Session Title: B-cell Biology and Targets in Autoimmune Disease
Session Type: Abstract Submissions (ACR)
Patients with the primary immunodeficiency Wiskott-Aldrich syndrome (WAS) have severe abnormalities in splenic marginal zone (MZ) anatomy and function. Consistent with this, WAS patients fail to develop T-independent antibody responses to polysaccharide antigens and are susceptible to Streptococcus pneumoniae infections. We previously showed that B cell entry into the MZ is unperturbed in WAS-/- mice, but that retention within the MZ is defective. However, the factors promoting WAS-/- B cell exit from the MZ have not been determined.
Proliferation of sorted wild-type (WT) and WAS-/- MZ B cells was quantified after stimulation with LPS (TLR4 agonist), CL294 (TLR7 agonist) or CPG (TLR9 agonist) for 72 hours. To study the impact of B cell intrinsic TLR signaling on WAS MZ homeostasis, mixed bone marrow chimeras were generated in which deficiency of WAS, WAS x Myd88, WAS x TLR7 or WAS x TLR9 was limited to the B cell lineage. Splenic MZ reconstitutition was then analyzed by flow-cytometry and immunohistochemistry. In vivo labeling of MZ cells at the vascular interface was performed by PE-labeled anti-CD19 antibody injection 5 minutes prior to sacrifice.
In addition to markedly decreased MZ size, we demonstrate by in vivo labeling that WAS-/- MZ B cells are not normally positioned at the MZ vascular interface. Unmanipulated WAS-/- mice also exhibit increased peripheral blood MZ B cells consistent with the idea that WAS-/- B cells are not appropriately retained within the MZ. Further, we show that sorted WAS-/- MZ B cells are hyper-responsive to multiple Toll-like receptor (TLR) ligands, including TLR4, TLR7 and TLR9. These combined findings suggested that TLR-mediated activation might directly promote WAS-/- B cell exit from the MZ.
To test this idea, we generated chimeras with B cells deficient in both WAS protein and the key TLR signaling adaptor, MyD88. Strikingly, the MZ B cell compartment was restored WT levels in WAS-/- x MyD88-/- chimeras. WAS-/- x MyD88-/- B cells were also appropriately positioned within the MZ both by in vivo labeling and immunohistochemistry. To address the impact of individual TLRs, we generated WAS-/- x TLR7-/- and WAS-/- x TLR9-/- chimeras. Although increased compared with WAS-/- controls, WAS-/- x TLR7-/- MZ size was reduced 50% compared with WT chimeras, suggesting a partial role for endogenous TLR7 ligands in driving WAS-/- B cells out of the MZ. In contrast, no rescue of MZ B cell numbers was noted in WAS-/- x TLR9-/- chimeras, implicating TLR9 in negatively regulating B cell activation and MZ retention, perhaps via downregulation of TLR7 signaling.
Our data demonstrate that activation by endogenous TLR ligands is sufficient to drive WAS-/- MZ B cells from the MZ and that B cell-intrinsic TLR7 and TLR9 signals have opposing impacts on MZ homeostasis. These data imply a previously unappreciated role for B cell intrinsic TLR signals in MZ B cell function; and have important implications regarding altered immune responses to blood-borne pathogens in WAS patients and for development of autoimmune disease in WAS and other clinical settings.
S. W. Jackson,
M. A. Schwartz,
D. J. Rawlings,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/marginal-zone-defects-in-wiskott-aldrich-syndrome-are-dependent-on-b-cell-intrinsic-toll-like-receptor-signals/