Biology of Blood and Marrow Transplantation
Volume 13, Issue 6 , Pages 644-654, June 2007

In Vitro Methotrexate as a Practical Approach to Selective Allodepletion

  • Atul Sathe

      Affiliations

    • Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas
    • ,
  • Sterling B. Ortega

      Affiliations

    • Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas
    • ,
  • Dorothy I. Mundy

      Affiliations

    • Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas
    • ,
  • Robert H. Collins

      Affiliations

    • Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas
    • ,
  • Nitin J. Karandikar

      Affiliations

    • Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, Texas
    • Corresponding Author InformationCorrespondence and reprint requests: Nitin J. Karandikar, MD, PhD, Department of Pathology, The University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9072.

Received 3 January 2007; accepted 21 January 2007. published online 20 March 2007.

Article Outline

Abstract 

Graft-versus-host disease (GVHD) is a major cause of transplant-related morbidity and mortality in recipients of allogeneic hematopoietic stem cell transplantation. As GVHD is mediated predominantly by alloreactive donor T cells, selective allodepletion from the graft may alleviate GVHD, whereas potentially maintaining other advantages conferred by donor T cells, such as graft survival, antiviral immunity, and graft-versus-leukemia effect. In this study, we evaluated the ability of methotrexate, a clinically approved antimetabolite drug, to deplete alloreactive T cells in HLA-mismatched mixed lymphocyte reactions (MLR). We observed that methotrexate could inhibit the proliferation of alloreactive T cells in primary in vitro MLR. On reexposure of methotrexate-treated cells to the same allostimulus, a significant reduction in the alloreactive immune response was observed, whereas responses to third-party allostimuli and viral antigens were preserved. Thus, our results provide preclinical evidence that in vitro methotrexate treatment results in specific allodepletion and may be used as an effective agent for preventing GVHD.

Key Words: Allodepletion, GVHD

 

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Introduction 

Allogeneic hematopoietic stem cell transplantation (HSCT) is used for treatment of leukemias and other hematopoietic disorders. Graft-versus-host-disease (GVHD) is a major complication of such transplants, leading to considerable morbidity and mortality [1, 2]. Treatment of GVHD requires immunosuppression leading to poor immune reconstitution, risk of infection, and prolonged recovery [3, 4, 5].

Although the pathogenesis of GVHD is multifactorial, its initiation depends on the presence of alloreactive donor T cells in the graft [6, 7, 8, 9, 10]. However, T cells cannot be completely eliminated from the graft because they are required for early engraftment [11, 12], immune recovery [13, 14], and the graft-versus-leukemia (GVL) effect [15]. The risk of relapse increases significantly when T cell-depleted grafts are used for transplantation [16]. Previous studies have shown that the GVL effect can be preserved without eliciting a GVH reaction [17, 18]; so targeted elimination of alloreactive T cells may prevent GVHD without losing the other benefits of grafted T cells.

There are several approaches being investigated to achieve selective allodepletion, including direct lysis of in vitro activated alloreactive T cells via targeting of surface markers or their elimination via cell sorting [19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29] or a combination of elimination and antiproliferative approaches, such as trimethrexate [30]. Some of these approaches have been scaled up for clinical trials, whereas others have remained preclinical, probably owing to very involved fractionation procedures. Thus, there is still the need for an effective but practical methodology that can achieve selective allodepletion.

Methotrexate (MTX) is an FDA-approved drug used for the therapy of neoplastic diseases, rheumatoid arthritis, and psoriasis. It is a folate analog that is actively transported into cells causing S-phase arrest and apoptosis. MTX chiefly inhibits the enzyme dihydrofolate reductase, which reduces dihydrofolate to tetrahydrofolate. Importantly, MTX has been shown to act on dividing T cells in vitro by interfering with DNA synthesis, repair, and cellular replication [31, 32]. Based on this knowledge, we hypothesized that this drug may have the potential as an in vitro agent of selective allodepletion. In this study, we evaluated this potential by studying the effects of MTX on in vitro mixed lymphocyte reactions (MLRs). Using a sensitive flow cytometry-based system, we could characterize the effect of MTX on both CD4+ and CD8+ T cell proliferation and activation in primary MLRs, as well as its utility in suppressing alloreactive responses in a specific manner. We present our preclinical results from these assays to show that MTX has excellent potential as an in vitro allodepleting agent, affording a practical methodology that can be scaled up for clinical use in the future.

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Materials and Methods 

Preparation and Isolation of Cells 

Peripheral blood mononuclear cells (PBMCs) from random healthy donors were isolated from buffy coats obtained from Carter Blood Care (Bedford, TX), using Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Sweden). The study was approved by the UT Southwestern IRB and was performed on deidentified specimens from a blood donation center.

Mixed Lymphocyte Reactions and Allodepletion 

PBMCs were activated in 1-way MLRs under various conditions. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based flow cytometric proliferation assays were used, as described [33, 34]. Briefly, CFSE-stained PBMCs (responder cells) were suspended in H5 medium (RPMI, Mediatech Inc., Herndon, VA, 5% heat-inactivated human AB serum (Gemini Bio-products West Sacramento, CA), and 1% mixture of penicillin, streptomycin, and L-glutamine (Invitrogen Corp., Grand Island, NY) at a concentration of 106 cells/mL. Stimulator PBMCs from an HLA-mismatched random donor were irradiated (30 Gy) and resuspended to the same concentration (of note, HLA testing was not specifically performed on these specimens, and thus, the degree of mismatch in each reaction was not known). These were mixed at a 1:1 ratio of responders to stimulators and incubated in T-75 (75 cm2) tissue culture flasks (BD Falcon, Lincoln Park, NJ) at 37°C in 5% CO2 for 7 days, unless otherwise indicated. In every experiment, an autologous reaction of CFSE-stained PBMCs and irradiated PBMCs from same donor served as a background control. This was an important control to ascertain that the MLRs observed were indeed alloantigen specific. For positive proliferation controls, MLRs were stimulated with 1 μg/mL of the superantigen, staphylococcal enterotoxin B (SEB, Sigma, St. Louis, MO).

For experiments involving allodepletion, reactions were conducted in the presence (or absence) of 10 μM MTX. For secondary MLRs, live cells were reisolated from the various primary reactions using Ficoll-Paque gradients. These cells were re-suspended in H5 medium, rested for 2 days, and then rechallenged in a secondary MLR. Cells from each primary reaction were stimulated in separate cultures with the following stimuli: PBMCs from same responder (autologous reaction), PBMCs from the same allostimulator as in the primary MLR, PBMCs from an unrelated third donor (“third party” stimulus), cytomegalovirus (CMV) antigen (Microbix Biosystems, Canada), or SEB. All stimulator cells were irradiated (30 Gy). These cultures were conducted in 24-well plates (Greiner Bio-one, Monroe, NC) because of smaller cell numbers, with all other conditions remaining the same (cell concentrations, etc.). The secondary MLR cells were harvested after 7 days and analyzed by flow cytometry.

Flow Cytometry 

In CFSE-based proliferation assays, dividing cells are detected by sequential halving of their fluorescence [35]. On the day of analysis, aliquots from MLRs were washed with FACS buffer (PBS with 1% BSA and 0.1% sodium azide) and stained with the following markers: anti-CD3-PerCP (peridinin chlorophyll protein), anti-CD14-PE (phycoerythrin), anti-CD4-PE Cy5.5 (phycoerythrin-cyanin5.5), anti-CD8-Pacific Blue, and anti-CD25-APC (allophycocyanin) (antibodies from BD Biosciences, San Diego, CA, or Caltag Laboratories, Burlingame, CA). Cells were washed twice in FACS buffer and fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) containing 0.1% EDTA (Fisher, Fairlane, NJ). Flow cytometric data (at least 100,000 nongated events) were acquired on a BD FACS-Calibur (4-color) or on a BD LSR II multicolor flow cytometer using BD FACS Diva software. FlowJo (TreeStar Inc., Ashland, OR) or BD FACS Diva (BD Biosciences) software were used for analysis. Cells were gated on CD14−/CD3+ T cells and further on the CD4+/CD8− or CD8+/CD4− populations [33]. The magnitude of proliferation was determined as the percentage of gated cells (CD4 or CD8) that were in the proliferating fraction on the day of analysis. Background proliferation was determined from the autologous MLR.

Statistical Analysis 

Wilcoxon’s signed rank test was used to evaluate quantitative differences between groups. A P value <.05 was considered significant.

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Results 

Subject Characteristics 

The median age of buffy coat donors was 26 years (range: 17-60 years). There were 11 male and 14 female donors. Of 14 responder-stimulator pairs used in MLRs, 10 donor pairs were gender matched (4 male pairs and 6 female pairs) and 4 were gender mismatched.

MTX Inhibits Proliferation of Allogeneic CD4+ and CD8+ T Cells in a Primary MLR with Little Effect on T Cell Activation 

Our first step in the evaluation of MTX as an allodepleting agent was to standardize culture conditions for a primary MLR, using a CFSE-based proliferation assay as a readout. In 5 replicate experiments, we evaluated various cell ratios, cell concentrations, and culture times. Importantly, an autologous background control was included in all our experiments to ascertain that the MLR readout was truly alloantigen specific. We observed optimal reactions (signal:noise discrimination) at a ratio of 1:1 and a total cell concentration of 1 × 106/mL (optimization data not shown). Across these experiments, a consistent kinetic of activation and growth of T cells was observed (Figure 1). On days 2 and 3 of the primary MLR, some alloreactive proliferation was detected. However, this growth was not consistently greater than the small amount of background proliferation present in autologous controls. By day 5, proliferation in the alloreaction increased markedly, and on day 7 of culture, alloreactive T cell proliferation was consistently robust, compared to the autologous control. This trend was similar for both CD4+ and CD8+ T cells (Figure 1).

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  • Figure 1. 

    Kinetics of T cell growth in MLR and MTX blockade. (A) Representative dot plots of a primary MLR. CFSE staining is shown on the X-axis and CD8 staining on Y-axis. The data are gated to show CD3+/CD4+/CD8− or CD3+/CD4−/CD8+ cells; thus, the CD8− populations represent CD4+ T cells. The gray populations to the right represent nondividing cells. The dividing cells are shown in red (CD4+ T cells) and green (CD8+ T cells). The numbers represent the proliferating fraction of CD4+ and CD8+ cells, respectively. As indicated, the top row is an autologous control, the middle row is an alloreaction, and the bottom row is the SEB-stimulated positive control. Each column represents the indicated day of culture. Data is representative of 5 replicate experiments. The lower 2 panels show the percent proliferation of CD4+ (B) and CD8+ (C) T cells at days 2, 3, 5, and 7 in an autologous control, an alloreaction, and an MTX-treated alloreaction. The mean (SEM) of 5 separate experiments is shown. Thus, MTX blocked the proliferation of both CD4+ and CD8+ T cells on all days.

Once this consistent pattern was standardized, we evaluated the effect of MTX on primary MLRs. Various concentrations of MTX were used in initial experiments, based on prior literature, with optimal effect seen at 10 μM. As expected, addition of MTX to the alloreaction suppressed proliferation on all days, and this was most significantly detected on days 5 and 7 of the culture (Figure 1B and C).

When measured on day 7 of culture in 14 replicate experiments (Figure 2), MTX treatment significantly and consistently reduced CD4+ T cell alloproliferation (proliferating fraction [PF] of 15.78% ± 4.86% reduced to 2.49% ± 0.65%, P < .001). Similarly, the PF of CD8+ T cells was also significantly reduced from 26.65% ± 7.3% to 1.72% ± 0.48%, P < .001. There was no significant difference between the autologous background proliferation and the MTX-treated alloreaction.

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  • Figure 2. 

    Methotrexate inhibits proliferation of alloreactive T-cells. (A) Representative dot plots of an autologous reaction, an alloreaction, and an MTX-treated alloreaction at days 5 and 7 of culture. The layout is similar to that of Figure 1, with numbers representing CD4+ and CD8+ proliferation. Data is representative of 14 replicate experiments. (B, C) The cumulative data from the 14 experiments, represented as mean ± SEM of CD4+ (B) and CD8+ (C) T cell proliferation. MTX significantly inhibited both CD4+ and CD8+ T cell proliferation in primary MLRs (P < .001).

This effect of MTX was not restricted to the alloreactions. In fact, the same concentration of MTX robustly inhibited proliferation of CD4+ and CD8+ T cells in superantigen-stimulated cultures (Figure 3). Thus, in vitro MTX treatment resulted in significant blockade of T cell proliferation.

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  • Figure 3. 

    Methotrexate suppresses proliferation of all dividing T-cells. (A) Representative dot plots demonstrating the proliferation of CD4+ and CD8+ T cells in SEB-stimulated cultures in the presence (bottom row) or absence (top row) of MTX. (B, C) Cumulative data from 14 experiments, demonstrating that MTX significantly inhibited SEB-induced proliferation of both CD4+ and CD8+ T-cells.

In addition to proliferation, the flow cytometric assay system also allowed to evaluate T cell activation in these cultures, as indicated by upregulation of CD25 expression. Interestingly, although MTX treatment had a dramatic effect on cell proliferation, it did not significantly inhibit CD25 upregulation on the nondividing CD4+ or CD8+ T cells, compared to the corresponding population in the alloreaction (Figure 4). Of note, these did not simply represent preculture CD25+ regulatory or activated T cells, as this population was upregulated in the MLR, but not seen in the autologous control (data not shown). Thus, MTX was unable to block initial activation of these cells, but clearly blocked their proliferation.

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  • Figure 4. 

    Methotrexate does not affect activation of T cells in MLR. (A) Representative dot plots of CD25 expression on T cells on day 7 of a primary MLR in the presence (bottom row) or absence (top row) of MTX. CD4+ T cells are shown in the left column, whereas CD8+ T cells are shown in the right column. CFSE staining is shown on the X-axis and CD25 staining on the Y-axis. The populations on the right represent nondividing cells. The threshold for CD25 expression was set using an isotype control antibody. The numbers represent the percentage of cells located in each quadrant. (B, C) Cumulative data from 14 replicate experiments. The graphs represent the mean (+SEM) percentage of CD25+/nondividing cells for the indicated culture conditions and cell types (n.s. = not significant).

MTX Treatment Results in Selective Allodepletion, with Preservation of Third-Party and Antiviral Responses 

The results described above showed that MTX could inhibit the proliferation but not the initial activation of alloreactive T cells. The persistence of these cells in the MLR cultures suggested that these cells may respond to the initial allostimulus once they were freed from the antiproliferative influence of MTX, in which case “allodepletion” would not have been accomplished.

To test this, we performed secondary MLRs using live cells obtained from the various primary MLRs after multiple washes. Thus, cells from the initial conditions (autologous control, MLR, and MLR + MTX) were each exposed to different stimuli, including autologous PBMCs, PBMCs from the initial allostimulus, PBMCs from a third-party allostimulus, CMV antigen, or SEB. In initial experiments, we performed a kinetic analysis, where cells were obtained from primary MLRs that were cultured for 2, 3, 5, and 7 days. Early cultures (days 2 and 3) showed little or no effect on the secondary MLR proliferation (data not shown). Most optimal effects were observed at 7 days of primary culture, and this time point was picked for future experiments.

In 12 replicate experiments, we observed that both CD4+ and CD8+ T cells from the “MLR + MTX” condition failed to respond to the initial allostimulus, whereas their response to a third-party stimulus, a viral antigen, or a superantigen was intact (Figure 5). Thus, even after the cultures were freed of the antiproliferative action of MTX, the cells from the MTX-treated alloreactions showed clear evidence of allodepletion. This was selective, as antiviral and antithird-party responses were preserved.

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  • Figure 5. 

    Methotrexate treatment of primary MLR results in selective allodepletion with preservation of third-party and antiviral responses. (A) Representative dot plots of a secondary MLR, using responder cells isolated from a primary alloreaction that was either untreated (top row) or treated with MTX (bottom row) for 7 days. The stimulators were either the autologous control, the same allostimulus , a “third-party” allostimulus, or CMV antigen (as indicated). (B, C) Cumulative data from 12 separate secondary MLRs. The mean proliferation (+SEM) of CD4+ (B) and CD8+ (C) T cells are shown, under the indicated culture conditions. Thus, MTX treatment resulted in selective inhibition of responses to the same allostimulus, whereas leaving intact responses to third-party stimuli as well as a viral antigen.

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Discussion 

Pretransplant in vitro allodepletion is a feasible option to reduce the incidence and severity of GVHD. In this study, we evaluated the potential use of methotrexate, an FDA-approved drug known to inhibit T cell proliferation, as an agent for specific in vitro allodepletion. In previous studies, MTX was shown to specifically kill/inhibit proliferating T cells. Additionally, MTX was shown to inhibit T cells that proliferate in response to a cell line and when these MTX-treated cells were re-exposed to the same cell line, they did not show any response. In contrast, when stimulated with a different cell line these cells were activated and responded by proliferating [36]. These properties of MTX suggest that it may be a suitable and practical agent for in vitro allodepletion. The potential use of MTX for allodepletion has been suggested in the past [37], but subsequent studies have been more focused on the role of MTX in autoimmune diseases, especially rheumatoid arthritis [38, 39]. Further rationale for the use of MTX is provided by prior studies using trimethrexate in combination with cell depletion approaches to achieve allodepletion [30].

In the current study, we used a sensitive flow cytometry-based proliferation assay to evaluate the allodepleting potential of MTX. The multiparameter flow cytometric approach allowed us to simultaneously measure proliferation and activation of both CD4+ and CD8+ T cell responses in a robust and well-controlled manner. This is a unique strength of this study. Moreover, the careful standardization of the assay, with the consistent inclusion of an autologous control adds specificity and confidence to our conclusions, as suggested by a recent study [40].

Using this assay system, we first show that addition of MTX results in significant inhibition of both CD4+ and CD8+ T cell proliferation in primary HLA-mismatched 1-way MLRs. In contrast, the initial activation of these T cells does not appear to be affected. However, we clearly demonstrate that these MTX-treated cells are unable to respond to the same initial allostimulus, consistent with allodepletion. This suggests that the initially activated cells likely incorporate MTX during the primary exposure and are anergic or undergo apoptosis during the re-exposure. Prior studies have demonstrated the ability of MTX (in contrast to several other immunosuppressive agents) to induce apoptosis of alloreactive T cells [41], and this mechanism likely contributes in large part to the lack of T cell response on re-exposure in our system. Importantly, antiviral and third-party responses remain intact in the MTX-treated cells. Thus, our studies show that MTX is an effective agent of selective in vitro allodepletion.

Although several strategies for in vitro allodepletion have been proposed, their transition from preclinical studies to clinical use is sometimes hindered because of choice of reagent or technique. Several allodepletion approaches use an activation marker such as CD69 or CD25 [23, 24, 25, 26, 27, 28] for targeted elimination of alloreactive T cells. In an MLR, different activation markers get preferentially upregulated at various time points, suggesting that different pathways for activation are being used by alloantigen-specific cells. Furthermore, all activated cells may not express the marker(s) of interest at a given time point [29]. These findings can be explained by the heterogeneous nature of the in vitro alloresponse and suggest that if a single marker is used for targeted elimination of alloreactive T cells, it is likely that some alloreactive precursors may escape depletion. Allodepletion using multiple markers is likely to overcome this problem [29]. However, in addition to the abovementioned activation markers, a large number of other markers like CD38, CD71, CD134, HLA-DR, etc., are upregulated by the alloreactive cells at various times in an MLR [22]. In the absence of comprehensive data determining the kinetics of expression and upregulation of these molecules in an MLR, it would be very difficult to narrow down to 1 or 2 prospective activation markers as being sufficient for allodepletion. Recent models of GVHD have shown that in vitro expansion of regulatory T cells (T regs) can ameliorate GVHD [42, 43]. In humans, T regs are distinguished by their CD4+/CD25high/FOXP3+ phenotype. Allodepletion targeting CD25 as an activation marker may deplete this beneficial cell population. However, in MTX-based allodepletion, the preculture as well as induced T regs (as measured by CD25 and FOXP3 coexpression) are relatively preserved (data not shown). Another drawback of cell targeting methods is the potential elimination of bystander and memory cells that have low expression of activation markers. This could reduce the available pool of nonalloreactive but immunocompetent T cells in the graft. As the action of MTX is specific for dividing cells regardless of surface phenotype, MTX-based allodepletion can steer clear of these problems.

Even though our findings demonstrate the potential of MTX for in vitro allodepletion, there are admittedly some drawbacks. First, MTX-based depletion is dependent on early and vigorous proliferation of alloreactive cells in an MLR. As MTX acts by blocking the folate pathway, unless the alloreactive T cell begins folate uptake and initiates cell division, it cannot be eliminated from the primary MLR. Thus, weak or delayed proliferation of T cells may lead to less efficient depletion of the alloreactive cells. This problem may be of more concern in an HLA-matched setting (or potentially in a haploidentical or locus-mismatched situations) because of potentially weaker or delayed proliferation. In ensuing studies, we intend to directly address this issue. Second, our study determined that it was necessary to culture a primary MLR for 5-7 days to ensure and maximize allodepletion by MTX (data not shown). A shorter period of culture may be more attractive clinically, but the variable proliferation at earlier time points meant that all alloreactive precursors were not actively dividing and MTX could not inhibit/eliminate them. However, this may be a minor concern as a 7-day culture can still be envisaged in the clinical setting. In our opinion, the advantages afforded by this agent appear to override the drawbacks, making it an attractive target for future studies.

In conclusion, our results provide strong preclinical evidence that in vitro treatment with MTX, a practical FDA-approved agent, results in specific allodepletion and may have excellent potential as an effective approach for preventing or minimizing GVHD.

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Acknowledgments 

These studies were supported, in part, by research grant awards from the NIH, The Leukemia and Lymphoma Society, Leukemia Texas, and the Ryan Gibson Foundation.

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PII: S1083-8791(07)00122-X

doi:10.1016/j.bbmt.2007.01.081

Biology of Blood and Marrow Transplantation
Volume 13, Issue 6 , Pages 644-654, June 2007

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