Interaction Of CTLA-4 Research Articles

IL-15 and cognate antigen successfully expand de novo-induced human antigen-specific regulatory CD4+ T cells that require antigen-specific activation for suppression.

An important prerequisite in using regulatory T cells for immunotherapy is their ex vivo expansion without loss of suppressor function. Human anergic regulatory T cells are expandable by Ag-specific stimulation in the presence of IL-2. IL-15, like IL-2, is a T cell growth factor that, in contrast to IL-2, stimulates survival of T cells. In this study, we examined whether IL-15 could be exploited as a superior growth factor of human CD4(+) anergic regulatory T cells that were generated by costimulation blockade. Next, IL-15, as compared with IL-2, was investigated with respect to expansion and function of these regulatory T cells. Optimal expansion required cognate allogeneic stimulation in the presence of exogenous IL-15. IL-15 resulted in enhanced survival that was paralleled by an increased number of Bcl-2-expressing cells. Moreover, IL-15 induced a distinct type of anergy characterized by hyperreactivity to IL-15, resulting in improved expansion. This is likely attributed to increased propensity of these cells to up-regulate both alpha- and gamma-chains of the IL-2 and IL-15 receptor. Notably, IL-15-expanded regulatory CD4(+) T cells suppressed both naive and memory T cells in a superior way. Immunosuppression required alloantigen-specific stimulation and appeared gamma-irradiation resistant and independent of IL-10, TGFbeta, or CTLA-4 interactions. These regulatory T cells were stable suppressors, mediating bystander suppression upon TCR stimulation, but leaving recall responses unaffected in the absence of cognate Ag. Finally, human naturally occurring regulatory CD4(+)CD25(+) T cells appeared important in generating regulatory T cells by costimulation blockade. In conclusion, IL-15-expanded, de novo-induced human anergic regulatory CD4(+) T cells are of interest in Ag-specific immunotherapy.

Cutting edge: transplantation tolerance through enhanced CTLA-4 expression.

Knockout and blocking studies have shown a critical role for CTLA-4 in peripheral tolerance, however, it is unknown whether augmenting CTLA-4 expression actually promotes tolerance. Here we demonstrate a specific and requisite role for CTLA-4 and its up-regulation in tolerance through anti-CD45RB. First, long-term murine islet allograft survival induced by anti-CD45RB is prevented by CTLA4-Ig, which interferes with B7:CTLA-4 interactions. Second, anti-CD45RB is ineffective in recipients lacking CTLA-4, B7-1, and B7-2. In contrast, CTLA4-Ig, which targets B7 on allogeneic cells, promotes long-term engraftment in these mice. Moreover, anti-CD45RB was effective in B7-deficient controls expressing CTLA-4. Finally, in wild-type mice, CTLA-4 expression returned to baseline 17 days after receiving anti-CD45RB, and was refractory to further increase. Transplantation and anti-CD45RB therapy at this time could neither augment CTLA-4 nor prolong engraftment. These data demonstrate a specific role for CTLA-4 in anti-CD45RB-mediated tolerance and indicate that CTLA-4 up-regulation can directly promote allograft survival.

Induction of CD4 T cell changes in murine AIDS is dependent on costimulation and involves a dysregulation of homeostasis.

Strong CD4 T cell activation and proliferation are seen in susceptible mice infected with the murine retroviral inoculum, LP-BM5, which produces an immunodeficiency syndrome called murine AIDS (MAIDS). We developed a short term adoptive transfer model of MAIDS to examine the requirements for the CD4 T cell response. Naive CD4 T cells from uninfected donors responded quickly after adoptive transfer into MAIDS-infected hosts, becoming activated and proliferating within several days. Using blocking mAbs to costimulatory ligands and CD4 T cells deficient in expression of their receptors, we found that the CD4 T cell response requires CD28:B7.1/B7.2 interactions, but not CTLA4 or CD40-CD40 ligand interactions. Naive CD4 T cells did not respond in H-2M-deficient mice with MAIDS, suggesting that disease requires recognition of self peptide-MHC complexes. The self MHC-dependent division and accumulation of large numbers of CD4 T cells suggest that MAIDS involves a disruption of the balance of homeostatic signals. Supporting this hypothesis, CD4 T cells from mice with MAIDS failed to regulate the homeostatic division of naive CD4 T cells in a cotransfer model. Thus, a combination of up-regulation of costimulatory ligands and disruption of homeostatic control may be responsible for CD4 lymphoproliferation in MAIDS.

Negative effect of CTLA-4 on induction of T-cell immunity in vivo to B7-1+, but not B7-2+, murine myelogenous leukemia

AbstractB7 molecules provide important costimulatory signals to T cells, and B7 genes have been introduced into B7-negative tumor cells to enhance their immunogenicity. However, the role of B7 molecules in inducing tumor immunity is controversial because of conflicting results and reports of differential signaling through the B7 molecules and their ligands CD28 and CTLA-4. In this study, we compared the effect of B7-1 (CD80) and B7-2 (CD86) on the induction of T-cell immunity to C1498, a murine myelogenous leukemia. When cultured with exogenous cytokines in vitro, C1498/B7-1 and C1498/B7-2 induced syngeneic CD8+ T cells to kill parental C1498. In vivo, C1498/B7-1 grew progressively after subcutaneous injection, whereas C1498/B7-2 completely regressed after transient growth in naive mice. Spontaneous rejection of C1498/B7-2 resulted in immunity to challenge doses of C1498 and C1498/B7-1. Antibody-depletion studies in vivo showed that CD8+ T cells rejected C1498/B7-2, whereas only natural killer cells affected the growth of C1498/B7-1. Two approaches were used to determine whether preferential interaction of B7-1 with CTLA-4 contributed to the failure of C1498/B7-1 to activate CD8+ T cells in vivo. First, CTLA-4 specific monoclonal antibody was used to block B7-1–CTLA-4 interaction. Second, CTLA-4 deletional mutant (−/−) bone marrow chimeras were used as tumor hosts. In both systems, there was a significant increase in the rate of rejection of C1498/B7-1 tumors. Resistance to C1498/B7-1 in CTLA-4−/− hosts was mediated by CD8+ T cells. Blocking or deletion of CTLA-4 did not affect the growth of parental C1498, indicating that B7-1 was important for the induction of CD8+ T-cell immunity in the absence of CTLA-4.

CTLA-4 is not required for induction of CD8(+) T cell anergy in vivo.

Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4(+) vs CD8(+) cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8(+) T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4(+) and CD8(+) T cells.

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.

A retro-inverso peptide mimic of CD28 encompassing the MYPPPY motif adopts a polyproline type II helix and inhibits encephalitogenic T cells in vitro.

Complete activation of T cells requires two signals: an Ag-specific signal delivered via the TCR by the peptide-MHC complex and a second costimulatory signal largely provided by B7:CD28/CTLA-4 interactions. Previous studies have shown that B7 blockade can either ameliorate experimental autoimmune encephalomyelitis by interfering with CD28 signaling or exacerbate the disease by concomitant blockade of CTLA-4 interaction. Therefore, we developed a functional CD28 mimic to selectively block B7:CD28 interactions. The design, synthesis, and structural and functional properties of the CD28 free peptide, the end group-blocked CD28 peptide, and its retro-inverso isomer are shown. The synthetic T cell-costimulatory receptor peptides fold into a polyproline type II helical structure commonly seen in regions of globular proteins involved in transient protein-protein interactions. The binding determinants of CD28 can be transferred onto a short peptide mimic of its ligand-binding region. The CD28 peptide mimics effectively block the expansion of encephalitogenic T cells in vitro suggesting the potential usefulness of the peptides for the treatment of autoimmune disease conditions requiring down-regulation of T cell responses.

Differentiating role of B7-CD28/CTLA-4 interaction in the induction of mucosal inflammation and regulatory T cell functions in chronic experimental colitis

Differentiating role of B7-CD28/CTLA-4 interaction in the induction of mucosal inflammation and regulatory T cell functions in chronic experimental colitis

Differentiating role of B7-CD28/CTLA-4 interaction in the induction of mucosal inflammation and regulatory T cell functions in chronic experimental colitis

Differentiating role of B7-CD28/CTLA-4 interaction in the induction of mucosal inflammation and regulatory T cell functions in chronic experimental colitis

Giving Form to CTLA-4 Interactions

Signals delivered by cell surface receptor CTLA-4 are critical for ensuring that T cells do not overstep their mark when responding to antigen. How this is accomplished is not fully understood, but it involves -- at least in part --direct inhibitory signals. The efficiency of these signals likely relies on the assembly of inhibitory signaling complexes at the interface between the antigen-presenting cell and the T cell. Ostrov et al. have solved the crystal structure of CTLA-4 and in so doing revealed how the organization of this receptor might facilitate the arrangement of such complexes. Their data suggest that CTLA-4 forms dimers that are unlike those made by other members of the immunolglobulin supergene family in that it can interact simultaneously with two of its natural B7 ligands. This serial organization could help to explain the proficiency of CTLA-4 in regulating the immune responses. Ostroy, D.A., Shi, W., Schwartz, J.-C.D., Almo, S.C., and Nathenson, S.G. (2000) Structure of murine CTLA-4 and its role in modulating T cell responsiveness. Science 290 : 816-819. [Abstract] [Full Text]

Giving Form to CTLA-4 Interactions

Signals delivered by cell surface receptor CTLA-4 are critical for ensuring that T cells do not overstep their mark when responding to antigen. How this is accomplished is not fully understood, but it involves -- at least in part --direct inhibitory signals. The efficiency of these signals likely relies on the assembly of inhibitory signaling complexes at the interface between the antigen-presenting cell and the T cell. Ostrov et al. have solved the crystal structure of CTLA-4 and in so doing revealed how the organization of this receptor might facilitate the arrangement of such complexes. Their data suggest that CTLA-4 forms dimers that are unlike those made by other members of the immunolglobulin supergene family in that it can interact simultaneously with two of its natural B7 ligands. This serial organization could help to explain the proficiency of CTLA-4 in regulating the immune responses.Ostroy, D.A., Shi, W., Schwartz, J.-C.D., Almo, S.C., and Nathenson, S.G. (2000) Structure of murine CTLA-4 and its role in modulating T cell responsiveness. Science 290: 816-819. [Abstract] [Full Text]

Giving Form to CTLA-4 Interactions

Giving Form to CTLA-4 Interactions

Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells.

Engagement of the T cell costimulatory receptor CTLA-4 can potently down-regulate an immune response. For example, in a T cell receptor transgenic mouse model of autoimmune diabetes, CTLA-4 interactions keep pancreatic islet-reactive T cells in check, evidenced by the finding that mAb blockade of CTLA-4 rapidly provokes diabetes in animals that would not normally succumb until many months later. Interestingly, this effect is only observed early in the course of disease, before insulitis is stably entrenched. Here, we have exploited a highly synchronous and easily manipulable transfer system to determine precisely when CTLA-4 must be engaged to check the diabetogenicity of islet-reactive T cells. Our results indicate that CTLA-4 interactions during initial priming of the T cells in the pancreatic lymph nodes are not determinant. Rather, the critical interactions occur when the T cells secondarily reencounter their antigen in the target organ, the pancreatic islets. In addition, we made use of CTLA-4-deficient mice to bolster our interpretation that CTLA-4 engagement has a dampening rather than an enhancing influence on diabetes progression.

Interaction of CTLA-4 (CD152) with CD80 or CD86 inhibits human T-cell activation.

Occupancy of CTLA-4 (cytotoxic T-lymphocyte antigen-4 or CD152) negatively regulates the activation of mouse T lymphocytes, as indicated by the fate of CTLA-4-deficient mice, by the impact of anti-CTLA-4 monoclonal antibodies (mAbs) on mouse T-cell activation in vitro and by the impact of CTLA-4 blockade on the course of experimental tumoral, autoimmune, alloimmune or infectious disease in this animal. The function of human CTLA-4, however, remains less clear. The expression and function of human CTLA-4 were further explored. CTLA-4 was expressed under mitogenic conditions only, its expression being, at least partially, dependent on the secretion of interleukin-2. Memory T cells expressed CTLA-4 with faster kinetics than naive T cells. The functional role of human CTLA-4 was assessed utilizing a panel of four anti-CTLA-4 mAbs that blocked the interaction between CTLA-4 and its ligands. These mAbs, in immobilized form, profoundly inhibited the activation of T cells by immobilized anti-CD3 mAb in the absence of anti-CD28 mAb, but co-stimulated T-cell activation in the presence of anti-CD28 mAb. Finally, and importantly, blockade of the interaction of CTLA-4 with its ligands using soluble anti-CTLA-4 mAbs, in intact form or as Fab fragments, enhanced T-cell activation in several polyclonal or alloantigen-specific CD80- or CD80/CD86-dependent assays, as measured by cytokine production, cellular proliferation or cytotoxic responses. It is concluded that interaction of CTLA-4 with its functional ligands, CD80 or CD86, can down-regulate human T-cell responses, probably by intracellular signalling events and independent of CD28 occupancy.

Expression of a soluble form of CTLA4 on macrophage and its biological activity.

Interaction between cytotoxic T lymphocyte-associated antigen-4 (CTLA4, CD152) and B7 molecules (B7-1 and B7-2) is of importance in the cellular events of lymphocyte, including antigen-specific T-cell activation and induction of autoreactive T-cell. We describe here the first introduction of a murine soluble CTLA4 gene, CTLA4Ig, to Mm1 cells, a macrophagic cell line. CTLA4Ig was successfully expressed on Mml cells and the expressed CTLA4Ig was found to be functionally active in their binding to B7 molecules by flow cytometry and immunofluorescence studies. The biological activity of CTLA4Ig from the transfected Mm1 cells was studied and showed inhibitory activity on mixed lymphocyte culture. A high CTLA4Ig producing macrophagic cell line was obtained. As Mm1 cells were regarded as difficult for gene transfection and there had so far been no report on expression of CTLA4Ig gene on Mm1 cells, these results suggested that the CTLA4Ig expressing Mm1 cells could be useful for analysis of CTLA4 and B7 molecule interaction in both macrophage and T-cell.

The role of B/T costimulatory signals in the immunopotentiating activity of neisserial porin.

A T cell-dependent immune response to group C meningococcal capsular polysaccharide (CPS) can be elicited when CPS is conjugated to the class 3 neisserial porin (CPS-porin). Treatment of CPS-porin-immunized mice with B7-2 blocking monoclonal antibody (MAb) caused a dramatic reduction in the CPS-specific IgG response, treatment with anti-B7-1 MAb had no effect, and concurrent blockade of B7-1 and B7-2 resulted in a synergistic abrogation of the CPS-specific IgG response while the CPS IgM response was unaffected. Anti-CD40L MAb treatment caused a significant reduction of both CPS-specific IgG and IgM levels. In contrast, blockade of CTLA4 interactions resulted in increases in both CPS IgG and IgM responses in CPS-porin-immunized mice. These data support the hypothesis that the ability of neisserial porins to improve the immune response to poorly immunogenic antigens (e.g., polysaccharides) is related to porin-induced increases in B7-2 expression on antigen-presenting cells and enhanced B/T cell interactions.

Blockade of CD28 During In Vitro Activation of Encephalitogenic T Cells or After Disease Onset Ameliorates Experimental Autoimmune Encephalomyelitis

Previous studies have shown complex roles for the B7 receptors in providing both positive and negative regulation of experimental autoimmune encephalomyelitis (EAE). B7 blockade can ameliorate clinical EAE by indirectly interfering with CD28 signaling. However, B7 blockade can also result in disease exacerbation, presumably by interfering with regulatory B7:CTLA-4 interactions. Therefore, we have directly targeted T cell CD28 with specific mAbs both during initial Ag priming and after the onset of clinical signs of EAE. We found that CD28 blockade ameliorated EAE during the efferent and afferent limbs of the immune response. Disease amelioration at disease onset was associated with suppression of TNF-alpha production. Finally, Ab blockade of T cell CD28 during the first disease episode resulted in significant attenuation of the subsequent disease course, with no significant relapses. In contrast to previous studies targeting APC B7 with CTLA4-Ig, reagents targeting CD28 can block ongoing disease. Therefore, the present results suggest a clinically relevant therapeutic scenario for human diseases, such as multiple sclerosis.

CTLA-4 blockade enhances the immune response induced by mycobacterial infection but does not lead to increased protection.

The murine immune response to a pulmonary mycobacterial infection is slow to develop, allowing bacterial numbers to increase in the lung for several weeks after infection. We sought to enhance the protective immune response induced during Mycobacterium bovis BCG infection by administering an antibody that blocks the interaction of CTLA-4 with its ligands, CD80 and CD86. We found that injection of anti-CTLA-4 monoclonal antibody (MAb) greatly enhanced and accelerated the immune response, as measured by increased cellularity of the draining mediastinal lymph nodes, and enhanced antigen-inducible proliferation and gamma interferon production by mediastinal lymphocytes in vitro. However, despite the apparently enhanced immune response in the mediastinal lymph node following treatment with anti-CTLA-4 MAb, there was no improvement in clearance of mycobacteria in the lungs, liver, or spleen. Examination of the primary site of infection, the lung, revealed that CTLA-4 blockade had no effect on the number or function of lymphocytes infiltrating the infected lung tissue. Taken together, these data suggest that in vivo CTLA-4 blockade enhances mycobacterial-infection-induced lymphocyte expansion and effector cell cytokine production in the draining lymph node but does not alter the number or function of lymphocytes at the primary site of infection and therefore does not lead to enhanced clearance of the infection.

Molecular basis of T cell inactivation by CTLA-4.

CTLA-4, a negative regulator of T cell function, was found to associate with the T cell receptor (TCR) complex zeta chain in primary T cells. The association of TCRzeta with CTLA-4, reconstituted in 293 transfectants, was enhanced by p56(lck)-induced tyrosine phosphorylation. Coexpression of the CTLA-4-associated tyrosine phosphatase, SHP-2, resulted in dephosphorylation of TCRzeta bound to CTLA-4 and abolished the p56(lck)-inducible TCRzeta-CTLA-4 interaction. Thus, CTLA-4 inhibits TCR signal transduction by binding to TCRzeta and inhibiting tyrosine phosphorylation after T cell activation. These findings have broad implications for the negative regulation of T cell function and T cell tolerance.

Regulation of T-cell activation by CD28 and CTLA-4

However, a second costimulatory signal is necessary to achieve complete activation. This can be provided by the accessory T cell molecule CD28 upon binding to its respective ligands B7-1 (CD80) or B7-2 (CD86). The same ligands bind to CTLA-4 (CD152), a receptor expressed at the surface of T cells previously activated for 2 to 3 days and capable of downregulating activation. IMMUNOSUPPRESSION BY CTLA-4Ig: A genetically engineered soluble fusion protein containing the extracellular domain of CTLA-4 and the Fc portion of an immunoglobulin heavy chain (CTLA-4Ig) prevents the interaction of CD28 and CTLA-4 with their B7 ligands, the subsequent activation of T cells and thereby eliminates or reduces unfavorable immune system activation in transplant rejection or autoimmunity. The importance of the regulatory system comprising CD28, CTLA-4 and the B7 molecules and its modulation by CTLA-4Ig has been demonstrated in a substantial number of animal models in recent years and holds promise as a novel approach for therapeutic immunomodulation in humans.