Defining a Role for T Regulatory Cell Expressed MyD88 During the Response to Allografts

Abstract

The myeloid differentiation primary response gene 88 (MyD88) is an adaptor protein proximally downstream of the Toll-like receptor (TLRs) and the IL-1 receptor family (IL-1R, IL- 18R and IL-33R). The TLR-MyD88 signaling pathway enables the innate immune system to sense inflammation and promote adaptive immune responses. TLRs are also expressed on T cells, and we have previously demonstrated that T cell intrinsic MyD88 signaling promotes T cell survival and is required for optimal responses to pathogens such as T. gondii and LCMV. Surprisingly however, we observed that mice with a targeted T cell deletion of MyD88 (MyD88fl/fl x CD4-Cre, termed MyD88-ΔT) reject bm12 cardiac allografts, and bm12 skin allografts when treated with αCD154 and rapamycin, at a higher frequency than wild type (WT) mice. As Tregs are critical for graft prolongation following costimulatory blockade in this model, we hypothesized a “defect” in MyD88-ΔT Tregs in the above model. As MyD88-ΔT mice do not allow us to differentiate the effect of MyD88 deletion specifically on Tregs versus the T cell compartment as a whole, we created mice with a targeted deletion of MyD88 specific to Tregs (MyD88fl/fl x FoxP3-Cre-YFP, MyD88-ΔTreg). MyD88-ΔTreg mice, similar to MyD88-ΔT mice have a similar Treg frequency when compared to WT mice and have no observable signs of autoimmunity. We found that MyD88-ΔTreg mice failed to accept bm12 skin allografts at the same frequency as WT control mice when given CoB, suggesting that the phenotype we observed in the MyD88-ΔT mice was likely caused by MyD88 deficient Tregs. As T cell intrinsic MyD88 is required for T cell survival during the response to pathogens, we thus hypothesized that the inability for MyD88 deficient Tregs to promote long-term allograft survival was due to an inability of these cells to survive. Surprisingly, through a series of in vivo and in vitro experiments, we observed that MyD88 deficient Tregs survive as well as WT Tregs under competitive conditions with WT Tregs, during activation with αCD3 and αCD28, as well as in the allograft 21 days post transplantation. In addition, MyD88 deficient Tregs survived as well as WT Tregs when activated and cultured with rapamycin and αCD154. In an attempt to reconcile the inability of MyD88-deficient Tregs to promote graft survival with their surprising ability to survive as well as WT Tregs, we next assessed the function of MyD88-deficient Tregs. In an in vitro suppression assay, MyD88-deficient Tregs suppressed effector T cell proliferation as well as WT Tregs at multiple Treg:T effector ratios. In addition, MyD88-deficient Tregs expressed similar levels of the Treg functional proteins CTLA-4, PD-1, Granzyme B, Lag-3, GITR, CD39, CD73 and CD28 as WT Tregs, suggesting that the function of MyD88-deficient Tregs may only be impaired during the response to alloantigen. Together, these data define MyD88 as having a divergent requirement for cell survival in non-Tregs and Tregs, and a yet-to-be defined functional requirement in Tregs during the response to alloantigen.