CD106 in tumor-specific exhausted CD8+ T cells mediates immunosuppression by inhibiting TCR signaling.

T cell exhaustion is a major contributor to immunosuppression in the tumor microenvironment (TME). Blockade of key regulators of T cell exhaustion, such as PD-1, can reinvigorate tumor-specific T cells and activate anti-tumor immunity in various types of cancer. Here, we identified that CD106 was specifically expressed in exhausted CD8+ T cells in the TME using single-cell RNA-sequencing. High CD106 expression in the TME in clinical samples corresponded to improved response to cancer immunotherapy. CD106 in tumor-specific T cells suppressed anti-tumor immunity both in vitro and in vivo, and loss of CD106 in CD8+ T cells suppressed tumor growth and improved response to PD-1 blockade. Mechanistically, CD106 inhibited T-cell receptor (TCR) signaling by interacting with the TCR/CD3 complex and reducing its surface expression. Together, these findings provide insights into the immunosuppressive role of CD106 expressed in tumor-specific exhausted CD8+ T cells, identifying it as a potential biomarker and therapeutic target for cancer immunotherapy.

Establishment of immune suppression by cancer cells in the tumor microenvironment.

With the clinical success of immune checkpoint inhibitors (ICIs), cancer immunotherapy has become an important pillar of cancer treatment in various types of cancer. However, more than half of patients fail to respond to ICIs, even in combination, uncovering a limited window of clinical responses. Therefore, it is essential to develop more effective cancer immunotherapies and to define biomarkers for stratifying responders and nonresponders by exploring the immunological landscape in the tumor microenvironment (TME). It has become clear that differences in immune responses in the TME determine the clinical efficacy of cancer immunotherapies. Additionally, gene alterations in cancer cells contribute to the development of the immunological landscape, particularly immune suppression in the TME. Therefore, integrated analyses of immunological and genomic assays are key for understanding diverse immune suppressive mechanisms in the TME. Developing novel strategies to control immune suppression in the TME from the perspective of immunology and the cancer genome is crucial for effective cancer immunotherapy (immune-genome precision medicine).

Stem-like progenitor and terminally differentiated TFH-like CD4+ T cell exhaustion in the tumor microenvironment.

Immune checkpoint inhibitors exert clinical efficacy against various types of cancer through reinvigoration of exhausted CD8+ T cells that attack cancer cells directly in the tumor microenvironment (TME). Using single-cell sequencing and mouse models, we show that CXCL13, highly expressed in tumor-infiltrating exhausted CD8+ T cells, induces CD4+ follicular helper T (TFH) cell infiltration, contributing to anti-tumor immunity. Furthermore, a part of the TFH cells in the TME exhibits cytotoxicity and directly attacks major histocompatibility complex-II-expressing tumors. TFH-like cytotoxic CD4+ T cells have high LAG-3/BLIMP1 and low TCF1 expression without self-renewal ability, whereas non-cytotoxic TFH cells express low LAG-3/BLIMP1 and high TCF1 with self-renewal ability, closely resembling the relationship between terminally differentiated and stem-like progenitor exhaustion in CD8+ T cells, respectively. Our findings provide deep insights into TFH-like CD4+ T cell exhaustion with helper progenitor and cytotoxic differentiated functions, mediating anti-tumor immunity orchestrally with CD8+ T cells.

Multiomic molecular characterization of the response to combination immunotherapy in MSS/pMMR metastatic colorectal cancer.

BACKGROUND: Immune checkpoint inhibitor (ICI) combinations represent an emerging treatment strategies in cancer. However, their efficacy in microsatellite stable (MSS) or mismatch repair-proficient (pMMR) colorectal cancer (CRC) is variable. Here, a multiomic characterization was performed to identify predictive biomarkers associated with patient response to ICI combinations in MSS/pMMR CRC for the further development of ICI combinations. METHODS: Whole-exome sequencing, RNA sequencing, and multiplex fluorescence immunohistochemistry of tumors from patients with MSS/pMMR CRC, who received regorafenib plus nivolumab (REGONIVO) or TAS-116 plus nivolumab (TASNIVO) in clinical trials were conducted. Twenty-two and 23 patients without prior ICI from the REGONIVO and TASNIVO trials were included in this study. A biomarker analysis was performed using samples from each of these studies. RESULTS: The epithelial-mesenchymal transition pathway and genes related to cancer-associated fibroblasts were upregulated in the REGONIVO responder group, and the G2M checkpoint pathway was upregulated in the TASNIVO responder group. The MYC pathway was upregulated in the REGONIVO non-responder group. Consensus molecular subtype 4 was significantly associated with response (p=0.035) and longer progression-free survival (p=0.006) in the REGONIVO trial. CD8+ T cells, regulatory T cells, and M2 macrophages density was significantly higher in the REGONIVO trial responders than in non-responders. Mutations in the POLE gene and patient response were significantly associated in the TASNIVO trial; however, the frequencies of other mutations or tumor mutational burden were not significantly different between responders and non-responders in either trial. CONCLUSIONS: We identified molecular features associated with the response to the REGONIVO and TASNIVO, particularly those related to tumor microenvironmental factors. These findings are likely to contribute to the development of biomarkers to predict treatment efficacy for MSS/pMMR CRC and future immunotherapy combinations for treatment.

Regulatory T cell-mediated immunosuppression orchestrated by cancer: towards an immuno-genomic paradigm for precision medicine.

Accumulating evidence indicates that aberrant signalling stemming from genetic abnormalities in cancer cells has a fundamental role in their evasion of antitumour immunity. Immune escape mechanisms include enhanced expression of immunosuppressive molecules, such as immune-checkpoint proteins, and the accumulation of immunosuppressive cells, including regulatory T (Treg) cells, in the tumour microenvironment. Therefore, Treg cells are key targets for cancer immunotherapy. Given that therapies targeting molecules predominantly expressed by Treg cells, such as CD25 or GITR, have thus far had limited antitumour efficacy, elucidating how certain characteristics of cancer, particularly genetic abnormalities, influence Treg cells is necessary to develop novel immunotherapeutic strategies. Hence, Treg cell-targeted strategies based on the particular characteristics of cancer in each patient, such as the combination of immune-checkpoint inhibitors with molecularly targeted agents that disrupt the immunosuppressive networks mediating Treg cell recruitment and/or activation, could become a new paradigm of cancer therapy. In this Review, we discuss new insights on the mechanisms by which cancers generate immunosuppressive networks that attenuate antitumour immunity and how these networks confer resistance to cancer immunotherapy, with a focus on Treg cells. These insights lead us to propose the concept of 'immuno-genomic precision medicine' based on specific characteristics of cancer, especially genetic profiles, that correlate with particular mechanisms of tumour immune escape and might, therefore, inform the optimal choice of immunotherapy for individual patients.

Metabolic advantages of regulatory T cells dictated by cancer cells.

Cancer cells employ glycolysis for their survival and growth (the "Warburg effect"). Consequently, surrounding cells including immune cells in the tumor microenvironment (TME) are exposed to hypoglycemic, hypoxic, and low pH circumstances. Since effector T cells depend on the glycolysis for their survival and functions, the metabolically harsh TME established by cancer cells is unfavorable, resulting in the impairment of effective antitumor immune responses. By contrast, immunosuppressive cells such as regulatory T (Treg) cells can infiltrate, proliferate, survive, and exert immunosuppressive functions in the metabolically harsh TME, indicating the different metabolic dependance between effector T cells and Treg cells. Indeed, some metabolites that are harmful for effector T cells can be utilized by Treg cells; lactic acid, a harmful metabolite for effector T cells, is available for Treg cell proliferation and functions. Deficiency of amino acids such as tryptophan and glutamine in the TME impairs effector T cell activation but increases Treg cell populations. Furthermore, hypoxia upregulates fatty acid oxidation via hypoxia-inducible factor 1α (HIF-1α) and promotes Treg cell migration. Adenosine is induced by the ectonucleotidases CD39 and CD73, which are strongly induced by HIF-1α, and reportedly accelerates Treg cell development by upregulating Foxp3 expression in T cells via A2AR-mediated signals. Therefore, this review focuses on the current views of the unique metabolism of Treg cells dictated by cancer cells. In addition, potential cancer combination therapies with immunotherapy and metabolic molecularly targeted reagents that modulate Treg cells in the TME are discussed to develop "immune metabolism-based precision medicine".

T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation.

Human leukocyte antigen (HLA)-DPB1 antigens are mismatched in approximately 70% of allogeneic hematopoietic stem cell transplantations (allo-HSCT) from HLA 10/10 matched unrelated donors. HLA-DP-mismatched transplantation was shown to be associated with an increase in acute graft-versus-host disease (GVHD) and a decreased risk of leukemia relapse due to the graft-versus-leukemia (GVL) effect. Immunotherapy targeting mismatched HLA-DP is considered reasonable to treat leukemia following allo-HCT if performed under non-inflammatory conditions. Therefore, we isolated CD4+ T cell clones that recognize mismatched HLA-DPB1 from healthy volunteer donors and generated T cell receptor (TCR)-gene-modified T cells for future clinical applications. Detailed analysis of TCR-T cells expressing TCR from candidate clone #17 demonstrated specificity to myeloid and monocytic leukemia cell lines that even expressed low levels of targeted HLA-DP. However, they did not react to non-hematopoietic cell lines with a substantial level of targeted HLA-DP expression, suggesting that the TCR recognized antigenic peptide is only present in some hematopoietic cells. This study demonstrated that induction of T cells specific for HLA-DP, consisting of hematopoietic cell lineage-derived peptide and redirection of T cells with cloned TCR cDNA by gene transfer, is feasible when using careful specificity analysis.

BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.

ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.

Protocol for acquiring samples to assess the impact of microplastics on immune microenvironments in the mouse intestine.

Environmental nano- and microplastics (NMPs) pose serious environmental issues, yet there is no established technique to assess their impact on health through oral ingestion. Here, we present a protocol to assess the impact of NMPs in the intestinal immune microenvironments by employing chronic exposure to NMPs in a mouse model. We describe steps for administration of NMPs, feces and tissue collection, and colonic gut digestion. We then detail procedures for isolation of intestinal immune cells and RNA isolation. For complete details on the use and execution of this protocol, please refer to Harusato et al.1.

Phase II Trial of Nivolumab in Metastatic Rare Cancer with dMMR or MSI-H and Relation with Immune Phenotypic Analysis (the ROCK Trial).

PURPOSE: Mismatch repair deficiency (dMMR)/microsatellite instability-high (MSI-H) are positive predictive markers for immune checkpoint inhibitors. However, data on the activity of nivolumab in advanced dMMR/MSI-H rare cancers and more accurate biomarkers are worth exploring. PATIENTS AND METHODS: We conducted a multicenter phase II, open-label, single-arm clinical trial to explore the effectiveness and safety of nivolumab monotherapy in patients with advanced rare cancers with dMMR/MSI-H, in parallel with immune phenotype analysis, to explore new biomarkers. A Bayesian adaptive design was applied. Characterization of peripheral blood mononuclear cells (PBMC) was characterized by multicolor flow cytometric analysis and CyTOF using samples collected before and after the intervention. The dMMR was identified by the complete loss of MLH1/MSH2/MSH6/PMS2. RESULTS: From May 2018 to March 2021, 242 patients were screened, and 11 patients were enrolled, of whom 10 were included in the full analysis. Median follow-up was 24.7 months (interquartile range, 12.4-31.5). Objective response rate was 60% [95% confidence interval (CI), 26.2-87.8] by central assessment and 70% (95% CI, 34.8-93.3) by local investigators. Median progression-free survival was 10.1 months (95% CI, 0.9-11.1). No treatment-related adverse events of grade 3 or higher were observed. Patients with a tumor mutation burden of ≥10/Mb showed a 100% response rate (95% CI, 47.8-100). Responders had increased T-bet+ PD-1+ CD4+ T cells in PBMC compared with nonresponders (P < 0.05). CONCLUSIONS: The trial met its primary endpoint with nivolumab, demonstrating clinical benefit in advanced dMMR/MSI-H rare solid cancers. Besides, the proportion of T-bet+ PD-1+ CD4+ T-cells may serve as a novel predictive biomarker.

Development of TCR-T cell therapy targeting mismatched HLA-DPB1 for relapsed leukemia after allogeneic transplantation.

Relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a significant challenge, with the re-emergence of the primary disease being the most frequent cause of death. Human leukocyte antigen (HLA)-DPB1 mismatch occurs in approximately 70% of unrelated allo-HSCT cases, and targeting mismatched HLA-DPB1 is considered reasonable for treating relapsed leukemia following allo-HSCT if performed under proper conditions. In this study, we established several clones restricted to HLA-DPB1*02:01, -DPB1*04:02, and -DPB1*09:01 from three patients who underwent HLA-DPB1 mismatched allo-HSCT using donor-derived alloreactive T cells primed to mismatched HLA-DPB1 in the recipient's body after transplantation. A detailed analysis of the DPB1*09:01-restricted clone 2A9 showed reactivity against various leukemia cell lines and primary myeloid leukemia blasts, even with low HLA-DP expression. T cell receptor (TCR)-T cells derived from clone 2A9 retained the ability to trigger HLA-DPB1*09:01-restricted recognition and lysis of various leukemia cell lines in vitro. Our study demonstrated that the induction of mismatched HLA-DPB1 specific T cell clones from physiologically primed post-allo-HSCT alloreactive CD4+ T cells and the redirection of T cells with cloned TCR cDNA by gene transfer are feasible as techniques for future adoptive immunotherapy.

Autologous peripheral blood stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia is safe but poses challenges for long-term maintenance of molecular remission: Results of the Auto-Ph17 study.

Autologous hematopoietic stem cell transplantation (SCT) is not a standard treatment option for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL); however, its position has been reassessed since the introduction of tyrosine kinase inhibitors (TKIs). We prospectively analyzed the efficacy and safety of autologous peripheral blood SCT (auto-PBSCT) for Ph+ALL patients aged between 55 and 70 years who had achieved complete molecular remission. Melphalan, cyclophosphamide, etoposide, and dexamethasone were used for conditioning. A total of 12 courses of maintenance therapy, including dasatinib, were performed. The required number of CD34+ cells was harvested in all five patients. No patient died within 100 days after auto-PBSCT, and no unexpected serious adverse events were observed. Although 1-year event-free survival was 100%, hematological relapse was observed in three patients at a median of 801 days (range, 389-1088 days) after auto-PBSCT. Molecular progressive disease was observed in the other two patients, although they maintained their first hematological remission at the last visit. Auto-PBSCT can be safely performed for Ph+ALL with TKIs. A limitation of auto-PBSCT was suggested, despite the increase in the intensity of a single treatment. The development of long-term therapeutic strategies by including new molecular targeted drugs is warranted to maintain long-term molecular remission.

Anti-tumor effects of anti-programmed cell death-1 antibody treatment are attenuated in streptozotocin-induced diabetic mice.

Hyperglycemia impairs immune response; however, it remains unknown whether the anti-tumor effects of anti-programmed cell death-1 antibody (PD-1-Ab) treatment are changed in hyperglycemic conditions. We analyzed the effect of PD-1-Ab on tumor growth in streptozotocin-induced diabetic mice (STZ-mice) subcutaneously inoculated with MC38 (a colon carcinoma cell line). Furthermore, we assessed the expression of chemokines by polymerase chain reaction (PCR) array in tumor-draining lymph nodes (dLNs) of these mice and MC38 cells cultured in different glucose concentrations. The suppressive effect of PD-1-Ab on tumor growth was attenuated. This was accompanied by fewer tumor-infiltrating CD8+ T cells, and STZ-mice had fewer tumor-infiltrating CD11c+ dendritic cells (DCs) than normoglycemic mice. mRNA expression levels of CXCL9, a chemokine recruiting CD8+ T cells, were lower in dLNs of STZ-mice than in normoglycemic mice after PD-1-Ab treatment, and its protein was expressed in DCs. In MC38 cells cultured with 25 mM glucose, mRNA expression of CCL7, a chemokine recruiting DCs, was decreased compared to cells cultured with 5 mM glucose. These results suggest that the STZ-induced hyperglycemia impairs the effect of PD-1-Ab treatment on MC38 tumor growth, and is accompanied by reduced infiltration of DCs and CD8+ T cells and decreased expression of CCL7 and CXCL9.

Impact of particulate microplastics generated from polyethylene terephthalate on gut pathology and immune microenvironments.

Environmental microplastics have emerged as a critical issue in maintaining the planetary ecosystem. In this study, we generated particulate microplastics from polyethylene terephthalate (PM-PET) and investigated their impact in the gut by using mouse models and implementing histological examinations, as well as multi-omics analysis for colonic immune cells and microbiota. As a result, histological approaches showed that chronic and physiological low dose exposure of PM-PET did not affect intestinal pathology and mucin barriers, respectively. Moreover, immunohistochemical analysis demonstrated that the numbers of T cells, B cells, macrophages, and granulocytes were not affected by the exposure to PM-PET. However, RNA-seq analysis revealed that PM-PET had a substantial impact on the transcriptome in gut immune cells and their metabolisms, while 16s rRNA metagenomic analysis showed that the composition of microbiota was modestly affected. These results suggest an unexpected role played by the PM-PET in affecting gut immune homeostasis without detectable inflammation.

Complete response to definitive chemoradiotherapy in unresectable locally advanced esophageal squamous cell carcinoma.

BACKGROUND: Although definitive chemoradiotherapy (CRT) is the standard therapy for patients with unresectable locally advanced esophageal squamous cell carcinoma (ESCC), poor survival has been reported. Although the complete response (CR) rate is strongly correlated with good prognosis, the predictive factors for CR have not been elucidated. METHODS: This registry study aimed to identify predictors of CR to definitive CRT in patients with unresectable locally advanced ESCC. "Unresectable" was defined as the primary lesion invading unresectable adjacent structures such as the aorta, vertebral body, and trachea (T4b), or the regional and/or supraclavicular lymph nodes invading unresectable adjacent structures (LNT4b). RESULTS: Overall, 175 patients who started definitive CRT between January 2013 and March 2020 were included. The confirmed CR (cCR) rate was 24% (42/175). The 2-year progression-free survival (PFS) and overall survival (OS) rates of cCR cases vs. non-cCR cases were 59% vs. 2% (log-rank p < 0.001) and 90% vs. 31% (log-rank p < 0.001), with a median follow-up period of 18.5 and 40.5 months, respectively. Multivariate analysis of clinicopathological factors revealed that tumor length ≥ 6 cm [odds ratio (OR) 0.446; 95% CI 0.220-0.905; p = 0.025] was a predictor of cCR. CONCLUSIONS: Favorable PFS and OS rates were observed in patients with cCR. Tumor length was a predictive factor for cCR.

Significance of regulatory T cells in cancer immunology and immunotherapy.

Immunosuppression in the tumour microenvironment (TME) attenuates antitumor immunity, consequently hindering protective immunosurveillance and preventing effective antitumor immunity induced by cancer immunotherapy. Multiple mechanisms including immune checkpoint molecules, such as CTLA-4, PD-1, and LAG-3, and immunosuppressive cells are involved in the immunosuppression in the TME. Regulatory T (Treg) cells, a population of immunosuppressive cells, play an important role in inhibiting antitumor immunity. Therefore, Treg cells in the TME correlate with an unfavourable prognosis in various cancer types. Thus, Treg cell is considered to become a promising target for cancer immunotherapy. Elucidating Treg cell functions in cancer patients is therefore crucial for developing optimal Treg cell-targeted immunotherapy. Here, we describe Treg cell functions and phenotypes in the TME from the perspective of Treg cell-targeted immunotherapy.

The cancer epigenome: Non-cell autonomous player in tumor immunity.

Dysregulation of the tumor-intrinsic epigenetic circuit is a key driver event for the development of cancer. Accumulating evidence suggests that epigenetic and/or genetic drivers stimulate intrinsic oncogenic pathways as well as extrinsic factors that modulate the immune system. These modulations indeed shape the tumor microenvironment (TME), allowing pro-oncogenic factors to become oncogenic, thereby contributing to cancer development and progression. Here we review the epigenetic dysregulation arising in cancer cells that disseminates throughout the TME and beyond. Recent CRISPR screening has elucidated key epigenetic drivers that play important roles in the proliferation of cancer cells (intrinsic) and inhibition of antitumor immunity (extrinsic), which lead to the development and progression of cancer. These epigenetic players can serve as promising targets for cancer therapy as a dual (two-in-one)-targeted approach. Considering the interplay between cancer and the immune system as a key determinant of immunotherapy, we discuss a novel lineage-tracing technology that enables longitudinal monitoring of cancer and immune phenotypic heterogeneity and fate paths during cancer development, progression, and therapeutic interventions.