The pentostatin plus cyclophosphamide nonmyeloablative regimen induces durable host T cell functional deficits and prevents murine marrow allograft rejection

Abstract

We describe a novel animal model of nonmyeloablative bone marrow transplantation (BMT) using the purine analog pentostatin. Other cohorts of mice received another purine analog, fludarabine, which we and others have previously evaluated in nonmyeloablative murine models. We evaluated pentostatin for its ability to (1) operate synergistically with cyclophosphamide to induce host T cell depletion; (2) induce host T cell suppression, as defined by modulation of cytokine secretion in vitro and abrogation of host-versus-graft reactivity in vivo; (3) constrain host T cell recovery post-therapy; and (4) prevent the rejection of T cell-depleted, fully major histocompatibility complex-mismatched bone marrow allografts. Relative to single-agent regimens, combination regimens with pentostatin and cyclophosphamide (PC) and with fludarabine and cyclophosphamide (FC) worked synergistically to deplete host CD4(+) and CD8(+) T cells. PC and FC regimens were developed that yielded similar levels of host T cell and myeloid cell depletion. In the setting of these generally comparable states of host T cell and myeloid cell depletion, the PC regimen was found to be highly immunosuppressive, as evidenced by a reduced host T cell capacity to secrete interleukin-2 and interferon-γ in vitro, to mediate host-versus-graft reactivity in vivo, and to recover numerically and functionally during a 2-week observation period after chemotherapy. Finally, using B6 hosts treated with the 14-day chemotherapy regimens, the PC regimen more consistently prevented the rejection of BALB/c T cell-depleted allografts compared with the FC regimen (rate of alloengraftment, 14/15 [93%] of PC-treated recipients vs 8/14 [57%] of FC-treated recipients; P < .05); similar results were observed using an 8-day conditioning regimen. These data suggest that host T cell suppression, distinct from T cell depletion, may be a critical determinant of engraftment after purine analog-based regimens and also may be preferentially attained by the use of pentostatin.

Published by Elsevier Inc.

Figures

Figure 1. Pentostatin and cyclophosphamide synergistically induce host immune depletion and suppression

BALB/c hosts were injected i.p. once daily for three days with either: HBSS (vehicle), pentostatin (P; 1mg/kg/d), fludarabine (F; 100mg/kg/d), cyclophosphamide (C50; 50 mg/kg/d), pentostatin plus cyclophosphamide (P/C50), fludarabine plus cyclophosphamide (F/C50) or treated with lethal total body irradiation (XRT; 950 cGy). (a) Three days post-therapy spleen cells were isolated, counted and analyzed by flow cytometry to determine the levels of depletion of myeloid cells (Gr-1+) and CD4+ and CD8+ T cells. (b) Spleen cells were co-stimulated and the resultant 24-hr supernatant was tested for IFN-γ and IL-2 content. (c) In a separate experiment, BALB/c hosts were lethally irradiated and then injected with syngeneic BALB/c host T cells (0.1 × 106) obtained from mice treated with either HBSS (control), lethal irradiation (XRT; 1050 cGy), combination pentostatin plus cyclophosphamide (P/C50), or combination fludarabine plus cyclophosphamide (F/C50). One day following injection of host T cells, the hosts were transplanted with fully MHC-mismatched T-cell depleted bone marrow from B6 mice (10 × 106 cells). On day 8 post-BMT, spleen cells were isolated and stimulated with syngeneic or allogeneic dendritic cells for 24 hours. The number of host CD4+ and CD8+ cells producing allospecific IFN-γ was determined by cytokine capture flow cytometry. (d) Resultant culture supernatants were also tested for cytokine content (allogeneic stimulation condition is shown). All results are shown as mean ± SEM of n=5–10 per cohort.

Figure 2. Host immune recovery after PC or FC therapy

(a) BALB/c hosts were transplanted with T-cell depleted B6 BM cells (10 × 106 cells) following conditioning with either lethal irradiation (XRT; 1050 cGy), combination pentostatin plus cyclophosphamide (P/C), or combination fludarabine plus cyclophosphamide (F/C). A graft rejection control group received lethal irradiation followed by an infusion of unmanipulated host T cells (0.1 × 106). Spleen cells were isolated on day 8 post-BMT and analyzed by flow cytometry for percent donor cells. (b) In addition, the total number of day 8 post-BMT host CD4+ and CD8+ T cells producing IFN-γ after 24h stimulation with syngeneic (BALB/c) or allogeneic (B6) DC was determined (results shown are with allogeneic stimulation). (c) In a separate experiment, BALB/c mice were injected i.p. once daily for three days with either: HBSS, pentostatin plus cyclophosphamide (P/C50), fludarabine plus cyclophosphamide (F/C50) or treated with lethal total body irradiation (XRT). Subsequently, spleen cells were obtained at days 3, 6 and 10 after therapy and analyzed by flow cytometry for number of CD4+ and CD8+ T-cells. (d) In addition, spleen cells at each time point were co-stimulated and the resultant supernatants tested for IFN-γ content. All results are shown as mean ± SEM of n=5 per cohort for each time.

Figure 3. Intensified PC and FC regimens result in profound immune depletion

(a) Host B6 mice were treated with a 14-day course of either pentostatin (P; 1mg/kg on days 1, 4, 8 and 12) or fludarabine (F; 100mg/kg on days 1, 4, 8. and 12) alone; daily cyclophosphamide alone (C100; 100 mg/kg/day); a combination of intermittent pentostatin or fludarabine with daily cyclophosphamide (P/C100 or F/C100); or treated with lethal total body irradiation (1150 cGy). Spleen cells were isolated and evaluated by flow cytometry for total number of CD4+ and CD8+ T cells and myeloid cells (Gr-1+). (b) Bone marrow cells were also analyzed for T cell composition and myeloid lineage. All results are shown as mean ± SEM of n=5 per cohort.

Figure 4. Intensified PC regimen effectively constrains host immune recovery

(a) Host B6 mice received either pentostatin (1 mg/kg on days 1, 4, 8 and 12) or fludarabine (100 mg/kg on days 1, 4, 8 and 12), with each drug administered in combination with cyclophosphamide (100 mg/kg/d; days 1 through 14). After treatment with these PC or FC regimens, spleen cells were harvested on days 0, 7 and 14 post-regimen and evaluated by flow cytometry for total number of CD4+ and CD8+ T cells and myeloid cells (Gr-1+). (b) Bone marrow cells were also harvested on days 0, 7 and 14 and analyzed for T cell composition and myeloid lineage. (c) On days 0, 7 and 14 post-regimen, spleen cells were stimulated with anti-CD3/CD28 beads and 24-hr supernatant was tested for IFN-γ and IL-2 content. All results are shown as mean ± SEM of n=5 per cohort for each time point.

Figure 5. Alloengraftment after intensified PC and FC regimens (14-day regimen)

(a) Host B6 mice received treatment with the 14-day PC or FC regimens and were subsequently transplanted with T-cell depleted BALB/c bone marrow (20 × 106 cells). The percentage of total donor cells in the blood was evaluated by flow cytometry at 30, 60 and 90 days after transplant. (b) The percentage of donor cells in the spleen and bone marrow was determined on day 90 post-BMT.

Figure 6. Alloengraftment after intensified PC and FC regimens (8-day regimen; increased fludarabine dosing)

Host B6 mice received either pentostatin (1 mg/kg on days 1, 4, and 7) or fludarabine (200 mg/kg on days 1, 4, and 7); each drug was administered in combination with cyclophosphamide (100 mg/kg/d, days 1 through 8; “F200C” regimen, “PC” regimen). After treatment, cohorts of mice were euthanized (n=10 in each cohort); spleen and marrow were harvested and the absolute numbers of post-conditioning CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and Gr-1+ myeloid cells were quantified by flow cytometry. “Host” indicates cell subset numbers in untreated host mice. Absolute cell numbers (mean values ± SEM) in the spleen are shown in (a) whereas absolute cell numbers in the marrow are shown in (b) (NS, not statistically significant). Other cohorts of mice treated with the F200C regimen (n=10) and the PC regimen (n=5) were subsequently transplanted with T-cell depleted BALB/c bone marrow (20 × 106 cells). The percentage of donor/host chimerism was then determined by flow cytometry at day 28 post-transplant for total cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, B220+ B cells, and Gr-1+ myeloid cells in the spleen (c) and in the bone marrow (d). “Host” and “Donor” values represent chimerism control values obtained from untreated host and donor mice; results are mean values ± SEM.