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A patient with a PD-L1-negative, TMB-low, KEAP1/STK11 co-mutated metastatic non-small cell lung cancer (NSCLC) experienced a multisite radiological progression at 3 months after initiation of chemoimmunotherapy as first-line treatment for metastatic disease. After the radiological progression, while she was not undergoing treatment, the patient had spontaneous lesions shrinkage and further achieved a prolonged complete response. Genomic and transcriptomic data collected at baseline and at the time of pseudoprogression allowed us to biologically characterize this rare response pattern. We observed the presence of a tumor-specific T-cell response against tumor-specific neoantigens (TNAs). Endogenous retroviruses (ERVs) expression following chemoimmunotherapy was also observed, concurrent with biological features of an anti-viral-like innate immune response with type I IFN signaling and production of CXCR3-associated chemokines. This is the first biological characterization of a NSCLC pseudoprogression under chemoimmunotherapy followed by a prolonged complete response in a PD-L1-negative, TMB-low, KEAP1/STK11 co-mutated NSCLC. These clinical and biological data underline that even patients with multiple factors of resistance to immune checkpoint inhibitors could trigger a tumor-specific immune response to tumor neoantigen, leading to complete eradication of the tumor and probably a vaccinal immune response., 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 Roussot, Thibaudin, Fumet, Daumoine, Hampe, Rébé, Limagne, Lagrange, Herreros, Lecuelle, Mananet, Ilie, Rageot, Boidot, Goussot, Comte, Jacob, Beltjens, Bergeron, Charon-Barra, Arnould, Derangère, Ladoire, Truntzer and Ghiringhelli.)

Background: Glioblastoma (GBM) is a primary brain tumor with a dismal prognosis, often resistant to immunotherapy and associated with immune suppression. This study aimed to assess the impact of steroids and Stupp-regimen treatment on peripheral blood immune parameters in GBM patients and their association with outcomes., Methods: Using cytometry panels and bioplex assays, we analyzed the immune phenotype and serum cytokines of 54 GBM patients and 21 healthy volunteers., Results: GBM patients exhibited decreased lymphoid cell numbers (CD4, CD8 T cells, NKT cells) with heightened immune checkpoint expression and increased myeloid cell numbers (especially neutrophils), along with elevated pro-inflammatory cytokine levels. Steroid use decreased T and NK cell numbers, while radio-chemotherapy led to decreased lymphoid cell numbers, increased myeloid cell numbers, and heightened immune checkpoint expression. Certain immune cell subsets were identified as potential outcome predictors., Conclusion: Overall, these findings shed light on the peripheral immune landscape in GBM, emphasizing the immunosuppressive effects of treatment. Baseline immune parameters may serve as prognostic indicators for treatment response., (© 2024. The Author(s).)

Here, we present a protocol for the simultaneous analysis of peripheral and intratumoral lymphocyte function by flow cytometry. Using tumor and blood samples from patients with colorectal cancer, we describe steps for tumor digestion, pre-labeling of the tumor-infiltrating leukocytes (TILs), and activation and labeling of the total cells (TILs and whole blood cells). For complete details on the use and execution of this protocol, please refer to Thibaudin et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although patients with microsatellite instable metastatic colorectal cancer (CRC) benefit from immune checkpoint blockade, chemotherapy with targeted therapies remains the only therapeutic option for microsatellite stable (MSS) tumors. The single-arm, phase 1b/2 MEDITREME trial evaluated the safety and efficacy of durvalumab plus tremelimumab combined with mFOLFOX6 chemotherapy in first line, in 57 patients with RAS-mutant unresectable metastatic CRC. Safety was the primary objective of phase Ib; no safety issue was observed. The phase 2 primary objective of efficacy in terms of 3-month progression-free survival (PFS) in patients with MSS tumors was met, with 3-month PFS of 90.7% (95% confidence interval (CI): 79.2-96%). For secondary objectives, response rate was 64.5%; median PFS was 8.2 months (95% CI: 5.9-8.6); and overall survival was not reached in patients with MSS tumors. We observed higher tumor mutational burden and lower genomic instability in responders. Integrated transcriptomic analysis underlined that high immune signature and low epithelial-mesenchymal transition were associated with better outcome. Immunomonitoring showed induction of neoantigen and NY-ESO1 and TERT blood tumor-specific T cell response associated with better PFS. The combination of durvalumab-tremelimumab with mFOLFOX6 was tolerable with promising clinical activity in MSS mCRC. Clinicaltrials.gov identifier: NCT03202758 ., (© 2023. The Author(s).)

Microsatellite stable colorectal cancers (MSS-CRC) are resistant to anti-PD-1/PD-L1 therapy but the combination of immune checkpoints inhibitors (ICI) could be a clue to reverse resistance. Our aim was to evaluate ex vivo the capacity of the combination of atezolizumab (anti-PD-L1) and tiragolumab (anti-TIGIT) to reactivate the immune response of tumor infiltrating lymphocytes (TILs) in MSS-CRC. We analysed CRC tumor tissue and the associated blood sample in parallel. For each patient sample, extensive immunomonitoring and cytokine production were tested. We generated an ex vivo assay to study immune reactivity following immune stimulation with checkpoint inhibitors of tumor cell suspensions. Three microsatellite instable (MSI) and 13 MSS-CRC tumors were analysed. To generalize our observations, bioinformatics analyses were performed on public data of single cell RNA sequencing of CRC TILs and RNA sequencing data of TCGA. Atezolizumab alone could only reactivate T cells from MSI tumors. Atezolizumab and tiragolumab reactivated T cells in 46% of MSS-CRC samples. Reactivation by ICK was observed in patients with higher baseline frequency of Th1 and Tc1 cells, and was also associated with higher baseline T cell polyfunctionality and higher CD96 expression. We showed that a high frequency of CD96 expression on T cells could be a surrogate marker of atezolizumab and tiragolumab efficacy. Together these data suggest that the association of atezolizumab and tiragolumab could restore function of CD4 and CD8 TILs in MSS-CRC and could be tested in a clinical trial in colorectal cancer patients with MSS status., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: T follicular helper cells (Tfh) are essential to shape B cell response during germinal center formation. Tfh accumulation has been reported in various human cancers, with positive or negative prognostic roles. However, the mechanisms explaining the accumulation of Tfh and their role in cancer remain obscure., Methods: In vitro differentiated and mouse cell sorted Tfh phenotype was evaluated by flow cytometry and quantitative PCR (qPCR). Antitumor effect of Tfh was evaluated by adoptive transfer in different tumor-bearing mice models. The involvement of immune cells, cytokines and chemokines was evaluated, using depleting antibodies. Chemokines and cytokines expression and production were evaluated by qPCR and ELISA. In human, the impact of immune cells and chemokines on survival was evaluated by analyzing transcriptomic data from public databases and from our own patient cohorts., Results: In this study, we show that Tfh exert an antitumor immune effect in a CD8 + -dependent manner. Tfh produce interleukin-21, which sustains proliferation, viability, cytokine production and cytotoxic functions of exhausted T cells. The presence of Tfh is required for efficacy of antiprogrammed cell death ligand-1 therapy. Tfh accumulate in the tumor bed and draining lymph nodes in different mouse cancer models. This recruitment is due to the capacity of transforming growth factor β to drive Chemokine (C-X-C motif) Ligand 13 expression, a chemoattractant of Tfh, by intratumor CD8 + T cells. Accumulation of Tfh and exhausted CD8 + T cells predicts cancer outcome in various cancer types. In patients treated with anti-programmed cell death-1 mAb, accumulation of Tfh and CD8 + at the tumor site is associated with outcome., Conclusion: This study provides evidence that CD8 + /Tfh crosstalk is important in shaping antitumor immune response generated by immunotherapy., Competing Interests: Competing interests: FG received speaker honoraria from Lilly, Sanofi, BMS, Astra Zeneca and Amgen, received funding for clinical trials from Astra Zeneca, received travel grants from Roche France, Amgen and Servier, and is an advisory board member for Merck Serano, Amgen, Roche France and Sanofi., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has caused a major pandemic. Patients with cancer are at higher risk of severe COVID-19. We aimed to describe and compare the immunological features of cancer patients hospitalised for COVID-19 or other concomitant, cancer-related illness., Methods: In this prospective study, the clinical and immunological characteristics of 11 cancer patients with COVID-19 and 11 non-COVID-19 cancer patients hospitalised in the same unit at the same period for other medical issues were analysed. We also used 10 healthy volunteers as controls. Peripheral immune parameters were analysed using multiparametric flow cytometry., Results: The median age of COVID-19-positive cancer patients was 71.1 years, and 66.4 years for controls. Compared with non-COVID-19 cancer patients, COVID-19-positive cancer patients had more extensive lymphopenia and hypoalbuminemia, with higher levels of C-reactive protein. In COVID-19 patients, elevated procalcitonin was associated with a higher risk of death. By phenotypic analysis, COVID-19-positive patients presented CD3 lymphopenia, with inversion of the CD4/CD8 ratio and modification of monocyte activation, with accumulation of mMDSC (monocytic Myeloid-Derived Suppressor Cells) -like cells and a decrease in activated monocytes. Analysis of the T-cell compartment revealed a T-dependent inflammatory response with accumulation of Th17 cells and cytotoxic CD8 T cells producing TNFα, a decrease in HLA-DR (Human Leukocyte Antigen - DR isotype)-positive CD8 T cells and Treg/CD8 ratio., Conclusion: SARS-CoV-2 infection in cancer patients is associated with CD4 T-cell lymphopenia with induction of an inflammatory T-cell response, accumulation of IFNγ + TNFα + CD8 T and Th17 cells, and a concomitant modification of monocyte activation status., Competing Interests: Conflict of interest statement F. Ghiringhelli reports receiving honoraria for oral communications from Lilly, Sanofi, Bristol-Myers Squibb, Astra Zeneca and Amgen; receiving funding for clinical trials from Astra Zeneca; receiving travel grants from Roche France, Amgen and Servier; and being an advisory board member for Merck Serano, Amgen, Roche France and Sanofi. The other authors have no potential conflict of interest to disclose., (Copyright © 2020. Published by Elsevier Ltd.)

Background and Purpose: Adiponectin, an adipokine possessing profound insulin-sensitizing and anti-inflammatory properties, is a potent biotherapeutic agent . The trimeric adiponectin subunit assembles into hexameric and functionally important higher molecular weight (HMW) forms, controlled by the endoplasmic reticulum protein 44 (ERp44). Obesity-induced ER stress decreases the HMW form in serum, contributing to the development of insulin resistance and Type 2 diabetes. In this study, a panel of synthetic peptides, designed to target ERp44-adiponectin interactions, were tested for their effects on circulating levels of HMW adiponectin., Experimental Approach: Peptides derived from the ERp44 binding region of adiponectin and immunoglobulin IgM were synthesized with or without a cell-penetrating sequence. Cultures of 3T3-L1 adipocytes were incubated with the peptides for assessing the assembly and secretion of HMW adiponectin. Mice given standard chow or a high-fat diet were treated acutely or chronically, with the peptides to investigate the therapeutic effects on insulin sensitivity and energy metabolism., Results: The designed peptides interfered with ERp44-adiponectin interactions and modulated adiponectin assembly and release from adipocytes. In particular, IgM-derived peptides facilitated the release of endogenous adiponectin (especially the HMW form) from adipose tissue, enhanced its circulating level and the ratio of HMW-to-total-adiponectin in obese mice. Long-term treatment of mice fed with high-fat diet by IgM-derived peptides reduced the circulating lipid levels and improved insulin sensitivity., Conclusions and Implications: Targeting ERp44-adiponectin interactions with short peptides represents an effective strategy to treat of obesity-related metabolic disorders, such as insulin resistance and Type 2 diabetes., (© 2017 The British Pharmacological Society.)

The human papillomavirus (HPV) capsid protein L2 is essential for viral entry. To gain a deeper understanding of the role of L2, we searched for novel cellular L2-interacting proteins. A yeast two-hybrid analysis uncovered the actin-depolymerizing factor gelsolin, the membrane glycoprotein dysadherin, the centrosomal protein 68 (Cep68), and the cytoskeletal adaptor protein obscurin-like 1 protein (OBSL1) as putative L2 binding molecules. Pseudovirus (PsV) infection assays identified OBSL1 as a host factor required for gene transduction by three oncogenic human papillomavirus types, HPV16, HPV18, and HPV31. In addition, we detected OBSL1 expression in cervical tissue sections and noted the involvement of OBSL1 during gene transduction of primary keratinocytes by HPV16 PsV. Complex formation of HPV16 L2 with OBSL1 was demonstrated in coimmunofluorescence and coimmunoprecipitation studies after overexpression of L2 or after PsV exposure. We observed a strong colocalization of OBSL1 with HPV16 PsV and tetraspanin CD151 at the plasma membrane, suggesting a role for OBSL1 in viral endocytosis. Indeed, viral entry assays exhibited a reduction of viral endocytosis in OBSL1-depleted cells. Our results suggest OBSL1 as a novel L2-interacting protein and endocytosis factor in HPV infection., Importance: Human papillomaviruses infect mucosal and cutaneous epithelia, and the high-risk HPV types account for 5% of cancer cases worldwide. As recently discovered, HPV entry occurs by a clathrin-, caveolin-, and dynamin-independent endocytosis via tetraspanin-enriched microdomains. At present, the cellular proteins involved in the underlying mechanism of this type of endocytosis are under investigation. In this study, the cytoskeletal adaptor OBSL1 was discovered as a previously unrecognized interaction partner of the minor capsid protein L2 and was identified as a proviral host factor required for HPV16 endocytosis into target cells. The findings of this study advance the understanding of a so far less well-characterized endocytic pathway that is used by oncogenic HPV subtypes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys(39) located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12-18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys(39) and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The chemical synthesis is described of a polypeptide construct possessing both the variable and the collagen-like domain of adiponectin, which can be used as a model system for probing the influence of the variable domain on multimerization of this important circulating hormone. Using a collagen domain repeat peptide unit derived from native adiponectin or a glutamic acid analogue was ineffective due to noncollagenous conformational properties in both cases. However, employing a collagen model peptide and linking this to the variable domain thioester peptide using native chemical ligation proved effective. The 63 residue peptide was characterized by circular dichroism and mass spectrometry which demonstrated that a collagen-like triple-helical structure was preserved., (© 2014 Wiley Periodicals, Inc.)