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

Fludarabine treatment attenuates the GVHD potential of allogeneic donor lymphocytes while maintaining graft-facilitating effects. B10.BR recipients underwent transplantation with C57BL/6 TCD-BM plus 0, 1, 3, 8, or 10 viable control DLI (A) or 1, 3, or 10 × 10⁶ viable F-DLI (B). Increasing thickness of lines indicates increasing numbers of DLI or F-DLI administered. Numbers within the graph indicate the number of viable T cells transplanted over the total number of splenocytes transplanted. There were 20 mice per group, except groups receiving no DLI (n = 45), 8 × 10⁶ control DLI (n = 10), and 10 × 10⁶ F-DLI (n = 35). (C) Day 60 survival × weight scores for recipients of TCD-BM plus untreated DLI or F-DLI. The number of viable T cells transplanted is shown on the x axis. (D) Peripheral blood analysis for donor- and recipient-derived T-cell chimerism 60 days after transplantation. Error bars represent the SEM. □, Recipient T cells; ▨, donor spleen T cells; ■, donor BM T cells. (E) Survival of B10.BR recipients transplanted with C57BL/6 TCD-BM plus 3.5 × 10⁶ B cell—depleted splenocytes containing 2.2 × 10⁶ viable T cells that were fludarabine cultured (2.2 Flud; upper solid line), cultured in media alone (2.2 Untr; short dashes), or cultured in media and then added to 10 × 10⁶ C57BL/6-irradiated (25 Gy) splenocytes (2.2 Untr + Irrard; irregular dashes). There were 10 mice per experimental group, and 5 mice received TCD-BM only (0; regular dashes).

Histologic and serum cytokine parameters of GVHD progression are reduced among recipients of F-DLI. (A and B) Sections of liver from B10.BR recipients of 10 × 10⁶ untreated DLI or 10 × 10⁶ F-DLI 16 days after transplantation. (A) Severe lymphocyte infiltration in the liver periportal regions in the recipient of untreated DLI. (C and D) Transverse sections of intestinal crypt glands from recipients of untreated DLI or F-DLI. (C) Granulation indicative of apoptotic cell death (arrows) in the recipient of untreated DLI. Original magnification, 400× for all images. (E) Day 16 histologic GVHD grade of liver and colon samples from mice that received TCD-BM alone (n = 5) or combined with 3 × 10⁶ untreated DLI (n = 4), 10 × 10⁶ untreated DLI (n = 8), or 10 × 10⁶ F-DLI (n = 3). (F) Day 16 serum levels of IFN-γ for the same recipients shown in (E). Error bars represent the SEM.

F-DLI retain GVL activity. Survival of B10.BR mice transplanted with C57BL/6 TCD-BM and untreated (open symbols) or fludarabine-treated (filled symbols) splenocytes from C57BL/6 donors. Groups of 10 mice received 11 Gy of radiation on day −2 and TCD-BM on day 0, and then they received single infusions of 0, 1, 3, or 10 × 10⁶ donor splenocytes on day 0 or 5 weekly infusions of 10 × 10⁶ donor splenocytes. For groups receiving LBRM, 3 × 10⁶ cells were administered on day −1. ○, untreated DLI; •, FDLI; □, untreated DLI + LBRM; ■, F-DLI + LBRM. Overlapping data points are offset for clarity. Note that symbols on the y axis (x = 0) represent groups that received TCD-BM without added splenocytes.

Effect of fludarabine treatment on alloreactivity of T cells in vitro. (A) LDA of frequency of alloreactive T cells in C57BL/6 → B10.BR 1-way MLR. Open circle indicates untreated DLI; filled square, F-DLI. The number of viable T cells plated per well is shown. LDA was performed using C57BL/6 responder splenocytes stimulated with irradiated B10.BR splenocytes. The frequencies of alloreactive cells in untreated and fludarabine-treated cultures are shown. The data are representative of 3 individual experiments. See Materials and Methods for details. (B) T cells from engrafted recipients are tolerant to both recipient- and donor-type alloantigens but retain reactivity to third-party—type antigen and mitogen. Proliferative responses are shown for spleen cells harvested from B10.BR mice 8 months after transplantation with C57BL/6 TCD-BM only or with C57BL/6 TCD-BM plus fludarabine-treated splenocytes. The proliferation of freshly isolated B10.BR splenocytes was analyzed for comparison. Proliferation was measured after stimulation with irradiated splenocytes from C57BL/6 (H-2^b) donor-type, B10.BR (H-2^k) recipient type or DBA/2 (H-2^d) third-party mice or concanavalin A. A total of 400,000 responding cells were cultured with 200,000 irradiated stimulators in a 7-day MLR. Error bars represent SD of 8 replicate wells.

F-DLI T cells proliferate in MHC-mismatched recipient spleen. (A) CFSE proliferation profiles of untreated or fludarabine-treated wild-type C57BL/6 H-2^b and 2CTCR transgenic donor T cells recovered from spleens of BALB/c scid/scid H-2^d recipients 40 and 64 hours after transplantation. Top 2 rows show wild-type (WT) CD4⁺ T cells. Middle 2 rows show WT CD8⁺ T cells. Bottom 2 rows show 2CTCR transgenic CD8⁺ T cells. (B) CFSE proliferation profiles and CD69 expression of CD4⁺C57BL6 T cells in unirradiated F1 (C57BL/6 × B10.BR) recipients at 24, 48, and 72 hours after transplantation. (Top) Untreated donor-derived CD4⁺ T cells. (Bottom) Fludarabine-treated donor-derived CD4⁺ T cells. (C) Expansion of untreated (open symbols) and fludarabine-treated (filled symbols) C57BL/6 donor T cells in B10.BR recipient spleens. (D) Expansion of untreated and fludarabine-treated C57BL/6 donor B cells in B10.BR recipient spleens. Total splenocyte counts were multiplied by the fraction of donor splenocyte-derived CD4⁺, CD8⁺, and CD19⁺ cells as determined by flow cytometric analyses. Three to 8 recipients per group were analyzed at each time point. Error bars represent the SEM.

Fludarabine treatment reduced the CD4⁺CD44^low donor T-cell population after transplantation or additional culture time and altered the cytokine production profile of donor CD4 cells. (A) CD25, CD44, CD62L, and CD122 expression profiles are shown for C57BL/6 donor CD4 (top) and CD8 (bottom) T cells recovered from B10.BR recipient spleen 40 hours after transplantation. Histograms representing untreated donor T cells are shown by a thin line and gray fill, and histograms representing fludarabine-treated donor T cells are shown by a thick line without fill. Results are representative of 2 individual experiments. U- and F- indicate the percentage of events in the right-hand gate for recipients of untreated and fludarabine-treated T-cells, respectively. Gates were set to discriminate CD4 or CD8 T cells with the highest level of expression for the various activation markers on the basis of the separation of these populations among untreated donor-derived T cells. (B) The pattern of CD44 expression for CD4⁺ T cells cultured 24 hours with fludarabine (heavy black line) or media alone (gray filled area) and then washed and cultured in fresh media without fludarabine for 40 or 64 hours. Histograms represent CD44 expression for the population of CD4 T cells surviving at the time of sampling, demonstrating the differential survival of CD44^low and CD44^high cells. U- and F- indicate the percentage of viable CD4⁺CD44⁺ cells among untreated and fludarabine-treated CD4⁺ cells, respectively. (C) Cytokine production and CD44/CD25 profiles of CD4 T cells in PMA/ionomycin-stimulated cultures of untreated or fludarabine-treated splenocytes. Results are representative of those obtained with splenocytes from 5 individual C57BL/6 mice.