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Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4+ and CD8+ T cells. LDRT elicited predominantly CD4+ cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4+ cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell–infiltrated tumors.Significance:Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4+ effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors.This article is highlighted in the In This Issue feature, p. 1

Supplementary Figure from Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy

Supplementary Data from Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy

Supplementary Table from Low-Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy

Supplementary Figure 2

Purpose:Characterization of tumor antigen–specific CD4 T-cell responses in healthy donors and malignant melanoma patients using an in vitro amplified T-cell library screening procedure.Patients and Methods:A high-throughput, human leukocyte antigen (HLA)-independent approach was used to estimate at unprecedented high sensitivity level precursor frequencies of tumor antigen- and neoantigen-specific CD4 T cells in healthy donors and patients with cancer. Frequency estimation was combined with isolation and functional characterization of identified tumor-reactive CD4 T-cell clones.Results:In healthy donors, we report frequencies of naïve tumor-associated antigen (TAA)-specific CD4 T cells comparable with those of CD4 T cells specific for infectious agents (Tetanus toxoid). Interestingly, we also identified low, but consistent numbers of memory CD4 T cells specific for several TAAs. In patients with melanoma, low frequencies of circulating TAA-specific CD4 T cells were detected that increased after peptide-based immunotherapy. Such antitumor TAA-specific CD4 T-cell responses were also detectable within the tumor-infiltrated tissues. TAA-specific CD4 T cells in patients displayed a highly polyfunctional state, with partial skewing to Type-2 polarization. Finally, we report the applicability of this approach to the detection and amplification of neoantigen-specific CD4 T cells.Conclusions:This simple, noninvasive, high-throughput screening of tumor- and neoantigen-specific CD4 T cells requires little biologic material, is HLA class II independent and allows the concomitant screening for a large number of tumor antigens of interest, including neoantigens. This approach will facilitate the immunomonitoring of preexisting and therapy-induced CD4 T-cell responses, and accelerate the development of CD4 T-cell–based therapies.

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.

The recognition of pathogen or cancer-specific epitopes by CD8

The recognition of pathogen or cancer-specific epitopes by CD8+ T cells is crucial for the clearance of infections and the response to cancer immunotherapy. This process requires epitopes to be presented on class I Human Leukocyte Antigen (HLA-I) molecules and recognized by the T-Cell Receptor (TCR). Machine learning models capturing these two aspects of immune recognition are key to improve epitope predictions. Here we assembled a high-quality dataset of naturally presented HLA-I ligands and experimentally verified neo-epitopes. We then integrated these data with new algorithmic developments to improve predictions of both antigen presentation and TCR recognition. Applying our tool to SARS-CoV-2 proteins enabled us to uncover several epitopes. TCR sequencing identified a monoclonal response in effector/memory CD8+ T cells against one of these epitopes and cross-reactivity with the homologous SARS-CoV-1 peptide.

SUMMARY In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53−/−Brca1−/− but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers., Graphical abstract, In brief Bruand et al. provide insights into the dual role of STING in promoting tumor-intrinsic mechanisms of both immunoreactivity, driven by DNA sensing and type I IFN, and also VEGF-A-driven immune resistance in BRCA1mut ovarian cancers. STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual ICB.

Developing strategies to inflame tumors is critical for increasing response to immunotherapy. Here, we report that low-dose radiotherapy (LDRT) of murine tumors promotes T-cell infiltration and enables responsiveness to combinatorial immunotherapy in an IFN-dependent manner. Treatment efficacy relied upon mobilizing both adaptive and innate immunity and depended on both cytotoxic CD4+ and CD8+ T cells. LDRT elicited predominantly CD4+ cells with features of exhausted effector cytotoxic cells, with a subset expressing NKG2D and exhibiting proliferative capacity, as well as a unique subset of activated dendritic cells expressing the NKG2D ligand RAE1. We translated these findings to a phase I clinical trial administering LDRT, low-dose cyclophosphamide, and immune checkpoint blockade to patients with immune-desert tumors. In responsive patients, the combinatorial treatment triggered T-cell infiltration, predominantly of CD4+ cells with Th1 signatures. Our data support the rational combination of LDRT with immunotherapy for effectively treating low T cell–infiltrated tumors.Significance:Low-dose radiation reprogrammed the tumor microenvironment of tumors with scarce immune infiltration and together with immunotherapy induced simultaneous mobilization of innate and adaptive immunity, predominantly CD4+ effector T cells, to achieve tumor control dependent on NKG2D. The combination induced important responses in patients with metastatic immune-cold tumors.This article is highlighted in the In This Issue feature, p. 1

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.

Reverse immunology has open the door to innovative cancer immunotherapy strategies such as immunogenic antigen-based vaccination and transgenic T cell receptor (TCR)-based adoptive cell transfer. This approach enables the identification of immunogenic tumor specific antigen derived peptides. One of the major challenges is the rapid selection of antigen-specific CD8+ T cell clones. Thus, IFNγ-producing CD8+ T cells magnetic sorting combined with limiting dilution cloning approach represents the most common method of specific T cell cloning. However, during plate setup several wells will not contain T cells whereas others will contain mixed population of T cells. In this case, a re-cloning step is required which make limiting dilution based cloning a laborious, inefficient, expensive and a time-consuming method. To address these obstacles, here we present a novel 2-step workflow combining simple, affordable and gentle magnetic cell separation followed by single cell isolation using a device called DispenCell-S1. We aimed to compare this new workflow with the traditional limiting dilution method using in vitro generated antigen-specific CD8+ T cells. Herein, we reported the reliability of DispenCell-S1 method and its efficiency in T cell clones isolation.

Background CD4 T cells have been implicated in cancer immunity for their helper functions. However, their direct cytotoxic potential remains elusive in cancer patients. Here, we aimed at assessing the presence, rate and cytotoxic function of tumor-specific Th-CTX directly in cancer patients. Methods We capitalized on published single cell transcriptomic analyses of patient samples, integrated with the direct phenotypic and functional characterization of clonal, tumor-specific CD4 T cell populations, using peptide-MHC class II multimers and a novel high-throughput single-cell cytotoxicity assay in picowell arrays. The direct tumor cell killing by cytolytic tumor-specific CD4 T cells in the arrays was monitored in a high-throughput manner by combining multi-channel time-lapse microscopy with deep neural networks. Results By mining single-cell RNA-seq datasets of tumor infiltrating lymphocytes, we identified CD4 T cells displaying cytotoxic phenotypes in different human tumors. The cytolytic CD4 T cells formed a distinct cluster and expressed genes related to classical cytotoxic functions, largely resembling CD8 T cell gene profiles. Using the peptide MHC class II multimer technology, we confirmed directly ex vivo the presence of cytolytic tumor antigen-specific CD4 T cells, both in the circulation and in the tumors of patients. We performed an integrated phenotypic and functional characterization of cytolytic tumor-specific CD4 T cells, down to the single cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells with sub-nanoliter volumes and machine learning. We demonstrated a direct, contact-dependent, granzyme-dependent cytotoxic activity against tumor cells, with delayed kinetics compared to classical cytotoxic lymphocytes. Lastly, we discovered that this cytotoxic activity was at least in part dependent on the expression of SLAMF7, a homophylic receptor known to regulate NK cell activity. Conclusions Our work provides a deep characterization of human Th-CTX in cancer and supports their role in tumor immunity. Moreover, our results showing that agonistic engagement of SLAMF7 enhances the cytolytic capacity of tumor-specific CD4 T cells, suggests that targeting these cells might prove synergistic with the use of other immunotherapies in cancer patients.

The recent developments of high-throughput bulk and single-cell sequencing technologies accelerated the understanding of the complexity of immune repertoire dynamics combined to transcriptomics. Also, profiling of cellular repertoires in health or disease requires statistical metrics to capture clonal diversity characterized by clones frequency, repertoire richness and convergence. Here we present the common technologies of bulk and single-cell sequencing of T-cell receptors (TCRs), discuss current knowledge regarding computational tools clustering and predicting specificity of TCR repertoires based on shared structural motifs and review main indices for repertoire diversity and convergence analyses. These tools represent potential biomarkers to decipher the fitness of immune repertoires in diseased or treated patients but also the presages and promises of computational approaches to revolutionize personalized immunotherapy.

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 mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer we show that tumor-specific CD8(+) TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1(+)CD8(+) TIL can be however polyfunctional. PD-1(+) TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8(+) TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APCs. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

Purpose: Characterization of tumor antigen–specific CD4 T-cell responses in healthy donors and malignant melanoma patients using an in vitro amplified T-cell library screening procedure. Patients and Methods: A high-throughput, human leukocyte antigen (HLA)-independent approach was used to estimate at unprecedented high sensitivity level precursor frequencies of tumor antigen- and neoantigen-specific CD4 T cells in healthy donors and patients with cancer. Frequency estimation was combined with isolation and functional characterization of identified tumor-reactive CD4 T-cell clones. Results: In healthy donors, we report frequencies of naïve tumor-associated antigen (TAA)-specific CD4 T cells comparable with those of CD4 T cells specific for infectious agents (Tetanus toxoid). Interestingly, we also identified low, but consistent numbers of memory CD4 T cells specific for several TAAs. In patients with melanoma, low frequencies of circulating TAA-specific CD4 T cells were detected that increased after peptide-based immunotherapy. Such antitumor TAA-specific CD4 T-cell responses were also detectable within the tumor-infiltrated tissues. TAA-specific CD4 T cells in patients displayed a highly polyfunctional state, with partial skewing to Type-2 polarization. Finally, we report the applicability of this approach to the detection and amplification of neoantigen-specific CD4 T cells. Conclusions: This simple, noninvasive, high-throughput screening of tumor- and neoantigen-specific CD4 T cells requires little biologic material, is HLA class II independent and allows the concomitant screening for a large number of tumor antigens of interest, including neoantigens. This approach will facilitate the immunomonitoring of preexisting and therapy-induced CD4 T-cell responses, and accelerate the development of CD4 T-cell–based therapies.

Natural killer (NK) cells have increasingly become a target of interest for immunotherapies1. NK cells express killer immunoglobulin-like receptors (KIRs), which play a vital role in immune response to tumors by detecting cellular abnormalities. The genomic region encoding the 16 KIR genes displays high polymorphic variability in human populations, making it difficult to resolve individual genotypes based on next generation sequencing data. As a result, the impact of polymorphic KIR variation on cancer phenotypes has been understudied. Currently, labor-intensive, experimental techniques are used to determine an individual’s KIR gene copy number profile. Here, we develop an algorithm to determine the germline copy number of KIR genes from whole exome sequencing data and apply it to a cohort of nearly 5000 cancer patients. We use a k-mer based approach to capture sequences unique to specific genes, count their occurrences in the set of reads derived from an individual and compare the individual’s k-mer distribution to that of the population. Copy number results demonstrate high concordance with population copy number expectations. Our method reveals that the burden of inhibitory KIR genes is associated with survival in two tumor types, highlighting the potential importance of KIR variation in understanding tumor development and response to immunotherapy.

We conducted a pilot clinical trial testing a personalized vaccine generated by autologous dendritic cells (DCs) pulsed with oxidized autologous whole-tumor cell lysate (OCDC), which was injected intranodally in platinum-treated, immunotherapy-naive, recurrent ovarian cancer patients. OCDC was administered alone (cohort 1, n = 5), in combination with bevacizumab (cohort 2, n = 10), or bevacizumab plus low-dose intravenous cyclophosphamide (cohort 3, n = 10) until disease progression or vaccine exhaustion. A total of 392 vaccine doses were administered without serious adverse events. Vaccination induced T cell responses to autologous tumor antigen, which were associated with significantly prolonged survival. Vaccination also amplified T cell responses against mutated neoepitopes derived from nonsynonymous somatic tumor mutations, and this included priming of T cells against previously unrecognized neoepitopes, as well as novel T cell clones of markedly higher avidity against previously recognized neoepitopes. We conclude that the use of oxidized whole-tumor lysate DC vaccine is safe and effective in eliciting a broad antitumor immunity, including private neoantigens, and warrants further clinical testing.

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.

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.

Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8+ T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5' UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5' UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5' UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions.

CD8αβ plays crucial roles in the thymic selection, differentiation, and activation of some, but not all, CD8(+) T cells, whereas CD8αα does not. To investigate these roles, we produced mice that expressed transgene P14 T-cell receptor β (TCRβ) chain and CD8β or did not (WT and KO mice, respectively). The primary CD8(+) T-cell response to acute lymphocytic choriomeningitis virus (LCMV) infection was predominantly D(b)/GP33 specific and CD8 independent in KO mice and was mostly CD8 dependent in WT mice. Cytotoxic T lymphocytes (CTL) from KO mice failed to mobilize intracellular Ca(2+) and to kill via perforin/granzyme. Their strong Fas/FasL-mediated cytotoxicity and IFN-γ response were signaled via a Ca(2+)-independent, PI3K-dependent pathway. This was also true for 15-20% of CD8-independent CTL found in WT mice. Conversely, the perforin/granzyme-mediated killing and IFN-γ response of CD8-dependent CTL were signaled via a Ca(2+), p56(lck), and nuclear factor of activated T cells-dependent pathway. Deep sequencing of millions of TCRα chain transcripts revealed that the TCR repertoires of preimmune CD8(+) T cells were highly diverse, but those of LCMV D(b)/GP33-specific CTL, especially from KO mice, were narrow. The immune repertoires exhibited biased use of Vα segments that encoded different complementary-determining region 1α (CDR1α) and CDR2α sequences. We suggest that TCR from WT CD8-independent T cells may engage MHC-peptide complexes in a manner unfavorable for efficient CD8 engagement and Ca(2+) signaling but permissive for Ca(2+)-independent, PI3K-dependent signaling. This duality of the CD8 compartment may provide organisms with broader protective immunity.

The bias of αβ T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.

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.

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.

An attractive treatment of cancer consists in inducing tumor-eradicating CD8+ CTL specific for tumor-associated Ags, such as NY-ESO-1 (ESO), a strongly immunogenic cancer germ line gene-encoded tumor-associated Ag, widely expressed on diverse tumors. To establish optimal priming of ESO-specific CTL and to define critical vaccine variables and mechanisms, we used HLA-A2/DR1 H-2−/− transgenic mice and sequential immunization with immunodominant DR1- and A2-restricted ESO peptides. Immunization of mice first with the DR1-restricted ESO123–137 peptide and subsequently with mature dendritic cells (DCs) presenting this and the A2-restriced ESO157–165 epitope generated abundant, circulating, high-avidity primary and memory CD8+ T cells that efficiently killed A2/ESO157–165+ tumor cells. This prime boost regimen was superior to other vaccine regimes and required strong Th1 cell responses, copresentation of MHC class I and MHC class II peptides by the same DC, and resulted in upregulation of sphingosine 1-phosphate receptor 1, and thus egress of freshly primed CD8+ T cells from the draining lymph nodes into circulation. This well-defined system allowed detailed mechanistic analysis, which revealed that 1) the Th1 cytokines IFN-γ and IL-2 played key roles in CTL priming, namely by upregulating on naive CD8+ T cells the chemokine receptor CCR5; 2) the inflammatory chemokines CCL4 (MIP-1β) and CCL3 (MIP-1α) chemoattracted primed CD4+ T cells to mature DCs and activated, naive CD8+ T cells to DC–CD4 conjugates, respectively; and 3) blockade of these chemokines or their common receptor CCR5 ablated priming of CD8+ T cells and upregulation of sphingosine 1-phosphate receptor 1. These findings provide new opportunities for improving T cell cancer vaccines.

Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that ∼50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that ∼40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

BackgroundRecent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background.MethodsWe describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out). For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation.ResultsHigh throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug.ConclusionThe technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of a particular drug in a living cell.

5519 Background: Personalized tumor lysate vaccines can encompass all putative antigens in tumors. Methods: A pilot clinical trial was conducted using an autologous oxidized whole tumor cell lysate...

The metabolic activity of the liver plays a central role in coordinating the supply of energy to peripheral organs, in particular the brain. It is at the heart of an adaptive system, which allows survival at times of food deprivation and governs the storage of excess energy when plenty of food is available. The liver controls the storage of energy first by converting glucose into glycogen and, second, by esterifying excess fatty acids (FA) to triacylglycerol, which is released as a component of the very low density lipoproteins (VLDL), finally to be stored in the form of triglycerides (TG) mainly in the adipose tissue. Conversely, during starvation, when plasma glycerol and free FA (FFA) levels are high due to TG lipolysis in the adipose tissue, the liver FA oxidative machinery participates in the production and release of ketone bodies that serve as lipid-derived fuel for the brain, muscle, kidney and other peripheral organs. Furthermore, plasma glucose levels are maintained by the depletion of the hepatic glycogen stores and then by de novo glucose synthesis. A dysfunction of the fine tuning of these metabolic pathways participates in ailments that affect millions of people, which are collectively known as metabolic syndrome. It is now well established that elevated circulating FFA levels, as seen in obesity, promote type 2 diabetes in part by stimulating hepatic gluconeogenesis and glucose output into the bloodstream. During the past decade, it has been recognized that FAs are potent modulators of gene expression via the transcription factors peroxisome proliferator activated receptors (PPARs). These receptors have been identified as key regulators of energy homeostasis when activated by FAs or FA derivatives that bind to them. Therefore, it is not surprising that synthetic PPAR ligands are used as drugs to treat metabolic disorders. Although the action of PPARs is obviously not limited to the liver, herein we will focus on their major hepatic effects in health and disease.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

The three peroxisome proliferator-activated receptors (PPARs) isotypes (PPAR alpha, beta/delta and gamma) belong to the nuclear hormone receptor family. During the last decade, they have been identified as anti-inflammatory transcription factors. Part of this regulation antiinflammatory is mediated through negative interference between PPARs and other nuclear factors such as NFkB, AP-1 and C/EBP, which regulate innate as well as adaptative immunity. In addition, the PPARs control the functions of macrophages, B cells and T cells. In this review, we summarise the pathways through which the PPARs control inflammatory responses. We also discuss the potential utilisation of PPAR specific ligands in the treatment of inflammatory diseases, such as inflammatory bowel diseases, atherosclerosis, Parkinson's and Alzheimer's diseases.

Ho and colleagues report that mitochondrial dysfunction and impaired mitophagy triggered by the tumor microenvironment lead to subsequent epigenetic changes and cause permanent T cell exhaustion and dysfunction. 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.