The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies.

Immune checkpoint blockade has provided a paradigm shift in cancer therapy, but the success of this approach is very variable; therefore, biomarkers predictive of clinical efficacy are urgently required. Here, we show that the frequency of PD-1+CD8+ T cells relative to that of PD-1+ regulatory T (Treg) cells in the tumor microenvironment can predict the clinical efficacy of programmed cell death protein 1 (PD-1) blockade therapies and is superior to other predictors, including PD ligand 1 (PD-L1) expression or tumor mutational burden. PD-1 expression by CD8+ T cells and Treg cells negatively impacts effector and immunosuppressive functions, respectively. PD-1 blockade induces both recovery of dysfunctional PD-1+CD8+ T cells and enhanced PD-1+ Treg cell-mediated immunosuppression. A profound reactivation of effector PD-1+CD8+ T cells rather than PD-1+ Treg cells by PD-1 blockade is necessary for tumor regression. These findings provide a promising predictive biomarker for PD-1 blockade therapies.

An Oncogenic Alteration Creates a Microenvironment that Promotes Tumor Progression by Conferring a Metabolic Advantage to Regulatory T Cells.

Only a small percentage of patients afflicted with gastric cancer (GC) respond to immune checkpoint blockade (ICB). To study the mechanisms underlying this resistance, we examined the immune landscape of GC. A subset of these tumors was characterized by high frequencies of regulatory T (Treg) cells and low numbers of effector T cells. Genomic analyses revealed that these tumors bore mutations in RHOA that are known to drive tumor progression. RHOA mutations in cancer cells activated the PI3K-AKT-mTOR signaling pathway, increasing production of free fatty acids that are more effectively consumed by Treg cells than effector T cells. RHOA mutant tumors were resistant to PD-1 blockade but responded to combination of PD-1 blockade with inhibitors of the PI3K pathway or therapies targeting Treg cells. We propose that the metabolic advantage conferred by RHOA mutations enables Treg cell accumulation within GC tumors, generating an immunosuppressive TME that underlies resistance to ICB.

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".

Three-year outcomes of preoperative chemoradiotherapy plus nivolumab in microsatellite stable and microsatellite instability-high locally advanced rectal cancer.

BACKGROUND: Preoperative chemoradiotherapy (CRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). We reported the short-term outcomes of the VOLTAGE trial that investigated the safety and efficacy of preoperative CRT followed by nivolumab and surgery. Here, we present the 3-year outcomes of this trial. METHODS: Thirty-nine patients with microsatellite stable (MSS) LARC and five patients with microsatellite instability-high (MSI-H) LARC underwent CRT (50.4 Gy) followed by five doses of nivolumab (240 mg) and surgery. The 3-year relapse-free survival (RFS), overall survival (OS), and associations with biomarkers were evaluated. RESULTS: The 3-year RFS rates in patients with MSS and MSI-H were 79.5% and 100%, respectively, and the 3-year OS rates were 97.4% and 100%, respectively. Of the MSS patients, those with pre-CRT PD-L1 positivity, pre-CRT high CD8 + T cell/effector regulatory T cell (eTreg) ratio, pre-CRT high expression of Ki-67, CTLA-4, and PD-1 had a trend toward better 3-year RFS than those without. CONCLUSIONS: Three-year outcomes of patients with MSI-H were better than those of patients with MSS. PD-L1 positivity, elevated CD8/eTreg ratio, and high expression of Ki-67, CTLA-4, and PD-1 could be positive predictors of prognosis in patients with MSS. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02948348.

Vaginal Transmission of Cancer from Mothers with Cervical Cancer to Infants.

Two cases of pediatric lung cancer (in 23-month-old and 6-year-old boys) resulting from mother-to-infant transmission of uterine cervical tumors were incidentally detected during routine next-generation sequencing of paired samples of tumor and normal tissue. Spontaneous regression of some lesions in the first child and slow growth of the tumor mass in the second child suggested the existence of alloimmune responses against the transmitted tumors. Immune checkpoint inhibitor therapy with nivolumab led to a strong regression of all remaining tumors in the first child. (Funded by the Japan Agency for Medical Research and Development and others; TOP-GEAR UMIN Clinical Trials Registry number, UMIN000011141.).

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).

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.

HLA Class I Analysis Provides Insight Into the Genetic and Epigenetic Background of Immune Evasion in Colorectal Cancer With High Microsatellite Instability.

BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

Regulatory T-cell development in the tumor microenvironment.

Regulatory T (Treg) cells are required for maintaining self-tolerance and preventing the development of autoimmune diseases. However, Treg cells are abundant in tumors and suppress antitumor immunity, contributing to tumor development and growth. Thus, the selective deletion of tumor-infiltrating Treg cells is important for successful Treg cell-targeted therapies, providing effective antitumor immunity without inducing deleterious autoimmune disorders. Advancements in sequencing technologies have exposed the diversity and heterogeneity of human Treg cells during activation and differentiation, further emphasizing the importance of understanding tumor-infiltrating Treg cells for the development of Treg cell-targeted therapies. This review provides an overview of the classification and function of Treg cells and summarizes recent knowledge on the activation and differentiation of Treg cells in the tumor microenvironment.

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.

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.

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.

CYBERTRACK2.0: zero-inflated model-based cell clustering and population tracking method for longitudinal mass cytometry data.

SUMMARY: Recent advancements in high-dimensional single-cell technologies, such as mass cytometry, enable longitudinal experiments to track dynamics of cell populations and identify change points where the proportions vary significantly. However, current research is limited by the lack of tools specialized for analyzing longitudinal mass cytometry data. In order to infer cell population dynamics from such data, we developed a statistical framework named CYBERTRACK2.0. The framework's analytic performance was validated against synthetic and real data, showing that its results are consistent with previous research. AVAILABILITY AND IMPLEMENTATION: CYBERTRACK2.0 is available at https://github.com/kodaim1115/CYBERTRACK2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genomic determinants impacting the clinical outcome of mogamulizumab treatment for adult T-cell leukemia/lymphoma.

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.

Engineering strategies for broad application of TCR-T- and CAR-T-cell therapies.

Adoptive-cell therapy, including the transfer of tumor-infiltrating T lymphocytes after in vitro expansion or T cells redirected to tumor antigens using antigen-specific transgenic T-cell receptor T cells (TCR-T cells) or chimeric antigen receptor T cells (CAR-T cells), has shown a significant clinical impact. Particularly, several types of CAR-T-cell therapies have been approved for the treatment of hematological malignancies. The striking success of CAR-T-cell therapies in hematological malignancies motivates their further expansion to a wide range of solid tumors, yet multiple obstacles, including the lack of proper target antigens exhibiting a tumor-specific expression pattern and the immunosuppressive tumor microenvironment (TME) impairing the effector functions of adoptively transferred T cells, have prevented clinical application. Gene engineering technologies such as the CRISPR/Cas9 system have enabled flexible reprogramming of TCR/CAR-T-cell signaling or loading genes that are targets of the tumor immunosuppression as a payload to overcome the difficulties. Here, we discuss recent advances in TCR/CAR-T-cell engineering: various promising approaches to enhance the anti-tumor activity of adoptively transferred T cells in the TME for maximizing the efficacy and the safety of adoptive-cell therapy are now being tested in the clinic, especially targeting solid tumors.

Newly emerged immunogenic neoantigens in established tumors enable hosts to regain immunosurveillance in a T-cell-dependent manner.

Tumor neoantigens derived from genetic alterations are potential T-cell targets for antitumor immunity. However, tumors develop immune escape mechanisms including loss of preexisting neoantigens and/or impairment of T-cell responses during tumor development and progression. Here, we addressed whether newly emerged immunogenic neoantigens in established tumors enabled hosts to inhibit tumor growth via controlling immune escape mechanisms. Using a doxycycline-driven gene expression system, we generated murine MC38, CT26 (colorectal cancer) and B16 (melanoma) cell lines with inducible expression of model immunogenic neoantigens such as chicken ovalbumin and human NY-ESO-1. A model neoantigen was induced by doxycycline administration in the tumors once tumors became palpable. Tumor growth was significantly inhibited upon induction of the neoantigen and this inhibition was abrogated in nude mice lacking T cells and in mice deprived of CD8+ T cells, indicating the critical role of CD8+ T cells in tumor regression. In addition, PD-1/PD-L1 blockade further augmented the antitumor immune response, resulting in a far stronger inhibition of tumor growth. Accordingly, newly emerged tumor neoantigen-specific CD8+ T cells with enhanced effector functions were significantly increased in mice treated with PD-1/PD-L1 blockade. We propose that a newly emerged neoantigen is sufficient to inhibit tumor growth via preventing immune escape in a T-cell-dependent manner. Our results imply that induction of immunogenic tumor neoantigens is a novel strategy to overcome the resistance to immune checkpoint blockade therapy.

Novel anti-GARP antibody DS-1055a augments anti-tumor immunity by depleting highly suppressive GARP+ regulatory T cells.

Regulatory T (Treg) cells, which are essential for maintaining self-tolerance, inhibit anti-tumor immunity, consequently hindering protective cancer immunosurveillance, and hampering effective anti-tumor immune responses in tumor-bearing hosts. Here, we show that depletion of Treg cells via targeting glycoprotein A repetitions predominant (GARP) induces effective anti-tumor immune responses. GARP was specifically expressed by highly suppressive Treg cells in the tumor microenvironment (TME) of multiple cancer types in humans. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation. DS-1055a, a novel afucosylated anti-human GARP monoclonal antibody, efficiently depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FoxP3+CD4+ T cells in the TME and exhibited remarkable anti-tumor activity in humanized mice bearing HT-29 tumors. We propose that DS-1055a is a new Treg-cell-targeted cancer immunotherapy agent with augmentation of anti-tumor immunity.

A multicenter, open-label, single-arm phase I trial of neoadjuvant nivolumab monotherapy for resectable gastric cancer.

BACKGROUND: Nivolumab monotherapy has demonstrated superior efficacy in advanced unresectable gastric cancer (GC), but its impact on resectable GC remains unknown. This phase I study aimed to evaluate safety, feasibility, and potential biomarkers of neoadjuvant nivolumab monotherapy in resectable GC. METHODS: Untreated, resectable, cT2 or more advanced gastric adenocarcinomas with clinical stage I, II, or III were treated with two doses of nivolumab before gastrectomy. Patients were excluded if their tumors may be applicable to neoadjuvant chemotherapy. The primary endpoint was the incidence of adverse event (AE) categories of special interest. RESULTS: All of the 31 enrolled patients completed 2 doses of nivolumab monotherapy. While 30 (97%) patients underwent surgery with curative intent, 1 patient discontinued before the planned surgical intervention because of a newly emerging liver metastasis. Seven patients (23%) had nivolumab treatment-related AEs, and one patient had a treatment-related AE of grade 3-4. The incidences of treatment-related AE categories of special interest ranged from 0 to 6%. Notable surgical complications included two cases of grade 3 anastomotic leakage and two cases of pancreatic fistula. The major pathologic response (MPR) assessed by the independent pathology review committee was achieved in five (16%) patients, of which one patient had a pathologic complete response. The MPR was mostly observed in patients with positive PD-L1 expression, high microsatellite instability, and/or high tumor mutation burden. CONCLUSIONS: Neoadjuvant nivolumab monotherapy is feasible with an acceptable safety profile and induces a MPR in certain patients with resectable GC. (Registration: clinicaltrials.jp, JapicCTI-183895).

Study protocol for JCOG1807C (DEEP OCEAN): a interventional prospective trial to evaluate the efficacy and safety of durvalumab before and after operation or durvalumab as maintenance therapy after chemoradiotherapy against superior sulcus non-small cell lung cancer.

BACKGROUND: Superior sulcus tumours (SSTs) are relatively uncommon and one of the most intractable lung cancers among non-small cell lung cancer (NSCLC). We planned a multicenter, single-arm confirmatory trial of new multidisciplinary treatment using immune-checkpoint inhibitor. The aim is to evaluate the safety and efficacy of new multidisciplinary treatment with perioperative durvalumab after chemoradiotherapy (CRT). METHODS: The primary endpoint is 3-year overall survival. Patients receive induction CRT with sequential two courses of durvalumab, followed by surgical resection for resectable SST. The regimen for CRT is two courses of cisplatin and S-1, and concurrent radiotherapy (66 Gy/33 Fr). After surgery, 22 courses of post-operative durvalumab therapy are administered. For unresectable SST, an additional 22 courses of durvalumab are administered after induction durvalumab. RESULTS: In two cases as a safety cohort, the safety of intervention treatment up to 30 days after surgery was examined, and there were no special safety signals. Patient enrollment has now resumed in the main cohort. CONCLUSIONS: The results of this study may contribute to the establishment of a new standard of care for SST, which is an intractable NSCLC.