Michael W. Boyer

Ex vivo fludarabine exposure inhibits graft-versus-host activity of allogeneic T cells while preserving graft-versus-leukemia effects

Allogeneic donor T cells in bone marrow transplantation (BMT) can contribute to beneficial graft-versus-leukemia (GVL) effects but can also cause detrimental graft-versus-host disease (GVHD). A successful method for the ex vivo treatment of donor T cells to limit their GVHD potential while retaining GVL activity would have broad clinical applications for patients undergoing allogeneic hematopoietic cell transplantation for malignant diseases. We hypothesized that donor lymphocyte infusions treated with fludarabine, an immunosuppressive nucleoside analog, would have reduced GVHD potential in a fully major histocompatibility complex-mismatched C57BL/6 --> B10.BR mouse BMT model. Recipients of fludarabine-treated donor lymphocyte infusions (F-DLI) had significantly reduced GVHD mortality, reduced histopathologic evidence of GVHD, and lower inflammatory serum cytokine levels than recipients of untreated DLI. Combined comparisons of GVHD incidence and donor-derived hematopoietic chimerism indicated that F-DLI had a therapeutic index superior to that of untreated DLI. Furthermore, adoptive immunotherapy of lymphoblastic lymphoma using F-DLI in the C57BL/6 --> B10.BR model demonstrated a broad therapeutic index with markedly reduced GVHD activity and preservation of GVL activity compared with untreated allogeneic T cells. Fludarabine exposure markedly reduced the CD4+CD44(low)-naive donor T-cell population within 48 hours of transplantation and altered the relative representation of cytokine-producing CD4+ T cells, consistent with T-helper type 2 polarization. However, proliferation of fludarabine-treated T cells in allogeneic recipient spleens was equivalent to that of untreated T cells. The results suggest that fludarabine reduces the GVHD potential of donor lymphocytes through effects on a CD4+CD44(low) T-cell population, with less effect on alloreactive T cells and CD4+CD44(high) memory T cells that are able to mediate GVL effects. Thus, F-DLI represents a novel method of immune modulation that may be useful to enhance immune reconstitution among allograft recipients with reduced risk of GVHD while retaining beneficial GVL effects.

Low risk of graft-versus-host disease with transplantation of CD34 selected peripheral blood progenitor cells from alternative donors for Fanconi anemia.

Objectives Transplant results for Fanconi anemia with alternative-donor bone marrow transplantation currently entail a high incidence of graft failure and graft-versus-host disease (GVHD). The authors sought to improve outcome in this disease category with alternative donors with a 5-6/6 antigen match by transplantation of highly purified peripheral blood progenitor cells (PBPC) using the Isolex 300i v2.5 device as a means of T-cell depletion to lessen the risk of GVHD. Methods All Fanconi anemia patients (n = 8) received the same preparative regimen that included total body irradiation (450 cGy), Cytoxan (20 mg/kg), ATGAM, and fludarabine (120 mg/m2). The cell dose of CD34+ cells was a median of 11.4 × 106/kg; the cell dose of CD3+ cells was a median of 1.9 × 104/kg. Primary engraftment was rapid in all patients, with neutrophil recovery occurring at a median of day 10 and platelet count more than 50,000 on day 27. Two patients subsequently had secondary graft failure. Despite lack of cyclosporine GVHD prophylaxis, only two patients developed acute GVHD (both grade I), and no patients developed chronic GVHD. Three patients died: one at day 59 secondary to disseminated fungal infection, the second at day 196 during a second transplant, and the third at day 202 due to graft failure. With a median follow-up of 12 months, the overall survival was 58 ± 18%. Conclusions Transplantation of CD34-selected PBPCs from alternative donors results in a very low risk of GVHD in patients with Fanconi anemia.

Transplantation of Unrelated Placental/Umbilical Cord Blood Cells for Leukocyte Adhesion Deficiency

Transplantation of Unrelated Placental/Umbilical Cord Blood Cells for Leukocyte Adhesion Deficiency

26 In vitro Cultured Allogeneic Cytotoxic T Cells Mediate GVL Without Systemic GVHD Despite Expression of Functional LPAM.

Background: Previous studies have shown that in vitro cultured alloreactive T cells had comparable GVL activity but decreased GVHD as compared to naïve cells. It has been shown that expression of LPAM on CD8 T cells is important for gut homing specificity in GVHD. Our preliminary data showed that the expression of LPAM is decreased upon in vitro culture of CTLs. Recently, retinoic acid (RA) has been shown to up regulate LPAM expression on naive T cells. We hypothesize that in vitro cultured CTLs without retinoic acid lack the ability to cause GVHD in part due to deficient LPAM expression.Methods: B6PL spleen/lymph node cells were stimulated against DBA splenocytes with IL-2 & IL-7 ± 100 nm RA. Day 14 CTL & CTLRA were infused into allogeneic recipients &compared to naïve T cells.Results: CTL & CTLRA showed CD8 LPAM expression of 0.7% & 61% (p<.01). Both groups had comparable in vitro migration towards to SDF, but CTLRA had ↑ migration towards TECK; 17.3% vs. 4.6% (p<.01). Homing analysis revealed ↑ migration of CTLRA to PP & MLN [Homing index (CTLRA/CTL) 2.3 & 2.5 respectively]. 600 cGy irradiated B6D2F1 mice were given 0.5x106 P815 murine mastocytoma cells on day 0 followed by B6 BM cells with either 5x106 CTLs or 10x106 naïve lymphocytes on day 1. CTLs and naïve lymphocytes mediated a potent GVL effect in spleen & BM. Recipients of naïve lymphocytes developed lethal GVHD with high clinical GVHD scores (median survival 17 days; =14) where as, CTL group had significantly improved survival due to attenuated GVHD (median 60 days; =10, p<0.001). In pure GVHD experiments, CTL, CTLRA & naive lymphocytes induced lethal BM aplasia (n= 10, mean Hb 3g% in all) in the absence of BM rescue at d12 as compared to rad controls (n= 8, mean Hb 7.5g%) without systemic GVHD in any group. With BM rescue, naïve cells resulted in lethal systemic GVHD at d20 (=8; Serum ALT 188 IU/L, clinical & histological GVHD scores of 8 & 11.5 respectively, p<.01). Where as recipients of CTL or CTLRA were alive >d150 (=8; ALT 60 IU/L, clinical GVHD score of 2) despite ↑ migration of CTLRA cells to gut in contrast to CTL without RA (p<.01).Conclusions: We demonstrate that in vitro cultured alloreactive CTLs mediate a rapid GVL effect that was equivalent to the infusion of naïve T cells but with attenuated sub clinical GVHD. We also showed that in vitro cultured CTLs have defective gut homing which could be corrected by up regulating LPAM expression with the addition of RA. Thus defective gut homing does not explain the lack of lethal GVHD seen with in vitro cultured CTL.