Caroline Addey

Dendritic cells permit identification of genes encoding MHC class II-restricted epitopes of transplantation antigens.

Minor or histocompatibility (H) antigens are recognized by CD4+ and CD8+ T lymphocytes as short polymorphic peptides associated with MHC molecules. They are the targets of graft versus host and graft versus leukemia responses following bone marrow transplantation between HLA-identical siblings. Several genes encoding class I-restricted minor H epitopes have been identified, but approaches used for these have proved difficult to adapt for cloning class II-restricted minor H genes. We have combined the unique antigen-presenting properties of dendritic cells and high levels of episomal expression following transfection of COS cells to identify a Y chromosome gene encoding two HY peptide epitopes, HYAb and HYEk.

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.

Role of Immunoproteasomes in Cross-Presentation

The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty246–254 epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7−/−) skin grafts by female LMP7−/− mice. Second, we ruled out an altered Uty246–254-specific T cell repertoire in LMP7−/− female mice and demonstrated efficient Uty246–254 presentation by re-expressing LMP7 in male LMP7−/− cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty246–254-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7−/− female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.

Identification of the Immunodominant HY H2-Dk Epitope and Evaluation of the Role of Direct and Indirect Antigen Presentation in HY Responses

Minor histocompatibility Ags derive from self-proteins and provoke allograft rejection and graft-vs-host disease in MHC-matched donor-recipient combinations. In this study, we define the HYDk epitope of the HY minor histocompatibility Ag as the 8mer peptide RRLRKTLL derived from the Smcy gene. Using HY tetramers, the response to this peptide was found to be immunodominant among the four characterized MHC class I-restricted HY epitopes (HYDkSmcy (defined here), HYKkSmcy, HYDbUty, and HYDbSmcy). Indirect presentation stimulated a robust primary HYDkSmcy response. Indirect presentation and priming of HY-specific CD8+ T cells is also operative in the presence of a full MHC mismatch. To determine whether the indirect route of Ag presentation is required for HY priming, female parent into F1 (H2bxk) female recipient bone marrow chimeras were immunized with male cells of the other parental haplotype, limiting presentation to the direct pathway. The dominant H2b HY response (HYDbUty) was dependent on indirect presentation. However, the dominant H2k HY response (HYDkSmcy) could be stimulated efficiently by the direct pathway. In contrast, secondary expansion of both HYDkSmcy and HYDbUty-specific CD8+ T cells was effective only when Ag was presented by the direct route. Transgenic overproduction of Smcy mRNA within the immunizing cells resulted in a corresponding increase in the HYDkSmcy, HYDbSmcy, and HYKkSmcy-specific CD8+ T cell responses when presented via the direct pathway but did not enhance indirect presentation demonstrating the independent regulation of MHC class I-peptide occupancy in the two Ag-processing pathways.

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.

Mice lacking C1q or C3 show accelerated rejection of minor H disparate skin grafts and resistance to induction of tolerance

Complement activation is known to have deleterious effects on organ transplantation. On the other hand, the complement system is also known to have an important role in regulating immune responses. The balance between these two opposing effects is critical in the context of transplantation. Here, we report that female mice deficient in C1q (C1qa−/−) or C3 (C3−/−) reject male syngeneic grafts (HY incompatible) at an accelerated rate compared with WT mice. Intranasal HY peptide administration, which induces tolerance to syngeneic male grafts in WT mice, fails to induce tolerance in C1qa−/− or C3−/− mice. The rejection of the male grafts correlated with the presence of HY DbUty‐specific CD8+ T cells. Consistent with this, peptide‐treated C1qa−/− and C3−/− female mice rejecting male grafts exhibited more antigen‐specific CD8+IFN‐γ+ and CD8+IL‐10+ cells compared with WT females. This suggests that accumulation of IFN‐γ‐ and IL‐10‐producing T cells may play a key role in mediating the ongoing inflammatory process and graft rejection. Interestingly, within the tolerized male skin grafts of peptide‐treated WT mice, IFN‐γ, C1q and C3 mRNA levels were higher compared to control female grafts. These results suggest that C1q and C3 facilitate the induction of intranasal tolerance.

Isolation of Human CD4/CD8 Double-Positive, Graft-Versus-Host Disease–Protective, Minor Histocompatibility Antigen–Specific Regulatory T Cells and of a Novel HLA-DR7–Restricted HY-Specific CD4 Clone

Minor histocompatibility (H) Ags are classically described as self-peptides derived from intracellular proteins that are expressed at the cell surface by MHC class I and class II molecules and that induce T cell alloresponses. We have isolated three different T cell populations from a skin biopsy of a patient suffering from acute graft-versus-host disease following sex-mismatched HLA-identical bone marrow transplantation. The first population was: 1) CD4+/CD8+ double-positive; 2) specific for an HLA class I–restricted autosomal Ag; 3) expressed a Tr1 profile with high levels of IL-10, but low IL-2 and IFN-γ; and 4) exerted regulatory function in the presence of recipient APCs. The second was CD8 positive, specific for an HLA class I–restricted autosomally encoded minor H Ag, but was only weakly cytotoxic. The third was CD4 single positive, specific for an HLA-DR7–restricted HY epitope and exerted both proliferative and cytotoxic functions. Identification of the peptide recognized by these latter cells revealed a new human HY epitope, TGKIINFIKFDTGNL, encoded by RPS4Y and restricted by HLA-DR7. In this paper, we show human CD4/CD8 double-positive, acute graft-versus-host disease–protective, minor H Ag–specific regulatory T cells and identify a novel HLA-DR7/ HY T cell epitope, encoded by RPS4Y, a potential new therapeutic target.

The roles of antigen-specificity, responsiveness to transforming growth factor-β and antigen-presenting cell subsets in tumour-induced expansion of regulatory T cells

In this study we investigated the impact of several factors on the expansion of natural regulatory T (nTreg) cells by tumours, including antigen specificity, transforming growth factor‐β (TGF‐β) signalling and the antigen‐presenting cell subsets responsible for expansion. We found that antigen non‐specific expansion of nTreg cells is tumour cell line‐dependent. Although both antigen‐specific and non‐specific pathways can contribute to expansion, the migration of activated nTreg cells to tumour tissues is strictly antigen‐dependent. Intact TGF‐β signalling on nTreg cells is also essential for tumour‐induced expansion. Finally, for stimulation of resting antigen‐specific CD4 T cells, CD11c+ cells purified from tumour‐draining lymph nodes were more potent than CD11b+ cells, suggesting that dendritic cells are the key antigen‐presenting cell subset involved in cross‐presentation of tumour antigens. This study not only provides an in vivo system in which cross‐talk between nTreg cells and tumours can be explored but also reveals novel aspects of tumour immune evasion.

Molecular Mechanisms of induction of antigen-specific allograft tolerance by intranasal peptide administration

We have previously shown that intranasal (i.n.) administration of a single MHC class II-restricted HY peptide to female mice induces tolerance to up to five additional epitopes expressed on test male grafts, a phenomenon known as linked suppression. In this study, we investigated the molecular mechanisms involved both in the induction phase following peptide administration and during linked suppression after grafting. We report that following initial i.n. administration, peptide is widely disseminated and is presented by functionally immature dendritic cells. These fail to cause optimal stimulation of the responding HY-specific CD4+ T cells that express genes characteristic of regulatory T cells. Following i.n. peptide plus LPS administration, causing immunization, HY-specific CD4+ T cells express genes characteristic of activated T cells. We further find that following male skin grafting, HY-specific CD8+ T cells from peptide-treated tolerant mice display both quantitative and qualitative differences compared with similar cells from untreated mice that reject their grafts. In tolerant mice there are fewer HY-specific CD8+ cells and they express several genes characteristic of exhausted T cells. Furthermore, associated with specific chemokine receptor and integrin expression, HY-specific CD8+ T cells show more limited migration from the graft draining lymph node into other tissues.

Distinct in vivo CD8 and CD4 T cell responses against normal and malignant tissues

Normal tissue and tumour grafts expressing the same alloantigens often elicit distinct immune responses whereby only normal tissue is rejected. To investigate the mechanisms that underlie these distinct outcomes, we compared the responses of adoptively transferred HY-specific conventional (CD8 and CD4) or regulatory T (Treg) cells in mice bearing HY-expressing tumour, syngeneic male skin graft or both. For local T cell priming, T cell re-circulation, graft localization and retention, skin grafts were more efficient than tumours. Skin grafts were also capable of differentiating CD4 T cells into functional Th1 cells. Donor T cell responses were inversely correlated with tumour progression. When skin graft and tumour transplants were performed sequentially, contemporary graft and tumour burden enhanced CD8 but reduced CD4 T cell responses causing accelerated skin-graft rejection without influencing tumour growth. Although both skin grafts and tumours were able to expand HY-specific Treg cells in draining lymph node (dLN), the proportion of tumour-infiltrating Treg cells was significantly higher than that within skin grafts, correlating with accelerated tumour growth. Moreover, there was a higher level of HY antigen presentation by host APC in tumour-dLN than in graft-dLN. Finally, tumour tissues expressed a significant higher level of IDO, TGFβ, IL10 and Arginase I than skin grafts, indicating that malignant but not normal tissue represents a stronger immunosuppressive environment. These comparisons provide important insight into the in vivo mechanisms that conspire to compromise tumour-specific adaptive immunity and identify new targets for cancer immunotherapy.