Understanding the CD28/CTLA-4 (CD152) Pathway and Its Implications for Costimulatory Blockade

T cell activation is a key event in the adaptive immune system and vital in the generation of protective cellular and humoral immunity. Activation is required to generate CD4 effector T cell responses and provide help for B cell and cytotoxic T cell responses. While defective T responses to foreign antigen result in infectious pathology, over-reactive T cell responses against self-antigens result in autoimmunity and, in a transplantation setting, tissue rejection. Understanding how T cell activation is normally regulated is critical to therapeutic intervention and the CD28/CTLA-4 (CD152) pathway represents the initial activation checkpoint in molecular terms. In particular, while the CTLA-4 pathway is well established as an essential regulator of self-reactivity, its mechanism of action is still uncertain. Such mechanistic issues are important given its central position in T cell activation and the increasing number of therapeutic modalities aimed at manipulating the CD28/CTLA-4 pathway. Here, we provide an updated view of CTLA-4 biology, reviewing the established features of the system and highlighting its interplay with CD28. We then discuss how recent progress in our understanding of this pathway affects our interpretations following intervention. T cell activation is a key event in the adaptive immune system and vital in the generation of protective cellular and humoral immunity. Activation is required to generate CD4 effector T cell responses and provide help for B cell and cytotoxic T cell responses. While defective T responses to foreign antigen result in infectious pathology, over-reactive T cell responses against self-antigens result in autoimmunity and, in a transplantation setting, tissue rejection. Understanding how T cell activation is normally regulated is critical to therapeutic intervention and the CD28/CTLA-4 (CD152) pathway represents the initial activation checkpoint in molecular terms. In particular, while the CTLA-4 pathway is well established as an essential regulator of self-reactivity, its mechanism of action is still uncertain. Such mechanistic issues are important given its central position in T cell activation and the increasing number of therapeutic modalities aimed at manipulating the CD28/CTLA-4 pathway. Here, we provide an updated view of CTLA-4 biology, reviewing the established features of the system and highlighting its interplay with CD28. We then discuss how recent progress in our understanding of this pathway affects our interpretations following intervention. CD28 is expressed on the surface of the majority of naïve CD4 and CD8 T cells and is the major costimulatory molecule in initial T cell activation. Together with engagement of the T cell receptor CD28 ligation results in the augmentation of many aspects of T cell–mediated immunity (1Linsley P Ledbetter J The role of the CD28 receptor during T cell responses to antigen.Annu Rev Immunol. 1993; 11: 191-212Crossref PubMed Scopus (1234) Google Scholar, 2Sansom DM CD28, CTLA-4 and their ligands: Who does what and to whom?.Immunology. 2000; 101: 169-177Crossref PubMed Scopus (234) Google Scholar, 3Keir ME Sharpe AH The B7/CD28 costimulatory family in autoimmunity.Immunol Rev. 2005; 204: 128-143Crossref PubMed Scopus (110) Google Scholar). 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 and immune memory. The immune stimulatory features of the CD28 pathway are triggered by engagement of two well-described ligands found on antigen-presenting cells (4Sansom DM Manzotti CN Zheng Y What’s the difference between CD80 and CD86?.Trends Immunol. 2003; 24: 313-318Abstract Full Text Full Text PDF Scopus (213) Google Scholar). The two ligands CD80 (B7/BB1 or B7-1) and CD86 (B7-2) were, until recently, thought to be the sole ligands for CD28 and CTLA-4; however, there are recent reports that human (but not mouse) CD28 and CTLA-4, can also bind to the ICOS ligand (5Yao S Zhu Y Zhu G et al.B7-h2 is a costimulatory ligand for CD28 in human.Immunity. 2011; 34: 729-740Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In addition, the PD-1 ligand, PD-L1 can also interact with CD80 (6Butte MJ Keir ME Phamduy TB Sharpe AH Freeman GJ Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses.Immunity. 2007; 27: 111-122Abstract Full Text Full Text PDF PubMed Scopus (1280) Google Scholar). The significance of these novel interactions is still emerging and will not be discussed further here. Importantly, the expression of CD80 and CD86 is up-regulated in response to inflammatory stimuli including Toll-like receptor stimulation. As such, up-regulation of ligands is seen as a key link between innate “danger” signals and the triggering of an effective adaptive immune response (7Joffre O Nolte MA Sporri R Reis e Sousa C Inflammatory signals in dendritic cell activation and the induction of adaptive immunity.Immunol Rev. 2009; 227: 234-247Crossref PubMed Scopus (436) Google Scholar). Despite structural and affinity differences (8Collins A Brodie D Gilbert R et al.The interaction properties of costimulatory molecules revisited.Immunity. 2002; 17: 201-210Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar) which would suggest functional differences, to date, the current view is that CD80 and CD86 have largely redundant or overlapping functions as represented in Figure 1 (9Lanier L O’Fallon S Somoza C et al.CD80(B7) and CD86(B70) provide similar costimulatory signals for T cell proliferation, cytokine production and generation of CTL.J Immunol. 1995; 154: 97-105Crossref PubMed Google Scholar,10Borriello F Sethna MP Boyd SD et al.B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation.Immunity. 1997; 6: 303-313Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). In addition to binding to CD28, both CD80 and CD86 also bind to the inhibitory protein CTLA-4, which is a CD28 homologue, expressed on activated T cells and especially regulatory T cells (Tregs) (11Walker LS Treg and CTLA-4: Two intertwining pathways to immune tolerance.J Autoimmun. 2013; 45: 49-57Crossref PubMed Scopus (277) Google Scholar). From a functional perspective, the most striking observation is that in contrast to CD28, where deficiency leads to lack of effective T cell responses, CTLA-4-deficient mice suffer from a fatal overactivated phenotype resulting in profound autoimmunity driven by self-reactive T cells (12Tivol EA Borriello F Schweitzer AN Lynch WP Bluestone JA Sharpe AH Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4.Immunity. 1995; 3: 541-547Abstract Full Text PDF PubMed Scopus (2412) Google Scholar,13Ise W Kohyama M Nutsch KM et al.CTLA-4 suppresses the pathogenicity of self antigen-specific T cells by cell-intrinsic and cell-extrinsic mechanisms.Nat Immunol. 2010; 11: 129-135Crossref PubMed Scopus (138) Google Scholar). This functional dichotomy between CD28 and CTLA-4 is even more striking given that these two diametrically opposite outcomes are controlled by interactions with the same ligands. The CD28/CTLA-4 pathway is therefore perhaps best viewed as an integrated system, which controls the balance between T cell activation and self-tolerance, in a process that is influenced by the expression of two shared ligands. At present, there are a large number of interpretations of how the CTLA-4 pathway functions based on a wide range of experiments. We will not try to cover all experimental observations, but concentrate on selected ideas in order to generate a conceptual framework for understanding how the various manipulations of the CD28/CTLA-4 pathway available clinically might impact immune function. Based predominantly on studies using agonistic anti-CTLA-4 antibodies, ideas relating to CTLA-4 function have frequently focused on the concept of an inhibitory signal preventing T cell activation (14Krummel MF Allison JP CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells.J Exp Med. 1996; 183: 2533-2540Crossref PubMed Scopus (780) Google Scholar,15Walunas TL Bakker CY Bluestone JA CTLA-4 ligation blocks CD28-dependent T cell activation.J Exp Med. 1996; 183: 2541-2550Crossref PubMed Scopus (700) Google Scholar). According to this concept, ligand binding to CTLA-4 (up-regulated on activated T cells) would be expected to generate intrinsic inhibitory signals that “switch off” T cell activation, proliferation and IL-2 production. This has been expanded to include roles for the direct recruitment of phosphatases (16Lee KM Chuang E Griffin M et al.Molecular basis of T cell inactivation by CTLA-4.Science. 1998; 282: 2263-2266Crossref PubMed Scopus (547) Google Scholar), effects on T cell adhesion and motility (17Schneider H Downey J Smith A et al.Reversal of the TCR stop signal by CTLA-4.Science. 2006; 313: 1972-1975Crossref PubMed Scopus (483) Google Scholar) and proposed roles for various splice variants of CTLA-4 (18Stumpf M Zhou X Bluestone JA The B7-independent isoform of CTLA-4 functions to regulate autoimmune diabetes.J Immunol. 2013; 190: 961-969Crossref PubMed Scopus (34) Google Scholar, 19Araki M Chung D Liu S et al.Genetic evidence that the differential expression of the ligand-independent isoform of CTLA-4 is the molecular basis of the Idd5.1 type 1 diabetes region in nonobese diabetic mice.J Immunol. 2009; 183: 5146-5157Crossref PubMed Scopus (63) Google Scholar, 20Vijayakrishnan L Slavik JM Illes Z et al.An autoimmune disease-associated CTLA-4 splice variant lacking the B7 binding domain signals negatively in T cells.Immunity. 2004; 20: 563-575Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) with presumed inhibitory signaling capacity. The concept that emerges from these studies is that CTLA-4 possesses an inhibitory signaling capacity. However, there are still questions over the extent to which cross-linked antibodies to CTLA-4 are a faithful mimic of CTLA-4 engagement by its ligands and the nature of inhibitory signals still remains uncertain. Therefore, while numerous cell-intrinsic concepts for CTLA-4 function have been proposed and discussed (21Wing K Yamaguchi T Sakaguchi S Cell-autonomous and non-autonomous roles of CTLA-4 in immune regulation.Trends Immunol. 2011; 32: 428-433Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 22Walker LS Sansom DM The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses.Nat Rev Immunol. 2011; 11: 852-863Crossref PubMed Scopus (496) Google Scholar, 23Rudd CE The reverse stop-signal model for CTLA4 function.Nat Rev Immunol. 2008; 8: 153-160Crossref PubMed Scopus (100) Google Scholar, 24Bour-Jordan H Esensten JH Martinez-Llordella M Penaranda C Stumpf M Bluestone JA Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of the CD28/B7 family.Immunol Rev. 2011; 241: 180-205Crossref PubMed Scopus (293) Google Scholar) the role of such signals in CTLA-4 function in vivo remains unestablished. A major challenge to the physiological importance of the cell-intrinsic signaling mechanisms mentioned above is a series of straightforward and widely repeated experiments in chimeric mice that possess both CTLA-4 WT and CTLA-4-deficient T cells. This reveals that mice containing mixtures of CTLA-4−/− and CTLA-4+/+ T cells fail to develop lethal lymphoproliferative disease and the CTLA-4−/− T cells maintain a normal nonactivated phenotype (25Bachmann MF Kohler G Ecabert B Mak TW Kopf M Cutting edge: Lymphoproliferative disease in the absence of CTLA-4 is not T cell autonomous.J Immunol. 1999; 163: 1128-1131Crossref PubMed Google Scholar, 26Homann D Dummer W Wolfe T et al.Lack of intrinsic CTLA-4 expression has minimal effect on regulation of antiviral T-cell immunity.J Virol. 2006; 80: 270-280Crossref PubMed Scopus (47) Google Scholar, 27Friedline RH Brown H et regulatory T cells CTLA-4 for the of tolerance.J Exp Med. 2009; PubMed Scopus Google Scholar). that the critical CTLA-4 required to are therefore T Such well with the of CTLA-4 in a such as an effector molecule on but are with direct inhibitory signaling in effector T cells. In of the concept of extrinsic regulation has that CTLA-4 is an important of Treg function. 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CTLA-4-deficient mice lacking both with CD28 deficiency or with ligand all have disease X F Sharpe AH A of autoimmune disease in mice on a CD28 that is required for in vivo S 2007; PubMed Scopus Google EA Boyd SD S et lymphoproliferation and fatal multiorgan tissue in CTLA-4-deficient mice.J Immunol. 1997; PubMed Google Scholar). the concept that emerges is that a key role of CTLA-4 is to control of the CD28 pathway its ligands. the of using CTLA-4 to inhibit CD28 function by preventing to ligands the of both and has not been widely thought of as a mechanism for CTLA-4 function However, we a novel molecular mechanism of CTLA-4 which this Based on using cell we that of ligands and between cells in with CTLA-4 cells Zheng Y K et of CD80 and A molecular basis for the cell-extrinsic function of CTLA-4.Science. 2011; PubMed Scopus Google Scholar). 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This process in vivo and is seen in T cells but not to and of ligands by can therefore be as a cell-extrinsic of ligand This process is of the mechanism by or with the difference that with ligand is as to with the concept of evidence that CTLA-4 is to the of CD80 and CD86 on antigen-presenting cells K Y P et al.CTLA-4 control over regulatory T cell 2008; PubMed Scopus Google Y Z Yamaguchi T Sakaguchi S regulatory T cells on dendritic cells in and inhibit their S 2008; PubMed Scopus Google Scholar, C L A F T of costimulatory molecules on dendritic cells in regulatory 2006; PubMed Scopus Google Scholar, W G et T cells control T cell effector IL-2 Immunol. 2011; PubMed Scopus Google Scholar). by CTLA-4 therefore a for how this can be as well as a CD28 and CTLA-4 ligands: this not the then CTLA-4 would be to control CD28 in this In of functional the in the of costimulation available CD28 be to activation of self-reactive T cells that from the and CTLA-4 expression can regulate an and model of T with a function for CTLA-4 on and which many of the features of CTLA-4 The above the that CD28 engagement represents a key checkpoint in the activation of T cells and that CTLA-4 this function. Therefore, CD28 and CTLA-4 as that function the pathway is best viewed as an integrated system with CTLA-4 a balance to CD28 function. of this balance can therefore have a number of on the functional mechanisms that are to be at we and how immune function in both and has to been the most widely for of the CD28/CTLA-4 pathway. antibodies to CD80 and CD86 have been as a for the CD28/CTLA-4 however, for immune responses, ligand has not been widely in a In contrast to of CD80 and of both ligands has been widely in the of the protein available clinically in two and and the affinity of CTLA-4 for its these have been to inhibit T cell responses in of autoimmune disease and in transplantation T-cell in Two 2013; PubMed Scopus Google T-cell costimulatory in Med. 2013; 3: PubMed Scopus Google Scholar). The of which a domain has been for the of to in have not to M M et and of of in with and an response to PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar) also that their the nature of the CTLA-4 in this model remains to be The is that CD28 the of inhibitory signals CTLA-4, which are then using anti-CTLA-4 As above this concept is not with of the on CTLA-4 in vivo LS Sansom DM The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses.Nat Rev Immunol. 2011; 11: 852-863Crossref PubMed Scopus (496) Google Scholar). is that anti-CTLA-4 affects Treg which the of effector responses CD28. while CD28 would to be a for immune in understanding mechanism of action are The two have progress in manipulating the immune response the CD28/CTLA-4 pathway for therapeutic This progress has of our understanding of how the pathway The mechanistic in our relating to CTLA-4, to be in order that for effector T cells while Treg functions can be The of this have of to as by the of