Activation and pro-inflammatory cytokine production by unswitched memory B cells during SARS-CoV-2 infection.

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.

Autoantibodies elicited with SARS-CoV-2 infection are linked to alterations in double negative B cells.

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-1ß), 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.