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Abstract Anti-angiogenic agents elicit considerable immune modulatory effects within the tumor microenvironment, underscoring the rationale for synergistic clinical development of VEGF and immune checkpoint inhibitors in advanced gastric cancer (AGC). Early phase studies involving Asian patients demonstrated encouraging anti-tumor efficacies. We report the results of the REGOMUNE phase II study, in which Caucasian patients were administered regorafenib, a multi-tyrosine kinase inhibitor, in combination with avelumab, a PD-L1-targeting monoclonal antibody. This therapeutic regimen resulted in deep and durable responses in 19% of patients, with the median duration of response not yet reached. Notwithstanding, a significant proportion of AGC patients exhibited no therapeutic advantage, prompting investigations into mechanisms of inherent resistance. Comprehensive biomarker profiling elucidated that non-responders predominantly exhibited an augmented presence of M2 macrophages within the tumor microenvironment and a marked overexpression of S100A10 by neoplastic cells, a protein previously implicated in macrophage chemotaxis. Additionally, peripheral biomarker assessments identified elevated levels of cytokines, including CSF-1, IL-4, IL-8, and TWEAK, correlating with adverse clinical outcomes, thereby accentuating the role of macrophage infiltration in mediating resistance. These insights furnish an invaluable foundation for elucidating, and potentially circumventing, resistance mechanisms in current AGC therapeutic paradigms, emphasizing the integral role of tumor microenvironmental dynamics and immune modulation.

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).

Abstract 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.

Summary: 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.

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.

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract 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.

Abstract 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.

Summary: 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.

Abstract Purpose FOXP3 + and CD8 + are recognized markers of tumor-infiltrating lymphocytes (TILs) for breast cancer. FOXP3 + TILs are composed of effector Tregs (eTregs) and other subpopulations that are classified by their differences in suppressive function. In this prospective study, we evaluated Treg subpopulations and CD8 + TILs in breast cancer. Methods 84 patients with breast cancer were enrolled. Fresh TILs were extracted andTregs were classified into eTregs (CD4+FOXP3highCD45RA−), other FOXP3+ Treg subsets (naïve and non-Tregs), and total CD8+CD4− TILs using flow cytometry. The suppression strength of each Treg subpopulation was analyzed. The association between TIL subpopulations, clinicopathological characteristics, and response to chemotherapy was evaluated. Results The mean CD8/eTreg ratio value was 7.86 (interquartile range: 4.08–12.80). The proliferation function of eTregs was significantly suppressed compared with that of the other subpopulations (proliferation rates: control: 89.3%, + naiiveTreg: 64.2%, + non-Treg: 78.2% vs eTreg 1.93%; all P

Elimination of regulatory T cells via the anti-CCR4 monoclonal antibody, mogamulizumab, is expected to augment anti-tumour immune response. Authors show here that although regulatory T cell targeting is successful, clinical improvement remains minimal in patients with solid tumours due to concomitant and unintended depletion of central memory CD8+ T cells.

Summary: Immunophenotyping of tumor-infiltrating lymphocytes (TILs) by flow cytometry can predict clinical efficacy of immunotherapy. However, several obstacles need to be overcome for developing a flow cytometry assay starting from solid tumor specimens. Here, we show a detailed enzyme-based protocol to isolate TILs from human tumor tissues. The protocol was optimized to obtain enough viable TILs from a biopsy tissue specimen for flow cytometry-based TIL immunophenotyping. Additionally, tissue samples could be preserved for up to 72 h for subsequent characterization.For complete details on the use and execution of this protocol, please refer to Kumagai et al. (2020, 2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Patients with non‐small cell lung cancer (NSCLC) harboring activating EGFR mutations are sensitive to epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) but inevitably develop resistance to the inhibitors mostly through acquisition of the secondary T790M mutation. Although third‐generation EGFR‐TKIs overcome this resistance by selectively inhibiting EGFR with EGFR‐TKI‐sensitizing and T790M mutations, acquired resistance to third‐generation EGFR‐TKIs invariably develops. Methods Next‐generation sequencing (NGS) and fluorescence in situ hybridization (FISH) analysis were performed in an EGFR T790M‐mutated NSCLC patient who had progressed after a third‐generation EGFR‐TKI, TAS‐121. EGFR‐mutated cell lines were subjected to a cell proliferation assay and western blotting analysis with EGFR‐TKIs and a heat shock protein 90 (HSP90) inhibitor. Results NGS and FISH analysis revealed EGFR amplification in the resistant cancer cells. While EGFR L858R/T90M‐mutated cell line was sensitive to osimertinib or TAS‐121 in vitro, EGFR‐overexpressing cell lines displayed resistance to these EGFR‐TKIs. Western blot analysis showed that EGFR phosphorylation and overexpression of EGFR in cell lines was not suppressed by third‐generation EGFR‐TKIs. In contrast, an HSP90 inhibitor reduced total and phosphorylated EGFR and inhibited the proliferation of resistant cell lines. Conclusions EGFR amplification confers resistance to third‐generation EGFR‐TKIs which can be overcome by HSP90 inhibition. The results provide a preclinical rationale for the use of HSP90 inhibitors to overcome EGFR amplification‐mediated resistance.

Immunity: exhausted T cells provide subtle long-term defenses A type of T cell previously thought to be defective could actually provide long-term mild immune responses for combating cancer. The unique environment created during chronic infections gives rise to functionally inert T cells known as ‘exhausted’ cells, which were once thought to impede immune responses. Wooseok Seo and co-workers at Nagoya University and the National Cancer Center in Tokyo, Japan, have reviewed recent studies suggesting that exhausted T cells may be generated by the body to provide mild immune responses. Such responses are safer over the long term than strong inflammatory defense mechanisms, where T cells may be overstimulated, leading to uncontrolled tissue damage and induced T cell death. The signaling pathways that generate and propagate exhausted cells are being revealed, shedding light on the developmental stages that lead to this subtle but effective response to chronic conditions.

Abstract Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.

In order to identify genomic biomarkers for the outcome of mogamulizumab-containing treatment, an integrated molecular analysis of adult T-cell leukemia/lymphoma (ATL) was conducted on 64 mogamulizumab-naïve patients. Among driver genes, CCR4 and CCR7 alterations were observed in 22% and 11% of the patients, respectively, both consisting of single nucleotide variants (SNV)/insertion-deletions (indels) in the C-terminus. Patients with CCR4 alterations or without CCR7 alterations exhibited a more favorable clinical response (complete response [CR] rate 93%, 13/14; P=0.024, and CR rate 71%, 40/56; P=0.036, respectively). Additionally, TP53, CD28, and CD274 alterations were identified in 35%, 16%, and 10% of the patients, respectively. TP53 alterations included SNV/indels or copy number variations (CNV) such as homozygous deletion; CD28 alterations included SNV, CNV such as amplification, or fusion; CD274 alterations included CNV such as amplification, or structural variants. Univariate analysis revealed that TP53, CD28 or CD274 alterations were associated with worse overall survival (OS) (hazard ratio [HR]: 2.330, 95% confidence interval [CI]: 1.183-4.589; HR: 3.191, 95% CI: 1.287- 7.911; HR: 3.301, 95% CI: 1.130-9.641, respectively) but that CCR4 alterations were associated with better OS (HR: 0.286, 95% CI: 0.087-0.933). Multivariate analysis indicated that in addition to performance status, TP53, CCR4 or CD274 alterations (HR: 2.467, 95% CI: 1.197-5.085; HR: 0.155, 95% CI: 0.031-0.778; HR: 14.393, 95% CI: 2.437-85.005, respectively) were independently and significantly associated with OS. The present study contributes to the establishment of precision medicine using mogamulizumab in ATL patients.

Abstract Background High-dimensional flow cytometry and mass cytometry allow systemic-level characterization of more than 10 protein profiles at single-cell resolution and provide a much broader landscape in many biological applications, such as disease diagnosis and prediction of clinical outcome. When associating clinical information with cytometry data, traditional approaches require two distinct steps for identification of cell populations and statistical test to determine whether the difference between two population proportions is significant. These two-step approaches can lead to information loss and analysis bias. Results We propose a novel statistical framework, called LAMBDA (Latent Allocation Model with Bayesian Data Analysis), for simultaneous identification of unknown cell populations and discovery of associations between these populations and clinical information. LAMBDA uses specified probabilistic models designed for modeling the different distribution information for flow or mass cytometry data, respectively. We use a zero-inflated distribution for the mass cytometry data based the characteristics of the data. A simulation study confirms the usefulness of this model by evaluating the accuracy of the estimated parameters. We also demonstrate that LAMBDA can identify associations between cell populations and their clinical outcomes by analyzing real data. LAMBDA is implemented in R and is available from GitHub ( https://github.com/abikoushi/lambda ).

Abstract Background The standard treatment for patients with unresectable locally advanced esophageal squamous cell carcinoma (ESCC) is definitive chemoradiotherapy (CRT) using 5-FU plus cisplatin. However, complete response (CR) rates are low at 11–25%, resulting in 9–10 months of median overall survival (OS). An improved therapeutic efficacy by combining immunotherapy with radiation has been reported in patients with locally advanced non-small cell lung cancer. The results using ESCC cell lines suggest sequential treatment with anti-PD-L1 agents soon after completion of CRT is the most effective combination. Methods TENERGY trial is a multicenter, phase II, proof-of-concept study to assess the efficacy and safety of atezolizumab following definitive CRT in patients with locally advanced ESCC. The main inclusion criteria are unresectable locally advanced ESCC without distant metastasis, completion of 60 Gy of radiation plus two concomitant cycles of chemotherapy (cisplatin 70 mg/m2 on day 1 and 5-FU 700 mg/m2 on days 1–4, every 28 days), and adequate organ function. Within 6 weeks after CRT, participants will start taking 1200 mg of atezolizumab every three weeks and continue until 12 months or disease progression. The primary endpoint is the confirmed CR rate by the investigator’s assessment. Secondary endpoints include overall response rate, progression-free survival (PFS), OS, adverse events, and confirmed CR rate by central assessment. We will enroll 50 patients (40 with primary locally advanced ESCC and 10 with postoperative locoregionally recurrent ESCC). We will obtain biopsies from the primary site and will collect blood at 3 time points (before CRT, after CRT, and four weeks after the start of atezolizumab) for an exploratory biomarker study. We will analyze the phenotype of immune-competent cells, neoantigens, tumor mutational burden, PD-L1 status, and Human Leukocyte Antigen haplotyping. Discussion The synergistic efficacies of the sequential combination of CRT and atezolizumab should improve the CR rate, resulting in survival improvement for patients with unresectable locally advanced ESCC. Because CRT is a standard treatment option for patients with early stage to locally advanced ESCC, the sequential combination of CRT and atezolizumab has the potential to change the standard ESCC treatments. Trial registration UMIN000034373 , 10/04/2018 and EPOC1802 .

Abstract Background Modern flow cytometry technology has enabled the simultaneous analysis of multiple cell markers at the single-cell level, and it is widely used in a broad field of research. The detection of cell populations in flow cytometry data has long been dependent on “manual gating” by visual inspection. Recently, numerous software have been developed for automatic, computationally guided detection of cell populations; however, they are not designed for time-series flow cytometry data. Time-series flow cytometry data are indispensable for investigating the dynamics of cell populations that could not be elucidated by static time-point analysis. Therefore, there is a great need for tools to systematically analyze time-series flow cytometry data. Results We propose a simple and efficient statistical framework, named CYBERTRACK (CYtometry-Based Estimation and Reasoning for TRACKing cell populations), to perform clustering and cell population tracking for time-series flow cytometry data. CYBERTRACK assumes that flow cytometry data are generated from a multivariate Gaussian mixture distribution with its mixture proportion at the current time dependent on that at a previous timepoint. Using simulation data, we evaluate the performance of CYBERTRACK when estimating parameters for a multivariate Gaussian mixture distribution, tracking time-dependent transitions of mixture proportions, and detecting change-points in the overall mixture proportion. The CYBERTRACK performance is validated using two real flow cytometry datasets, which demonstrate that the population dynamics detected by CYBERTRACK are consistent with our prior knowledge of lymphocyte behavior. Conclusions Our results indicate that CYBERTRACK offers better understandings of time-dependent cell population dynamics to cytometry users by systematically analyzing time-series flow cytometry data.

With the broad application of cancer immunotherapies such as immune checkpoint inhibitors in multiple cancer types, the immunological landscape in the tumor microenvironment (TME) has become enormously important for determining the optimal cancer treatment. Tumors can be immunologically divided into two categories: inflamed and non-inflamed based on the extent of immune cell infiltration and their activation status. In general, immunotherapies are preferable for the inflamed tumors than for non-inflamed tumors. Regulatory T cells (Tregs), an immunosuppressive subset of CD4+ T cells, play an essential role in maintaining self-tolerance and immunological homeostasis. In tumor immunity, Tregs compromise immune surveillance against cancer in healthy individuals and impair the antitumor immune response in tumor-bearing hosts. Tregs, therefore, accelerate immune evasion by tumor cells, leading to tumor development and progression in various types of cancer. Therefore, Tregs are considered to be a crucial therapeutic target for cancer immunotherapy. Abundant Tregs are observed in the TME in many types of cancer, both in inflamed and non-inflamed tumors. Diverse mechanisms of Treg accumulation, activation, and survival in the TME have been uncovered for different tumor types, indicating the importance of understanding the mechanism of Treg infiltration in each patient when selecting the optimal Treg-targeted therapy. Here, we review recent advances in the understanding of mechanisms leading to Treg abundance in the TME to optimize Treg-targeted therapy. Furthermore, in addition to the conventional strategies targeting cell surface molecules predominantly expressed by Tregs, reagents targeting molecules and signaling pathways specifically employed by Tregs for infiltration, activation, and survival in each tumor type are illustrated as novel Treg-targeted therapies. The effectiveness of immune precision therapy depends on conditions in the TME of each cancer patient.

Patients with colorectal cancers (CRCs) generally exhibit improved survival through intensive lymph node (LN) dissection. However, recent progress in cancer immunotherapy revisits the potential importance of regional LNs, where T cells are primed to attack tumor cells. To elucidate the role of regional LN, we investigated the immunological status of nonmetastatic regional LN lymphocytes (LNLs) in comparison with those of the tumor microenvironment (tumor-infiltrating lymphocytes; TILs) using flow cytometry and next-generation sequencing. LNLs comprised an intermediate level of the effector T cell population between peripheral blood lymphocytes (PBLs) and TILs. Significant overlap of the T cell receptor (TCR) repertoire was observed in microsatellite instability–high/mismatch repair–deficient (MSI-H/dMMR) CRCs with high tumor mutation burden (TMB), although limited TCRs were shared between nonmetastatic LNs and primary tumors in microsatellite stable/MMR proficient (MSS/pMMR) CRC patients with low TMB. In line with the overlap of the TCR repertoire, an excessive LN dissection did not provide a positive impact on long-term prognosis in our MSI-H/dMMR CRC cohort (n = 130). We propose that regional LNs play an important role in antitumor immunity, particularly in MSI-H/dMMR CRCs with high TMB, requiring care to be taken regarding excessive nonmetastatic LN dissection in MSI-H/dMMR CRC patients.

Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRβ) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRβ signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients’ repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients.

Abstract Background Several studies have established a correlation between the VEGF–VEGFR2 axis and an immunosuppressive microenvironment; this immunosuppression can be overcome by anti-angiogenic reagents, such as ramucirumab (RAM). However, little is known about the immunological impact of anti-angiogenic reagents within the tumor microenvironment in human clinical samples. This study aimed at investigating the effects of RAM on the tumor microenvironmental immune status in human cancers. Methods We prospectively enrolled 20 patients with advanced gastric cancer (GC) who received RAM-containing chemotherapy. We obtained paired samples from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in primary tumors both pre- and post-RAM therapy to assess immune profiles by immunohistochemistry and flow cytometry. Results Within the tumor microenvironment, both PD-L1 expression and CD8+ T-cell infiltration increased after RAM-containing therapies. In addition, CD45RA−FOXP3highCD4+ cells (effector regulatory T cells [eTreg cells]) and PD-1 expression by CD8+ T cells were significantly reduced in TILs compared with PBMCs after RAM-containing therapies. Patients with partial response and longer progression-free survival had significantly higher pre-treatment eTreg frequencies in TILs than those with progressive disease. In in vitro analysis, VEGFR2 was highly expressed by eTreg cells. Further, VEGFA promoted VEGFR2+ eTreg cell proliferation, and this effect could be inhibited by RAM. Conclusions This study suggests that the frequency of eTreg cells in TILs could be a biomarker for stratifying clinical responses to RAM-containing therapies. Further, we propose that RAM may be employed as an immuno-modulator in combination with immune checkpoint blockade.

The purpose of this trial was to evaluate the efficacy of 2-year consolidation therapy with nilotinib, at a dose of 300 mg twice daily, for achieving treatment-free remission in chronic myeloid leukemia patients with a deep molecular response (BCR-ABL1IS ≤0.0032%). Successful treatment-free remission was defined as no confirmed loss of deep molecular response. We recruited 96 Japanese patients, of whom 78 sustained a deep molecular response during the consolidation phase and were therefore eligible to discontinue nilotinib in the treatment-free remission phase; of these, 53 patients (67.9%; 95% confidence interval: 56.4–78.1%) remained free from molecular recurrence in the first 12 months. The estimated 3-year treatment-free survival was 62.8%. Nilotinib was readministered to all patients (n=29) who experienced a molecular recurrence during the treatment-free remission phase. After restarting treatment, rapid deep molecular response returned in 25 patients (86.2%), with 50% of patients achieving a deep molecular response within 3.5 months. Tyrosine kinase inhibitor withdrawal syndrome was reported in 11/78 patients during the early treatment-free remission phase. The treatment-free survival curve was significantly better in patients with undetectable molecular residual disease than in patients without (3-year treatment-free survival, 75.6 versus 48.6%, respectively; P=0.0126 by the log-rank test). There were no significant differences in treatment-free survival between subgroups based on tyrosine kinase inhibitor treatment before the nilotinib consolidation phase, tyrosine kinase inhibitor-withdrawal syndrome, or absolute number of natural killer cells. The results of this study indicate that it is safe and feasible to stop tyrosine kinase inhibitor therapy in patients with chronic myeloid leukemia who have achieved a sustained deep molecular response with 2 years of treatment with nilotinib. This study was registered with UMIN-CTR (UMIN000005904).

Fulminant type 1 diabetes (FT1D) is a distinct subtype of type 1 diabetes and is fatal without immediate diagnosis and treatment. At present, there are no biomarkers for early and predictive detection of FT1D.First, we analyzed a total of 6 serum samples from 3 patients with FT1D (1 sample in the acute and 1 in the sub-acute phases from each patient) by seromic analysis. Second, titres of the antibody were measured by ELISA in sera from 30 patients with FT1D (both in the acute and sub-acute phases), 13 patients with FT1D in the chronic phase, 32 patients with autoimmune type 1 (type 1A) diabetes (T1AD), 30 patients with type 2 diabetes (T2D), 23 patients with autoimmune thyroid disease (AITD) and 31 healthy control subjects (HC).Seromic analysis revealed 9 antibodies which showed high signals from all 3 patients with FT1D in the acute phase. Among them, the titre of anti-CD300e antibody was significantly higher in FT1D patients in the acute phase than that in T1AD, T2D, AITD patients and HC, as determined by ELISA (P