Mechanisms of female-biased autoimmunity in human immune organoids

Stanford University, Stanford CA

Investigators

Abstract

Project 2 Summary A critical problem in understanding sex differences in human immunology generally is how to pursue important observations in depth, testing hypotheses and defining mechanisms. Currently the typical way this is done is by utilizing mouse models, but inbred mice vary considerably from human beings in terms of genetic heterogeneity, microbial experience and longevity, and thus there has long been a need for an alternative system that could more closely mimic often highly variable human responses. Thus, for some years we have been developing human immune organoids for dissociated tonsils and spleens, which we find respond well to various vaccines, exhibiting the hallmarks of specific B and T cell mobilization, antibody secretion and affinity maturation and class switching. We have also been able to use CRISPR/Cas9 to delete specific genes efficiently with both T and B cells in these organoids. Very relevant to this proposal is that we find that deleting FoxP3 in T cells allows the cultures to allow autoreactive B cells to proliferate and make autoantibodies and in the case of a Granzyme B deletion, which attenuates CD8 Tregs, the cultures with this deletion now allow autoreactive T cells to proliferate. Here we will use this new technology to investigate in depth the effects of Xist on male and female splenic organoids, particularly whether it can adjuvant cultures where either FoxP3 or Granzyme B have been deleted in most T cells (Aim 1). We will also insert the recently described TLR7 gain-of-function mutant that causes a Lupus-like syndrome into splenic B cells in these cultures and analyze its effect on autoreactive B and T cells specific for Lupus antigens (Aim 2). This will also be another opportunity to test the effect of Xist and its components on the status of autoreactivity and innate parameters. Lastly, we are able to get a steady supply of spleens in the 20-60+ age range and by making single cell suspensions we can freeze 30-70 aliquots per spleen. This gives us the opportunity to create a unique spleen bank of 200 or so donors that we can characterize for autoreactive T and B cells and share with other groups, so that they can test other reagents or hypotheses regarding why females are more susceptible to autoimmunity (Aim 3). We have already observed that females often have elevated levels of autoreactive B cells vs males [32], and expect with a larger survey we will find a broader range of female donors, some of whom may be on the verge of disease, and thus particularly susceptible to putative triggers of autoimmunity like the Xist RNP or TLR7 stimulation. We have also obtained one spleen from a male Lupus patient and hope to obtain a small number of spleens from female donors with Lupus or other forms of autoimmunity, and together with the range of autoreactive T and B cells we expect to see in the other donors, this may illustrate key steps in the progression to disease.