SARS-CoV-2 vaccination may mitigate dysregulation of IL-1/IL-18 and gastrointestinal symptoms of the post-COVID-19 condition.

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-1ß 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.

SAM-Competitive EZH2-Inhibitors Induce Platinum Resistance by EZH2-Independent Induction of ABC-Transporters.

T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

Immune signatures in variant syndromes of primary biliary cholangitis and autoimmune hepatitis.

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.

Reduced proliferation of bone marrow MSC after allogeneic stem cell transplantation is associated with clinical outcome.

Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function after alloSCT; however, the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill-defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N = 196) who had undergone alloSCT. This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared with pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients after alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished in vitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways, and altered impact on cytokine release upon contact with hematopoietic cells. To avoid in vitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT colony-forming unit fibroblast-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution after alloSCT and its therapeutic manipulation.

Resistance to BRAF Inhibitors: EZH2 and Its Downstream Targets as Potential Therapeutic Options in Melanoma.

Activating BRAF mutations occurs in 50-60% of malignant melanomas. Although initially treatable, the development of resistance to BRAF-targeted therapies (BRAFi) is a major challenge and limits their efficacy. We have previously shown that the BRAFV600E signaling pathway mediates the expression of EZH2, an epigenetic regulator related to melanoma progression and worse overall survival. Therefore, we wondered whether inhibition of EZH2 would be a way to overcome resistance to vemurafenib. We found that the addition of an EZH2 inhibitor to vemurafenib improved the response of melanoma cells resistant to BRAFi with regard to decreased viability, cell-cycle arrest and increased apoptosis. By next-generation sequencing, we revealed that the combined inhibition of BRAF and EZH2 dramatically suppresses pathways of mitosis and cell cycle. This effect was linked to the downregulation of Polo-kinase 1 (PLK1), a key regulator of cell cycle and proliferation. Subsequently, when we inhibited PLK1, we found decreased cell viability of melanoma cells resistant to BRAFi. When we inhibited both BRAF and PLK1, we achieved an improved response of BRAFi-resistant melanoma cells, which was comparable to the combined inhibition of BRAF and EZH2. These results thus reveal that targeting EZH2 or its downstream targets, such as PLK1, in combination with BRAF inhibitors are potential novel therapeutic options in melanomas with BRAF mutations.

Liquid biomarkers of macrophage dysregulation and circulating spike protein illustrate the biological heterogeneity in patients with post-acute sequelae of COVID-19.

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.

The IL-1ß, IL-6, and TNF cytokine triad is associated with post-acute sequelae of COVID-19.

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-1ß, 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.

Efficacy of Ipilimumab vs FOLFOX in Combination With Nivolumab and Trastuzumab in Patients With Previously Untreated ERBB2-Positive Esophagogastric Adenocarcinoma: The AIO INTEGA Randomized Clinical Trial.

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.

Rapid Hypermutation B Cell Trajectory Recruits Previously Primed B Cells Upon Third SARS-Cov-2 mRNA Vaccination.

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.

Azacitidine-induced reconstitution of the bone marrow T cell repertoire is associated with superior survival in AML patients.

Hypomethylating agents (HMA) like azacitidine are licensed for the treatment of acute myeloid leukemia (AML) patients ineligible for allogeneic hematopoietic stem cell transplantation. Biomarker-driven identification of HMA-responsive patients may facilitate the choice of treatment, especially in the challenging subgroup above 60 years of age. Since HMA possesses immunomodulatory functions that constitute part of their anti-tumor effect, we set out to analyze the bone marrow (BM) immune environment by next-generation sequencing of T cell receptor beta (TRB) repertoires in 51 AML patients treated within the RAS-AZIC trial. Patients with elevated pretreatment T cell diversity (11 out of 41 patients) and those with a boost of TRB richness on day 15 after azacitidine treatment (12 out of 46 patients) had longer event-free and overall survival. Both pretreatment and dynamic BM T cell metrics proved to be better predictors of outcome than other established risk factors. The favorable broadening of the BM T cell space appeared to be driven by antigen since these patients showed significant skewing of TRBV gene usage. Our data suggest that one course of AZA can cause reconstitution to a more physiological T cell BM niche and that the T cell space plays an underestimated prognostic role in AML.Trial registration: DRKS identifier: DRKS00004519.

Translational analysis and final efficacy of the AVETUX trial - Avelumab, cetuximab and FOLFOX in metastatic colorectal cancer.

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).

Maturation trajectories and transcriptional landscape of plasmablasts and autoreactive B cells in COVID-19.

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.

Landscape of T-cell repertoires with public COVID-19-associated T-cell receptors in pre-pandemic risk cohorts.

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.

High-Resolution Cartography of the Transcriptome and Methylome Landscapes of Diffuse Gliomas.

Molecular mechanisms of lower-grade (II-III) diffuse gliomas (LGG) are still poorly understood, mainly because of their heterogeneity. They split into astrocytoma- (IDH-A) and oligodendroglioma-like (IDH-O) tumors both carrying mutations(s) at the isocitrate dehydrogenase (IDH) gene and into IDH wild type (IDH-wt) gliomas of glioblastoma resemblance. We generated detailed maps of the transcriptomes and DNA methylomes, revealing that cell functions divided into three major archetypic hallmarks: (i) increased proliferation in IDH-wt and, to a lesser degree, IDH-O; (ii) increased inflammation in IDH-A and IDH-wt; and (iii) the loss of synaptic transmission in all subtypes. Immunogenic properties of IDH-A are diverse, partly resembling signatures observed in grade IV mesenchymal glioblastomas or in grade I pilocytic astrocytomas. We analyzed details of coregulation between gene expression and DNA methylation and of the immunogenic micro-environment presumably driving tumor development and treatment resistance. Our transcriptome and methylome maps support personalized, case-by-case views to decipher the heterogeneity of glioma states in terms of data portraits. Thereby, molecular cartography provides a graphical coordinate system that links gene-level information with glioma subtypes, their phenotypes, and clinical context.

PD-L1 targeting and subclonal immune escape mediated by PD-L1 mutations in metastatic colorectal cancer.

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.

Evolutionary clonal trajectories in nodular lymphocyte-predominant Hodgkin lymphoma with high risk of transformation.

The B-cell architecture of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is complex since it is composed of malignant lymphocyte-predominant cells along with a rich B-cell bystander environment. To gain insight into molecular determinants of disease transformation, we studied B-cell evolutionary trajectories in lymphoma tissue from diagnosis to relapse or transformation to non- Hodgkin lymphoma by next-generation sequencing of immunoglobulin heavy chains. Patients with NLPHL that later transformed were older and showed IgD negativity, absence of the characteristic IGHV3/IGHD3/IGHJ6 lymphocyte-predominant rearrangement and high repertoire clonality. We constructed phylogenetic trees within the compartment of the malignant clone to investigate clonal evolution. In all relapsing cases, the lymphocyte-predominant rearrangement was identical at diagnosis and relapse. NLPHL cases with transformation showed more complex trajectories with strong intraclonal diversification. The dominant founder clone in transformations showed clonal evolution if derived from the same cell of origin, or arose from a different cell of origin. Together, our data point to a significant role of antigenic drive in the transformation of NLHPL and identify high B-cell repertoire clonality with dominant intraclonal lymphocyte-predominant cell diversification as a hallmark of transformation. Sequencing of initial paraffin-embedded tissue may therefore be applied diagnostically to identify NLPHL cases with high risk of transformation.

Deciphering the Transcriptomic Heterogeneity of Duodenal Coeliac Disease Biopsies.

Coeliac disease (CD) is a clinically heterogeneous autoimmune disease with variable presentation and progression triggered by gluten intake. Molecular or genetic factors contribute to disease heterogeneity, but the reasons for different outcomes are poorly understood. Transcriptome studies of tissue biopsies from CD patients are scarce. Here, we present a high-resolution analysis of the transcriptomes extracted from duodenal biopsies of 24 children and adolescents with active CD and 21 individuals without CD but with intestinal afflictions as controls. The transcriptomes of CD patients divide into three groups-a mixed group presenting the control cases, and CD-low and CD-high groups referring to lower and higher levels of CD severity. Persistence of symptoms was weakly associated with subgroup, but the highest marsh stages were present in subgroup CD-high, together with the highest cell cycle rates as an indicator of virtually complete villous atrophy. Considerable variation in inflammation-level between subgroups was further deciphered into immune cell types using cell type de-convolution. Self-organizing maps portrayal was applied to provide high-resolution landscapes of the CD-transcriptome. We find asymmetric patterns of miRNA and long non-coding RNA and discuss the effect of epigenetic regulation. Expression of genes involved in interferon gamma signaling represent suitable markers to distinguish CD from non-CD cases. Multiple pathways overlay in CD biopsies in different ways, giving rise to heterogeneous transcriptional patterns, which potentially provide information about etiology and the course of the disease.

SLAMF receptors negatively regulate B cell receptor signaling in chronic lymphocytic leukemia via recruitment of prohibitin-2.

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 SLAMFlow 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.

SARS-CoV-2-specific antibody rearrangements in prepandemic immune repertoires of risk cohorts and patients with COVID-19.

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.

Next-Generation Immunosequencing Reveals Pathological T-Cell Architecture in Autoimmune Hepatitis.

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.