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.

Description: In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.

Description: Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (TEM) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

Description: Acute graft-versus-host disease (aGVHD) results from alloreactive donor derived T cells attacking targets in the gastrointestinal tract, liver and skin. We observed the initiation and rapid kinetics of aGVHD in a murine model [FVB/N (H-2q) into irradiated Balb/c (H-2d)] using in vivo bioluminescence imaging. The transition from the initiation to the effector phase of aGVHD (day 3–4) was characterized by rapid T cell proliferation and upregulation of gut homing receptors alpha4beta7, alphaEbeta7 and CCR9 on alloreactive T cells in Peyer’s patches (PP), mesenteric lymph nodes (LN) and spleen, but not peripheral LNs. Therefore we asked whether the lack of specific lymphoid priming sites would lead to decreased alloreactive T cell infiltration in the gut compared to the liver and skin. Using PP deficient mice, we observed that mesenteric LN and spleen compensate for the lack of PP as alloreactive priming sites. Transplantation of PP and LN deficient mice (TNFalpha-/-) showed that the spleen alone was sufficient to cause the complete profile of aGVHD with a time course similar to that of wildtype mice. Splenectomized mice with intact secondary lymphoid organs also developed aGVHD. Strikingly, treatment of splenectomized recipients with blocking antibodies against the lymphoid homing receptors L-selectin and MAdCAM-1 prevented GVHD with 100% survival (〉120 d, p

Description: Acute graft-versus-host disease (aGVHD) is caused by alloreactive effector T cells attacking the gastrointestinal tract, liver and skin after allogeneic hematopoietic cell transplantation (aHCT). The mechanism by which alloreactive T cells target these organs and not others remains elusive. Recently, we reported that different secondary lymphoid organs (SLOs), as alloreactive priming sites, can imprint distinct homing phenotypes on evolving alloreactive effector cells in vivo. However, preventing access to selected lymphoid organs (via the use of blocking antibodies or recipient mice lacking Peyer’s patches (PP), PP and lymph nodes (LN) or spleens) did not alter the aGVHD organ manifestation. These findings not only suggested a high redundancy of SLOs as induction sites of aGVHD, but also questioned whether homing instruction of alloreactive T cells by these sites can explain the mechanism of aGVHD target organ manifestation. To test the homing instruction model we transplanted transgenic luciferase+ (luc+) FVB/N (H-2q, Thy1.1+) splenocytes into conditioned (2×400rad) Balb/c recipients (H-2d, Thy1.2+). On day+3 we isolated luc+ donor lymphocytes from peripheral LN, mesenteric LN, or spleens and transferred them into conditioned secondary allogeneic recipients. 16 hours later, bioluminescence imaging revealed that allogeneic luc+ T cells irrespective of their original priming site targeted the intestinal tract and liver. Subsequently, we compared aHCT of conditioned with non-conditioned secondary Balb/cRag−/− cγ-Chain−/− recipients. Surprisingly, we found allogeneic luc+ T cells accumulating in SLOs in non-conditioned recipients in contrast to intestinal and hepatic tissues in conditioned recipients. These in vivo findings establish that alloreactive effector cells migrate to aGVHD target tissues because of attraction to these sites rather than specific instruction by SLOs. Therefore, we propose a signal hierarchy model of alloreactive cell trafficking whereby inflammatory signal/ligand interactions dominate over organ-specific homing receptor/ligand interactions.