Cutting edge: cell-extrinsic immune regulation by CTLA-4 expressed on conventional T cells

The CTLA-4 pathway is a key regulator of T cell activation and a critical failsafe against autoimmunity. Although 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-extrinsic, has been an ongoing paradox in the field. Although CTLA-4 expressed on conventional T cells (Tconv) can mediate inhibitory function, it is unclear why this fails to manifest as an intrinsic effect. In this study, 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 regulatory T cells, were able to regulate the response of CTLA-4(-/-) T cells in cotransfer experiments. This observation provides a potential resolution to the above paradox and suggests CTLA-4 function on both Tconv and regulatory T cells can be achieved through cell-extrinsic mechanisms.     

Reduced ultraviolet-induced carcinogenesis in mice with a functional disruption in B7-mediated costimulation

Immunosuppression by UV light contributes significantly to the induction of skin cancer by suppressing the cell-mediated immune responses which control the development of carcinogenesis. The B7/CD28-CTLA-4 signaling pathway provides costimulatory signals essential for Ag-specific T cell activation. To investigate the role of this pathway in photocarcinogenesis, we utilized transgenic (Tg) mice which constitutively express CTLA-4Ig, a high-affinity CD28/CTLA-4 antagonist that binds to both B7-1 and B7-2. The transgene is driven by a skin-specific promoter yielding high levels of CTLA-4Ig in the skin and serum. Chronic UV exposure of CTLA-4Ig Tg mice resulted in significantly reduced numbers of skin tumors, when compared to control mice. In addition, Tg mice were resistant to UV-induced suppression of delayed-type hypersensitivity responses to alloantigens. Most importantly, upon stimulation with mitogens and alloantigens, T cells isolated from CTLA-4Ig Tg mice produced significantly less IL-4 but more IFN-gamma compared to control T cells, suggesting an impaired Th2 response and a relative increase of Th1-type immunity. Together, these data show that overall B7 engagement directs immune responses toward the Th2 pathway. Moreover, they point out the crucial role of Th1 immune reactions in the protection against photocarcinogenesis.     

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.     

B7-independent inhibition of T cells by CTLA-4

CTLA-4 is an inhibitory molecule that regulates T cell expansion and differentiation. CTLA-4 binding to B7-1/B7-2 is believed to be crucial for its inhibitory signal both by competing for CD28 binding to the same ligands and aggregating CTLA-4 to deliver negative signals. In this study, we demonstrate that B7 binding is not essential for CTLA-4 activity. CTLA-4 knockout T cells are hyperresponsive compared with wild-type T cells in B7-free settings. Expression of a B7-nonbinding CTLA-4 mutant inhibited T cell proliferation, cytokine production, and TCR-mediated ERK activation in otherwise CTLA-4-deficient T cells. Finally, transgenic expression of the ligand-nonbinding CTLA-4 mutant delayed the lethal lymphoproliferation observed in CTLA-4-deficient mice. These results suggest that ligand binding is not essential for the CTLA-4 function and supports an essential role for CTLA-4 signaling during T cell activation.     

Cutting edge: the relative distribution of T cells responding to chemically dominant or minor epitopes of lysozyme is not affected by CD40-CD40 ligand and B7-CD28-CTLA-4 costimulatory pathways

We examined the frequencies and specificities of the CD4+ T cell responses to the protein hen egg white lysozyme in mice deficient in the CD40-CD40 ligand or B7-CD28 costimulatory pathways. The frequency of T cells was decreased by between 3- and 4-fold in CD40-/- mice, and 12-fold in B7-1/B7-2-/- mice, but surprisingly, the relative distribution of T cells responding to peptides that were presented at levels that differed by >250-fold was similar. We also examined the CD4 response after blocking the regulatory molecule CTLA-4 during immunization. We observed no difference in either the frequency or specificity of the CD4+ T cell response if CTLA-4 was blocking during priming. Thus, the T cell response was generated toward the constellation of chemically dominant and subdominant epitopes as a whole, and did not discriminate among them based on their relative abundance.     

Spontaneous colitis occurrence in transgenic mice with altered B7-mediated costimulation

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.     

Established T cell-driven germinal center B cell proliferation is independent of CD28 signaling but is tightly regulated through CTLA-4

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.     

Cutting edge: an NK cell-independent role for Slamf4 in controlling humoral autoimmunity

Several genes within a syntenic region of human and mouse chromosome 1 are associated with predisposition to systemic lupus erythematosus. Analyses of lupus-prone congenic mice have pointed to an important role for the signaling lymphocyte activation molecule family (slamf)6 surface receptor in lupus pathogenesis. In this article, we demonstrate that a second member of the Slamf gene family, Slamf4 (Cd244), contributes to lupus-related autoimmunity. B6.Slamf4(-/-) mice spontaneously develop activated CD4 T cells and B cells and increased numbers of T follicular helper cells and a proportion develop autoantibodies to nuclear Ags. B6.Slamf4(-/-) mice also exhibit markedly increased autoantibody production in the B6.C-H-2bm12/KhEg → B6 transfer model of lupus. Although slamf4 function is best characterized in NK cells, the enhanced humoral autoimmunity of B6.Slamf4(-/-) mice is NK cell independent, as judged by depletion studies. Taken together, our findings reveal that slamf4 has an NK cell-independent negative regulatory role in the pathogenesis of lupus a normally non-autoimmune prone genetic background.     

Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells

Ex vivo treatment of bone marrow-derived dendritic cells (DCs) with TNF-alpha has been previously shown to induce partial maturation of DCs that are able to suppress autoimmunity. In this study, we demonstrate that i.v. administration of TNF-alpha-treated, semimature DCs pulsed with thyrogloblin (Tg), but not with OVA Ag, inhibits the subsequent development of Tg-induced experimental autoimmune thyroiditis (EAT) in CBA/J mice. This protocol activates CD4(+)CD25(+) T cells in vivo, which secrete IL-10 upon specific recognition of Tg in vitro and express regulatory T cell (Treg)-associated markers such as glucocorticoid-induced TNFR, CTLA-4, and Foxp3. These CD4(+)CD25(+) Treg cells suppressed the proliferation and cytokine release of Tg-specific, CD4(+)CD25(-) effector cells in vitro, in an IL-10-independent, cell contact-dependent manner. Prior adoptive transfer of the same CD4(+)CD25(+) Treg cells into CBA/J hosts suppressed Tg-induced EAT. These results demonstrate that the tolerogenic potential of Tg-pulsed, semimature DCs in EAT is likely to be mediated through the selective activation of Tg-specific CD4(+)CD25(+) Treg cells and provide new insights for the study of Ag-specific immunoregulation of autoimmune diseases.     

Costimulation requirements of induced murine systemic autoimmune disease

Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl2-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L-/- mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.     

Specific T regulatory cells display broad suppressive functions against experimental allergic encephalomyelitis upon activation with cognate antigen

To date, very few Ag-based regimens have been defined that could expand T regulatory (Treg) cells to reverse autoimmunity. Additional understanding of Treg function with respect to specificity and broad suppression should help overcome these limitations. Ig-proteolipid protein (PLP)1, an Ig carrying a PLP1 peptide corresponding to amino acid residues 139-151 of PLP, displayed potent tolerogenic functions and proved effective against experimental allergic encephalomyelitis (EAE). In this study, we took advantage of the Ig-PLP1 system and the PLP1-specific TCR transgenic 5B6 mouse to define a regimen that could expand Ag-specific Treg cells in vivo and tested for effectiveness against autoimmunity involving diverse T cell specificities. The findings indicate that in vivo exposure to aggregated Ig-PLP1 drives PLP1-specific 5B6 TCR transgenic cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3 and producing IL-10. These Treg cells were able to suppress PLP1 peptide-induced EAE in both SJL/J and F(1) (SJL/J x C57BL/6) mice. However, despite being effective against disease induced with a CNS homogenate, the Treg cells were unable to counter EAE induced by a myelin basic protein or a myelin oligodendrocyte glycoprotein peptide. Nevertheless, activation with Ag before transfer into the host mice supports suppression of both myelin oligodendrocyte glycoprotein- and myelin basic protein peptide-induced EAE. Thus, it is suggested that activation of Treg cells by the cognate autoantigen is necessary for operation of broad suppressive functions.     

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RB(high)CD4(+) T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RB(high)CD4(+) cells from CD28(-/-) mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RB(high)CD4(+) cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-gamma by lamina propria CD4(+) cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25(+)CD4(+) cells from CD28(-/-) mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RB(high) cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.     

CTLA-4+CD8+ T cells that encounter B7-2+ iris pigment epithelial cells express their own B7-2 to achieve global suppression of T cell activation

Pigment epithelial (PE) cells cultured from the eye possess the novel property of suppressing TCR-dependent activation of T cells in vitro. Iris PE (IPE) cells accomplish this suppression by a direct cell contact mechanism in which B7-2 expressed by the PE cells interacts with CTLA-4 on responding T cells. Because CTLA-4 expression is constitutively expressed on a very small proportion of naive splenic T cells and since exposure of splenic T cells to IPE leads to global T cell suppression, we have inquired into the mechanism by which suppression is achieved. Using splenic T cells and IPE from donor mice with disrupted genes for CD80 (B7-1), CD86 (B7-2), CTLA-4, and/or CD28, we report that B7-2(+) IPE in the presence of anti-CD3 supported selectively the activation of CTLA-4(+) CD8(+) T cells that express their own B7-2 and secrete enhanced amounts of active TGFbeta. By contrast, activation of CTLA-4-negative T cells, especially CD4(+) cells, in these cultures was profoundly suppressed. Because global suppression of T cell activation in these cultures was obtained only when both IPE and T cells possessed B7-2 genes and expressed the costimulators as surface molecules, we propose that T cells activated in the presence of parenchymal cells from the eye (an immune privileged site) express B7-2 in a manner that equips them to suppress bystander T cells. Thus, B7-2 expression on T cells participates in their eventual ability to function as regulators in vitro.     

TIRC7 deficiency causes in vitro and in vivo augmentation of T and B cell activation and cytokine response

The membrane protein T cell immune response cDNA 7 (TIRC7) was recently identified and was shown to play an important role in T cell activation. To characterize the function of TIRC7 in more detail, we generated TIRC7-deficient mice by gene targeting. We observed disturbed T and B cell function both in vitro and in vivo in TIRC7(-/-) mice. Histologically, primary and secondary lymphoid organs showed a mixture of hypo-, hyper-, and dysplastic changes of multiple lymphohemopoietic compartments. T cells from TIRC7(-/-) mice exhibited significantly increased proliferation and expression of IL-2, IFN-gamma, and IL-4 in response to different stimuli. Resting T cells from TIRC7(-/-) mice exhibited decreased CD62L, but increased CD11a and CD44 expression, suggesting an in vivo expansion of memory/effector T cells. Remarkably, activated T cells from TIRC7(-/-) mice expressed lower levels of CTLA-4 in comparison with wild-type cells. B cells from TIRC7-deficient mice exhibited significantly higher in vitro proliferation following stimulation with anti-CD40 Ab or LPS plus IL-4. B cell hyperreactivity was reflected in vivo by elevated serum levels of various Ig classes and higher CD86 expression on B cells. Furthermore, TIRC7 deficiency resulted in an augmented delayed-type hypersensitivity response that was also reflected in increased mononuclear infiltration in the skin obtained from TIRC7-deficient mice food pads. In summary, the data strongly support an important role for TIRC7 in regulating both T and B cell responses.     

CD4+CD25+ T cell-dependent inhibition of autoimmunity in transgenic mice overexpressing human Bcl-2 in T lymphocytes

Regulation of lymphocyte survival is essential for the maintenance of lymphoid homeostasis preventing the development of autoimmune diseases. Recently, we described a systemic lupus erythematosus associated with an IgA nephropathy in autoimmune-prone (NZW x C57BL/6)F(1) overexpressing human Bcl-2 (hBcl-2) in B cells (transgenic (Tg) 1). In the present study, we analyze in detail a second line of hBcl-2 Tg mice overexpressing the transgene in all B cells and in a fraction of CD4(+) and CD8(+) T cells (Tg2). We demonstrate here that the overexpression of hBcl-2 in T cells observed in Tg2 mice is associated with a resistance to the development of lupus disease and collagen type II-induced arthritis in both (NZW x C57BL/6)F(1) and (DBA/1 x C57BL/6)F(1) Tg2 mice, respectively. The disease-protective effect observed in autoimmune-prone Tg2 mice is accompanied by an increase of peripheral CD4(+)CD25(+) hBcl-2(+) regulatory T cells (T(regs)), expressing glucocorticoid-induced TNFR, CTLA-4, and FoxP3. Furthermore, the in vivo depletion of CD4(+)CD25(+) T(regs) in (DBA/1 x C57BL/6)F(1) Tg2 mice promotes the development of a severe collagen type II-induced arthritis. Taken together, our results indicate that the overexpression of hBcl-2 in CD4(+) T cells alters the homeostatic mechanisms controlling the number of CD4(+)CD25(+) T(regs) resulting in the inhibition of autoimmune diseases.     

Local Effects of Regulatory T Cells in MUC1 Transgenic Mice Potentiate Growth of MUC1 Expressing Tumor Cells In Vivo

MUC1 transgenic (MUC1.Tg) mice have widely been used as model recipients of cancer immunotherapy with MUC1. Although MUC1.Tg mice have previously been shown to be immunologically tolerant to MUC1, the involvement of regulatory T (Treg) cells in this phenotype remains unclear. Here, we showed that numbers of Treg cells in MUC1-expressing tumors were greater in MUC1.Tg mice than in control C57BL/6 (B6) mice, and that the growth of tumor cells expressing MUC1, but not that of control cells, in MUC1. Tg mice was faster than in B6 mice. The MUC1.Tg mice appeared to develop MUC1-specific peripheral tolerance, as transferred MUC1-specific T cells were unable to function in MUC1.Tg mice but were functional in control B6 mice. The suppressive function of CD4⁺CD25^high cells from MUC1.Tg mice was more potent than that of cells from control B6 mice when Treg cell activity against MUC1-specific T cells was compared in vitro. Therefore, the enhanced growth of MUC1-expressing tumor cells in MUC1.Tg mice is likely due to the presence of MUC1-specific Treg cells.     

Activity and safety of CTLA-4 blockade combined with vaccines in cynomolgus macaques

The immune modulatory molecule CTLA-4 (CD152), through interactions with the B7 costimulatory molecules, has been shown to be a negative regulator of T cell activation in various murine model systems. Abs that block CTLA-4 function can enhance immune responses that mediate potent antitumor activity. However, CTLA-4 blockade can also exacerbate autoimmune disease. The safety and activity of anti-CTLA-4 Abs in primates has not been addressed. To that end, we generated human Abs against CTLA-4 using transgenic mice expressing human Ig genes. A high affinity Ab (10D1) that blocked the binding of CTLA-4 to the B7-1 and B7-2 ligands and had cross-reactivity with macaque CTLA-4 was chosen for further development. Administration of 10D1 to cynomolgus macaques significantly enhanced Ab responses to hepatitis surface Ag and a human melanoma cell vaccine. Anti-self Ab responses as measured by immunoassays using lysate from melanocyte-rich tissues were elicited in those animals receiving the melanoma cell vaccine and anti-CTLA-4 Ab. Remarkably, chronic administration of 10D1 did not result in measurable polyclonal T cell activation, significant alteration of the lymphocyte subsets, or induce clinically observable autoimmunity. Repeated dosing of the 10D1 did not elicit monkey anti-human Ab responses in the monkeys. These observations support the development of CTLA-4 blockade for human immunotherapy.     

Preferential costimulation by CD80 results in IL-10-dependent TGF-beta1(+) -adaptive regulatory T cell generation

Costimulatory ligands CD80 and CD86 have different binding preferences and affinities to their receptors, CD28 and CTLA-4. Earlier, we demonstrated that CD80 binds to CTLA-4 with higher affinity and has a role in suppressing T cell response. The current study demonstrates that not only did blockade of CD86 upon Ag presentation by bone marrow-derived dendritic cells (DC) to OVA-specific T cells result in induction of hyporesponsive T cells but also that these T cells could suppress the proliferative response of effector T cells. These T cells showed TGF-beta1 on their surface and secreted TGF-beta1 and IL-10 upon restimulation. Although blockade of CTLA-4 and neutralization of IL-10 profoundly inhibited the induction of these TGF-beta1(+) T cells, their ability to suppress the effector T cell proliferation was abrogated by neutralization of TGF-beta1 alone. Induction of TGF-beta1(+) and IL-10(+) T cells was found to be independent of natural CD4(+)CD25(+) regulatory T cells, demonstrating that preferential ligation of CTLA-4 by CD80 induced IL-10 production by effector T cells, which in turn promoted the secretion of TGF-beta1. Treatment of prediabetic NOD mice with islet beta cell Ag-pulsed CD86(-/-) DCs, but not CD80(-/-) DCs, resulted in the induction of TGF-beta1- and IL-10-producing cells, significant suppression of insulitis, and delay of the onset of hyperglycemia. These observations demonstrate not only that CD80 preferentially binds to CTLA-4 but also that interaction during Ag presentation can result in IL-10-dependent TGF-beta1(+) regulatory T cell induction, reinstating the potential of approaches to preferentially engage CTLA-4 through CD80 during self-Ag presentation in suppressing autoimmunity.     

Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4high T regulatory cells with target (allo)antigen specificity

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.