Similarities In the Initial Events of Murine Gvhd Across Minor. as Compared to Major, Histocompatibility Barriers.

Abstract 3750

Graft-vs-Host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation (HCT). Murine models have been critically important to define the biological mechanisms and potential pathways of intervention of GVHD prevention and treatment. Although it is well recognized that GVHD occurs in response to minor histocompatibility antigens, little is know about the kinetics of donor T cell proliferation and homing in minor mismatch models. This is in contrast to models across major histocompatibility barriers where the early development of GVHD has been more thoroughly characterized. In prior studies across major barriers, we have defined an initiation phase within the first 3 days where conventional CD4⁺ and CD8⁺ T cells (Tcon) home to secondary lymphoid tissues, proliferate and up-regulate key homing markers allowing for entry into GVHD target tissues during the effector phase (Beilhack, et al. Blood 106:1113, 2005). Since minor models are more similar to clinical HCT, it is critical to understand the timecourse of GVHD development across minor histocompatibility barriers. Since the manifestations of GVHD in recipients of minor mismatch transplants are delayed, it is possible that disease development has altered kinetics. To investigate the temporal and spatial events of donor T cell activation and homing, side-by-side transplants were conducted using T cell depleted bone marrow (TCD BM) and Tcon from donor C57BL/6 (H2^b) mice into either major mismatched Balb.c (H2^d), or minor mismatch Balb.b (H2^b) recipients. Balb.c mice received 1×10⁶ Tcon while Balb.b mice were given 15×10⁶ Tcon, based on previous titration experiments. Recipient mice were regularly scored for GVHD symptoms and monitored for at least 100 days for survival. Additionally, donor Tcon proliferation and migration were monitored longitudinally using in vivo and ex vivo bioluminescent imaging (BLI) by quantitating photons emitted by luciferase (luc⁺) expressing donor Tcon isolated from luc⁺ transgenic mice. Donor Tcon were also labeled with CFSE to determine proliferation kinetics at selected timepoints. The upregulation of T cell activation and tissue specific homing markers was examined using flow cytometric analysis of donor CD4⁺ and CD8⁺ T cells re-isolated from the secondary lymphoid tissues of transplanted mice. In both models, T cells initially home to secondary nodal sites by 3 days post-transplant, with an exodus into the tissues by day 6, albeit to a lesser extent in recipients of minor mismatch transplants. Additionally, similar levels of global donor CD4⁺ and CD8⁺ T cell proliferation between the models were observed using both BLI and CFSE staining as early as 3 days after transplant (BLI, p>0.05, n=9). More noticeable reductions in minor mismatch recipients were apparent by day 6 (BLI p<0.0001, n=9). The expression profiles of several T cell activation and tissue homing markers, such as CD44, CD69, a₄b₇, and P- and E-selectin, were also quite similar when measured at timepoints within one week of transplant, although there was some variability in expression between tissues as well as between CD4 and CD8 T cells. However re-isolated donor T cells from recipients of minor mismatched transplants did consistently show delays in the up-regulation of CD25 and the down-regulation of CD62L, as compared to that noted in major mismatch transplant recipients. Together, these data support the conclusion that the early events of donor T cell activation in the initiation phase of GVHD, particularly spatially, are quite similar to those previously documented in major mismatch models of GVHD, reinforcing the usefulness of both models as translational research tools. More importantly, the data suggest that the delay in visible GVHD onset noted in transplants across minor barriers arises from temporal differences in the effector phase of T cell migration and proliferation, rather than delays in the initiation phase. As such, these findings support the targeting of very early events in T cell activation as the most effective method of reducing GVHD in the clinical setting.