Signaling Profiles of T Cells during Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract In this study we characterized the signaling profiles of T cells in murine allogeneic bone marrow transplantation (BMT) models. We used a novel high-throughput technique to analyze activation of signaling pathways by flow cytometry. We first studied normal splenic T cells (Table 1) and found that relative to naive CD4 and CD8 cells, effector and memory cells displayed nearly global increases in activation of signaling pathways including the MAP kinases and STATs. We observed similar increases in signaling activity in effector/memory vs. naive T cells in the mesenteric and peripheral lymph nodes (PLN) and the liver. Signaling in the C57BL/6 spleen CD4 CD8 Naive (CD44lo CD62Lhi) Effector (CD44hi CD62Llo) Memory (CD44hi CD62Lhi) Naive (CD44lo) Effector/Memory (CD44hi) c-Raf (pS259) 1 2.2 1.8 1 1.2 MEK 1/2 (pS217/pS221) 1 2.7 9.7 1 2.2 ERK 1/2 (pT202/pY204) 1 1.6 1.8 1 1.2 JNK (pT183/pY185) 1 1.1 6.5 1 2.3 Pan-p38 (pT180/pY182) 1 2.1 3.0 1 1.8 Pan-Akt (pT308) 1 2.1 3.8 1 1.4 Pan-Akt (pS473) 1 2.6 3.9 1 1.7 Pan-PKC ( β II pS660) 1 2.7 10.1 1 1.4 STAT-1 (pY701) 1 0.9 6.3 1 2.4 STAT-3 (pY705) 1 1.4 1.8 1 1.3 STAT-3 (pS727) 1 1.2 1.3 1 1.3 STAT-4 (pY693) 1 2.0 2.3 1 1.2 STAT-5A (pY694) 1 1.3 1.7 1 1.3 S6 Ribosomal Protein 1 3.0 6.3 1 1.8 (pS235/pS236) Histone H2B (pS14) 1 2.6 4.2 1 1.8 Cleaved Caspase 3 (D175) 1 2.0 3.8 1 1.3 PIP2 1 1.3 0.4 1 0.6 Ratios of activation of signaling molecules in effector or memory CD4 or CD8 cells compared with naive cells. In the thymus, there was a general lack of signaling activity in CD4+CD8+ (DP) cells, and these responded poorly to cytokine stimulation. By contrast, CD4−CD8− (DN) cells of the DN1 and DN2 stage had the greatest signaling activity of all thymocytes. We then studied alloreactive and homeostatically proliferating T cells by infusion of carboxyfluorescein (CFSE) labeled T cells into irradiated hosts. We noted a gradual and global increase in signaling activity with each cell division in the spleen, liver, and PLN. Peak increases relative to the undivided CFSEhi population of up to 2.5-fold (ERK 1/2) in the CD4 cells by the 4th cell division, with similar increases in CD8 cells, were found. Even the undivided CD4 CFSEhi population itself displayed large increases in signal (~4 fold for p38 and ERK 1/2) relative to naive splenic CD4 cells. We defined slow-cycling cells as homeostatically expanding and fast-cycling cells as alloreactive, and found a distinct signaling pattern in homeostatically expanding T cells, with lower levels of MAP kinase pathway activity, JAK/STAT pathway activity, and pro-apoptotic proteins such as cleaved caspase 3 and phosphorylated histone H2B. However, PKC phosphorylation appeared to be higher in homeostatically proliferating cells than alloreactive T cells. In the setting of T-cell depleted bone marrow transplantation, we found in several models that newly generated, donor-derived splenic T cells had signaling levels generally comparable with that of the naive populations in a normal spleen, but with a marked deficiency in STAT-3 (pS727) phosphorylation. In the thymus, we observed defects in Akt and STAT-3 (pS727 and pY705) signaling in the DN, DP, CD4, and CD8 subsets relative to signaling levels in the normal thymus. We conclude that the high-throughput analysis of signaling activity in T cells during allogeneic BMT allows for the definition of distinct signaling profiles. These signaling signatures can be used for the development of targeted therapies to enhance or inhibit specific T cell subsets.