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Anti-PD-1, trastuzumab, and chemotherapy are used in the treatment of patients with advanced HER2-positive esophagogastric adenocarcinoma (EGA), but long-term survival remains limited. Herein, we report extended follow-up data from the INTEGA trial (NCT03409848), which investigated the efficacy of the anti-PD-1 nivolumab, trastuzumab, and FOLFOX chemotherapy (FOLFOX arm) in comparison to a chemotherapy-free regimen involving nivolumab, trastuzumab, and the anti-CTLA-4 ipilimumab (Ipi arm) in the first-line setting for advanced disease. The 12-month overall survival (OS) showed no statistical difference between the arms, with 57% OS (95% CI: 41%-71%) in the Ipi arm and 70% OS (95% CI: 54%-82%) in the FOLFOX arm. Crossing of the survival curves indicated a potential long-term benefit for some patients within the Ipi arm, but early progressors in the Ipi arm underlined the need for biomarker-guided strategies to optimize treatment selection. To this end, metabolomic and cytokine analysis demonstrated elevated levels of normetanephrine, cortisol, and interleukin 6 (IL-6) in immunotherapy-unresponsive patients in the Ipi arm, suggesting a role for systemic inflammatory stress in modulating antitumor immune responses. Patients with this signature also showed an increased neutrophil-to-lymphocyte ratio (NLR) that persisted in the Ipi arm, but not in the FOLFOX arm, and strongly correlated with survival. Furthermore, a low NLR characterized patients benefiting from immune- and targeted therapy without the need for additional chemotherapy. This data suggests that patient selection based on inflammatory stress-driven immune changes could help to customize first-line treatment in patients with advanced HER2-positive EGA to potentially improve long-term survival.

While T cell lymphomas are classified as mature neoplasms, emerging evidence indicates that malignant transformation may occur at an earlier stage of T cell maturation. In this study, we determined clonal architectures in a broad range of T cell lymphomas. Our multidimensional profiling indicates that a large part of these lymphomas in fact emerge from an immature lymphoid T cell precursor at a maturation stage prior to V(D)J rearrangement that undergoes branching evolution. Consequently, at single cell resolution we observed considerable clonal tiding under selective therapeutic pressure. T cell receptor next-generation sequencing suggested a highly biased usage of TRBV20-1 gene segments as part of multiple antigen receptor rearrangements per patient. The predominance of TRBV20-1 was found across all major T cell lymphoma subtypes analyzed. This suggested that this particular V gene - independently of complementarity-determining region 3 (CDR3) configuration - may represent a driver of malignant transformation. Together, our data indicate that T cell lymphomas derive from immature lymphoid precursors and display considerable intratumoral heterogeneity that may provide the basis for relapse and resistance in these hard-to-treat cancers.

Genetic TNFAIP3 (A20) inactivation is a classical somatic lymphoma lesion and the genomic trait in haploinsufficiency of A20 (HA20). In a cohort of 34 patients with HA20, we show that heterozygous TNFAIP3 loss skews immune repertoires toward lymphocytes with classical self-reactive antigen receptors typically found in B and T cell lymphomas. This skewing was mediated by a feed-forward tumor necrosis factor (TNF)/A20/nuclear factor κB (NF-κB) loop that shaped pre-lymphoma transcriptome signatures in clonally expanded B ( CD81 , BACH2 , and NEAT1 ) or T ( GATA3 , TOX , and PDCD1 ) cells. The skewing was reversed by anti-TNF treatment but could also progress to overt lymphoma. Analysis of conditional TNFAIP3 knock-out mice reproduced the wiring of the TNF/A20/NF-κB signaling axis with permissive antigen receptors and suggested a distinct regulation in B and T cells. Together, patients with the genetic disorder HA20 provide an exceptional window into A20/TNF/NF-κB-mediated control of immune homeostasis and early steps of lymphomagenesis that remain clinically unrecognized.

Background: Chronic HCV infection leads to a complex interplay with adaptive immune cells that may result in B cell dyscrasias like cryoglobulinemia or lymphoma. While direct-acting antiviral therapy has decreased the incidence of severe liver damage, its effect on extrahepatic HCV manifestations such as B cell dyscrasias is still unclear., Methods: We sequenced B cell receptor (BCR) repertoires in patients with chronic HCV mono-infection and patients with HCV with a sustained virological response (SVR) after direct-acting antiviral therapy. This data set was mined for highly neutralizing HCV antibodies and compared to a diffuse large B cell lymphoma data set. The TKO model was used to test the signaling strength of selected B-BCRs in vitro. Single-cell RNA sequencing of chronic HCV and HCV SVR samples was performed to analyze the transcriptome of B cells with HCV-neutralizing antigen receptors., Results: We identified a B cell fingerprint with high richness and somatic hypermutation in patients with chronic HCV and SVR. Convergence to specific immunoglobulin genes produced high-connectivity complementarity-determining region 3 networks. In addition, we observed that IGHV1-69 CDR1 and FR3 mutations characterizing highly neutralizing HCV antibodies corresponded to recurrent point mutations found in clonotypic BCRs of high-grade lymphomas. These BCRs did not show autonomous signaling but a lower activation threshold in an in vitro cell model for the assessment of BCR signaling strength. Single-cell RNA sequencing revealed that B cells carrying these point mutations showed a persisting oncogenic transcriptome signature with dysregulation in signaling nodes such as CARD11, MALT1, RelB, MAPK, and NFAT., Conclusions: We provide evidence that lymphoma-like cells derive from the anti-HCV immune response. In many patients, these cells persist for years after SVR and can be interpreted as a mechanistic basis for HCV-related B cell dyscrasias and increased lymphoma risk even beyond viral elimination., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

The classification of B cell lymphomas-mainly based on light microscopy evaluation by a pathologist-requires many years of training. Since the B cell receptor (BCR) of the lymphoma clonotype and the microenvironmental immune architecture are important features discriminating different lymphoma subsets, we asked whether BCR repertoire next-generation sequencing (NGS) of lymphoma-infiltrated tissues in conjunction with machine learning algorithms could have diagnostic utility in the subclassification of these cancers. We trained a random forest and a linear classifier via logistic regression based on patterns of clonal distribution, VDJ gene usage and physico-chemical properties of the top-n most frequently represented clonotypes in the BCR repertoires of 620 paradigmatic lymphoma samples-nodular lymphocyte predominant B cell lymphoma (NLPBL), diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL)-alongside with 291 control samples. With regard to DLBCL and CLL, the models demonstrated optimal performance when utilizing only the most prevalent clonotype for classification, while in NLPBL-that has a dominant background of non-malignant bystander cells-a broader array of clonotypes enhanced model accuracy. Surprisingly, the straightforward logistic regression model performed best in this seemingly complex classification problem, suggesting linear separability in our chosen dimensions. It achieved a weighted F1-score of 0.84 on a test cohort including 125 samples from all three lymphoma entities and 58 samples from healthy individuals. Together, we provide proof-of-concept that at least the 3 studied lymphoma entities can be differentiated from each other using BCR repertoire NGS on lymphoma-infiltrated tissues by a trained machine learning model., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schmidt-Barbo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Optimizing functional outcomes and securing long-term remissions are key goals in managing patients with locally advanced rectal cancer. In this proof-of-concept study, we set out to further optimize neoadjuvant therapy by integrating the radiosensitizer trifluridine/tipiracil and explore the potential of cell free tumor DNA (ctDNA) to monitor residual disease., Methods: About 10 patients were enrolled in the phase I dose finding part which followed a 3 + 3 dose escalation design. Tipiracil/trifluridine was administered concomitantly to radiotherapy. ctDNA monitoring was performed before and after chemoradiation with patient-individualized digital droplet PCRs., Results: No dose-limiting toxicities were observed at the maximum tolerated dose level of 2 × 35 mg/m² trifluridine/tipiracil. There were 9 grade 3 adverse events, of which 8 were hematologic with anemia and leukopenia. Chemoradiation yielded a pathological complete response in 1 out of 8 assessable patients, downstaging in nearly all patients, and 1 clinical complete response referred for watchful waiting. Three of 4 assessable patients with residual tumor cells at pathological assessment remained liquid biopsy positive after chemoradiation, but 1 turned negative., Conclusion: In this exploratory phase I trial, the novel combination of neoadjuvant trifluridine/tipiracil and radiotherapy proved to be feasible, tolerable, and effective. However, the application of liquid biopsy as a potential marker for therapeutic de-escalation in the neoadjuvant setting requires additional research and prospective validation. The trial was registered at ClinicalTrials.gov: NCT04177602., Competing Interests: Disclosures Alexander Stein: Institutional research funding and advisory role Servier; Jörn Rüssel: Advisory role Servier; Marianne Sinn: Advisory role: Servier/ Astra Zeneca/ Amgen/ MSD/ Sanofi, honoraria: Servier/ BMS/ Incyte/ Pfizer, research funding: Servier/ Astra Zeneca/ Bayer/ BMS/ MSD/ Roche/ Amgen/ Incyte/ Pierre Fabre (institution); Mascha Binder: Institutional research funding and advisory role Servier; All other authors report no conflict of interest related to this trial., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although natural killer (NK) cells are recognized for their modulation of immune responses, the mechanisms by which human NK cells mediate immune regulation are unclear. Here, we report that expression of human leukocyte antigen (HLA)-DP, a ligand for the activating NK cell receptor NKp44, is significantly upregulated on CD8 + effector T cells, in particular in human cytomegalovirus (HCMV) + individuals. HLA-DP + CD8 + T cells expressing NKp44-binding HLA-DP antigens activate NKp44 + NK cells, while HLA-DP + CD8 + T cells not expressing NKp44-binding HLA-DP antigens do not. In line with this, frequencies of HLA-DP + CD8 + T cells are increased in individuals not encoding for NKp44-binding HLA-DP haplotypes, and contain hyper-expanded CD8 + T cell clones, compared to individuals expressing NKp44-binding HLA-DP molecules. These findings identify a molecular interaction facilitating the HLA-DP haplotype-specific editing of HLA-DP + CD8 + T cell effector populations by NKp44 + NK cells and preventing the generation of hyper-expanded T cell clones, which have been suggested to have increased potential for autoimmunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Globally, head and neck squamous cell carcinoma (HNSCC) is the seventh most common malignancy. Despite aggressive multimodal treatment approaches, recurrent and/or metastatic (R/M) disease develops in >50% of patients. In this setting, pembrolizumab was approved for patients with PD-L1 expression. However, response rates with checkpoint inhibitor monotherapy remain limited and strategies to strengthen tumor-directed immune responses are needed., Objective: The FOCUS trial is designed to estimate the effectiveness of UV1 vaccination in combination with pembrolizumab versus pembrolizumab as a single agent in patients with R/M HNSCC., Methods and Analysis: The FOCUS trial is a two-armed, randomized, multicenter phase II study which was designed to evaluate the efficacy and feasibility of the hTERT-targeted cancer vaccine UV1 as add-on to pembrolizumab in the 1st line treatment of patients with R/M PD-L1 positive (combined positive score ≥1) HNSCC. Secondary objectives are the exploration of patient subgroups most likely deriving benefit from this novel combination and the establishment of liquid biopsy tumor monitoring in HNSCC., Ethics and Dissemination: This clinical study was designed and will be conducted in compliance with Good Clinical Practice and in accordance with the Declaration of Helsinki. It is intended to publish the results of this study in peer-reviewed scientific journals and to present its content at academic conferences., Conclusions: A significant number of patients with R/M HNSCC are frail and may not tolerate chemotherapy, these patients may only be suitable for pembrolizumab monotherapy. However, long term disease stabilizations remain the exception and there is a need for the development of efficacious combination regimens for this patient population. The FOCUS study aims to optimize treatment of R/M HNSCC patients with this promising new treatment approach., Clinical Trial Registration: https://clinicaltrials.gov/study/NCT05075122, identifier NCT05075122., Competing Interests: Author AH was employed by the company Clinical Cancer Research Consulting CCRC. AS received research funding from MSD and serves as an advisory board member for MSD. UM-R serves as a consultant or advisor and/or received honoraria from AstraZeneca, BioNTech, BMS, KuraOncology, Merck, MSD, Novartis, and Sanofi. JA serves as an advisor for AstraZeneca, MSD, Novartis, Roche, BMS, Janssen, and Merck and received honoraria from AstraZeneca, BMS, Roche, and Boehringer Ingelheim. KK serves as a consultant or advisor and/or received honoraria from MSD, Merck, BMS, Roche, Novartis, Sanofi, Bayer, BioNTech, Boehringer Ingelheim, and onkowissen. 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Brandt, Schultheiss, Klinghammer, Schafhausen, Busch, Blaurock, Hinke, Tometten, Dietz, Müller-Richter, Hahn, Alt, Stein and Binder.)

The rapid development of safe and effective vaccines helped to prevent severe disease courses after SARS-CoV-2 infection and to mitigate the progression of the COVID-19 pandemic. While there is evidence that vaccination may reduce the risk of developing post-COVID-19 conditions (PCC), this effect may depend on the viral variant. Therapeutic effects of post-infection vaccination have been discussed but the data for individuals with PCC remains inconclusive. In addition, extremely rare side effects after SARS-CoV-2 vaccination may resemble the heterogeneous PCC phenotype. Here, we analyze the plasma levels of 25 cytokines and SARS-CoV-2 directed antibodies in 540 individuals with or without PCC relative to one or two mRNA-based COVID-19 vaccinations as well as in 20 uninfected individuals one month after their initial mRNA-based COVID-19 vaccination. While none of the SARS-CoV-2 naïve individuals reported any persisting sequelae or exhibited PCC-like dysregulation of plasma cytokines, we detected lower levels of IL-1β and IL-18 in patients with ongoing PCC who received one or two vaccinations at a median of six months after infection as compared to unvaccinated PCC patients. This reduction correlated with less frequent reporting of persisting gastrointestinal symptoms. These data suggest that post-infection vaccination in patients with PCC might be beneficial in a subgroup of individuals displaying gastrointestinal symptoms., (© 2024. The Author(s).)

The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21 R110 ) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21 R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21 G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21 R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells., (© 2024. The Author(s).)

Objectives: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Although an acute SARS-CoV-2 infection mainly presents with respiratory illness, neurologic symptoms and sequelae are increasingly recognised in the long-term treatment of COVID-19 patients. The pathophysiology and the neuropathogenesis behind neurologic complications of COVID-19 remain poorly understood, but mounting evidence points to endothelial dysfunction either directly caused by viral infection or indirectly by inflammatory cytokines, followed by a local immune response that may include virus-specific T cells. However, the type and role of central nervous system-infiltrating T cells in COVID-19 are complex and not fully understood., Methods: We analysed distinct anatomical brain regions of patients who had deceased as a result of COVID-19-associated pneumonia or complications thereof and performed T cell receptor Vβ repertoire sequencing. Clonotypes were analysed for SARS-CoV-2 association using public TCR repertoire data., Results: Our descriptive study demonstrates that SARS-CoV-2-associated T cells are found in almost all brain areas of patients with fatal COVID-19 courses. The olfactory bulb, medulla and cerebellum were brain regions showing the most SARS-CoV-2 specific sequence patterns. Neuropathological workup demonstrated primary CD8 + T-cell infiltration with a perivascular infiltration pattern., Conclusion: Future research is needed to better define the relationship between T-cell infiltration and neurological symptoms and its long-term impact on patients' cognitive and mental health., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Importance: Adding immune checkpoint inhibitors to chemotherapy has been associated with improved outcomes in metastatic esophagogastric adenocarcinoma, but treatment combinations and optimal patient selection need to be established., Objective: To investigate the efficacy and tolerability of the programmed cell death ligand 1 (PDL-1) inhibitor avelumab with paclitaxel plus ramucirumab., Design, Setting, and Participants: This multicenter, single-group, phase 2 nonrandomized controlled trial was conducted among patients with second-line metastatic esophagogastric adenocarcinoma. Patients pretreated with platinum plus fluoropyrimidine between April 2019 and November 2020 across 10 German centers (median follow-up, 27.4 months [95% CI 22.0-32.9 months]) were included. Data analysis was performed from January to December 2022., Interventions: Patients received ramucirumab at 8 mg/kg on days 1 and 15, avelumab at 10 mg/kg on days 1 and 15, and paclitaxel at 80 mg/m2 on days 1, 8, and 15 every 4 weeks., Main Outcomes and Measures: The prespecified primary end point was overall survival (OS) rate at 6 months, with the experimental therapy considered insufficiently active with an OS rate of 50% or less and a promising candidate with an OS rate of 65% or greater., Results: Of 60 enrolled patients, 59 patients (median [range] age, 64 [18-81] years; 47 males [70.7%]) were evaluable, including 30 patients with metastatic adenocarcinoma of the stomach and 29 patients with gastroesophageal junction. All patients were pretreated with platinum plus fluoropyrimidine, and 40 patients (67.8%) had received prior taxanes; 24 of 56 evaluable patients (42.9%) had a PDL-1 combined positive score (CPS) of 5 or greater, centrally assessed. The OS rate at 6 months was 71.2% (95% CI, 61.5%-83.7%). The median OS in the intention-to-treat population (59 patients) was 10.6 months (95% CI, 8.4-12.8 months) overall. Among patients assessable by central pathology, median OS was 9.4 months (95% CI, 7.2-11.7 months) in 32 patients with a PDL-1 CPS less than 5 and 14.0 months (95% CI, 6.0-22.1 months) in 24 patients with a PDL-1 CPS of 5 or greater (P = .25). Treatment was generally well tolerated, without unexpected toxicities. Patients with higher vs lower than median T cell repertoire richness showed an increased median OS of 20.4 months (95% CI, 7.7-33.0 months) compared with 8.3 months (95% CI, 3.7-12.9 months; hazard ratio, 0.43; 95% CI, 0.23-0.81; P = .008). Patients with lower vs higher than median cell-free DNA burden had a median OS of 19.2 months (95% CI, 8.9-29.6 months) compared with 7.3 months (95% CI, 3.2-11.4 months; hazard ratio, 0.30; 95% CI, 0.16-0.59; P < .001)., Conclusions and Relevance: In this study, the combination of avelumab with paclitaxel plus ramucirumab showed favorable efficacy and tolerability in the second-line treatment for metastatic esophagogastric adenocarcinoma. A PDL-1 CPS score of 5 or greater, cell-free DNA level less than the median, and T cell repertoire richness greater than the median were associated with increased median OS., Trial Registration: ClinicalTrials.gov Identifier: NCT03966118.

Autoreactive B cells are considered pathogenic drivers in many autoimmune diseases; however, it is not clear whether autoimmune B cells are invariably pathogenic or whether they can also arise as bystanders of T cell-driven autoimmune pathology. Here, we studied the B cell response in an autoantigen- and CD4 + T cell-driven model of autoimmune hepatitis (AIH), the Alb-iGP&#95;Smarta mouse in which expression of a viral model antigen (GP) in hepatocytes and its recognition by GP-specific CD4 + T cells causes spontaneous AIH-like disease. T cell-driven AIH in Alb-iGP&#95;Smarta mice was marked by autoantibodies and hepatic infiltration of plasma cells and B cells, particularly of isotype-switched memory B cells, indicating antigen-driven selection and activation. Immunosequencing of B cell receptor repertoires confirmed B cell expansion selectively in the liver, which was most likely driven by the hepatic GP model antigen, as indicated by branched networks of connected sequences and elevated levels of IgG antibodies to GP. However, intrahepatic B cells did not produce increased levels of cytokines and their depletion with anti-CD20 antibody did not alter the CD4 + T cell response in Alb-iGP&#95;Smarta mice. Moreover, B cell depletion did not prevent spontaneous liver inflammation and AIH-like disease in Alb-iGP&#95;Smarta mice. In conclusion, selection and isotype-switch of liver-infiltrating B cells was dependent on the presence of CD4 + T cells recognizing liver antigen. However, recognition of hepatic antigen by CD4 + T cells and CD4 + T cell-mediated hepatitis was not dependent on B cells. Thus, autoreactive B cells can be bystanders and need not be drivers of liver inflammation in AIH., (© 2023 The Authors. Immunology published by John Wiley & Sons Ltd.)

We set out to gain insight into peripheral blood B and T cell repertoires from 120 infants of the LoewenKIDS birth cohort to investigate potential determinants of early life respiratory infections. Low antigen-dependent somatic hypermutation of B cell repertoires, as well as low T and B cell repertoire clonality, high diversity, and high richness especially in public T cell clonotypes reflected the immunological naivety at 12 months of age when high thymic and bone marrow output are associated with relatively few prior antigen encounters. Infants with inadequately low T cell repertoire diversity or high clonality showed higher numbers of acute respiratory infections over the first 4 years of life. No correlation of T or B cell repertoire metrics with other parameters such as sex, birth mode, older siblings, pets, the onset of daycare, or duration of breast feeding was noted. Together, this study supports that-regardless of T cell functionality-the breadth of the T cell repertoire is associated with the number of acute respiratory infections in the first 4 years of life. Moreover, this study provides a valuable resource of millions of T and B cell receptor sequences from infants with available metadata for researchers in the field., (© 2023. The Author(s).)

Background: The addition of nivolumab to trastuzumab and chemotherapy in first-line unresectable or metastatic HER2 positive esophagogastric adenocarcinoma (HER2+ EGA) results in long progression-free and overall survival as shown by the INTEGA (ipilimumab or FOLFOX in combination with nivolumab and trastuzumab in HER2 positive esophagogastric adenocarcinoma) trial. This trial suggested that the chemotherapy backbone is needed in an unselected HER2+ patient population. Yet, it remains an open question if there are specific patient subsets that may benefit from an enhanced immunotherapeutic but chemotherapy-free approach., Methods: We analyzed blood T cell repertoire metrics determined by next-generation sequencing, circulating tumor cell (CTC) counts detected by CellSearch and their expression of HER2 and PD-L1 as potential liquid biomarkers predicting outcomes on ipilimumab versus FOLFOX (folinic acid, FOL, fluorouracil, F, oxaliplatin, OX) chemotherapy added to a backbone of trastuzumab and nivolumab in patients with HER2+ EGA in the INTEGA trial population., Results: Patients with two out of three baseline-determined liquid biomarkers-high T cell repertoire richness, absence of CTCs or HER2-expression on CTCs-made up approximately 44% of HER2+ EGA cases and did not show compromise in efficacy if treated with a chemotherapy-free regimen. Long-term responders showing a progression-free survival of >12 months were enriched in this biomarker triad, especially if treated on the chemotherapy-free arm., Conclusion: Prospective validation of this liquid biomarker triad is needed to molecularly define HER2+ EGA patient subsets with different needs in the first-line systemic treatment setting., Competing Interests: Competing interests: AS received institutional research funding for this trial from and serves as an advisory board member for Bristol-Myers Squibb. All other authors report no potential conflict of interest related to this trial., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces B and T cell responses, contributing to virus neutralization. In a cohort of 2,911 young adults, we identified 65 individuals who had an asymptomatic or mildly symptomatic SARS-CoV-2 infection and characterized their humoral and T cell responses to the Spike (S), Nucleocapsid (N) and Membrane (M) proteins. We found that previous infection induced CD4 T cells that vigorously responded to pools of peptides derived from the S and N proteins. By using statistical and machine learning models, we observed that the T cell response highly correlated with a compound titer of antibodies against the Receptor Binding Domain (RBD), S and N. However, while serum antibodies decayed over time, the cellular phenotype of these individuals remained stable over four months. Our computational analysis demonstrates that in young adults, asymptomatic and paucisymptomatic SARS-CoV-2 infections can induce robust and long-lasting CD4 T cell responses that exhibit slower decays than antibody titers. These observations imply that next-generation COVID-19 vaccines should be designed to induce stronger cellular responses to sustain the generation of potent neutralizing antibodies., Competing Interests: CD, MSc, BS, PZ, FH, AR, Y-JH, GL, and HB are employees of Miltenyi Biotec B.V. & Co. KG. LP is an employee of Humabs BioMed SA, a subsidiary of Vir Biotechnology. 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 © 2023 Marcinkevics, Silva, Hankele, Dörnte, Kadelka, Csik, Godbersen, Goga, Hasenöhrl, Hirschi, Kabakci, LaPierre, Mayrhofer, Title, Shu, Baiioud, Bernal, Dassisti, Saenz-de-Juano, Schmidhauser, Silvestrelli, Ulbrich, Ulbrich, Wyss, Stekhoven, Al-Quaddoomi, Yu, Binder, Schultheiβ, Zindel, Kolling, Goldhahn, Seighalani, Zjablovskaja, Hardung, Schuster, Richter, Huang, Lauer, Baurmann, Low, Vaqueirinho, Jovic, Piccoli, Ciesek, Vogt, Sallusto, Stoffel and Ulbrich.)

Background: Variant syndromes of autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) share diagnostic features of both entities, but their immunological underpinnings remain largely unexplored., Methods: We performed blood profiling of 23 soluble immune markers and immunogenetics in a cohort of 88 patients with autoimmune liver diseases (29 typical AIH, 31 typical PBC and 28 with clinically PBC/AIH variant syndromes). The association with demographical, serological and clinical features was analyzed., Results: While T and B cell receptor repertoires were highly skewed in variant syndromes compared to healthy controls, these biases were not sufficiently discriminated within the spectrum of autoimmune liver diseases. High circulating checkpoint molecules sCD25, sLAG-3, sCD86 and sTim-3 discriminated AIH from PBC on top of classical parameters such as transaminases and immunoglobulin levels. In addition, a second cluster of correlated soluble immune factors encompassing essentially TNF, IFNγ, IL12p70, sCTLA-4, sPD-1 and sPD-L1 appeared characteristic of AIH. Cases with complete biochemical responses to treatment generally showed a lower level of dysregulation. Unsupervised hierarchical clustering of classical and variant syndromes identified two pathological immunotypes consisting predominantly of either AIH or PBC cases. Variant syndromes did not form a separate group, but clustered together with either classical AIH or PBC. Clinically, patient with AIH-like variant syndromes were less likely to be able discontinue immunosuppressive treatment., Conclusions: Our analyses suggest that variants of immune mediated liver diseases may represent an immunological spectrum from PBC to AIH-like disease reflected by their pattern of soluble immune checkpoint molecules rather than separate entities., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Tumors shed cell-free DNA (cfDNA) into the plasma. "Liquid biopsies" are a diagnostic test to analyze cfDNA in order to detect minimal residual cancer, profile the genomic tumor landscape, and monitor cancers non-invasively over time. This technique may be useful in patients with head and neck squamous cell carcinoma (HNSCC) due to genetic tumor heterogeneity and limitations in imaging sensitivity. However, there are technical challenges that need to be overcome for the widespread use of liquid biopsy in the clinical management of these patients. In this review, we discuss our current understanding of HNSCC genetics and the role of cfDNA genomic analyses as an emerging precision diagnostic tool.

We introduce a simple diagnostic test for assessing the overall or partial goodness of fit of a linear causal model with errors being independent of the covariates. In particular, we consider situations where hidden confounding is potentially present. We develop a method and discuss its capability to distinguish between covariates that are confounded with the response by latent variables and those that are not. Thus, we provide a test and methodology for partial goodness of fit. The test is based on comparing a novel higher-order least squares principle with ordinary least squares. In spite of its simplicity, the proposed method is extremely general and is also proven to be valid for high-dimensional settings. Supplementary materials for this article are available online., Competing Interests: The authors report there are no competing interests to declare., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Post-acute sequelae of COVID-19 (PASC) are long-term consequences of SARS-CoV-2 infection that can substantially impair the quality of life. Underlying mechanisms ranging from persistent viruses to innate and adaptive immune dysregulation have been discussed. Here, we profiled the plasma of 181 individuals from the cohort study for digital health research in Germany (DigiHero), including individuals after mild to moderate COVID-19 with or without PASC and uninfected controls. We focused on soluble factors related to monocyte/macrophage biology and on circulating SARS-CoV-2 spike (S1) protein as a potential biomarker for persistent viral reservoirs. At a median time of 8 months after infection, we found pronounced dysregulation in almost all tested soluble factors, including both pro-inflammatory and pro-fibrotic cytokines. These immunological perturbations were remarkably independent of ongoing PASC symptoms per se, but further correlation and regression analyses suggested PASC-specific patterns involving CCL2/MCP-1 and IL-8 that either correlated with sCD162, sCD206/MMR, IFN-α2, IL-17A and IL-33, or IL-18 and IL-23. None of the analyzed factors correlated with the detectability or levels of circulating S1, indicating that this represents an independent subset of patients with PASC. These data confirm prior evidence of immune dysregulation and persistence of viral protein in PASC and illustrate its biological heterogeneity that still awaits correlation with clinically defined PASC subtypes., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Introduction: In metastatic colorectal cancer (mCRC), the efficacy of immune checkpoint blockade (ICB) has so far been limited to patients with microsatellite instability high tumors (MSI-H). Unfortunately, most mCRC patients suffer from non-immunogenic microsatellite stable (MSS) tumors. Therefore, new combinatorial strategies are urgently needed to enhance the immunogenicity of MSS tumors to finally increase the number of patients benefiting from ICB., Methods: The AVETUX trial aimed to combine the PD-L1 antibody avelumab with the standard of care chemotherapy combination FOLFOX and the anti-EGFR antibody cetuximab. Furthermore, we performed a central radiological review of the pre- and on-treatment computed tomography scans to better define the individual response to treatment., Results and Discussion: In total, 43 patients were treated of which 39 patients were confirmed as RAS/BRAF wildtype in central tissue review and finally response evaluated. A final progression-free survival (PFS) of 11.1 (range: 0.8 to 22.3 months) and a herein updated final overall survival (OS) of 32.9 months (range: 0.8 to 47.1 months) was reached. We observed a strong median depth of response of 67.5% tumor shrinkage and deepness of response correlated significantly with survival. On the other hand, early tumor shrinkage was not an indicator of better outcome at a cut-off of 20% (median values). In a next step, we correlated the individual best radiological response with potential ICB response biomarkers and found that the clonality and diversity, but not frequency of tumor infiltrating lymphocytes (TiLs) and peripheral blood mononuclear cells (PBMCs), strongly correlated with response. In summary, we report the final overall survival of the AVETUX trial and propose T cell clonality and diversity as a potential marker to predict response to chemo-immunotherapy combinations in MSS mCRC by performing a central radiological review., Clinical Trial Registration: ClinicalTrials.gov, identifier (NCT03174405)., Competing Interests: AS received institutional research grants from Merck, BMS, Roche, Sanofi, Servier and honoraria for lectures and advisory board meetings by Merck, Roche, Amgen, Lilly, Sanofi- Aventis, Servier, Bayer, BMS, MSD and Sirtex. S-EA-B has an advisory role with Merck, Roche, Celgene, Lilly, Nordic Pharma, Bristol- Myers Squibb, Astellas and MSD Sharp & Dohme; is a speaker for Roche, Celgene, Lilly, Nordic Pharma, AIO gGmbH, MCI, promedicis, Forum für Medizinische Fortbildung and Taiho pharma; he is CEO/founder of IKF Klinische Krebsforschung GmbH at Northwest Hospital; and has received research grants from Sanofi, Merck, Roche, Celgene, Vifor, Medac, Hospira, Lilly, Eurozyto, German Cancer Aid (Krebshilfe), German Research Foundation and the Federal Ministry of Education and Research. UP received institutional research grants from Celgene, BMS, Amgen, Lilly, Roche, Sanofi and Servier and honoraria for lectures and advisory board meetings by Roche, Celgene, Amgen, Lilly, Sanofi- Aventis, Servier, Bayer and BMS. AH received honoraria for lectures from Roche. CB received institutional research grants and honoraria for lectures and advisory board meetings from Merck, BMS, Roche, Sanofi, Servier, Bayer, BMS, Astrazeneca, Lilly, Mundipharma, Hexal, MSD and GSO. MBi received institutional research grants from Merck, BMS, Hexal, German Cancer Aid (Krebshilfe), German Research Foundation and the Federal Ministry of Education and Research as well as honoraria for lectures and advisory board meetings by Celgene, Janssen, Gilead, Merck, Roche, Amgen, Sanofi-Aventis and BMS., (Copyright © 2022 Tintelnot, Ristow, Sauer, Simnica, Schultheiß, Scholz, Goekkurt, von Wenserski, Willscher, Paschold, Lorenzen, Riera-Knorrenschild, Depenbusch, Ettrich, Dörfel, Al-Batran, Karthaus, Pelzer, Hinke, Bauer, Massa, Seliger, Wickenhauser, Bokemeyer, Hegewisch-Becker, Binder and Stein.)

Importance: In metastatic esophagogastric adenocarcinoma (EGA), the addition of programmed cell death 1 (PD-1) inhibitors to chemotherapy has improved outcomes in selected patient populations., Objective: To investigate the efficacy of trastuzumab and PD-1 inhibitors with cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors or FOLFOX in first-line treatment of advanced ERBB2-positive EGA., Design, Setting, and Participants: This phase 2 multicenter, outpatient, randomized clinical trial with 2 experimental arms compared with historical control individually was conducted between March 2018 and May 2020 across 21 German sites. The reported results are based on a median follow-up of 14.3 months. Patients with previously untreated, metastatic ERBB2-positive (local immunohistochemistry score of 3+ or 2+/in situ hybridization amplification positive) EGA, adequate organ function, and eligibility for immunotherapy were included. Data analysis was performed from June to September 2021., Interventions: Patients were randomized to trastuzumab and nivolumab (1 mg/kg × 4/240 mg for up to 12 months) in combination with mFOLFOX6 (FOLFOX arm) or ipilimumab (3 mg/kg × 4 for up to 12 weeks) (ipilimumab arm)., Main Outcomes and Measures: The primary end point was survival improvement with a targeted increase of the 12-month overall survival rate from 55% (trastuzumab/chemotherapy-ToGA regimen) to 70% in each arm., Results: A total of 97 patients were enrolled, and 88 were randomized (18 women, 70 men; median [range] age, 61 [41-80] years). Baseline Eastern Cooperative Oncology Group performance status was 0 in 54 patients (61%) and 1 in 34 patients (39%); 66 patients (75%) had EGA localized in the esophagogastric junction and 22 in the stomach (25%). Central post hoc biomarker analysis (84 patients) showed PD-1 ligand 1 (PD-L1) combined positive score of 1 or greater in 59 patients (72%) and 5 or greater in 46 patients (56%) and confirmed ERBB2 positivity in 76 patients. The observed overall survival rate at 12 months was 70% (95% CI, 54%-81%) with FOLFOX and 57% (95% CI, 41%-71%) with ipilimumab. Treatment-related grade 3 or greater adverse events (AEs) and serious AEs occurred in 29 and 15 patients in the FOLFOX arm and in 20 and 17 patients in the ipilimumab arm, respectively, with a higher incidence of autoimmune-related AEs in the ipilimumab arm and neuropathy in the FOLFOX arm. Liquid biopsy analyses showed strong correlation of early cell-free DNA increase with shorter progression-free and overall survival and emergence of truncating and epitope-loss ERBB2 resistance sequence variations with trastuzumab treatment., Conclusions and Relevance: In this randomized clinical trial, trastuzumab, nivolumab, and FOLFOX showed favorable efficacy compared with historical data and trastuzumab, nivolumab, and ipilimumab in ERBB2-positive EGA. The ipilimumab arm yielded similar OS compared with the ToGA regimen., Trial Registration: ClinicalTrials.gov Identifier: NCT03409848.

The two most common antibody targeting principles in oncology are the induction of direct antitumor effects and the release of antitumor T cell immunity by immune checkpoint blockade. These two principles, however, may be overlapping if the targeted checkpoint molecule is not located on the immune cell but on the tumor cell itself. Secondary resistance by epitope escape may therefore remain a challenge in both settings. We previously reported epitope escape through L88S and truncating programmed cell death ligand 1 (PD-L1) gene mutations in colorectal cancer patients on selective pressure with avelumab, a PD-L1-directed checkpoint blocker that-in addition to T cell disinhibition-allows direct tumor cell killing via its unmodified Fc portion. Here, we confirmed this principle by liquid biopsy monitoring in a colorectal cancer patient from an independent clinical trial. In this patient, both PD-L1 L88E and L88fs mutations emerged under selective pressure with avelumab. By ectopically expressing PD-L1 L88E, we show that this mutation leads to a reduction of full-length glycosylated PD-L1 and greatly reduced avelumab surface binding. Further experiments indicated that PD-L1 L88E represents a phosphomimetic variant of PD-L1 L88S leading to loss of protein stability and increased proteasomal degradation. The association of this PD-L1 mutation with the high-affinity FCGR3A single nucleotide polymorphism rs396991 confirms prior evidence that patients harboring this polymorphism experience the strongest selective pressure by avelumab. Together, position 88 of PD-L1 is a hotspot residue critically regulating PD-L1 cell surface expression with clinical significance in the context of immune checkpoint blockade., Competing Interests: SS received honoraria for talks and advisory board role from Amgen, Astra-Zeneca, Bayer, BMS, ESAI, LEO Pharma, Lilly, Merck KGaA, MSD, Pierre-Fabre, Roche, Sanofi, Servier, Taiho, and Takeda and research funding from Merck KGaA, Pierre-Fabre, Servier, and Roche. VH received honoraria for talks and advisory board role from Merck, Amgen, Roche, Sanofi, Sirtex, Servier, Pfizer, Pierre-Fabre, AstraZeneca, BMS, MSD, Novartis, Boehringer Ingelheim, Pierre-Fabre, Celgene, Terumo, Oncosil, and Seagen and research funding from Merck, Amgen, Roche, Sanofi, Pfizer, Boehringer-Ingelheim, Sirtex, and Servier. 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 © 2022 Claaß, Schultheiß, Scholz, Paschold, Simnica, Heinemann, Stintzing and Binder.)

B cells are central for the adaptive immune system to mount successful immune responses not only as antibody producers but also as regulators of cellular immunity. These multifaceted features are also reflected in autoimmunity where autoreactive B cells can fuel disease by production of cytotoxic autoantibodies, presentation of autoantigens to autoreactive T cells, and secretion of cytokines and chemokines that either promote detrimental immune activation or impair regulatory T and B cells. The role of B cells and autoantibodies in autoimmune hepatitis (AIH) have been controversially discussed, with typical autoantibodies and hypergammaglobulinemia indicating a key role, while strong HLA class II association suggests T cells as key players. In this review, we summarize current knowledge on B cells in AIH and how different B cell subpopulations may drive AIH progression beyond autoantibodies. We also discuss recent findings of B cell-directed therapies in AIH., (© 2022. The Author(s).)

Post-acute sequelae of COVID-19 (PASC) is emerging as global problem with unknown molecular drivers. Using a digital epidemiology approach, we recruited 8,077 individuals to the cohort study for digital health research in Germany (DigiHero) to respond to a basic questionnaire followed by a PASC-focused survey and blood sampling. We report the first 318 participants, the majority thereof after mild infections. Of those, 67.8% report PASC, predominantly consisting of fatigue, dyspnea, and concentration deficit, which persists in 60% over the mean 8-month follow-up period and resolves independently of post-infection vaccination. PASC is not associated with autoantibodies, but with elevated IL-1β, IL-6, and TNF plasma levels, which we confirm in a validation cohort with 333 additional participants and a longer time from infection of 10 months. Blood profiling and single-cell data from early infection suggest the induction of these cytokines in COVID-19 lung pro-inflammatory macrophages creating a self-sustaining feedback loop., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The COVID-19 pandemic shows that vaccination strategies building on an ancestral viral strain need to be optimized for the control of potentially emerging viral variants. Therefore, aiming at strong B cell somatic hypermutation to increase antibody affinity to the ancestral strain - not only at high antibody titers - is a priority when utilizing vaccines that are not targeted at individual variants since high affinity may offer some flexibility to compensate for strain-individual mutations. Here, we developed a next-generation sequencing based SARS-CoV-2 B cell tracking protocol to rapidly determine the level of immunoglobulin somatic hypermutation at distinct points during the immunization period. The percentage of somatically hypermutated B cells in the SARS-CoV-2 specific repertoire was low after the primary vaccination series, evolved further over months and increased steeply after boosting. The third vaccination mobilized not only naïve, but also antigen-experienced B cell clones into further rapid somatic hypermutation trajectories indicating increased affinity. Together, the strongly mutated post-booster repertoires and antibodies deriving from this may explain why the third, but not the primary vaccination series, offers some protection against immune-escape variants such as Omicron B.1.1.529., 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 © 2022 Paschold, Klee, Gottschick, Willscher, Diexer, Schultheiß, Simnica, Sedding, Girndt, Gekle, Mikolajczyk and Binder.)

In parasite and viral infections, aberrant B cell responses can suppress germinal center reactions thereby blunting long-lived memory and may provoke immunopathology including autoimmunity. Using COVID-19 as model, we set out to identify serological, cellular, and transcriptomic imprints of pathological responses linked to autoreactive B cells at single-cell resolution. We show that excessive plasmablast expansions are prognostically adverse and correlate with autoantibody production but do not hinder the formation of neutralizing antibodies. Although plasmablasts followed interleukin-4 (IL-4) and BAFF-driven developmental trajectories, were polyclonal, and not enriched in autoreactive B cells, we identified two memory populations (CD80 + /ISG15 + and CD11c + /SOX5 + /T-bet +/- ) with immunogenetic and transcriptional signs of autoreactivity that may be the cellular source of autoantibodies in COVID-19 and that may persist beyond recovery. Immunomodulatory interventions discouraging such adverse responses may be useful in selected patients to shift the balance from autoreactivity toward long-term memory., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Objectives: T cells have an essential role in the antiviral defence. Public T-cell receptor (TCR) clonotypes are expanded in a substantial proportion of COVID-19 patients. We set out to exploit their potential use as read-out for COVID-19 T-cell immune responses., Methods: We searched for COVID-19-associated T-cell clones with public TCRs, as defined by identical complementarity-determining region 3 (CDR3) beta chain amino acid sequence that can be reproducibly detected in the blood of COVID-19 patients. Of the different clonotype identification algorithms used in this study, deep sequencing of brain tissue of five patients with fatal COVID-19 delivered 68 TCR clonotypes with superior representation across 140 immune repertoires of unrelated COVID-19 patients., Results: Mining of immune repertoires from subjects not previously exposed to the virus showed that these clonotypes can be found in almost 20% of pre-pandemic immune repertoires of healthy subjects, with lower representation in repertoires from risk groups like individuals above the age of 60 years or patients with cancer., Conclusion: Together, our data show that at least a proportion of the SARS-CoV-2 T-cell response is mediated by public TCRs that are present in repertoires of unexposed individuals. The lower representation of these clones in repertoires of risk groups or failure to expand such clones may contribute to more unfavorable clinical COVID-19 courses., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Cleavage of double-stranded RNA is described as an evolutionary conserved host defense mechanism against viral infection. Small RNAs are the product and triggers of post transcriptional gene silencing events. Up until now, the relevance of this mechanism for SARS-CoV-2-directed immune responses remains elusive. Herein, we used high throughput sequencing to profile the plasma of active and convalescent COVID-19 patients for the presence of small circulating RNAs. The existence of SARS-CoV-2 derived small RNAs in plasma samples of mild and severe COVID-19 cases is described. Clusters of high siRNA abundance were discovered, homologous to the nsp2 3'-end and nsp4 virus sequence. Four virus-derived small RNA sequences have the size of human miRNAs, and a target search revealed candidate genes associated with ageusia and long COVID symptoms. These virus-derived small RNAs were detectable also after recovery from the disease. The additional analysis of circulating human miRNAs revealed differentially abundant miRNAs, discriminating mild from severe cases. A total of 29 miRNAs were reduced or absent in severe cases. Several of these are associated with JAK-STAT response and cytokine storm.

Background: Renal cell carcinomas (RCC) are characterized by the deregulation of several hundred hyperosmolality-responsive genes. High expression of a subset of these genes including the Ran binding protein 3 like (RANBP3L) is linked to a favorable prognostic outcome in RCC. However, the cellular function of RANBP3L remains largely unknown., Methods: We used CRISPR/Cas9-mediated gene editing to generate functional deletions of the Ranbp3l and nuclear factor of activated T cells 5 (Nfat5) gene loci in a murine renal cell line. The NFAT5-KO cells were used to assess the regulation of Ranbp3l by NFAT5 using immunofluorescence, RNA-Seq and promoter assays. RANBP3L-deficient cells were analyzed for changes in cell morphology, proliferation, migration and colony-forming capacity using immunofluorescence and live cell imaging. RANPB3L-dependent changes in gene expression were identified by RNA-Seq., Results: We show that NFAT5 directly regulates Ranpb3l under hyperosmotic conditions by binding its promoter. Functional analysis of RANBP3L-deficient cells revealed a loss of epithelial structure, an increased cell migration behavior and colony forming capacity, accompanied by massive alterations in gene expression, all of which are hallmarks for tumor cells. Strikingly, a RANBP3L dependent signature of 60 genes separated samples with clear cell carcinoma (KIRC) from papillary (KIRP), chromophobe renal carcinoma (KICH) and healthy tissue., Conclusions: Loss of RANBP3L induces a tumor like phenotype resembles RCC, especially KIRC, on the morphological and gene expression level and might promote tumor development and progression. Therapeutic reconstitution or elevation of osmoregulated RANBP3L expression might represent a novel treatment strategy for RCC or KIRC.

Background: In patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC), immune checkpoint blockade is ineffective, and combinatorial approaches enhancing immunogenicity need exploration., Methods: We treated 43 patients with predominantly microsatellite stable RAS/BRAF wild-type mCRC on a phase II trial combining chemotherapy with the epidermal growth factor receptor antibody cetuximab and the programmed cell death ligand 1 (PD-L1) antibody avelumab. We performed next-generation gene panel sequencing for mutational typing of tumors and liquid biopsy monitoring as well as digital droplet PCR to confirm individual mutations. Translational analyses included tissue immunohistochemistry, multispectral imaging and repertoire sequencing of tumor-infiltrating T cells. Detected PD-L1 mutations were mechanistically validated in CRISPR/Cas9-generated cell models using qRT-PCR, immunoblotting, flow cytometry, complement-dependent cytotoxicity assay, antibody-dependent cytotoxicity by natural killer cell degranulation assay and LDH release assay as well as live cell imaging of T cell mediated tumor cell killing., Results: Circulating tumor DNA showed rapid clearance in the majority of patients mirroring a high rate of early tumor shrinkage. In 3 of 13 patients expressing the high-affinity Fcγ receptor 3a (FcγR3a), tumor subclones with PD-L1 mutations were selected that led to loss of tumor PD-L1 by nonsense-mediated RNA decay in PD-L1 K162fs and protein degradation in PD-L1 L88S. As a consequence, avelumab binding and antibody-dependent cytotoxicity were impaired, while T cell killing of these variant clones was increased. Interestingly, PD-L1 mutant subclones showed slow selection dynamics reversing on avelumab withdrawal and patients with such subclones had above-average treatment benefit. This suggested that the PD-L1 mutations mediated resistance to direct antitumor effects of avelumab, while at the same time loss of PD-L1 reduced biological fitness by enhanced T cell killing limiting subclonal expansion., Conclusion: The addition of avelumab to standard treatment appeared feasible and safe. PD-L1 mutations mediate subclonal immune escape to avelumab in some patients with mCRC expressing high-affinity FcγR3a, which may be a subset experiencing most selective pressure. Future trials evaluating the addition of avelumab to standard treatment in MSS mCRC are warranted especially in this patient subpopulation., Trial Registration Number: NCT03174405., Competing Interests: Competing interests: AS received institutional research grants from Merck, BMS, Roche, Sanofi, Servier and honoraria for lectures and advisory board meetings by Merck, Roche, Amgen, Lilly, Sanofi-Aventis, Servier, Bayer, BMS, MSD and Sirtex. S-E A-B has an advisory role with Merck, Roche, Celgene, Lilly, Nordic Pharma, Bristol-Myers Squibb, Astellas and MSD Sharp & Dohme; is a speaker for Roche, Celgene, Lilly, Nordic Pharma, AIO gGmbH, MCI, promedicis, Forum für Medizinische Fortbildung and Taiho pharma; he is CEO/founder of IKF Klinische Krebsforschung GmbH at Northwest Hospital; and has received research grants from Sanofi, Merck, Roche, Celgene, Vifor, Medac, Hospira, Lilly, Eurozyto, German Cancer Aid (Krebshilfe), German Research Foundation and the Federal Ministry of Education and Research. UP received institutional research grants from Celgene, BMS, Amgen, Lilly, Roche, Sanofi and Servier and honoraria for lectures and advisory board meetings by Roche, Celgene, Amgen, Lilly, Sanofi-Aventis, Servier, Bayer and BMS. AH received honoraria for lectures from Roche. CB received institutional research grants and honoraria for lectures and advisory board meetings from Merck, BMS, Roche, Sanofi, Servier, Bayer, BMS, Astrazeneca, Lilly, Mundipharma, Hexal, MSD and GSO. MB received institutional research grants from Merck, BMS, Hexal, German Cancer Aid (Krebshilfe), German Research Foundation and the Federal Ministry of Education and Research as well as honoraria for lectures and advisory board meetings by Celgene, Janssen, Gilead, Merck, Roche, Amgen, Sanofi-Aventis and BMS., (© 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.)

Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. Superantigen specificity for different Vβ chains results in Vβ skewing, whereby T cells with specific Vβ chains and diverse antigen specificity are overrepresented in the T cell receptor (TCR) repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCRβ variable gene 11-2 (TRBV11-2), with up to 24% of clonal T cell space occupied by TRBV11-2 T cells, which correlated with MIS-C severity and serum cytokine levels. Analysis of TRBJ gene usage and complementarity-determining region 3 (CDR3) length distribution of MIS-C expanded TRBV11-2 clones revealed extensive junctional diversity. Patients with TRBV11-2 expansion shared HLA class I alleles A02, B35, and C04, indicating what we believe is a novel mechanism for CDR3-independent T cell expansion. In silico modeling indicated that polyacidic residues in the Vβ chain encoded by TRBV11-2 (Vβ21.3) strongly interact with the superantigen-like motif of SARS-CoV-2 spike glycoprotein, suggesting that unprocessed SARS-CoV-2 spike may directly mediate TRBV11-2 expansion. Overall, our data indicate that a CDR3-independent interaction between SARS-CoV-2 spike and TCR leads to T cell expansion and possibly activation, which may account for the clinical presentation of MIS-C.

We identified a subset of Chronic Lymphocytic Leukemia (CLL) patients with high Signaling Lymphocytic Activation Molecule Family (SLAMF) receptor-related signaling that showed an indolent clinical course. Since SLAMF receptors play a role in NK cell biology, we reasoned that these receptors may impact NK cell-mediated CLL immunity. Indeed, our experiments showed significantly decreased degranulation capacity of primary NK cells from CLL patients expressing low levels of SLAMF1 and SLAMF7. Since the SLAMF low signature was strongly associated with an unmutated CLL immunoglobulin heavy chain (IGHV) status in large datasets, we investigated the impact of SLAMF1 and SLAMF7 on the B cell receptor (BCR) signaling axis. Overexpression of SLAMF1 or SLAMF7 in IGHV mutated CLL cell models resulted in reduced proliferation and impaired responses to BCR ligation, whereas the knockout of both receptors showed opposing effects and increased sensitivity toward inhibition of components of the BCR pathway. Detailed molecular analyzes showed that SLAMF1 and SLAMF7 receptors mediate their BCR pathway antagonistic effects via recruitment of prohibitin-2 (PHB2) thereby impairing its role in signal transduction downstream the IGHV-mutant IgM-BCR. Together, our data indicate that SLAMF receptors are important modulators of the BCR signaling axis and may improve immune control in CLL by interference with NK cells.

Background and Aims: Autoimmune hepatitis (AIH) is a chronic liver disease that regularly relapses when immunosuppression is tapered. It is thought to be driven by T-cells, whereas the etiologic impact of an apparently deregulated B lineage system, as evidenced by hypergammaglobulinemia and autoantibodies, remains elusive. We set out to investigate T and B cell repertoires supporting chronic inflammation in AIH., Approach and Results: T and B cell receptor (TCR/BCR) and human leukocyte antigen (HLA) next-generation immunosequencing were used to record immune signatures from a cohort of 60 patients with AIH and disease controls. Blood and liver B lineage immune metrics were not indicative of a dominant directional antigen selection apart from a slight skewing of IGHV-J genes. More importantly, we found strong AIH-specific TRBV-J skewing not attributable to the HLA-DRB1 specificities of the cohort. This TCR repertoire bias was generated as a result of peripheral T cell (de)selection and persisted in disease remission. Using a clustering algorithm according to antigenic specificity, we identified liver TCR clusters that were shared between patients with AIH but were absent or deselected in patients with other liver pathologies., Conclusions: Patients with AIH show profound and persisting T-cell architectural changes that may explain high relapse rates after tapering immunosuppression. Liver T-cell clusters shared between patients may mediate liver damage and warrant further study., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Hyperinflammation contributes to lung injury and subsequent acute respiratory distress syndrome (ARDS) with high mortality in patients with severe coronavirus disease 2019 (COVID-19). To understand the underlying mechanisms involved in lung pathology, we investigated the role of the lung-specific immune response. We profiled immune cells in bronchoalveolar lavage fluid and blood collected from COVID-19 patients with severe disease and bacterial pneumonia patients not associated with viral infection. By tracking T cell clones across tissues, we identified clonally expanded tissue-resident memory-like Th17 cells (Trm17 cells) in the lungs even after viral clearance. These Trm17 cells were characterized by a a potentially pathogenic cytokine expression profile of IL17A and CSF2 (GM-CSF). Interactome analysis suggests that Trm17 cells can interact with lung macrophages and cytotoxic CD8 + T cells, which have been associated with disease severity and lung damage. High IL-17A and GM-CSF protein levels in the serum of COVID-19 patients were associated with a more severe clinical course. Collectively, our study suggests that pulmonary Trm17 cells are one potential orchestrator of the hyperinflammation in severe COVID-19., (Copyright © 2021, American Association for the Advancement of Science.)

A considerable fraction of B cells recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with germline-encoded elements of their B cell receptor, resulting in the production of neutralizing and nonneutralizing antibodies. We found that antibody sequences from different discovery cohorts shared biochemical properties and could be retrieved across validation cohorts, confirming the stereotyped character of this naive response in coronavirus disease 2019 (COVID-19). While neutralizing antibody sequences were found independently of disease severity, in line with serological data, individual nonneutralizing antibody sequences were associated with fatal clinical courses, suggesting detrimental effects of these antibodies. We mined 200 immune repertoires from healthy individuals and 500 repertoires from patients with blood or solid cancers - all acquired prior to the pandemic - for SARS-CoV-2 antibody sequences. While the largely unmutated B cell rearrangements occurred in a substantial fraction of immune repertoires from young and healthy individuals, these sequences were less likely to be found in individuals over 60 years of age and in those with cancer. This reflects B cell repertoire restriction in aging and cancer, and may to a certain extent explain the different clinical courses of COVID-19 observed in these risk groups. Future studies will have to address if this stereotyped B cell response to SARS-CoV-2 emerging from unmutated antibody rearrangements will create long-lived memory.

We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a repository of currently >14 million B and T cell receptor (BCR and TCR) sequences from the blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires were associated with interferon type I and III responses, early CD4 + and CD8 + T cell activation, and counterregulation by the co-receptors BTLA, Tim-3, PD-1, TIGIT, and CD73. Tfh, Th17-like, and nonconventional (but not classical antiviral) Th1 cell polarizations were induced. SARS-CoV-2-specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our data provide fundamental insight into adaptive immunity to SARS-CoV-2 with the actively updated repository providing a resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Epidermal growth factor receptor (EGFR) antibodies may have detrimental effects in patients with metastatic colorectal cancer expressing oncogenic Rat sarcoma (RAS). Since a significant number of patients acquire RAS-mediated resistance during EGFR-directed treatment, understanding the molecular mechanism underlying these antibody-mediated tumor-promoting effects is of relevance to design more resistance-preventive treatment approaches. To test this, we set up a Ba/F3 cellular model system transformed to EGFR/RAS dependency to be able to study proliferation, RAS activity as well as MAPK signaling upon inhibition of wild-type RAS isoforms by therapeutic EGFR antibodies. Here, we show that the EGFR antibodies cetuximab and panitumumab induce paradoxical stimulation and enhance proliferation in cells expressing oncogenic RAS (KRAS G12V). These experiments clearly showed that the stimulatory effect is a direct result of the antibody-EGFR interaction leading to prolonged mitogen-activated protein-Kinase (MAPK) signaling. The effect was also induced by antibody-chemotherapy combinations but always depended on simultaneous low-level ligand-dependent EGFR pathway activation. Moreover, we observed significant growth retardation of RAS mutant cells after antibody withdrawal compatible with a drug-addiction phenotype. Our data suggests that EGFR antibodies paradoxically sustain MAPK signaling downstream of oncogenic RAS thereby driving proliferation of RAS mutant tumors or tumor subclones. The observed drug-addiction encourages fixed-duration or liquid-biopsy-guided drug holiday concepts to preventively clear RAS mutant subclones selected under EGFR-directed therapeutic pressure., (Copyright © 2020 Tintelnot, Metz, Trentmann, Oberle, von Wenserski, Schultheiß, Braig, Kriegs, Fehse, Riecken, Bokemeyer, Stein and Binder.)

Autoimmune cytopenias (AIC) such as immune thrombocytopenia or autoimmune hemolytic anemia are claimed to be essentially driven by a dysregulated immune system. Using next-generation immunosequencing we profiled 59 T and B cell repertoires ( TRB and IGH ) of 25 newly diagnosed patients with primary or secondary (lymphoma-associated) AIC to test the hypothesis if these patients present a disease-specific immunological signature that could reveal pathophysiological clues and eventually be exploited as blood-based biomarker. Global TRB and IGH repertoire metrics as well as VJ gene usage distribution showed uniform characteristics for all lymphoma patients (high clonality and preferential usage of specific TRBV - and TRBJ genes), but no AIC-specific signature. Since T cell immune reactions toward antigens are unique and polyclonal, we clustered TCRβ clones in-silico based on target recognition using the GLIPH (grouping of lymphocyte interactions by paratope hotspots) algorithm. This analysis revealed a considerable lack of physiological T cell clusters in patients with primary AIC. Interestingly, this signature did not discriminate between the different subentities of AIC and was also found in an independent cohort of 23 patients with active autoimmune hepatitis. Taken together, our data suggests that the identified T cell cluster signature could represent a blood biomarker of autoimmune conditions in general and should be functionally validated in future studies.

Epidermal growth factor receptor (EGFR) ectodomain variants mediating primary resistance or secondary treatment failure in cancer patients treated with cetuximab or panitumumab support the need for more resistance-preventive or personalized ways of targeting this essential pathway. Here, we tested the hypothesis that the EGFR nanobody 7D12 fused to an IgG1 Fc portion (7D12-hcAb) would overcome EGFR ectodomain-mediated resistance because it targets a very small binding epitope within domain III of EGFR. Indeed, we found that 7D12-hcAb bound and inhibited all tested cell lines expressing common resistance-mediating EGFR ectodomain variants. Moreover, we assessed receptor functionality and binding properties in synthetic mutants of the 7D12-hcAb epitope to model resistance to 7D12-hcAb. Because the 7D12-hcAb epitope almost completely overlaps with the EGF-binding site, only position R377 could be mutated without simultaneous loss of receptor functionality, suggesting a low risk of developing secondary resistance toward 7D12-hcAb. Our binding data indicated that if 7D12-hcAb resistance mutations occurred in position R377, which is located within the cetuximab and panitumumab epitope, cells expressing these receptor variants would retain sensitivity to these antibodies. However, 7D12-hcAb was equally ineffective as cetuximab in killing cells expressing the cetuximab/panitumumab-resistant aberrantly N-glycosylated EGFR R521K variant. Yet, this resistance could be overcome by introducing mutations into the Fc portion of 7D12-hcAb, which enhanced immune effector functions and thereby allowed killing of cells expressing this variant. Taken together, our data demonstrate a broad range of activity of 7D12-hcAb across cells expressing different EGFR variants involved in primary and secondary EGFR antibody resistance., (©2019 American Association for Cancer Research.)

This study provides first insights into the biosynthesis, structure, biochemistry and complex processing of the proteins encoded by hNOT/ALG3, the human counterpart of the Drosophila Neighbour of TID (NOT) and the yeast asparagine linked glycosylation 3 gene (ALG3), which encodes a mannosyltransferase. Unambiguous evidence that both the fly and human proteins act as mannosyltransferases has not been provided yet. Previously, we showed that hNOT/ALG3 encodes two alternatively spliced main transcripts, hNOT-1/ALG3-1 and hNOT-4/ALG3-4, and their 15 truncated derivatives that lack diverse sets of exons and/or carry point mutations that result in premature termination codons. Here we show that the truncated transcripts are not translated. The two main forms hNOT-1/ALG3-1 and -4, distinguishable by alternative exon 1, encode full-length precursors that undergo a complex posttranslational processing. To specifically detect the two full-length hNOT/ALG3 proteins and their distinct derivatives and to examine their expression profiles and cellular location we generated polyclonal antibodies against diverse parts of the putative full-length proteins. We provide experimental evidence for the N-glycosylation of the two precursors. This modification seems to be a prerequisite for their sequential cleavage resulting in derivatives destined to distinct cellular compartments and links them with the N-glycosylation machinery not as its functional component but as molecules functionally dependent on its action. We present the expression profiles and subcellular location of the two full-length proteins, their N-glycosylated forms and distinct cleavage products. Furthermore, using diverse bioinformatics tools, we characterize the properties and predict the 2D and 3D structure of the two proteins and, for comparative purposes, of their Drosophila counterpart.

This study provides first insights into the involvement of hNOT/ALG3, the human counterpart of the Drosophila Neighbour of TID and yeast ALG3 gene, in various putative molecular networks. HNOT/ALG3 encodes two translated transcripts encoding precursor proteins differing in their N-terminus and showing 33% identity with the yeast asparagine-linked glycosylation 3 (ALG3) protein. Experimental evidence for the functional homology of the proteins of fly and man in the N-glycosylation has still to be provided. In this study, using the yeast two-hybrid technique we identify 17 molecular partners of hNOT-1/ALG3-1. We disclose the building of hNOT/ALG3 homodimers and provide experimental evidence for its in vivo interaction with the functionally linked proteins OSBP, OSBPL9 and LRP1, the SYPL1 protein and the transcription factor CREB3. Regarding the latter, we show that the 55 kDa N-glycosylated hNOT-1/ALG3-1 molecule binds the N-glycosylated CREB3 precursor but does not interact with CREB3's proteolytic products specific to the endoplasmic reticulum and to the nucleus. The interaction between the two partners is a prerequisite for the proteolytic activation of CREB3. In case of the further binding partners, our data suggest that hNOT-1/ALG3-1 interacts with both OSBPs and with their direct targets LRP1 and VAMP/VAP-A. Moreover, our results show that various partners of hNOT-1/ALG3-1 interact with its diverse post translationally processed products destined to distinct cellular compartments. Generally, our data suggest the involvement of hNOT-1/ALG3-1 in various molecular contexts determining essential processes associated with distinct cellular machineries and related to various pathologies, such as cancer, viral infections, neuronal and immunological disorders and CDG.