*Purpose: Bystander B cell differentiation into antibody (Ab) secreting cells (ASCs) may augment alloimmunity in the HLA-sensitized patient. Recently, a population of FcrL5⁺IgD^negB cells that express the master transcriptional regulator T-bet has been defined in autoimmunity as “pre-ASCs.” Without BCR ligation, this population directly forms ASCs in vitro in the presence of TLR7/8 and IFN-γcytokine signals. Therefore, this population may directly mediate bystander B cell responses after inflammatory events (e.g. vaccination). We recently identified expansion of a similar population within one month of flu vaccination in healthy individuals. Here we test the hypothesis that vaccinated subjects who mount a T-bet^hi(Fcrl5⁺) IgD^neg B cell response may also be at risk to activate bystander alloimmunity.

*Methods: We enrolled 19 subjects in the 2015-2016 flu vaccine season, administered Fluzone and processed blood at serial time points after vaccination (days 0, 7, 14, 21, 28, and 120). We enumerated the total T-bet^hi as well as the hemagglutinin (HA)-specific T-bet^hi IgD^neg B cells at these timepoints using flow cytometry and HA B cells tetramer reagents. We used FlowPRA^TM Screening beads to assess the development of HLA-specific Abs (HLA-Abs) after vaccination. Analyses were performed with FlowJo v9 and v10 and Prism v7 software.

*Results: Using increase in FlowPRA^TMscreen as an indicator of broadened HLA-Ab reactivity, we divided our subjects into those who did (Group 1, n= 7) or did not (Group 2, N=12) have an increase in FlowPRA^TM screen within 14 days of vaccination. 50% of Group 1 and 30% of Group 2 subjects had positive baseline FlowPRA^TM screens. In comparison to Group 2 subjects, we found that Group 1 subjects were significantly more likely to have an expansion in total and HA-specific day 14 T-bet^hiIgD^negB cells (p = 0.02 and p = 0.01, respectively). Three Group 1 subjects developed de novo HLA-Abs at day 14 that were detectable at 120 days after vaccine.

*Conclusions: We found a significant association between breadth of HLA-Abs and the expansion of T-bet^hi IgD^neg B cells after vaccination. Our next steps are to perform HLA single antigen bead testing on serum from these subjects to assess HLA-Ab reactivities. We will also test for pre-existing HLA-specific pre-ASCs in vaccinated Group 1 subjects who developed de novo HLA-Abs by sort-purifying day 0 IgD^negB cells by T-bet (FcrL5) expression status for HLA ELISPOT assays.

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