Soluble immune checkpoint factors reflect exhaustion of antitumor immunity and response to PD-1 blockade.

BACKGROUNDPrecise stratification of patients with non-small cell lung cancer (NSCLC) is needed for appropriate application of PD-1/PD-L1 blockade therapy.METHODSWe measured soluble forms of the immune-checkpoint molecules PD-L1, PD-1, and CTLA-4 in plasma of patients with advanced NSCLC before PD-1/PD-L1 blockade. A prospective biomarker-finding trial (cohort A) included 50 previously treated patients who received nivolumab. A retrospective observational study was performed for patients treated with any PD-1/PD-L1 blockade therapy (cohorts B and C), cytotoxic chemotherapy (cohort D), or targeted therapy (cohort E). Plasma samples from all patients were assayed for soluble immune-checkpoint molecules with a highly sensitive chemiluminescence-based assay.RESULTSNonresponsiveness to PD-1/PD-L1 blockade therapy was associated with higher concentrations of these soluble immune factors among patients with immune-reactive (hot) tumors. Such an association was not apparent for patients treated with cytotoxic chemotherapy or targeted therapy. Integrative analysis of tumor size, PD-L1 expression in tumor tissue (tPD-L1), and gene expression in tumor tissue and peripheral CD8+ T cells revealed that high concentrations of the 3 soluble immune factors were associated with hyper or terminal exhaustion of antitumor immunity. The combination of soluble PD-L1 (sPD-L1) and sCTLA-4 efficiently discriminated responsiveness to PD-1/PD-L1 blockade among patients with immune-reactive tumors.CONCLUSIONCombinations of soluble immune factors might be able to identify patients unlikely to respond to PD-1/PD-L1 blockade as a result of terminal exhaustion of antitumor immunity. Our data suggest that such a combination better predicts, along with tPD-L1, for the response of patients with NSCLC.TRIAL REGISTRATIONUMIN000019674.FUNDINGThis study was funded by Ono Pharmaceutical Co. Ltd. and Sysmex Corporation.

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.

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.

Hepatitis C virus NS5B triggers an MDA5-mediated innate immune response by producing dsRNA without the replication of viral genomes.

During the replication of viral genomes, RNA viruses produce double-stranded RNA (dsRNA), through the activity of their RNA-dependent RNA polymerases (RdRps) as viral replication intermediates. Recognition of viral dsRNA by host pattern recognition receptors - such as retinoic acid-induced gene-I (RIG-I)-like receptors and Toll-like receptor 3 - triggers the production of interferon (IFN)-ß via the activation of IFN regulatory factor (IRF)-3. It has been proposed that, during the replication of viral genomes, each of RIG-I and melanoma differentiation-associated gene 5 (MDA5) form homodimers for the efficient activation of a downstream signalling pathway in host cells. We previously reported that, in the non-neoplastic human hepatocyte line PH5CH8, the RdRp NS5B derived from hepatitis C virus (HCV) could induce IFN-ß expression by its RdRp activity without the actual replication of viral genomes. However, the exact mechanism by which HCV NS5B produced IFN-ß remained unknown. In the present study, we first showed that NS5B derived from another Flaviviridae family member, GB virus B (GBV-B), also possessed the ability to induce IFN-ß in PH5CH8 cells. Similarly, HCV NS5B, but not its G317V mutant, which lacks RdRp activity, induced the dimerization of MDA5 and subsequently the activation of IRF-3. Interestingly, immunofluorescence analysis showed that HCV NS5B produced dsRNA. Like HCV NS5B, GBV-B NS5B also triggered the production of dsRNA and subsequently the dimerization of MDA5. Taken together, our results show that HCV NS5B triggers an MDA5-mediated innate immune response by producing dsRNA without the replication of viral genomes in human hepatocytes.

Immunologic Significance of CD80/CD86 or Major Histocompatibility Complex-II Expression in Thymic Epithelial Tumors.

Introduction: Unresectable or recurrent thymic epithelial tumors (TETs) have a poor prognosis, and treatment options are limited. This study aimed to investigate the immunologic significance of CD80/CD86 or major histocompatibility complex class II (MHC-II) expression in TETs, as potential predictive biomarkers for immune checkpoint inhibitors (ICIs). Methods: We analyzed CD80, CD86, MHC class I (MHC-I), and MHC-II expression in TETs using immunohistochemistry and investigated their association with T-cell infiltration or ICI efficacy. In addition, we generated CD80- or MHC-II-expressing mouse tumors, evaluated the effects of ICIs, and analyzed tumor-infiltrating lymphocytes. We also performed tumor-rechallenge experiments in vivo. Results: We found that approximately 50% and 30% of TETs had high expression of CD80/CD86 and MHC-II in tumor cells, respectively, and that this expression was related to T-cell infiltration in clinical samples. In mouse models, both CD80 and MHC-II increase the effects of ICIs. In addition, senescent T cells and long-lived memory precursor effector T cells were significantly decreased and increased, respectively, in tumor-infiltrating lymphocytes from CD80-expressing tumors, and rechallenged tumors were completely rejected after the initial eradication of CD80-expressing tumors by programmed cell death protein 1 blockade. Indeed, patients with CD80-high thymic carcinoma had longer progression-free survival with anti-programmed cell death protein 1 monoclonal antibody. Conclusions: Half of the TETs had high expression of CD80/CD86 or MHC-II with high T-cell infiltration. These molecules could potentially increase the effects of ICIs, particularly inducing a durable response. CD80/CD86 and MHC-II can be predictive biomarkers of ICIs in TETs, promoting the development of drugs for such TETs.

Combination therapy with hydrogen peroxide and irradiation promotes an abscopal effect in mouse models.

Hydrogen peroxide (H2 O2 ) induces oxidative stress and cytotoxicity, and can be used for treating cancers in combination with radiotherapy. A product comprising H2 O2 and sodium hyaluronate has been developed as a radiosensitizer. However, the effects of H2 O2 on antitumor immunity remain unclear. To investigate the effects of H2 O2 , especially the abscopal effect when combined with radiotherapy (RT), we implanted murine tumor cells simultaneously in two locations in mouse models: the hind limb and back. H2 O2 mixed with sodium hyaluronate was injected intratumorally, followed by irradiation only at the hind limb lesion. No treatment was administered to the back lesion. The H2 O2 /RT combination significantly reduced tumor growth at the noninjected/nonirradiated site in the back lesion, whereas H2 O2 or RT individually did not reduce tumor growth. Flow cytometric analyses of the tumor-draining lymph nodes in the injected/irradiated areas showed that the number of dendritic cells increased significantly with maturation in the H2 O2 /RT combination group. In addition, analyses of tumor-infiltrating lymphocytes showed that the number of CD8+ (cluster of differentiation 8) T cells and the frequency of IFN-γ+ (interferon gamma) CD8+ T cells were higher in the noninjected/nonirradiated tumors in the H2 O2 /RT group compared to those in the other groups. PD-1 (programmed death receptor 1) blockade further increased the antitumor effect against noninjected/nonirradiated tumors in the H2 O2 /RT group. Intratumoral injection of H2 O2 combined with RT therefore induces an abscopal effect by activating antitumor immunity, which can be further enhanced by PD-1 blockade. These findings promote the development of H2 O2 /RT therapy combined with cancer immunotherapies, even for advanced cancers.

High Expression of MHC Class I Overcomes Cancer Immunotherapy Resistance Due to IFNγ Signaling Pathway Defects.

IFNγ signaling pathway defects are well-known mechanisms of resistance to immune checkpoint inhibitors. However, conflicting data have been reported, and the detailed mechanisms remain unclear. In this study, we have demonstrated that resistance to immune checkpoint inhibitors owing to IFNγ signaling pathway defects may be primarily caused by reduced MHC-I expression rather than by the loss of inhibitory effects on cellular proliferation or decreased chemokine production. In particular, we found that chemokines that recruit effector T cells were mainly produced by immune cells rather than cancer cells in the tumor microenvironment of a mouse model, with defects in IFNγ signaling pathways. Furthermore, we found a response to immune checkpoint inhibitors in a patient with JAK-negative head and neck squamous cell carcinoma whose HLA-I expression level was maintained. In addition, CRISPR screening to identify molecules associated with elevated MHC-I expression independent of IFNγ signaling pathways demonstrated that guanine nucleotide-binding protein subunit gamma 4 (GNG4) maintained MHC-I expression via the NF-κB signaling pathway. Our results indicate that patients with IFNγ signaling pathway defects are not always resistant to immune checkpoint inhibitors and highlight the importance of MHC-I expression among the pathways and the possibility of NF-κB-targeted therapies to overcome such resistance. See related Spotlight by Haugh and Daud, p. 864.

Activated CTLA-4-independent immunosuppression of Treg cells disturbs CTLA-4 blockade-mediated antitumor immunity.

Combination therapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) monoclonal antibodies (mAbs) has dramatically improved the prognosis of patients with multiple types of cancer, including renal cell carcinoma (RCC). However, more than half of RCC patients fail to respond to this therapy. Regulatory T cells (Treg cells) are a subset of highly immunosuppressive CD4+ T cells that promote the immune escape of tumors by suppressing effector T cells in the tumor microenvironment (TME) through various mechanisms. CTLA-4 is constitutively expressed in Treg cells and is regarded as a key molecule for Treg-cell-mediated immunosuppressive functions, suppressing antigen-presenting cells by binding to CD80/CD86. Reducing Treg cells in the TME with an anti-CTLA-4 mAb with antibody-dependent cellular cytotoxicity (ADCC) activity is considered an essential mechanism to achieve tumor regression. In contrast, we demonstrated that CTLA-4 blockade without ADCC activity enhanced CD28 costimulatory signaling pathways in Treg cells and promoted Treg-cell proliferation in mouse models. CTLA-4 blockade also augmented CTLA-4-independent immunosuppressive functions, including cytokine production, leading to insufficient antitumor effects. Similar results were also observed in human peripheral blood lymphocytes and tumor-infiltrating lymphocytes from patients with RCC. Our findings highlight the importance of Treg-cell depletion to achieve tumor regression in response to CTLA-4 blockade therapies.

Somatic mutations can induce a noninflamed tumour microenvironment via their original gene functions, despite deriving neoantigens.

BACKGROUND: Identifying biomarkers to predict immune checkpoint inhibitor (ICI) efficacy is warranted. Considering that somatic mutation-derived neoantigens induce strong immune responses, patients with a high tumour mutational burden reportedly tend to respond to ICIs. However, there are several conflicting data. Therefore, we focused on the original function of neoantigenic mutations and their impact on the tumour microenvironment (TME). METHODS: We evaluated 88 high-frequency microsatellite instability (MSI-H) colorectal cancers and analysed the function of the identified neoantigenic mutations and their influence on programmed cell death 1 (PD-1) blockade efficacy. The results were validated using The Cancer Genome Atlas (TCGA) datasets. RESULTS: We identified frameshift mutations in RNF43 as a common neoantigenic gene mutation in MSI-H tumours. However, loss-of-function RNF43 mutations induced noninflamed TME by activating the WNT/ß-catenin signalling pathway. In addition, loss of RNF43 function induced resistance to PD-1 blockade even in neoantigen-rich tumours. TCGA dataset analyses demonstrated that passenger rather than driver gene mutations were related to the inflamed TME in diverse cancer types. CONCLUSIONS: We propose a novel concept of "paradoxical neoantigenic mutations" that can induce noninflamed TME through their original gene functions, despite deriving neoantigens, suggesting the significance of qualities as well as quantities in neoantigenic mutations.

Resistance to immune checkpoint inhibitors and the tumor microenvironment.

Immune checkpoint inhibitors (ICIs) have contributed significantly to the treatment of various types of cancer, including skin cancer. However, not all patients respond; some patients do not respond at all (primary resistance), while others experience recurrence after the initial response (acquired resistance). Therefore, overcoming ICI resistance is an urgent priority. Numerous ICI resistance mechanisms have been reported. They are seemingly quite complex, varying from patient to patient. However, most involve T-cell activation processes, especially in the tumor microenvironment (TME). ICIs exert their effects in the TME by reactivating suppressed T cells through inhibition of immune checkpoint molecules, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1). Thus, this review focuses on the resistance mechanisms based on the T-cell activation process. Here, we classify the main mechanisms of ICI resistance into three categories based on (1) antigen recognition, (2) T-cell migration and infiltration, and (3) effector functions of T cells. By identifying and understanding these resistance mechanisms individually, including unknown mechanisms, we seek to contribute to the development of novel treatments to overcome ICI resistance.

[Clonal Evolution in the Tumor Microenvironment].

Cancer immunotherapy has shown efficacy in many types of cancer. However, there are many challenges, such as the difficulty in predicting therapeutic efficacy. In the tumor microenvironment, tumor cells evolve to escape the anti-tumor immune response and have capacity of proliferation, whereas immune cells also evolve along with tumor cells. Elucidating the detailed clonal evolution is helpful for the development of biomarkers for prediction of therapeutic effects and novel therapies. To elucidate clonal evolution in the tumor microenvironment, analyses at the single-cell level, rather than in bulks, is necessary for heterogeneous and highly diverse cell populations. Recently, single-cell sequencing can be used to analyze comprehensive gene expression, or it is possible to focus on specific regions, such as T-cell or B-cell receptor sequences. In addition, technologies have been developed that allow spatial analyses by a single-cell level while preserving tissue location information. Recently, new findings have been clarified using pre- and post-treatment samples from same patients to analyze the clonal progression of the tumor cells themselves and immune cells based on sequential changes in the tumor microenvironment.

Mechanisms of resistance to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) are effective for various types of cancer, and their application has led to paradigm shifts in cancer treatment. While many patients can obtain clinical benefits from ICI treatment, a large number of patients are primarily resistant to such treatment or acquire resistance after an initial response. Thus, elucidating the resistance mechanisms is warranted to improve the clinical outcomes of ICI treatment. ICIs exert their antitumor effects by activating T cells in the tumor microenvironment. There are various resistance mechanisms, such as insufficient antigen recognition by T cells, impaired T-cell migration and/or infiltration, and reduced T-cell cytotoxicity, most of which are related to the T-cell activation process. Thus, we classify them into three main mechanisms: resistance mechanisms related to antigen recognition, T-cell migration and/or infiltration, and effector functions of T cells. In this review, we summarize these mechanisms of resistance to ICIs related to the T-cell activation process and progress in the development of novel therapies that can overcome resistance.

A variety of 'exhausted' T cells in the tumor microenvironment.

In T-cell biology, 'exhaustion' was initially described as a hyporesponsive state in CD8+ T cells during chronic infections. Recently, exhaustion has been recognized as a T-cell dysfunctional state in the tumor microenvironment (TME). The term 'exhaustion' is used mainly to refer to effector T cells with a reduced capacity to secrete cytokines and an increased expression of inhibitory receptors. The up-regulation of exhaustion-related inhibitory receptors, including programmed cell death protein 1 (PD-1), in such T cells has been associated with the development of tumors, prompting the development of immune checkpoint inhibitors. In addition to CD8+ T cells, CD4+ T cells, including the regulatory T (Treg) cell subset, perform a wide variety of functions within the adaptive immune system. Up-regulation of the same inhibitory receptors that are associated with CD8+ T-cell exhaustion has also been identified in CD4+ T cells in chronic infections and cancers, suggesting a similar CD4+ T-cell exhaustion phenotype. For instance, high expression of PD-1 has been observed in Treg cells in the TME, and such Treg cells can play an important role in the resistance to PD-1 blockade therapies. Furthermore, recent progress in single-cell RNA sequencing has shown that CD4+ T cells with cytotoxic activity are also vulnerable to exhaustion. In this review, we will discuss novel insights into various exhausted T-cell subsets, which could reveal novel therapeutic targets and strategies to induce a robust anti-tumor immune response.

PD-1 blockade therapy promotes infiltration of tumor-attacking exhausted T cell clonotypes.

PD-1 blockade exerts clinical efficacy against various types of cancer by reinvigorating T cells that directly attack tumor cells (tumor-specific T cells) in the tumor microenvironment (TME), and tumor-infiltrating lymphocytes (TILs) also comprise nonspecific bystander T cells. Here, using single-cell sequencing, we show that TILs include skewed T cell clonotypes, which are characterized by exhaustion (Tex) or nonexhaustion signatures (Tnon-ex). Among skewed clonotypes, those in the Tex, but not those in the Tnon-ex, cluster respond to autologous tumor cell lines. After PD-1 blockade, non-preexisting tumor-specific clonotypes in the Tex cluster appear in the TME. Tumor-draining lymph nodes (TDLNs) without metastasis harbor a considerable number of such clonotypes, whereas these clonotypes are rarely detected in peripheral blood. We propose that tumor-infiltrating skewed T cell clonotypes with an exhausted phenotype directly attack tumor cells and that PD-1 blockade can promote infiltration of such Tex clonotypes, mainly from TDLNs.

Preoperative Chemoradiotherapy plus Nivolumab before Surgery in Patients with Microsatellite Stable and Microsatellite Instability-High Locally Advanced Rectal Cancer.

PURPOSE: Preoperative chemoradiotherapy (CRT) and surgical resection are the standard treatment for locally advanced rectal cancer (LARC). Combining immune checkpoint inhibitors with radiation suggests a promising approach for enhancing efficacy. We investigated the efficacy of CRT followed by nivolumab and surgery in patients with LARC. PATIENTS AND METHODS: In phase I, we investigated the feasibility of sequentially combined CRT, 5 cycles of nivolumab, and radical surgery. In phase II, patients with microsatellite stable (MSS) and microsatellite instability-high (MSI-H) LARC were evaluated. RESULTS: Three patients in phase I received full courses of CRT and nivolumab without dose modification; the schedule was recommended for phase II. A pathologic complete response (pCR) was centrally confirmed in 30% [11/37; 90% confidence interval (CI), 18%-44%] and 60% (3/5) of the MSS and exploratory MSI-H cohorts, respectively. While immune-related severe adverse events were observed in 3 patients, no treatment-related deaths were observed. In 38 patients with MSS who underwent surgery, pCR rates of 75% (6/8) and 17% (5/30; P = 0.004, Fisher exact test) were observed in those with programmed cell death ligand 1 (PD-L1) tumor proportion score ≥1% and <1%, respectively; IHC staining was performed using pre-CRT samples. In 24 patients with MSS, pre-CRT samples were analyzed by flow cytometry; pCR rates of 78% (7/9) and 13% (2/15; P = 0.003, Fisher exact test) were observed for CD8+ T cell/effector regulatory T cell (CD8/eTreg) ratios of ≥2.5 and <2.5, respectively, in tumor-infiltrating lymphocytes. CONCLUSIONS: CRT followed by consolidation nivolumab could increase pCR. PD-L1 expression and an elevated CD8/eTreg ratio were positive predictors in patients with MSS LARC.

Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments.

The balance of programmed death-1 (PD-1)-expressing CD8+ T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector T cells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors. Under low-glucose environments via glucose consumption by tumor cells, Treg cells actively absorbed lactic acid (LA) through monocarboxylate transporter 1 (MCT1), promoting NFAT1 translocation into the nucleus, thereby enhancing the expression of PD-1, whereas PD-1 expression by effector T cells was dampened. PD-1 blockade invigorated the PD-1-expressing Treg cells, resulting in treatment failure. We propose that LA in the highly glycolytic TME is an active checkpoint for the function of Treg cells in the TME via upregulation of PD-1 expression.

Mixed Response to Cancer Immunotherapy is Driven by Intratumor Heterogeneity and Differential Interlesion Immune Infiltration.

Some patients experience mixed response to immunotherapy, whose biological mechanisms and clinical impact have been obscure. We obtained two tumor samples from lymph node (LN) metastatic lesions in a same patient. Whole exome sequencing for the both tumors and single-cell sequencing for the both tumor-infiltrating lymphocytes (TIL) demonstrated a significant difference in tumor clonality and TILs' characteristics, especially exhausted T-cell clonotypes, although a close relationship between the tumor cell and T-cell clones were observed as a response of an overlapped exhausted T-cell clone to an overlapped neoantigen. To mimic the clinical setting, we generated a mouse model of several clones from a same tumor cell line. Similarly, differential tumor clones harbored distinct TILs, and one responded to programmed cell death protein 1 (PD-1) blockade but the other did not in this model. We further conducted cohort study (n = 503) treated with PD-1 blockade monotherapies to investigate the outcome of mixed response. Patients with mixed responses to PD-1 blockade had a poor prognosis in our cohort. Particularly, there were significant differences in both tumor and T-cell clones between the primary and LN lesions in a patient who experienced tumor response to anti-PD-1 mAb followed by disease progression in only LN metastasis. Our results underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome. Significance: Several patients experience mixed responses to immunotherapies, but the biological mechanisms and clinical significance remain unclear. Our results from clinical and mouse studies underscore that intertumoral heterogeneity alters characteristics of TILs even in the same patient, leading to mixed response to immunotherapy and significant difference in the outcome.