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Murine syngeneic graft-versus-host disease (SGVHD) initiates colon and liver inflammation following lethal irradiation, reconstitution with syngeneic bone marrow transplantation, and therapy with the immunosuppressive agent cyclosporine A. Previous studies have demonstrated that the inducible disease is mediated by CD4(+) T cells with increased reactivity of peripheral and liver-associated lymphocytes against intestinal microbial Ags. In the current report, studies were performed to analyze the specificity of the CD4(+) T cell response of T cells isolated from diseased animals and to determine the in vivo role of the microbiota to the development of SGVHD. Increased major histocompatibility Ag (MHC) class II-restricted responsiveness of SGVHD CD4(+) T cells against microbial Ags isolated from the ceca of normal animals was observed. The enhanced proliferative response was observed in the CD62L(-) memory population of CD4(+) T cells. To determine the role of the bacterial microbiota in the development of murine SGVHD, control and CsA-treated bone marrow transplantation animals were treated with broad-spectrum antibiotics (metronidazole, ciprofloxacin) after transplantation. Cyclosporine A-treated animals that were given antibiotic therapy failed to develop clinical symptoms and pathological lesions in the target tissues characteristic of SGVHD. Furthermore, the reduction in intestinal bacteria resulted in the elimination of the enhanced antimicrobial CD4(+) T cell response and significantly reduced levels of the inflammatory cytokines, IFN-γ, IL-17, and TNF-α. The elimination of the disease-associated inflammatory immune responses and pathology by treatment with broad-spectrum antibiotics definitively links the role of the microbiota and microbial-specific immunity to the development of murine SGVHD.

Curcumin (diferuloylmethane), a component of dietary spice turmeric (Curcuma longa), has been shown in recent studies to have therapeutic potential in the treatment of cancer, diabetes, arthritis, and osteoporosis. We investigated the ability of curcumin to modulate the growth of B lymphomas. Curcumin inhibited the growth of both murine and human B lymphoma in vitro and murine B lymphoma in vivo. We also demonstrate that curcumin-mediated growth inhibition of B lymphoma is through inhibition of the survival kinase Akt and its key target Bad. However, in vitro kinase assays show that Akt is not a direct target of curcumin. We identified a novel target for curcumin in B lymphoma viz spleen tyrosine kinase (Syk). Syk is constitutively activated in primary tumors and B lymphoma cell lines and curcumin down-modulates Syk activity accompanied by down-regulation of Akt activation. Moreover, we show that overexpression of Akt, a target of Syk, or Bcl-x(L), a target of Akt can overcome curcumin-induced apoptosis of B lymphoma cells. These observations suggest a novel growth promoting role for Syk in lymphoma cells.

B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti-idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igalpha or Igbeta-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas.

Syngeneic graft-vs-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a 21-day course of the immunosuppressive agent cyclosporin A (CsA). Following cessation of CsA, this inducible disease is characterized by weight loss, diarrhea, and development of inflammation in the colon and liver. Although nonspecific effector cells and Th1 cytokines have been shown to participate in disease induction, the role of T cells has not been fully elucidated. Initial studies demonstrated significant increases in CD4+ T cells, but not other T cell populations in the colons of diseased animals relative to transplant control animals. To demonstrate a functional linkage between increases in colonic CD4+ T cells and disease induction, in vivo T cell depletion studies were performed. Beginning on the day of bone marrow transplantation, groups of control and CsA-treated animals were treated with mAb against either CD4 or CD8 for 21 days. Treatment with anti-CD4, but not anti-CD8, eliminated clinical symptoms and colon pathology. Interestingly, neither anti-CD4 nor anti-CD8 therapy affected the development of liver pathology associated with SGVHD. These findings demonstrated that CD4+ T cells initiate development of the intestinal inflammation associated with murine SGVHD.

Syngeneic graft-vs-host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow cells followed by a short course of therapy with the immunosuppressive agent cyclosporine A. Following cessation of cyclosporine A therapy, animals develop clinical symptoms of SGVHD: weight loss, runting, and diarrhea. While it has been suggested that T cells are responsible for the induction and effector phases of SGVHD, the role of nonspecific effector cells and cytokine mediators has yet to be examined in the disease process. Mice with SGVHD had increased levels of message for IL-12p40, IFN-gamma, and TNF-alpha in the target organs of SGVHD as compared with transplant controls and asymptomatic cyclosporine A-treated mice. Concomitant with the increase in Th1 cytokines was an enhanced cellular infiltrate in the target organs of SGVHD mice as determined by histological analysis. To directly examine the role of IL-12 in the development of SGVHD, in vivo neutralization of IL-12 was performed. Treatment of mice with Abs to IL-12 inhibited SGVHD-mediated tissue pathology and mortality. Because IL-12 has been shown to activate both T cells and NK cells to secrete IFN-gamma and to become more cytolytic, studies were initiated to ascertain which lymphocyte populations play a role in the development of murine SGVHD. Depletion of NK cells inhibited clinical symptoms of SGVHD. In contrast, T cell depletion did not alter the disease process. Therefore, these findings collectively demonstrate a role for IL-12 and NK cells in the effector phase of murine SGVHD.

Graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation is associated with significant morbidity and mortality. T cell depletion of the marrow graft, which is currently used to prevent GVHD, has been shown to result in increased graft failure and leukemia relapse. To explore the feasibility of controlling GVHD, transgenic mice with the herpes simplex virus thymidine kinase suicide gene linked to the IL-2 promoter were used as a source of T cells to induce GVHD, which would be modulated with ganciclovir. Injection of herpes simplex virus thymidine kinase transgenic B10.A(5R) spleen cells into lethally irradiated DBA/2 mice resulted in severe acute GVHD. Treatment of the recipient mice with ganciclovir significantly inhibited GVHD-mediated weight loss and mortality and reduced the severity of inflammation in the target organs of GVHD.