Abstract 3539Poster Board III-476Pentostatin has been utilized clinically in combination with irradiation for host conditioning prior to reduced-intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, murine models utilizing pentostatin to facilitate engraftment across fully MHC-disparate barriers have not been developed. To address this deficit in murine modeling, we first compared the immunosuppressive and immunodepleting effects of pentostatin (P) plus cyclophosphamide (C) to a regimen of fludarabine (F) plus (C) that we previously described. Cohorts of mice (n=5-10) received a three-day regimen consisting of P alone (1 mg/kg/d), F alone (100 mg/kg/d), C alone (50 mg/kg/d), or combination PC or FC. Combination PC or FC were each more effective at depleting and suppressing splenic T cells than either agent alone (depletion was quantified by flow cytometry; suppression was quantified by cytokine secretion after co-stimulation). The PC and FC regimens were similar in terms of yielding only modest myeloid suppression. However, the PC regimen was more potent in terms of depleting host CD4+ T cells (p<0.01) and CD8+ T cells (p<0.01), and suppressing their function (cytokine values are pg/ml/0.5×106 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-g (13±5 vs. 48±12), IL-2 (59±44 vs. 258±32), IL-4 (34±10 vs. 104±12), and IL-10 (15±3 vs. 34±5). Next, we evaluated whether T cells harvested from PC-treated and FC-treated hosts were also differentially immune suppressed in terms of capacity to mediate an alloreactive host-versus-graft rejection response (HVGR) in vivo when transferred to a secondary host. BALB/c hosts were lethally irradiated (1050 cGy; day -2), reconstituted with host-type T cells from PC- or FC-treated recipients (day -1; 0.1 × 106 T cells transferred), and challenged with fully allogeneic transplant (B6 donor bone marrow, 10 × 106 cells; day 0). In vivo HVGR was quantified on day 7 post-BMT by cytokine capture flow cytometry: absolute number of host CD4+ T cells secreting IFN-g in an allospecific manner was ([x 106/spleen]) 0.02 ± 0.008 in recipients of PC-treated T cells and 1.55 ± 0.39 in recipients of FC-treated cells (p<0.001). Similar results were obtained for allospecific host CD8+ T cells (p<0.001). Our second objective was to characterize the host immune barrier for engraftment after PC treatment. BALB/c mice were treated for 3 days with PC and transplanted with TCD B6 bone marrow. Surprisingly, such PC-treated recipients developed alloreactive T cells in vivo and ultimately rejected the graft. Because the PC-treated hosts were heavily immune depleted at the time of transplantation, we reasoned that failure to engraft might be due to host immune T cell reconstitution after PC therapy. In an experiment performed to characterize the duration of PC-induced immune depletion and suppression, we found that although immune depletion was prolonged, immune suppression was relatively transient. To develop a more immune suppressive regimen, we extended the C therapy to 14 days (50 mg/Kg) and provided a longer interval of pentostatin therapy (administered on days 1, 4, 8, and 12). This 14-day PC regimen yielded CD4+ and CD8+ T cell depletion similar to recipients of a lethal dose of TBI, more durable immune depletion, but again failed to achieve durable immune suppression, therefore resulting in HVGR and ultimate graft rejection. Finally, through intensification of C therapy (to 100 mg/Kg for 14 days), we were identified a PC regimen that was both highly immune depleting and achieved prolonged immune suppression, as defined by host inability to recover T cell IFN-g secretion for a full 14-day period after completion of PC therapy.Finally, our third objective was to determine with this optimized PC regimen might permit the engraftment of MHC disparate, TCD murine allografts. Indeed, using a BALB/c-into-B6 model, we found that mixed chimerism was achieved by day 30 and remained relatively stable through day 90 post-transplant (percent donor chimerism at days 30, 60, and 90 post-transplant were 28 ± 8, 23 ± 9, and 21 ± 7 percent, respectively). At day 90, mixed chimerism in myeloid, T, and B cell subsets was observed in the blood, spleen, and bone marrow compartments. Pentostatin therefore synergizes with cyclophosphamide to deplete, suppress, and limit immune reconstitution of host T cells, thereby allowing engraftment of T cell-depleted allografts across MHC barriers. Disclosures:No relevant conflicts of interest to declare.
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Apr 1, 2024
Rong Li + 12
Open Access
