease,

which could be prevented by the presence of wild-type Treg (Zhang

et al., 2013). It was also observed that CD28-deficient Treg express lower

levels of suppressive molecules including CTLA-4 which may account

for their functional impairment. Interestingly, the reliance on CD28 sig-

naling by Treg is also observed, albeit indirectly, in CTLA-4-deficient

mice where the proliferation of Foxp3 expressing Treg is extremely marked.

Initially, this large expansion of CD4+ CD25+ T cells seen in CTLA-4

knockouts (Waterhouse et al., 1995) was presumed to be solely activated

T cells but more recent analysis confirms the expansion of a large cohort

of Foxp3-expressing cells (Schmidt et al., 2009). Here, the most likely

explanation is that loss of CTLA-4 allows increased CD28 signaling which

in conjunction with the more self-reactive repertoire possessed by Treg

results in their expansion. This reveals the tight connection between

CD28 and CTLA-4 function. Nonetheless, due to their lack of CTLA-4

expression, such expanded Treg populations are functionally impaired

and still associated with fatal autoimmunity mediated by uncontrolled

T cell activation.

The strategy of using CD28 costimulation to expand Treg was initially

behind the ill-fated TGN1412 trial (Hunig, 2012). However, recent studies

have continued to explore CD28 superagonists and the data indicate that

selective Treg expansion is nonetheless possible with such an approach

(Tabares et al., 2013). Again this highlights the importance of CD28 in Treg

homeostasis, an area which is becoming more clinically relevant with the

emergence of higher affinity ligand antagonists, such as belatacept, as well

as specific antagonistic CD28 antibodies (Yeung, Najafian, & Sayegh,

2014). Using these antagonistic approaches, there are indications that Treg

numbers may be reduced following treatment, as would be predicted from

mouse studies (Riella et al., 2012). However, in contrast to the CTLA-4-

deficient setting, the lack of Treg in these settings is also accompanied by

a compensatory decrease in the ability to activate effector T cells. This is

due to impaired CD28 costimulation, resulting from ligand blockade, such

that this Treg deficiency does not inevitably cause disease. This likely also

explains the general lack of autoimmune problems in CD28-deficient mice

and once again exemplifies the underlying “ying-yang” balance that is con-

tinually observed within the CD28/CTLA-4 system. Nonetheless, for such

a reduction in Treg to be nonpathogenic relies on effector responses being

dependent on ligand-CD28 costimulation. In situations where responses are


not CD28 dependent, the impairment of Treg caused by CD28 blockade or

deficiency may be expected to have deleterious consequences. An example

of this problem is seen in CD28-deficient NODmice, which develop exac-

erbated diabetes compared to CD28-sufficient mice (Lenschow et al., 1996;

Salomon et al., 2000) and would be predicted in many settings where robust

TCR stimulation can occur.

2.3. CD28 signals and T cell activationThe key intracellular signaling events associated with CD28 ligation are

still being elucidated. However, it is well recognized that during contact

with its ligands CD28 relocates to the immune synapse where it delivers

activation signals. A number of pathways appear to be involved and /or

recruited to CD28 including PI3 kinase (Pages et al., 1994; Ward,

Westwick, Hall, & Sansom, 1993), lck (Raab et al., 1995), ITK (Liao

et al., 1997; Marengere et al., 1997), GRB2 (Kim, Tharayil, & Rudd,

1998; Okkenhaug & Rottapel, 1998) PKC theta (Kong et al., 2011;

Yokosuka et al., 2010), and GADS (Boomer & Green, 2010; Riha &

Rudd, 2010). However, the relative importance of each signaling pathway

is still rather unclear and the CD28 pathways that are required may ulti-

mately depend on the outcome being assessed (Crooks et al., 1995;

Garcon et al., 2008; Okkenhaug et al., 2001; Pagan, Pepper, Chu,

Green, & Jenkins, 2012). One recent intriguing study of a mutant of

LAT has led to the identification of an actin uncapping pathway downstream

of CD28. Here, the protein Rlptr appears to be required to connect CD28

in the cSMAC with PKC theta and subsequently Carma 1 (Liang et al.,

2013), thereby positioning CD28 upstream of the NFkB pathway and

AP-1 in line with other studies (Boulougouris et al., 1999; Edmead et al.,

1996; Su et al., 1994; Takeda et al., 2008; Watanabe et al., 2012). Further

elegant in vivo approaches have identified roles for both tyrosine- and non-

tyrosine-basedmotifs in proliferation and IL-2 production and survival using

an in vivo transgenic approach (Dodson et al., 2009; Ogawa et al., 2013).

These studies indicated a role for the YMNMmotif in CD28 costimulation

but specifically in naı̈ve T cells. Other studies have revealed T cell responses

to be largely unimpaired without the YVKM motif in response to

antigen expressed by bacteria. However, a role was revealed for the YMNM

motif in the presence of weak stimulation in the absence of adjuvants (Pagan

et al., 2012). In these studies, it was clear that complete loss of the CD28

cytoplasmic domain or CD28-deficiency had a profound impact on the


generation of memory T cells highlighting the critical role of possibly as yet

unidentified pathways in CD28 signaling.

A further striking observation has highlighted the importance of ITK sig-

naling, which is downstream of CD28, as being required for correct tissue

migration following T cell activation. Accordingly, deficiency of ITK can

prevent the fatal pathology in CTLA-4 knockout mice (which is CD28

driven) by causing T cells to accumulate in LN but not infiltrate tissues

( Jain et al., 2013). This therefore identifies a clear and profound role for

CD28 in T cell migration and egress from lymph nodes.

2.4. CD28 and T cell differentiationCD28 has frequently been indicated to play an important role in T-helper

cell differentiation and in particular there are several reports of an involve-

ment for CD28 in Th2 responses (Lenschow et al., 1996; Tao, Constant,

Jorritsma, & Bottomly, 1997). Notably, weak TCR signaling in combina-

tion with strong CD28 costimulation appears to drive Th2 cytokines.

Recent microarray studies have provided support for this conclusion, dem-

onstrating that T cell responses resulting from CD28 engagement in the

presence of relatively weak calcium flux clearly upregulate genes associa-

ted with Th2 responses (Smeets et al., 2012). Consistent with these

concepts, a relative increase in CD28 signaling, such as may be seen in

CTLA-4-deficient mice or in the presence of CTLA-4 blockade, has also

been reported to drive a predominantly Th2 response (Khattri, Auger,

Griffin, Sharpe, & Bluestone, 1999; Oosterwegel et al., 1999; Walunas &

Bluestone, 1998). Thus, it would appear that CD28 costimulation may pre-

dominantly be important in differentiation towards Th2 fates although this is

seemingly not absolutely required (Brown et al., 1996).

Aside from T cell-intrinsic defects, a major feature of CD28- and ligand-

deficient mice is their lack of effective provision of B cell help in germinal

center formation (Borriello et al., 1997; Shahinian et al., 1993); conse-

quently, these mice have highly impaired class switching and affinity

maturation. This may relate to control of ICOS expression (Hutloff et al.,

1999) which is involved in T follicular helper differentiation, and is

CD28 dependent (Linterman et al., 2009; Walker et al., 1999). In addition

to the role of CD28 in T effector responses, there have been a number of

reports relating to the role of CD28 in the induction of iTreg. While

there seems to be some support for a positive role for CD28 signals in induc-

ing iTreg (Gabrysova et al., 2011; Guo, Iclozan, Suh, Anasetti, & Yu, 2008)


there are also opposite findings (Etemire, Krull, Hasenberg, Reichardt, &

Gunzer, 2013; Ma, Ding, Fang, Wang, & Sun, 2012; Semple et al.,

2011), suggesting CD28 signals inhibit induction of Treg. Thus, the role

of CD28 in Treg differentiation is not straightforward and seems to depend

on other conditions of activation particularly the level of TCR engagement

(Molinero, Miller, Evaristo, & Alegre, 2011) and cytokine environment.

2.5. CD28 and memory responsesAnother important question in CD28 biology is the requirement for CD28

costimulation in the generation and activation of memory T cells. While a

role for CD28 costimulation is generally thought to be important for naı̈ve

T cells, whether CD28 is similarly required for the activation of memory

T cells is less well established since memory T cells appear to have lower

activation thresholds (London, Lodge, & Abbas, 2000; Veiga-Fernandes,

Walter, Bourgeois, McLean, & Rocha, 2000). However, it is worth noting

that this concept has recently been challenged at least for CD8+ T cells

(Mehlhop-Williams & Bevan, 2014). Thus, varying views exist on the

requirement for CD28, and again it seems likely that differences may depend

on the nature and intensity of stimulation used (Arens et al., 2011). For

example, it may be relatively easy to trigger a proliferative response without

costimulation, but more demanding longer-term outcomes such as the full

range of effector responses and memory generation may still require CD28

(Boesteanu & Katsikis, 2009; Borowski et al., 2007; Pagan et al., 2012).

While generalizable requirements for CD28 costimulation seem difficult

to predict there does nonetheless seem to be evidence that CD28 is impor-

tant for effective T cell memory responses (Dooms & Abbas, 2006; Eberlein

et al., 2012; Ndlovu et al., 2014). It is also likely that CD28 is important to

effectively upregulate downstream proteins that are also significant for T cell

memory such as OX40 (Croft, So, Duan, & Soroosh, 2009; Withers

et al., 2011).

Interestingly, while CD28 expression generally persists on T cells follow-

ing activation in the short term, it appears to be ultimately downregulated

from the surface of highly differentiated mature CD8 cells with a similar effect

on CD4 cells. Accordingly these CD28�negative T cells have been found to

be associatedwith chronic disease andwith viral infections (Broux,Markovic-

Plese, Stinissen, & Hellings, 2012). Ultimately, how this downregulation of

CD28 expression affects cellular activation, survival, and indeed control by

Treg is not completely understood but may well be important in disease.


2.6. CD28 and anergyAn established concept in CD28 biology is the idea that CD28 provides a

“second signal” in addition to TCR engagement, which is important to pro-

ductive T cell activation. In this model, the engagement of TCR in the

absence of costimulation is thought to lead to an unresponsive state known

as T cell anergy (Schwartz, 2003). The recognition that CD28 costimulation

could prevent anergy induction initially focused attention on this pathway as

a regulatory checkpoint. Costimulation provided via engagement of the

CD28 receptor by its ligands, CD80 and CD86, on APCs is thought to pre-

vent anergy due to the production of IL-2 and induction of cell division

(Wells, 2009). Importantly, since expression of CD28 ligands is linked to

inflammatory signals, including cytokines and TLR ligation, this provides

context information for T cell activation. Accordingly, high levels of

costimulatory ligand expression can be seen as “dangerous” and therefore

provide discrimination on whether T cell activation is likely to be against

pathogen-associated antigens or self-antigens. Thus, in the absence of suffi-

cient ligand expression and therefore weak CD28 costimulation the induc-

tion of anergy can be seen as a mechanism of maintaining self-tolerance.

Initial reports indicated that blocking CD28 ligands could therefore be used

to induce tolerance or long-term anergy in disease models (Lenschow et al.,

1992). However, the ability to reliably induce anergy or tolerance clinically

has been more difficult (Larsen, Knechtle, Adams, Pearson, & Kirk, 2006;

Pilat, Sayegh, & Wekerle, 2011). A number of specific anergy genes have

been identified as being upregulated in anergic cells and appear to play a role

in maintaining this nonresponsive state including diacylglycerol kinase and

EGR2 (Safford et al., 2005; Wells, 2009; Zheng, Zha, Driessens, Locke, &

Gajewski, 2012). Experimentally, the induction of T cell anergy has tradi-

tionally utilized several approaches predominantly based on generating

strong signals via the calcium/calcinuerin/NFAT pathways downstream

of the TCR, while blocking pathways that are downstream of CD28

costimulation such as AP-1/NFkB, PKC. Accordingly, the use of calcium

ionophores has been widely used to induce anergy and has been reported to

result in the upregulation of a number of ubiquitin ligases such as Cbl,

ITCH, and GRAIL (Heissmeyer & Rao, 2004; Safford et al., 2005). Nota-

bly, defects in all of these ligases are known to result in autoimmune phe-

notypes and appear to result in resistance to anergy induction (Lin &

Mak, 2007).


It is also noteworthy that natural Treg are described as being anergic

in vitro due to their inability to proliferate in response to normally effective

T cell stimuli. Despite this, in vivo Treg are readily observed to be highly

active and many of them in cell cycle most likely in a self-antigen-dependent

manner. This proliferative capacity is strongly influenced by CD28 engage-

ment as discussed above. Given that anergy relates to a block in the produc-

tion of IL-2, it is perhaps unsurprising that in vitro purified Treg are unable to

produce IL-2 and are therefore appear anergic. In contrast, in vivo, IL-2 is

provided by other T cells in the local environment thereby allowing the

observed Treg proliferation and survival (Fehervari, Yamaguchi, &

Sakaguchi, 2006;Walker, Chodos, Eggena, Dooms, & Abbas, 2003). While

it is clear that CD28 contributes to Treg homeostasis whether this is via

maintenance of CD25 expression is controversial. An impact of CD28

engagement on CD25 levels is observed in some settings (Tang et al.,

2003) but not in T cells with conditionally deleted CD28 (Gogishvilli

et al., 2013; Zhang et al., 2013), suggesting the impact on CD25 is relatively

mild. Overall, CD28 interacting with its two ligands provides key activating

signals that are important in the avoidance of anergy during normal T cell

activation and which are important for the maintenance of Treg.

2.7. CD28 and metabolismStudies initially emerging from the study of T cell anergy have begun to

reveal links between CD28 costimulation and the coordination of metabolic

pathways (Zheng, Delgoffe, Meyer, Chan, & Powell, 2009). Much atten-

tion has now focused on the mTOR pathway which is influenced by PI3

kinase signals generated downstream of CD28 and which is essential in pre-

paring T cells metabolically for division. It had been previously observed that

CD28 costimulation is important in promoting the generation of ATP via

glycolysis as a source of energy (Frauwirth et al., 2002) a process required to

meet the various metabolic demands of rapid cell division. It is now clear that

CD28 signaling is an upstream control point for the expression of nutrient

receptors such as amino acid sensors, transferrin receptors, or the GLUT-1

glucose transporters, which also generate metabolic signals sensed via

mTOR. A number of metabolic pathways are therefore involved in activat-

ing mTOR that are important in allowing T cells to proceed through cell

cycle (Cobbold, 2013; Powell, Pollizzi, Heikamp, & Horton, 2012).


Interestingly, recent studies of mice selectively deficient in different compo-

nents of the mTOR pathway (affecting TORC1 and TORC2 complexes

selectively) reveal substantial effects on T cell differentiation including reg-

ulatory T cell induction (Delgoffe et al., 2011). Strikingly, deficiency in the

TORC1 complex prevented T cells from becoming Th1 cells whereas defi-

ciency in TORC2 prevented Th2 differentiation. In addition, complete

mTORdeficiency led to the generation of Foxp3+Treg possibly suggesting

that Treg differentiation is a default pathway in the absence of properly coor-

dinated activation. Interestingly, mTOR activity does appear to be required

for Treg function (Zeng et al., 2013).

The above data provide key examples of how the CD28 checkpoint

controls a large number of downstream pathways that are critical for effec-

tive T cell activation, cytokine production, proliferation, migration, differ-

entiation, survival, effector, and memory pathways. Moreover, blocking or

limiting CD28 signals are associated with inhibitory (anergic) or regulatory

outcomes. Importantly, CD28 costimulation does not only affect T cells

intrinsically, but it also has profound impacts on B cell responses as well

as cytotoxic T cell responses via T cell help. Together, this demonstrates

how CD28 can act as an apical checkpoint for a very wide array of immu-

nological effector outcomes and therefore is a key control point for immune

regulation.

3. CD80 AND CD86: THE LIGANDS FOR CD28AND CTLA-4

All the features of the CD28 signaling pathways outlined above are

thought to be triggered upon engagement by two well-described ligands

found on antigen-presenting cells, making control of ligand expression

another obvious checkpoint for T cell activation. The first identified ligand

CD80 (B7/BB1 or B7-1) was described by Linsley et al. (1991). This was

followed by the identification of a second ligand CD86 (B7-2) (Azuma,

Ito, et al., 1993; Freeman et al., 1993). Until recently, these two ligands were

thought to be the sole ligands for CD28 and CTLA-4. However, there are

reports that human CD28 and CTLA-4 (but not mouse) can bind to the

ICOS ligand (Yao et al., 2011) and it has also been observed that the PD-1

ligand PD-L1 can interact with CD80 (Butte, Keir, Phamduy, Sharpe, &

Freeman, 2007). The significance of these novel interactions is still emerging

and will not be discussed further here.


In terms of expression, CD80 and CD86 are both found on the cell sur-

face of APCs (Freedman, Freeman, Rhynhart, & Nadler, 1991; Freeman

et al., 1991). In the absence of inflammatory or infectious stimuli, CD86

appears more constitutively expressed and found at moderate level on

B cells, monocytes, and DCs (Azuma, Ito, et al., 1993; Caux et al., 1994;

Hathcock et al., 1993; Inaba et al., 1994; Inaba et al., 1995; Larsen et al.,

1994). Upregulation of CD86 is generally more rapid and at higher levels

than that of CD80 upon induction by activating stimuli such as TLR ligation

or inflammatory cytokine signaling (Boussiotis, Freeman, Gribben, &

Nadler, 1996; Zheng et al., 2004).

CD80 is a 45–60 kDa type I transmembrane glycoprotein which con-

tains two extracellular domains, a membrane distal Ig variable-like domain

and a membrane proximal Ig constant-like domain. A similar organization is

seen for CD86 and the two ligands appear to have emerged from a common

ancestor as a result of a gene duplication (Collins, Ling, & Carreno, 2005).

Despite their common origin and shared receptor binding, there is surpris-

ingly little amino acid sequence conservation between the CD80 and CD86

which share around 30% identity at the protein level (Collins et al., 2005). In

addition to amino acid sequence differences, CD80 and CD86 display clear

differences in oligomerization and receptor-binding affinities (Collins et al.,

2002). Accordingly, the affinity of CD80 for both CD28 and CTLA-4 is

substantially greater than CD86 making CD80 a potentially more potent

ligand. Additionally, CD80 appears to be a dimer, whereas CD86 is a mono-

mer and while both CD28 and CTLA-4 are dimers CD28 appears to be

monovalent in terms of binding to its ligands (Collins et al., 2002). In con-

trast, the CTLA-4 dimer appears to be capable of binding to CD80 or CD86

molecules bivalently. These differences potentially translate into marked dif-

ferences in avidity between the different ligand–receptor complexes, which

can be viewed in a number of ways depending on the functional context. In

isolation, CD80 would be expected to be a more potent ligand than CD86

for CD28 stimulation. Such differences are revealed during activation of

naı̈ve CD4 T cells, where CTLA-4 is initially absent and CD28

costimulation is unopposed (Manzotti et al., 2006). However, when one

considers stimulation in the presence of CTLA-4 then it is possible that

CD86 is less inhibited by CTLA-4 (due to inferior CTLA-4 binding) and

may therefore be a more effective CD28 ligand in this context. Such cir-

cumstances may occur during the stimulation of Treg where CTLA-4

expression is constitutive and CD86 appears to be the preferred ligand for

stimulation. Thus, depending on the context of stimulation CD80 and


CD86 may have different abilities to costimulate CD28. The crystallo-

graphic structures of CD80 and CD86 in contact with their receptors are

known (Ikemizu et al., 2000; Schwartz, Zhang, Fedorov, Nathenson, &

Almo, 2001; Zhang, Schwartz, Almo, & Nathenson, 2003), and the inter-

actions between CD80/CD86 and their receptors well characterized at the

biophysical level. Accordingly, much is known about CD28/CTLA-4

ligand interactions yet despite this, the differences between the ligands at

the functional level are still rather unclear.

To date, the general view has been that CD80 and CD86 have largely

redundant or overlapping functions (Borriello et al., 1997; Lanier et al.,

1995) and it is clear that both molecules can provide effective CD28

costimulation. Where differences between ligands have been observed it

is often difficult to dissociate differences in expression pattern or level of

expression from proposed functional capacities. Thus, the fundamental dif-

ferences between the two ligands are still poorly understood.

An interesting feature of CD86 is its tight regulation and control by

ubiquitin ligases MARCH-1 and MARCH-8 (Corcoran et al., 2011).

Targeting of CD86 by these ligases can result in rapid degradation and

impaired stimulation by DC. Interestingly, CD83, which is on mature DC,

may act as a decoy protecting CD86 from downregulation (Tze et al.,

2011). In addition, CD86 appears to be a target for both IL-10 and TGFb

which inhibits its expression (Buelens et al., 1997; Chattopadhyay &

Shevach, 2013; Geissmann et al., 1999). The number of mechanisms involved

in controlling CD86 which appears to underscore its importance in triggering

T cell costimulation.

While CD80 is generally found to be less abundant, and upregulated at

later time points on APC, one place where CD80 is highly expressed is on

mTEC in the thymus possibly indicating a specific role in T cell selection or

possibly development of the medullary functions (Irla et al., 2012; Rossi

et al., 2007). In addition, both CD80 and CD86 can be upregulated on

T cells in response to activation, although the significance of this is still

unclear (Azuma, Yssel, et al., 1993; Sansom & Hall, 1993).

Attempts to distinguish between CD80 and CD86 function in vivo have

been made using CD80�/� and CD86�/� mice. CD86�/� mice have a

phenotype similar to mice deficient in both ligands and immunization given

without adjuvant results in a lack of T cell help for B cell responses as mea-

sured by class switching and germinal center formation (Borriello et al.,

1997). This perhaps suggests that CD86 may be the dominant ligand for ini-

tiating T cell responses; however, CD80 can compensate for these functions


when induced by adjuvants or inflammation. In contrast, the CD80�/�

mice reveal a relatively mild phenotype as far as antibody responses are con-

cerned (Borriello et al., 1997), and while there are differences in outcome

between CD86 and CD86 knockouts neither seems to be obligatory for

Th subset differentiation (Schweitzer, Borriello, Wong, Abbas, & Sharpe,

1997; Schweitzer & Sharpe, 1998).

Current data indicate that CD86 is the dominant ligand utilized in ini-

tiating CD28-dependent T cell response in vivo and the subdominant role of

CD80 in initiating T cell responses is further highlighted by studies compar-

ing different immunogens (Santra, Barouch, Sharpe, & Letvin, 2000). To

some extent, this bias has been a rationale for developing new blockade

drugs such as belatacept, which binds significantly more avidly to CD86

and is accordingly a more potent immunosuppressive compound (Yeung

et al., 2014). Interestingly, in vitro the results comparing ligands directly using

model systems do not suggest that CD80 has limited costimulation potential

and we have observed earlier commitment to T cell activation with CD80

when comparing ligands at equivalent levels (Manzotti et al., 2006). This

concept of CD80 being a more potent CD28 ligand is in keeping with

its higher affinity for CD28. Thus, the reason for the apparent subdominant

role of CD80 in stimulating T cell responses in vivo is not completely clear.

Aside from simple expression differences, there may also be differences

between ligands in spatial localization at the immune synapse (Pentcheva-

Hoang, Egen, Wojnoonski, & Allison, 2004). The recruitment of ligands

and receptors within the immune synapse may largely reflect the affinity

of ligand–receptor binding; however, it is possible that CD80 and CD86

may have different distributions or motilities on the cell surface by virtue

of their different cytoplasmic domains. It would be useful to get direct com-

parisons between ligands using the TIRF microscopy approaches that have

been revealing so far (Yokosuka et al., 2008, 2010; Yokosuka & Saito, 2009).

Indeed, evidence has suggested that the cytoplasmic domain can have

important effects on initiation of T cell costimulation and dimerization state

has been reported to influence costimulation (Bhatia, Sun, Almo,

Nathenson, & Hodes, 2010; Doty & Clark, 1998; Girard et al., 2012).

Another difference between the ligands relates to possible signaling via

their cytoplasmic domains. While both ligands possess relatively short cyto-

plasmic tails, there are suggestions that the ligands may possess signaling

properties into the antigen-presenting cell via PI3 kinase (Koorella et al.,

2014). Signaling has also been reported to trigger IDO activity

(Grohmann et al., 2002; Munn, Sharma, & Mellor, 2004) where it has been


suggested that engagement of ligands via CTLA-4-Ig or by Treg can trigger

the induction of the tryptophan-degrading enzyme IDO, with resultant

immune suppression (Fallarino et al., 2003). Whether CTLA-4-Ig consis-

tently performs this function is unclear (Mayer et al., 2013; Pree et al.,

2007; Sucher et al., 2012) and it is possible that the Fc region of the reagents

used can have impacts on APC (Davis, Nadler, Stetsko, & Suchard, 2008).

One study was unable to identify changes in gene expression subsequent to

CTLA-4-Ig binding (Carman et al., 2009). There is nonetheless consider-

able evidence that in vivo IDO is important to tolerance (Mellor & Munn,

2004). What remains to be fully clarified is whether CTLA-4-Ig binding to

B7 is required for IDO induction or perhaps alternatively, whether in

tolerogeneic states where CTLA-4-Ig plays a role, there is an associated

involvement of amino acid-degrading enzymes such as IDO which partic-

ipate in generating a tolerising milieu (Cobbold &Waldmann, 2013; Sucher

et al., 2012). While the concept of reverse signaling is attractive, it is worth

noting there is little or no conservation of cytoplasmic domains between

either of the human and mouse ligands, which argues against a conserved

ligand signaling function. Moreover, as yet there do not appear to be any

well-established signaling motifs within the cytoplasmic domains nor evi-

dence of the identity of proximal signaling machinery recruited to CD80

or CD86. Further precise studies are therefore required to fully establish

the nature and extent of ligand signaling and how CD28 compared to

CTLA-4 binding is detected.

While knowledge of CD80 and CD86 has grown considerably in the last

two decades, there still remains a major conceptual issue in the field:Why do

we have two biophysically and structurally distinct ligands and what are their

biological functions? Unraveling the precise functions of each ligand in this

process will undoubtedly help our understanding of both T cell activation

and regulation in disease settings.

4. CTLA-4

CTLA-4 is a type I transmembrane glycoprotein homologous to

CD28 (Harper et al., 1991). Despite this, CD28 and CTLA-4 share limited

identity at protein level being only �30% identical at the amino acid level.

Both CD28 and CTLA-4 are colocated on human chromosome 2 along

with the ICOS gene as a result of duplication. Notably, both CD28 and

CTLA-4 share a conserved hexamer motif MYPPPY that forms part of

the ligand-binding site shared by both ligands (Yu et al., 2011). While


CD28 and CTLA-4 are both expressed by T cells, CD28 is constitutive,

whereas CTLA-4 is expressed in a more restricted fashion limited to acti-

vated T cells and Treg. From a functional perspective, the most striking

observation is that in contrast to CD28-deficient mice, which lack effective

T cell responses, CTLA-4-deficient mice suffer from a fatal lympho-

proliferative disease driven by self-reactive T cells (Ise et al., 2010; Tivol

et al., 1995). This dichotomy of function is even more striking given that

these two diametrically opposing outcomes controlled by CD28 and

CTLA-4 result from binding to the same ligands. Thus, at its most basic

level, CTLA-4 represents an essential nonredundant pathway for preventing

self-reactive T cells from triggering autoimmunity. However, the mecha-

nisms by which this is achieved and how this integrates with CD28 function

have been extremely challenging to elucidate.

4.1. Cell biology of CTLA-4A striking molecular feature of CTLA-4 is the almost complete conservation

of its cytoplasmic domain in all mammals (Walker & Sansom, 2011). This

contrasts with the lesser degree of conservation of the extracellular (ligand

binding) domain and suggests that this region of CTLA-4 is under strong

selective pressure and encodes important biological functions. While a con-

served cytoplasmic domain is consistent with a signaling function, the cyto-

plasmic domain also controls a highly characteristic intracellular trafficking

pattern (Fig. 4.2). Accordingly, CTLA-4 protein is largely intracellular in

location with dynamic trafficking to and from the cell surface (Qureshi

et al., 2012). This pattern of protein expression is somewhat surprising since

the function CTLA-4 is largely dependent on engaging cell surface mem-

brane anchored ligands. A number of studies have shown CTLA-4 in

perinuclear intracellular vesicles which relocate to the site of TCR engage-

ment (Egen & Allison, 2002; Linsley et al., 1996). Subsequently, it was

found that CTLA-4 is a robust target for recruitment by the clathrin adaptor

AP-2 which interacts with an “YVKM” motif found in its cytoplasmic tail

resulting in clathrin-mediated endocytosis (Chuang et al., 1997; Schneider

et al., 1999; Shiratori et al., 1997). More recent studies have further charac-

terized CTLA-4 traffic (Qureshi et al., 2012) between plasma membrane

and a recycling endosomal compartment in T cells in the absence of ligand

binding, resulting in a steady state where �90% of CTLA-4 is intracellular.

These features of endocytosis and recycling are also conserved and evident in

birds and amphibians, although not in fish (Kaur, Qureshi, & Sansom, 2013).


Thus, in this respect, CTLA-4 resembles a number of clathrin-mediated

endocytic and recycling receptors such as EGFR and transferrin receptor

(Grant & Donaldson, 2009; Madshus & Stang, 2009). Consistent with this

highly endocytic pattern of expression, CTLA-4 is also targeted to lysosomes

and neutralization of lysosome pH using ammonium chloride or

bafilomycin results in increased CTLA-4 expression (Kaur et al., 2013).

Thus, the core cell biology of unligated CTLA-4 appears to be that of a con-

stitutively endocytic protein of relatively short half-life which recycles to the

plasma membrane and undergoes rapid degradation in lysosomes.

Figure 4.2 Cell biology of CTLA-4. A central feature of CTLA-4 biology is its intracel-lular trafficking to and from the cell surface. Following its synthesis on rough endo-plasmic reticulum (ER), CTLA-4 matures in the Golgi into a functional homodimer andis transported to the plasma membrane. At the cell surface, clathrin adaptor proteinAP-2 recognizes the YVKM motif in the cytoplasmic domain of CTLA-4, resulting inrapid clathrin-mediated endocytosis. Subsequently, CTLA-4 either recycles back tothe cell surface or goes to the lysosomes for rapid degradation. As a consequenceof its rapid internalization, around 90% of CTLA-4 is intracellular at any given time.CTLA-4 is therefore a highly dynamic trafficking protein with a short half-life; how-ever, the factors that govern CTLA-4 recycling or degradation remain to be largelydetermined.


Following T cell activation of conventional T cells or stimulation of

Treg, CTLA-4 expression is increased both transcriptionally and post-

translationally. As well as increased transcription due to activation

CTLA-4 is specifically targeted to the plasma membrane in a manner stim-

ulated by TCR signals (Egen & Allison, 2002; Linsley et al., 1996). Scaffold-

ing proteins such as TRIM (Valk et al., 2006) and vesicle transport operating

via a PLD-dependent mechanism (Mead et al., 2005) are also thought to be

involved. Accordingly, following T cell activation the levels of CTLA-4 at

the cell surface increase. However, this increase appears to be mainly due

to the higher overall cellular expression, resulting in a proportional increase

at the cell surface. Although it has been also proposed that T cell activation

can disengage AP-2-mediated internalization via CTLA-4 phosphorylation

(Shiratori et al., 1997), it is difficult to observe stabilization of CTLA-4 at the

cell surface in activated T cells (Qureshi et al., 2012). Nonetheless, it is very

clear that mutation of the AP-2-interacting motif, YVKM, results in abla-

tion of normal trafficking resulting in dramatically increased cell surface

CTLA-4 which is indeed relatively stable (Iida et al., 2000; Mead et al.,

2005; Valk, Rudd, & Schneider, 2008). Interestingly, internalization defec-

tive and even cytoplasmic deletion mutants of CTLA-4 are surprisingly not

devoid of functional capacity (Masteller, Chuang, Mullen, Reiner, &

Thompson, 2000; Yamaguchi et al., 2013; Yi, Hajialiasgar, & Chuang,

2004) and can still ameliorate autoimmunity in some settings. This suggests

that the ectodomain, in the absence of presumed signaling or normal

clathrin-mediated endocytosis still has functional capacity. This is most

likely mediated by cell surface competition for ligand binding based on its

higher affinity for ligands compared to CD28. However, it should be noted

that the YVKMmutants are not completely devoid of endocytic activity and

continue to have slow internalization rates. Moreover, whether such muta-

tions ablate transendocytosis (see later) remains to be established.

4.2. CTLA-4 functionA number of cell-intrinsic and non-intrinsic mechanisms for CTLA-4 func-

tion have been proposed (Bour-Jordan et al., 2011; Rudd, 2008; Walker &

Sansom, 2011; Wing, Yamaguchi, & Sakaguchi, 2011). There have been

proposed roles for all possible splice variants (Araki et al., 2009; Liu et al.,

2012; Stumpf, Zhou, & Bluestone, 2013; Vijayakrishnan et al., 2004),

including both ligand-independent and soluble variants. In addition, there

are also more traditional inhibitory signaling mechanisms proposed for


CTLA-4 (Krummel & Allison, 1996; Lee et al., 1998) as well as effects on

T cell:APC interactions via adhesion effects (Schneider et al., 2006). Given

such a diverse array of mechanisms, it has generally proved difficult generate

a cohesive set of ideas that are useful in predicting the functional behavior of

CTLA-4 observed during T cell responses. Since the many possible mech-

anisms have been extensively reviewed elsewhere (Bour-Jordan et al., 2011;

Rudd, 2008;Walker & Sansom, 2011;Wing et al., 2011), it is not our inten-

tion to discuss them again here. Instead, we will focus specifically on tran-

sendocytosis, a feature of CTLA-4 biology that we have recently identified

(Qureshi et al., 2011). We discuss this mechanism in the light of the broader

literature pertaining to CTLA-4 function and attempt to highlight how such

a mechanism can explain interactions with their ligands and the observed

functional inter-dependence within the CD28/CTLA-4 system.

4.2.1 A cell-extrinsic function for CTLA-4 in vivoThe profound nature of CTLA-4 function has been widely recognized since

it was observed that CTLA-4�/� mice die at 3–4 weeks after birth because

of severe spontaneous lymphoproliferative disorder resulting in multiple

organ infiltration (Tivol et al., 1995; Waterhouse et al., 1995). More recent

data have established that CTLA-4 is required to prevent self-reactive T cells

from initiating autoimmunity (Ise et al., 2010) against identifiable self-

antigens. Importantly, it is evident that the fatal phenotype occurs as a result

of CD28-dependent and ligand-dependent T cell activation, which can be

prevented by interfering with these pathways. Accordingly, mice lacking

both ligands, having CD28-deficiency or those treated with CTLA-4-Ig

all have substantially ameliorated disease (Tai, Van Laethem, Sharpe, &

Singer, 2007; Tivol et al., 1997). Thus, the key concept that emerges is that

a major role of CTLA-4 is to regulate CD28 stimulation by its natural ligands

(Mandelbrot, McAdam, & Sharpe, 1999; Tai et al., 2007; Tivol et al., 1997).

Given that preventing such CD28–B7 interactions largely cures CTLA-4-

deficient mice, this suggest some limitations on the functional capabilities of

ligand-independent CTLA-4 splice variants as has been recently observed

(Stumpf et al., 2013).

Based predominantly on studies using agonistic anti-CTLA-4 antibodies,

concepts for CTLA-4 function initially focused on the generation of an

inhibitory signal preventing T cell activation (Krummel & Allison, 1996;

Walunas, Bakker, & Bluestone, 1996). Accordingly, ligand binding to

CTLA-4 (upregulated as a result of T cell activation) is then presumed to

generate intrinsic inhibitory signals that “switch off” T cell activation,


proliferation, and IL-2 production. The extent to which this approach of

using cross-linked antibodies to CTLA-4 is a mimic of CTLA-4 in contact

with its ligands has not been established. However, the physiological impor-

tance of such cell-intrinsic signaling is largely challenged by straightforward

and widely repeated experiments using mice that possess both CTLA-4

wild-type and CTLA-4-deficient T cells. This reveals that mice-containing

mixtures of CTLA-4�/� and CTLA-4+/+ T cells fail to develop lethal lym-

phoproliferative disease (Bachmann, Kohler, Ecabert, Mak, & Kopf, 1999;

Friedline et al., 2009; Homann et al., 2006). This indicates that CTLA-4�/�

cells, which are responsible for the lethal phenotype, can be extrinsically

controlled by the presence of normal CTLA-4-expressing T cells, predom-

inantly Treg. It follows that the critical CTLA-4 functions, required to pre-

vent systemic autoimmunity, are therefore T cell-extrinsic. Such results are

difficult to explain if CTLA-4 is viewed as an inhibitory signal responsible

for autonomous T cell control but fit well with the possibility of CTLA-4

acting in a suppressive manner such as might be required for example, as an

effector molecule on Treg.

4.2.2 CTLA-4 and TregAlongside the development of this cell-extrinsic concept, it has become clear

that CTLA-4 is demonstrably an important component of Treg function.

Following on from early experiments (Read, Malmstrom, & Powrie,

2000; Takahashi et al., 2000) which indicated such a possibility, this issue

has been decisively tested recently in experiments where Treg deficient

for CTLA-4 derived from healthy mice were examined (Schmidt et al.,

2009) or CTLA-4 was conditionally deleted only in Treg (Wing et al.,

2008). This shows CTLA-4 to be critical for Treg function and prevention

of autoimmunity. While it is absolutely clear that CTLA-4 is not the sole

mechanism by which Treg suppress (Vignali, Collison, & Workman,

2008; Walker, 2013), it is nonetheless a nonredundant and arguably major

part of their function. Moreover, compared to the aggressive and systemic

autoimmune phenotype seen in mice where Treg are CTLA-4-deficient,

IL-10-deficient Treg give rise to a more limited and largely mucosal pheno-

type (Rubtsov et al., 2008), indicating that different mechanisms of Treg

suppression are important in different settings. Interestingly, scurfy mice

which have a deficiency in Foxp3 and therefore Treg also suffer from a dis-

ease which is largely CD28-dependent consistent with possibility that scurfy

features may also relate to a loss of CTLA-4 function due to Treg deficiency

(Singh et al., 2007).


An important point when discussing cell-intrinsic and cell-extrinsic

mechanisms of CTLA-4 function is that this does not simply equate to

expression on conventional T cells versus Treg, respectively. Accordingly,

it is also clear that CTLA-4 expression on conventional T cells can also act in

a cell-extrinsic manner to suppress T cell responses (Corse & Allison, 2012;

Wang et al., 2012; Zheng et al., 2008). Thus, it can be reasonably argued that

CTLA-4 can function in the same manner on both conventional and reg-

ulatory T cells, with Treg having the advantage of higher levels and consti-

tutive cellular expression compared to the inducible, activation-dependent

expression of CTLA-4 seen in conventional T cells. Moreover, it may also

reasonable to view any CTLA-4-expressing cells as potentially having some

suppressive capacity. In support of this concept, recent highly informative

studies from the Sakaguchi lab have shown that expression of CTLA-4

and repression of IL-2 expression are two essential components in generating

Treg characteristics in conventional T cells (Yamaguchi et al., 2013). They

also observe that CTLA-4 expression is a key element in allowing the

expression of a self-reactive TCR repertoire in Treg cells in keeping with

the self-reactive repertoire observed in Treg. This fits with the possibility

that by limiting CD28 signals, which seem to promote negative selection,

CTLA-4 expression may allow Treg to survive despite the presence of more

highly self-reactive TCRs. Perhaps most strikingly, this CTLA-4 effect

could be mediated without an intact cytoplasmic domain suggesting that

cell-intrinsic competition for ligand binding between CD28 and CTLA-4

at the surface of Treg is sufficient for these effects (Yamaguchi et al., 2013).

5. TRANSENDOCYTOSIS AS A MODEL OF CTLA-4FUNCTION

The above data make a convincing case for a T cell-extrinsic mech-

anism of suppression mediated by CTLA-4 predominantly expressed by

Treg. However, until recently, mechanisms that could deliver such a func-

tion have been generally lacking. Based initially on observations, using trans-

fected cell models, we recently observed robust transfer of ligands could

occur into CTLA-4-expressing cells (Qureshi et al., 2011). This observation

raised the possibility that CTLA-4 could potentially act as a ligand capture

device, thereby depleting its shared CD28 ligands fromAPC (Fig. 4.3). Such

depletion of ligand would therefore result in cell-extrinsic control of CD28

costimulation. Surprisingly, our subsequent experiments revealed that the

entire ligand (either CD80 or CD86) including a cytoplasmic domain


GFP-tag could be transferred from the donor (ligand expressing) cell into a

CTLA-4-expressing recipient cell. Moreover, we observed that internalized

ligands were ultimately degraded inside the CTLA-4-expressing cells

(Qureshi et al., 2011) as revealed by blocking lysosomal degradation with

bafilomycin. In more physiological T cell systems, this process was seen only

in CD4+CD25+ T cells (i.e., either Treg or activated T cells) and occurred

in vivo upon peptide stimulation. Taken together, these data provide a

simple model of antigen-specific, T cell-extrinsic suppression compatible

with a function for CTLA-4 on Treg. At present, the molecular details

Figure 4.3 Transendocytosis as a model of CTLA-4 function. Following stimulation ofCTLA-4 expressing T cells including regulatory T cells (Tregs), CTLA-4 is targeted towardthe immune synapse in TCR-dependent manner, where it interacts with its ligands(CD80 and CD86). CTLA-4 internalization can then occur together with its intact boundligands from APCs in the process termed transendocytosis. Vesicles containing ligandbound to CTLA-4 (CD86 is shown) appear to fuse with lysosomes where ligand isdegraded. Potentially, CTLA-4 may recycle back to the cell surface based on analogyto some other trafficking receptors. CTLA-4 therefore can function to reduce the avail-ability of ligands for CD28 binding. Such a model is consistent with CTLA-4 functioningas an effector molecule for Treg suppression. Recognition of APCs which have beendepleted of costimulatory ligands results in impaired T cell responses or, theoretically,in T cell anergy in conventional T cells.


underpinning this process remain to be fully elucidated; however, transfer of

ligands from one cell to another is clearly not limited to CTLA-4 (Davis,

2007). We use the term transendocytosis for this process in keeping with

other systems where ligand transfer occurs between cells resulting in intra-

cellular localization of transferredmolecules in recipient cells (Kusakari et al.,

2008; Marston, Dickinson, & Nobes, 2003). There is also a large literature

that describes the transfer of molecules between immune cells in a manner

referred to as trogocytosis (Daubeuf et al., 2010). It is notable that in nearly

all cases this transfer is measured by surface detection of the transferred pro-

tein. In our view, transendocytosis likely differs from this process in that it

results in intracellular transfer with little detection on the recipient cell sur-

face. We therefore currently draw a distinction between trogocytosis and

transendocytosis but such issues await further mechanistic understanding.

In the discussion below, we evaluate the extent to which the concept of

transendocytosis of ligands by CTLA-4 fits with the available data on

immune functions of CTLA-4.

5.1. CTLA-4-expressing cells can reduce the levels of ligandon APC

The core concept behind transendocytosis is that it depletes the levels of

CD28 ligands fromAPC thereby preventing costimulation. In line with this,

there is in fact substantial evidence indicating that CTLA-4 expression is able

to alter the levels of CD80 and CD86 on APCs (Kastenmuller et al., 2011;

Oderup, Cederbom, Makowska, Cilio, & Ivars, 2006; Onishi, Fehervari,

Yamaguchi, & Sakaguchi, 2008; Schildknecht et al., 2010; Schmidt et al.,

2009; Wing et al., 2008). Given these observations, transendocytosis can

provide a satisfying explanation for how this could be achieved in a

CTLA-4-dependent manner. In terms of functional significance, it is then

plausible that a reduction in the level of costimulation available through

CD28 is sufficient to suppress the activation of weakly self-reactive

T cells that populate the peripheral repertoire. Accordingly, in the absence

of CTLA-4, the level of costimulation would rise, rendering such cells capa-

ble of driving autoimmunity as is readily seen in the models discussed above.

Thus, the loss of Treg themselves (Fontenot, Gavin, & Rudensky, 2003) or

conditional deletion of CTLA-4 from Treg (Wing et al., 2008) is sufficient

to reveal the presence of self-reactive T cells which are ligand and CD28

dependent. One intriguing possibility is that aside from simply preventing

T cell activation due to lack of costimulation, transendocytosis could


stimulate the induction of anergy as predicted for T cells stimulated in such a

costimulation-independent manner (Schwartz, 2003).

5.2. Transendocytosis exploits the biophysical ligand-bindingcharacteristics of CTLA-4

Competition between CTLA-4 and CD28 for binding to their ligands is

predicted by their known biophysical characteristics (Collins et al., 2002).

Despite this, the context where direct intrinsic competition between

CD28 and CTLA-4 actually occurs is less clear. The concept of tran-

sendocytosis modifies our view of ligand competition in that it represents

a form of cell-extrinsic competition. That is, CTLA-4 can remove ligands

thereby competing with CD28 which is expressed by other T cells. Poten-

tially, this process can be separated temporally, such that CTLA-4 (for exam-

ple, on Treg) can compete for ligands before other T cells arrive on the same

APC. In addition to cell-extrinsic competition, CTLA-4 and CD28 com-

pete on the surface of T cells where both proteins are concomitantly

expressed. Themost likely arena for such direct competition is on the surface

of Treg. Here, the superior ligand binding ability of CTLA-4 is beneficial in

order for to carry out transendocytosis in the face of cell surface CD28

expression. Thus, the ability of CTLA-4 to carry out transendocytosis when

expressed on a CD28-expressing cell effectively demands that it must possess

higher affinity than CD28 for both ligands. Interestingly, it is evident that

CTLA-4 on Treg does not completely eliminate CD28 ligation which is still

required for Treg homeostasis. Indeed, CD28 signals for Treg homeostasis

are generated in spite of high levels of CTLA-4 expression and can be

observed by studying the impact of CTLA-4 deletion on increasing Treg

proliferation (Schmidt et al., 2009; Wing et al., 2008). One intriguing pos-

sibility is that the affinity and avidity of CTLA-4 binding to CD80 and

CD86 are specifically set within a precise range such that ligands (particularly

CD86) are never fully occupied by CTLA-4.

5.3. Transendocytosis explains the requirement for ligandsharing by CD28 and CTLA-4

The fact that arguably the most important activating receptor in T cell

immunity and the most important inhibitor of autoimmunity share the same

ligands provides an interesting conceptual paradox. From a signaling per-

spective, it is difficult to envisage how the “on switch” and the “off switch”

would work without having separate and controllable triggers. At present,

there is no model for understanding when CD28 would be triggered


compared to CTLA-4. Thus, the reason for the observed ligand sharing

between CD28 and CTLA-4 therefore requires further explanation. This

paradox is even more obvious when considering the fact that ligand sharing

has been deliberately maintained, despite extensive sequence divergence

between the two ligands (Collins et al., 2005) and between CD28 and

CTLA-4 during evolution. This evolutionary divergence has offered ample

opportunity for discrete functions to evolve yet ligand sharing has been

deliberately maintained. The most obvious explanation is that ligand sharing

between CD28 and CTLA-4 is actually required for function. While from a

signaling perspective, ligand sharing is difficult to rationalize, from the per-

spective of transendocytosis ligand sharing is an absolutely essential require-

ment for CTLA-4 to be able to control CD28 function (Qureshi et al.,

2011). Accordingly, the superior binding of CTLA-4 to both ligands

(Collins et al., 2002) and the ligand sharing with CD28 are features in a tran-

sendocytosis model that are required in order to make CTLA-4 an effective

“molecular hoover” for controlling CD28 ligand levels, but present substan-

tial difficulties in many other models.

5.4. Transendocytosis is a cell-extrinsic, ligand-dependent,CD28-dependent mechanism

The CTLA-4 in vivo experiments described above specify a number of char-

acteristics of CTLA-4 function which would seem to be necessary as part of

any cohesive model. Principal among these is that a critical function of

CTLA-4 must be cell extrinsic. Given the ability of CTLA-4 tran-

sendocytosis to effectively suppress APC costimulation for T cell activation,

then this criterion easily is satisfied.Moreover, the fact that disease in CTLA-

4-deficient mice is effectively cured by blocking CD28 signals, also demands

such a CTLA-4 function must impact on the functions of both CD28 and its

ligands. Again these criteria are met using a transendocytosis model. Indeed,

we observe that in vitro T cell responses that are not driven by CD28 ligands

(e.g., by using CD3 and CD28 antibody-coated beads) are not controllable

by CTLA-4, a feature that has significant implications for the design of

in vitro Treg assays. There are nonetheless other alternatives for T cell-

extrinsic, ligand-dependent CTLA-4 functions such as CTLA-4 triggering

the production of IDO. While it has been reported that CTLA-4-Ig treat-

ment as well as Treg can induce IDO activity in APCs (Fallarino et al., 2003;

Grohmann et al., 2002), the importance of IDO to CTLA-4 function is still

rather unclear. It has been shown that neither IDO-1 nor IDO-2 knockout

mice phenocopy CTLA-4 knockouts (Baban et al., 2004; Metz et al., 2014)


and while some level of redundancy is possible, it seems increasingly unlikely

that IDO activity is responsible for the major in vivo activity of CTLA-4

(Munn & Mellor, 2013). Thus, while IDO remains an important immune

regulatory pathway in its own right, its relationship and importance to

CTLA-4 function remains unclear.

5.5. Transendocytosis exploits the complex traffickingbehavior in CTLA-4

One of the most obvious features of CTLA-4 is its intracellular trafficking pat-

tern, which involves not only endocytosis but also recycling and degradation.

These obvious and conserved features are generally not well accounted for in

models of CTLA-4 function. In a transendocytosis model, it is immediately

obvious how such features might be required for efficient depletion of ligands

via transendocytosis. It is notable that trafficking of CTLA-4 is itself stimulated

by engagement of the TCR (Egen & Allison, 2002; Linsley et al., 1996;

Qureshi et al., 2012), which is in line with the fact that Treg suppression is

thought to be contact dependent and antigen driven. Other features such

as the cycling nature of CTLA-4 between the plasma membrane and cyto-

plasm combined with its strong avidity raises the possibility that CTLA-4

can act as a “pump” removing its ligands from APCs targeting them for deg-

radation and then possibly returning to the cell surface to continue this pro-

cess. Such recycling is typical of other receptors such as the transferrin receptor

(Grant&Donaldson, 2009). The reason for the trafficking ofCTLA-4 to lyso-

somes is also evident in this model and provides CTLA-4 with the ability to

directly target its ligands for degradation. Importantly, the ligand removal pro-

cess is inevitably time dependent and therefore would be expected to benefit

from the prolonged and more stable interactions that have been observed

between DC and Tregs (Onishi et al., 2008; Tang et al., 2006). Such stable

interactions would also be enhanced by their increased affinity for self-antigens

and by Neuropilin-1 which is expressed on Tregs (Sarris, Andersen, Randow,

Mayr, & Betz, 2008). Thus, through their enhanced self-reactivity and con-

stitutive expression of CTLA-4, Treg would be able to directly deliver

CTLA-4 to the synapse in a stable antigen-specific manner and have the

potential to efficiently utilize transendocytosis to suppress APCs.

5.6. Suppression by transendocytosis is easily overriddenAnother important aspect of CTLA-4 transendocytosis is that it essentially

sets up a mechanism of immune control that is quantitative. Accordingly,


the inhibitory function of CTLA-4 is dependent on the balance between the

overall amount of costimulatory molecules (CD80 and CD86) available on

APCs and the number of CTLA-4 molecules in Treg cells. Thus, increased

ligand density or increased numbers of APCs would have the capacity to

simply override CTLA-4 inhibitory capacity, making CTLA-4 function

unimportant in some settings. Thus, in the context of immunization using

adjuvants or strong responses to infectious agents, the inhibitory impact of

CTLA-4 is likely to be very limited as the process is simply overwhelmed by

excess ligand stimulated via TLR signaling. This concept fits well with

observations where responses have been observed to proceed in a manner

not greatly affected by CTLA-4 blockade or CTLA-4 deficiency

(Bachmann et al., 2001, 1998; Homann et al., 2006). Indeed, outside of

autoimmunity, it has been generally difficult to identify immune settings

where the profound nature of CTLA-4 inhibition can be predictably

observed. Transendocytosis as a mechanism of action predicts such contexts

where the functional capacity of CTLA-4 will be saturated by excess levels

of ligand expression. Thus, in settings where the effect of ligand removal

cannot keep pace with ligand expression, then T cell activation will ensue

in a manner which is CD28 dependent but where the impact of CTLA-4

cannot be observed despite it being intact. It is interesting to note that such

a quantitative threshold mechanism is ideally suited to controlling basal

immune responses to self-antigens without obviously interfering with the

establishment of immune responses to pathogens.

6. AN INTEGRATED PERSPECTIVE ON CD28 AND CTLA-4

The generation of a large and diverse TCR repertoire presents the

immune systemwith an enormous challenge. On one hand, sufficient recep-

tor diversity is required in order recognize and destroy potential pathogens,

whereas on the other self-reactive T cells must remain under strict control.

Given that in terms of molecular recognition by the TCR, there is no dif-

ference between peptides derived from self-antigens and those from patho-

gens, the regulation of the T cell activation relies on discrimination of

context (Matzinger, 2002). The control of CD80/CD86 ligand expression

provides one possible mechanism and the CD28/CTLA-4 pathways are ide-

ally placed at this key decision point ( Janeway, 2001). The ability of

CTLA-4 to carry out transendocytosis of its ligands generally fits well with

many functional observations, particularly those generated in vivo. However,


whether transendocytosis represents a major component of CTLA-4 func-

tion in vivo still remains difficult to determine. This is currently true for the

functional significance of most of the proposed CTLA-4 mechanisms since

in vivo genetic manipulation approaches frequently impact on multiple

potential mechanisms. This will doubtless be resolved by more refined

experiments, but at present, it is still useful to examine whether givenmodels

have value in predicting outcomes.

Reagents manipulating the CD28/CTLA-4 pathways have become

extremely promising therapeutic candidates in many areas of medicine.

These reagents have been used promote graft survival after transplanta-

tion control allergies, curb autoimmunity by ligand blockade (abatacept,

belatacept, anti-CD28), expand Treg (CD28 agonists), and augment

protective immunity to cancers (anti-CTLA-4). Understanding the precise

molecular mechanisms of the CD28/CTLA-4 pathway and how this is

influenced by interactions with natural ligands will continue to offer fresh

opportunities and novel interventional strategies either alone or in combi-

nation with existing treatment methods. It is nonetheless clearly emerging

that the CD28–CTLA-4 pathway operates as a finely balanced and highly

integrated system and as such, perturbations inevitably impact on all the

players within the system.

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[Advances in Immunology] Volume 124 || A Transendocytosis Perspective on the CD28/CTLA-4 Pathway

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