FcRγ promotes T cell apoptosis in Fas-deficient mice.

Deficiency of Fas or its ligand leads to lymphocyte accumulation, lymphadenopathy, splenomegaly, and autoimmunity in mice and humans. Although the Fas pathway is important for limiting the number of peripheral T cells, inactivation of other pro-apoptotic molecules can also perturb T cell homeostasis independently of and/or in concert with Fas deficiency. Here, we show that combined deficiency of Fas and the Fc receptor common γ signaling chain (FcRγ) results in worsened T cell accumulation in comparison with mice lacking Fas alone, with a particularly marked increase in the number of TCRαß(+)CD4(-)CD8(-) double negative (DN) T cells. LPR FcRγ(-/-) mice exhibited reduced survival due to progressive lymphadenopathy. We further investigated the mechanisms whereby FcRγ deficiency promotes lymphoproliferative disease in Fas-mutant mice. Interestingly, there were no significant differences in T cell proliferation between LPR FcRγ(+/+) and LPR FcRγ(-/-) mice in vivo and in vitro. However, FcRγ deletion resulted in significantly decreased peripheral T cell apoptosis. Importantly, the observed increase in apoptosis was restricted to a subset of FcRγ(+) T cells. These T cells, but not those lacking FcRγ expression, exhibited increased activation of caspases 3 and 9, indicating a role for FcRγ in driving their apoptosis. FcRγ(+) DN T cells also showed enhanced sensitivity to TCR restimulation-induced cell death (RICD) despite lacking Fas, suggesting that TCR stimulation of autoreactive T cells in vivo may serve to eliminate FcRγ(+) T cells and thus exert partial control over lymphoproliferative disease. Hence, our data reveal a novel role for FcRγ in promoting peripheral T cell apoptosis in Fas-deficient mice, which may be of significant value in understanding autoimmune lymphoproliferative syndromes.

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients.

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restrictions.

Lentivirally Transduced Recipient-derived Dendritic Cells Serve to Ex Vivo Expand Functional FcRγ-sufficient Double-negative Regulatory T Cells.

αßTCR+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress antigen-specific immune responses mediated by CD8+ and CD4+ T cells in mice and humans. In this study, we developed a system to expand DN Treg cells for transplantation therapy that exclusively uses recipient-derived immune cells and confers a high degree of safety as the protocol does not involve the direct injection of lentiviral vectors. Recipient-derived dendritic cells (DCs) were transduced with lentiviral vectors that express major histocompatibility complex class I Ld antigen (LV-Ld), which is expressed by the donor graft but is allogeneic to the graft recipient. LV-Ld-transduced mature DCs (mDCs) were able to expand effectively both FcRγ-/- and FcRγ+/+ DN T cells. After expansion with LV-Ld-transduced mDCs, only the FcRγ+/+ DN Treg cells maintained their ability to suppress CD8+ T cells in vitro. In addition, adoptive transfer of the FcRγ+/+ex vivo expanded DN Treg cells significantly prolonged the survival of Ld+ skin grafts. This study is the first description of successful ex vivo expansion of antigen-specific DN Treg cells using genetically modified syngeneic DCs for adoptive immunotherapy and demonstrates that although FcRγ-/- DN T cells can be expanded, they do not gain regulatory ability.

Double-negative regulatory T cells: non-conventional regulators.

The crucial role of regulatory T (Treg) cells in self-tolerance and downregulating immune responses has been clearly established. Numerous different Treg subsets have been identified that possess distinct phenotypes and functions in various disease models. Among these subsets, alphabeta-TCR+CD3+CD4-CD8- double-negative (DN) Treg cells have been shown to be able to inhibit a variety of immune responses in part via direct killing of effector T cells in an antigenspecific manner in both mice and humans. This was shown to occur at least partially by acquisition of MHC-peptide complexes from antigen-presenting cells (APCs) and subsequent Fas/Fas-ligand interactions. In addition, DN Treg cells have been shown to express several molecules uncommon to other Treg cell subsets, such as IFN-gamma, TNF-alpha, Ly6A, FcRgamma, and CXCR5, which may contribute to their unique regulatory ability. Understanding the development and regulatory functions of DN Treg cells may elucidate the etiology for loss of self-tolerance and serve as a therapeutic modality for various diseases. This review will summarize the characteristics, developmental pathways, and mechanisms of action of DN Treg cells, as well as their role in transplant tolerance, autoimmunity, and anticancer immunity.

FcRγ controls the fas-dependent regulatory function of lymphoproliferative double negative T cells.

Patients with autoimmune lymphoproliferative syndrome (ALPS) and lymphoproliferation (LPR) mice are deficient in Fas, and accumulate large numbers of αß-TCR(+), CD4(-), CD8(-) double negative (DN) T cells. The function of these DN T cells remains largely unknown. The common γ subunit of the activating Fc receptors, FcRγ, plays an important role in mediating innate immune responses. We have shown previously that a significant proportion of DN T cells express FcRγ, and that this molecule is required for TCR transgenic DN T cells to suppress allogeneic immune responses. Whether FcRγ plays a critical role in LPR DN T cell-mediated suppression of immune responses to auto and allo-antigens is not known. Here, we demonstrated that FcRγ(+), but not FcRγ(-) LPR DN T cells could suppress Fas(+) CD4(+) and CD8(+) T cell proliferation in vitro and attenuated CD4(+) T cell-mediated graft-versus host disease. Although FcRγ expression did not allow LPR DN T cells to inhibit the expansion of Fas-deficient cells within the LPR context, adoptive transfer of FcRγ(+), but not FcRγ(-), DN T cells inhibited lymphoproliferation in generalized lymphoproliferative disease (GLD) mice. Furthermore, FcRγ acted in a cell-intrinsic fashion to limit DN T cell accumulation by increasing the rate of apoptosis in proliferated cells. These results indicate that FcRγ can confer Fas-dependent regulatory properties on LPR DN T cells, and suggest that FcRγ may be a novel marker for functional DN Tregs.

Lentivirally transduced recipient-derived dendritic cells serve to ex vivo expand functional FcRgamma-sufficient double-negative regulatory T cells.

alphabetaTCR(+)CD4(-)CD8(-) double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress antigen-specific immune responses mediated by CD8(+) and CD4(+) T cells in mice and humans. In this study, we developed a system to expand DN Treg cells for transplantation therapy that exclusively uses recipient-derived immune cells and confers a high degree of safety as the protocol does not involve the direct injection of lentiviral vectors. Recipient-derived dendritic cells (DCs) were transduced with lentiviral vectors that express major histocompatibility complex class I L(d) antigen (LV-L(d)), which is expressed by the donor graft but is allogeneic to the graft recipient. LV-L(d)-transduced mature DCs (mDCs) were able to expand effectively both FcRgamma(-/-) and FcRgamma(+/+) DN T cells. After expansion with LV-L(d)-transduced mDCs, only the FcRgamma(+/+) DN Treg cells maintained their ability to suppress CD8(+) T cells in vitro. In addition, adoptive transfer of the FcRgamma(+/+) ex vivo expanded DN Treg cells significantly prolonged the survival of L(d+) skin grafts. This study is the first description of successful ex vivo expansion of antigen-specific DN Treg cells using genetically modified syngeneic DCs for adoptive immunotherapy and demonstrates that although FcRgamma(-/-) DN T cells can be expanded, they do not gain regulatory ability.

Eosinophilia is induced in the colon of Th2-sensitized mice upon exposure to locally expressed antigen.

Eosinophilic inflammation is a feature of a variety of gastrointestinal (GI) disorders including eosinophil-associated GI disorder, allergy, inflammatory bowel disease, and parasite infection. Elucidating the mechanisms of eosinophil infiltration into the GI tract is important to the understanding of multiple disease processes. We hypothesize that eosinophilia in the large intestine (colon) can be induced by an antigen in a host that is associated with Th2-skewed antigen-specific immune responses. To investigate the importance of antigenic triggering, we established polarized antigen-specific Th2 type responses in BALB/c mice, using ovalbumin in conjunction with aluminum hydroxide. Upon challenge at the colonic mucosa through transient (3-4 days) expression of the antigen gene encoded in an adenovirus vector, sensitized animals developed significant subepithelial colonic inflammation, characterized by marked eosinophilic infiltration, and the presence of enlarged and increased numbers of lymphoid follicles. The alterations peaked around day 5 and resolved over the next 5-10 days, and no epithelial cell damage was detected through the entire course. Administration of a control (empty) adenovirus vector did not lead to any pathological changes. These data suggest that colonic eosinophilia can be induced by exposure to an antigen associated with preexisting Th2-skewed responses. Thus the model established here may provide a useful tool to study GI and, in particular, colonic inflammation with respect to underlying mechanisms involved in the recruitment and the immediate function of eosinophils.

FcR gamma presence in TCR complex of double-negative T cells is critical for their regulatory function.

TCRalphabeta+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells in humans and mice. Our previous study using cDNA microarray analysis of global gene expression showed that FcRgamma was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRgamma-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRgamma-sufficient DN T cells, FcRgamma-deficient DN T cells were unable to prolong donor-specific allograft survival when adoptively transferred to recipient mice. Protein analyses indicate that in addition to FcRgamma, DN Treg cell clones also express higher levels of TCRbeta, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRgamma associates with the TCR complex and that both FcRgamma and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRgamma expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRgamma deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRgamma plays a role in mediating TCR signaling in DN Treg cells.

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival.

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.