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.

Recent studies have demonstrated that both mouse and human αβTCR+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 FcεRIγ 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.

The large intestinal mucosa contains immunological structures that may potentially serve as a site for induction of mucosal immunity against infections. Adenovirus (Ad), which is effective in gene transfer to epithelia, may be an ideal antigen delivery system for vaccination at the large intestinal mucosa. To investigate this potential, we immunized mice with recombinant replication-deficient Ad through a single intracolorectal (ICR) administration. Effective transfer of encoded genes was found in both the epithelial layer and lamina propria of the colorectal mucosa. Dendritic cells were able to transfer antigen to the draining lymph nodes, where antigen-specific CD8(+) T cells were primed. Functional antigen-specific CD8(+) T cells and IgA-specific antibodies were detected during the effector phase in the large intestine. Compared to other immunization routes (intranasal, subcutaneous), ICR immunization induced stronger colorectal immune responses and more potent protection against rectal challenge with pathogenic viruses. Further, this immunization strategy provided vaginal protection, more potent than that induced by vaccination in the nose or skin. Therefore, large intestine mucosal immunization using Ad represents an effective vaccination strategy against virus infection at both rectal and vaginal mucosal tissue sites.

TCRαβ+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 FcRγ was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRγ-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRγ-sufficient DN T cells, FcRγ-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 FcRγ, DN Treg cell clones also express higher levels of TCRβ, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRγ associates with the TCR complex and that both FcRγ and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRγ expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRγ deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRγ plays a role in mediating TCR signaling in DN Treg cells.

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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.

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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.