STAT-3 and ERK 1/2 phosphorylation are critical for T-cell alloactivation and graft-versus-host disease

Abstract

Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets. [1][2][3][4][5][6][7] This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phosphospecific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD. (Blood. 2008;112:5254-5258) IntroductionAnalysis of in vivo T-cell signaling with Western blots is limited by requirements for large numbers of cells and the inability to access subpopulations. We used a flow cytometric method for measuring phosphorylated epitopes in single cells [1][2][3][4][5][6][7] to establish a signaling profile for alloactivated T cells in vivo in murine graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. We identify and validate molecular targets important for GVHD pathobiology and demonstrate the importance of ERK1/2 and STAT-3 phosphorylation for T-cell alloactivation in vivo. Finally, we confirm that small molecule inhibitors of STAT-3 phosphorylation can attenuate T-cell activation, proliferation, and GVHD. MethodsBone marrow transplantation, transfer of CFSE-labeled T cells, and GVHD assessment were described previously 8,9 and discussed in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). In vitro stimulation of T cells with cytokines, anti-CD3, anti-CD3 ϩ CD28 antibody, or irradiated stimulator cells were previously described 1,9 and further discussed in Document S1. All protocols were approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center.Cell-surface staining, intracellular phospho-specific staining, flow cytometry, and data analysis were previously described 2,4-6 and further discussed in Document S1. The T(X) population comparison metric was described previously 10 and its use is further discussed in the Document S1.Finally, a list of phospho-specific antibodies and inhibitors used in this study can also be found in Document S1. Results and discussionWe first validated the antibodies for this study (Figure S1A-C and accompanying text), and then examined subsets of splenic T cells in the normal mouse. When we divided CD4 T cells into naive (CD44 lo CD62L hi ), effector memory (CD44 hi CD62L lo ), and central memory (C...

The IL-33/ST2 axis augments effector T-cell responses during acute GVHD

“…[32][33][34]71 Downregulation of IL-18R in ST2-deficient T cells may be due to an impaired MAPK-dependent pathway, 15,17,32 since the regulation of the receptor has been shown to be MAPK-dependent. 22,36,37 Also, the activation of stress-activated protein kinases such as MAPK 72 and ERK 73 were previously shown to be relevant for the development of aGVHD.…”

MEK inhibitors selectively suppress alloreactivity and graft-versus-host disease in a memory stage-dependent manner

“…16 Single-cell analysis of ERK1/2 phosphorylation in murine T cells suggested that ex vivo MEK inhibition inhibited alloreactivity, suggesting the potential to ameliorate GVHD. 17 MEK1/2 inhibitors are being tested for efficacy in multiple cancers dependent on RAS/MEK/ERK signaling, with little apparent hematologic toxicity reported in over 60 ongoing human clinical trials. 18,19 Recently, extremely promising results have been evident in multiple cancer trials, either using MEK inhibition alone or with other targeted inhibitors.…”

SOCS3 regulates graft-versus-host disease

“…47 That study showed the presence of phosphorylated STAT3 in dividing cells after BMT. 48 Furthermore, the preincubation of donor T cells with a small molecule inhibitor of STAT3 could reduce donor CD8 T-cell expansion and …”