The metabolic challenges present in tumors attenuate the metabolic fitness and antitumor activity of tumor-infiltrating T lymphocytes (TILs). However, it remains unclear whether persistent metabolic ...insufficiency can imprint permanent T cell dysfunction. We found that TILs accumulated depolarized mitochondria as a result of decreased mitophagy activity and displayed functional, transcriptomic and epigenetic characteristics of terminally exhausted T cells. Mechanistically, reduced mitochondrial fitness in TILs was induced by the coordination of T cell receptor stimulation, microenvironmental stressors and PD-1 signaling. Enforced accumulation of depolarized mitochondria with pharmacological inhibitors induced epigenetic reprogramming toward terminal exhaustion, indicating that mitochondrial deregulation caused T cell exhaustion. Furthermore, supplementation with nicotinamide riboside enhanced T cell mitochondrial fitness and improved responsiveness to anti-PD-1 treatment. Together, our results reveal insights into how mitochondrial dynamics and quality orchestrate T cell antitumor responses and commitment to the exhaustion program.
The identification of patient-specific tumor antigens is complicated by the low frequency of T cells specific for each tumor antigen. Here we describe NeoScreen, a method that enables the sensitive ...identification of rare tumor (neo)antigens and of cognate T cell receptors (TCRs) expressed by tumor-infiltrating lymphocytes. T cells transduced with tumor antigen-specific TCRs identified by NeoScreen mediate regression of established tumors in patient-derived xenograft mice.
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.
CD4+ T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on class II major histocompatibility complex (MHC-II) molecules. The high ...polymorphism of MHC-II genes represents an important hurdle toward accurate prediction and identification of CD4+ T cell epitopes. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry. This enabled us to precisely determine the binding motifs of 88 MHC-II alleles across humans, mice, cattle, and chickens. Analysis of these binding specificities combined with X-ray crystallography refined our understanding of the molecular determinants of MHC-II motifs and revealed a widespread reverse-binding mode in HLA-DP ligands. We then developed a machine-learning framework to accurately predict binding specificities and ligands of any MHC-II allele. This tool improves and expands predictions of CD4+ T cell epitopes and enables us to discover viral and bacterial epitopes following the aforementioned reverse-binding mode.
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•Analysis of MHC-II peptidomics data identifies >600,000 MHC-II ligands•Motif deconvolution determines high-resolution binding motifs for 88 MHC-II alleles•Structural analysis uncovers alternative binding modes of MHC-II ligands•MixMHC2pred improves predictions of CD4+ T cell epitopes
CD4+ T cells recognize peptides displayed on MHC-II molecules. Racle et al. curate >600,000 MHC-II ligands and derive high-resolution motifs for 88 MHC-II alleles. These motifs reveal a widespread reverse-binding mode for HLA-DP ligands and improve predictions of CD4+ T cell epitopes with MixMHC2pred.
Immunotherapy directed against private tumor neo-antigens derived from non-synonymous somatic mutations is a promising strategy of personalized cancer immunotherapy. However, feasibility in low ...mutational load tumor types remains unknown. Comprehensive and deep analysis of circulating and tumor-infiltrating lymphocytes (TILs) for neo-epitope specific CD8
T cells has allowed prompt identification of oligoclonal and polyfunctional such cells from most immunotherapy-naive patients with advanced epithelial ovarian cancer studied. Neo-epitope recognition is discordant between circulating T cells and TILs, and is more likely to be found among TILs, which display higher functional avidity and unique TCRs with higher predicted affinity than their blood counterparts. Our results imply that identification of neo-epitope specific CD8
T cells is achievable even in tumors with relatively low number of somatic mutations, and neo-epitope validation in TILs extends opportunities for mutanome-based personalized immunotherapies to such tumors.
The success of cancer immunotherapy depends in part on the strength of antigen recognition by T cells. Here, we characterize the T cell receptor (TCR) functional (antigen sensitivity) and structural ...(monomeric pMHC-TCR off-rates) avidities of 371 CD8 T cell clones specific for neoantigens, tumor-associated antigens (TAAs) or viral antigens isolated from tumors or blood of patients and healthy donors. T cells from tumors exhibit stronger functional and structural avidity than their blood counterparts. Relative to TAA, neoantigen-specific T cells are of higher structural avidity and, consistently, are preferentially detected in tumors. Effective tumor infiltration in mice models is associated with high structural avidity and CXCR3 expression. Based on TCR biophysicochemical properties, we derive and apply an in silico model predicting TCR structural avidity and validate the enrichment in high avidity T cells in patients' tumors. These observations indicate a direct relationship between neoantigen recognition, T cell functionality and tumor infiltration. These results delineate a rational approach to identify potent T cells for personalized cancer immunotherapy.
Chromatin remodeling at specific genomic loci controls lymphoid differentiation. Here, we investigated the role played in this process by Kruppel‐associated box (KRAB)‐associated protein 1 (KAP1), ...the universal cofactor of KRAB‐zinc finger proteins (ZFPs), a tetrapod‐restricted family of transcriptional repressors. T‐cell‐specific Kap1‐deleted mice displayed a significant expansion of immature thymocytes, imbalances in CD4+/CD8+ cell ratios, and altered responses to TCR and TGFβ stimulation when compared to littermate KAP1 control mice. Transcriptome and chromatin studies revealed that KAP1 binds T‐cell‐specific cis‐acting regulatory elements marked by the H3K9me3 repressive mark and enriched in Ikaros/NuRD complexes. Also, KAP1 directly controls the expression of several genes involved in TCR and cytokine signaling. Among these, regulation of FoxO1 seems to play a major role in this system. Likely responsible for tethering KAP1 to at least part of its genomic targets, a small number of KRAB‐ZFPs are selectively expressed in T‐lymphoid cells. These results reveal the so far unsuspected yet important role of KAP1‐mediated epigenetic regulation in T‐lymphocyte differentiation and activation.—Santoni de Sio, F. R., Barde, I., Offner, S., Kapopoulou, A., Corsinotti, A., Bojkowska, K., Genolet, R., Thomas, J. H., Luescher, I. F., Pinschewer, D., Harris, N., Trono, D. KAP1 regulates gene networks controlling T‐cell development and responsiveness. FASEB J. 26, 4561–4575 (2012). www.fasebj.org
Label-free approaches to assess cell properties ideally suit the requirements of cell-based therapeutics, since they permit to characterize cells with minimal perturbation and manipulation, at the ...benefit of sample recovery and re-employment for treatment. For this reason, label-free techniques would find sensible application in adoptive T cell-based immunotherapy. In this work, we describe the label-free and single-cell detection of in vitro activated T lymphocytes in flow through an electrical impedance-based setup. We describe a novel platform featuring 3D free-standing microelectrodes presenting passive upstream and downstream extensions and integrated into microfluidic channels. We employ such device to measure the impedance change associated with T cell activation at electrical frequencies maximizing the difference between non-activated and activated T cells. Finally, we harness the impedance signature of unstimulated T cells to set a boundary separating activated and non-activated clones, so to characterize the selectivity and specificity of the system. In conclusion, the strategy here proposed highlights the possible employment of impedance to assess T cell activation in label-free.
•Activated T cells detected for the first time in flow by electrical impedance.•Arrays of 3D platinum microelectrodes integrated with SU-8 microfluidic elements.•Sensitivity for single activated CD69+ T cells is 94% and specificity is 74%.
Polysomal messenger RNA (mRNA) populations change rapidly in response to alterations in the physiological status of the cell. For this reason, translational regulation, mediated principally at the ...level of initiation, plays a key role in the maintenance of cellular homeostasis. In an earlier translational profiling study, we followed the impact of rapamycin on polysome re-seeding. Despite the overall negative effect on transcript recruitment, we nonetheless observed that some mRNAs were significantly less affected. Consequently, their relative polysomal occupancy increased in the rapamycin-treated cells. The behaviour of one of these genes, mdm2, has been further analysed. Despite the absence of internal ribosome entry site activity we demonstrate, using a dual reporter assay, that both the reported mdm2 5'-UTRs confer resistance to rapamycin relative to the 5'-UTR of β-actin. This relative resistance is responsive to the downstream targets mTORC1 but did not respond to changes in the La protein, a reported factor acting positively on MDM2 translational expression. Furthermore, extended exposure to rapamycin in the presence of serum increased the steady-state level of the endogenous MDM2 protein. However, this response was effectively reversed when serum levels were reduced. Taken globally, these studies suggest that experimental conditions can dramatically modulate the expressional output during rapamycin exposure.
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