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In the context of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), despite a range of possibilities, the antigen specificities of tumor-infiltrating T cells and their relevance to control of is largely unknown. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver and tumor tissues from 46 HCC patients, we detected 91 different CD8 T cell populations specific for epitopes derived from HBV, tumor-associated and neoantigens (NeoAg), as well as disease-unrelated antigens. Parallel high-dimensional analysis delineated distinct tissue-resident memory T cells (TRM) populations among other highly diverse lymphocytes profiles observed in these compartments. Intratumoral and intrahepatic HBV-specific T cells were particularly enriched for three TRM phenotypes and the majority expressed relatively low levels of PD-1 receptor and TOX gene, inconsistent with reported terminal exhausted T cells (TEX) despite being clonally expanded within tumors. High frequencies of terminal TEX in these tumors was uncommon, whereas patients who had detectable and less exhausted tumor-infiltrating HBV- or NeoAg-specific CD8 TRM had superior long-term relapse-free survival. Thus, non-terminally-exhausted tumor-resident HBV-specific CD8 TRM show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.

Summary Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating T cells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8+ T cell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific T cells were enriched for two Trm cell subsets that were PD-1loTOXlo, despite being clonally expanded. High frequencies of intratumoral terminally exhausted T cells were uncommon. Patients with tumor-infiltrating HBV-specific CD8+ Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8+ Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.

Summary Growing evidence indicates a role for the gut microbiota in modulating anti-tumor treatment efficacy in human cancer. Here we study mucosa-associated invariant T (MAIT) cells to look for evidence of bacterial antigen recognition in human colon, lung, and kidney carcinomas. Using mass cytometry and single-cell mRNA sequencing, we identify a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3 and observe high expression of CD39 on MAIT cells from colorectal cancer (CRC) only, which we show in vitro to be expressed specifically after TCR stimulation. We further reveal that these cells are phenotypically and functionally exhausted. Sequencing data show high bacterial infiltration in CRC tumors and highlight an enriched species, Fusobacteria nucleatum, with capability to activate MAIT cells in a TCR-dependent way. Our results provide evidence of a MAIT cell response to microbial antigens in CRC and could pave the way for manipulating MAIT cells or the microbiome for cancer therapy., Graphical Abstract, Highlights Comprehensive profiling of tumor-infiltrating MAIT cells in colorectal cancer (CRC) Identification of a tumor-infiltrating MAIT cell subset expressing CD4 and Foxp3 TCR-driven CD39 expression is enriched in tumor-infiltrating MAIT cells CRC-related Fusobacterium nucleatum can activate MAIT cells in a TCR-dependent way, By investigating human colon, lung, and kidney carcinomas, Li et al. propose MAIT cells as part of the missing link between the gut microbiota and tumor biology. This work will open new avenues in cancer therapy by manipulating MAIT cells or MAIT-related bacterial ligands in the context of microbiota-influenced cancers.

Deep-immunophenotyping of the circulatory T cell compartment using mass cytometry and unsupervised clustering analysis, identifies phenotypic and functional differences in patients with atopic dermatitis (AD) and psoriasis, highlighting the stronger systemic component associated with AD.

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types1–4. Patients and tumours may respond unpredictably to immunotherapy partly owing to heterogeneity of the immune composition and phenotypic profiles of tumour-infiltrating lymphocytes (TILs) within individual tumours and between patients5,6. Although there is evidence that tumour-mutation-derived neoantigen-specific T cells play a role in tumour control2,4,7–10, in most cases the antigen specificities of phenotypically diverse tumour-infiltrating T cells are largely unknown. Here we show that human lung and colorectal cancer CD8+ TILs can not only be specific for tumour antigens (for example, neoantigens), but also recognize a wide range of epitopes unrelated to cancer (such as those from Epstein–Barr virus, human cytomegalovirus or influenza virus). We found that these bystander CD8+ TILs have diverse phenotypes that overlap with tumour-specific cells, but lack CD39 expression. In colorectal and lung tumours, the absence of CD39 in CD8+ TILs defines populations that lack hallmarks of chronic antigen stimulation at the tumour site, supporting their classification as bystanders. Expression of CD39 varied markedly between patients, with some patients having predominantly CD39− CD8+ TILs. Furthermore, frequencies of CD39 expression among CD8+ TILs correlated with several important clinical parameters, such as the mutation status of lung tumour epidermal growth factor receptors. Our results demonstrate that not all tumour-infiltrating T cells are specific for tumour antigens, and suggest that measuring CD39 expression could be a straightforward way to quantify or isolate bystander T cells. Human lung and colorectal tumours contain a population of tumour-infiltrating lymphocytes that are specific for tumour-unrelated antigens and, unlike tumour-antigen-specific tumour-infiltrating lymphocytes, do not express CD39.

Objectives Lymphoepithelioma‐like carcinoma (LELC) is an uncommon lung cancer, typically observed in young, non‐smoking Asian populations. LELC is associated with Epstein–Barr virus (EBV) infection of lung tumor cells of epithelial origin, suggesting a carcinogenic role of EBV as observed in nasopharyngeal carcinoma (NPC). Here, we studied the antigen specificity and phenotype of EBV‐specific CD8+ T cells in blood and tumor of one LELC patient positive for EBV infection in lung tumor cells. Methods Using multiplex MHC class I tetramers, mass cytometry and mRNA sequencing, we studied EBV‐specific CD8+ T cells at the transcriptomic and phenotypic levels in blood and tumor tissues of the LELC patient. Results Lymphoepithelioma‐like carcinoma lung tumor cells were positive for EBV infection. In both blood and tumor tissues, we detected two populations of EBV‐specific CD8+ T cells targeting the EBV lytic cycle proteins: BRLF1 and BMLF1. Transcriptomic analyses of these two populations in the tumor, which can be considered as tumor‐specific, revealed their distinct exhausted profile and polyclonal TCR repertoire. High‐dimensional phenotypical analysis revealed the distinct phenotype of these cells between blood and tumor tissues. In tumor tissue, EBV‐specific CD8+ TILs were phenotypically heterogeneous, but consistently expressed CD39. Unexpectedly, although the LELC tumor cells expressed abundant PD‐L1, these tumor‐specific CD8+ tumor‐infiltrating lymphocytes (TILs) mostly did not express PD‐1. Conclusion Epstein–Barr virus‐specific CD8+ TILs in EBV‐driven tumor are heterogeneous and partially lack PD‐1 expression, suggesting that anti‐PD1/PD‐L1 immunotherapy may not be an appropriate strategy for disinhibiting EBV‐specific cells in the treatment of LELC patients., We studied the antigen specificity and phenotype of CD8+ tumor‐infiltrating lymphocytes (TILs) in one LELC patient positive for EBV infection in lung tumor cells. Transcriptomic analyses of EBV‐specific CD8+ TILs reveal their distinct exhausted profiles and polyclonal TCR repertoire. Although the LELC tumor cells expressed abundant PD‐L1, EBV‐specific CD8+ TILs mostly did not express PD‐1, but express CD39.

Lymphoepithelioma-like carcinoma (LELC) is an uncommon lung cancer, typically observed in young, non-smoking Asian populations. LELC is associated with Epstein-Barr virus (EBV) infection of lung tumor cells of epithelial origin, suggesting a carcinogenic role of EBV as observed in nasopharyngeal carcinoma (NPC). Here, we studied the antigen specificity and phenotype of CD8+tumor infiltrating lymphocytes (TILs) in one LELC patient positive for EBV infection in lung tumor cells. Using MHC class I tetramers, we detected two populations of EBV-specific CD8+TILs, which can be considered as tumor-specific CD8+T cells, in the tumor of this patient. Transcriptomic analyses of these two populations reveal their distinct exhausted profiles and polyclonal TCR repertoire. High dimensional analyses at single cell level using mass cytometry showed showed that populations of tumor specific CD8+TILs are phenotypically heterogeneous, although they consistently express CD39. Unexpectedly, although the LELC tumor cells expressed abundant PD-L1, these tumor-specific CD8+TILs mostly did not express PD-1, suggesting that anti-PD1/PD-L1 immunotherapy may not be an appropriate strategy for disinhibiting EBV-specific cells for the treatment of LELC patients. These results might also help to explain low rates of checkpoint blockade immunotherapy response for NPC, despite the antigenicity of EBV for both tumor types.

Malaria remains a significant infectious disease with even artemisinin-based therapies now facing resistance in the field. Development of new therapies is urgently needed, either by finding new compounds with unique modes of action, or by reversing resistance towards known drugs with 'chemosensitizers' or 'chemoreversal' agents (CRA). Concerning the latter, we have focused on the resistance mechanisms developed against chloroquine (CQ). We have synthesized a series of compounds related to previously identified CRAs, and found promising novel compounds. These compounds show encouraging results in a coumarin labeled chloroquine uptake assay, exhibiting a dose response in resensitising parasites to the antimalarial effects of chloroquine. Selected compounds show consistent potency across a panel of chloroquine and artemisinin sensitive and resistant parasites, and a wide therapeutic window. This data supports further study of CRAs in the treatment of malaria and, ultimately, their use in chloroquine-based combination therapies.

Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge. Reversal of acquired resistance can be achieved using agents that resensitize resistant parasites to a previously efficacious therapy. Building on our initial work describing novel chemoreversal agents (CRAs) that resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA scaffold to chloroquine produces hybrid compounds with restored potency toward a range of resistant malaria parasites. A preferred compound, compound 35, showed broad activity and good potency against seven strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane permeability, and in vitro toxicity in a hepatocyte line and a cardiomyocyte line indicates that compound 35 has a good therapeutic window and favorable drug-like properties. This study provides initial support for CQ-CRA hybrid compounds as a potential treatment for resistant malaria.