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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.1-7 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 phospho-specific 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.

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

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 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.

Differential scanning calorimetry (DSC) experiments have been carried out on the desorption of water from polyadenylic acid and deoxyribonucleic acid in the region of less than 10 water molecules per monomer unit. The enthalpy of the desorption process increases from a value essentially that of the vaporization of liquid water to approximately 16,000 cal per mole as the quantity of absorbed water decreases from 10 to 2 water molecules per monomer unit. Equilibrium vapor pressure studies together with the DSC measurements on polyadenylic acid permit calculation of the entropy of the absorbed water as essentially that of liquid water.