Cynthia A. Chambers

Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the antitumor activity of anti–CTLA-4 antibodies

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is a critical negative regulator of immune responses. Uniquely among known inhibitory receptors, its genetic ablation results in a fulminating and fatal lymphoproliferative disorder. This central regulatory role led to the development of antibodies designed to block CTLA-4 activity in vivo, aiming to enhance immune responses against cancer. Despite their preclinical efficacy and promising clinical activity against late stage metastatic melanoma, the critical cellular targets for their activity remains unclear. In particular, debate has focused on whether the effector T cell (Teff) or regulatory T cell (T reg cell) compartment is the primary target of antibody-mediated blockade. We developed a mouse expressing human instead of mouse CTLA-4, allowing us to evaluate the independent contributions of CTLA-4 blockade of each T cell compartment during cancer immunotherapy in an in vivo model of mouse melanoma. The data show that although blockade on effector cells significantly improves tumor protection, unicompartmental blockade on regulatory cells completely fails to enhance antitumor responses. However, concomitant blockade of both compartments leads to a synergistic effect and maximal antitumor activity. We conclude that the combination of direct enhancement of Teff cell function and concomitant inhibition of T reg cell activity through blockade of CTLA-4 on both cell types is essential for mediating the full therapeutic effects of anti–CTLA-4 antibodies during cancer immunotherapy.

Co-stimulation in T cell responses

Antigen-specific T cell responses have primarily been considered in terms of activation signals delivered through the TCR and the co-stimulatory molecule CD28. In the past few years, studies have demonstrated the critical importance of inhibitory signals for regulating lymphocyte activation. CD28 and its homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) share the same counter-receptors on antigen-presenting cells, but recent experiments have shown that CD28 and CTLA-4 have opposite effects on T cell activation. The mechanisms responsible for integrating these activation and inhibitory signals at the cellular and molecular levels are just beginning to be elucidated.

Lymphoproliferation in CTLA-4–Deficient Mice Is Mediated by Costimulation-Dependent Activation of CD4+ T Cells

CTLA-4-deficient animals develop a fatal lymphoproliferative disorder. The cellular mechanism(s) responsible for this phenotype have not been determined. Here, we show that there is a preferential expansion of CD4+ T cells in CTLA-4(-/-) mice, which results in a skewing of the CD4/CD8 T cell ratio. In vivo antibody depletion of CD8+ T cells from birth does not alter the onset or the severity of the CD28-dependent lymphoproliferative disorder. In contrast, CD4+ T cell depletion completely prevents all features characteristic of the lymphoproliferation observed in CTLA-4-deficient mice. These results demonstrate that CD4+ T cells initiate the phenotype in the CTLA-4(-/-) mice. Further, these results suggest that the role of CTLA-4 in peripheral CD4+ versus CD8+ T cell homeostasis is distinct.

The expanding world of co-stimulation: the two-signal model revisited

The crucial role for CD28, its homolog CTLA-4 and their binding partners B7-1 and B7-2 in the generation of effective T-cell responses has been well documented. Recently, two new pairs of the CD28/B7 families were identified. The ability of these molecules to regulate T-cell expansion and effector function and the dynamic integration of the co-stimulatory and T-cell receptor signals are just beginning to be explored. Understanding these processes will be crucial for designing clinically relevant approaches to manipulate the adaptive immune system.

The Role of CTLA‐4 in the Regulation and Initiation of T‐Cell Responses

The generation of an effective immune response involves antigen-specific T-cell expansion and difTerentiation of effector function. This process involves at least two signals: one mediated by interaction of the T-cell antigen receptor (TCR) with specific antigen in association with major histocompatibility complex (MHC) molecules, and the second an antigen-independent, costimulatory signal. Over the past few years it has become established that the primary source of costimulatory signals is the interaction between CD28 on the T cell with members of the B7 family (CD80, CD86) on the antigen-presenting cell (APC). For several years the functional role of the enigmatic CD28 homologue CTLA-4 in T-cell activation has been both obscure and controversial. In this article we review recent results from our laboratory and others that provide compelling support for an inhibitory role for CTLA-4 in the regulation of T-cell responses. We discuss these results in the context of the two-signal model of T-cell activation, and propose that T-cell responses are regulated by a complex and dynamic interplay of TCR, CD28 and CTLA-4 signals at both early and late stages of T-cell activation. We also discuss the potential offered by emerging information concerning CTLA-4 for the development of new strategies for the manipulation of immune responses in tumour immunolherapy and other clinical situations.

CD4+ regulatory T cells require CTLA-4 for the maintenance of systemic tolerance

Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a critical role in negatively regulating T cell responses and has also been implicated in the development and function of natural FOXP3+ regulatory T cells. CTLA-4–deficient mice develop fatal, early onset lymphoproliferative disease. However, chimeric mice containing both CTLA-4–deficient and –sufficient bone marrow (BM)–derived cells do not develop disease, indicating that CTLA-4 can act in trans to maintain T cell self-tolerance. Using genetically mixed blastocyst and BM chimaeras as well as in vivo T cell transfer systems, we demonstrate that in vivo regulation of Ctla4−/− T cells in trans by CTLA-4–sufficient T cells is a reversible process that requires the persistent presence of FOXP3+ regulatory T cells with a diverse TCR repertoire. Based on gene expression studies, the regulatory T cells do not appear to act directly on T cells, suggesting they may instead modulate the stimulatory activities of antigen-presenting cells. These results demonstrate that CTLA-4 is absolutely required for FOXP3+ regulatory T cell function in vivo.

Dual function of CTLA-4 in regulatory T cells and conventional T cells to prevent multiorgan autoimmunity

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory receptor on T cells essential for maintaining T cell homeostasis and tolerance to self. Mice lacking CTLA-4 develop an early onset, fatal breakdown in T cell tolerance. Whether this autoimmune disease occurs because of the loss of CTLA-4 function in regulatory T cells, conventional T cells, or both is unclear. We show here that lack of CTLA-4 in regulatory T cells leads to aberrant activation and expansion of conventional T cells. However, CTLA-4 expression in conventional T cells prevents aberrantly activated T cells from infiltrating and fatally damaging nonlymphoid tissues. These results demonstrate that CTLA-4 has a dual function in maintaining T cell tolerance: CTLA-4 in regulatory T cells inhibits inappropriate naïve T cell activation and CTLA-4 in conventional T cells prevents the harmful accumulation of self-reactive pathogenic T cells in vital organs.

SECONDARY BUT NOT PRIMARY T CELL RESPONSES ARE ENHANCED IN CTLA-4-DEFICIENT CD8+ T CELLS

Negative as well as positive co‐stimulation appears to play an important role in controlling T cell activation. CTLA‐4 has been proposed to negatively regulate T cell responses. CTLA‐4‐deficient mice develop a lymphoproliferative disorder, initiated by the activation and expansion of CD4+ T cells. To assess the function of CTLA‐4 on CD8+ T cells, CTLA‐4−/‐ animals were crossed to an MHC class I‐restricted 2C TCR transgenic mouse line. We demonstrate that although the primary T cell responses were similar, the CTLA‐4‐deficient 2C TCR+ CD8+ T cells displayed a greater proliferative response upon secondary stimulation than the 2C TCR+ CD8+ T cells from CTLA‐4 wild‐type mice. These results suggest that CTLA‐4 regulates antigen‐specific memory CD8+ T cell responses.

Lack of a role for transforming growth factor-β in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-β1 (TGF-β1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-β production. Here, we examine the role of TGF-β in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-β production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-β does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-β1−/−, and Smad3−/− T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-β by CD4+ T cells. These results indicate that CTLA-4 ligation does not regulate TGF-β production and that CTLA-4-mediated inhibition can occur independently of TGF-β. Collectively, these data demonstrate that CTLA-4 and TGF-β represent distinct mechanisms for regulation of T cell responses.

STAT5 is required for thymopoiesis in a development stage-specific manner.

Diverse cytokines necessary for normal lymphopoiesis and lymphocyte homeostasis activate STAT5 in responder cells. Although STAT5 has been suggested to be a central molecular effecter of IL-7 function, its essential role during IL-7-dependent T cell development in vivo remained unclear. Using Stat5−/− mice we now show that STAT5 is essential for various functions ascribed to IL-7 in vivo. STAT5 is required for embryonic thymocyte production, TCRγ gene transcription, and Peyer’s patch development. In sharp contrast, normal STAT5 is dispensable for adult thymopoiesis. In peripheral lymphocytes, STAT5 is primarily required for the generation and/or maintenance of γδ T cells and TCRγδ+ intraepithelial lymphocytes. Collectively, these results demonstrate that STAT5 is critical for many, but not all, aspects of steady state lymphoid lineage development and maintenance and suggest the existence of previously undocumented cytokine signaling traits and/or cytokine milieu during adult thymopoiesis.