Loss of FLVCR, a Heme Export Protein, Blocks Thymic T Cell Development at the CD4⁺CD8⁺ Double-Positive Stage.

Abstract 913

Cats viremic with the feline leukemia virus C (FeLV-C) develop profound anemia with an early block in erythroid development. In infected cells, the FeLV-C envelope protein sequesters its receptor, feline leukemia virus subgroup Creceptor (FLVCR), in the endosomal compartment and prevents FLVCR trafficking to the cell surface. FLVCR is a transmembrane protein and major facilitator superfamily member, which we previously showed exports heme from cells [Cell(2004)118:57]. FLVCR-null mice died in utero with failed definitive erythropoiesis [Science(2008)319:825]. Conditionally-deleted mice (Flvcr^flox/flox;Mx-cre) were then generated, and neonatal or adult deletion caused progressive anemia similar to that seen in cats infected with FeLV-C. In addition to its role as an oxygen carrier in hemoglobin, heme has many other important and diverse roles, including regulation of several transcription and translation factors, microRNA processing, and N-end rule ubiquitination.

Early reports noted that cats viremic with FeLV-C had thymic aplasia and lymphopenia. However, it was unclear whether these findings were due to a thymus-intrinsic defect, or a secondary effect of severe anemia and resultant stress. In order to study the role of FLVCR in lymphocyte development, we transplanted limiting numbers (2 × 10⁶) of FLVCR-deleted (FLVCR KO) or control (FLVCR ^{+/+ or +/flox};Mx-cre littermate) bone marrow cells into sub-lethally irradiated (500 cGy) Rag 1-deficient (Rag1 KO) mice. Rag 1KO mice cannot recombine T cell receptor (TCR) or immunoglobulin genes and have no T or B cells. The use of sub-lethal irradiation and transplantation of low numbers of FLVCR KO bone marrow cells allowed endogenous Rag1KO hematopoietic cells to contribute to the erythroid lineage, preventing anemia. However, any T or B cells that developed had to arise from the transplanted FLVCR KO bone marrow.

As expected, the FLVCR KO-transplanted Rag1 KO mice were not anemic. However, very few T cells were found in the peripheral blood, spleen, or lymph nodes of FLVCR KO-transplanted mice compared to controls (>90% reduction). In contrast, B cell numbers were preserved. Thus, the previously observed thymic aplasia and lymphopenia seen in FeLV-C viremic cats was not due to severe anemia or stress, but to a direct effect of FLVCR loss on T cell development. Since thymic epithelial cells and the majority of myeloid cells were derived from the Rag1 KO host, the block in T cell development was caused by a cell-intrinsic defect in developing FLVCR KO thymocytes. The requirement for FLVCR was at a developmental stage downstream of the common lymphoid progenitor because B cells developed normally.

We then analyzed thymic development in Rag 1 KO mice transplanted with FLVCR KO or control bone marrow. While there were similar numbers of CD4^-CD8^- double-negative thymocytes (DN) and a slight diminution in the number of CD4⁺CD8⁺ double-positive thymocytes (DP), there was a dramatic decrease in the number of CD4⁺ and CD8⁺ single positive thymocytes (SP) in FLVCR KO-transplanted mice compared to controls (82% reduction). Analysis of surface TCR expression as well as other maturation markers on developing thymocytes showed that TCR beta rearrangement occurred in FLVCR KO-transplanted mice. However, there was progressive loss of FLVCR KO-derived thymocytes at each subsequent stage. Thus, FLVCR is required for T cell development beyond the DP stage. Following TCR beta rearrangement and surface expression, thymocytes proliferate extensively, rearrange TCR alpha genes, and then undergo positive and negative selection. Our findings suggest that excessive heme caused by FLVCR loss impairs T cell development during these stages, and are intriguing because thus far, heme has not been shown to play a role in T cell development. Whether direct heme toxicity impairs thymocyte proliferation and/or induces apoptosis, or whether excessive heme derails the T cell developmental transcriptional/translational programs, remains to be discovered.