A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Autoimmunity Against Surfactant Protein B Is Associated with Pneumonitis During Checkpoint Blockade.

Rationale: Immune checkpoint inhibitor (ICI)-related pneumonitis is a serious autoimmune event affecting as many as 20% of patients with non-small-cell lung cancer (NSCLC), yet the factors underpinning its development in some patients and not others are poorly understood. Objectives: To investigate the role of autoantibodies and autoreactive T cells against surfactant-related proteins in the development of pneumonitis. Methods: The study cohort consisted of patients with NSCLC who provided blood samples before and during ICI treatment. Serum was used for proteomics analyses and to detect autoantibodies present during pneumonitis. T-cell stimulation assays and single-cell RNA sequencing were performed to investigate the specificity and functionality of peripheral autoreactive T cells. The findings were confirmed in a validation cohort comprising patients with NSCLC and patients with melanoma. Measurements and Main Results: Across both cohorts, patients in whom pneumonitis developed had higher pretreatment levels of immunoglobulin G autoantibodies targeting surfactant protein (SP)-B. At the onset of pneumonitis, these patients also exhibited higher frequencies of CD4+ IFN-γ-positive SP-B-specific T cells and expanding T-cell clonotypes recognizing this protein, accompanied by a proinflammatory serum proteomic profile. Conclusions: Our data suggest that the cooccurrence of SP-B-specific immunoglobulin G autoantibodies and CD4+ T cells is associated with the development of pneumonitis during ICI therapy. Pretreatment levels of these antibodies may represent a potential biomarker for an increased risk of developing pneumonitis, and on-treatment levels may provide a diagnostic aid.

Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Autoreactive T cells targeting type II pneumocyte antigens in COVID-19 convalescent patients.

BACKGROUND: The role of autoreactive T cells on the course of Coronavirus disease-19 (COVID-19) remains elusive. Type II pneumocytes represent the main target cells of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autoimmune responses against antigens highly expressed in type II pneumocytes may influence the severity of COVID-19 disease. OBJECTIVE: The aim of this study was to investigate autoreactive T cell responses against self-antigens highly expressed in type II pneumocytes in the blood of COVID-19 patients with severe and non-severe disease. METHODS: We collected blood samples of COVID-19 patients with varying degrees of disease severity and of pre-pandemic controls. T cell stimulation assays with peptide pools of type II pneumocyte antigens were performed in two independent cohorts to analyze the autoimmune T cell responses in patients with non-severe and severe COVID-19 disease. Target cell lysis assays were performed with lung cancer cell lines to determine the extent of cell killing by type II PAA-specific T cells. RESULTS: We identified autoreactive T cell responses against four recently described self-antigens highly expressed in type II pneumocytes, known as surfactant protein A, surfactant protein B, surfactant protein C and napsin A, in the blood of COVID-19 patients. These antigens were termed type II pneumocyte-associated antigens (type II PAAs). We found that patients with non-severe COVID-19 disease showed a significantly higher frequency of type II PAA-specific autoreactive T cells in the blood when compared to severely ill patients. The presence of high frequencies of type II PAA-specific T cells in the blood of non-severe COVID-19 patients was independent of their age. We also found that napsin A-specific T cells from convalescent COVID-19 patients could kill lung cancer cells, demonstrating the functional and cytotoxic role of these T cells. CONCLUSIONS: Our data suggest that autoreactive type II PAA-specific T cells have a protective role in SARS-CoV-2 infections and the presence of high frequencies of these autoreactive T cells indicates effective viral control in COVID-19 patients. Type II-PAA-specific T cells may therefore promote the killing of infected type II pneumocytes and viral clearance.

BP180-specific IgG is associated with skin adverse events, therapy response, and overall survival in non-small cell lung cancer patients treated with checkpoint inhibitors.

BACKGROUND: Anti-programmed cell death protein 1 (PD1)/programmed death-ligand 1(PD-L1) therapy frequently entails immune-related adverse events (irAEs), and biomarkers to predict irAEs are lacking. Although checkpoint inhibitors have been found to reinvigorate T cells, the relevance of autoantibodies remains elusive. OBJECTIVE: Our aim was to explore whether IgG autoantibodies directed against coexpressed antigens by tumor tissue and healthy skin correlate with skin irAEs and therapy outcome. METHODS: We measured skin-specific IgG via enzyme-linked immunosorbent assay in patients with non-small cell lung cancer (NSCLC) who received anti-PD1/PD-L1 treatment between July 2015 and September 2017 at the Kantonsspital St. Gallen. Sera were sampled at baseline and during therapy after 8 weeks. RESULTS: Analysis of publicly available tumor expression data revealed that NSCLC and skin coexpress BP180, BP230, and type VII collagen. A skin irAE developed in 16 of 40 recruited patients (40%). Only elevated anti-BP180 IgG at baseline significantly correlated with the development of skin irAEs (P = .04), therapy response (P = .01), and overall survival (P = .04). LIMITATIONS: The patients were recruited in a single tertiary care center. CONCLUSIONS: Our data suggest that the level of anti-BP180 IgG of NSCLC patients at baseline is associated with better therapy response and overall survival and with a higher probability to develop skin irAEs during anti-PD1/PD-L1 treatment.

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

BACKGROUND: Leucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive. RESULTS: The LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants. CONCLUSIONS: LRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

Immune signatures predict development of autoimmune toxicity in patients with cancer treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are among the most promising treatment options for melanoma and non-small cell lung cancer (NSCLC). While ICIs can induce effective anti-tumor responses, they may also drive serious immune-related adverse events (irAEs). Identifying biomarkers to predict which patients will suffer from irAEs would enable more accurate clinical risk-benefit analysis for ICI treatment and may also shed light on common or distinct mechanisms underpinning treatment success and irAEs. METHODS: In this prospective multi-center study, we combined a multi-omics approach including unbiased single-cell profiling of over 300 peripheral blood mononuclear cell (PBMC) samples and high-throughput proteomics analysis of over 500 serum samples to characterize the systemic immune compartment of patients with melanoma or NSCLC before and during treatment with ICIs. FINDINGS: When we combined the parameters obtained from the multi-omics profiling of patient blood and serum, we identified potential predictive biomarkers for ICI-induced irAEs. Specifically, an early increase in CXCL9/CXCL10/CXCL11 and interferon-γ (IFN-γ) 1 to 2 weeks after the start of therapy are likely indicators of heightened risk of developing irAEs. In addition, an early expansion of Ki-67+ regulatory T cells (Tregs) and Ki-67+ CD8+ T cells is also likely to be associated with increased risk of irAEs. CONCLUSIONS: We suggest that the combination of these cellular and proteomic biomarkers may help to predict which patients are likely to benefit most from ICI therapy and those requiring intensive monitoring for irAEs. FUNDING: This work was primarily funded by the European Research Council, the Swiss National Science Foundation, the Swiss Cancer League, and the Forschungsförderung of the Kantonsspital St. Gallen.

Autoreactive napsin A-specific T cells are enriched in lung tumors and inflammatory lung lesions during immune checkpoint blockade.

Cancer treatment with immune checkpoint blockade (ICB) often induces immune-related adverse events (irAEs). We hypothesized that proteins coexpressed in tumors and normal cells could be antigenic targets in irAEs and herein described DITAS (discovery of tumor-associated self-antigens) for their identification. DITAS computed transcriptional similarity between lung tumors and healthy lung tissue based on single-sample gene set enrichment analysis. This identified 10 lung tissue-specific genes highly expressed in the lung tumors. Computational analysis was combined with functional T cell assays and single-cell RNA sequencing of the antigen-specific T cells to validate the lung tumor self-antigens. In patients with non-small cell lung cancer (NSCLC) treated with ICB, napsin A was a self-antigen that elicited strong CD8+ T cell responses, with ICB responders harboring higher frequencies of these CD8+ T cells compared with nonresponders. Human leukocyte antigen (HLA) class I ligands derived from napsin A were present in human lung tumors and in nontumor lung tissues, and napsin A tetramers confirmed the presence of napsin A-specific CD8+ T cells in blood and tumors of patients with NSCLC. Napsin A-specific T cell clonotypes were enriched in lung tumors and ICB-induced inflammatory lung lesions and could kill immortalized HLA-matched NSCLC cells ex vivo. Single-cell RNA sequencing revealed that these T cell clonotypes expressed proinflammatory cytokines and cytotoxic markers. Thus, DITAS successfully identified self-antigens, including napsin A, that likely mediate effective antitumor T cell responses in NSCLC and may simultaneously underpin lung irAEs.

Defects in Cell Wall Differentiation of the Arabidopsis Mutant rol1-2 Is Dependent on Cyclin-Dependent Kinase CDK8.

Plant cells are encapsulated by cell walls whose properties largely determine cell growth. We have previously identified the rol1-2 mutant, which shows defects in seedling root and shoot development. rol1-2 is affected in the Rhamnose synthase 1 (RHM1) and shows alterations in the structures of Rhamnogalacturonan I (RG I) and RG II, two rhamnose-containing pectins. The data presented here shows that root tissue of the rol1-2 mutant fails to properly differentiate the cell wall in cell corners and accumulates excessive amounts of callose, both of which likely alter the physical properties of cells. A surr (suppressor of the rol1-2 root developmental defect) mutant was identified that alleviates the cell growth defects in rol1-2. The cell wall differentiation defect is re-established in the rol1-2 surr mutant and callose accumulation is reduced compared to rol1-2. The surr mutation is an allele of the cyclin-dependent kinase 8 (CDK8), which encodes a component of the mediator complex that influences processes central to plant growth and development. Together, the identification of the surr mutant suggests that changes in cell wall composition and turnover in the rol1-2 mutant have a significant impact on cell growth and reveals a function of CDK8 in cell wall architecture and composition.

Keratinocyte differentiation antigen-specific T cells in immune checkpoint inhibitor-treated NSCLC patients are associated with improved survival.

Immune checkpoint inhibitors (ICIs) have improved the survival of patients with non-small cell lung cancer (NSCLC) by reinvigorating tumor-specific T cell responses. However, the specificity of such T cells and the human leukocyte antigen (HLA)-associated epitopes recognized, remain elusive. In this study, we identified NSCLC T cell epitopes of recently described NSCLC-associated antigens, termed keratinocyte differentiation antigens. Epitopes of these antigens were presented by HLA-A 03:01 and HLA-C 04:01 and were associated with responses to ICI therapy. Patients with CD8+ T cell responses to these epitopes had improved overall and progression-free survival. T cells specific for such epitopes could eliminate HLA class I-matched NSCLC cells ex vivo and were enriched in patient lung tumors. The identification of novel lung cancer HLA-associated epitopes that correlate with improved ICI-dependent treatment outcomes suggests that keratinocyte-specific proteins are important tumor-associated antigens in NSCLC. These findings improve our understanding of the mechanisms of ICI therapy and may help support the development of vaccination strategies to improve ICI-based treatment of these tumors.

Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines.

The requisite analyses on bone marrow biopsies are increasing: Molecular analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR are demanded in addition to morphology and immunohistochemistry to improve diagnostic accuracy. Moreover, analysis of certain molecular prognostic or predictive biomarkers is increasingly mandatory in the assessment of hematologic diseases. In some circumstances, only formalin fixed, bone-containing tissue is available for molecular analysis. Because various fixation and decalcification procedures can impair DNA and RNA quality, there is an urgent need for standardized decalcification protocols which allow FISH and PCR analysis. In this study we developed a routinely applicable decalcification protocol to optimize the molecular analysis method although preserving morphology and immunohistochemical results. Therefore, we compared 2 different approaches including ultrasonic decalcification versus nonultrasonic procedures and ethylenediaminetetraacetate-based reagents versus acid-based ones. In our hands, the combined use of ultrasound and ethylenediaminetetraacetate-based reagents permits successful interphase FISH, PCR, and RT-PCR analysis whereas concomitantly preserving morphology and antigeneicity.

Identification of the genetic defect in the original Wagner syndrome family.

PURPOSE: The aim of the present study was to determine the genetic defect in Wagner syndrome, a rare disorder belonging to the group of hereditary vitreoretinal degenerations. This disease has been genetically mapped to chromosome 5q14.3. METHODS: Molecular analysis was performed in the progeny of the original pedigree described by Wagner in 1938. We searched for pathogenic mutations and their effects in two candidate genes, CSPG2 and EDIL3, which locate to the critical chromosomal interval. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was used to investigate potential splice defects of CSPG2 transcripts. RESULTS: While no alterations were detected in the exons of EDIL3, several changes were identified in the CSPG2 gene. Only one of the novel changes, a heterozygous G to A substitution of the first nucleotide in intron 8, cosegregates with the disease phenotype. This change disrupts the highly conserved splice donor sequence. In blood cells of an index patient, we found CSPG2 transcripts with normally spliced exon 8/9 junction but also two additional CSPG2 transcripts, which were not detected in the control. One lacks the entire exon 8, while the other is missing only the last 21 bp of exon 8. CONCLUSIONS: CSPG2 encodes versican, a large proteoglycan, which is an extracellular matrix component of the human vitreous and participates in the formation of the vitreous gel. The splice site mutation described here may lead to a complete lack of exon 8 in CSPG2 transcripts, which shortens the predicted protein by 1754 amino acids and leads to severe reduction of glycosaminoglycan attachment sites.

Improved detection of bladder carcinoma cells in voided urine by standardized microsatellite analysis.

Successful treatment of bladder cancer depends largely on early diagnosis of primary and recurrent disease. Sensitive, specific and noninvasive procedures for detection are especially needed for grade 1 and 2 bladder tumors, because of the relatively low sensitivity of cytology. Here we introduce a novel strategy to improve the sensitivity and reliability of microsatellite analyses by employing marker-specific threshold values for loss-of-heterozygosity (LOH) at 10 loci. These individual cut-offs were experimentally determined with 35 normal control tissues and subsequently validated in a retrospective study with bladder cancer biopsies from 86 patients. In a prospective analysis of voided urines samples and matched blood probes from 91 patients, LOH-analysis, UroVysion FISH and conventional urine cytology were compared with histological findings of consecutive transurethral biopsies. Whereas all samples could be analyzed by our LOH assay, only 56 samples were suitable for all 3 analyses. The highest sensitivity was obtained with our LOH-assay/cytology approach (G1-2: 72%; G3: 96%) being only surpassed by a combination of all 3 techniques (G1-2: 83%; G3: 100%). Since over 93% of the patients with recurrent disease were identified by LOH/cytology-analyses of their voided urine samples, a monitoring scheme alternating cystoscopy with LOH/cytology-examination could now be envisioned to reduce invasive interventions and consequently improve follow-up compliance, especially in patients with low grade tumors.

High frequency of t(14;18)-translocation breakpoints outside of major breakpoint and minor cluster regions in follicular lymphomas: improved polymerase chain reaction protocols for their detection.

The detection of t(14;18) translocations is widely used for the diagnosis and monitoring of follicular lymphomas displaying a high prevalence for this aberration. Cytogenetics, Southern blotting, and polymerase chain reaction (PCR) are commonly used techniques. It is generally believed that the vast majority of the breakpoints occurs on chromosome 18 in the major breakpoint region (mbr) and the minor cluster region (mcr). Yet, by improving long-distance PCR protocols we identified half of the breakpoints outside of these clusters. Our study included biopsies from 59 patients with follicular lymphoma. Seventy-one percent carried translocations detectable with our long-distance PCR protocol. The novel primer sets were derived from the hitherto uncharacterized 25-kb-long stretch between mbr and mcr that we have sequenced for this purpose. Sequence analysis of the novel breakpoints reveals a wide distribution between mbr and mcr displaying some clustering 16 kb downstream from the BCL2 gene. By including a primer for this intermediate cluster region in standard PCRs we could also improve the detection of t(14;18) translocations in formalin-fixed and paraffin-embedded biopsies. Our new PCRs are highly sensitive, easy to perform, and thus well suited for routine analysis of t(14;18) translocations for the primary diagnosis of follicular lymphoma and surveillance of minimal residual disease.