Severe SARS-CoV-2 infection is associated with strong inflammation and autoantibody production against diverse self-antigens, suggesting a system-wide defect in B cell tolerance. BND cells are a B cell subset in healthy individuals harboring autoreactive but anergic B lymphocytes. In vitro evidence suggests inflammatory stimuli can breach peripheral B cell tolerance in this subset. We asked whether SARS-CoV-2–associated inflammation impairs BND cell peripheral tolerance. To address this, PBMCs and plasma were collected from healthy controls, individuals immunized against SARS-CoV-2, or subjects with convalescent or severe SARS-CoV-2 infection. We demonstrate that BND cells from severely infected individuals are significantly activated, display reduced inhibitory receptor expression, and restored BCR signaling, indicative of a breach in anergy during viral infection, supported by increased levels of autoreactive antibodies. The phenotypic and functional BND cell alterations significantly correlate with increased inflammation in severe SARS-CoV-2 infection. Thus, autoreactive BND cells are released from peripheral tolerance with SARS-CoV-2 infection, likely as a consequence of robust systemic inflammation.

Double negative (DN) B cells (CD27-IgD-) comprise a heterogenous population of DN1, DN2, and the recently described DN3 and DN4 subsets. In autoimmune disease, DN2 cells are reported to be precursors to autoreactive antibody secreting cells and expansion of DN2 cells is linked to elevated interferon levels. Severe SARS-CoV-2 infection is characterized by elevated systemic levels of pro-inflammatory cytokines and serum autoantibodies and expansion of the DN2 subset in severe SARS-CoV-2 infection has been reported. However, the activation status, functional capacity and contribution to virally-induced autoantibody production by DN subsets is not established. Here, we validate the finding that severe SARS-CoV-2 infection is associated with a reduction in the frequency of DN1 cells coinciding with an increase in the frequency of DN2 and DN3 cells. We further demonstrate that with severe viral infection DN subsets are at a heightened level of activation, display changes in immunoglobulin class isotype frequency and have functional BCR signaling. Increases in overall systemic inflammation (CRP), as well as specific pro-inflammatory cytokines (TNFα, IL-6, IFNγ, IL-1β), significantly correlate with the skewing of DN1, DN2 and DN3 subsets during severe SARS-CoV-2 infection. Importantly, the reduction in DN1 cell frequency and expansion of the DN3 population during severe infection significantly correlates with increased levels of serum autoantibodies. Thus, systemic inflammation during SARS-CoV-2 infection drives changes in Double Negative subset frequency, likely impacting their contribution to generation of autoreactive antibodies.

Background: Soft tissue sarcoma is a rare form of cancer primarily affecting connective tissue in adults. Standard chemotherapy includes the anthracycline doxorubicin which has previously been shown in a variety of cancer models to induce immunogenic cell death (ICD). ICD is driven, in part, by the upregulation and/or translocation of molecules such as calreticulin and annexin I, known as damage associated molecular patterns (DAMPs). We aimed to investigate the ICD capacity of anthracyclines doxorubicin and mitoxantrone and explore activity in combination with anti-PD1 and CTLA-4 checkpoint inhibitors.Methods: We treated a variety of human osteosarcoma cell lines and MCA-205 mouse fibrosarcoma cells with doxorubicin and mitoxantrone and measured expression of DAMPs and immunosuppressive markers via multiplex flow cytometry. We also measured anti-tumor activity of doxorubicin with and without PD1/CTLA4 blockade in the murine sarcoma model MCA-205. After tumors were harvested, we analyzed tumor-infiltrating lymphocytes to determine the effects of treatment on phenotype, quantity, activation status, function, clonality, and expression of immune checkpoint proteins.Results: We found expression of Annexin I and TRAIL-R2 on the cell surface of osteosarcoma cell lines treated with anthracyclines and significant inhibition of tumor growth with the combination of doxorubicin plus PD1/CTLA4 blockade compared to either doxorubicin or checkpoint blockade alone. Profiling of TILs revealed a marked increase in CD8:CD4 ratio and increased expression of checkpoint proteins including PD1, 41BB and TIM3 in the combination of doxorubicin plus checkpoint blockade. Finally, increased preponderance of the TCRVb 5.1-5.2 clonotype was observed with the combination in TIL, which was not observed in spleen or peripheral blood samples.Conclusion: We have observed early evidence of improved anti-tumor effects with combination doxorubicin plus PD1/CTLA4 along with reshaping of the TIL repertoire, suggesting that doxorubicin may improve immunogenic cell death and supporting clinical investigation in human sarcoma patients. Citation Format: Nicholas R Therrien, Kyle Powers, Lindsey Kemp, Cristiam Moreno Tellez, Breelyn A Wilky. Evaluation of doxorubicin and PD-1/CTLA-4 immune blockade combination therapy in the MCA-205 murine model [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B64.