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Supplementary Figures S1-S5 Supplementary Tables S1-S3 revised

Patients with cancer, in particular patients with hematologic malignancies, are at increased risk for critical illness upon COVID-19. We here assessed antibody as well as CD4+ and CD8+ T-cell responses in unexposed and SARS-CoV-2–infected patients with cancer to characterize SARS-CoV-2 immunity and to identify immunologic parameters contributing to COVID-19 outcome. Unexposed patients with hematologic malignancies presented with reduced prevalence of preexisting SARS-CoV-2 cross-reactive CD4+ T-cell responses and signs of T-cell exhaustion compared with patients with solid tumors and healthy volunteers. Whereas SARS-CoV-2 antibody responses did not differ between patients with COVID-19 and cancer and healthy volunteers, intensity, expandability, and diversity of SARS-CoV-2 T-cell responses were profoundly reduced in patients with cancer, and the latter associated with a severe course of COVID-19. This identifies impaired SARS-CoV-2 T-cell immunity as a potential determinant for dismal outcome of COVID-19 in patients with cancer.Significance:This first comprehensive analysis of SARS-CoV-2 immune responses in patients with cancer reports on the potential implications of impaired SARS-CoV-2 T-cell responses for understanding pathophysiology and predicting severity of COVID-19, which in turn might allow for the development of therapeutic measures and vaccines for this vulnerable patient population.See related commentary by Salomé and Horowitz, p. 1877.This article is highlighted in the In This Issue feature, p. 1861

Immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pediatric patients with malignant disease may be affected by tumor therapy. Here, we report the case of a child with rhabdomyosarcoma and recurrent SARS-CoV-2 infection. Immunologic responses, analyzed by T-cell activity and anti-viral IgG levels, were impaired and not durable as a result of intensive radiochemotherapy.

Systemic sclerosis (SSc) is a connective tissue disease with poorly understood pathogenesis and limited treatment options. Patient mortality is rooted predominantly in the development of pulmonary and cardiac complications. The overactivated immune system is assumed to sustain the inflammatory signature of this autoimmune disease. Here, we investigate the potential of immunoregulatory invariant natural killer T (iNKT) cells to inhibit proinflammatory B cell responses in an in vitro model of inflammation.B cells from healthy volunteers (n = 17) and patients with SSc (n = 15) were used for functional testing upon lipopolysaccharide (LPS) stimulation in a co-culture system with third-party iNKT cells. Cytokine production was measured with antibody-based immunoassays (ELISA) and intracellular cytokine staining.iNKT cells strongly inhibited the production of proinflammatory interleukin-6 by B cells upon stimulation with LPS in both healthy volunteers and patients with SSc. In a Transwell assay, cell contact between B cells and iNKT cells proved necessary for this inhibitory effect. Similarly, blocking of CD1d on the surface of B cells abolished the immunoregulatory effect of iNKT cells on B cells. B cell subsets with higher expression of CD1d, namely unswitched memory B cells, were more susceptible to iNKT cell inhibition.Our in vitro data underline the potential of iNKT cells in the control of SSc and provide a rationale for the use of novel iNKT cell-based therapeutic strategies in the context of autoimmune diseases.

G protein-coupled receptors (GPCR) are involved in various physiological and pathophysiological processes. Functional autoantibodies targeting GPCRs have been associated with multiple disease manifestations in this context. Here we summarize and discuss the relevant findings and concepts presented in the biennial International Meeting on autoantibodies targeting GPCRs (the 4th Symposium), held in Lübeck, Germany, 15-16 September 2022. The symposium focused on the current knowledge of these autoantibodies' role in various diseases, such as cardiovascular, renal, infectious (COVID-19), and autoimmune diseases (e.g., systemic sclerosis and systemic lupus erythematosus). Beyond their association with disease phenotypes, intense research related to the mechanistic action of these autoantibodies on immune regulation and pathogenesis has been developed, underscoring the role of autoantibodies targeting GPCRs on disease outcomes and etiopathogenesis. The observation repeatedly highlighted that autoantibodies targeting GPCRs could also be present in healthy individuals, suggesting that anti-GPCR autoantibodies play a physiologic role in modeling the course of diseases. Since numerous therapies targeting GPCRs have been developed, including small molecules and monoclonal antibodies designed for treating cancer, infections, metabolic disorders, or inflammatory conditions, anti-GPCR autoantibodies themselves can serve as therapeutic targets to reduce patients' morbidity and mortality, representing a new area for the development of novel therapeutic interventions.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18–80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency., A phase I open-label trial evaluating the immunogencity, reactogenicity and safety of a peptide-based SARS-CoV-2 vaccine candidate to induce SARS-CoV-2-specific T cell responses.

Patients with cancer, in particular patients with hematologic malignancies, are at increased risk for critical illness upon COVID-19. We here assessed antibody as well as CD4+ and CD8+ T-cell responses in unexposed and SARS-CoV-2–infected patients with cancer to characterize SARS-CoV-2 immunity and to identify immunologic parameters contributing to COVID-19 outcome. Unexposed patients with hematologic malignancies presented with reduced prevalence of preexisting SARS-CoV-2 cross-reactive CD4+ T-cell responses and signs of T-cell exhaustion compared with patients with solid tumors and healthy volunteers. Whereas SARS-CoV-2 antibody responses did not differ between patients with COVID-19 and cancer and healthy volunteers, intensity, expandability, and diversity of SARS-CoV-2 T-cell responses were profoundly reduced in patients with cancer, and the latter associated with a severe course of COVID-19. This identifies impaired SARS-CoV-2 T-cell immunity as a potential determinant for dismal outcome of COVID-19 in patients with cancer. Significance: This first comprehensive analysis of SARS-CoV-2 immune responses in patients with cancer reports on the potential implications of impaired SARS-CoV-2 T-cell responses for understanding pathophysiology and predicting severity of COVID-19, which in turn might allow for the development of therapeutic measures and vaccines for this vulnerable patient population. See related commentary by Salomé and Horowitz, p. 1877. This article is highlighted in the In This Issue feature, p. 1861

Objectives To evaluate the effect of autologous stem cell transplantation (aSCT) on functional antibodies (abs) to the angiotensin II type-1-receptor (AT1R) and topoisomerase-I (topo-I) in SSc-patients and to analyse their prognostic relevance. Material and methods Forty-three SSc-patients in whom aSCT was performed were analysed. Thirty-one patients had a favourable outcome after aSCT (group 1), 12 patients showed no response or relapse (group 2). Patients’ sera were tested for anti-AT1R and anti-topo-I antibodies by ELISA and in a luminometric assay (LA) using AT1R-expressing Huh7-cells for inhibitory or stimulatory anti-AT1R antibodies before and after aSCT (4–217 months, median 28 months). Anti-topo-I antibodies were also analysed for their capacity to inhibit enzyme function. Results A total of 70% of the SSc patients had anti-topo-I- and 51% anti-AT1R antibodies in the ELISA before aSCT. In all instances, anti-topo-I antibodies inhibited topo-I-enzyme function. In the LA, 40% had stimulatory and 12% inhibitory anti-AT1R antibodies. Anti-topo-I- and anti-AT1R-reactivity (ELISA) significantly decreased after aSCT. Before aSCT, anti-topo-I-reactivity was significantly higher in group 2 patients than in group 1 patients (P Conclusions Reactivity of functionally active anti-AT1R antibodies was not influenced by aSCT, while anti-topo-I antibodies decreased after aSCT. The fact that anti-topo-I antibodies inhibited enzyme function in all instances supports the hypothesis of a pathogenetic role of the topo-I antigen/antibody-system in SSc. High anti-topo-I reactivity before aSCT was associated with an unfavourable, presence of stimulatory anti-AT1R antibodies with a favourable course after aSCT.

Summary Background Vegan diet (VD) has improved inflammatory activity in patients with rheumatoid arthritis (RA) in several small controlled trials. The underlying mechanism remains widely unclear. We investigated the effect of a VD in comparison to a meat-rich diet (MD) on markers of inflammation (which have been shown to be relevant in patients with RA) in healthy volunteers. Methods 53 healthy, omnivore subjects were randomized to a controlled VD (n = 26) or MD (n = 27) for 4 weeks following a pre-treatment phase of a one week controlled mixed diet. Primary parameters of interest were sialylation of immunoglobulins, percentage of regulatory T-cells and level of interleukin 10 (IL10). Usual care immune parameters used in patients with RA and amino acid serum levels as well as granulocytes and monocytes colony stimulating factor (GM-CSF) serum levels were secondary parameters. Results In the VD group, total leukocyte, neutrophil, monocyte and platelet counts decreased and after four weeks they were significantly lower compared to the MD group (ANCOVA: leukocytes p = 0.003, neutrophils p = 0.001, monocytes p = 0.032, platelets p = 0.004). Leukocytes, neutrophils, monocytes, and platelets correlated with each other and likewise conform with serum levels of branched-chain amino acids, which were significantly lower in the VD compared to the MD group. The primary parameters did not differ between the groups and BMI remained stable in the two groups. Conclusion Four weeks of a controlled VD affected the number of neutrophils, monocytes and platelets but not the number or function of lymphocytes. The relation with branched-chain amino acids and GM-CSF suggests a mode of action via the mTOR signaling pathway. Registered at http://www.drks.de (German Clinical Trial register) at DRKS00011963.

Summary Dendritic cells (DCs) are sentinels of the immune system that bridge innate and adaptive immunity. By capturing antigens in peripheral tissue, processing and presenting them with concurrent expression of co-stimulatory molecules and cytokine secretion they control and modulate immune reactions. Through pattern recognition receptors, DCs sense molecules that are associated with infection or tissue damage, frequently resulting in the formation of inflammasomes upon intracellular stimulation. The inherited autoinflammatory familial Mediterranean fever (FMF) is associated with deregulated activity of the pyrin inflammasome leading to acute inflammatory episodes. However, differentiation and function of DCs in this disease are as yet unclear. Therefore, we first determined DC subpopulation frequency in peripheral blood of a cohort of FMF patients. Joint evaluation without classification according to specific patient characteristics, such as mutational status, did not disclose significant differences compared to healthy controls. For the further examination of phenotype and function, we used immature and mature monocyte-derived DCs (imMo-DCs, mMo-DCs) that were generated in vitro from FMF patients. Immunophenotypical analysis of imMo-DCs revealed a significantly elevated expression of CD83, CD86 and human leukocyte antigen D-related (HLA-DR) as well as a significant down-regulation of CD206, CD209 and glycoprotein NMB (GPNMB) in our FMF patient group. Furthermore, FMF imMo-DCs presented a significantly higher capacity to migrate and to stimulate the proliferation of unmatched allogeneic T cells. Finally, the transition towards a more mature, and therefore activated, phenotype was additionally reinforced by the fact that peripheral blood DC populations in FMF patients exhibited significantly increased expression of the co-stimulatory molecule CD86.

Objective Systemic sclerosis (SSc) is a connective tissue disease with a significant morbidity and reduced survival of patients. Effective treatment and clinical control of the disease remain challenging. In particular, the development of pulmonary and cardiac fibrosis and pulmonary hypertension are severe complications responsible for excessive mortality. Currently available treatment strategies only alleviate symptoms and slow disease progression. Here, we investigated the therapeutic potential of ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor used in B cell malignancies, to alter B cell pathology in SSc in an in vitro model of autoimmunity. Methods PBMCs and sorted B cells of 24 patients with SSc were used for functional testing after stimulation with hypomethylated DNA fragments (CpG) to induce an innate immune response. The effects of ibrutinib on cytokine production, autoantibody release, and activation of the transcription factor NFκB were evaluated. Results Ibrutinib was able to reduce the production of the profibrotic hallmark cytokines IL-6 and TNF-α mainly from the effector B cell population in patients with SSc. Importantly, small doses of ibrutinib (0.1 μM) preserved the production of immunoregulatory IL-10 while effectively inhibiting hyperactivated, profibrotic effector B cells. In a flow cytometry analysis of phosphorylated NFκB, an important transcription factor in the induction of innate immune responses in B cells, significantly less activation was observed with ibrutinib treatment. Conclusion Our data could pave the avenue for a clinical application of ibrutinib for patients with SSc as a novel treatment option for the underlying pathogenetic immune imbalance contributing to disease onset and progression.

T-cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T-cell epitopes derived from various viral proteins, combined with the toll-like receptor 1/2 agonist XS15 emulsified in MontanideTM ISA51 VG, aiming to induce superior SARS-CoV-2 T-cell immunity to combat COVID-19. We conducted a Phase I open-label trial, including 36 participants aged 18 to 80 years, who received one single subcutaneous CoVAC-1 vaccination. The primary endpoint was safety analyzed until day 56. Immunogenicity in terms of CoVac-1-induced T-cell response was analyzed as main secondary endpoint until day 28. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study subjects, while systemic reactogenicity was absent or mild. SARS-CoV-2-specific T-cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T-helper 1 CD4+ and CD8+ T cells. CoVac-1-induced interferon-γ T-cell responses by far surpassed those detected in COVID-19 convalescents and were unaffected by current SARS-CoV-2 variants of concern (VOC). Together, CoVac-1 showed a favorable safety profile and induced broad, potent, and VOC-independent T-cell responses, supporting the presently ongoing evaluation in a Phase II trial for patients with B-cell/antibody deficiency. Funded by the Ministry of Science, Research and the Arts Baden-Württemberg, Germany; ClinicalTrials.gov number, NCT04546841.

Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry–based approach to identify naturally presented HLA class I– and class II–restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL– and ABL-BCR–derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell–based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare tumor disease, which affects children and adolescents without history of primary liver disease. Beside surgical resection established treatment options are lacking for FL-HCC. Recently, the DNAJB1-PRKACA fusion transcript was identified as the oncogenic driver of tumor pathogenesis in 100% of FL-HCC patients. Here, we investigated the role of the DNAJB1-PRKACA fusion protein as a source for immunogenic neoepitopes and showed first immunotherapeutic application of these antigens in a FL-HCC patient.HLA class I- and class II-presented neoantigens derived from the DNAJB1-PRKACA fusion protein were predicted in silico using NetMHCpan 4.1 and SYFPEITHI 1.0, or NetMHCIIpan 4.0, respectively. With this workflow nine binding cores of nine amino acid length for a total of 1290 different HLA class II alleles, as well as 13 HLA class I ligands for the 20 most frequent HLA class I allotypes (European population, iedb.org) were identified. Cellular processing and HLA presentation of DNAJB1-PRKACA-derived peptides was proven by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) of DNAJB1-PRKACA-transduced HCC cell lines. Immunogenicity of DNAJB1-PRKACA-derived peptides was assessed for the HLA class II peptide (PII-1) and the HLA-A*24 peptide (PA*24) by in vitro priming experiments which showed an induction of multifunctional peptide-specific CD4+ and CD8+ T cells, respectively, with expression of CD107a, IFNγ, and TNF upon peptide-pulsing. Furthermore, PA*24-specific T cells showed antigen-specific lysis of autologous peptide-loaded target cells and single-cell next-generation sequencing (10x Genomics) of PA*24-specific CD8+ T cells further enabled the identification of DNAJB1-PRKACA-reactive T cell receptors. Based on these preclinical data we applied a peptide vaccine, consisting of three HLA class I ligands (PA*02, PB*44, and PC*05) and PII-1 spanning the DNAJB1-PRKACA fusion region, to a 15-year old patient with histologically confirmed FL-HCC, who experienced multiple tumor relapses after early liver transplant due to unresectable FL-HCC not responsive to chemotherapy. After two vaccinations in vivo induction of multifunctional CD4+ T cells targeting PII-1 and PB*44 was observed by IFNγ ELISPOT. Single-cell RNA sequencing of vaccine-induced CD4+ T cells revealed distinct gene expression clusters of T cell activation and high TCR clonality. DNAJB1-PRKACA-specific T cells persisted in peripheral blood and were accompanied by relapse free survival of the patient until now, more than one year post vaccination. These findings identified the DNAJB1-PRKACA fusion transcript as novel prime source for broadly applicable neoepitopes and corresponding TCRs and provide first evidence for their application in cancer immunotherapy of FL-HCC. Citation Format: Jens Bauer, Natalie Köhler, Yacine Maringer, Philip Bucher, Tatjana Bilich, Melissa Zwick, Severin Dicks, Annika Nelde, Marissa Dubbelaar, Jonas Scheid, Marcel Wacker, Jonas J. Heitmann, Sarah Schroeder, Jonas Rieth, Monika Denk, Marion Richter, Reinhild Klein, Irina Bonzheim, Julia Luibrand, Ursula Holzer, Martin Ebinger, Ines B. Brecht, Michael Bitzer, Melanie Boerries, Helmut R. Salih, Hans-Georg Rammensee, Stephan Hailfinger, Juliane S. Walz. The oncogenic fusion protein DNAJB1-PRKACA can be actively targeted by peptide-based immunotherapy in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2008.

Low human leukocyte antigen (HLA)-DR expression might compromise CD4+ T-cell-mediated anti-tumor immunity. Its immunological and clinical significance however remain undefined in non-promyelocytic acute myeloid leukemia (AML). Taking advantage of mass spectrometry-based immunopeptidome analysis of primary AML samples (n = 31), we studied the implications of low HLA-DR expression for antigen presentation and analyzed its association with disease characteristics and survival within a cohort of 399 AML patients. Remarkably, overall HLA-DR/DQ immunopeptidome diversity was preserved in AML with low HLA-DR expression (HLA-DRlow AML) and was associated with a shift in HLA-DR/DQ allotype abundances (HLA-DQ to HLA-DR/DQ ligand ratio 0.36 vs 0.19 in HLA-DRlow and HLA-DRhigh patients, respectively). Consistent with unimpaired antigenicity, survival was similar in HLA-DRlow and HLA-DRhigh patients. Demonstrating for the first time that overall HLA-DR/DQ antigen presentation is preserved in HLA-DRlow AML, our findings provide a rationale for the non-inferior outcome observed in HLA-DRlow AML patients.

Cancer patients, in particular patients with hematological malignancies, are at increased risk for critical illness upon COVID-19. We here assessed antibody as well as CD4+ and CD8+ T cell responses in unexposed and SARS-CoV-2-infected cancer patients to characterize SARS-CoV‑2 immunity and to identify immunological parameters contributing to COVID-19 outcome. Unexposed patients with hematological malignancies presented with reduced prevalence of pre-existing SARS-CoV-2 cross-reactive CD4+ T cell responses and signs of T cell exhaustion when compared to solid tumor patients and healthy volunteers. Whereas SARS-CoV-2 antibody responses did not differ between COVID-19 cancer patients and healthy volunteers, intensity, expandability, and diversity of SARS-CoV-2 T cell responses were profoundly reduced in cancer patients, and the latter associated with a severe course of COVID-19. This identifies impaired SARS-CoV-2 T cell immunity as determinant for dismal outcome of COVID-19 in cancer patients.

COVID-19 disease increases interleukin (IL)-1β release. Anti-IL-1-treatment is effective in IL-1-mediated autoinflammatory diseases (AID). This case series presents COVID-19 in patients with IL-1-mediated and unclassified AID with immunosuppressive therapy (IT). Patient 1 is a 34-year-old woman with an unclassified AID and methotrexate. Patients 2 and 3 (14-year-old girl and 12-year-old boy, respectively) have a Cryopyrin-Associated Periodic Syndrome (NLRP3 p.Q703K heterozygous, CAPS) treated with canakinumab 150 mg/month since three and five years, respectively. Patient 4 is a 15-year-old girl who has had familial Mediterranean fever (MEFV p.M694V homozygous) for 3 years treated with canakinumab 150 mg/month and colchicine. All patients had a mild acute COVID-19 course, particularly the adolescent patients. A few weeks after COVID-19 recovery, both CAPS patients developed increased AID activity, necessitating anti-IL-1-treatment intensification in one patient. At day 100, one out of four patients (25%) showed positive antibody response to SARS-CoV-2. This is one of the first reports providing follow-up data about COVID-19 in AID. The risk for severe acute COVID-19 disease was mild/moderate, but increased AID activity post-COVID-19 was detected. Follow-up data and data combination are needed to expand understanding of COVID-19 and SARS-CoV-2 immunity in AID and the role of IT.

The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, we treated 2 patients with defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved during treatment. Moreover, coagulation parameters indicating hypofibrinolysis and complement activation normalized.The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, 2 patients received defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved and hypofibrinolysis/complement activation normalized during treatment.

The SARS-CoV-2 pandemic calls for the rapid development of diagnostic, preventive, and therapeutic approaches. CD4+ and CD8+ T cell-mediated immunity is central for control of and protection from viral infections[1-3]. A prerequisite to characterize T-cell immunity, but also for the development of vaccines and immunotherapies, is the identification of the exact viral T-cell epitopes presented on human leukocyte antigens (HLA)[2-8]. This is the first work identifying and characterizing SARS-CoV-2-specific and cross-reactive HLA class I and HLA-DR T-cell epitopes in SARS-CoV-2 convalescents (n = 180) as well as unexposed individuals (n = 185) and confirming their relevance for immunity and COVID-19 disease course. SARS-CoV-2-specific T-cell epitopes enabled detection of post-infectious T-cell immunity, even in seronegative convalescents. Cross-reactive SARS-CoV-2 T-cell epitopes revealed preexisting T-cell responses in 81% of unexposed individuals, and validation of similarity to common cold human coronaviruses provided a functional basis for postulated heterologous immunity[9] in SARS-CoV-2 infection[10,11]. Intensity of T-cell responses and recognition rate of T-cell epitopes was significantly higher in the convalescent donors compared to unexposed individuals, suggesting that not only expansion, but also diversity spread of SARS-CoV-2 T-cell responses occur upon active infection. Whereas anti-SARS-CoV-2 antibody levels were associated with severity of symptoms in our SARS-CoV-2 donors, intensity of T-cell responses did not negatively affect COVID-19 severity. Rather, diversity of SARS-CoV-2 T-cell responses was increased in case of mild symptoms of COVID-19, providing evidence that development of immunity requires recognition of multiple SARS-CoV-2 epitopes. Together, the specific and cross-reactive SARS-CoV-2 T-cell epitopes identified in this work enable the identification of heterologous and post-infectious T-cell immunity and facilitate the development of diagnostic, preventive, and therapeutic measures for COVID-19.

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.

Here, we describe the preliminary results of an experimental vaccination of a self-experimenting healthy volunteer with eight SARS-CoV-2-derived peptides: five predicted to bind to HLA class I molecules (CD8 peptides) and three predicted to bind to HLA-DR molecules (CD4 peptides). The vaccine formulation also included one long and one short CMV-pp65-derived peptide that had previously been administered to the same individual and could thus act as positive controls. It further contained the new adjuvant XS15 and was administered as an emulsion in Montanide as a single subcutaneous (s.c.) injection. Peripheral blood mononuclear cells (PBMCs) isolated from blood drawn on day 36 before vaccination and day 19 after vaccination were assessed using an ex vivo Interferon-γ ELISpot assay. We detected strong vaccine-induced T-cell responses against all four CD4 peptides and against the recall CMV CD8 epitope, but found no immune responses against the five predicted SARS-CoV-2 CD8 peptides. Antibody reactivity against all the SARS-CoV-2 CD4 peptides, as detected using ELISA, was negative or marginal. We interpret these results in terms of the prospects of a therapeutic vaccine to be applied in symptomatic COVID-19 patients. An advantage of this approach is the possibility to assess efficacy or failure within a short time after vaccination.

Background: Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) constitute rare chronic inflammatory biliary diseases which likely comprise genetic, environmental and autoimmune factors. Specific inhibitory (auto-) antibodies against the muscarinic acetylcholine receptor type 3 (mAChR3 auto-ab) may contribute to the pathogenesis of chronic biliary inflammation by modulating mAChR3− mediated signaling. Aims: The aim of this study was to analyze the prevalence and relevance of inhibitory mAChR3 auto-ab (mAChR3inh+ auto-ab) in a large cohort of PBC patients from two independent tertiary centers in Berlin and Leipzig in comparison to a large PSC cohort. Baseline parameters and response rates to standard treatment with ursodeoxycholic acid (UDCA) were characterized with respect to the individual mAChR3 auto-ab status. Methods: In total, the study population comprised 437 PBC patients, 187 PSC patients and 80 healthy controls. Clinical and laboratory baseline characteristics were retrieved from medical records. The response to ursodeoxycholic acid (UDCA) therapy after 12 months of treatment was available in 176 PBC and 45 PSC patients. Results: The prevalence of mAChR3inh+ auto-ab was significantly higher among PBC patients (11.2%, 49/437; p = 0.008 vs. healthy controls) and PSC patients (33.6%, 63/187; p < 0.0001 vs. healthy controls) compared to healthy controls (2.5%, 2/80), respectively. PBC patients with mAChR3inh+ auto-ab exhibited significantly higher levels of alkaline phosphatase (ALP) and bilirubin, which constitute established parameters for PBC risk stratification. Moreover, mAChR3inh+ PBC patients tended to show decreased response rates to UDCA therapy compared to PBC patients without mAChR3inh+ auto-ab (mAChR3− PBC). In contrast, PSC patients with mAChR3inh+ auto-ab showed no significant differences in laboratory findings compared to mAChR3 auto-ab negative (mAChR3−) PSC patients. Conclusion: MAChR3inh+ auto-ab might be involved in the pathogenesis and treatment response of chronic biliary inflammation in patients with PBC but not in patients with PSC.

Background Systemic sclerosis (SSc) is a potentially fatal autoimmune disease that leads to extensive fibrosis of the skin and internal organs. Invariant natural killer T (iNKT) cells are potent immunoregulatory T lymphocytes being able to orchestrate dysregulated immune responses. The purpose of this study was to evaluate numbers and function of iNKT cells in patients with SSc and to analyze their correlation with disease parameters. Methods Human iNKT cells from 88 patients with SSc and 33 healthy controls were analyzed by flow cytometry. Their proliferative capacity and cytokine production were investigated following activation with CD1d ligand α-galactosylceramide (α-GalCer). Results We observed an absolute and relative decrease of iNKT cells in patients with SSc compared with healthy controls. Interestingly, the subtype of SSc, disease severity, or treatment with immunosuppressive drugs did not affect iNKT cell numbers. However, T helper (Th) cell immune polarization was biased towards a Th17 immunophenotype in SSc patients. Moreover, iNKT cells from patients with SSc showed a significantly decreased expansion capacity upon stimulation with α-GalCer. Conclusion iNKT cells are deficient and functionally impaired in patients with SSc. Therefore, adoptive transfer strategies using culture-expanded iNKT cells could be a novel approach to treat SSc patients.

BACKGROUND Determination and quantification of serum free light chains (FLC) is essential for the diagnosis and monitoring of patients with monoclonal gammopathies. Currently, the Freelite assay (The Binding Site, United Kingdom) and the N Latex FLC assay (Siemens Healthineers, Germany) are available for FLC determination. Data concerning stability of FLC following long-term storage are limited. Therefore, the aim of the present study is to investigate the stability of FLC in frozen samples determined by the N latex FLC assay. METHODS One hundred eighty-eight serum samples from 47 patients with monoclonal gammopathies were analyzed at the beginning and after long-term storage (-20°C, 193 - 568 days). Serum free light chain kappa (κFLC) and lambda (λFLC) concentrations were measured, and the corresponding kappa/lambda (κ/λ FLC) ratios were calculated. All samples were analyzed with the N latex FLC assay on a single BNII System. RESULTS Comparison analyses between fresh and stored samples revealed very strong correlations for the determination of κFLC (r = 0.993), λFLC (r = 0.998) and the calculated κ/λ FLC ratio (r = 0.997). Median percentage changes of κFLC (-0.5%) and λFLC (-0.8%) measurements and the calculated κ/λ FLC ratios (-3.7%) were within the calculated analytical imprecision of the FLC assay. Changes of κFLC (r = 0.17, p = 0.02) and λFLC (r = 0.28, p < 0.01) concentrations and of the κ/λ FLC ratio (r = 0.18, p = 0.01) over time were very weak, but statistically significant. CONCLUSIONS Serum free light chains in serum samples are sufficiently stable following long-term frozen storage and are therefore suitable to be measured with the N Latex FLC assay.

Im Abstand von 2 Wochen stellten sich 2 Kinder aus derselben Nachbarschaft mit Granuloma anulare (GA) und Uveitis intermedia (UI) vor. Ein Zufall schien unwahrscheinlich, da UI bei Kindern und zusatzlich in Zusammenhang mit GA sehr selten ist. Eine ausfuhrliche Diagnostik wurde durchgefuhrt, um andere assoziierte Erkrankungen auszuschliesen. Keine systemischen Erkrankungen, Besonderheiten bei Impfungen oder Medikation, keine Infektion oder toxische Ursache wurden gefunden. Eine Assoziation von GA und UI, die auf anderen, bisher nicht nachgewiesenen Ursachen beruht, ist dennoch moglich. Daher sollte bei der Untersuchung einer neu diagnostizierten UI auch bezuglich GA gefragt und untersucht werden.

Importance Achromatopsia linked to variations in theCNGA3gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available. Objective To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encodingCNGA3(AAV8.CNGA3) in patients withCNGA3-linked achromatopsia. Design, Setting, and Participants This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants inCNGA3were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018. Intervention Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017). Main Outcomes and Measures Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment. Results Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor–mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13;P = .006, 2-sidedttest paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log;P = .003, 2-sidedttest paired samples). Conclusions and Relevance Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains. Trial Registration ClinicalTrials.gov Identifier:NCT02610582

It is not known whether food additives associated with western lifestyle, such as the widely used carrageenan, also play a role in the pathogenesis of diabetes. Animal data suggest that increased carrageenan consumption causes insulin resistance and diabetes, mainly by interfering with hepatic insulin signaling. This is the first trial in humans that tested whether carrageenan added to the diet affects key metabolic traits. We conducted a randomized, double-blind, placebo-controlled, cross-over trial (registered as NCT02629705). Healthy males (N=20) were randomly allocated to 14 days of carrageenan (250 mg twice daily) or matching placebo. After a washout-period of 30±7 days, they received the other compound. At the end of each treatment phase, participants underwent an oral glucose tolerance test (OGTT), a hyperinsulinemic-euglycemic clamp with labeled glucose, whole-body MR-tomography and 1H-MR-spectroscopy, blood immune cell phenotyping and a lactulose-mannitol test for investigating intestinal permeability. Participants had a mean (±SD) age of 27.6±4.8 years and a BMI of 24.4±2.6 kg/m2. Intestinal permeability significantly increased after carrageenan vs. placebo exposure (lactulose-mannitol-ratio 0.0196 vs. 0.015, p=0.03). Whole-body insulin sensitivity index (ISI) did not differ between placebo and carrageenan exposure (p=0.5 for both ISIclamp and ISIOGTT). No changes of hepatic fat content, body fat mass, distribution, liver transaminases and inflammatory cytokines were observed (all p>0.4). In young healthy males, short-term carrageenan intake resulted in increased gut permeability. This was not accompanied by changes of whole-body insulin sensitivity, body fat mass, distribution and liver fat content. Further investigations of hepatic and brain insulin sensitivity, immune phenotyping will reveal whether carrageenan intake affected these variables. Disclosure R. Wagner: None. J. Büttner: None. M. Heni: Research Support; Self; Boehringer Ingelheim GmbH. Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Merck Sharp & Dohme Corp., Novo Nordisk Inc.. L. Fritsche: None. S. Kullmann: None. M. Wagmüller: None. A. Peter: None. H. Preissl: None. J. Machann: None. U. Pape: None. G. van Hall: None. P. Plomgaard: None. R. Klein: None. A. Fritsche: Advisory Panel; Self; Sanofi-Aventis Deutschland GmbH, Novo Nordisk A/S, Eli Lilly and Company, Boehringer Ingelheim GmbH. H. Haering: None. N. Stefan: Consultant; Self; Merck Sharp & Dohme Corp.. Speaker's Bureau; Self; Novo Nordisk A/S. Research Support; Self; AstraZeneca. Speaker's Bureau; Self; AstraZeneca, OmniaMed Ltd..

PURPOSE. To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients. METHODS. Three groups of six NHP each received subretinal injections (high dose: 1 x 10(12) vector genomes [vg], low dose: 1 x 10(11) vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 x 10(12) vg). Three patients received 1 x 10(10) vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients. RESULTS. Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no humoral immune response in patients following subretinal delivery of 1 x 10(10) vg. Conclusion: S. No significant induction of ADA occurred in NHP when mimicking the clinical scenario of subretinal delivery with a clinical-grade rAAV8 and concomitant immunosuppression. Likewise, clinical data showed no humoral immune response in patients. In contrast, intravitreal delivery was associated with a substantial humoral immune response. Subretinal delivery might be superior to an intravitreal application regarding immunologic aspects.

To evaluate the effect of autologous stem cell transplantation (aSCT) on antibody (ab) reactivity towards linear epitopes of topoisomerase-I (topo-I/Scl70) in patients with systemic sclerosis (SSc) and to correlate antibody reactivities with clinical outcome after aSCT.Fourteen anti-topo-I/Scl70-positive SSc-patients were analysed before and after non-myeloablative aSCT. Five patients showed ongoing good response (group 1), 9 had primarily responded but later relapsed or did not respond (group 2). Patients' sera were tested by ELISA against full length (fl) topo-I and 45 overlapping 25-mer peptides. Furthermore, for comparison sera from patients with anti-topo-I-negative SSc (n=12), other collagen disorders (n=6), and from 21 healthy controls (HC) were analysed.Anti-topo-I-positive SSc-sera showed significantly higher IgG-reactivity as compared to HC towards 34 of the 45 peptides. Especially peptide 39 (aa647-671) emerged as a immunodominant epitope being recognised predominantly by anti-topo-I-positive SSc-sera. Reactivity towards 17 of the 45 peptides decreased after aSCT in group 1- and 2-patients. Before aSCT, group 1-patients had lower antibody reactivity towards peptide 39 than group 2-patients. There was no change in peptide-specificity after aSCT.Reactivity towards topo-I-epitopes is heterogeneous in SSc, but peptide 39 (aa647-671) may be another immunodominant epitope besides the published epitope aa489-573. Antibody reactivity to this peptide 39 was higher in group 2- than in group 1-patients. Peptide recognition pattern did not change after aSCT.