Visualizing Allogeneic Bone Marrow (BM) Graft Rejection.

Abstract

The process by which bone marrow is rejected by host T cells has not been able to be directly visualized to date. To study the process of allogeneic bone marrow rejection and the effects of therapeutic interventions, we created 2 models that allow us to 1) quantify the specific expansion of host-type alloreactive T cells early post-transplant in response to allogeneic BM infusion and to 2) image host T cells in vivo during BM rejection. For the first model, 2C and TEa lymph node (LN) cells were adoptively transferred into syngeneic C57BL/6 (B6) Rag deficient mice on d-2. 2C CD8+ and TEa CD4+ T cell receptor transgenic T cells are reactive against BALB/c alloantigen. Mice were irradiated with 200 cGy on d-1 and BALB/c BM was infused on d0. Controls included mice that received 2C/TEa LN cells but no BM. Ten days later, spleen analysis revealed that 2C CD8+ and TEa CD4+ T cells had expanded 322-fold and 33-fold (ave of 6 exp.), respectively, in mice receiving BALB/c BM compared to controls that did not receive BM. Expanded T cells were activated as determined by flow cytometric parameters and cell surface antigens. Data indicate that host alloreactive T cell expansion was inhibited by >95% by combined, but not single, costimulatory pathway blockade. Studies are in progress to analyze in vitro host anti-donor responses of adoptively transferred T cells. To visualize the response of host T cells to donor BM in vivo, we developed a rejection model for imaging involving the adoptive transfer of green fluorescent protein (GFP) T cells (obtained from GFP transgenic mice) into syngeneic non-GFP B6 recipients immediately following sublethal irradiation (500 cGy). Allogeneic BALB/c or syngeneic B6 BM was infused the following day. The syngeneic BMT controls allowed for the distinction of homeostatic vs alloreactive expansion of GFP+ T cells. Transplanted mice not receiving GFP T cells served as negative controls to verify lack of autofluorescence. Cohorts were imaged d4 to d18 post BMT. By d4, low numbers of GFP+ cells were evident in femoral BM cavity, peripheral and mesenteric LNs, spleen, Peyer's patches (PP) and to a lesser extent, lung. By d7, massive expansion of GFP+ cells could be visualized throughout the body of recipients of allogeneic BM. LNs, spleen, PP (peri-follicular area) and BM cavity increased dramatically in GFP intensity from d4 to d7. On d7 to d14, there were large foci of GFP+ cells in the lung, liver, skin, gingiva, kidney, uterus, and colon in allogeneic BMT recipients. Compared with allogeneic BMT recipients, syngeneic BMT recipients had greatly reduced numbers of GFP+ T cells in lymphoid organs and only rare cells were noted in liver, kidney, skin, BM and gingiva. In both allogeneic and syngeneic BMT recipients, lengths of ileum were diffusely infiltrated while other sections contained discrete foci of GFP+ cells. These imaging data provide a vivid illustration of the massive expansion and multi-organ distribution of host anti-donor T cells in vivo. Recent generation of GFP+ 2C and GFP+ TEa mice will permit the imaging of alloantigen-specific T cells during a rejection response. Additional imaging experiments are planned to study the fate of GFP+ BM transferred to allogeneic recipients under conditions of engraftment vs rejection. These models provide a unique platform for the testing of therapeutic interventions.