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Abstract Background Cost-containment strategies are required to face the challenge of rising drug expenditures in Oncology. Drug wastage leads to economic loss, but little is known about the size of the problem in this field. Methods Starting January 2005 we introduced a day-to-day monitoring of drug wastage and an accurate assessment of its costs. An internal protocol for waste minimisation was developed, consisting of four corrective measures: 1. A rational, per pathology distribution of chemotherapy sessions over the week. 2. The use of multi-dose vials. 3. A reasonable rounding of drug dosages. 4. The selection of the most convenient vial size, depending on drug unit pricing. Results Baseline analysis focused on 29 drugs over one year. Considering their unit price and waste amount, a major impact on expense was found to be attributable to six drugs: cetuximab, docetaxel, gemcitabine, oxaliplatin, pemetrexed and trastuzumab. The economic loss due to their waste equaled 4.8% of the annual drug expenditure. After the study protocol was started, the expense due to unused drugs showed a meaningful 45% reduction throughout 2006. Conclusion Our experience confirms the economic relevance of waste minimisation and may represent a feasible model in addressing this issue. A centralised unit of drug processing, the availability of a computerised physician order entry system and an active involvement of the staff play a key role in allowing waste reduction and a consequent, substantial cost-saving.

Background: Cost-containment strategies are required to face the challenge of rising drug expenditures in Oncology. Drug wastage leads to economic loss, but little is known about the size of the problem in this field.Methods: Starting January 2005 we introduced a day-to-day monitoring of drug wastage and an accurate assessment of its costs. An internal protocol for waste minimisation was developed, consisting of four corrective measures: 1. A rational, per pathology distribution of chemotherapy sessions over the week. 2. The use of multi-dose vials. 3. A reasonable rounding of drug dosages. 4. The selection of the most convenient vial size, depending on drug unit pricing.Results: Baseline analysis focused on 29 drugs over one year. Considering their unit price and waste amount, a major impact on expense was found to be attributable to six drugs: cetuximab, docetaxel, gemcitabine, oxaliplatin, pemetrexed and trastuzumab. The economic loss due to their waste equaled 4.8% of the annual drug expenditure. After the study protocol was started, the expense due to unused drugs showed a meaningful 45% reduction throughout 2006.Conclusion: Our experience confirms the economic relevance of waste minimisation and may represent a feasible model in addressing this issue.A centralised unit of drug processing, the availability of a computerised physician order entry system and an active involvement of the staff play a key role in allowing waste reduction and a consequent, substantial cost-saving. [ABSTRACT FROM AUTHOR]

Introduction: Advanced cutaneous melanoma is a skin cancer characterized by a poor prognosis and high metastatic potential. During metastatic spread, melanoma cells often undergo dedifferentiation toward an invasive phenotype, resulting in reduced expression of microphthalmia-associated transcription factor (MITF)-dependent melanoma antigens and facilitating immune escape. Tumor Necrosis Factor (TNF) is known to be a key factor in melanoma dedifferentiation. Interestingly, accumulating evidence suggests that TNF may play a role in melanoma progression and resistance to immunotherapies. Additionally, TNF has been identified as a potent regulator of sphingolipid metabolism, which could contribute to melanoma aggressiveness and the process of melanoma dedifferentiation., Methods: We conducted RNA sequencing and mass spectrometry analyses to investigate TNF-induced dedifferentiation in two melanoma cell lines. In vitro experiments were performed to manipulate sphingolipid metabolism using genetic or pharmacologic alterations in combination with TNF treatment, aiming to elucidate the potential involvement of this metabolism in TNF-induced dedifferentiation. Lastly, to evaluate the clinical significance of our findings, we performed unsupervised analysis of plasma sphingolipid levels in 48 patients receiving treatment with immune checkpoint inhibitors, either alone or in combination with anti-TNF therapy., Results: Herein, we demonstrate that TNF-induced melanoma cell dedifferentiation is associated with a global modulation of sphingolipid metabolism. Specifically, TNF decreases the expression and activity of acid ceramidase (AC), encoded by the ASAH1 gene, while increasing the expression of glucosylceramide synthase (GCS), encoded by the UGCG gene. Remarkably, knockdown of AC alone via RNA interference is enough to induce melanoma cell dedifferentiation. Furthermore, treatment with Eliglustat, a GCS inhibitor, inhibits TNF-induced melanoma cell dedifferentiation. Lastly, analysis of plasma samples from patients treated with immune checkpoint inhibitors, with or without anti-TNF therapy, revealed significant predictive sphingolipids. Notably, the top 8 predictive sphingolipids, including glycosphingolipids, were associated with a poor response to immunotherapy., Discussion: Our study highlights that ceramide metabolism alterations are causally involved in TNF-induced melanoma cell dedifferentiation and suggests that the evolution of specific ceramide metabolites in plasma may be considered as predictive biomarkers of resistance to immunotherapy., Competing Interests: NM reports grants and personal fees from Bristol-Myers Squibb and MSD, and personal fees from Roche, Novartis, Pierre Fabre, Sanofi, Sun Pharma, and AbbVie outside the submitted work. VP reports grants from Pierre Fabre outside of the submitted work. BS reports grants and personal fees from Bristol-Myers Squibb outside the submitted work. J-PD reports being advisory board member for BMS, MSD, Pierre Fabre, Roche and Amgen, and received research grant from Amgen, Astra Zeneca, BMS, Genentech, MSD, Transgene outside the submitted work. BS, CD, MG, AM, TL, NM, J-PD, and NA-A declare a patent pending application number: EP22306318; filled in Sept 6, 2022. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dufau, Genais, Mucher, Jung, Garcia, Montfort, Tosolini, Clarke, Medin, Levade, Delord, Meyer, Pancaldi, Andrieu-Abadie and Ségui.)

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αβ-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27 + Ly6C - cells convert into CD27 + Ly6C + cells, and these CD27 + Ly6C + cells control cancer progression in mice, while the CD27 + Ly6C - cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27 + Ly6C + cells and human Vδ2 + cells, while IL-27 is dispensable for mouse CD27 + Ly6C - cell and human Vδ1 + cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology., (© 2024. The Author(s).)

Cancer metabolic reprogramming has been recognized as one of the cancer hallmarks that promote cell proliferation, survival, as well as therapeutic resistance. Up-to-date regulation of metabolism in T-cell lymphoma is poorly understood. In particular, for human angioimmunoblastic T-cell lymphoma (AITL) the metabolic profile is not known. Metabolic intervention could help identify new treatment options for this cancer with very poor outcomes and no effective medication. Transcriptomic analysis of AITL tumor cells, identified that these cells use preferentially mitochondrial metabolism. By using our preclinical AITL mouse model, mimicking closely human AITL features, we confirmed that T follicular helper (Tfh) tumor cells exhibit a strong enrichment of mitochondrial metabolic signatures. Consistent with these results, disruption of mitochondrial metabolism using metformin or a mitochondrial complex I inhibitor such as IACS improved the survival of AITL lymphoma-bearing mice. Additionally, we confirmed a selective elimination of the malignant human AITL T cells in patient biopsies upon mitochondrial respiration inhibition. Moreover, we confirmed that diabetic patients suffering from T-cell lymphoma, treated with metformin survived longer as compared to patients receiving alternative treatments. Taking together, our findings suggest that targeting the mitochondrial metabolic pathway could be a clinically efficient approach to inhibit aggressive cancers such as peripheral T-cell lymphoma., (© 2024. The Author(s).)

Background: Angioimmunoblastic T-cell lymphoma (AITL) is a malignancy with very poor survival outcome, in urgent need of more specific therapeutic strategies. The drivers of malignancy in this disease are CD4 + follicular helper T cells (Tfh). The metabolism of these malignant Tfh cells was not yet elucidated. Therefore, we decided to identify their metabolic requirements with the objective to propose a novel therapeutic option., Methods: To reveal the prominent metabolic pathways used by the AITL lymphoma cells, we relied on metabolomic and proteomic analysis of murine AITL (mAITL) T cells isolated from our established mAITL model. We confirmed these results using AITL patient and healthy T cell expression data., Results: Strikingly, the mAITL Tfh cells were highly dependent on the second branch of the Kennedy pathway, the choline lipid pathway, responsible for the production of the major membrane constituent phosphatidylcholine. Moreover, gene expression data from Tfh cells isolated from AITL patient tumors, confirmed the upregulation of the choline lipid pathway. Several enzymes involved in this pathway such as choline kinase, catalyzing the first step in the phosphatidylcholine pathway, are upregulated in multiple tumors other than AITL. Here we showed that treatment of our mAITL preclinical mouse model with a fatty acid oxydation inhibitor, significantly increased their survival and even reverted the exhausted CD8 T cells in the tumor into potent cytotoxic anti-tumor cells. Specific inhibition of Chokα confirmed the importance of the phosphatidylcholine production pathway in neoplastic CD4 + T cells, nearly eradicating mAITL Tfh cells from the tumors. Finally, the same inhibitor induced in human AITL lymphoma biopsies cell death of the majority of the hAITL PD-1 high neoplastic cells., Conclusion: Our results suggest that interfering with choline metabolism in AITL reveals a specific metabolic vulnerability and might represent a new therapeutic strategy for these patients., (© 2024. The Author(s).)

1,8-Naphthalimides (NIs) represent a class of organic dyes with interesting optical properties that has been extensively explored in the last decades in lighting devices, chemosensors, optical probes or medicinal chemistry. However, despite their remarkable potential, reports on organometallic dyes bearing NIs are scarce and virtually inexistent regarding palladium(II) complexes. Herein, we report the synthesis of NIs bearing phosphine and amine chelating moieties and the characterization of their optical properties both as single molecules and when complexed on Pd(II) ions. It is shown that the introduction of phosphine moieties in the naphthalimide core results in a marked increase in non-radiative processes, leading to a significant reduction of the emission efficiency and lifetime of these dyes, compared to amine-bearing counterparts. The complexation to Pd(II) sequesters the electronic contribution of chelating moieties, with complexes assuming an optical behavior similar to that of unsubstituted 1,8-naphthalimide. The complexation significantly increases the acidity of chelating secondary amines, giving rise to an unexpected intramolecular reaction that results in the formation of a novel 1,8-naphthalimide dye bearing a cyclic phosphorylamide moiety. The new dye exhibits good emission quantum yield, long fluorescence lifetime and sensitivity to basic media, evidencing potential for application in optical imaging and sensing scenarios., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8 + -exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stress granules (SGs) and processing bodies (PBs) are membraneless cytoplasmic assemblies regulating mRNAs under environmental stress such as viral infections, neurological disorders, or cancer. Upon antigen stimulation, T lymphocytes mediate their immune functions under regulatory mechanisms involving SGs and PBs. However, the impact of T cell activation on such complexes in terms of formation, constitution, and relationship remains unknown. Here, by combining proteomic, transcriptomic, and immunofluorescence approaches, we simultaneously characterized the SGs and PBs from primary human T lymphocytes pre and post stimulation. The identification of the proteomes and transcriptomes of SGs and PBs indicate an unanticipated molecular and functional complementarity. Notwithstanding, these granules keep distinct spatial organizations and abilities to interact with mRNAs. This comprehensive characterization of the RNP granule proteomic and transcriptomic landscapes provides a unique resource for future investigations on SGs and PBs in T lymphocytes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The development of single-cell technologies yields large datasets of information as diverse and multimodal as transcriptomes, immunophenotypes, and spatial position from tissue sections in the so-called 'spatial transcriptomics'. Currently however, user-friendly, powerful, and free algorithmic tools for straightforward analysis of spatial transcriptomic datasets are scarce., Results: Here, we introduce Single-Cell Spatial Explorer, an open-source software for multimodal exploration of spatial transcriptomics, examplified with 9 human and murine tissues datasets from 4 different technologies., Conclusions: Single-Cell Spatial Explorer is a very powerful, versatile, and interoperable tool for spatial transcriptomics analysis., (© 2023. The Author(s).)

Despite the success of standard front-line chemotherapy, 20% of classical Hodgkin lymphoma (cHL) patients still relapse or have refractory disease (r/r), and a subset of them die due to disease progression. There is a critical lack of predictive factors for early identification of those r/r patients who may benefit from new therapeutic strategies. This study aimed to evaluate the dynamic expression of 586 immune-related genes in a cohort of 42 cHL patients including 30 r/r cHL after first-line chemotherapy. Gene expression profiling (GEP) using NanoString technology identified a 19-gene immune signature at diagnosis predictive of cHL relapse, but dependent on histological subtypes. Genes related to tumor survival were found upregulated while genes related to B-lineage were downregulated at diagnosis in r/r nodular sclerosis cHL. In contrast to the mixed-cellularity subtype, comparative GEP analyses between paired diagnosis/relapse biopsies of nodular sclerosis cHL showed 118 differentially expressed genes, supporting an immune contexture switch at relapse with upregulation of immunosuppressive cytokines, such as LGALS1 and TGFB1 , and downregulation of the T-cell co-stimulatory receptor ICOS . These results indicate that the predictive value of immune signature in cHL is strongly influenced by histological subtype which should be considered when assessing new immunotherapy target strategies.

Follicular lymphoma (FL) is the most common indolent lymphoma. Despite the clear benefit of CD20-based therapy, a subset of FL patients still progress to aggressive lymphoma. Thus, identifying early biomarkers that incorporate PET metrics could be helpful to identify patients with a high risk of treatment failure with Rituximab. We retrospectively included a total of 132 untreated FL patients separated into training and validation cohorts. Optimal threshold of baseline SUVmax was first determined in the training cohort (n=48) to predict progression-free survival (PFS). The PET results were investigated along with the tumor and immune microenvironment, which were determined by immunochemistry and transcriptome studies involving gene set enrichment analyses and immune cell deconvolution, together with the tumor mutation profile. We report that baseline SUVmax >14.5 was associated with poorer PFS than baseline SUVmax ≤14.5 (HR=0.28; p=0.00046). Neither immune T-cell infiltration nor immune checkpoint expression were associated with baseline PET metrics. By contrast, FL samples with Ki-67 staining ≥10% showed enrichment of cell cycle/DNA genes (p=0.013) and significantly higher SUVmax values (p=0.007). Despite similar oncogenic pathway alterations in both SUVmax groups of FL samples, 4 out of 5 cases harboring the infrequent FOXO1 transcription factor mutation were seen in FL patients with SUVmax >14.5. Thus, high baseline SUVmax reflects FL tumor proliferation and, together with Ki-67 proliferative index, can be used to identify patients at risk of early relapse with R-chemotherapy.

The detailed characterization of human γδ T lymphocyte differentiation at the single-cell transcriptomic (scRNAseq) level in tumors and patients with coronavirus disease 2019 (COVID-19) requires both a reference differentiation trajectory of γδ T cells and a robust mapping method for additional γδ T lymphocytes. Here, we incepted such a method to characterize thousands of γδ T lymphocytes from ( n = 95) patients with cancer or adult and pediatric COVID-19 disease. We found that cancer patients with human papillomavirus-positive head and neck squamous cell carcinoma and Epstein-Barr virus-positive Hodgkin's lymphoma have γδ tumor-infiltrating T lymphocytes that are more prone to recirculate from the tumor and avoid exhaustion. In COVID-19, both TCRVγ9 and TCRVγnon9 subsets of γδ T lymphocytes relocalize from peripheral blood mononuclear cells (PBMC) to the infected lung tissue, where their advanced differentiation, tissue residency, and exhaustion reflect T cell activation. Although severe COVID-19 disease increases both recruitment and exhaustion of γδ T lymphocytes in infected lung lesions but not blood, the anti-IL6R therapy with Tocilizumab promotes γδ T lymphocyte differentiation in patients with COVID-19. PBMC from pediatric patients with acute COVID-19 disease display similar γδ T cell lymphopenia to that seen in adult patients. However, blood γδ T cells from children with the COVID-19-related multisystem inflammatory syndrome are not lymphodepleted, but they are differentiated as in healthy PBMC. These findings suggest that some virus-induced memory γδ T lymphocytes durably persist in the blood of adults and could subsequently infiltrate and recirculate in tumors.

Therapy resistance represents a major clinical challenge in acute myeloid leukemia (AML). Here we define a 'MitoScore' signature, which identifies high mitochondrial oxidative phosphorylation in vivo and in patients with AML. Primary AML cells with cytarabine (AraC) resistance and a high MitoScore relied on mitochondrial Bcl2 and were highly sensitive to venetoclax (VEN) + AraC (but not to VEN + azacytidine). Single-cell transcriptomics of VEN + AraC-residual cell populations revealed adaptive resistance associated with changes in oxidative phosphorylation, electron transport chain complex and the TP53 pathway. Accordingly, treatment of VEN + AraC-resistant AML cells with electron transport chain complex inhibitors, pyruvate dehydrogenase inhibitors or mitochondrial ClpP protease agonists substantially delayed relapse following VEN + AraC. These findings highlight the central role of mitochondrial adaptation during AML therapy and provide a scientific rationale for alternating VEN + azacytidine with VEN + AraC in patients with a high MitoScore and to target mitochondrial metabolism to enhance the sensitivity of AML cells to currently approved therapies., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

γδ T lymphocytes diverge from conventional T CD8 lymphocytes for ontogeny, homing, and antigen specificity, but whether their differentiation in tumors also deviates was unknown. Using innovative analyses of our original and ~150 published single-cell RNA sequencing datasets validated by phenotyping of human tumors and murine models, here we present the first high-resolution view of human γδ T cell differentiation in cancer. While γδ T lymphocytes prominently encompass TCRVγ9 cells more differentiated than T CD8 in healthy donor's blood, a different scenario is unveiled in tumors. Solid tumors and lymphomas are infiltrated by a majority of TCRVγnon9 γδ T cells which are quantitatively correlated and remarkably aligned with T CD8 for differentiation, exhaustion, gene expression profile, and response to immune checkpoint therapy. This cancer-wide association is critical for developing cancer immunotherapies., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro-metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kα-selective inhibition prevented macro-metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the produced lipid second messenger PIP 3 , with a selective decrease of C36:2 PI-3,4,5-P 3 . Tumoural PI3Kα inactivation prevented the accumulation of pro-tumoural CD206-positive macrophages in the tumour-adjacent tissue. Tumour cell-intrinsic PI3Kα promotes pro-metastatic features that could be pharmacologically targeted to delay macro-metastatic evolution., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3 , catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8 + tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro , small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8 + T-cell-dependent immune responses and overcome resistance to anti-PD-1., (©2021 American Association for Cancer Research.)

High-definition transcriptomic studies through single-cell RNA sequencing (scRNA-Seq) have revealed the heterogeneity and functionality of the various microenvironments across numerous solid tumors. Those pioneer studies have highlighted different cellular signatures correlated with clinical response to immune checkpoint inhibitors. scRNA-Seq offers also a unique opportunity to unravel the intimate heterogeneity of the ecosystems across different lymphoma entities. In this review, we will first cover the basics and future developments of the technology, and we will discuss its input in the field of translational lymphoma research, from determination of cell-of-origin and functional diversity, to monitoring of anti-cancer targeted drugs response and toxicities, and how new improvements in both data collection and interpretation will further foster precision medicine in the upcoming years., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ysebaert, Quillet-Mary, Tosolini, Pont, Laurent and Fournié.)

Tumor antigen-specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

Background: Ibrutinib, an irreversible Bruton Tyrosine Kinase (BTK) inhibitor, has revolutionized Chronic Lymphocytic Leukemia (CLL) treatment, but resistances to ibrutinib have emerged, whether related or not to BTK mutations. Patterns of CLL evolution under ibrutinib therapy are well characterized for the leukemic cells but not for their microenvironment., Methods: Here, we addressed this question at the single cell level of both transcriptome and immune-phenotype. The PBMCs from a CLL patient were monitored during ibrutinib treatment using Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq) technology., Results: This unveiled that the short clinical relapse of this patient driven by BTK mutation is associated with intraclonal heterogeneity in B leukemic cells and up-regulation of common signaling pathways induced by ibrutinib in both B leukemic cells and immune cells. This approach also pinpointed a subset of leukemic cells present before treatment and highly enriched during progression under ibrutinib. These latter exhibit an original gene signature including up-regulated BCR, MYC-activated, and other targetable pathways. Meanwhile, although ibrutinib differentially affected the exhaustion of T lymphocytes, this treatment enhanced the T cell cytotoxicity even during disease progression., Conclusions: These results could open new alternative of therapeutic strategies for ibrutinib-refractory CLL patients, based on immunotherapy or targeting B leukemic cells themselves.

Anaplastic large cell lymphoma (ALCL) is a mature T cell neoplasm that often expresses the CD4+ T cell surface marker. It usually harbors the t(2;5) (p23;q35) translocation, leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. We demonstrated that in vitro transduction of normal human CD4+ T lymphocytes with NPM-ALK results in their immortalization and malignant transformation. The tumor cells displayed morphological and immunophenotypical characteristics of primary patient-derived anaplastic large cell lymphomas. Cell growth, proliferation, and survival were strictly dependent on NPM-ALK activity and include activation of the key factors STAT3 and DNMT1 and expression of CD30 (the hallmark of anaplastic large-cell lymphoma). Implantation of NPM-ALK-transformed CD4+ T lymphocytes into immunodeficient mice resulted in the formation of tumors indistinguishable from patients' anaplastic large cell lymphomas. Integration of "Omic" data revealed that NPM-ALK-transformed CD4+ T lymphocytes and primary NPM-ALK+ ALCL biopsies share similarities with early T cell precursors. Of note, these NPM-ALK+ lymphoma cells overexpress stem cell regulators (OCT4, SOX2, and NANOG) and HIF2A, which is known to affect hematopoietic precursor differentiation and NPM-ALK+ cell growth. Altogether, for the first time our findings suggest that NPM-ALK could restore progenitor-like features in mature CD30+ peripheral CD4+ T cells, in keeping with a thymic progenitor-like pattern.

Immunotherapies have achieved clinical benefit in many types of cancer but remain limited to a subset of patients in colorectal cancer (CRC). Resistance to immunotherapy can be attributed in part to tissue-specific factors constraining antitumor immunity. Thus, a better understanding of how the colon microenvironment shapes the immune response to CRC is needed to identify mechanisms of resistance to immunotherapies and guide the development of novel therapeutics. In an orthotopic mouse model of MC38-CRC, tumor progression was monitored by bioluminescence imaging and the immune signatures were assessed at a transcriptional level using NanoString and at a cellular level by flow cytometry. Despite initial tumor growth in all mice, only 25% to 35% of mice developed a progressive lethal CRC while the remaining animals spontaneously rejected their solid tumor. No tumor rejection was observed in the absence of adaptive immunity, nor when MC38 cells were injected in non-orthotopic locations, subcutaneously or into the liver. We observed that progressive CRC tumors exhibited a protumor immune response, characterized by a regulatory T-lymphocyte pattern, discernible shortly post-tumor implantation, as well as suppressive myeloid cells. In contrast, tumor-rejecting mice presented an early inflammatory response and an antitumor microenvironment enriched in CD8 + T cells. Taken together, our data demonstrate the role of the colon microenvironment in regulating the balance between anti or protumor immune responses. While emphasizing the relevance of the CRC orthotopic model, they set the basis for exploring the impact of the identified signatures in colon cancer response to immunotherapy., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Although understanding of T-cell exhaustion is widely based on mouse models, its analysis in patients with cancer could provide clues indicating tumor sensitivity to immune checkpoint blockade (ICB). Data suggest a role for costimulatory pathways, particularly CD28, in exhausted T-cell responsiveness to PD-1/PD-L1 blockade. Here, we used single-cell transcriptomic, phenotypic, and functional approaches to dissect the relation between CD8 + T-cell exhaustion, CD28 costimulation, and tumor specificity in head and neck, cervical, and ovarian cancers. We found that memory tumor-specific CD8 + T cells, but not bystander cells, sequentially express immune checkpoints once they infiltrate tumors, leading, in situ , to a functionally exhausted population. Exhausted T cells were nonetheless endowed with effector and tumor residency potential but exhibited loss of the costimulatory receptor CD28 in comparison with their circulating memory counterparts. Accordingly, PD-1 inhibition improved proliferation of circulating tumor-specific CD8 + T cells and reversed functional exhaustion of specific T cells at tumor sites. In agreement with their tumor specificity, high infiltration of tumors by exhausted cells was predictive of response to therapy and survival in ICB-treated patients with head and neck cancer. Our results showed that PD-1 blockade-mediated proliferation/reinvigoration of circulating memory T cells and local reversion of exhaustion occur concurrently to control tumors., (©2020 American Association for Cancer Research.)