Transfer of T cells to freshly irradiated allogeneic recipients leads to their rapid recruitment to nonlymphoid tissues, where they induce graft-versus-host disease (GVHD). In contrast, when donor T cells are transferred to established mixed chimeras (MCs), GVHD is not induced despite a robust graft-versus-host (GVH) reaction that eliminates normal and malignant host hematopoietic cells. We demonstrate here that donor GVH-reactive T cells transferred to MCs or freshly irradiated mice undergo similar expansion and activation, with similar up-regulation of homing molecules required for entry to nonlymphoid tissues. Using dynamic two-photon in vivo microscopy, we show that these activated T cells do not enter GVHD target tissues in established MCs, contrary to the dogma that activated T cells inevitably traffic to nonlymphoid tissues. Instead, we show that the presence of inflammation within a nonlymphoid tissue is a prerequisite for the trafficking of activated T cells to that site. Our studies help to explain the paradox whereby GVH-reactive T cells can mediate graft-versus-leukemia responses without inducing GVHD in established MCs.
Following bone marrow transplantation, delayed donor leukocyte infusions (DLIs) can induce graft-versus-leukemia (GVL) effects without graft-versus-host disease (GVHD). These antitumor responses are maximized by the presence of host hematopoietic antigen-presenting cells (APCs) at the time of DLI. Using a tumor-protection model, we demonstrate here that GVL activity following administration of DLIs to established mixed chimeras is dependent primarily on reactivity to allogeneic MHC antigens rather than minor histocompatibility or tumorassociated antigens. CD8 ؉ T-celldependent GVL responses against an MHC class II-negative tumor following delayed DLI require CD4 ؉ T-cell help and are reduced significantly when host APCs lack MHC class II expression. CD4 ؉ T cells primed by host APCs were required for maximal expansion of graft-versus-host reactive CD8 ؉ T cells but not their synthesis of IFN-␥. In contrast, the GVL requirement for CD4 ؉ T-cell help was bypassed almost completely when DLI was administered to freshly irradiated recipients, indicating that the host environment is a major factor influencing the cellular mechanisms of GVL. IntroductionFollowing allogeneic bone marrow transplantation (BMT), donor T-cell alloreactivity can be co-opted to generate powerful antitumor activity, an effect termed the graft-versus-leukemia (GVL) response. 1,2 The GVL effect is associated with the presence of graft-versus-host disease (GVHD) and is linked to the degree of major MHC disparity and the presence of T cells within the graft, indicating that graft-versus-host (GVH) alloreactive donor T cells are important for this effect. 3 We have previously shown that administration of delayed donor leukocyte infusion (DLI) to established mixed chimeras (MCs; in which hematopoietic elements from both the donor and recipient are present) produces dramatically improved GVL effects compared with those seen following delayed DLI to full chimeras (FCs). 4 Host hematopoietic antigen-presenting cells (APCs) expressing MHC class I molecules are necessary for this optimization of GVL effects in MCs. 4 The importance of host APCs in inducing GVL has recently been confirmed in freshly irradiated mice, 5 and previous studies have shown their importance in inducing GVHD under such conditions. 6,7 The marked overlap of GVL and GVHD limit the wider application of allogeneic BMT, especially in those individuals who lack an HLA-identical donor. However, GVL can be achieved without GVHD by administration of DLI to established MCs that lack proinflammatory stimuli from recent conditioning. 4,8 A precise definition of the mechanisms that underlie the GVL effect of DLI in MCs will be important for the rational development of this strategy for achieving maximal GVL effects without GVHD in humans. Using a tumor protection model, we demonstrate here that GVL responses of DLI are due to alloresponses against recipient MHC antigens. We also demonstrate a requirement for CD4 ϩ T-cell help in generating maximal CD8 ϩ T-cell-mediated GVL activity against MH...
Surprisingly, antitumor responses can occur in patients who reject donor grafts following nonmyeloablative hemopoietic cell transplantation. In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism, IFN-γ production, and antitumor responses against host-type tumors. However, the mechanisms behind this phenomenon remain to be determined. We now demonstrate that the effects of RLI are mediated by distinct and complex mechanisms. Donor marrow rejection is induced by RLI-derived alloactivated T cells, which activate non-RLI-derived, recipient IFN-γ-producing cells. RLI-derived CD8 T cells induce the production of IFN-γ by both RLI and non-RLI-derived recipient cells. The antitumor responses of RLI involve mainly RLI-derived IFN-γ-producing CD8 T cells and recipient-derived CD4 T cells and do not involve donor T cells. The pathways of donor marrow and tumor rejection lead to the development of tumor-specific cell-mediated cytotoxic responses that are not due to bystander killing by alloreactive T cells.
We have examined how the host environment influences the graft-vs-leukemia (GVL) response following transfer of donor T cells to allogeneic chimeras. Donor T cells induce significant GVL when administered in large numbers to established mixed chimeras (MC). However, when using limiting numbers of T cells, we found that late transfer to MC induced less GVL than did early transfer to freshly irradiated allogeneic recipients. Late donor T cell transfer to MC was associated with marked accumulation of anti-host CD8 cells within the spleen, but delayed kinetics of differentiation, reduced expression of effector molecules including IFN-␥, impaired cytotoxicity, and higher rates of sustained apoptosis. Furthermore, in contrast to the spleen, we observed a significant delay in donor CD8 cell recruitment to the bone marrow, a key location for hematopoietic tumors. donor T cell alloreactivity can be exploited to generate a powerful anti-leukemia effect, a phenomenon termed the graft-vs-leukemia (GVL) response (1, 2). Unfortunately, under conditions in which donor T cells are transferred immediately to lethally irradiated recipients, GVL responses are often associated with the development of graft-vs-host disease (GVHD). Delaying the timing of donor T cell administration by 2-3 wk may reduce the risk of GVHD in full allogeneic chimeras and still permit the induction of GVL, but by 4 -5 wk, graft-vs-host (GVH) reactivity cannot be induced (3, 4). In sharp contrast, the GVL response remains intact when donor T cells are infused (at Ͼ8 wk) into established mixed chimeras (MC), where hematopoietic elements from both the donor and recipient are present at the time of transfer (5-7). This finding relates to the continued presence of large numbers of host hematopoietic APCs, which are required for maximal priming of donor T cells recognizing recipient class I (7) and class II (5) following MHC-mismatched transplantation.Although high numbers of donor T cells activated in MC do not cause GVHD, they do so readily upon transfer to secondary, irradiated allogeneic recipients (8). Moreover, the application of a local or systemic TLR stimulus allows such donor T cells to cause local or systemic GVHD, respectively (8). These studies indicate that donor leukocyte infusion (DLI)-derived GVH-reactive T cell populations activated in MC have no absolute, intrinsic functional defect. Rather, these and other data (9 -14) argue that extrinsic factors such as inflammation within the host environment are critical for the recruitment of activated T cells to the epithelial target tissues and hence the development of GVHD. It is still not known, however, whether antihost CTL arising in established MC or freshly irradiated allogeneic (TBI-allo) recipients are fully equivalent in terms of functional activity and their capacity to induce GVL. Potentially important differences between the two host environments include the duration of direct Ag presentation by professional APC, the levels of lymphopenia-induced proliferation, the extent of suppression m...
Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). DLI given immediately following lethal irradiation lead to severe GVHD. We examined the proliferation, expansion, differentiation and survival of GVH-reactive T cells following delayed DLI and compared the outcomes to those observed when identical DLI were administered early following lethal irradiation (TBI). MC recipients were prepared by TBI of BALB/c mice and reconstitution with mixed BALB/c and CD45.2 C57BL/6 (B6) T-cell depleted bone marrow (TCD BM). 10 weeks later, we transferred 1 x 107 CD45.1 B6 and 5 x 106 2C transgenic (tg) splenocytes. CD8+ T cells from 2C tg mice bear TCR specific for recipient class I MHC Ld. Polyclonal DLI-derived CD4+/CD8+ T cell responses were monitored by gating on CD45.1+ events and clonal 2C CD8+ T cell responses tracked using a clonotypic marker. For comparison, identical DLI together with TCD BM was administered to freshly irradiated BALB/c or B6 CD45.2 syngeneic recipients. MC recipients of delayed DLI developed a GVH reaction, as indicated by increases in donor chimerism, but no GVHD. In contrast, allogeneic recipients receiving DLI immediately following TBI developed lethal GVHD (median survival 32d vs. >100d post-delayed DLI, p<0.0001). By day 6, donor CD4+/CD8+ T cells had undergone almost equivalent proliferation as monitored by CFSE-dilution in MC and TBI allogeneic recipients. However, the kinetics and distribution of donor T cell expansion were distinct. Following delayed DLI, marked expansion of donor CD4+ cells (peak day 10) preceded expansion of CD8+ cells (peak day 13) in the spleen, with less accumulation in the lymph nodes, BM, liver and lung, and no accumulation in the gut. Histology revealed transient, mild lymphocytic infiltrates within the lung/liver but no evidence of colitis. In contrast, the kinetics of donor CD4+/CD8+ T cell expansion were more rapid in freshly irradiated recipients with CD4+/CD8+ responses peaking on day 4–7. The distribution was also different with major increases in donor CD4+/CD8+ numbers in the gut but less accumulation in the spleen. Histology confirmed severe colitis. The kinetics of proliferation, expansion and distribution of tg 2C CD8+ T cells showed a similar pattern to the polyclonal donor CD8+ T cell population. Following delayed DLI, 2C CD8+ T cells acquired a memory/activation phenotype (CD44hi, CD45RBlo, CD62Llo, CD49d+, CD27+) with similar kinetics to those observed in TBI mice developing GVHD. Despite the marked differences in trafficking to the gut, 2C CD8+ T cells in both groups of allogeneic recipients expressed equivalent levels of the gut homing receptor, α4β7. However, we also observed important differences: 1) IL-2Rαexpression was absent on 2C CD8+ T cells following delayed DLI, but was expressed at high levels (>50% by day +3) in freshly irradiated DLI recipients; 2) 2C CD8+ T cells showed greater reductions in expression of IL-7Rαfollowing delayed DLI; and 3) high rates of 2C CD8+ T cell apoptosis, as indicated by annexin V staining, were observed following delayed DLI with absolute numbers up to 6-fold greater than in TBI recipients. Thus, in contrast to responses in freshly irradiated mice that develop GVHD, GVH reactions induced by delayed DLI are characterized by delayed kinetics, a distinct distribution, marked apoptosis and reduced expression of cytokine receptors important for CD8+ T-cell survival.
One potential approach for the effective disengagement of graft-versus-leukemia (GVL) effects from graft-versus-host disease (GVHD) following BMT is the use of nonmyeloablative conditioning as a platform for the adoptive transfer of donor T cells. In pre-clinical models, donor CD8+ T cells can induce powerful responses against tumors of host origin, but the effect lacks durability such that a re-challenge with tumor inevitability leads to tumor progression and death. This deficit is associated with the failure of functional CD8+ effector/memory T cells (TE/M) to survive long-term post-DLI. To examine the fate of GVH-reactive CD8+ T cells following DLI, we established mixed hematopoietic chimeras (MC) in a parent →F1 model using a nonmyeloablative protocol that incorporates co-stimulatory molecule blockade. B6D2F1 mice received 3Gy TBI and intra-peritoneal injections of anti-CD154 and anti-CD8 mAb on day 0 followed by infusion of 2 x 107 C57BL/6 bone marrow cells. 10 weeks later, when mAb had cleared from the circulation, MC received DLI that included CD8+ T cells from 2C transgenic mice that bear TCR specific for recipient class I MHC Ld. Using a clonotypic marker to monitor the response, we observed expansion of 2C CD8+ cells, peaking in the spleen on day 7 and then rapidly declining such that 2C CD8+ T cells were <0.1% of splenocytes by day 60. The decline in GVH-reactive T cells was associated with marked apoptosis and a sustained reduction in the expression of IL-7Rα. By day 60, no CTL activity against host cells was detectable. We reasoned that strategies that augment the survival of GVH TE/M might enhance the durability of the GVL response and, in the absence of tissue inflammation induced by conditioning, might not lead to GVHD. Co-stimulation through the tumor necrosis family receptor, OX40, which is expressed on activated T cells, is anti-apoptotic and enhances recruitment of TE/M to the memory pool. Following DLI, OX40 expression on 2C CD8+ T cells peaked on day 7 with somewhat earlier and sustained expression on DLI-derived CD4+ T cells. Since OX40 expression was specific for GVH-reactive T cells, we examined the effect of giving agonistic anti-OX40 antibody on day +5 following DLI. This was associated with rapid and complete conversion to full donor chimerism by day +14, whereas DLI + control antibody recipients had only partially converted by day +28. By day 60 post-DLI, anti-host CTL activity was clearly detectable in anti-OX40 recipients but not in controls. No clinical evidence of GVHD was observed, although histological examination revealed transient mild lymphocytic infiltration of the lamina propria on day +13, which resolved completely by day +18. In further experiments, anti-OX40 administration was associated with marked increases in the numbers of 2C CD8+ T cells in spleen, lymph node and bone marrow following DLI. Furthermore, effector differentiation, as assayed by intracellular expression of interferon-γ by 2C CD8+ T cells, was increased in recipients of anti-OX40 antibody. Of note, we observed a complete inhibition IL-7Rα down-regulation that is normally observed on activated CD8+ T cells following DLI. We conclude that OX40 co-stimulation following delayed DLI to established MC represents a potential means to enhance the magnitude and duration of a GVH reaction without the induction of significant GVHD.
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