Hiroyoshi Nishikawa

Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota

Manipulation of the gut microbiota holds great promise for the treatment of inflammatory and allergic diseases. Although numerous probiotic microorganisms have been identified, there remains a compelling need to discover organisms that elicit more robust therapeutic responses, are compatible with the host, and can affect a specific arm of the host immune system in a well-controlled, physiological manner. Here we use a rational approach to isolate CD4+FOXP3+ regulatory T (Treg)-cell-inducing bacterial strains from the human indigenous microbiota. Starting with a healthy human faecal sample, a sequence of selection steps was applied to obtain mice colonized with human microbiota enriched in Treg-cell-inducing species. From these mice, we isolated and selected 17 strains of bacteria on the basis of their high potency in enhancing Treg cell abundance and inducing important anti-inflammatory molecules—including interleukin-10 (IL-) and inducible T-cell co-stimulator (ICOS)—in Treg cells upon inoculation into germ-free mice. Genome sequencing revealed that the 17 strains fall within clusters IV, XIVa and XVIII of Clostridia, which lack prominent toxins and virulence factors. The 17 strains act as a community to provide bacterial antigens and a TGF-β-rich environment to help expansion and differentiation of Treg cells. Oral administration of the combination of 17 strains to adult mice attenuated disease in models of colitis and allergic diarrhoea. Use of the isolated strains may allow for tailored therapeutic manipulation of human immune disorders.

Regulatory T cells in tumor immunity

Recent studies have revealed that Foxp3+CD25+CD4+ regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self‐tolerance, play critical roles for the control of antitumor immune responses. For example, a large number of Foxp3+Tregs infiltrate into tumors, and systemic removal of Foxp3+Tregs enhances natural as well as vaccine‐induced antitumor T‐cell responses. Tregs are recruited to tumor tissues via chemokines, such as CCL22 binding to CCR4 expressed by Tregs. They appear to expand and become activated in tumor tissues and in the draining lymph nodes by recognizing tumor‐associated antigens as well as normal self‐antigen expressed by tumor cells. These results indicate that cancer vaccines targeting tumor‐associated self‐antigens may potentially expand/activate Tregs and hamper effective antitumor immune responses, and that tumor immunity can therefore be enhanced by depleting Tregs, attenuating Treg suppressive function, or rendering effector T cells refractory to Treg‐mediated suppression. Recent attempts have indeed demonstrated that combinations of monoclonal antibodies capable of modulating Treg functions synergistically enhance antitumor activity and are more effective than a single monoclonal antibody therapy. Combination therapy targeting a variety of molecules expressed in antigen‐presenting cells, effector T cells and Tregs is envisaged to be a promising anticancer immunotherapy.

Regulatory T cells in cancer immunotherapy.

FOXP3(+)CD25(+)CD4(+) regulatory T (Treg) cells, crucial for the maintenance of immunological self-tolerance, are abundant in tumors. Most of them are chemo-attracted to tumor tissues, expanding locally and differentiating into a Treg-cell subpopulation that strongly suppresses the activation and expansion of tumor-antigen-specific effector T cells. Several cancer immunotherapies targeting FOXP3(+)CD4(+) Treg cells, including depletion of Treg cells, are currently being tested in the clinic. In addition, clinical benefit of immune-checkpoint blockade, such as anti-CTLA-4 monoclonal antibody therapy, could be attributed at least in part to depletion of FOXP3(+)CD4(+) Treg cells from tumor tissues. Thus, optimal strategies need to be established for reducing Treg cells or attenuating their suppressive activity in tumor tissues, together with activating and expanding tumor-specific effector T cells.

NY-ESO-1: review of an immunogenic tumor antigen.

In the 9 years since its discovery, cancer-testis antigen NY-ESO-1 has made one of the fastest transitions from molecular, cellular, and immunological description to vaccine and immunotherapy candidate, already tested in various formulations in more than 30 clinical trials worldwide. Its main characteristic resides in its capacity to elicit spontaneous antibody and T-cell responses in a proportion of cancer patients. An overview of immunological findings and immunotherapeutic approaches with NY-ESO-1, as well the role of regulation in NY-ESO-1 immunogenicity, is presented here.

Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans

Significance Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a critical role in suppressing antitumor immune responses. Here we found that, compared with peripheral blood T cells, tumor-infiltrating T cells contained a higher frequency of effector Tregs, which are defined as FOXP3hi and CD45RA−, terminally differentiated, and most suppressive. Effector Treg cells, but not FOXP3lo and CD45RA+ naïve Treg cells, predominantly expressed C-C chemokine receptor 4 (CCR4) in both cancer tissues and peripheral blood. In vivo or in vitro anti-CCR4 mAb treatment selectively depleted effector Treg cells and efficiently induced tumor-antigen-specific CD4+ and CD8+ T cells. Thus, cell-depleting anti-CCR4 mAb therapy is instrumental for evoking and enhancing tumor immunity in humans via selectively removing effector-type FOXP3+ Treg cells. CD4+ Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show that CCR4 was specifically expressed by a subset of terminally differentiated and most suppressive CD45RA−FOXP3hiCD4+ Treg cells [designated effector Treg (eTreg) cells], but not by CD45RA+FOXP3loCD4+ naive Treg cells, in peripheral blood of healthy individuals and cancer patients. In melanoma tissues, CCR4+ eTreg cells were predominant among tumor-infiltrating FOXP3+ T cells and much higher in frequency compared with those in peripheral blood. With peripheral blood lymphocytes from healthy individuals and melanoma patients, ex vivo depletion of CCR4+ T cells and subsequent in vitro stimulation of the depleted cell population with the cancer/testis antigen NY-ESO-1 efficiently induced NY-ESO-1–specific CD4+ T cells. Nondepletion failed in the induction. The magnitude of the responses was comparable with total removal of FOXP3+ Treg cells by CD25+ T-cell depletion. CCR4+ T-cell depletion also augmented in vitro induction of NY-ESO-1–specific CD8+ T cells in melanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly reduced the eTreg-cell fraction and augmented NY-ESO-1–specific CD8+ T-cell responses in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1. Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for evoking and augmenting antitumor immunity in cancer patients by selectively depleting eTreg cells.

Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation.

B cells can suppress autoimmunity by secreting interleukin-10 (IL-10). Although subpopulations of splenic B lineage cells are reported to express IL-10 in vitro, the identity of IL-10-producing B cells with regulatory function in vivo remains unknown. By using IL-10 reporter mice, we found that plasmablasts in the draining lymph nodes (dLNs), but not splenic B lineage cells, predominantly expressed IL-10 during experimental autoimmune encephalomyelitis (EAE). These plasmablasts were generated only during EAE inflammation. Mice lacking plasmablasts by genetic ablation of the transcription factors Blimp1 or IRF4 in B lineage cells developed an exacerbated EAE. Furthermore, IRF4 positively regulated IL-10 production that can inhibit dendritic cell functions to generate pathogenic T cells. Our data demonstrate that plasmablasts in the dLNs serve as IL-10 producers to limit autoimmune inflammation and emphasize the importance of plasmablasts as IL-10-producing regulatory B cells.

Two FOXP3+CD4+ T cell subpopulations distinctly control the prognosis of colorectal cancers

CD4+ T cells that express the forkhead box P3 (FOXP3) transcription factor function as regulatory T (Treg) cells and hinder effective immune responses against cancer cells. Abundant Treg cell infiltration into tumors is associated with poor clinical outcomes in various types of cancers. However, the role of Treg cells is controversial in colorectal cancers (CRCs), in which FOXP3+ T cell infiltration indicated better prognosis in some studies. Here we show that CRCs, which are commonly infiltrated by suppression-competent FOXP3hi Treg cells, can be classified into two types by the degree of additional infiltration of FOXP3lo nonsuppressive T cells. The latter, which are distinguished from FOXP3+ Treg cells by non-expression of the naive T cell marker CD45RA and instability of FOXP3, secreted inflammatory cytokines. Indeed, CRCs with abundant infiltration of FOXP3lo T cells showed significantly better prognosis than those with predominantly FOXP3hi Treg cell infiltration. Development of such inflammatory FOXP3lo non-Treg cells may depend on secretion of interleukin (IL)-12 and transforming growth factor (TGF)-β by tissues and their presence was correlated with tumor invasion by intestinal bacteria, especially Fusobacterium nucleatum. Thus, functionally distinct subpopulations of tumor-infiltrating FOXP3+ T cells contribute in opposing ways to determining CRC prognosis. Depletion of FOXP3hi Treg cells from tumor tissues, which would augment antitumor immunity, could thus be used as an effective treatment strategy for CRCs and other cancers, whereas strategies that locally increase the population of FOXP3lo non-Treg cells could be used to suppress or prevent tumor formation.

In vivo antigen delivery by a Salmonella typhimurium type III secretion system for therapeutic cancer vaccines

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratumoral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-specific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.

Tumor-infiltrating IL-17-producing γδ T cells support the progression of tumor by promoting angiogenesis

Based on the evidence that IL‐17 is a key cytokine involved in various inflammatory diseases, we explored the critical role of IL‐17‐producing γδ T cells for tumor development in tumor‐bearing mouse model. IL‐17−/− mice exhibited a significant reduction of tumor growth, concomitantly with the decrease of vascular density at lesion area, indicating a pro‐tumor property of IL‐17. Among tumor‐infiltrating lymphocytes (TIL), γδ T cells were the major cellular source of IL‐17. Analysis of TCR repertoires in TIL‐γδ T cells showed that circulating γδ T cells, but not skin resident Vγ5+γδ T cells, produced IL‐17. Neutralizing antibodies against IL‐23, IL‐6, and TGF‐β, which were produced within the tumor microenvironment, inhibited the induction of IL‐17‐producing γδ T cells. IL‐17 production by tumor‐infiltrating γδ T cells was blocked by anti‐γδTCR or anti‐NKG2D antibodies, indicating that these ligands, expressed within the tumor microenvironment, are involved in γδ T‐cell activation. The IL‐17‐producing TIL‐γδ T cells exhibited reduced levels of perforin mRNA expression, but increased levels of COX‐2 mRNA expression. Together, our findings support the novel concept that IL‐17‐producing γδ T cells, generated in response to tumor microenvironment, act as tumor‐promoting cells by inducing angiogenesis.

Definition of target antigens for naturally occurring CD4+ CD25+ regulatory T cells

The antigenic targets recognized by naturally occurring CD4+ CD25+ regulatory T cells (T reg cells) have been elusive. We have serologically defined a series of broadly expressed self-antigens derived from chemically induced mouse sarcomas by serological identification of antigens by recombinant expression cloning (SEREX). CD4+ CD25+ T cells from mice immunized with SEREX-defined self-antigens had strong suppressive activity on peptide-specific proliferation of CD4+ CD25− T cells and CD8+ T cells. The suppressive effect was observed without in vitro T cell stimulation. Foxp3 expression in these CD4+ CD25+ T cells from immunized mice was 5–10 times greater than CD4+ CD25+ T cells derived from naive mice. The suppressive effect required cellular contact and was blocked by anti-glucocorticoid–induced tumor necrosis factor receptor family–related gene antibody. In vitro suppressive activity essentially disappeared 8 wk after the last immunization. However, it was regained by in vitro restimulation with cognate self-antigen protein but not with control protein. We propose that SEREX-defined self-antigens such as those used in this study represent self-antigens that elicit naturally occurring CD4+ CD25+ T reg cells.