BACKGROUND:Patients with advanced malignant adrenal tumors face poor prognoses with limited treatment options. Emerging data suggest that these rare tumors exhibit immunogenicity, potentially benefiting from intensified immunotherapy. METHODS:The SPENCER trial (NCT04187404) evaluates EO2401, an off-the-shelf in vivo peptide immunotherapy containing three synthetic HLA-A*02 restricted-peptides called OncoMimic and derived from human gut commensals that exhibit molecular mimicry to tumor-associated antigens (BIRC5, FOXM1, IL13RA2) combined with nivolumab in patients with locally advanced or metastatic adrenocortical carcinoma (ACC) or malignant pheochromocytoma/ paraganglioma (PPGL). We performed T cell receptor (TCR) repertoire sequencing to profile the TCR architecture in ACC and characterize vaccination imprints. RESULTS:Here we show that ACC patients are characterized by a restricted peripheral T cell richness. EO2401 does not expand highly shared TCR clones across patients but diversifies pre-existing T cell clusters. These clusters are not converging on known neoantigen-specific TCRs but exhibit generation probabilities characteristic of public clones some of which being shared across patients. CONCLUSIONS:These findings suggest that EO2401-based peptide immunotherapy combined with checkpoint inhibition can broaden and activate public, pre-existing T cell clusters, potentially enhancing tumor-specific immune responses.

BACKGROUND/OBJECTIVES:Vestibular schwannoma (VS) is the most common benign tumor in the cerebellopontine angle. In preliminary studies, macrophage infiltration has been suggested to influence disease progression. However, the infiltration of other immune cells in VS remains largely unexplored. The aim of this study was to comprehensively characterize the immune cells in sporadic VS. METHODS:Cryosections of five tumor samples from VS patients with different tumor volumes were examined. The abundance of fourteen immune-cell markers, one vascular marker, and two tumor markers were detected using multi-epitope ligand cartography (MELC). This enabled the spatial distribution and colocalization of immune- and tumor cell markers to be examined. Furthermore, using qPCR and bulk RNAseq, the mRNA levels of the immune-cell markers were examined in 204 VS samples of different tumor sizes. RESULTS:VSs with greater tumor volumes showed an increased number of immune cells, more precisely T-helper cells (TH cells), cytotoxic T cells (Tc cells), CD68+, and CD163+ macrophages, as well as CD279+ (PD-1) and CTLA4+ cells (p < 0.05). In addition, an increased number of CD274+ (PD-L1) tumor cells were detected in VSs with higher tumor volume (p < 0.05). CONCLUSIONS:These results indicate that an increased diversity of immune-cell subtypes influences VS tumor size. Thus, novel diagnostic and therapeutic options could be developed by targeting the tumor-associated immune-cell populations in VSs.

Anaplastic thyroid carcinoma (ATC) is a rare thyroid malignancy with poor prognosis and very limited treatment options. Therefore, the development of novel therapies is urgently needed. Here, we identified the Receptor Tyrosine Kinase like Orphan Receptor 1 (ROR1) protein as a specific CAR T cell target for ATC. ROR1 is part of the Wnt signalling pathway, mainly expressed during embryogenesis, but mostly absent in differentiated adult tissue. In ATCs, ROR1 is strongly overexpressed (RNA/protein level, surface expression) and high expression levels are associated with reduced survival. ROR1 CAR Ts specifically target ATC cell lines in 2D and 3D spheroid cultures, reduce the quantity of circulating tumour cells and block tumour metastases in different ATC mouse models. While small tumours were completely eliminated by ROR1 CAR T cells alone, larger tumours required the combination of ROR1 CAR T cells with the multikinase inhibitor lenvatinib. Lenvatinib blocked primary tumour growth, reduced the quantity of immunosuppressive cancer associated fibroblasts (CAF-S1) in the microenvironment and enhanced CAR T cell functionality and activation. Overall, we validated ROR1 as a prime target for CAR T cell therapies in ATC and identified lenvatinib as highly valuable combination partner, which is able to improve CAR T cell functionality.

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 architecture in a broad range of T-cell lymphomas. Our multidimensional profiling indicates that many of these lymphomas do 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 configuration - may represent a driver of malignant transformation. Together, our data indicate that T-cell lymphomas are derived from immature lymphoid precursors and display considerable intratumoral heterogeneity that may provide the basis for relapse and resistance in these hard-to-treat cancers.

Introduction Peripheral T-cell lymphomas (PTCL) are rare and highly heterogenous malignancies. The diagnosis can be challenging for pathologists. Accurate subtyping of PTCL is crucial for therapy decision making and clinical outcome prediction. Here we demonstrate that a deep learning (DL) model can reliably identify common subgroups of PTCL using histopathological images. Methods We trained a DL model on 223 H&E stained whole slides images (WSIs) from the German National T-Cell Lymphoma Registry and Biobank of the GLA and OSHO study groups. The training cohort included 144 patients with the following subtypes: anaplastic large cell T-cell lymphoma (ALCL) n=31; peripheral T-cell lymphoma with T follicular-helper phenotype (TFH-NHL) n=46; peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) n=23; other subtypes n=44. We trained a weakly supervised, open-source DL pipeline comprising UNI model as feature extractor and a vision transformer architecture for multiclass prediction. The DL model was trained to differentiate between the four subgroups: ALCL, TFH-NHL, PTCL-NOS and a group of other T-NHL entities. External validation was performed using an independent cohort of 118 WSIs from 118 patients (ALCL n=25; TFH-NHL n=75; PTCL-NOS n=18). To assess the quality of the model we used area under the receiver operating characteristic curve (AUROC). Heatmaps highlighting the most important image areas leading to correctly and incorrectly predicted cases were evaluated by hematopathologists to identify patterns for incorrectly and correctly categorized cases. Results In the five-fold cross-validation the DL model significantly distinguished the subgroups with an AUROC of 0.9 (95% confidence interval 0.84-0.96) for ALCL, TFH-NHL with 0.77 (0.59-0.95), PTCL-NOS with 0.55 (0.41-0.69) and other subtypes with 0.81 (0.71-0.90), even in different tissues types. When applied to the external validation cohort, the model maintained high predictive performance with AUROCs of 0.85 (0.74-0.95) for ALCL, 0.87 (0.79-0.94) for PTCL-TFH and 0.69 (0.52-0.85) for PTCL-NOS. A comparison of the most important image areas for DL-based classification was carried out with reference pathologists. This showed that the DL model successfully recognized the characteristic morphological areas, resulting in highly accurate classification for ALCL and PTCL-TFH. Conclusions The DL model was able to classify main PTCL subtypes on H&E stainings with high accuracy. This model needs prospective validation as a supportive tool for PTCL diagnosis. HE-based DL-based subtyping could assist pathologists in decision-making and help to guide effective diagnostics, addressing a critical unmet need.

Nuclear factor of activated T-cells 5 (NFAT5) is a transcription factor known for its role in osmotic stress adaptation in the renal inner medulla, due to the osmotic gradient that is generated between the renal cortex and renal inner medulla. However, its broader implications in kidney injury and chronic kidney disease (CKD) are less understood. Here we used two different Cre deleter mice (Ksp1.3-Cre and Aqp2-Cre) to generate tubule segment and even cell type-specific NFAT5-deficient mice and performed extensive gene expression profiling. In both Nfat5 knockout models, we observed massive changes in gene expression pattern, with heightened inflammatory responses and renal injury, culminating in renal fibrosis. Interestingly, inflammatory responses were much more prominent in the Aqp2Cre+/-Nfat5fl/fl mice that lack NFAT5 only in the collecting duct. By analyzing gene expression in the medullary and cortical regions of the kidney separately, we confirmed that the loss of NFAT5 results in kidney injury that extends beyond hypertonic areas. Renal injury correlates with the expression level of genes involved in inflammatory response, injury severity, and cytokine signaling. Thus, NFAT5 is essential not only for adapting to osmotic stress but also for its loss of function, which induces activation of inflammatory response and cytokine signaling that might affect regions with functional NFAT5 expression.

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.

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.

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.

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.

Key Points • Allogenic SCT leads to damage of the proliferative capacity of BM-MSC.• This damage correlates with clinical outcome of alloSCT, reduced patient survival, and impaired engraftment of allogeneic hematopoiesis.

Abstract Chronic hepatitis C virus (HCV) infection leads to a complex interplay with adaptive immune cells that may result in cryoglobulinemia or lymphoma. Although direct-acting antiviral (DAA) therapy has decreased the incidence of severe liver damage, its effect on extrahepatic HCV manifestations such as B cell dyscrasias is still unclear. Here, we used immunosequencing to analyze HCV imprinting on B cell receptor (BCR) repertoires in patients with chronic HCV mono-infection or a sustained virological response (SVR) after DAA therapy. The majority of patients had a B cell signature with high somatic hypermutation and richness. Convergence to specific immunoglobulin genes produced high-connectivity complementarity-determining region 3 (CDR3) networks. Interestingly, IGHV1-69 CDR1 and CDR3 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. 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. Collectively, this study provides evidence that lymphoma-like cells may result from the anti-HCV immune response and may persist for years after SVR, leading to HCV-related B cell dyscrasias and increased lymphoma risk beyond viral elimination.

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.

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.

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.

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.

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.

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.