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The accessibility of CAR-T cells in centralized production models faces significant challenges, primarily stemming from logistical complexities and prohibitive costs. However, European Regulation EC No. 1394/2007 introduced a pivotal provision known as the hospital exemption. This exemption enables academic institutions to produce ATMPs, including autologous CAR-T cells, under GMP conditions tailored to specific needs. In response to this regulatory framework, our work group has launched a guideline initiative. This endeavor aims to bolster academic CAR T-cell manufacturing by implementing strategic workshops that serve a dual purpose: standardizing production processes and ensuring both efficacy and safety standards are met. This inaugural focused on delineating criteria for the release of fresh CAR-T cell batches. Key emphases included thorough product characterization, stringent safety and potency evaluations. These criteria are essential for compliance with regulatory mandates and aligning with industry best practices. Notably, the release of initial CAR T-cell batches will be facilitated through provisional certification, with final certification contingent upon the acquisition of comprehensive analytical control results. This procedural framework adheres to methodologies endorsed by the SFGM-TC and the EBMT. Such adherence underscores a commitment to harmonizing practices across academic manufacturing facilities, thus fortifying the accessibility, efficacy, and safety of point-of-care units., Competing Interests: Competing interests: OB received research funding and/or honoraria from Argenx, BMS, CSL Behring, Egle Tx, OGD2 and UCB. DB: Declares no conflict of interest in the field of CAR T cells. HB: Licensing fees and royalties from Medac; research support from Erydel, Miltenyi, Sandoz-Hexal (a Novartis company); honoraria and speaker fees from Medac, Miltenyi, Novartis and Terumo BCT; consultancy and membership of advisory boards for Boehringer-Ingelheim, Celgene (a BMS company), Medac, Novartis and Sandoz-Hexal; stock ownership in Healthineers CC: received research funding and honoraria (institutional or personal) for participation in advisory boards and speakers bureau from BMS, Janssen Pharmaceuticals, Kite/Gilead, Miltenyi Biotec, Novartis. BC: Declares no conflict of interest in the field of CAR T cells. AC: Declares no conflict of interest. GD: Declares no conflict of interest in the field of CAR T cells. JSD: Declares no conflict of interest in the field of CAR T cells. SD: Declares no conflict of interest in the field of CAR T cells. JDV: Declares no conflict of interest in the field of CAR T cells. AD: Declares no conflict of interest in the field of CAR T cells. LD: Declares no conflict of interest in the field of CAR T cells. CF: shareholder and employee Advesya. EF: received honoraria for participation in advisory boards and speaker’s bureau from Kite/Gilead, Novartis, Alexion, Astellas, GSK. JG: Declares no conflict of interest in the field of CAR T cells. AG: Declares no conflict of interest in the field of CAR T cells. CG: Declares no conflict of interest in the field of CAR T cells. JM: Declares no conflict of interest in the field of CAR T cells. CM: Declares no conflict of interest. JL: received honoraria from Novartis, Kite/Gilead, BMS, Janssen, Miltenyi Biotec. LR: Declares no conflict of interest in the field of CAR T cells. SV: Declares no conflict of interest. IYA: received honorarium from Novartis, BMS, KITE and Milteny Biomedecine. MD: shareholder and employee Advesya., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

Chimeric antigen receptor (CAR) T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen. scFv selection should involve safety profiling with evaluation of the efficacy/toxicity balance, especially when the target antigen also is expressed on healthy cells. Here, to assess differences in terms of efficacy and on-target/off-tumor effects, we generated five different CARs targeting CD123 by substituting only the scFv. In in vitro models, T cells engineered to express three of these five CD123 CARs were effectively cytotoxic on leukemic cells without increasing lysis of monocytes or endothelial cells. Using the IncuCyte system, we confirmed the low cytotoxicity of CD123 CAR T cells on endothelial cells. Hematotoxicity evaluation using progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR T cells were less cytotoxic on hematopoietic stem cells. Using a humanized mouse model, we confirmed that CD123- cells were not eliminated by the CD123 CAR T cells. Two CD123 CAR T cells reduced tumor infiltration and increased the overall survival of mice in three in vivo models of blastic plasmacytoid dendritic cell neoplasm. In an aggressive version of this model, bulk RNA sequencing analysis showed that these CD123 CAR T cells upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Together, these results emphasize the importance of screening different scFvs for the development of CAR constructs to support selection of cells with the optimal risk-benefit ratio for clinical development., (©2024 American Association for Cancer Research.)

While CAR-T and tgTCR-T therapies have exhibited noteworthy and promising outcomes in hematologic and solid tumors respectively, a set of distinct challenges remains. Consequently, the quest for novel strategies has become imperative to safeguard and more effectively release the full functions of engineered T cells. These factors are intricately linked to the success of adoptive cell therapy. Recently, CRISPR-based technologies have emerged as a major breakthrough for maintaining T cell functions. These technologies have allowed the discovery of T cells' negative regulators such as specific cell-surface receptors, cell-signaling proteins, and transcription factors that are involved in the development or maintenance of T cell dysfunction. By employing a CRISPR-genic invalidation approach to target these negative regulators, it has become possible to prevent the emergence of hypofunctional T cells. This review revisits the establishment of the dysfunctional profile of T cells before delving into a comprehensive summary of recent CRISPR-gene invalidations, with each invalidation contributing to the enhancement of engineered T cells' antitumor capacities. The narrative unfolds as we explore how these advancements were discovered and identified, marking a significant advancement in the pursuit of superior adoptive cell therapy., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Engineered T cell-based adoptive immunotherapies met promising success for the treatment of hematological malignancies. Nevertheless, major hurdles remain to be overcome regarding the management of relapses and the translation to solid tumor settings. Properties of T cell-based final product should be appropriately controlled to fine-tune the analysis of clinical trial results, to draw relevant conclusions, and finally to improve the efficacy of these immunotherapies. For this purpose, we addressed the existence of atypical T cell subsets and deciphered their phenotypic and functional features in an HPV16-E7 specific and MHC II-restricted transgenic-TCR-engineered T cell setting. To note, atypical T cell subsets include mismatched MHC/co-receptor CD8 or CD4 and miscommitted CD8+ or CD4+ T cells. We generated both mismatched and appropriately matched MHC II-restricted transgenic TCR on CD8 and CD4-expressing T cells, respectively. We established that CD4+ cultured T cells exhibited miscommitted phenotypic cytotoxic pattern and that both interleukin (IL)-2 or IL-7/IL-15 supplementation allowed for the development of this cytotoxic phenotype. Both CD4+ and CD8+ T cell subsets, transduced with HPV16-E7 specific transgenic TCR, demonstrated cytotoxic features after exposure to HPV-16 E7-derived antigen. Ultimately, the presence of such atypical T cells, either mismatched MHC II-restricted TCR/CD8+ T cells or cytotoxic CD4+ T cells, is likely to influence the fate of patient-infused T cell product and would need further investigation., 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 © 2024 Mercier-Letondal, Kumar, Marton, Bonnefoy, Fredon, Boullerot, Dehecq, Adotévi, Godet and Galaine.)

Competing Interests: Conflicts of interest all authors declare no conflicts of interest.

Engineered T-cell therapies have proven to be successful in cancer and their clinical effectiveness is directly correlated with the infused T-cell differentiation profile. Indeed, stem cell memory and central memory T cells proliferate and persist longer in vivo compared with more-differentiated T cells, while conferring enhanced antitumor activity. Here, we propose an optimized process using cord blood (CB) to generate minimally differentiated T-cell products in terms of phenotype, function, gene expression, and metabolism, using peripheral blood (PB)-derived T cells cultured with IL-2 as a standard. Phenotypically, CB-derived T cells, particularly CD4 T cells, are less differentiated than their PB counterparts when cultured with IL-2 or with IL-7 and IL-15. Furthermore, culture with IL-7 and IL-15 enables better preservation of less-differentiated CB-derived T cells compared with IL-2. In addition, transcriptomic and metabolic assessments of CB-derived transgenic T cells cultured with IL-7 and IL-15 point out their naivety and stemness signature. These relatively quiescent transgenic T cells are nevertheless primed for secondary stimulation and cytokine production. In conclusion, our study indicates that CB may be used as a source of early differentiated T cells to develop more effective adoptive cancer immunotherapy., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Chimeric Antigen Receptor (CAR) therapy has led to great successes in patients with leukemia and lymphoma. Umbilical Cord Blood (UCB), stored in UCB banks, is an attractive source of T cells for CAR-T production. We used a third generation CD123 CAR-T (CD28/4-1BB), which was previously developed using an adult's Peripheral Blood (PB), to test the ability of obtaining CD123 CAR-T from fresh or cryopreserved UCB. We obtained a cell product with a high and stable transduction efficacy, and a poorly differentiated phenotype of CAR-T cells, while retaining high cytotoxic functions in vitro and in vivo. Moreover, CAR-T produced from cryopreserved UCB are as functional as CAR-T produced from fresh UCB. Overall, these data pave the way for the clinical development of UCB-derived CAR-T. UCB CAR-T could be transferred in an autologous manner (after an UCB transplant) to reduce post-transplant relapses, or in an allogeneic setting, thanks to fewer HLA restrictions which ease the requirements for a match between the donor and recipient.

T cell-based adoptive cell transfer therapy is now clinically used to fight cancer with CD19-targeting chimeric antigen receptor T cells. The use of other T cell-based immunotherapies relying on antigen-specific T cells, genetically modified or not, is expanding in various neoplastic diseases. T cell manufacturing has evolved through sophisticated processes to produce T cells with improved therapeutic potential. Clinical-grade manufacturing processes associated with these therapies must meet pharmaceutical requirements and therefore be standardized. Here, we focus on the use of cytokines to expand minimally differentiated T cells, as well as their standardization and harmonization in research and clinical settings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization., Results: We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions., Conclusion: The validation of the method consisting in a JURKAT cell line experimental incorporation as a control material contributes to improve the Seahorse technology's robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.

Immune checkpoint blockade has proven its efficacy in hypermutated subtypes of metastatic colorectal cancers (mCRC). Immunogenic potential can also be observed with conventional chemotherapies, but this property has never been explored thoroughly in CRC patients. The CRC therapeutic arsenal includes oxaliplatin, a well-characterized platinum drug already described as immunogenic. Here, we investigated the impact of the oxaliplatin-based treatment on mCRC immunopeptidome. We demonstrated that oxaliplatin-resistant CRC cell lines overexpressed telomerase reverse transcriptase (TERT), colorectal-associated-tumor antigen-1 (COA-1) and mesothelin tumor-associated antigens. We identified new HLA class-II-restricted and promiscuous peptides derived from COA-1 and mesothelin. The two naturally processed peptides COA-1 331-345 and Meso 366-380 appear to be the most immunogenic in mCRC patients. A prospective cohort of 162 mCRC patients enabled us to explore the impact of oxaliplatin exposure on the antitumor-specific immune response. Interestingly, chemotherapy-naive mCRC patients present high immune CD4 T-cell responses directed against TERT, COA-1 and mesothelin-derived peptides. These antitumor T-cell responses were maintained after 3 months of oxaliplatin-based treatment. Altogether, these findings highlight the interest of immunostimulatory agents to improve the management of chemoresistant mCRC patients. Finally, the high frequency of immune responses targeting the new immunogenic peptides derived from COA-1 and mesothelin support their use in immunomonitoring strategies., (© 2019 UICC.)

Several distinct innate lymphoid cell (ILC) populations have been recently identified and shown to play a critical role in the immediate immune defense. In the context of tumors, there is evidence to support a dual role for ILCs with pro- or antitumor effects, depending on the ILC subset and the type of cancer. This ambivalent role has been particularly well-described in colorectal cancer models (CRC), but the presence and the evolution of ILCs in the peripheral blood of metastatic CRC (mCRC) patients have not yet been explored. Here, we investigated the distribution of ILC subsets in 96 mCRC patients who were prospectively included in the "Epitopes-CRC02" trial. Peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry at metastatic diagnosis and after 3-months of treatment. The treatments consisted of Oxaliplatin-based chemotherapies for 76% of the patients or Folfiri (5FU, Irinotecan) chemotherapies for 14% of patients. Compared to healthy donors, the frequency of total ILCs was dramatically increased at metastatic diagnosis. CD56 + ILC1-like cells were expanded, whereas ILC2, NCR - ILCP and NCR + ILCP subsets were decreased. Combined analysis with the systemic anti-telomerase hTERT Th1 CD4 response revealed that patients with low anti-TERT Th1 CD4 responses had the highest frequencies of total ILCs at diagnosis. Of those, 91% had synchronous metastases, and their median progression-free survival was 7.43 months (vs. 9.17 months for the other patients). In these patients, ILC1 and ILC2 were significantly decreased, whereas CD56 + ILC1-like cells were significantly increased compared to patients with low frequency of total ILCs and high anti-TERT responses. After treatment, the NCR + ILCP were further decreased irrespective of the chemotherapy regimen, whereas the balance between ILC1 and CD56 + ILC1-like cells was modulated mainly by the Folfiri regimen in favor of ILC1. Altogether our results describe the effects of different chemotherapies on ILCs in mCRC patients. We also establish for the first time a link between frequency of ILCs and anti-tumor CD4 T cell responses in cancer patients. Thus, our study supports an interest in monitoring ILCs during cancer therapy to possibly identify predictive biomarkers in mCRC.

Car-T Cells: BIOLOGY, CONCEPTS AND PRINCIPLES: The development of new anti-tumor immunotherapy approaches has recently dramatically increased. Progresses made in molecular biology and the development of various genetic manipulation tools allow the "reprogrammation" of T cells in order to make them express a chimeric receptor including the variable part of an immunoglobulin capable of recognizing a tumor antigen along with the expression of molecules involved in T-lymphocyte activation signaling. Genetically modified T-cells, called "CAR (chimeric antigen receptors) -T cells", have yielded impressive clinical results in the treatment of relapsed or refractory lymphoid hematological malignancies after conventional treatments and are in development in solid tumors. Different generations of CAR-T cells have been developed and technological progress makes it possible to envisage modulations of gene constructs that could further optimize the efficacy and tolerance of CAR-T cells. The first challenge of these approaches concerns the identification of specific tumor antigen targets in order to limit the on-target/off-tumor effects and the loss of expression of the target. Approaches i) targeting several antigens or ii) limiting the duration of expression of CAR in lymphocytes or iii) destroying CAR-T cells by a suicide gene. Interesting approaches are the second objective of improvement concerns the accessibility of CAR-T cells to tumor sites and the control of the immune escape mechanisms of tumor cells to the cytotoxicity of CAR-T cells. This issue is currently under the way of search of innovative strategies that should improve the clinical effectiveness of CAR-T cells, especially in solid tumors. Cet article fait partie du numéro supplément Les cellules CAR-T : une révolution thérapeutique ? réalisé avec le soutien institutionnel des partenaires Gilead : Kite et Celgene., (© 2018 Société Française du Cancer. Publié par Elsevier Masson SAS. Tous droits réservés.)

Sorafenib, a multi-targeted kinase inhibitor, is the current standard systemic treatment for advanced hepatocellular carcinoma. Sorafenib has anti-angiogenic and anti-proliferative properties and is also known to favor anti-tumor T cell responses by reducing the population of immunosuppressive cells such as Treg and MDSC. Anti-tumor immune responses, especially mediated by CD4+ T-cells, are critical for tumor cells eradication and therapies modulating those responses are appealing in a growing number of cancers. Here, we report and investigate the case of a patient diagnosed with an advanced HCC treated by sorafenib who experienced a complete histological response. We aimed to identify immunogenic peptides derived from tumor mutated proteins that stimulated CD4+ T cells responses thus favoring the exceptional recovery process of this patient. Tumor neoantigens were identified using whole exome sequencing of normal and tumor tissue and peptide MHC binding prediction algorithms. Among 442 tumor-specific somatic variants, 50 missense mutations and 20 neoepitopes predicted to bind MHC-II were identified. Candidate neoepitopes immunogenicity was assessed by IFN-γ ELISpot after culture of patient's PBMCs in presence of synthetic neopeptides. CD4+ memory T cell responses were detected against a mutated IL-1β S230F peptide and two additional neoepitopes from HELZ2 V241M and MLL2 A4458V suggesting that efficient anti-tumor immune response occurred in this patient. These results showed that T cells can recognize neoantigens and may lead to the cancer elimination after immunomodulation in the tumor-microenvironment induced by sorafenib. This observation indicates that other immunotherapies in combination with sorafenib could potentially increase the response rate in HCC at advanced stage., Competing Interests: CONFLICTS OF INTEREST The authors declare no financial or commercial conflicts of interest.

Natural killer (NK) cells are innate effector lymphocytes widely involved in cancer immunosurveillance. In this study, we described three circulating NK cell subsets in patients with non-small cell lung cancer (NSCLC). Compared to healthy donors (HD), lower rate of the cytotoxic CD56 dim CD16 + NK cells was found in NSCLC patients (76.1% vs 82.4%, P  = 0.0041). In contrast, the rate of CD56 bright NK cells was similar between patients and HD. We showed in NSCLC patients a higher rate of a NK cell subset with CD56 dim CD16 - phenotype (16.7% vs 9.9% P  = 0.0001). The degranulation property and cytokines production were mainly drive by CD56 dim CD16 - NK cell subset in patients. Analysis of natural cytotoxicity receptors (NCRs) expression identified four distinct clusters of patients with distinct NK cell subset profiles as compared to one major cluster in HD. Notably the cluster characterized by a low circulating level of NKp46 + NK cell subsets was absent in HD. We showed that the rate of circulating NKp46 + CD56 dim CD16 + NK cells influenced the patients' survival. Indeed, the median overall survival in patients exhibiting high versus low level of this NK cell subset was 16 and 27 months respectively ( P  = 0.02). Finally, we demonstrated that blocking NKp46 receptor in vitro was able to restore spontaneous tumor specific T cell responses in NSCLC patients. In conclusion, this study showed a distinct distribution and phenotype of circulating NK cell subsets in NSCLC. It also supports the regulatory role of NKp46 + NK cell subset in NSCLC patients.

High-risk human papillomavirus (HPV) infection is a causal factor in oropharyngeal and gynecological malignancies, and development of HPV-targeted immunotherapy could be used to treat patients with these cancers. T cell-mediated adoptive immunotherapy targeting E6 and E7, two HPV16 proteins consistently expressed in tumor cells, appears to be both attractive and safe. However, isolation of HPV-specific T cells is difficult owing to the low frequency of these cell precursors in the peripheral blood. In addition, HPV-positive cancer cells often down-regulate major histocompatibility complex (MHC) class I expression ex vivo, limiting the efficacy of MHC class I-restricted approaches. Of particular interest is that both CD4 and CD8 T cells can mediate the responses. Given that CD4 T cells play a critical role in coordinating effective antitumor responses, the generation of a T helper response in patients with HPV16-associated malignancies would unleash the ultimate potential of immunotherapy. In this view, T-cell receptor (TCR) gene transfer could be a relevant strategy to generate HPV16-E7-specific and MHC class II-restricted T cells in sufficient numbers. An HPV16-E7/HLA-DRB1*04 TCR has been isolated from a cancer patient with complete response, and retroviral particles encoding this TCR have been produced. The transgenic TCR is highly expressed in transduced T cells, with a functional inducible caspase-9 suicide gene safety cassette. TCR transgenic T cells are HPV16-E7 70-89 specific and HLA-DRB1*04 restricted, as determined by interferon (IFN)-γ secretion. CD8 and CD4 T cells are equivalently transduced and secrete interleukin-2 and IFN-γ when cultured with appropriate targets. We also demonstrate that TCR transgenic T cells recognize the endogenously processed and presented HPV16-E7 70-89 peptide. In conclusion, our data indicate that the production of MHC class II-restricted HPV16-E7-specific T cells is feasible through TCR gene transfer and could be used for immunotherapy.

Purpose : To investigate the immunoprevalence of SALL4-derived peptides in healthy volunteers and cancer patients. Experimental Design : A multistep approach including prediction algorithms was used to design in silico SALL4-derived peptides theoretically able to bind on common HLA-DR and HLA-A/B molecules. The presence of T-cell responses after a long term T-cell assay (28 days) against SALL4 was monitored in 14 healthy donors and the presence of T-cell responses after a short term T-cell assay (10 days) was monitored in 67 cancer patients using IFN-γ ELISPOT assay. A T-cell clone specific for the immunoprevalent A18 K-derived peptide was isolated, characterized and used as a tool to characterize the natural processing of A18 K. Results : A SALL4 specific T-cell repertoire was present in healthy donors (8/14) and cancer patients (29/67) after short term T-cell assay. We further identified two immunoprevalant SALL4-derived peptides, R18 A and A18 K, which bind MHC-class II. In parallel, an A18 K specific Th1 clone recognized monocyte derived Dendritic Cell (moDC) loaded with SALL4 containing cell lysate. The level of IFN-γ secreted by specific T-cell clone was greater in presence of moDC loaded with SALL4 containing cell lysate (49.23 ± 14.02%) than with moDC alone (18.03 ± 3.072%) (p = 0.0477) Conclusion : These results show for the first time immunogenicity of SALL4 oncogenic protein-derived peptides, especially A18 K and R18 A peptides and make them potential targets for personalized medicine. Thus, SALL4 possess major characteristics of a tumor antigen.

HLA-A*0201/DRB1*0101 transgenic mice (A2/DR1 mice) have been developed to study the immunogenicity of tumor antigen-derived T cell epitopes. To extend the use and application of this mouse model in the field of antitumor immunotherapy, we described a tumor cell line generated from a naturally occurring tumor in A2/DR1 mouse named SARC-L1. Histological and genes signature analysis supported the sarcoma origin of this cell line. While SARC-L1 tumor cells lack HLA-DRB1*0101 expression, a very low expression of HLA-A*0201 molecules was found on these cells. Furthermore they also weakly but constitutively expressed the programmed death-ligand 1 (PD-L1). Interestingly both HLA-A*0201 and PD-L1 expressions can be increased on SARC-L1 after IFN-γ exposure in vitro. We also obtained two genetically modified cell lines highly expressing either HLA-A*0201 or both HLA-A*0201/ HLA-DRB1*0101 molecules referred as SARC-A2 and SARC-A2DR1 respectively. All the SARC-L1-derived cell lines induced aggressive subcutaneous tumors in A2DR1 mice in vivo. The analysis of SARC-L1 tumor microenvironment revealed a strong infiltration by T cells expressing inhibitory receptors such as PD-1 and TIM-3. Finally, we found that SARC-L1 is sensitive to several drugs commonly used to treat sarcoma and also susceptible to anti-PD-L1 monoclonal antibody therapy in vivo. Collectively, we described a novel syngeneic tumor model A2/DR1 mice that could be used as preclinical tool for the evaluation of antitumor immunotherapies.

T cells activation is a finely regulated process to establish an effective anti-infectious or antitumor immune response while avoiding harmful autoimmune reactions. Although T cells are considered to be the main protagonists of the antitumor immune response, they act in interaction with other immune cells. The meeting of naive T cells with dendritic cells induces their differentiation into effector cells following the recognition of the peptide-MHC complex by the T cell receptor. The interaction of costimulatory molecules present on the surface of T cells with their ligand (s) expressed by mature dendritic cells contribute to the optimal T cell activation and to the formation of the immunological synapse. Conversely, engagement of inhibitory receptors expressed by T cells induces a negative feedback involved in the T cells homeostasis but also in the tumor escape from the immune system. The integration of stimulatory signals contributes to the proliferation, the survival and the differentiation of T cells whereas the inhibitory signals permit their regulation. The better understanding of T cell activation mechanisms has led to the development of therapeutic strategies aimed at stimulating the antitumor immune response or alleviating the immunosuppression., (© 2016 Société Française du Cancer. Publié par Elsevier Masson SAS. Tous droits réservés.)

Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR-restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT's uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT's internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells., (Copyright © 2016 by The American Association of Immunologists, Inc.)

NK cells are critical for innate immunity-mediated protection. The main roles of NK cells rely on their cytotoxic functions or depend on the tuning of Th1 adaptive immunity by IFN-γ. However, the precise influence of inflammatory cytokines on NK cell and CD4 T lymphocyte interactions was never investigated. In this study, we provide evidence that IL-21, a cytokine produced during chronic inflammation or infectious diseases, promotes the differentiation of a specific subset of NK cells coexpressing CD86 and HLA-DR and lacking NKp44. More importantly, IL-21-propagated HLA-DR(+) NK cells produce macrophage migration inhibitory factor and provide costimulatory signaling during naive CD4(+) T cell priming inducing the differentiation of uncommitted central memory T cells. Central memory T cells expanded in the presence of HLA-DR(+) NK cells are CXCR3(+)CCR6(-)CCR4(-)CXCR5(-) and produce IL-2, as well as low levels of TNF-α. Costimulation of CD4(+) T cells by HLA-DR(+) NK cells prevents the acquisition of effector memory phenotype induced by IL-2. Moreover, we identified this population of NK HLA-DR(+) macrophage migration inhibitory factor(+) cells in inflammatory human appendix. Collectively, these results demonstrate a novel function for IL-21 in tuning NK and CD4(+) T cell interactions promoting a specific expansion of central memory lymphocytes., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Cumulative evidence supports that CD4(+) Th1 cells play a key role in antitumor immunity. We previously reported the presence of spontaneous HLA-DR-restricted CD4(+) Th1 responses against telomerase reverse transcriptase (TERT) in various cancers by using promiscuous HLA-DR epitopes. Here, we described novel highly immunogenic HLA-DP4-binding epitopes from TERT named TERT541-555, TERT573-587, TERT613-627 and TERT911-925 and addressed the question about the immunoprevalence and magnitude of the naturally occurring antitumor CD4(+) T cell responses restricted by HLA-DP4 or HLA-DR, the two most common HLA class II. Direct comparative study of spontaneous anti-TERT CD4(+) T cell responses in a cohort of 87 lung cancer patients showed that HLA-DP4 and HLA-DR sustained specific Th1 responses in 10.1% and 25.2% of cancer patients respectively (p = 0.01). The magnitude of the HLA-DR-restricted responses was two to three times significantly higher than HLA-DP one (p = 0.005). Similar results were found in other cancers such as melanoma, breast cancer, renal cell carcinoma and colon cancer. Thus, our results describe for the first time in a large cohort of cancer patients a high immunoprevalence of HLA-DR-restricted spontaneous anti-TERT Th1 immunity compared to HLA-DP restriction. These results provide a new tool for comprehensive monitoring of antitumor CD4(+) Th1 response in various cancers.

Cancer-specific splice variants gain significant interest as they generate neo-antigens that could be targeted by immune cells. CD20, a membrane antigen broadly expressed in mature B cells and in B cell lymphomas, is subject to an alternative splicing named D393-CD20 leading to loss of membrane expression of the spliced isoform. D393-CD20 expression is detectable in transformed B cells and upregulated in various lymphoma B cells. In this study, we show that D393-CD20 is translated in malignant B cells and that D393-CD20 specific CD4 T cells producing IFN-γ are present in B-cell lymphoma patients. Then, we have investigated whether the 20mer D393-CD20 peptide spanning the splicing site might be targeted by the immune system and we have shown that D393-CD20-specific CD4 Th1 clones could directly recognize malignant B cell lines and kill autologous lymphoma B cells indicating that D393-CD20-derived epitopes are naturally processed and presented on tumor cells. Finally, D393-CD20 peptide-based vaccination induced specific CD8 and CD4 T cell responses in HLA-humanized transgenic mice suggesting the presentation of D393-CD20 derived peptides on both HLA Class-I and -II. These findings support further investigations on the potential use of D393-CD20 directed specific immunotherapy in B cell malignancies., (© 2014 UICC.)

Nowadays, immunotherapy represents one promising approach for cancer treatment. Recently, spectacular results of cancer immunotherapy clinical trials have confirmed the crucial role of immune system in cancer regression. Therapeutic cancer vaccine represents one widely used immunotherapy strategy to stimulate tumor specific T cell responses but clinical impact remains disappointing in targeting CD8 T cells. Although CD8 T cells have been initially considered to be the main protagonists, it is now clear that CD4 T cells also play a critical role in antitumor response. In this article, we discuss the role of tumor antigen-specific CD4 T cell responses and how we can target these cells to improve cancer vaccines.

Current cancer immunotherapies predominantly rely on CD8(+) T cells to fight against tumors. However accumulative evidence showed that proinflammatory CD4(+) helper T cells are critical determinants of effective antitumor immunity. The utilization of universal tumor-reactive helper peptides from telomerase represents a powerful approach to the fully use of CD4(+) T cell-based immunotherapy.

Background: Early in life, patients with cystic fibrosis (CF) are infected with microorganisms including bacteria and fungi, particularly Pseudomonas aeruginosa and Aspergillus fumigatus. Since recent research has identified the anti-inflammatory properties of statins (besides their lipid-lowering effects), we investigated the effect of fluvastatin on the production of the potent neutrophil chemoattractant chemokine, IL-8, in whole blood from CF patients, stimulated by Pseudomonas aeruginosa (LPS) and Aspergillus fumigatus (AFA) antigens., Results: Whole blood from adult patients with CF and from healthy volunteers was collected at the Rennes University Hospital (France). Blood was pretreated for 1 h with fluvastatin (0-300 µM) and incubated for 24 h with LPS (10 µg/mL) and/or AFA (diluted 1/200). IL-8 protein levels, quantified by ELISA, were increased in a concentration-dependent manner when cells were stimulated by LPS or AFA. Fluvastatin strongly decreased the levels of IL-8, in a concentration-dependent manner, in whole blood from CF patients. However, its inhibitory effect was decreased or absent in whole blood from healthy subjects. Furthermore, the inhibition induced by fluvastatin in CF whole blood was reversed in the presence of intermediates within the cholesterol biosynthesis pathway, mevalonate, farnesyl pyprophosphate or geranylgeranyl pyrophosphate that activate small GTPases by isoprenylation., Conclusions: For the first time, the inhibitory effects of fluvastatin on CF systemic inflammation may reveal the important therapeutic potential of statins in pathological conditions associated with the over-production of pro-inflammatory cytokines and chemokines as observed during the manifestation of CF. The anti-inflammatory effect could be related to the modulation of the prenylation of signalling proteins.