Search

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4+ T cells, but mechanistic insights into how these CD4+ T cells trigger and perpetuate colitis have remained elusive. Here, using single-cell transcriptomic analysis, we found that several CD4+ T-cell subsets are present in the intestines of colitic mice, including an interferon (IFN)-γ-producing subset. In vivo challenge of primed mice with Red 40 promoted rapid activation of CD4+ T cells and caused marked intestinal epithelial cell (IEC) apoptosis that was attenuated by depletion of CD4+ cells and blockade of IFN-γ. Ex vivo experiments showed that intestinal CD4+ T cells from colitic mice directly promoted apoptosis of IECs and intestinal enteroids. CD4+ T cell-mediated cytotoxicity was contact-dependent and required FasL, which promoted caspase-dependent cell death in target IECs. Genetic ablation of IFN-γ constrained IL-23- and Red 40-induced colitis development, and blockade of IFN-γ inhibited epithelial cell death in vivo. These results advance the understanding of the mechanisms regulating colitis development caused by IL-23 and food colorants and identify IFN-γ+ cytotoxic CD4+ T cells as a new potential therapeutic target for colitis.

Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder resulting from mutations in an endolysosomal cholesterol transporter, NPC1. Despite typically presenting with pronounced neurological manifestations, NP-C1 also resembles long-term congenital immunodeficiencies that arise from impairment of cytotoxic T lymphocyte (CTL) effector function. CTLs kill their targets through exocytosis of the contents of lysosome-like secretory cytotoxic granules (CGs) that store and ultimately release the essential pore-forming protein perforin and proapoptotic serine proteases, granzymes, into the synapse formed between the CTL and target cell. We discovered that NPC1 deficiency increases CG lipid burden, impairs autophagic flux through stalled trafficking of the transcription factor EB (TFEB), and dramatically reduces CTL cytotoxicity. Using a variety of immunological and cell biological techniques, we found that the cytotoxic defect arises specifically from impaired perforin pore formation. We demonstrated defects of CTL function of varying severity in patients with NP-C1, with the greatest losses of function associated with the most florid and/or earliest disease presentations. Remarkably, perforin function and CTL cytotoxicity were restored in vitro by promoting lipid clearance with therapeutic 2-hydroxypropyl-β-cyclodextrin; however, restoration of autophagy through TFEB overexpression was ineffective. Overall, our study revealed that NPC1 deficiency has a deleterious impact on CTL (but not natural killer cell) cytotoxicity that, in the long term, may predispose patients with NP-C1 to atypical infections and impaired immune surveillance more generally.

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.

Familial forms of the severe immunoregulatory disease Haemophagocytic Lymphohistiocytosis (HLH) arise from bi-allelic mutations in the PRF1, UNC13D, STXBP2 and STX11 genes. Early and accurate diagnosis of the disease is important to determine the most appropriate treatment option, including potentially curative stem cell transplantation. The diagnosis of familial HLH (FHL) is traditionally based on finding bi-allelic mutations in patients with HLH symptoms and reduced natural killer (NK) cell cytotoxicity. However, patients often have a low NK cell count or receive immunosuppressive therapies that may render the NK cytotoxicity assay unreliable. Furthermore, to fully understand the nature of a disease it is critical to directly assess the effect of mutations on cellular function; this will help to avoid instances where carriers of innocuous mutations may be recommended for invasive procedures including transplantation. To overcome this diagnostic problem, we have developed a rapid and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11 and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously replacing them with their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of otherwise cryptic cases of FHL/HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. This novel approach provides valuable new information to enable more accurate diagnosis and treatment of HLH/FHL patients who inherit mutations of undetermined pathogenicity.

Chimeric antigen receptor (CAR)-T-cell therapy is a promising approach for the treatment of childhood cancers, particularly high-risk tumors that fail to respond to standard therapies. CAR-T cells have been highly successful in treating some types of hematological malignancies. However, CAR-T cells targeting solid cancers have had limited success so far for multiple reasons, including their poor long-term persistence and proliferation. Evidence is emerging to show that maintaining CAR-T cells in an early, less-differentiated state in vitro results in superior persistence, proliferation, and antitumor effects in vivo. Children are ideal candidates for receiving less-differentiated CAR-T cells, because their peripheral T-cell pool primarily comprises naïve cells that could readily be harvested in large numbers to generate early-phenotype CAR-T cells. Although several studies have reported different approaches to successfully generate early CAR-T cells, there are only a few clinical trials testing these in adult patients. No trials are currently testing early CAR-T cells in children. Here, we summarize the different strategies used to maintain CAR-T cells in an early phenotypic stage and present evidence suggesting that this approach may be particularly relevant to treating childhood cancers.

SARS-CoV-2 is a single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Given its acute and often self-limiting course, components of the innate immune system are likely central in controlling virus replication thereby determining clinical outcome. Natural killer (NK) cells are innate lymphocytes with notable activity against a broad range of viruses, including RNA viruses1,2. NK cell function may be altered during COVID-19 despite increased representation of NK cells with an activated and ‘adaptive’ phenotype3,4. Here we show that viral load decline in COVID-19 correlates with NK cell status and that NK cells can control SARS-CoV-2 replication by recognizing infected target cells. In severe COVID-19, NK cells show remarkable defects in virus control, cytokine production and cell-mediated cytotoxicity despite high expression of cytotoxic effector molecules. Single-cell RNA-sequencing (scRNA-seq) of NK cells along the time course of the entire COVID-19 disease spectrum reveals a unique gene expression signature. Transcriptional networks of interferon-driven NK cell activation are superimposed by a dominant TGFβ response signature with reduced expression of genes related to cell-cell adhesion, granule exocytosis and cell-mediated cytotoxicity. In severe COVID-19, serum levels of TGFβ peak during the first 2 weeks of infection, and serum obtained from these patients profoundly inhibits NK cell function in a TGFβ-dependent manner. Our data reveal that untimely production of TGFβ is a hallmark of severe COVID-19 and may inhibit NK cell function and early virus control.

Background Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. Methods We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. Results A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. Conclusions Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

This study explored the novel immune checkpoint poliovirus receptor- related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced naural killer (NK)-cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK-cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK-cell PVRIG expression levels were not increased. In order to understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK-cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumor cell recognition, cytokines (interleukin 2 [IL-2] and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti-PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIG-PVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.

For most researchers, the time they spend as a postdoc stands out as one of challenge, but also enormous personal and professional growth. This Words of Advice is intended to guide the choice of postdoctoral position to help make the venture a success and to launch the first chapter of a happy and fulfilling professional life.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11–deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

Cancer tissue is not homogenous, and individual metastases at different anatomical locations can differ from the primary tumor and from one another in both their morphology and cellular composition, even within an individual patient. Tumors are composed of cancer cells and a range of other cell types, which, together with a variety of secreted molecules, collectively comprise the tumor microenvironment (TME). Cells of the TME can communicate with each other and with distant tissues in a form of molecular cross-talk to influence their growth and function. Cross-talk between cancer cells and local immune cells is well described and can lead to the induction of local immunosuppression. Recently, it has become apparent that tumors located remotely from each other, can engage in cross-talk that can influence their responsiveness to various therapies, including immunotherapy. In this article, we review studies that describe how tumors systemically communicate with distant tissues through motile cells, extracellular vesicles, and secreted molecules that can affect their function. In addition, we summarize evidence from mouse studies and the clinic that indicate an ability of some tumors to influence the progression and therapeutic responses of other tumors in different anatomical locations.

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here, we present a 4-Å resolution cryo–electron microscopy structure of the perforin pore, revealing previously unidentified inter- and intramolecular interactions stabilizing the assembly. During pore formation, the helix-turn-helix motif moves away from the bend in the central β sheet to form an intermolecular contact. Cryo–electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerization and membrane insertion, and explain how several pathogenic mutations affect function.

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4

Despite aggressive surgery, chemotherapy, and radiotherapy, survival of children and adolescents and young adults (AYAs) with sarcoma has not improved significantly in the past four decades. Immune checkpoint inhibitors (ICIs) are an exciting type of immunotherapy that offer new opportunities for the treatment of paediatric and AYA sarcomas. However, to date, most children do not derive a benefit from this type of treatment as a monotherapy. The immunosuppressive tumour microenvironment is a major barrier limiting their efficacy. Combinations of ICIs, such as anti-PD-1 therapy, with targeted molecular therapies that have immunomodulatory properties may be the key to breaking through immunosuppressive barriers and improving patient outcomes. Preclinical studies have indicated that several receptor tyrosine kinase inhibitors (RTKi) can alter the tumour microenvironment and boost the efficacy of anti-PD-1 therapy. A number of these combinations have entered phase-1/2 clinical trials, mostly in adults, and in most instances have shown efficacy with manageable side-effects. In this review, we discuss the status of ICI therapy in paediatric and AYA sarcomas and the rationale for co-treatment with RTKis. We highlight new opportunities for the integration of ICI therapy with RTK inhibitors, to improve outcomes for children with sarcoma.

Patients with refractory relapsed multiple myeloma (RRMM) respond to Elotuzumab (Elo) and lenalidomide (Len) combination treatment. The mechanisms underlying this observation are not fully understood. Furthermore, predictive biomarkers of response have not been revealed to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human NK cells to show that Elo and Len treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that PBMCs from a subset of RRMM patients were effective killers of OPM2 myeloma cells when treated with Elo and Len, and this was associated with significantly increased expression of CD54 expression on OPM2 cells. Furthermore, Elo and Len induced OPM2 cell killing and increased OPM2 CD54 expression was dependent on both monocytes and NK cells, and was not mediated by soluble factors alone. At the transcript level, Elo and Len treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that MM patients require Elo and Len-mediated upregulation of CD54 on the autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumour response. Further, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to Elo and Len treatment.

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with pro-apoptotic granzymes, to bind to the plasma membrane of the target cell where it oligomerises and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here we present a 4 Å resolution cryo-EM structure of the perforin pore, revealing new inter- and intra-molecular interactions stabilising the pore. During assembly and pore formation, the helix-turn-helix motif at the bend in the central β-sheet moves away from the bend to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerisation and membrane insertion, and explain how several pathogenic mutations affect function.

The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses., Cas13b can be harnessed to target and degrade RNA transcripts inside a cellular environment. Here the authors reprogram Cas13b to target SARSCoV-2 transcripts in infected mammalian cells and reveal its resilience to variants thanks to single mismatch tolerance.

Immune checkpoint inhibitors and related molecules can achieve tumour regression, and even prolonged survival, for a subset of cancer patients with an otherwise dire prognosis. However, it remains unclear why some patients respond to immunotherapy and others do not. PET imaging has the potential to characterise the spatial and temporal heterogeneity of both immunotherapy target molecules and the tumor immune microenvironment, suggesting a tantalising vision of personally-adapted immunomodulatory treatment regimens. Personalised combinations of immunotherapy with local therapies and other systemic therapies, would be informed by immune imaging and subsequently modified in accordance with therapeutically induced immune environmental changes. An ideal PET imaging biomarker would facilitate the choice of initial therapy and would permit sequential imaging in time-frames that could provide actionable information to guide subsequent therapy. Such imaging should provide either prognostic or predictive measures of responsiveness relevant to key immunotherapy types but, most importantly, guide key decisions on initiation, continuation, change or cessation of treatment to reduce the cost and morbidity of treatment while enhancing survival outcomes. We survey the current literature, focusing on clinically relevant immune checkpoint immunotherapies, for which novel PET tracers are being developed, and discuss what steps are needed to make this vision a reality.

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16, the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.

We have previously reported that ICOS-Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4+ CD25+ Foxp3+ T cells and specific xenograft prolongation. In the present study we isolated and purified CD4+ CD25+ T cells from ICOS-Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4+ CD25+ T cells isolated from xenografts secreting ICOS-Ig were analysed by flow cytometry and gene expression by real-time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre-existing Foxp3+ Treg, IL-10, perforin and granzyme B. CD4+ CD25+ Foxp3+ T cells isolated from xenografts secreting ICOS-Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

International audience; Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

A new technology, SUGAR-seq, enables simultaneous detection of surface glycans, epitopes, transcripts, and TCR repertoire., Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

Fully functional, tumor-specific, SLAMF7-expressing cytotoxic CD4 T cells directly mediate immunity against human cancer., CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies.

BackgroundPatients with BRAF-mutant and wild-type melanoma have different response rates to immune checkpoint blockade therapy. However, the reasons for this remain unknown. To address this issue, we investigated the precise immune composition resulting from BRAF mutation in treatment-naive melanoma to determine whether this may be a driver for different response to immunotherapy.MethodsIn this study, we characterized the treatment-naive immune context in patients with BRAF-mutant and BRAF wild-type (BRAF-wt) melanoma using data from single-cell RNA sequencing, bulk RNA sequencing, flow cytometry and immunohistochemistry (IHC).ResultsIn single-cell data, BRAF-mutant melanoma displayed a significantly reduced infiltration of CD8+ T cells and macrophages but also increased B cells, natural killer (NK) cells and NKT cells. We then validated this finding using bulk RNA-seq data from the skin cutaneous melanoma cohort in The Cancer Genome Atlas and deconvoluted the data using seven different algorithms. Interestingly, BRAF-mutant tumors had more CD4+ T cells than BRAF-wt samples in both primary and metastatic cohorts. In the metastatic cohort, BRAF-mutant melanoma demonstrated more B cells but less CD8+ T cell infiltration when compared with BRAF-wt samples. In addition, we further investigated the immune cell infiltrate using flow cytometry and multiplex IHC techniques. We confirmed that BRAF-mutant melanoma metastases were enriched for CD4+ T cells and B cells and had a co-existing decrease in CD8+ T cells. Furthermore, we then identified B cells were associated with a trend for improved survival (p=0.078) in the BRAF-mutant samples and Th2 cells were associated with prolonged survival in the BRAF-wt samples.ConclusionsIn conclusion, treatment-naive BRAF-mutant melanoma has a distinct immune context compared with BRAF-wt melanoma, with significantly decreased CD8+ T cells and increased B cells and CD4+ T cells in the tumor microenvironment. These findings indicate that further mechanistic studies are warranted to reveal how this difference in immune context leads to improved outcome to combination immune checkpoint blockade in BRAF-mutant melanoma.

The mechanisms by which cells respond and adapt to oxidative stress are largely unknown but are key to developing a rationale for cancer therapies that target antioxidant pathways. APR-246 is a mutant-p53 targeted therapeutic currently under clinical investigation in myeloid dysplastic syndrome (MDS) and acute myeloid leukemia1. Whilst the mechanism of action of APR-246 is thought to be reactivation of wild-type p53 activity through covalent modification of cysteine residues in the core domain of mutant-p53 protein2,3, here we report that the anti-neoplastic capacity of APR-246 lies predominantly in the conjugation of free cysteine. Genome-wide CRISPR perturbation screening, metabolite profiling and proteomics in response to APR-246 treatment in mutant-p53 cancer cells highlighted the role of GSH and mitochondrial metabolism in determining APR-246 efficacy. APR-246 sensitivity was increased through loss of key enzymes in mitochondrial one-carbon metabolism,SHMT2andMTHFD1L, due to diminished glycine supply forde novoGSH synthesis. Critically, we show that APR-246 induces iron-dependent, apoptotic machinery-independent cell death, ferroptosis. Whole-cell proteomics analyses indicated an upregulation of proteins involved in iron-sulfur cluster biogenesis (eg. FDX1). GSH, acetyl-CoA and NADH levels were also depleted in APR-246 treated cells. Importantly, we found that APR-246 inhibits iron-sulfur cluster biogenesis in the mitochondria of cancer cells through cysteine conjugation. This work not only details novel determinants of APR-246 activity in cancer cells, but also provides a clinical roadmap for targeting antioxidant pathways in tumours - beyond targeting mutant-p53 tumours.

Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.

Background Chimeric antigen receptor (CAR-T) cells are a promising new therapy for patients with cancer. However, in contrast to their success in B cell malignancies, CAR-T cells targeting solid cancers have had limited success so far due to their poor proliferation and poor long-term persistence in vivo. To address this issue, we used naive T cells to generate second-generation CAR-T cells recognizing the tumor antigen Lewis Y (LeY), termed ‘early’ CAR-T cells. Methods Purified naive T cells were activated by CD3/CD28 soluble tetrameric antibody complex, retrovirally transduced (LeY scFv-CD3z-CD28 CAR) and expanded in IL-7/IL-15. The early LeY CAR-T cell function was tested in vitro for cytotoxicity (Cr-release and degranulation), proliferation, and cytokine secretion by CBA, either de novo or following chronic stimulation for 1 month. Finally, early CAR-T cell persistence and anti-tumor efficacy was assessed in the OVCAR3-NSG model, in the presence or absence of anti-PD-1. Results The early-CAR-T cells comprised stem cell memory-like (CD95+, CD62L+, CD45RA+) and central memory phenotype (CD95+, CD62L+, CD45RA-) T cells with increased expression of ICOS, Ki67, TCF7 and CD27 (Figure 1). The early-CAR-T cells retained potent antigen-specific cytotoxicity, and secreted significantly higher levels of cytokines (IFN-?, TNF-a and IL-2) and increased proliferation compared to conventional CAR-T cells. Importantly, early-CAR-T cells had a significantly higher proliferative capacity after long-term chronic stimulation compared to conventional CAR-T cells (figure 2), and CD4+ CAR-T cells were critical for effective early CD8+ CAR-T cell proliferation capacity in vitro (figure 3). Early CAR-T cells had significantly better in vivo tumor control compared to conventional CAR-T cells (Figure 4), this was associated with increased CAR-T cell persistence. Because chronically stimulated early-LeY-CAR-T cells expressed PD-1 (figure 2), and OVCAR-3 cells expressed PD-L1 when co-cultured with LeY-CAR-T cells (figure 5), we combined early LeY-CAR-T cells with anti-PD-1 therapy and observed complete tumour regression in these mice. Interestingly, early LeY-CAR-T cell plus anti-PD-1 treatment also enhanced the percentage of circulating stem-cell memory like CAR-T cells in vivo (figure 5). Conclusions Our early CAR-T cells have better cytokine secretion and proliferation than conventional CAR-T cells. Early CAR-T cells also have superior anti-tumor efficacy in vivo, they have better persistence and maintain the circulating T cell memory pool. Importantly, low dose early-LeY-CAR-T cells combined with anti-PD1-treatment leads to complete clearance of LeY+ solid tumors in vivo. The early CAR-T cell production protocol is directly translatable for improving CAR-T cell efficacy in clinical trials for patients with solid tumors.

Immunotherapy has revolutionized the treatment of cancer. Nevertheless, the majority of patients do not respond to therapy, meaning a deeper understanding of tumor immune evasion strategies is required to boost treatment efficacy. The vast majority of immunotherapy studies have focused on how treatment reinvigorates exhausted CD8+ T cells within the tumor. In contrast, how therapies influence regulatory processes within the draining lymph node is less well studied. In particular, relatively little has been done to examine how tumors may exploit peripheral CD8+ T cell tolerance, an under-studied immune checkpoint that under normal circumstances prevents detrimental autoimmune disease by blocking the initiation of T cell responses. Here we review the therapeutic potential of blocking peripheral CD8+ T cell tolerance for the treatment of cancer. We first comprehensively review what has been learnt about the regulation of CD8+ T cell peripheral tolerance from the non-tumor models in which peripheral tolerance was first defined. We next consider how the tolerant state differs from other states of negative regulation, such as T cell exhaustion and senescence. Finally, we describe how tumors hijack the peripheral tolerance immune checkpoint to prevent anti-tumor immune responses, and argue that disruption of peripheral tolerance may contribute to both the anti-cancer efficacy and autoimmune side-effects of immunotherapy. Overall, we propose that a deeper understanding of peripheral tolerance will ultimately enable the development of more targeted and refined cancer immunotherapy approaches.

Chronic stimulation of CD8

Objectives Adoptive transfer of chimeric antigen receptor (CAR)‐modified T cells is a form of cancer immunotherapy that has achieved remarkable efficacy in patients with some haematological cancers. However, challenges remain in CAR T‐cell treatment of solid tumours because of tumour‐mediated immunosuppression. Methods We have demonstrated that CAR T‐cell stimulation through T‐cell receptors (TCRs) in vivo can generate durable responses against solid tumours in a variety of murine models. Since Clec9A‐targeting tailored nanoemulsion (Clec9A‐TNE) vaccine enhances antitumour immune responses through selective activation of Clec9A+ cross‐presenting dendritic cells (DCs), we hypothesised that Clec9A‐TNE could prime DCs for antigen presentation to CAR T cells through TCRs and thus improve CAR T‐cell responses against solid tumours. To test this hypothesis, we used CAR T cells expressing transgenic TCRs specific for ovalbumin (OVA) peptides SIINFEKL (CAROTI) or OVA323‐339 (CAROTII). Results We demonstrated that the Clec9A‐TNEs encapsulating full‐length recombinant OVA protein (OVA‐Clec9A‐TNE) improved CAROT T‐cell proliferation and inflammatory cytokine secretion in vitro. Combined treatment using the OVA‐Clec9A‐TNE and CAROT cells resulted in durable responses and some rejections of tumours in immunocompetent mice. Tumour regression was accompanied by enhanced CAROT cell proliferation and infiltration into the tumours. Conclusion Our study presents Clec9A‐TNE as a prospective avenue to enhance CAR T‐cell efficacy for solid cancers., Clec9A‐targeting tailored nanoemulsion (Clec9A‐TNE) induced extensive proliferation, persistence and activation of chimeric antigen receptor (CAR) T cells, leading to the eradication of solid tumours in murine models. This novel approach could lead to the development of new CAR T‐cell therapies against some common solid tumour types in patients.

Pediatric cancers, particularly high-risk solid tumors, urgently need effective and specific therapies. Their outlook has not appreciably improved in decades. Immunotherapies such as immune checkpoint inhibitors offer much promise, but most are only approved for use in adults. Though several hundred clinical trials have tested immune-based approaches in childhood cancers, few have been guided by biomarkers or clinical-grade assays developed to predict patient response and, ultimately, to help select those most likely to benefit. There is extensive evidence in adults to show that immune profiling has substantial predictive value, but few studies focus on childhood tumors, because of the relatively small disease population and restricted use of immune-based therapies. For instance, only one published study has retrospectively examined the immune profiles of pediatric brain tumors after immunotherapy. Furthermore, application and integration of advanced multiplex techniques has been extremely limited. Here, we review the current status of immune profiling of pediatric solid tumors, with emphasis on tumor types that represent enormous unmet clinical need, primarily in the context of immune checkpoint inhibitor therapy. Translating optimized and informative immune profiling into standard practice and access to personalized combination therapy will be critical if childhood cancers are to be treated effectively and affordably.

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during the immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and the negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity., Physical membrane properties play a determining role in defining the sensitivity of membranes to the immune effector perforin.

In a population-based analysis including a large database restricted to patients over age 70, the authors demonstrate that the A91V polymorphism in the familial hemophagocytic lymphohistiocytosis–related gene is a nonpathological polymorphism that confers no increase in cancer, death, or immunopathology.

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1–driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. Significance: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity.

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis., CD8 T cells can protect the liver from viral infection, but can also result in severe liver damage and organ failure. Here, the authors develop a mouse model reflecting fulminant CD8 T cell mediated viral hepatitis, which occurs in a perforin-dependent manner that is protected by the use of perforin inhibitors.

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3− cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3− T cell–mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3− cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069–81. ©2018 AACR.