Betty E. Caywood

Induction of a syngeneic graft-versus-host disease-like syndrome in DBA/2 mice.

Syngeneic graft-versus-host disease has been shown to occur in syngeneic rat radiation chimeras after treatment with a short course of CsA. However, data concerning this model have been controversial in murine systems. We have successfully induced a GVHD-like syndrome in syngeneic mouse radiation chimeras treated transiently with CsA. Lethally irradiated (950 rads) DBA/2 mice were reconstituted with syngeneic bone marrow and treated daily, i.p. with 15 mg/kg CsA in olive oil for 21 days. Within 1 week after discontinuing CsA, animals developed clinical signs of GVHD including runting, hunched posture, and severe diarrhea. This disease was fatal for greater than 80% of treated animals within 4 weeks after cessation of CsA. Furthermore, the induction of syngeneic GVHD did not appear to be linked to a particular MHC haplotype. Histologically, there was pronounced lymphoid atrophy of the spleen and thymus. Sections of large intestine showed an acute inflammatory process involving the mucosal layer ranging from single-cell destruction to complete mucosal ulceration. This murine model of GVHD should provide new opportunities for studying the development and regulation of autoimmune processes.

Rejection of an MHC class II negative tumor following induction of murine syngeneic graft-versus-host disease

Cyclosporin A (CsA) has been used clinically to induce graft-versus-host disease following autologous bone marrow transplantation in an attempt to destroy residual leukemia cells and reduce relapse. To analyze the antitumor potential of murine syngeneic graft-versus-host disease (SGVHD), C3H/HeN mice were lethally irradiated, reconstituted with T cell-depleted syngeneic bone marrow (ATBM) and treated with CsA for 21 days. Graft-versus-leukemia activity was assessed by challenging groups of olive oil-treated control ATBM (OO-ATBM) and CsA-treated (CsA-ATBM) mice 1 week after CsA therapy with graded doses of the syngeneic 38C13 B cell lymphoma. Following CsA treatment, up to 70% of CsA-ATBM developed SGVHD and more than 70% of the animals injected with 500 38C13 cells exhibited long-term survival (MST >80 days). In contrast, none of the OO-ATBM control mice developed SGVHD, and more than 75% of these mice died following injection of 500 38C13 tumor cells (MST = 34 days). Long-term survivors were not resistant to tumor challenge suggesting that tumor-specific immunity did not develop. Finally, class II negative 38C13 cells cultured in IL-4 or IL-10 were not inducible for MHC class II molecules, demonstrating that class II-independent antitumor mechanisms exist in SGVHD mice.

Thy1+ bone marrow cells regulate the induction of murine syngeneic graft-versus-host disease

A syngeneic graft-versus-host disease (GVHD)-like syndrome has been shown to be inducible in some strains of mice after lethal irradiation, reconstitution with syngeneic bone marrow (BM), and treatment with a short course of CsA therapy. Since Thy1+ BM cells have been shown to regulate the development of other experimental autoimmune diseases, it was important to determine their role in the inducibility of syngeneic GVHD (SGVHD) in different strains of mice. Lethally irradiated mice were reconstituted with either syngeneic BM or T cell-depleted syngeneic BM, then treated with CsA or diluent. Removal of Thy1+ cells from BM before reconstitution of an inducible strain, C3H/HeN, exacerbated SGVHD when compared with animals given whole BM cells before CsA treatment. Furthermore, a noninducible strain, C57BL/6 mice, developed SGVHD when reconstituted with T cell-depleted syngeneic BM but not BM before CsA therapy. These results suggest that Thy1+ BM cells may regulate the development of SGVHD, and be of importance in controlling autoreactivity after bone marrow transplantation.

Enhanced graft-versus-host disease in older recipient mice following allogeneic bone marrow transplantation

The incidence and severity of GVHD following bone marrow transplantation increases with recipient age. The role of recipient age on the development of GVHD was analyzed in a semi-allogeneic (C57BL/6 → (C57BL/6 × DBA/2)F1) murine GVHD system. Young adult (2 months) and old (12–14 months) recipient mice were lethally irradiated and reconstituted with young adult T cell-depleted bone marrow (ATBM) or ATBM and young spleen cells. A significantly higher percentage of old vs young recipients developed lethal GVHD. Furthermore, while pre-transplant conditioning with irradiation was not required to observe increased mortality in old recipients, irradiation predisposed the older animals for a more severe course of GVHD, suggesting that GVHD occurred in old compared to young animals in the absence of pre-transplant conditioning but was exacerbated by irradiation. Histologically, the immunological responses in the GVHD target organs were more severe in the old GVHD animals. In support of this observation, increased spontaneous proliferation was observed using lymphoid cells isolated from old vs young GVHD mice. These findings demonstrate that old recipients develop a more severe course of GVHD following BMT, and may present a unique opportunity to study age-related factors in the generation of GVHD.

CD4+ T Cells Mediate Murine Syngeneic Graft-versus-Host Disease-Associated Colitis

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.

In vivo reactivity of T cell clones isolated from mice with syngeneic graft-versus-host disease

Syngeneic graft-versus-host disease (SGVHD) has been shown to occur in murine syngeneic radiation bone marrow chimeras following a short course of cyclosporine. To analyze the effector mechanisms present in diseased animals, four T cell clones (1D5, 1D8, 1C10, 2D8) were isolated from the spleens of C3H/HeN mice late in the disease course by cloning on irradiated syngeneic spleen cells. These clones were CD4+, alpha beta TCR+ and responded to I-Kk in vitro. In addition to I-Ek reactivity, three of the clones exhibited crossreactivity with the superantigen MIs 1a (mtv 7). Clones 1D5 and 1C10 were found to express TCR V beta chains (V beta 4 and V beta 8.1, respectively), which are normally present in the T cell repertoire of C3H/HeN mice. All SGVHD clones were found to be autoreactive in that they responded to syngeneic stimulator cells in the absence of xenogeneic serum proteins. To test in vivo activity, the 1D5 SGVHD clone was injected into the hind footpad of mice where it was shown to induce footpad swelling in a cell dose-dependent, I-Ek-specific manner in sublethally irradiated, but not normal mice. Histological analysis indicated that the clone induced dermal and subcutaneous edema that correlated directly with injection of 1D5 and not the control clone. Preliminary experiments suggested that the other three autoreactive clones behaved in a similar manner. These data are consistent with the involvement of a self-class II-specific CD4+ T cell in murine SGVHD.

Induction of syngeneic graft-versus-host disease in LPS hyporesponsive C3H/HeJ mice

Syngeneic GVHD (SGVHD) develops following syngeneic bone marrow transplantation and treatment with cyclosporine A. Previous studies have demonstrated a role for IL‐12, IFN‐γ, and TNF‐α in the development of murine SGVHD. Macrophages can be activated to secrete IL‐12 and TNF‐α via a T‐cell‐dependent or T‐cell‐independent pathway (LPS or bacterial products). Studies were designed to determine if LPS participated in the development of SGVHD in C3H/HeN (LPS‐responsive) and C3H/HeJ (LPS‐hyporesponsive) mice. C3H/HeJ and C3H/HeN mice had similar levels of disease induction and pathology. Following induction of SGVHD, treatment of C3H/HeN, but not C3H/HeJ, mice with a sublethal dose of LPS resulted in mortality. However, neutralization of IL‐12 abrogated the development of disease in C3H/HeJ mice, demonstrating that activated macrophages and their products participated in the development of SGVHD in these animals. These data suggested that LPS responsiveness was not a predisposing factor for SGVHD induction.

Adoptive transfer of murine syngeneic graft‐vs.‐host disease by CD4+ T cells

Syngeneic graft‐vs.‐host disease (SGVHD) develops in rodents following the treatment of lethally irradiated, bone marrow (BM) reconstituted animals with a short course of the immunosuppressive agent cyclosporine A (CsA). Using an in vivo depletion approach, we recently demonstrated that CD4+, but not CD8+, T cells participated in inducing SGVHD. Studies were therefore undertaken to adoptively transfer SGVHD into lethally irradiated, syngeneic BM reconstituted secondary recipients. Whole T cell populations as well as purified CD4+T cells isolated from SGVHD, but not normal or transplant control, animals mediated the transfer of SGVHD into secondary recipients. These cells have an apparent specificity for enteric bacterial antigens. The pathologic process that developed was identical to that observed in the animals with de novo SGVHD after syngeneic BMT and CsA therapy. It was shown that a radiation‐sensitive mechanism prevented the transfer of SGVHD into normal, nonirradiated secondary recipients. The ability to reproducibly transfer SGVHD into secondary recipients will enhance our ability to study regulatory mechanisms that are altered during CsA therapy and permit the development of murine CsA‐induced SGVHD.

Induction of murine syngeneic graft-versus-host disease by cells of recipient origin

Background. Syngeneic graft-versus-host disease (SGVHD) develops after lethal irradiation, reconstitution with syngeneic bone marrow (BM), and treatment with a 21-day course of the immunosuppressant cyclosporine A (CsA). Clinical symptoms of SGVHD appear 2-3 weeks after CsA treatment, with inflammation in the colon and liver. It has been demonstrated that CD4+ T cells and a T helper cell type 1 cytokine response (Th1) are involved in the development of SGVHD associated intestinal inflammation. The immune response associated with SGVHD is thought to be the result of the reconstitution of the recipient immune system with the syngeneic donor BM. However, definitive studies have not addressed this issue experimentally. Methods. To determine the origin of the effector cells that participate in SGVHD, C3H/HeN recipient mice were lethally irradiated and transplanted with BM from normal immunocompetent mice or from immunodeficient, severe combined immune deficient, or Rag-2−/− animals. Results. CsA-treated animals, but not control animals, developed inflammation characteristic of SGVHD in the colon and liver regardless of the source of the donor marrow. Furthermore, immunologically, all CsA treated animals responded similarly with increased production of inflammatory cytokines and an increase in activated CD4+ T cells in the periphery and colon relative to controls. Conclusion. These results demonstrate that after lethal irradiation and in the absence of donor T cells, T cells of recipient origin can expand and mediate the induction of CsA-induced SGVHD.

In vivo regulation of the murine syngeneic mixed lymphocyte reaction.

Previous work from this laboratory has suggested that a CD8+ T suppressor (Ts) cell network regulated the murine syngeneic mixed lymphocyte reaction (SMLR). We have attempted to disrupt this network by the inoculation of anti-CD8 monoclonal antibodies (mAb) in vivo. Intraperitoneal inoculation of three mAbs resulted in a marked increase in the proliferation of CD4+, self-Ia-reactive splenic T cells in vitro to syngeneic, but not to allogeneic, spleen cells. Suppression was not limited to a specific mouse strain as the enhanced SMLR was reproducible following anti-CD8 treatment of three strains of mice. In vivo depletion of CD8+ T cells was not a prerequisite for enhancement of the SMLR as several mAb to CD8 augmented the SMLR independent of their capacity to cause CD8 T cell depletion. Moreover, enhancement of the SMLR could be mimicked in vitro by inclusion of anti-CD8 mAb in in vitro cultures of responder T cells and irradiated Ia+ syngeneic stimulators. Since the in vitro SMLR was enhanced following mAb treatment, it was expected that the in vivo SMLR would also be increased. However, no evidence of increased in vivo autoreactivity could be detected following in vivo treatment with anti-CD8 mAb, indicating that other mechanisms in addition to CD8+ regulatory T cells acted to regulate the in vivo activity of autoreactive T cells.