Takuro Saito

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.

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.

Detection of self-reactive CD8+ T cells with an anergic phenotype in healthy individuals

Immunological tolerance to self requires naturally occurring regulatory T (Treg) cells. Yet how they stably control autoimmune T cells remains obscure. Here, we show that Treg cells can render self-reactive human CD8+ T cells anergic (i.e., hypoproliferative and cytokine hypoproducing upon antigen restimulation) in vitro, likely by controlling the costimulatory function of antigen-presenting cells. Anergic T cells were naïve in phenotype, lower than activated T cells in T cell receptor affinity for cognate antigen, and expressed several coinhibitory molecules, including cytotoxic T lymphocyte–associated antigen-4 (CTLA-4). Using these criteria, we detected in healthy individuals anergic T cells reactive with a skin antigen targeted in the autoimmune disease vitiligo. Collectively, our results suggest that Treg cell–mediated induction of anergy in autoimmune T cells is important for maintaining self-tolerance. Healthy individuals harbor “silenced” self-reactive T cells. For the immune system, silence is golden For the immune system, balance is key. Immune cells must learn to eliminate invading pathogens but tolerate self. A cell type called regulatory T cells (Tregs) help to maintain this balance, but how they do so, particularly in humans, is unclear. Maeda et al. now report that Tregs “silence” T cells with modest reactivity to self. After culture with Tregs, the silenced T cells proliferated very little and produced almost no cytokines in response to antigen. The authors then examined T cells from healthy donors and from people with an autoimmune disease. Only healthy donors harbored silenced T cells, suggesting that if silencing goes awry, autoimmunity may result. Science, this issue p. 1536

High expression of MAGE-A4 and MHC class I antigens in tumor cells and induction of MAGE-A4 immune responses are prognostic markers of CHP-MAGE-A4 cancer vaccine.

PURPOSE We conducted a cancer vaccine clinical trial with MAGE-A4 protein. Safety, clinical response, and antigen-specific immune responses were analyzed and the prognostic factors by vaccination were investigated. EXPERIMENTAL DESIGN Twenty patients with advanced esophageal, stomach or lung cancer were administered MAGE-A4 vaccine containing 300μg protein subcutaneously once every 2 weeks in six doses. Primary endpoints of this study were safety and MAGE-A4 immune responses. RESULTS The vaccine was well tolerated. Fifteen of 20 patients completed one cycle of vaccination and two patients showed SD. A MAGE-A4-specific humoral immune response was observed in four patients who had high expression of MAGE-A4 and MHC class I on tumor cells. These four patients showed significantly longer overall survival than patients without an antibody response after vaccination (p=0.009). Patients with tumor cells expressing high MAGE-A4 or MHC class I antigen showed significantly longer overall survival than those with low expression. Induction of CD4 and CD8T cell responses was observed in three and six patients, respectively, and patients with induction of MAGE-A4-specific IFNγ-producing CD8T cells, but not CD4T cells, lived longer than those without induction. CONCLUSIONS The CHP-MAGE-A4 vaccine was safe. Expression of MAGE-A4 and MHC class I in tumor tissue and the induction of a MAGE-A4-specific immune response after vaccination would be feasible prognostic markers for patients vaccinated with MAGE-A4.

Which is a more reliable indicator of survival after gastric cancer surgery: Postoperative complication occurrence or C-reactive protein elevation?

The impact of postoperative complications on long‐term outcome has been reported in several types of malignancies. However, it is unclear why postoperative complications affect long‐term outcome. The aim of this study is evaluating whether postoperative complication occurrence or C‐reactive protein (CRP) elevation better reflects long‐term outcome in gastric cancer patients.

Subtotal gastrectomy for gastric tube cancer after esophagectomy: A safe procedure preserving the proximal part of gastric tube based on intraoperative ICG blood flow evaluation

Recent improvements in the survival of patients after esophagectomy have led to an increase in the occurrence of gastric tube cancer (GTC). Total resection of the gastric tube with lymphadenectomy is a standard and reliable treatment for GTC, but problems may arise during or after surgery, such as laryngeal nerve injury, reduced selection of organs for reconstruction, and impaired swallowing function. We recently performed a less invasive procedure, subtotal gastrectomy with preservation of the upper region of the gastric tube, in two patients. In these patients, blood supply to the gastric tube was evaluated by indocyanine green fluorescence imaging. Blood flow was confirmed as passing from the remnant esophagus to the upper region of the gastric tube through the esophago‐gastric anastomotic site by indocyanine green fluorescence imaging. Therefore, we resected the gastric tube while preserving the upper region of the gastric tube. There was no necrosis of the remnant gastric tube or anastomotic leakage postoperatively, and postoperative swallowing and eating functions were quite good in both patients. In summary, subtotal gastrectomy as a treatment for GTC is potentially safe, curative, and beneficial for the patients quality of life. J. Surg. Oncol. 2012; 106:107–110.

Abstract B174: FOXP3+CD4+ T-cell subpopulations distinctly control the prognosis of colorectal cancers

Introduction; Abundant FOXP3+CD4+ regulatory T cell (Tregs) infiltration into tumors is significantly associated with poor clinical outcomes in various types of cancer. However, the role of Tregs is controversial in colorectal cancers (CRCs), in which FOXP3+ T-cell infiltration indicated better prognosis in some studies. We previously reported that FOXP3+ T cells are composed of three functionally distinct subpopulations (Miyara et al, Immunity 2009): CD45RA+FOXP3lo+ naive Tregs, CD45RA-FOXP3hi+ effector Tregs, and CD45RA-FOXP3lo+ non-Tregs. By analyzing the Treg subpopulation in tumor infiltrating lymphocytes (TILs) of CRCs, we aimed to precisely assess the impact of Treg infiltration on CRC prognosis. Procedures; TILs from 35 CRCs were analyzed by flow cytometry. CRCs were thus divided into two groups: Type A (low % of non-Tregs in CD4+ T cells) and Type B (high % of non-Tregs in CD4+ T cells) CRCs. mRNA expression of tumor tissues was analyzed with microarray analysis and quantitative polymerase chain reaction (PCR) analysis to assess the difference between Type A and Type B tumors. In-vitro naive T cell culture was performed to confirm the effect of observed cytokines to induce FOXP3+ T cells. Shorty naive T cells were sorted by flow cytometry and cultured with anti-CD3/CD28 TCR stimulation with or without the observed cytokines. Based on observed differences in gene expression, the impact of FOXP3 mRNA levels on survival was evaluated using mRNA from another set of 109 CRCs. Finally, to clarify the association of bacteria existence with the difference of the CRC type, bacteria infiltration was assessed by fluorescence in situ hybridization (FISH) analysis with Formalin-Fixed Paraffin-Embedded (FFPE) tumor tissues. Results; More than half of 35 CRCs were classified into Type B CRCs. Inflammatory and immune response related genes, especially IL12A, TGFB1 and TNF mRNA expression levels were significantly upregulated in Type B relative to Type A CRCs. Of these cytokines, TGF-β effectively induced FOXP3hi+ T cells from naive T cells in vitro and the combination of IL12 and TGF-β mostly induced FOXP3lo+ T cells. Tumor tissue mRNA gene expression showed that Type A CRCs were largely IL12Alo and TGFB1lo, whereas Type B CRCs were either IL12Ahi or TGFB1hi, or both. With this cytokine-dependent classification of CRC tissues, we evaluated the impact of FOXP3+ T cells on prognosis with another cohort of 109 CRC patients. High FOXP3 mRNA expression was associated with poor prognosis in Type A where FOXP3+ T cells were mostly Tregs. In contrast, high FOXP3 mRNA expression was not associated with prognosis in Type B as these tumors contained both non-Tregs and effecter Tregs. As regard to bacterial existence, intestinal bacteria deeply invading tumor tissues were found at a significant frequency by the FISH method in Type B CRC tissues relative to Type A CRC tissues. Conclusion; We hypothesize that functionally distinct subpopulations of tumor-infiltrating FOXP3+ T cells oppositely contribute to the prognosis of CRCs. Non-Treg infiltration is caused by inflammatory response of CRCs possibly induced by bacteria infiltration. Our results also indicate that Treg-cell depletion may augment anti-tumor immunity and provide clinical benefits in Type A CRCs and also other cancers with predominant FOXP3hi Treg-cell infiltration. Citation Format: Takuro Saito, Hiroyoshi Nishikawa, Hisashi Wada, Masaki Mori, Yuichiro Doki, Shimon Sakaguchi. FOXP3+CD4+ T-cell subpopulations distinctly control the prognosis of colorectal cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B174.