Biology of Blood and Marrow Transplantation
Volume 9, Issue 10 , Pages 616-632, October 2003

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

  • Cynthia R Giver

      Affiliations

    • Winship Cancer Institute, Hematology/Oncology Department, Emory University, Atlanta, Georgia, USA
    • ,
  • Richard O Montes

      Affiliations

    • Winship Cancer Institute, Hematology/Oncology Department, Emory University, Atlanta, Georgia, USA
    • ,
  • Stephen Mittelstaedt

      Affiliations

    • Winship Cancer Institute, Hematology/Oncology Department, Emory University, Atlanta, Georgia, USA
    • ,
  • Jian-Ming Li

      Affiliations

    • Winship Cancer Institute, Hematology/Oncology Department, Emory University, Atlanta, Georgia, USA
    • ,
  • David L Jaye

      Affiliations

    • Department of Pathology, Emory University, Atlanta, Georgia, USA
    • ,
  • Sagar Lonial

      Affiliations

    • Winship Cancer Institute, Hematology/Oncology Department, Emory University, Atlanta, Georgia, USA
    • ,
  • Michael W Boyer

      Affiliations

    • Columbus Children’s Hospital, Columbus, Ohio, USA
    • ,
  • Edmund K Waller

      Affiliations

    • Corresponding Author InformationCorrespondence and reprint requests: Edmund K. Waller, MD, PhD, Emory University, Department of Hematology/Oncology, 1639 Pierce Dr., Ste. 1003, Atlanta, GA 30322, USA
    • Winship Cancer Institute, Hematology/Oncology Department, Emory University, Atlanta, Georgia, USA

Received 22 October 2002; accepted 26 June 2003.

Article Outline

Abstract 

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+CD44low-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+CD44low T-cell population, with less effect on alloreactive T cells and CD4+CD44high 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.

Keywords:  Graft-versus-host disease, Naive T cells, Allogeneic transplantation, Fludarabine

 

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Introduction 

Graft-versus-host disease (GVHD) initiated by donor T cells is a major complication of allogeneic bone marrow transplantation (BMT). Because allogeneic T cells also provide beneficial graft-versus-leukemia effects (GVL) and antimicrobial immunity, there is great interest in developing a method for separating these 2 linked activities. The ideal graft would include sufficient donor hematopoietic progenitor cells for rapid engraftment and donor T cells capable of contributing to posttransplantation immune reconstitution and GVL effects without causing GVHD. We have previously demonstrated that irradiated donor T cells have the ability to facilitate engraftment of allogeneic bone marrow (BM) cells without causing GVHD in the recipients [1]. This approach has been applied in clinical trials, with promising results [2], but it may have limited clinical application because infusions of large numbers of irradiated splenocytes (SP) result in persistent mixed chimerism in a murine model and do not contribute to long-term immune reconstitution [1]. Recipients of allogeneic transplants with persistent mixed (host + donor) chimerism in the T-cell compartment have less of the GVL effect than recipients that achieve full donor chimerism [3].

To achieve more consistent donor chimerism and better survival among recipients of allogeneic T cells, we have evaluated the use of an immunosuppressive agent, fludarabine, as an alternative method of treating T cells to limit their potential for GVHD while preserving GVL activity. Fludarabine (9-β-d-arabinosyl-2-fluoroadenine) is an adenine nucleoside analog that inhibits DNA synthesis when incorporated into a replicating chain 4, 5. Fludarabine also inhibits the proliferative responses of human CD4+ and CD8+ T cells to mitogen and alloantigen [6] and has potent immunosuppressive effects, including profound depletion of CD4+ T cells [7] and induction of apoptosis in resting lymphocytes [8]. Fludarabine is known to inhibit STAT1 signaling in resting T cells, leading to decreased proliferation in response to cytokine activation [9]. We hypothesized that fludarabine pretreatment of donor T cells would render them susceptible to activation-induced cell death, thus selectively eliminating antigen-specific T cells responding to alloantigen in the host. This would potentially spare donor T cells that could contribute to reconstitution of the naive T-cell repertoire.

Results of major histocompatibility complex (MHC)-mismatched allogeneic murine transplant experiments demonstrate that adoptive immunotherapy with fludarabine-treated donor lymphocytes indeed resulted in significantly reduced GVHD morbidity and mortality compared with untreated donor lymphocytes while preserving graft-facilitating effects. It is important to note that GVL activity against a transplanted T-cell lymphoma cell line was retained. However, fludarabine pre-exposure did not reduce the alloreactivity of T cells in both in vitro and short-term in vivo transfer experiments. Contrary to our original hypothesis, the major immunomodulatory effect of fludarabine treatment on donor lymphocyte infusions (DLI) seemed to involve selective reduction of a donor CD4+CD44low-naive T-cell population and alteration of the T-cell cytokine secretion profile consistent with T-helper type 2 (Th-2) polarization. These effects did not change the proliferative response of fludarabine-treated donor T cells in allogeneic recipient spleens, suggesting that donor CD4+CD44low cells play an important role in GVHD progression, independent of donor T-cell expansion. Thus, fludarabine-treated lymphocytes may represent a novel method for reducing the GVHD risk of DLI in allogeneic BMT while preserving therapeutic GVL effects.

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Materials and methods 

Animals 

Male B10.BR mice (H-2k, CD45.2, and Thy1.2) and a congenic strain of C57BL/6 mice (H-2b, CD45.1, and Thy1.2), aged 8 to 10 weeks, were purchased from Jackson Laboratories (Bar Harbor, ME). A Thy1.1 congenic strain of C57BL/6 (H-2b, CD45.2, and Thy1.1) was obtained from Miriam Lieberman (Stanford University, CA), and a third congenic strain of C57BL/6 (H-2b, CD45.1, and Thy1.1) was bred and maintained at Emory University. Some experiments used DBA/2 (H-2d), BALB/c scid/scid (H-2d), and 2CTCR transgenic mice (C57BL/6 background) with T cells allospecific against H-2d[10], purchased from Jackson Laboratories. Mice were housed in microisolator cages in the Emory University Animal Care Facility, with acidified water and standard chow available at all times. Animal handling and experimental procedures were in concordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996) and approved by the Emory University Institutional Animal Care and Use Committee.

T cell—depleted BM 

Femora and tibiae were removed from C57BL/6 (H-2b, CD45.1, and Thy1.1) mice, and BM cells were expelled by flushing sterile phosphate-buffered saline (PBS) containing 1% heat-inactivated fetal bovine serum (FBS) through the shaft. CD3+ T cells were depleted by using immunomagnetic separation techniques (MACS, Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany). T cell—depleted (TCD) BM was analyzed by flow cytometry and routinely contained <0.5% residual T cells.

Fludarabine-treated SP 

Spleens from C57BL/6 (H-2b, CD45.1, and Thy1.2 or H-2b, CD45.2, and Thy1.1) mice were perfused with sterile PBS/FBS. Spleen contents were gently removed from the capsule, washed, and strained to obtain a single-cell suspension. SP (10 × 106/mL) were then incubated in complete media (RPMI supplemented with 10% heat-inactivated FBS, 50 μM 2-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 292 μg/mL l-glutamine, 1 mM sodium pyruvate, and nonessential amino acids [Biowhittaker, Walkersville, MD]). Fludarabine (Berlex Laboratories, Richmond, CA) was added to a final concentration of 20 μg/mL; control SP were cultured without fludarabine. Cultures were maintained at 37°C in a 5% CO2 incubator for 24 hours. Overall viability was assessed by counting ethidium bromide/acridine orange—stained cells under fluorescence microscopy. The representation of T- and B-cell populations among surviving SP was determined by flow cytometric analyses by using fluorochrome-conjugated antibodies against mouse CD3, CD4, CD8, and CD19. Propidium iodide staining was used to exclude dead cells from the analysis. In all subsequent transplant and in vitro experiments, the number of fludarabine-treated or -untreated SP used was based on the viable cell count. SP were washed and resuspended to prepare DLI and fludarabine-treated DLI (F-DLI).

Conditioning and transplantation 

B10.BR recipient mice were irradiated (2 × 5.5 Gy, 3-hour interval) 1 day before transplantation by using a cesium 137 source at a rate of 1.24 Gy/min. Irradiated mice were given drinking water containing antibiotics (1.1 mg/mL neomycin sulfate and 1000 U/mL polymixin sulfate) for 4 weeks after BMT. Donor cell infusions were prepared in 0.2 mL of Hanks balanced salt solution/1% FBS per recipient. For GVHD and graft facilitation experiments, 1 × 106 TCD-BM cells were administered alone or mixed with DLI or F-DLI. The number of DLI or F-DLI transplanted varied for dose-escalation studies. Some experiments also used mixtures of TCD-BM, B cell—depleted (BCD) SP (MACS [immunomagnetic separation techniques] depletion of CD19+ cells resulted in approximately 3% residual B cells), and irradiated (7.5 Gy) SP. All transplants were administered via tail vein injection.

Assessment of GVHD in transplant recipients 

Recipient mice were monitored daily for survival; moribund animals were killed and their deaths recorded. Animals were weighed twice a week for the first month after transplantation and then weekly until termination of survival experiments. For GVHD assessment experiments, groups of 3 to 8 mice were killed at predetermined time points for analysis of serum cytokine levels, donor cell proliferation in recipient spleen (see below), and histologic examination of liver and colon sections. Tissues were removed, fixed in 10% buffered formalin, and embedded in paraffin, and sections were stained with hematoxylin and eosin for histologic examination. Slides were coded and scored for evidence of GVHD (grade 0–4) without knowledge of treatment conditions by a trained pathologist (D.L.J.) by using previously reported criteria 1, 11. Histologic evidence of GVHD included infiltration of lymphocytes in the periportal regions of the liver and apoptotic cells in the intestinal epithelium of the colon, particularly in the crypts, along with foci of epithelial ulceration.

Measurement of serum cytokine levels/enzyme-linked immunosorbent assay 

Recipient mice were anesthetized by isoflurane inhalation, and peripheral blood was collected by retro-orbital bleeding into Microtainer serum separation tubes (Beckton Dickinson, Franklin Lakes, NJ). Mice were killed immediately after bleeding. Serum was obtained and stored frozen at −80°C until use. Tumor necrosis factor (TNF)-α and interferon (IFN)-γ levels were assayed by using OptEIA enzyme-linked immunosorbent assay sets (Pharmingen, San Diego, CA), and reaction dishes were analyzed with a SpectraMax 340PC spectrophotometer (Molecular Devices, Sunnyvale, CA). All samples were assayed in duplicate wells.

Analysis of hematopoietic chimerism 

Peripheral blood (0.2 mL) was collected by tail vein nick at 1 to 4 months after BMT. Red blood cells were lysed by brief treatment with an ammonium chloride solution. Leukocytes were then washed and resuspended in Hanks balanced salt solution containing 2 mM EDTA and 3% FBS. Donor and host-derived leukocytes and T cells were distinguished by flow cytometric analysis by using fluorochrome-conjugated monoclonal antibodies specific for H-2b and H-2k MHC, as well as specific leukocyte markers (CD3, CD4, CD8, Thy1.1, Thy1.2, CD45.1, and CD45.2; Pharmingen). Similar procedures were used to determine the donor-derived T- and B-cell content of recipient spleens.

Leukemic cell line and GVL transplant experiments 

A radiation-induced splenic lymphoma cell line from the B10.BR mouse, LBRM [12] was obtained from the American Type Culture Collection (Rockville, MD) and used here as a transplantable leukemic tumor in GVL experiments. B10.BR recipient mice were irradiated on day −2, administered 3 × 106 (>10 times the median lethal dose [LD50]) LBRM tumor cells via tail vein injection on day −1, and transplanted with allogeneic TCD-BM and DLI on day 0. Depending on the experimental protocol, additional infusions of DLI were administered on a weekly basis as indicated.

Proliferative response to alloantigen (mixed lymphocyte reaction) and mitogen 

A total of 4 × 106 cells per milliliter of responder cells (SP obtained from C57BL/6 → B10.BR transplant recipients or fresh B10.BR SP) were cocultured with 2 × 106 irradiated (25 Gy) stimulator cells per milliliter (C57.BL6, B10.BR, or DBA/2) or 10 μg/mL concanavalin A in complete media with 10 U/mL interleukin (IL)-2 (Chiron Corporation, Emeryville, CA) and 10 ng/mL IL-7 (PeproTech Inc., Rocky Hill, NJ) in triplicate wells of a 96-well plate. Proliferation was monitored by measuring DNA thymidine incorporation. Cultures were pulsed with 1 μCi per well of tritiated thymidine (NEN Life Sciences, Boston, MA) on day 6, harvested onto filtermats by using a Tomtec Harvester (Hamden, CT) on day 7, and read on a MicroBeta TriLux counter (PerkinElmer, Wallac Inc., Gaithersburg, MD).

Limiting dilution analysis 

The frequency of alloreactive cells was measured by using a limiting dilution assay [13]. Briefly, 20,000 C57BL/6 responder cells (untreated or fludarabine-treated SP) were 3-fold serially diluted in 96-well plates with 12 replicate wells per dilution. Then 10,000 irradiated B10.BR stimulator cells were added to each well in a combined total volume of 200 μL of complete media. After 7 days, the proliferation of responder cells was measured by using tritiated thymidine incorporation. The number of responder cells was plotted versus the natural logarithm of the percentage of negative wells (ie, wells with counts per minute readings less than media control [average + 3 SD]). The reciprocal of the slope of the regression line yielded the frequency of alloreactive T cells.

5-[and-6]-carboxyfluorescein diacetate, succinimidyl esther in vivo proliferation analysis 

5-[and-6]-Carboxyfluorescein diacetate, succinimidyl esther (CFSE; Molecular Probes, Eugene, OR) staining was performed by using a slight modification of methods outlined by Lyons [14]. After 24-hour culture in standard or fludarabine-containing media, donor SP (C57BL/6 alone or combined with transgenic 2CTCR T cells [10]) were washed and resuspended at 5 × 107/mL in PBS containing 0.5% FCS. CFSE was added to a final concentration of 3 μM, and cells were incubated 10 minutes at 37°C and then washed twice. Then 3 × 107 CFSE-labeled cells were administered to irradiated (3 Gy) BALB/c (H-2d) scid/scid mice, unirradiated F1(C57BL/6 × B10.BR), or irradiated (11 Gy) B10.BR recipient mice via tail vein injection. Recipient mice were killed 1 to 4 days after transplantation, and SP harvested for flow cytometric analysis of donor cell proliferation. R-Phycoerythrin (PE), PerCP, or allophycocyanin (APC) conjugated antibodies against T-cell surface markers were used for simultaneous analysis of T-cell subsets.

Intracellular cytokine analysis 

SP were removed from 24-hour culture with or without fludarabine, washed, and placed in fresh media for 16 hours. Phorbol 12-myristate 13-acetate (PMA)/ionomycin and a protein transport inhibitor (GolgiStop; Pharmingen) were then added for an additional 5 hours by using conditions specified by the supplier. Cells were assayed for simultaneous expression of T-cell phenotypic markers (CD4 or CD8, CD44, and CD25) and intracellular cytokine production (IL-2, IL-10, IFN-γ, or TNF-α) by using appropriate fluorochrome-conjugated monoclonal antibodies, isotype controls, and Cytofix/Cytoperm reagents (Pharmingen). Labeled cells were analyzed by flow cytometry.

T-cell purification 

Splenic T cells were purified with an immunodepletion protocol similar to that specified for the Miltenyi Pan T-Cell Isolation Kit to remove cells expressing B220, Ter-119, DX5, and CD11b. T-cell purity was determined with flow cytometric analysis for CD3+ cells and was routinely shown to be >97%.

Statistical analysis 

Kaplan-Meier analysis was used to analyze the survival of the animals. The differences in survival among different treatment groups were tested with the Wilcoxon log-rank test [15]. The Student t test was used to compare in vitro assay results obtained with fludarabine-treated versus -untreated cells and to determine the significance of blinded histologic scoring results from recipients of fludarabine-treated versus -untreated cells.

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Results 

T cells are relatively resistant to killing by fludarabine compared with B cells 

Experiments were conducted to determine the yield of viable SP after 24 hours of culture in media containing graded doses of fludarabine (0, 3, 10, 20, or 40 μg/mL) and to characterize the GVHD potential of treated SP in the C57BL/6 → B10.BR transplant model. Increasing loss of viability among cultured cells was dose dependent up to 10 μg/mL fludarabine, and a plateau was seen at higher fludarabine doses (data not shown). Cultures treated with 20 μg/mL fludarabine for 24 hours had approximately 42% overall cell viability, whereas control cultures had approximately 75% viability compared with the starting cell population (Table 1). The viability of B cells after fludarabine treatment was significantly reduced compared with control cultures. T-cell viability was also lower after fludarabine exposure versus control for both the CD4 and CD8 populations, but these differences did not reach statistical significance (Table 1). Allogeneic transplant experiments demonstrated that mice receiving TCD-BM plus SP treated with 20 μg/mL fludarabine had the most favorable 40-day survival rate compared with groups receiving control SP or SP treated with other doses of fludarabine (data not shown). Hence, this dose was selected for further studies. Note that for all subsequent transplant and in vitro experiments, the number of fludarabine-treated or -untreated SP used was based on the number of viable cells in the culture after the incubation period.

Table 1. Cell Viability after 24 Hours of Culture with or without 20 μg/mL Fludarabine
Cell population0 μg/mL Fludarabine (%)20 μg/mL Fludarabine (%)P Value
All leukocytes74.5 ± 2.941.6 ± 7.8.008
CD4+ T cells84.3 ± 4.669.0 ± 9.9.232
CD8+ T cells81.2 ± 3.762.1 ± 12.9.204
B cells67.7 ± 0.530.5 ± 4.1.006

Results show the percentages of T-cell and B-cell populations that remained viable after the culture period. The data represent the mean ± SEM for 4 individual experiments. P values were calculated with a 2-tailed t test.

Treatment of allogeneic lymphocytes with fludarabine reduced GVHD and facilitated engraftment in the MHC-mismatched C57BL/6→ B10.BR transplant model 

A dramatic improvement in survival was observed among B10.BR mice transplanted with graded doses of F-DLI in combination with 1 × 106 TCD-BM from C57BL/6 donors, compared with recipients of untreated DLI. In this model system, the dose of TCD-BM is low enough that the absence of additional donor SP leads to approximately 50% mortality from aplasia and graft rejection (Figure 1A), with autologous reconstitution of host-type hematopoiesis or mixed chimerism in surviving mice. Recipients of 10 × 106 viable F-DLI (containing 4.8 × 106 viable T cells) had approximately 65% survival at 140 days after transplantation (Figure 1B), compared with no long-term survival among recipients of TCD-BM plus 10 × 106 untreated DLI (containing 3.8 × 106 viable T cells; P < .0005; Figure 1A). Transplantation of graded doses of F-DLI in combination with TCD-BM resulted in higher overall survival at all F-DLI cell doses tested versus untreated DLI, with the LD50 for F-DLI not reached at a viable cell dose of 10 × 106 donor cells. Because the major toxicity of DLI is GVHD and the most direct manifestations of GVHD are weight loss and mortality, the product of the day +60 average weight of survivors (as percentage of weight at day 0) and the fraction surviving was calculated as a survival score for each dose of viable T cells transplanted (Figure 1C). Increasing numbers of untreated DLI produced a steep decline in the score, with greater than 3-fold more toxicity per viable transplanted T cell (the slope of the line) compared with the GVHD activity of F-DLI (Figure 1C). The addition of DLI or F-DLI to the TCD-BM graft increased the level of donor T-cell chimerism in surviving animals (Figure 1D) and produced full donor-derived myeloid chimerism (not shown). The therapeutic index (defined as the donor T-cell dose that was associated with a toxic effect [weight × survival score of 80] divided by the dose of T cells that produced a beneficial effect [>5% of donor spleen-derived T cells in the blood at day +60]) was 3-fold higher for F-DLI (2.6 = 3.6 × 106/1.4 × 106) than for untreated DLI (0.9 = 1.2 × 106/1.1 × 106). An additional study showed that the survival difference between transplant recipient groups was more pronounced when donor SP were cultured with fludarabine for 24 hours and then in fresh media without fludarabine for 24 hours, versus 48 hours of untreated culture. For both groups, DLI were prepared so that 3 × 106 viable T cells were administered per recipient. The F-DLI group exhibited 80% survival at +100 days after transplantation (n = 15), compared with rapid GVHD mortality after transplantation with untreated SP (0% survival at day 15; n = 11; data not shown).

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  • Figure 1. 

    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).

B-cell depletion of allogeneic donor SP, or mixing apoptotic or dead donor cells with live donor T cells, did not reduce GVHD activity 

Our initial hypothesis predicted that fludarabine pre-exposure would have an immunosuppressive effect on donor T cells, ultimately resulting in reduced GVHD potential. Although the preceding experiments demonstrated reduced GVHD with F-DLI, the results also raised the possibility that effects on B cells in the F-DLI may have contributed to GVHD reduction [16] or, alternatively, that the large dead-cell population infused with F-DLI may have provided a GVHD-mitigating effect [17]. To address these questions, B10.BR mice were transplanted with C57BL/6 TCD-BM combined with untreated or fludarabine-treated B-cell—depleted SP (BCD-SP) or a mixture of BCD-SP and irradiated (25 Gy) SP. BCD-SP was prepared so that 2.2 × 106 viable T cells (untreated or fludarabine treated) were administered per recipient. The survival rate for recipients of TCD-BM alone was 20% at day 40. All mice that received TCD-BM with untreated BCD-SP were dead by day 20 (Figure 1E). The addition of 1 × 107 irradiated SP seemed to mitigate the rapid lethality of BCD-SP, with a survival rate of 40% at day 40, but all recipients in this group were dead at day 84 (Figure 1E). All mice that received fludarabine-treated BCD-SP survived beyond 100 days (Figure 1E), with a >10% increase in body weight, indicating normal growth (data not shown). Analysis of day 42 blood samples demonstrated that recipients of fludarabine-treated BCD-SP had complete donor chimerism, with normal levels of T and B cells, chiefly derived from the donor BM (data not shown). The data suggest that the reduced GVHD potential of F-DLI (Figure 1A–C) was not attributable to effects on the donor B-cell population or to co-infusion of dead or apoptotic SP.

Recipients of F-DLI had less GVHD histopathology and lower levels of inflammatory serum cytokines 

We confirmed that the survival advantage of F-DLI was due to a reduction in GVHD by histologic assessment of GVHD in transplant recipients. B10.BR mice were transplanted with C57BL/6 TCD-BM alone or combined with 3 × 106 untreated DLI, 10 × 106 untreated DLI, or 10 × 106 F-DLI as in the previous experiments. Three to 8 mice per group were killed 14 to 16 days after transplantation, and tissues were harvested for histopathologic assessment of GVHD. Evidence of GVHD was consistently reduced in recipients of F-DLI compared with either dose of untreated DLI. Recipients of untreated DLI had marked infiltration of lymphocytes in the liver periportal regions (Figure 2A), whereas liver sections from recipients of F-DLI retained a normal appearance with minimal infiltration (Figure 2B). In recipients of untreated DLI, numerous apoptotic cells were observed in the intestinal epithelium (Figure 2C), particularly in the crypts, along with foci of epithelial ulceration. All signs of intestinal GVHD were less evident among recipients of F-DLI (Figure 2D). The average day 14 to 16 histologic GVHD grades for liver and colon are shown in Figure 2E on a scale of 0 to 4. Compared with the 10 × 106 untreated DLI group, the F-DLI group had significantly reduced symptoms of GVHD in both liver (P = .02) and colon epithelial (P = .01) tissues. The reduced histologic evidence of GVHD at day 14 to 16 among recipients of F-DLI was accompanied by lower IFN-γ levels in serum (versus the 3 × 106 untreated group, P = .02; and the 10 × 106 untreated group, P = .01; Figure 2F). A similar trend was observed for serum levels of TNF-α, although the data were not statistically significant (data not shown).

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  • Figure 2. 

    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 have superior GVL activity without dose-limiting GVHD 

Transplantation of C57BL/6 TCD-BM into B10.BR recipients with graded numbers of untreated C57BL/6 donor SP (0, 1 × 106, 3 × 106, or 10 × 106) on the day of BMT, or a total of 50 × 106 SP (in 5 weekly injections), resulted in 50%, 100%, 80%, 0%, and 0% survival at 200 days after transplantation, respectively (Figure 3, n = 10 mice per group). Mice that received more than 3 × 106 donor SP died of GVHD on the basis of clinical and histologic staging (data not shown). Transplantation with the same numbers of F-DLI resulted in 50%, 100%, 100%, 80%, and 80% survival at day 200, demonstrating that F-DLI is well tolerated in this transplant model over a large dose range. To test the GVL activity of F-DLI, a second series of BMT experiments was performed in parallel, incorporating a transplanted lymphoblastic lymphoma tumor cell line, LBRM [12]. Transplantation of TCD-BM alone into mice inoculated on day −1 with 3 × 106 LBRM (>10 times the LD50) led to 0% survival at 60 days after transplantation, demonstrating the lethality of the transplanted tumor (Figure 3). The addition of graded doses of untreated donor SP to recipients of supralethal doses of LBRM showed GVL activity over a narrow dose range, with 70% and 40% day 200 survival among recipients of 1 × 106 and 3 × 106 donor SP, respectively, and 0% survival seen with larger doses of untreated SP due to dose-limiting GVHD. Transplantation of F-DLI in the LBRM model resulted in a broader survival-versus-dose curve, with day 200 survival rates of 80%, 90%, 70%, and 60% for doses of 1, 3, 10, and 50 × 106 F-DLI, respectively (Figure 3). Chimerism analyses of mice surviving to day +30 showed a dose-dependent increase in DLI-derived donor T cells, with recipients of 50 × 106 viable F-DLI having 60% of T cells derived from donor spleen versus recipients of the same number of untreated DLI, in which only 30% of blood T cells were derived from donor spleen (data not shown). The day +200 survival and enhanced immune reconstitution seen among recipients of F-DLI support the hypothesis that ex vivo fludarabine treatment effectively separates GVL activity from GVHD in an MHC-mismatched BMT model.

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  • Figure 3. 

    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.

Fludarabine treatment did not reduce the alloreactive T-cell frequency, but recipients engrafted with F-DLI were tolerant to host-type alloantigen 

The immediate effect of fludarabine exposure on the frequency of alloreactive T cells was measured by limiting-dilution analysis (LDA). The frequency of fludarabine-treated C57BL/6 T cells reactive to irradiated B10.BR SP (1/1000) was unchanged compared with the frequency of alloreactive cells cultured in media alone (1/910; Figure 4A). The results demonstrate that the frequency of alloreactive T cells was not directly affected by 24-hour ex vivo fludarabine treatment. Furthermore, donor-derived T cells recovered from stably engrafted, fully chimeric B10.BR recipients of C57BL/6 TCD-BM plus F-DLI were tolerant to host-type (B10.BR) and donor-type (C57BL/6) stimulators in a 1-way mixed lymphocyte reaction (MLR) but retained proliferative activity against a DBA/2 (H-2d) third-party stimulator (Figure 4B), indicating that prior fludarabine exposure had not produced a global effect on the proliferative responses of donor-derived T cells. Recipients of TCD-BM alone had graft rejection with autologous reconstitution; hence, the recipients’ T cells remained nonreactive to host antigen and reactive to donor and third-party alloantigen. Results were similar to those obtained with wild-type B10.BR SP as responders (Figure 4B). Equivalent data are not available for recipients of untreated DLI because of 100% GVHD mortality.

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  • Figure 4. 

    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-2b) donor-type, B10.BR (H-2k) recipient type or DBA/2 (H-2d) 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.

Fludarabine-treated donor T cells retained proliferative capacity in vivo 

The potential effect of fludarabine exposure on the short-term proliferation of allogeneic T cells in vivo was examined by using CFSE dye tracking. The proliferation of wild-type C57BL/6 (H-2b) T cells and anti-H-2d transgenic 2CTCR T cells (C57BL/6 background [10]) was compared in BALB/c (H-2d) scid/scid mice. The use of scid recipients allowed for a low signal-to-noise ratio. C57BL/6 and 2CTCR SP were combined in a 10:1 ratio before 24-hour culture in standard or fludarabine-containing medium and then labeled with CFSE after the culture period. Recipient mice were irradiated with 2.5 Gy, underwent transplantation with 3 × 107 CFSE-labeled cells, and then were killed after 40, 64, or 88 hours. Recipient SP suspensions were analyzed by flow cytometry; the CFSE fluorescence intensity of labeled donor cells was decreased by half after each cell division (Figure 5A). The percentage of C57BL/6 CD4+ and CD8+ T cells undergoing 1 or more rounds of cell division was determined at 40 and 64 hours after transplantation [18] and is summarized in Table 2. Both untreated and fludarabine-treated C57BL/6 T cells proliferated equally in the first 4 days after transplantation in BALB/c scid/scid mice, with approximately 30% of T cells undergoing 1 to 8 cell divisions within the first 64 hours after transplantation (Figure 5A; Table 2). Most of the fludarabine-treated and -untreated 2CTCR CD8+ allogeneic T cells had undergone 5 to 8 cell divisions during the same time period (Figure 5A; note that 2CTCR transgenic mice have very few CD4+ T cells). The fraction of dividing T cells could not be accurately determined by using this method at the 88-hour time point, because most of the donor-derived T cells had divided more than 8 times, with CFSE intensity reduced to background levels (data not shown). Of note, untreated and fludarabine-treated T cells showed no significant difference in the fraction of original cells that divided or in the number of cell divisions measured by CFSE during the analysis period. Similar CFSE proliferation profiles were obtained from recipients of C57BL/6 → B10.BR transplants and recipients of C57BL/6 → BALB/c scid/scid transplants without added transgenic allospecific T cells (data not shown). These results demonstrate that fludarabine treatment did not have a specific inhibitory effect on homeostatic proliferation of allogeneic donor T cells [19] or on allospecific CD8+ transgenic T cells.

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  • Figure 5. 

    F-DLI T cells proliferate in MHC-mismatched recipient spleen. (A) CFSE proliferation profiles of untreated or fludarabine-treated wild-type C57BL/6 H-2b and 2CTCR transgenic donor T cells recovered from spleens of BALB/c scid/scid H-2d 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.

Table 2. Percentage of C57BL/6 T Cells Proliferating in BALB/c scid/scid Recipient Spleen∗
Hours after TransplantationCD4CD8
Untreated DLIFludarabine DLIUntreated DLIFludarabine DLI
408%7%10%6%
6423%29%30%32%

∗ The percentage of donor T cells proliferating in allogeneic recipient spleen was determined by the method of Suchin et al. [18]. Briefly, the numbers of undivided cells (Pundiv) and those produced through 1 to 8 rounds of cell division were obtained from the CFSE staining profile. The calculated number of precursor cells necessary to account for all the divided cells is Pdiv. Hence, the total number of precursors (Ptot) is Pdiv + Pundiv, and the percentage of precursors undergoing division is determined as Pdiv/Ptot. The data represent the mean of 2 individual experiments.

To specifically examine the effect of fludarabine treatment on C57BL/6 alloreactive cell proliferation in response to alloantigen exposure in vivo, CFSE-labeled DLI or F-DLI cells were administered to unirradiated F1(C57BL/6 × B10.BR) recipients. In the parent → unirradiated F1 system, only alloreactive donor cells proliferate [18], in contrast to the combination of alloreactive and homeostatic proliferation observed in irradiated allogeneic recipients [19]. Analysis of early donor T-cell proliferation in the spleens of recipient animals 48 hours after transplantation demonstrated similar frequencies of dividing cells among untreated (average, 12%) and fludarabine-treated T cells (average, 7%; Figure 5B). At 72 hours, the dividing cell frequency was 7% for both untreated and fludarabine-treated T cells. Concurrent analysis of CFSE proliferation profiles and CD69 expression, as an indicator of early alloactivation, demonstrated upregulation of CD69 on both untreated and fludarabine-treated CD4+ donor T cells 24 hours after transplantation, early division of CD69+ T cells at 48 hours, and reduced CD69 expression among divided cells at 72 hours after transplantation (Figure 5B). The data suggest that fludarabine pre-exposure does not inhibit early proliferation of alloreactive donor T cells in response to alloantigen in vivo.

Fludarabine pre-exposure did not affect long-term donor T-cell proliferation in vivo 

Additional experiments were conducted to quantify donor-derived lymphocyte populations in allogeneic recipient spleens at time points up to 36 days after transplantation (when recipients of untreated DLI showed significant GVHD symptoms). B10.BR recipients underwent transplantation with 1 × 106 C57BL/6 (CD45.1) TCD-BM and 1 × 107 untreated C57BL/6 (CD45.2) DLI or F-DLI. Three to 8 recipients per group were killed at 2, 6, 10, 15, 21, and 36 days after transplantation. Total SP counts and flow cytometry analyses allowed determination of the numbers of T and B cells derived from donor BM (H-2b and CD45.1), donor SP (H-2b and CD45.2), and B10.BR recipients (H-2k and CD45.2). T cells from untreated DLI or F-DLI showed similar cell numbers and proliferation rates, with a peak of 200-fold CD8 cell expansion at day 15, followed by a gradual reduction in cell number (Figure 5C). CD4 cells expanded 50-fold by day 15 (Figure 5C). The number of F-DLI—derived T cells in recipient spleens averaged 70% of that found in recipients of untreated DLI for 2 to 21 days after transplantation, and no significant difference was found at any time point. In contrast, the number of F-DLI—derived B cells at 2 days after transplantation was only 12% of that from untreated DLI and increased to 32% at 15 days after transplantation (Figure 5D). At day 36, the proliferation of F-DLI—derived B cells in recipient spleens surpassed that for recipients of untreated DLI.

Fludarabine exposure led to loss of CD4+CD44low cells 

We tested whether there were subsets of activated, naı̈ve, or memory T cells that were more sensitive to fludarabine and that might have contributed to the altered immunoreactivity observed with F-DLI. B10.BR recipient mice underwent transplantation with 3 × 106 untreated or fludarabine-treated purified C57BL/6 Thy1.1+ T cells. Mice were killed on day 2 after transplantation, and SP were analyzed for the expression of Thy1.1 (donor T-cell marker), CD4, CD8, and CD25, CD44, CD62L, or CD122. The most notable difference between donor T cells recovered from recipients of fludarabine-treated or -untreated T cells was the relative lack of the CD4+CD44low-naive T-cell population in recipients of fludarabine-treated T cells (Figure 6A). A reduction in CD4+CD62Lhigh T cells was also observed, consistent with a loss of CD4+-naive T cells (Figure 6A). Because there are no significant differences in the absolute numbers of donor CD4 and CD8 T cells in spleens of F-DLI versus untreated DLI recipients at 2 days after transplantation (Figure 5C), the T-cell subset profiles presented here may be considered representative of donor T-cell populations found in vivo after allogeneic transplantation. In a separate experiment, T cells were cultured in the presence or absence of fludarabine for 24 hours and then washed and placed in fresh media. Fludarabine did not alter the relative representation of CD4+CD44low or CD4+CD44high cells after the initial 24-hour culture, but after an additional 1 to 2 days of culture in the absence of fludarabine, the CD4+CD44low population was markedly reduced (Figure 6B), thus corroborating the in vivo findings.

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  • Figure 6. 

    Fludarabine treatment reduced the CD4+CD44low 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 CD44low and CD44high 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.

Additional experiments to investigate the immunoregulatory roles of untreated and fludarabine-treated CD4+CD44high versus CD44low cells used intracellular cytokine analysis to determine the potential for these populations to produce IL-2, TNF-α, IFN-γ, and IL-10 in vitro (Figure 6C). As described previously, SP were incubated with or without fludarabine for 24 hours and then were washed and placed in fresh media overnight before 5 hours of stimulation with PMA/ionomycin. Cytokine production analysis of CD4+ T cells is shown in Figure 6C. Striking differences in the levels of IL-2 and TNF-α production were observed. In the untreated cultures, both CD4+CD44low and CD4+CD44high T cells produced these cytokines, and because CD4+CD44low cells were eliminated or apoptotic in the fludarabine-treated culture, IL-2 and TNF-α production were not observed for this population. Fludarabine-treated CD4+CD44high cells continued to produce these cytokines. Note that CD4+CD44high cells produced IL-10, whereas CD44low cells did not. Hence, the fludarabine-treated culture contained proportionally more IL-10-producing CD4 cells (8%) than the untreated culture (4%). A similar trend was observed for IFN-γ—producing cells. Although the cytokine production capability may differ in the transplant setting versus PMA/ionomycin stimulation, these results suggest a skewing of the T-cell population from a Th-1 to a Th-2 phenotype in the fludarabine-treated culture. The representation of CD4+CD44+CD25+ cells after untreated or fludarabine-treated culture was also examined (Figure 6C). In the untreated culture, approximately 60% of CD4 cells were CD44low, 32% were CD44highCD25−, and 6% were CD44highCD25+. In the fludarabine-treated culture, only 34% of CD4 T cells were CD44low, whereas 52% were CD44highCD25− and 11% were CD44highCD25+. Thus, the fludarabine-treated culture had a slightly larger representation of CD4+CD25+ T cells, a population that may include activated T cells and regulatory T cells [20].

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Discussion 

Multiple strategies of BMT engineering that involve depletion of T cells from the graft have reduced the incidence of GVHD, but because of higher rates of infection and relapse, they have not resulted in significant improvements in survival outcomes [21]. Several approaches to reduce the GVHD activity of allogeneic donor T cells while preserving the contribution of donor T cells to immune reconstitution have been investigated: thymidine kinase (TK) transduction 22, 23, 24; removal of alloreactive CD25+ or CD69+ T cells after MLR [25]; ex vivo tolerance by using monoclonal antibodies or soluble receptors that block the second signals necessary for T-cell activation 26, 27; in vitro exposure to IL-2 and anti-CD3 as a nonspecific method to generate activated, but nonalloreactive, donor T cells [28]; “cytokine starvation” in mixed lymphocyte cultures with third-party allogeneic stimulators [29]; and elimination of alloactivated T cells from MLR by incubation with 4,5-dibromorhodamine 123 and exposure to light [30]. We have previously described ex vivo treatment of donor T cells with radiation so that their proliferative capacity in vivo is impaired to the extent that they are not capable of producing GVHD [1]. Ex vivo exposure of T cells to psoralen and UV-A light nonspecifically blocks the proliferative capacity of T cells in vivo 31, 32, whereas treatment with leucyl-leucine methyl ester inactivates perforin activity in CD8+ T cells [33]. These approaches are based on nonspecific T-cell inactivation or deletion/tolerance of alloreactive T-cell subsets. The relative advantage of one method of reducing donor T-cell alloreactivity versus another remains to be determined. Clinical studies to date have not demonstrated the clear superiority of any approach because of limited GVL activity of TK-transduced [23] or irradiated [34] donor T cells and the persistence of alloreactivity after ex vivo co-stimulatory blockade [26]. This study describes a novel approach, ex vivo fludarabine pretreatment of allogeneic T cells, that seems to act by targeting a GVHD-promoting CD4 T-cell subset while preserving the overall ability of treated T cells to proliferate and contribute to posttransplantation immunity.

The fully MHC-mismatched C57BL/6 → B10.BR model is more clinically relevant than transplantation systems that use minor histocompatibility antigen strains because compared with mice, humans have a higher propensity for developing GVHD when they are administered low numbers of allogeneic T cells. Using the C57BL/6 → B10.BR model, we have shown that ex vivo fludarabine treatment specifically reduced the GVHD potential of allogeneic lymphocytes, thereby improving the therapeutic index of DLI (Figure 1C and D). This finding is consistent with analyses of histologic and serum cytokine manifestations of GVHD: recipients of 3 × 106 and 10 × 106 DLI had similar levels of these GVHD symptoms, whereas recipients of 10 × 106 F-DLI had reduced levels of liver and colon GVHD pathology and inflammatory serum cytokines (Figure 2). The data support the hypothesis that fludarabine pretreatment specifically reduces the GVHD potential of allogeneic T cells and that reduced GVHD activity is not attributable to a simple reduction in the number of allogeneic T cells administered. It is important to note that transplant experiments incorporating the LBRM leukemic cell line show that allogeneic F-DLI retain enough type 1 immune activity to mediate GVL activity against the transplanted lymphoma with low GVHD mortality over a broad DLI dose range and with a therapeutic index clearly superior to that of untreated DLI (Figure 3). Additional studies indicate that F-DLI contributes to posttransplantation immunity against the murine cytomegalovirus in allogeneic recipients without causing significant GVHD (E.K.W., unpublished data).

Our initial hypothesis for the reduced GVHD activity of fludarabine-treated allogeneic T cells was limited long-term survival of alloreactive donor T cells in vivo, similar to the effect of irradiation [1] or psoralen treatment [32]. Surprisingly, fludarabine-treated donor T cells expanded and persisted in recipient spleens at levels indistinguishable from untreated donor T cells (Figure 5). Further analyses of donor T-cell subsets showed that fludarabine pretreatment reduced the CD4+CD44low population both in allogeneic recipient spleens and in vitro (Figure 6A and B). Fludarabine exposure increased the relative numbers of CD4+CD44high memory-type donor cells that are associated with predominance of Th-2 immune responses 35, 36. Specifically, CD4 T cells that remained viable after fludarabine treatment produced proportionally less IL-2 and TNF-α (Th-1 cytokines) and more IL-10 (a Th-2 cytokine) when stimulated in vitro with PMA/ionomycin (Figure 6C). The increased numbers of CD4 T cells with a Th-2 phenotype may explain, in part, the reduced acute GVHD activity observed after fludarabine treatment [37]. A concurrent modest enrichment of the CD4+CD25+ population was also observed (Figure 6C). Of note, CD4+CD25+ T cells include activated as well as regulatory populations [20]. The finding that the immunosuppressive effect of fludarabine was more pronounced on naive T cells than on memory T cells is consistent with results obtained in membranous nephropathy patients treated with fludarabine [38]. A similar reduction in CD4+CD44low T cells was observed by Kishimoto and Sprent [39] after murine T cells were cultured overnight with plate-bound anti-T-cell receptor monoclonal antibodies. The authors showed that CD4+CD44low cells were susceptible to Fas-mediated apoptosis triggered by T-cell receptor engagement. Desbarats et al. [40] also showed that CD4+CD44low-naive murine T cells died as a result of simultaneous exposure to anti-CD3 antibody and Fas ligation, whereas CD4+CD44high memory T cells were stimulated to proliferate under the same conditions, and Hartwig et al. [41] showed that in vitro Fas-mediated activation-induced cell death eliminated alloreactive T cells and prevented GVHD in allogeneic recipients. Although our data bear some interesting similarities to these studies, and in vitro exposure to fludarabine has been shown to increase FasL expression and Fas messenger RNA levels in leukemic cell lines 42, 43, preliminary experiments of Fas expression on fludarabine-treated SP in vitro failed to show upregulation on CD4-naive T cells, militating against involvement of this mechanism as responsible for the specific sensitivity of CD4+CD44low T cells to fludarabine-induced apoptosis (Giver et al., unpublished data).

Because the role of CD4+CD44high donor T cells in the initiation of GVHD has been described [44], it may seem paradoxical that a reduction in CD4+CD44low donor T cells reduces GVHD activity. Our results suggest that CD4+CD44low-naive donor T cells contribute to the Th-1 inflammatory response in the allogeneic recipient and show that reduction of this cell type by using fludarabine pretreatment results in reduced GVHD morbidity and mortality without altering the overall proliferation, engraftment, and GVL activity of allogeneic donor T cells. We have not determined whether any potential functional alteration of the remaining CD4+CD44low T cells that survive 40 to 64 hours after fludarabine treatment (Figure 6B) contributes to reduced GVHD capacity. The reduction in viable CD4+CD44low donor T cells may lessen the incidence and severity of chronic GVHD, because naive CD44low T cells represent one of the major contributors to posttransplantation homeostatic proliferation after adoptive transfer [45]. However, CFSE labeling studies showed that initial proliferation of CD4+ donor T cells from F-DLI was not significantly different from proliferation of untreated donor CD4+ T cells in spleens of recipients treated with ablative doses of radiation (Figure 5A), conditions in which homeostatic proliferation of T cells will predominate [19]. Recent reports emphasize that donor T-cell proliferation in allogeneic recipient spleen does not necessarily correspond to GVHD progression in target organs and focus on donor T-cell activity and inflammatory responses in the intestinal tissues as critical events in GVHD progression 44, 46, 47. Our data also show that fludarabine pretreatment did not alter early donor T-cell proliferation in a parental → nonirradiated F1 transplant model (Figure 5B), in which donor T-cell proliferation is limited to alloreactive clones that recognize the nonshared haplotype [18]. The data are consistent with in vitro LDA studies showing that the frequency of alloreactive cells is not affected by fludarabine pretreatment (Figure 4A).

Other potential factors that could have contributed to the reduced GVHD activity of F-DLI, including the presence of dead cells in the treated cell preparation [17] and the relative reduction in B cells compared with untreated DLI [16], were examined. However, neither the use of BCD-SP nor the coadministration of T cells with irradiated SP resulted in reduced GVHD mortality (Figure 1E). The key effect of fludarabine exposure seems to be the selective loss of the CD4+CD44low population that occurred in vitro in cultures of purified T cells in the absence of any interaction with alloantigen or antigen presenting cells (Figure 6B), and in vivo after transfer of F-DLI into allogeneic recipients (Figure 6A). This is supported by additional C57BL/6 → B10.BR BMT experiments with fluorescence-activated cell sorter-purified T-cell subsets. Recipients of 1 × 106 CD8 plus 2 × 106 CD4+CD44high T cells have a 90% day +100 survival rate, normal weight gain, and full donor-derived T-cell chimerism. In contrast, all recipients of TCD-BM plus 1 × 106 CD8 and 2 × 106 total CD4+ T cells (containing 1.7 × 106 CD4+CD44low and 0.3 × 106 CD4+CD44high) die rapidly because of acute GVHD (Giver and Waller, unpublished data). A recent publication by Anderson et al. [48] demonstrates that memory CD4+ T-cells do not induce GvHD in a MiHA-incompatible murine transplant system, and similar results have been reported by Chen et al. [49], who used allogeneic transfer of CD3+ naive T-cells (rapid GvHD) versus CD3+ memory T-cells (high survival rate). These data further support our hypothesis that fludarabine-induced reduction of donor naive CD4+ T-cells, while sparing memory CD4+ T-cells, reduces the GvHD potential of allogeneic DLI. Whereas physical separation of donor T-cell subsets is expensive and time consuming, fludarabine pretreatment is simple and relatively inexpensive. This method for selective depletion of the CD4+CD44low donor cell population from allogeneic DLI represents an interesting and potentially clinically relevant approach for limiting GVHD while preserving immune reconstitution, graft-facilitating effects, and GVL activity of allogeneic donor T cells.

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Acknowledgements 

This work was supported by National Institutes of Health grant nos. R01 CA-74364-03 (E.K.W.) and DK-60647 (D.L.J.). C.R.G. was supported by NIH Institutional Research and Academic Cancer Development Award no. 5K12-GM-00680 to Emory University School of Medicine. We thank John Roback, MD, and John Gorechlad for assistance with these experiments, and Peter Jensen, MD, for careful critique of the manuscript.

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References 

  1. Waller EK, Ship AM, Mittelstaedt S, et al.  Irradiated donor leukocytes promote engraftment of allogeneic bone marrow in major histocompatibility complex mismatched recipients without causing graft-versus-host disease. Blood. 1999;94:3222–3233
  2. Waller EK, Boyer M. New strategies in allogeneic stem cell transplantation (immunotherapy using irradiated allogeneic T cells). Bone Marrow Transplant. 2000;(suppl 2):S20–S24
  3. Roux E, Abdi K, Speiser D, et al.  Characterization of mixed chimerism in patients with chronic myeloid leukemia transplanted with T-cell—depleted bone marrow (involvement of different hematologic lineages before and after relapse). Blood. 1993;81:243–248
  4. Avramis VI, Plunkett W. 2-fluoro-ATP (a toxic metabolite of 9-beta-D-arabinosyl-2-fluoroadenine). Biochem Biophys Res Commun. 1983;113:35–43
  5. Huang P, Chubb S, Plunkett W. Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity. J Biol Chem. 1990;265:16617–16625
  6. Priebe T, Platsoucas CD, Seki H, Fox FE, Nelson JA. Purine nucleoside modulation of functions of human lymphocytes. Cell Immunol. 1990;129:321–328
  7. Cheson BD. Infectious and immunosuppressive complications of purine analog therapy. J Clin Oncol. 1995;13:2431–2448
  8. Sandoval A, Consoli U, Plunkett W. Fludarabine-mediated inhibition of nucleotide excision repair induces apoptosis in quiescent human lymphocytes. Clin Cancer Res. 1996;2:1731–1741
  9. Frank DA, Mahajan S, Ritz J. Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling. Nat Med. 1999;5:444–447
  10. Sha WC, Nelson CA, Newberry RD, Kranz DM, Russell JH, Loh DY. Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature. 1988;335:271–274
  11. Cooke KR, Hill GR, Crawford JM, et al.  Tumor necrosis factor-alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J Clin Invest. 1998;102:1882–1891
  12. Gillis S, Mizel SB. T-cell lymphoma model for the analysis of interleukin 1-mediated T-cell activation. Proc Natl Acad Sci U S A. 1981;78:1133–1137
  13. Taswell C. Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J Immunol. 1981;126:1614–1619
  14. Lyons AB. Divided we stand (tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester). Immunol Cell Biol. 1999;77:509–515
  15. Cox DR, Oakes D. Proportional hazards model. In:  Cox DR,  Oakes D editor. Analysis of Survival Data. New York: Chapman and Hall; 1984;p. 104–106
  16. Schultz KR, Paquet J, Bader S, HayGlass KT. Requirement for B cells in T cell priming to minor histocompatibility antigens and development of graft-versus-host disease. Bone Marrow Transplant. 1995;16:289–295
  17. Bittencourt MC, Perruche S, Contassot E, et al.  Intravenous injection of apoptotic leukocytes enhances bone marrow engraftment across major histocompatibility barriers. Blood. 2001;98:224–230
  18. Suchin EJ, Langmuir PB, Palmer E, Sayegh MH, Wells AD, Turka LA. Quantifying the frequency of alloreactive T cells in vivo (new answers to an old question). J Immunol. 2001;166:973–981
  19. Maury S, Salomon B, Klatzmann D, Cohen JL. Division rate and phenotypic differences discriminate alloreactive and nonalloreactive T cells transferred in lethally irradiated mice. Blood. 2001;98:3156–3158
  20. Taylor PA, Lees CJ, Blazar BR. The infusion of ex vivo activated and expanded CD4+CD25+ immune regulatory cells inhibits graft-versus-host disease lethality. Blood. 2002;99:3493–3499
  21. Ho VT, Soiffer RJ. The history and future of T-cell depletion as graft-versus-host disease prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood. 2001;98:3192–3204
  22. Bordignon C, Bonini C, Verzeletti S, et al.  Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. Hum Gene Ther. 1995;6:813–819
  23. Bonini C, Bordignon C. Potential and limitations of HSV-TK-transduced donor peripheral blood lymphocytes after allo-BMT. Hematol Cell Ther. 1997;39:273–274
  24. Bonini C, Ferrari G, Verzeletti S, et al.  HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science. 1997;276:1719–1724
  25. van Dijk AM, Kessler FL, Stadhouders-Keet SA, Verdonck LF, de Gast GC, Otten HG. Selective depletion of major and minor histocompatibility antigen reactive T cells (towards prevention of acute graft-versus-host disease). Br J Haematol. 1999;107:169–175
  26. Guinan EC, Boussiotis VA, Neuberg D, et al.  Transplantation of anergic histoincompatible bone marrow allografts. N Engl J Med. 1999;340:1704–1714
  27. Taylor PA, Panoskaltsis-Mortari A, Noelle RJ, Blazar BR. Analysis of the requirements for the induction of CD4+ T cell alloantigen hyporesponsiveness by ex vivo anti-CD40 ligand antibody. J Immunol. 2000;164:612–622
  28. Drobyski WR, Majewski D, Ozker K, Hanson G. Ex vivo anti-CD3 antibody-activated donor T cells have a reduced ability to cause lethal murine graft-versus-host disease but retain their ability to facilitate alloengraftment. J Immunol. 1998;161:2610–2619
  29. Bachar-Lustig E, Reich-Zeliger S, Gur H, Zhao Y, Krauthgamer R, Reisner Y. Bone marrow transplantation across major genetic barriers (the role of megadose stem cells and nonalloreactive donor anti-third party CTLS). Transplant Proc. 2001;33:2099–2100
  30. Chen BJ, Cui X, Liu C, Chao NJ. Prevention of graft-versus-host disease while preserving graft-versus-leukemia effect after selective depletion of host-reactive T cells by photodynamic cell purging process. Blood. 2002;99:3083–3088
  31. Grass JA, Wafa T, Reames A, et al.  Prevention of transfusion-associated graft-versus-host disease by photochemical treatment. Blood. 1999;93:3140–3147
  32. Truitt R, Johnson B, Hanke C, Talib S, Hearst J. Photochemical treatment with S-59 psoralen and ultraviolet A light to control the fate of naive or primed T lymphocytes in vivo after allogeneic bone marrow transplantation. J Immunol. 1999;163:5145–5156
  33. Hsieh MH, Varadi G, Flomenberg N, Korngold R. Leucyl-leucine methyl ester-treated haploidentical donor lymphocyte infusions can mediate graft-versus-leukemia activity with minimal graft-versus-host disease risk. Biol Blood Marrow Transplant. 2002;8:303–315
  34. Ship AM, Waller EK. Clinical use of irradiated donor lymphocytes in bone marrow transplantation. Cancer Treat Res. 1999;101:267–282
  35. Aguado E, Richelme S, Nunez-Cruz S, et al.  Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science. 2002;296:2036–2040
  36. Coudert JD, Foucras G, Demur C, et al.  Lethal host-versus-graft disease and hypereosinophilia in the absence of MHC I-T-cell interactions. J Clin Invest. 2000;105:1125–1132
  37. Fowler DH, Gress RE. Th2 and Tc2 cells in the regulation of GVHD, GVL, and graft rejection (considerations for the allogeneic transplantation therapy of leukemia and lymphoma). Leuk Lymphoma. 2000;38:221–234
  38. Boumpas DT, Tassiulas IO, Fleisher TA, et al.  A pilot study of low-dose fludarabine in membranous nephropathy refractory to therapy. Clin Nephrol. 1999;52:67–75
  39. Kishimoto H, Sprent J. Strong TCR ligation without costimulation causes rapid onset of Fas-dependent apoptosis of naive murine CD4+ T cells. J Immunol. 1999;163:1817–1826
  40. Desbarats J, Wade T, Wade WF, Newell MK. Dichotomy between naive and memory CD4(+) T cell responses to Fas engagement. Proc Natl Acad Sci U S A. 1999;96:8104–8109
  41. Hartwig UF, Robbers M, Wickenhauser C, Huber C. Murine acute graft-versus-host disease can be prevented by depletion of alloreactive T lymphocytes using activation-induced cell death. Blood. 2002;99:3041–3049
  42. Villunger A, Egle A, Kos M, et al.  Drug-induced apoptosis is associated with enhanced Fas (Apo-1/CD95) ligand expression but occurs independently of Fas (Apo-1/CD95) signaling in human T-acute lymphatic leukemia cells. Cancer Res. 1997;57:3331–3334
  43. Jones DT, Ganeshaguru K, Virchis AE, et al.  Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation. Blood. 2001;98:2800–2807
  44. Snider D, Liang H. Early intestinal Th1 inflammation and mucosal T cell recruitment during acute graft-versus-host reaction. J Immunol. 2001;166:5991–5999
  45. Cho BK, Rao VP, Ge Q, Eisen HN, Chen J. Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells. J Exp Med. 2000;192:549–556
  46. Cooke KR, Gerbitz A, Crawford JM, et al.  LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation. J Clin Invest. 2001;107:1581–1589
  47. Clouthier SG, Ferrara JL, Teshima T. Graft-versus-host disease in the absence of the spleen after allogeneic bone marrow transplantation. Transplantation. 2002;73:1679–1681
  48. Anderson BE, McNiff J, Yan J, et al.  Memory CD4+ T cells do not induce graft-versus-host disease. J Clin Invest. 2003;112:101–108
  49. Chen B, Cui J, Chao NJ. Differential effects of naive and non-naive T-cells on GvHD (implications in separating GvL from GvHD). Blood. 2002;100:70a; (abstr.)

PII: S1083-8791(03)00229-5

doi:10.1016/S1083-8791(03)00229-5

Biology of Blood and Marrow Transplantation
Volume 9, Issue 10 , Pages 616-632, October 2003

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