Jian-Guo Chai

Anergic T Cells Inhibit the Antigen-Presenting Function of Dendritic Cells

The phenomena of infectious tolerance and linked-suppression are well established, but the mechanisms involved are incompletely defined. Anergic T cells can inhibit responsive T cells in vitro and prolong skin allograft survival in vivo. In this study the mechanisms underlying these events were explored. Allospecific mouse T cell clones rendered unresponsive in vitro inhibited proliferation by responsive T cells specific for the same alloantigens. The inhibition required the presence of APC, in that the response to coimmobilized anti-CD3 and anti-CD28 Abs was not inhibited. Coculture of anergic T cells with bone marrow-derived dendritic cells (DC) led to profound inhibition of the ability of the DC to stimulate T cells with the same or a different specificity. After coculture with anergic T cells expression of MHC class II, CD80 and CD86 by DC were down-regulated. These effects did not appear to be due to a soluble factor in that inhibition was not seen in Transwell experiments, and was not reversed by addition of neutralizing anti-IL-4, anti-IL-10, and anti-TGF-β Abs. Taken together, these data suggest that anergic T cells function as suppressor cells by inhibiting Ag presentation by DC via a cell contact-dependent mechanism.

Anergic T cells act as suppressor cells in vitro and in vivo.

The potential suppressive effects of allospecific anergic T cells were investigated both in vitro and in vivo. Allospecific T cells were rendered unresponsive in vitro using immobilized anti‐CD3 mAb. These anergic T cells profoundly inhibited proliferation of responsive T cells in an antigen‐specific manner. The observed inhibition did not appear to be due to the release of inhibitory cytokines in that secretion of IL‐2, IFN‐γ, IL‐4, IL‐10 and TGF‐β was greatly reduced following the induction of anergy, and neutralizig mAb specific for IL‐4, IL‐10 and TGF‐β failed to reverse the inhibition. Furthermore, the suppression mediated by anergic T cells required cell to cell contact. In vivo, adoptive transfer of anergic T cells into recipients of allogeneic skin grafts led to prolonged skin graft survival. Consistent with the lack of inhibitory cytokine production by the anergic cells, prolongation of skin allograft rejection was not influenced by the simultaneous administration of a neutralizing anti‐IL‐4 antibody. These results indicate that anergic T cells can function as antigen‐specific suppressor cells both in vitro and in vivo.

Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy

In vitro, engagement of GITR on Treg cells by the agonistic anti-GITR mAb, DTA-1, appears to abrogate their suppressive function. The consequence of in vivo engagement of GITR by DTA-1 is, however, less clear. In this study, we show that Treg cells isolated from DTA-1-treated mice were as potent as those from untreated mice in suppressing conventional CD4 T cells in vitro, indicating that in vivo GITR ligation does not disable Treg cells. Treatment of Foxp3/GFP knock-in mice with DTA-1 led to a selective reduction of circulating Treg cells, suggesting that DTA-1 is a depleting mAb which preferentially targets Treg cells. In tumour-bearing mice, DTA-1-mediated depletion of Treg cells was most marked in tumours but not in tumour-draining lymph node. These features were confirmed in an adoptive transfer model using tumour antigen-specific Treg cells. Interestingly, Treg cells detected in tumour tissues expressed much higher levels of GITR than those in tumour-draining lymph nodes, indicating that the efficiency of depletion might be correlated with the level of GITR expression. Finally, in vivo labelling of GITR in naive or tumour-bearing mice demonstrated that Treg cells constitutively expressed higher levels of GITR than conventional T cells, independent of location and activation state, consistent with the preferential in vivo depletion of Tregs by DTA-1. Thus, depletion of Treg cells represents a previously unrecognised in vivo activity of DTA-1 which has important implications for the application of anti-GITR antibodies in cancer immunotherapy.

The contributions of T-cell anergy to peripheral T-cell tolerance

Intrathymic deletion of thymocytes with high affinity for self antigen cells plays a crucial role in contracting the autoreactive T-cell repertoire. However, this is manifestly an incomplete process. Not all self proteins are effectively presented in the thymus, including those that are expressed well after the bulk of the T-cell repertoire has been formed, and it is relatively easy to detect autoreactive T cells following immunization with self antigens. For this reason mechanisms of regulating peripheral T cells with unwanted specificity are crucial to survival. There are several mechanisms of peripheral T-cell unresponsiveness including ignorance, deletion by apoptosis, and cytokine-mediated regulation. The topic of this review is a further mechanism, T-cell anergy. Data will be highlighted, which suggests that the induction of T-cell anergy is an important contributor to peripheral T-cell tolerance, and that anergic T-cells are not passive, but may play an important role as regulatory cells.

CD4+CD25+ T cells as immunoregulatory T cells in vitro.

We have further characterized the in vitro phenotype and function of anergic and suppressive CD4+25+ T cells. Following TCR ligation, DO.11.10 CD4+25+ T cells suppress the activation of OT‐1 CD8+25– T cells in an antigen nonspecific manner. Although suppression was seen when using a mixture of APC from both parental strains, it was very much more marked when using F1 APC. APC pretreated with, and then separated from CD4+25+ T cells did not have diminished T cell costimulatory function, suggesting that APC are not the direct targets of CD4+25+ T cell regulation. CTLA‐4 blockade failed to abrogate suppression by CD4+25+ T cells in mixing experiments. Although CD4+25+ T cells failed to respond following cross‐linking of TCR, they could be induced to proliferate following the addition of exogenous IL‐2, allowing the generation of a T cell line from CD4+25+ T cells. After the first in vitro restimulation, CD4+25+ T cells were still anergic and suppressive following TCR engagement. However, after three rounds of restimulation, their anergic and suppressive status was abrogated.

In Vitro Expansion Improves In Vivo Regulation by CD4+CD25+ Regulatory T Cells

CD4+CD25+ T regulatory cells (Tregs) can actively suppress immune responses and thus have substantial therapeutical potential. Clinical application is, however, frustrated by their scarcity, anergic status, and lack of defined specificity. We found that a single injection of a small number of expanded but not fresh HY-specific Tregs protected syngeneic male skin grafts from rejection by immune-competent recipients. The expanded Tregs were predominantly located in the grafts and graft-draining lymph nodes. In vitro expanded Tregs displayed a phenotype of CD25highCD4lowFoxp3+CTLA4+, and also up-regulated IL10 and TGFβ while down-regulating IFN-γ, GM-CSF, IL5, and TNF-α production. Furthermore, expanded Tregs appeared to express a reduced level of Foxp3, which could be prevented by adding TGFβ to the culture, and they also tended to lose Foxp3 following the repeated stimulation. Finally, a proportion of expanded HY-specific Tregs secreted IL2 in response to their cognate peptide, and this finding could be confirmed using Tregs from Foxp3GFP reporter mice. We not only demonstrated that expanded Tregs are superior to fresh Tregs in suppressing T cell responses against alloantigens, but also revealed some novel immunobiological properties of expended Tregs which are very instructive for modifying current Treg expansion procedures.

Ig gene-like molecule CD31 plays a nonredundant role in the regulation of T-cell immunity and tolerance

CD31 is an Ig-like molecule expressed by leukocytes and endothelial cells with an established role in the regulation of leukocyte trafficking. Despite genetic deletion of CD31 being associated with exacerbation of T cell-mediated autoimmunity, the contribution of this molecule to T-cell responses is largely unknown. Here we report that tumor and allograft rejection are significantly enhanced in CD31-deficient mice, which are also resistant to tolerance induction. We propose that these effects are dependent on an as yet unrecognized role for CD31-mediated homophilic interactions between T cells and antigen-presenting cells (APCs) during priming. We show that loss of CD31 interactions leads to enhanced primary clonal expansion, increased killing capacity, and diminished regulatory functions by T cells. Immunomodulation by CD31 signals correlates with a partial inhibition of proximal T-cell receptor (TCR) signaling, specifically Zap-70 phosphorylation. However, CD31-deficient mice do not develop autoimmunity due to increased T-cell death following activation, and we show that CD31 triggering induces Erk-mediated prosurvival activity in T cells either in conjunction with TCR signaling or autonomously. We conclude that CD31 functions as a nonredundant comodulator of T-cell responses, which specializes in sizing the ensuing immune response by setting the threshold for T-cell activation and tolerance, while preventing memory T-cell death.

CD152 Ligation by CD80 on T Cells Is Required for the Induction of Unresponsiveness by Costimulation-Deficient Antigen Presentation

Two apparently contradictory observations have been made concerning peripheral T cell tolerance; costimulation-deficient Ag presentation leads to unresponsiveness, and CTLA4 (CD152) ligation is required for unresponsiveness to be induced. This issue was addressed using a CD80− CD86low B cell line to present Ag to DO.11.10 naive CD4+ T cells. Proliferation was substantially enhanced by anti-CD80 or anti-CD152, but was inhibited by anti-CD86. Furthermore, anti-CD80 partially, and anti-CD152 totally protected cloned DO.11.10 T cells from the induction of unresponsiveness following culture with peptide and Chinese hamster ovary H2-Ad+ CD80− CD86− cells. Fab of anti-CD80 caused similar enhancement, and coimmobilized anti-CD80 failed to costimulate the anti-CD3 response of purified T cells, indicating that direct signaling by anti-CD80 was not responsible for these effects. The possibility that anti-CD80 liberated CD28 molecules that were sequestered by the T cell-expressed CD80, enabling them to coaggregate with TCR:CD3 complexes was excluded by finding that anti-CD80 and anti-CD152 individually caused maximal enhancement, rather than having additive effects. These data suggest that T cell-expressed CD80 has a regulatory function and plays a key role in the induction of unresponsiveness due to costimulation-deficient Ag presentation by the ligation of CD152 on neighboring, or even the same, T cell.

Functional Plasticity of Antigen-Specific Regulatory T Cells in Context of Tumor

Although polyclonal regulatory T cells (Tregs) that once expressed Foxp3 (ex-Tregs) derived from Foxp3+ Tregs have been described in homeostatic and autoimmune settings, little is known regarding the influence of the tumor environment on ex-Treg development. After adoptive transfer of HY-specific green Tregs (peripheral or thymic) to Rag2−/− B6 female mice bearing syngeneic HY-expressing MB49 tumors, a significant fraction rapidly lost expression of Foxp3. On the second transfer to a Rag2−/− B6 male environment, these ex-Tregs expanded strongly, whereas Tregs that maintained expression of Foxp3 expression did not. Both FACS and quantitative real-time-PCR analysis revealed that ex-Tregs upregulated genes characteristic of a Th1 effector-memory phenotype including IFN-γ and downregulated a panel of Treg-specific genes. Peripheral HY-specific green Tregs were adoptively transferred to Rag2−/− B6 male mice, to dissect the factors regulating ex-Treg differentiation. Development of ex-Tregs was more efficient in the mesenteric lymph node (mLN) than peripheral lymph node environment, correlating with a much greater level of IL-6 mRNA in mLN. In addition, the preferential development of ex-Tregs in mLN was significantly impaired by cotransfer of HY-specific naive CD4 T cells. Collectively, our study not only demonstrates the plasticity of Ag-specific Tregs in the context of the tumor environment, but also defines key molecular and cellular events that modulate ex-Treg differentiation.

The T-cell receptor is not hardwired to engage MHC ligands

The bias of αβ T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.