Purpose Tumor overexpression of cyclooxygenase-2 (COX-2) has been associated with worse outcome in non-small-cell lung cancer (NSCLC). In Cancer and Leukemia Group B (CALGB) 30203, we found that the selective COX-2 inhibitor celecoxib in addition to chemotherapy in advanced NSCLC improved progression-free and overall survival in patients with moderate to high COX-2 expression by immunohistochemistry (IHC). CALGB 30801 (Alliance) was designed to prospectively confirm that finding. Patients and Methods Patients with NSCLC (stage IIIB with pleural effusion or stage IV according to American Joint Committee on Cancer [sixth edition] criteria) were preregistered, and biopsy specimens were analyzed for COX-2 by IHC. Patients with COX-2 expression ≥ 2, performance status of 0 to 2, and normal organ function were eligible. Chemotherapy was determined by histology: carboplatin plus pemetrexed for nonsquamous NSCLC and carboplatin plus gemcitabine for squamous histology. Patients were randomly assigned to celecoxib (400 mg twice per day; arm A) or placebo (arm B). The primary objective was to demonstrate improvement in progression-free survival in patients with COX-2 index ≥ 4 with hazard ratio of 0.645 with approximately 85% power at two-sided significance level of .05. Results The study was halted for futility after 312 of the planned 322 patients with COX-2 index ≥ 2 were randomly assigned. There were no significant differences between the groups (hazard ratio, 1.046 for COX-2 ≥ 4). Subset analyses evaluating histology, chemotherapy regimen, and incremental COX-2 expression did not demonstrate any advantage for COX-2 inhibition. Elevation of baseline urinary metabolite of prostaglandin E2, indicating activation of the COX-2 pathway, was a negative prognostic factor. Values above the third quartile may have been a predictive factor. Conclusion COX-2 expression by IHC failed to select patients who could benefit from selective COX-2 inhibition. Urinary metabolite of prostaglandin E2 may be able to identify patients who could benefit from COX-2 inhibition.

Objective Patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) therapy are at higher risk of poor COVID‐19 outcomes and show substantially impaired humoral immune response to anti–SARS–CoV‐2 vaccine. However, the complex relationship between antigen‐specific B cells and T cells and the level of B cell repopulation necessary to achieve anti‐vaccine responses remain largely unknown. Methods Antibody responses to SARS–CoV‐2 vaccines and induction of antigen‐specific B and CD4/CD8 T cell subsets were studied in 19 patients with rheumatoid arthritis (RA) or antineutrophil cytoplasmic antibody–associated vasculitis receiving RTX, 12 patients with RA receiving other therapies, and 30 healthy controls after SARS–CoV‐2 vaccination with either messenger RNA or vector‐based vaccines. Results A minimum of 10 B cells per microliter (0.4% of lymphocytes) in the peripheral circulation appeared to be required for RTX‐treated patients to mount seroconversion to anti‐S1 IgG upon SARS–CoV‐2 vaccination. RTX‐treated patients who lacked IgG seroconversion showed reduced receptor‐binding domain–positive B cells (P = 0.0005), a lower frequency of Tfh‐like cells (P = 0.0481), as well as fewer activated CD4 (P = 0.0036) and CD8 T cells (P = 0.0308) compared to RTX‐treated patients who achieved IgG seroconversion. Functionally relevant B cell depletion resulted in impaired interferon‐γ secretion by spike‐specific CD4 T cells (P = 0.0112, r = 0.5342). In contrast, antigen‐specific CD8 T cells were reduced in both RA patients and RTX‐treated patients, independently of IgG formation. Conclusion In RTX‐treated patients, a minimum of 10 B cells per microliter in the peripheral circulation is a candidate biomarker for a high likelihood of an appropriate cellular and humoral response after SARS–CoV‐2 vaccination. Mechanistically, the data emphasize the crucial role of costimulatory B cell functions for the proper induction of CD4 responses propagating vaccine‐specific B cell and plasma cell differentiation.

Objectives: Patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) therapy show substantially impaired anti-SARS-CoV-2 vaccine humoral but partly inducible cellular immune responses. However, the complex relationship between antigen-specific B and T cells and the level of B cell repopulation necessary to achieve anti-vaccine responses remain largely unknown. Methods: Antibody responses to SARS-CoV-2 vaccines and induction of antigen-specific B and CD4/CD8 T cell subsets were studied in 19 rheumatoid arthritis (RA) and ANCA-associated vasculitis (AAV) patients receiving RTX, 12 RA patients on other therapies and 30 healthy controls after SARS-CoV-2 vaccination with either mRNA or vector based vaccines. Results: A minimum of 10 B cells/uL in the peripheral circulation was necessary in RTX patients to mount seroconversion to anti-S1 IgG upon SARS-CoV-2 vaccination. RTX patients lacking IgG seroconversion showed reduced antigen-specific B cells, lower frequency of TfH-like cells as well as less activated CD4 and CD8 T cells compared to IgG seroconverted RTX patients. Functionally relevant B cell depletion resulted in impaired IFNgamma secretion by spike-specific CD4 T cells. In contrast, antigen-specific CD8 T cells were reduced in patients independently of IgG formation. Conclusions: Patients receiving rituximab with B cell numbers above 10 B cells/ul were able to mount humoral and more robust cellular responses after SARS-CoV-2 vaccination that may permit optimization of vaccination in these patients. Mechanistically, the data emphasize the crucial role of co-stimulatory B cell functions for the proper induction of CD4 responses propagating vaccine-specific B and plasma cell differentiation.

The interferon pathway, a key antiviral defense mechanism, is being considered as a therapeutic target in COVID‐19. Both, substitution of interferon and JAK/STAT inhibition to limit cytokine storms have been proposed. However, little is known about possible abnormalities in STAT signaling in immune cells during SARS‐CoV‐2 infection. We investigated downstream targets of interferon signaling, including STAT1, STAT2, pSTAT1 and 2, and IRF1, 7 and 9 by flow cytometry in 30 patients with COVID‐19, 17 with mild, and 13 with severe infection. We report upregulation of STAT1 and IRF9 in mild and severe COVID‐19 cases, which correlated with the IFN‐signature assessed by Siglec‐1 (CD169) expression on peripheral monocytes. Interestingly, Siglec‐1 and STAT1 in CD14+ monocytes and plasmablasts showed lower expression among severe cases compared to mild cases. Contrary to the baseline STAT1 expression, the phosphorylation of STAT1 was enhanced in severe COVID‐19 cases, indicating a dysbalanced JAK/STAT signaling that fails to induce transcription of interferon stimulated response elements (ISRE). This abnormality persisted after IFN‐α and IFN‐γ stimulation of PBMCs from patients with severe COVID‐19. Data suggest impaired STAT1 transcriptional upregulation among severely infected patients may represent a potential predictive biomarker and would allow stratification of patients for certain interferon‐pathway targeted treatments.

BackgroundVaccination is considered as most efficient strategy in controlling SARS-CoV-2 pandemic spread. Nevertheless, patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) are at increased risk to fail humoral and cellular responses upon vaccination. The ability to predict vaccination responses is essential to guide adequate safety and optimal protection in these patients.MethodsB- and T- cell data before vaccination were evaluated for characteristics predicting vaccine responses in altogether 15 patients with autoimmune inflammatory rheumatic diseases receiving RTX. Eleven patients with rheumatoid arthritis (RA) on other therapies, 11 kidney transplant recipients (KTR) on regular immunosuppression and 15 healthy controls (HC) served as controls. A multidimensional analysis of B cell subsets via UMAP algorithm and a correlation matrix were performed in order to identify predictive markers of response in patients under RTX therapy.ResultsSignificant differences regarding absolute B cell counts and specific subset distribution pattern between the groups were identified at baseline. In this context, the majority of B cells from vaccination responders of the RTX group (RTX IgG+) were naïve and transitional B cells, whereas vaccination non-responders (RTX IgG-) carried preferentially plasmablasts and double negative (CD27-IgD-) B cells. Moreover, there was a positive correlation between neutralizing antibodies and B cells expressing HLA-DR and CXCR5 as well as an inverse correlation with CD95 expression and CD21low expression by B cells among vaccination responders.SummarySubstantial repopulation of the naïve B cell compartment after RTX therapy appeared to be essential for an adequate vaccination response, which seem to require the additional capability of antigen presentation and germinal center formation. Moreover, expression of exhaustion markers represent negative predictors of vaccination responses.

Antibody-secreting cells (ASCs) contribute to immunity through production of antibodies and cytokines. Identification of specific markers of ASC would allow selective targeting of these cells in several disease contexts. Here, we performed an unbiased, large-scale protein screening, and identified twelve new molecules that are specifically expressed by murine ASCs. Expression of these markers, particularly CD39, CD81, CD130, and CD326, is stable and offers an improved resolution for ASC identification. We accessed their expression in germ-free conditions and in T cell deficient mice, showing that at least in part their expression is controlled by microbial- and T cell-derived signals. Further analysis of lupus mice revealed the presence of a subpopulation of LAG-3– plasma cells, co-expressing high amounts of CD39 and CD326 in the bone marrow. This population was IgM+ and correlated with IgM anti-dsDNA autoantibodies in sera. Importantly, we found that CD39, CD81, CD130, and CD326 are also expressed by human peripheral blood and bone marrow ASCs. Our data provide innovative insights into ASC biology and function in mice and human, and identify an intriguing BM specific CD39++CD326++ ASC subpopulation in autoimmunity.

The interferon pathway represents a key antiviral defense mechanism and is being considered as a therapeutic target in COVID-19. Both, substitution of interferon and blocking interferon signaling through JAK STAT inhibition to limit cytokine storms have been proposed. However, little is known so far about possible abnormalities in STAT signaling in immune cells during SARS-CoV-2 infection. In the current study, we investigated downstream targets of interferon signaling, including STAT1, pSTAT1 and 2 and IRF1, 7 and 9 by flow cytometry in 30 patients with COVID-19, 17 with mild and 13 with severe infection. We report an upregulation of STAT1 and IRF9 in mild and severe COVID-19 cases, which correlated with the IFN-signature assessed by Siglec-1 (CD169) expression on peripheral monocytes. Most interestingly, Siglec-1 and STAT1 in CD14+ monocytes and plasmablasts showed lower expression among severe COVID-19 cases compared to mild cases. Contrary to the baseline whole protein STAT1 expression, the phosphorylation of STAT1 was enhanced in severe COVID-19 cases, indicating a dysbalanced JAK STAT signaling that fails to induce transcription of interferon stimulated response elements (ISRE). This abnormality persisted after IFN-α and IFN-γ stimulation of PBMCs from patients with severe COVID-19. The data suggest impaired STAT1 transcriptional upregulation among severely infected patients which may represent a potential predictive biomarker and may allow stratification of patients for certain interferon-pathway targeted treatments.

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