Abstract
Although fludarabine and pentostatin are variably utilized for conditioning prior to clinical allogeneic transplantation, limited data exists with respect to their relative efficacy in terms of host immune T cell depletion and T cell suppression. To directly compare these agents in vivo in a murine model, we compared a regimen of fludarabine plus cyclophosphamide (FC) similar to one that we previously developed (Petrus et al, BBMT, 2000) to a new regimen of pentostatin plus cyclophosphamide (PC). 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. Similar to our previous data, administration of P, F, or C alone yielded minimal host T cell depletion (as measured by enumeration of splenic CD4+ and CD8+ T cells) and minimal T cell suppression (as determined by CD3, CD28 co-stimulation of a constant number of remaining splenic T cells and measuring resultant cytokine secretion by multi-analyte assay). The PC and FC regimens were similar in terms of myeloid suppression (p=.2). However, the PC regimen was more potent in terms of depleting host CD4+ T cells (remaining host CD4 number [× 10^6/spleen], 2.1±0.3 [PC] vs. 4.4±0.6 [FC], p<0.01) and CD8+ T cells (remaining host CD8 number, 1.7±0.2 [PC] vs. 2.4±0.5 [FC], p<0.01). Moreover, the PC regimen yielded greater T cell immune suppression than the FC regimen (cytokine values are pg/ml/0.5×10^6 cells/ml; all comparisons p<0.05) with respect to capacity to secrete IFN-γ (13±5 [PC] vs. 48±12 [FC]), IL-2 (59±44 [PC] vs. 258±32 [FC]), IL-4 (34±10 [PC] vs. 104±12 [FC]), and IL-10 (15±3 [PC] vs. 34±5 [FC]). In light of this differential in both immune T cell depletion and suppression of T cell effector function, we hypothesized that T cells from PC-treated recipients would have reduced capacity to mediate a host-versus-graft rejection response (HVGR) relative to FC-treated recipients. To directly test this hypothesis, we utilized a host T cell add-back model of rejection whereby 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 × 10^6 T cells transferred), and finally challenged with fully MHC-disparate transplantation (B6 donor bone marrow cells, 10 × 10^6 cells; day 0). In vivo HVGR was quantified by the following method at day 7 post-BMT: harvest of splenic T cells, stimulation with host- or donor-type dendritic cells, and use of six-color flow cytometry to detect host T cells, CD4 and CD8 subsets, and cytokine secretion by capture method. Consistent with our hypothesis, PC-treated cells acquired greatly reduced alloreactivity in vivo relative to FC-treated cells: the percentage of host CD4+ T cells secreting IFN-γ in an allospecific manner was 2.3±0.8% in recipients of PC-treated T cells and 62.7±13.4% in recipients of FC-treated cells (p<0.001). Similarly, the percentage of host CD8+ T cells secreting IFN-γ in an allospecific manner was 8.6±2.8% in recipients of PC-treated T cells and 92.7±4.1% in recipients of FC-treated T cells (p<0.001). We therefore conclude that at similar levels of myeloid suppression, the PC regimen is superior to the FC regimen in terms of murine T cell depletion, suppression of global T cell cytokine secretion, and inhibition of in vivo capacity to acquire allospecificity in response to fully genetically disparate marrow allografts. These data provide a rationale to develop PC regimens as an alternative to currently utilized FC regimens.
Rescue of Notch-1 Signaling in Antigen-Specific CD8+ T Cells Overcomes Tumor-Induced T-cell Suppression and Enhances Immunotherapy in Cancer