by negative magnetic separation (EasySep Human Naive CD4 + T cell Enrichment Kit, Stem Cell Technologies). Cells were plated at a density of 100,000 per well in a 96-well round bottom plate and stimulated with anti-CD3/28 beads (Human T-Activator Dynabeads, Life Technologies) for 5 days. Where indicated, cultures were treated with 10 ng/ml IL-12 and/or 20 ng/ml IL-2 (both from Peprotech). Statistics. Data were analyzed using Prism statistical software. Statistical significance was assessed using the Mann-Whitney test. Paired data were analyzed using a 2-tailed paired Student's t test. A P value of less than 0.05 was considered significant.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is an essential regulator of T-cell responses, and its absence precipitates lethal T-cell hyperactivity. However, whether CTLA-4 acts simply to veto the activation of certain clones or plays a more nuanced role in shaping the quality of T-cell responses is not clear. Here we report that T cells in CTLA-4-deficient mice show spontaneous Tfollicular helper (T FH ) differentiation in vivo, and this is accompanied by the appearance of large germinal centers (GCs). Remarkably, short-term blockade with anti-CTLA-4 antibody in wild-type mice is sufficient to elicit T FH generation and GC development. The latter occurs in a CD28-dependent manner, consistent with the known role of CTLA-4 in regulating the CD28 pathway. CTLA-4 can act by down-regulating CD80 and CD86 on antigen presenting cells (APCs), thereby altering the level of CD28 engagement. To mimic reduced CD28 ligation, we used mice heterozygous for CD28, revealing that the magnitude of CD28 engagement is tightly linked to the propensity for T FH differentiation. In contrast, other parameters of T-cell activation, including CD62L down-regulation and Ki67 expression, were relatively insensitive to altered CD28 level. Altered T FH generation as a result of graded reduction in CD28 was associated with decreased numbers of GC B cells and a reduction in overall GC size. These data support a model in which CTLA-4 control of immunity goes beyond vetoing T-cell priming and encompasses the regulation of T FH differentiation by graded control of CD28 engagement.ontrol of the magnitude and nature of adaptive immune responses is critical for health. The cytotoxic T-lymphocyteassociated antigen-4 (CTLA-4)/CD28 axis has long been known to control the magnitude of T-cell responses, however whether it also influences their nature has not been clear. Early studies suggested that CD28 may be particularly important for Th2 differentiation (1, 2), although others identified roles for CD28 in both Th1 and Th2 responses (3, 4). It is known that CD28 is an absolute requirement for the differentiation of follicular helper T cells (T FH s) that support germinal center (GC) formation (5, 6). However, these studies generally make use of CD28-deficient T cells, and therefore, results may reflect a failure of the cells to properly activate, proliferate, or survive, particularly given the known contribution of CD28 to these processes.A key outstanding question is whether CD28 costimulation in vivo is more complex than a binary checkpoint for T-cell priming. It is clear that expression of costimulatory ligands on antigen presenting cells (APCs) fluctuates in response to environmental stimuli, being up-regulated by inflammatory cytokines and TLR agonists and down-regulated by Treg-expressed CTLA-4 (7-11). Thus, variable levels of costimulatory ligands will be available for CD28 binding depending on the microenvironmental context. However, whether this simply alters the number of T cells that achieve the required threshold to commit to a resp...

The CTLA-4 pathway is recognized as a major immune inhibitory axis and is a key therapeutic target for augmenting antitumor immunity or curbing autoimmunity. CTLA-4-deficient mice provide the archetypal example of dysregulated immune homeostasis, developing lethal lymphoproliferation with multiorgan inflammation. In this study, we show that surprisingly these mice have an enlarged population of Foxp3+ regulatory T cells (Treg). The increase in Treg is associated with normal thymic output but enhanced proliferation of Foxp3+ cells in the periphery. We confirmed the effect of CTLA-4 deficiency on the Treg population using OVA-specific Treg which develop normally in the absence of CTLA-4, but show increased proliferation in response to peripheral self-Ag. Functional analysis revealed that Ag-specific Treg lacking CTLA-4 were unable to regulate disease in an adoptive transfer model of diabetes. Collectively, these data suggest that the proliferation of Treg in the periphery is tuned by CTLA-4 signals and that Treg expression of CTLA-4 is required for regulation of pancreas autoimmunity.

The activity of regulatory T cells (Treg) is widely accepted to play a central role in preventing pathogenic immune responses against self-Ags. However, it is not clear why such regulation breaks down during the onset of autoimmunity. We have studied self-Ag-specific Treg during the induction of spontaneous diabetes. Our data reveal a shift in the balance between regulatory and pathogenic islet-reactive T cells in the pancreas-draining lymph nodes during disease onset. Treg function was not compromised during disease initiation, but instead conventional T cells showed reduced susceptibility to Treg-mediated suppression. Release from Treg suppression was associated with elevated levels of IL-21 in vivo, and provision of this cytokine abrogated Treg suppression in vitro and in vivo. These data suggest that immunological protection of a peripheral tissue by Treg can be subverted by IL-21, suggesting new strategies for intervention in autoimmunity.

IntroductionAlthough regulation is essential for homeostatic control of the immune system, it also presents a barrier to effective tumor surveillance. Professional immune regulation is orchestrated by T cells expressing the transcription factor Foxp3, which fixes a genetic program that imparts suppressive function. The majority of Foxp3-expressing regulatory T cells appear to arise in the thymus and represent, in the main, a remarkably stable population committed to providing life-long immune regulation. 1 Regulatory T cells (Tregs) can call on a wide range of different mechanisms to exert suppressive activity, including pathways involving IL-10, TGF-␤, and CTLA4.In recent years, it has become apparent that the ability of Tregs to elicit effective suppression can be modified, not just by the ratio of suppressors to target cells, but also by the local cytokine environment. Multiple cytokines have reported to interfere with Treg suppression, including 2,3 TNF,3,[4][5][6][7]4,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]7 This raises the possibility that the way in which T cells differentiate may dictate the extent to which a given T-cell response may be regulated. Consistent with this notion, it has been shown that human Th17 clones are significantly less susceptible to Treg suppression than those exhibiting a Th1 or Th2 phenotype. 8 Understanding the rules that govern professional immune regulation has important implications for our ability to alter the magnitude of immune responses therapeutically, for example, to augment antitumor immunity or diminish autoimmunity. In this study, we have focused on the cytokine IL-21, which we and others have previously shown can counteract Treg suppression in vitro and in vivo. 6,7 We have explored the mechanism underlying the capacity of IL-21 to alter Treg suppression and have pinpointed which cell population needs to receive IL-21 signals to permit this effect. We find that IL-21 signaling to Tregs does not impair their suppressive capacity, but instead IL-21 signaling to conventional T cells is responsible for the abrogation of suppression. Our work reveals a novel feedback loop by which IL-21 impacts on Treg homeostasis by down-regulating IL-2 production from conventional T cells. Thus, in addition to inhibiting de novo Treg differentiation, an affect that has been well documented for IL-21, 9-11 this cytokine also negatively regulates the homeostasis of natural Tregs. Methods Mice DO11.10 TCR transgenic and BALB/c mice were purchased from The Jackson Laboratory. IL-21R Ϫ/Ϫ mice were provided by M.K. Rat insulin promoter (RIP)-mOVA mice on a BALB/c background expressing a membrane-bound form of OVA under the control of the RIP (from line 296-1B) were a gift from W. Heath (Walter and Eliza Hall Institute, Melbourne, Australia). DO11.10 mice and RIP-mOVA were crossed as previously described. 12 Mice were housed at the University of Birmingham Biomedical Services Unit and used according to Home Office and institutional guidelines under Home Office Project L...

CTLA-4 is a critical negative regulator of the immune system and a major target for immunotherapy. Yet precisely how it functions in vivo to maintain immune homeostasis is not clear. As a highly endocytic molecule, CTLA-4 can capture costimulatory ligands from opposing cells by a process of transendocytosis (TE). By restricting costimulatory ligand expression in this manner, CTLA-4 controls the CD28-dependent activation of T cells. Regulatory T cells (Tregs) constitutively express CTLA-4 at high levels and in its absence show defects in TE and suppressive function. Activated conventional T cells (Tconv) are also capable of CTLA-4-dependent TE, however the relative use of this mechanism by Tregs and Tconv in vivo remains unclear. Here we set out to characterize both the perpetrators and cellular targets of CTLA-4 TE in vivo. We found that Tregs showed constitutive cell surface recruitment of CTLA-4 ex vivo and performed TE rapidly following TCR stimulation. Tregs outperformed activated Tconv at TE in vivo, and expression of ICOS marked Tregs with this capability. Using TCR-transgenic Tregs that recognise a protein expressed in the pancreas, we showed that presentation of tissue-derived self-antigen could trigger Tregs to capture costimulatory ligands in vivo. Finally, we identified migratory dendritic cells (DCs) as the major target for Treg-based CTLA-4-dependent regulation in the steady state. These data support a model in which CTLA-4 expressed on Tregs dynamically regulates the phenotype of DCs trafficking to lymph nodes from peripheral tissues in an antigen-dependent manner.

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Whilst it is clear that CTLA-4 is a critical regulator of T cell responses, the immunological contexts in which CTLA-4 controls immune responses are not well defined. Here we show that whilst CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression, in this context CTLA-4 blocking antibodies had no impact on the response. In contrast, in settings where CTLA-4+ cells were present as “regulators”, inhibition of resting T cell responses was dependent on CTLA-4 expression and specifically related to the number of antigen presenting cells. At low numbers of APC or low levels of ligand, CTLA-4-dependent suppression was highly effective whereas at higher APC numbers or high levels of ligand, inhibition was lost. Accordingly, the degree of suppression correlated with the level of CD86 expression remaining on the antigen presenting cells. These data reveal clear rules for the inhibitory function of CTLA-4 on Treg which are predicted by its ability to remove ligands from antigen presenting cells.

The CTLA-4 pathway is a key regulator of T cell activation and a critical failsafe against autoimmunity. While early models postulated that CTLA-4 transduced a negative signal, in vivo evidence suggests that CTLA-4 functions in a cell-extrinsic manner. That multiple cell-intrinsic mechanisms have been attributed to CTLA-4, yet its function in vivo appears to be cell-extrinisic, has been an ongoing paradox in the field. Whilst CTLA-4 expressed on conventional T cells (Tconv) can mediate inhibitory function, it is unclear why this fails to manifest as an intrinsic effect. Here we show that Tconv-expressed CTLA-4 can function in a cell-extrinsic manner in vivo. CTLA-4+/+ T cells, from DO11/rag−/− mice that lack Treg, were able to regulate the response of CTLA4−/− T cells in co-transfer experiments. This observation provides a potential resolution to the above paradox, and suggests CTLA-4 function on both Tconv and Treg can be achieved through cell-extrinsic mechanisms.