Introduction: Pharmacological immunosuppression after transplantation increases morbidity and mortality, and thus alternative immunotherapies require investigation. Regulatory T cells (Tregs) are essential for maintaining immune tolerance and are great candidates for cellular therapy; understanding and modulating their function is therefore of formidable value. Iron restriction has been shown to impair effector T and B cell responses to vaccines and infections, but its effect on Tregs is largely unknown. A mutation in the transferrin receptor gene Tfrc (Tfrc^Y20H/Y20H) leads to combined immunodeficiency in patients, caused by poor iron import via this transferrin receptor, but its effect on Tregs has not been studied.

Methods: Mice receiving fully mismatched skin grafts were injected with a hepcidin mimetic to lower serum iron, and Tregs from peripheral blood, spleens, and graft-draining or contralateral lymph nodes were phenotyped via flow cytometry. Treg phenotype was also assessed in a systemic infection model in malaria-infected Tfrc^Y20H/Y20H mice. To study cell-intrinsic iron limitation in Tregs, RAG1^- mice with a fully mismatched skin allograft were adoptively transferred with CD4⁺CD25^- conventional T cells (Tconvs) alongside CD4⁺CD25⁺ Tregs from either wildtype mice or iron-impaired TfrcY^20H/Y20H mice. Mixed wildtype and Tfrc^Y20H/Y20H bone marrow chimeras were created for analysis of these mice's immune composition and Treg phenotype, both at resting state and during skin transplant rejection. Cell-extrinsic and intrinsic hypoferremia in Tregs and Tconvs was also studied in vitro with activation, proliferation, migration, and suppression assays.

Results: Hepcidin mimetic injection impaired alloresponses in T cells and prolonged graft rejection, but Tregs seemed to be more resistant to iron limitation than Tconvs as shown by Tconv:Treg ratio, and Tregs even showed increased CD25 expression after hepcidin treatment. Similarly, Tfrc^Y20H/Y20H mice had impaired immune responses to malaria but Tregs were preferentially less impaired and better activated than other cells such as Tconvs. However, Tfrc^Y20H/Y20H Tregs were unable to control allorejection when adoptively transferred, and migration assays with Tfrc^Y20H/Y20H Tregs suggest that this was not due to impaired migration to the graft. Mixed bone marrow chimeras are still in progress, but offer a direct comparison of wildtype and Tfrc^Y20H/Y20H Tregs within the exact same environment at steady state and after allogenic challenge.

Conclusions: Tregs and Tconvs often differ in their metabolism or response to the same stimulus, and pathways that affect them differentially show great promise in tipping the immune balance toward tolerance or immune activation. These results on preferential Treg resistance to iron deprivation highlight iron as a candidate for manipulating the Treg:Tconv ratio, which could have tremendous therapeutic applications.