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.

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.

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.

In patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC), immune checkpoint blockade is ineffective. However, data from the AVETUX trial suggests PD-L1 epitope escape upon avelumab treatment in MSS-mCRC patients expressing the high affinity SNP of FcγR3a (rs396991). Biomarker substudy of a patient on the FIRE-6 (AIO KRK-0118) trial with histologically confirmed, treatment-naïve, RAS/RAF wild-type metastatic colorectal cancer (MSS). The patient received 4 cycles of FOLFIRI/cetuximab, followed by 4 cycles FOLFIRI/cetuximab + avelumab, followed by avelumab maintenance treatment for 10 weeks until end of treatment (EOT) due to progression in week 19. Mutational profiling of cell-free DNA (cfDNA) was performed with custom gene panels and liquid biopsy monitoring by digital droplet PCR (ddPCR). Identified PD-L1 variants were functionally validated in cell culture models using standard molecular genetics tools. We confirmed the principle of PD-L1 immune escape in MSS-mCRC patients expressing the high affinity SNP of FcγR3a under avelumab treatment. In the monitored patient PD-L1 L88E and L88fs mutations emerged under selective pressure with avelumab. Functional validation of PD-L1 in cell models suggests that L88E represents a phosphomimetic variant of L88S identified in the AVETUX trial. Both variants exhibit loss of protein stability and proteasomal degradation. PD-L1 mutations mediate subclonal immune escape to avelumab in some patients with mCRC expressing high-affinity FcγR3a. Leucine 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. Together with the emergence of truncated PD-L1 variants, regulation of PD-L1 availabilty but not epitope disruption appears to be the key mechanistic principle towards PD-L1 escape. Future trials confirming this principle in other entities are warranted especially in patient subpopulations with rs396991.

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).

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein–Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail.

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.

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.

We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a living repository of currently >14 million B and T cell receptor (BCR/TCR) sequences from 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 was associated with interferon type I and III responses, early CD4+/CD8+ activation and counterregulation by BTLA, Tim-3, PD-1, TIGIT and CD73. Tfh, Th17-like and nonconventional (but not classical anti-viral) Th1 polarizations were induced. COVID-19 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 cohort – especially the subset with effective viral clearance – reveals fundamental insight into adaptive immunity to SARS-CoV-2 with the living repository providing a bottleneck resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development.Conflict of Interest: The authors declare no competing interests.Ethical Approval: Blood collection was performed under institutional review board approvals number 2020-039 and 11/17.