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...

BackgroundAllogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models.MethodsWe studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1−/− T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25hi, CD62Llo). Additionally, Ceacam1−/− CD8 T cells had greater expression of the gut-trafficking integrin α4β7, though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1−/− recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1−/− mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1+ lymphoma model was improved in animals receiving Ceacam1−/− vs. control T cells.ConclusionsWe conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.

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Carcinoembryonic antigen associated cell adhesion molecule 1 (CEACAM-1) belongs to a family of carcinoembryonic antigen-associated glycoproteins. It is expressed on leukocytes, endothelium, and epithelium. Microarray analysis showed that CEACAM-1 mRNA is increased in the small bowel during gut graft-versus-host-disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). Using CEACAM-1−/− mice as recipients or sources of donor bone marrow or T cells caused significantly worse GVHD mortality (p<0.05) compared to wildtype (WT) controls. Histopathological analysis of GVHD target organs from CEACAM-1−/− recipients of WT T cells or WT recipients of CEACAM-1−/− T cells revealed increased GVHD of the large bowel (p<0.05) but not liver or small bowel compared to WT control. Alloreactive splenic CD8 CEACAM-1−/− T cells from recipients with GVHD had increased levels of α4β7 integrin compared to WT controls. We also found increased numbers of small bowel intraepithelial lymphocytes and mesenteric lymph node cellularity in CEACAM-1−/− recipients of WT T cells and WT recipients of CEACAM-1−/− T cells, with a corresponding decrease of cellularity in peripheral lymph nodes and the liver. Adoptive transfer of CFSEhi CEACAM-1−/− T cells into WT hosts, or of WT T cells into CEACAM 1−/− hosts revealed more profound activation of T cells in CEACAM-1 deficient settings, shown by increased early CD25 expression and CD62L down-regulation on splenic CFSEdim alloreactive T cells. We found no significant differences in serum levels of TNF or IFNγ, T cell proliferation kinetics upon adoptive transfer, percentages of alloactivated CD4 or CD8 cells, intracellular levels of T-bet or IFNγ, CD8 T cell cytolytic efficiency, percentages of splenic regulatory T cells, or levels of T cell apoptosis in WT recipients of CEACAM-1−/− T cells or CEACAM-1−/− recipients of WT T cells with GVHD as compared with controls. Finally, irradiation of non-transplanted CEACAM-1−/− mice revealed increased radiation sensitivity, shown by earlier and greater lethality and increased small bowel crypt apoptosis, suggesting a role for CEACAM-1 in conditioning-related toxicity and subsequent GVHD amplification. We conclude that CEACAM-1 deficiency on donor T cells or transplant recipients results in increased gut and systemic GVHD due to increased T cell activation and elevated expression of the gut homing integrin α4β7. This suggests that the use of CEACAM-1 agonists could be a novel theraputic strategy for ameliorating acute intestinal and systemic graft-versus-host-disease.

Graft-versus-host-disease (GVHD) is a serious complication of allogeneic bone marrow transplantation (allo-BMT). The physiology of GVHD is dominated by alloactivated donor T cells, yet current treatments are often nonspecific and offer limited efficacy with relatively high toxicities. We screened for novel drug targets in alloactivated T cells in a murine allo-BMT model using a flow cytometric technique for the in vivo analysis of intracellular signaling. We defined the signaling profile of normal T cells and alloreactive T cells during GVHD, focusing on pathways involved in T cell receptor (TCR), costimulatory, and cytokine signaling. This analysis revealed that although proteins in multiple pathways (MAP kinases, PI3K, Jak/STAT signaling) were all heavily phosphorylated in alloactivated T cells, phosphorylation of STAT-3 and ERK1/2 were particularly prominently increased in donor alloactivated CD4 T cells. We further analyzed the importance of STAT-3 and ERK1/2 signaling in alloactivated T cells via the use of small-molecule inhibitors of STAT-3 (curcurbitacin E/I) and ERK1/2 phosphorylation (SL327). Treatment with these inhibitors attenuated T cell proliferation in response to anti-CD3+CD28 stimulation and in mixed leukocyte reactions in vitro in a dose-dependent fashion (figure 1). Figure 1 Figure 1. Pre-incubation of donor splenocytes with cucurbitacin E significanly reduced T cell activation (CD25, CD69) at 24 hours in adoptive transfer experiments in vivo (p<0.05). To rule out any direct toxicity, we analyzed recovered cells for apoptosis by Annexin-V staininga dn detected no significant toxicity (figure 2). Figure 2 Figure 2. We conclude that flow cytometric analysis of signaling pathways in single cells represents a novel methodology to assess the in vivo signaling profiles of specific cell populations in order to select drug targets for further study. STAT-3 and ERK1/2 phosphorylation may also represent potential targets to selectively inhibit donor T cell alloactivation and proliferation in GVHD.