ALBERT

Description: To study the complex pathophysiology of aGvHD in allogeneic hematopoietic cell transplantation (HCT) we transplanted transgenic luciferase expressing T cell populations into lethally irradiated HCT recipients (murine MHC major mismatch model, H-2q into H-2d). Tracking of light emitting donor T cells in living animals and detailed studies by multi color immunofluorescence microscopy (IFM) and FACS revealed the tight links of spatial and temporal evolution in this complex immune process. Donor derived T cells migrate to T cell areas in lymphoid tissues within a period of 12 hours. In the initial periods donor CD4+ T cells appear first with CD8+ T cell infiltration at later time points. Donor T cells start proliferating in lymphatic tissues on day 2 after transfer, as observed by BrdU stainings. Although alloreactive T cells are similarly activated in all lymphoid organs, they only up-regulate gut homing molecules after more than 5 cell divisions (CFSE proliferation analysis by FACS) in certain lymphoid organs (Peyer’s patches, mesenteric LN and spleen). Abruptly on day 4 after HCT, T cells migrate into intestinal sites. These findings strongly suggested, that specific priming sites are required for alloreactive T cells to induce a distinct type of tissue tropism in GvHD. In contrast to previous reports peformed without host conditioning, depletion of certain lymphoid organs (e.g. Peyer’s patches) before HCT or antibody blocking experiments did not control aGVHD. BLI showed, that anti-L-selectin or anti-MAdCAM-1 antibody treatment alone or in combination was effective in blocking donor T cell migration to lymph nodes and Peyer’s patches, while redirecting these cells to liver and spleen. Subsequently cells proliferated predominantly in the spleen until day 3 after HCT. Surprisingly we observed a full picture of gut infiltration on day 4 and skin involvement on day 5–6, similar in dynamics and strength to the aGvHD isotype control group. These findings demonstrated, that other lymphoid organs can functionally compensate for inducing gut and skin homing of alloreactive T cells. Of importance, we demonstrated that T cells that lacked homing molecules for secondary lymphoid organs had alloreactive properties in vitro, yet did not cause aGVHD in vivo. In summary, the activation of alloreactive T cells in specific sites throughout the body is complex and involves the acquisition of homing molecule expression. Transplantation of T cells with defined homing properties therefore, appears to be a promising alternative in conferring protective immunity early after HCT without the risk of aGvHD.

Description: Abstract 2451 Poster Board II-428 Hematopoietic cell transplantation (HCT) is a curative therapy for a variety of malignancies. HCT provides disease eradication through both the high-dose conditioning regimen and an allogeneic graft versus tumor effect (GVT), however graft-versus-host disease (GVHD) remains a major obstacle. In a murine aHCT model of bioluminescence imaging (BLI) we have previously demonstrated that acute GVHD can be separated to a GVHD initiation phase confined to secondary lymphoid organs and a subsequent GVHD effector phase in peripheral target tissues. It has been proposed that host conditioning may not only be crucial in the activation of alloreactive T cells but also determine acute GVHD organ manifestation in the effector phase. Here we wanted to investigate how the host conditioning regimen affects the host target tissues in terms of inflammatory cytokines and their role in donor T cell recruitment. We compared lethally irradiated (8Gy) vs. non-irradiated Balb/c wild type or Balb/c Rag-/-cGC-/- (H-2d) -DKO mice that received allogeneic luciferase+ FVB/N T cells (H-2q). Surprisingly, we did not observe marked differences in the donor T cell proliferation (BLI, CFSE), acquisition of activation markers (CD25, CD44, CD69) and homing receptors (a4b7, aEb7, P-selectin ligand, E-selecting ligand) in conditioned, non-conditioned Balb/c Rag-/-cGC-/-. Despite the upregulation of these homing receptors on donor T cells, infiltration of target tissues (intestinal tract, liver and skin) was significantly accelerated in conditioned and delayed in non-conditioned hosts. As T cell recruitment may have occurred as a result of alterations of the milieu inflammatory cytokines in GVHD target tissues, we compared the cytokine profile in conditioned vs. non-conditioned recipients. At days 3 and 6 after transplantation tissues were harvested and cytokines from the target tissues; liver, large bowel, small bowel, peripheral blood and a non target tissue: kidney were analyzed for a TH1/TH2/Th17a cytokines. At day 3 high levels of INF-γ and TNF were detected in the Balb/c WT conditioned host compared to the non-conditioned host in all target tissues (SB, LB, and liver) and most markedly in peripheral blood and the large bowel. More importantly the Balb/c Rag-/-cGC-/- conditioned host displayed about 5 times higher levels of both inflammatory cytokines compared to the non conditioned DKO hosts and to the Balb/c WT. Similar results with a lesser levels were observed both for IL-2 and IL17a. By day 6 similar results are seen but with a much reduced expression of the cytokines, indicating that the cytokine storm peak was maybe at day 3. In summary host conditioning is not a requirement for alloreactive T cell activation rather induced inflammatory cytokines such as TNF and INF-γ are the determinant factors for effector T cell recruitment to GVHD target tissues. JB and AB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.