Activation and trafficking of insulin-binding B cells to the pLN in nPOD donors

Mia Smith (Barbara Davis Center for Diabetes)

Zachary Stensland, Hali Broncucia, Adam Magera, Peter Gottlieb, Mia Smith

In healthy individuals autoreactive B cells that escape central tolerance mechanisms are normally silenced by anergy, a type of B cell tolerance wherein autoreactive cells occupy peripheral lymphoid organs but are antigen unresponsive. Previously we have found that young new-onset T1D subjects exhibit a loss of anergic insulin-binding B cells (IBCs) in their peripheral blood. Given that an increase in B cells in both the blood and pancreas is associated with the aggressive form of T1D that is seen in younger patients, we hypothesize that loss of anergic IBCs in the peripheral blood reflects their activation and trafficking to tissues rich in autoantigen (e.g. pancreas and pancreatic lymph node (pLN)) where they participate in disease pathogenesis.

Since antigen-specific B cells occur in very low frequency in the periphery, we developed a magnetic nanoparticle-based scheme to enrich for IBCs from blood and tissue. Aided by enrichment, we explored the frequency of various IBC and non-IBC subsets and their activation status in the peripheral blood of T1D subjects and spleen and pLN of nPOD donors using both mass cytometry and spectral flow cytometry.

Summary of Results
Both manual gating and unsupervised clustering algorithms identified ‘anergic’ B cells in the peripheral blood of T1D donors are highly activated (e.g. increased expression of CD86, CD11c, and CXCR3), particularly in insulin-binding B cells compared to non-insulin-binding B cells. Analysis of the spleen and pLN of nPOD donors revealed similar findings with these B cell subsets further enriched in these tissues, especially the pLN. Trajectory and phenotypic analysis suggests these activated ‘anergic’ B cells could be precursors to extrafollicular antibody secreting cells, as well as act as potent antigen-presenting cells to diabetogenic T cells.

Our results suggest that autoreactive B cells that are normally silenced by anergy in healthy individuals, become activated in T1D subjects and traffic to the pLN, where they likely participate in disease pathogenesis through antibody secretion and/or antigen presentation to T cells.