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.

Conditional and inducible Cre-loxP systems are used to target gene deletion to specific cell lineages and tissues through promoter-restricted expression of the bacterial DNA recombinase, Cre. Although Cre-loxP systems are widely used to target gene deletion in lung macrophages, limited data are published on the specificity and efficiency of "macrophage targeting" Cre lines. Using R26-stopfl/fl-TdTomato and tetOn-GFP reporter lines, we assessed the specificity and efficiency of four commercially available Cre driver lines that are often considered "macrophage specific." We evaluated two conditional (Csf1r-Cre and LysM-Cre) and two inducible [CX3CR1-estrogen receptor-Cre (ERCre) and CD68-rtTA] lines. We assessed Cre activation in six resident lung myeloid populations, as well as activation in lung leukocytes, lung epithelial and endothelial cells, peripheral blood leukocytes, and tissue macrophages of the spleen, bone marrow, and peritoneal cavity. Although Csf1r-Cre and LysM-Cre target resident alveolar macrophages (ResAM) and interstitial macrophages (IM) with high efficiency, neither line is specific for macrophages. Csf1r-Cre targets all leukocyte populations, while LysM-Cre targets dendritic cell, neutrophils, monocytes, and a quarter of lung epithelial cells. CX3CR1-ERCre and CD68-rtTA both target IM, but do not target ResAM. Further, although neither line is specific for macrophages, a pulse-wait administration of tamoxifen or doxycycline can be used to significantly improve IM specificity in these inducible lines. In summary, while Cre-loxP remains a powerful tool to study macrophage function, numerous pitfalls exist. Herein, we document strengths and weaknesses of Csf1r-Cre, LysM-Cre, CX3CR1-ERCre, and CD68-rtTA systems for targeting specific macrophage populations in the lungs and provide data that will aid investigators in selecting the proper strain.

Memory B cells are comprised of unswitched (CD27+IgD+) and switched (CD27+IgD-) subsets. The origin and function of unswitched human memory B cells are debated in the literature, whereas switched memory B cells are primed to respond to recurrent infection. Unswitched memory B cells have been described to be reduced in frequency with severe SARS-CoV2 infection and here we characterize their activation status, BCR functionality, and contribution to virally-induced cytokine production. Analyses of whole blood from healthy individuals, people immunized against SARS-CoV2, and those who have had mild and severe SARS-CoV2 infection, confirm a reduction in the frequency of unswitched memory B cells during severe SARS-CoV2 infection and demonstrate this reduction is associated with increased levels of systemic TNFα. We further document how severe viral infection is associated with an increased frequency of 'IgD+' only memory B cells that correlate with increased IgG autoantibody levels. Unswitched and switched memory B cells from severe SARS-CoV2 infection displayed evidence of heightened activation with a concomitant reduction in the expression of the inhibitory receptor CD72. Functionally, both populations of memory B cells from severe SARS-COV2 infection harbored a signaling-competent BCR that displayed enhanced BCR signaling activity in the unswitched population. Finally, we demonstrate that B cells from mild SARS-CoV2 infection are poised to secrete pro-inflammatory cytokines IL-6 and TNFα. Importantly, unswitched memory B cells were a major producer of IL-6 and switched memory B cells were a major producer of TNFα in response to viral TLR ligands. Together these data indicate that B cells contribute to the inflammatory milieu during viral infection.