121: Interaction of CD4 +CD25 +Foxp3 + Regulatory T Cells with Hematopoietic Progenitor Cells Reveals Two Distinct Pathways of Regulation

Abstract

Naturally occurring CD4+CD25+Foxp3+ regulatory T (Treg) cells are responsible for a physiologically essential peripheral mechanism to maintain self tolerance. Several Treg cytokines which mediate immune regulation concomitantly possess hematopoietic modulating activity, for example: transforming growth factor β1 (TGF- β1) and interleukin 9 (IL-9) have been found to have multiple effects during early and late stages of myelopoiesis. Based on the inhibition of CFU-IL3 observed after co-culture of BMC with anti-CD3/CD28+IL-2 activated Tregs, we hypothesize that this regulation occurs in a contact dependent manner through a mechanism involving TGF-β1. Inhibition was not observed in transwell cultures and we found that Tregs fail to inhibit CFU-IL3 activity from MHC class II deficient BMC in vitro. Interestingly, CFU-IL3 numbers were enhanced following transplant of syngeneic B6-wt but not B6-Class II-/- BM in lethally conditioned recipients depleted of Tregs, further supporting the notion that cognate interaction is required for this negative regulation to take place. IL-9 is a T cell factor which can promote both growth and survival signals capable of augmenting erythroid progenitor cell (PC) activity. Activated Treg cells co-cultured with syngeneic BMC populations as well as Lin- fractions routinely resulted in a 3–4 × augmentation of CFU-E vs. control cultures lacking Tegs. Anti-IL-9 specific mab but not isotype control Ig abolished the enhancement of CFU-E activity. Moreover, Tregs from IL-9 KO mice inhibited CFU-IL3 activity, but lost the capacity to enhance CFU-E. Additionally, a requirement for IL-9R signaling (IL-9Rα/γc) in BMC was supported by the absence of Treg CFU-E enhancement of γc KO marrow. Finally, Treg augmentation of CFU-E in transwell cultures using B6-wt and B6-Class II-/- Lin- populations showed that this enhancing activity: a) was not contact dependent and b) did not require MHC class II expression on the targeted populations. These findings reveal two distinct pathways of regulation, i.e. inhibition and enhancement as defined by cytokines, contact dependency and an MHC class II requirement. Current studies have found neither perforin or fasL are required to mediate either of these pathways. In total, these results demonstrate for the first time that Tregs can modulate activity which is critically important to successful engraftment in clinical BMT, in addition to adaptive and innate immune responses. Naturally occurring CD4+CD25+Foxp3+ regulatory T (Treg) cells are responsible for a physiologically essential peripheral mechanism to maintain self tolerance. Several Treg cytokines which mediate immune regulation concomitantly possess hematopoietic modulating activity, for example: transforming growth factor β1 (TGF- β1) and interleukin 9 (IL-9) have been found to have multiple effects during early and late stages of myelopoiesis. Based on the inhibition of CFU-IL3 observed after co-culture of BMC with anti-CD3/CD28+IL-2 activated Tregs, we hypothesize that this regulation occurs in a contact dependent manner through a mechanism involving TGF-β1. Inhibition was not observed in transwell cultures and we found that Tregs fail to inhibit CFU-IL3 activity from MHC class II deficient BMC in vitro. Interestingly, CFU-IL3 numbers were enhanced following transplant of syngeneic B6-wt but not B6-Class II-/- BM in lethally conditioned recipients depleted of Tregs, further supporting the notion that cognate interaction is required for this negative regulation to take place. IL-9 is a T cell factor which can promote both growth and survival signals capable of augmenting erythroid progenitor cell (PC) activity. Activated Treg cells co-cultured with syngeneic BMC populations as well as Lin- fractions routinely resulted in a 3–4 × augmentation of CFU-E vs. control cultures lacking Tegs. Anti-IL-9 specific mab but not isotype control Ig abolished the enhancement of CFU-E activity. Moreover, Tregs from IL-9 KO mice inhibited CFU-IL3 activity, but lost the capacity to enhance CFU-E. Additionally, a requirement for IL-9R signaling (IL-9Rα/γc) in BMC was supported by the absence of Treg CFU-E enhancement of γc KO marrow. Finally, Treg augmentation of CFU-E in transwell cultures using B6-wt and B6-Class II-/- Lin- populations showed that this enhancing activity: a) was not contact dependent and b) did not require MHC class II expression on the targeted populations. These findings reveal two distinct pathways of regulation, i.e. inhibition and enhancement as defined by cytokines, contact dependency and an MHC class II requirement. Current studies have found neither perforin or fasL are required to mediate either of these pathways. In total, these results demonstrate for the first time that Tregs can modulate activity which is critically important to successful engraftment in clinical BMT, in addition to adaptive and innate immune responses.