The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rettig, M. P.
Right arrow Articles by DiPersio, J. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rettig, M. P.
Right arrow Articles by DiPersio, J. F.
The Journal of Immunology, 2004, 173: 3620-3630.
Copyright © 2004 by The American Association of Immunologists

Kinetics of In Vivo Elimination of Suicide Gene-Expressing T Cells Affects Engraftment, Graft-versus-Host Disease, and Graft-versus-Leukemia after Allogeneic Bone Marrow Transplantation1

Michael P. Rettig*, Julie K. Ritchey*, Julie L. Prior{dagger},{ddagger}, Jeffrey S. Haug*, David Piwnica-Worms{dagger},{ddagger} and John F. DiPersio2,*

* Division of Oncology, Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63110; and {dagger} Molecular Imaging Center, Mallinckrodt Institute of Radiology, and {ddagger} Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Suicide gene therapy is one approach being evaluated for the control of graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). We recently constructed a novel chimeric suicide gene in which the entire coding region of HSV thymidine kinase (HSV-tk) was fused in-frame to the extracellular and transmembrane domains of human CD34 ({Delta}CD34-tk). {Delta}CD34-tk is an attractive candidate as a suicide gene in man because of the ensured expression of HSV-tk in all selected cells and the ability to rapidly and efficiently purify gene-modified cells using clinically approved CD34 immunoselection techniques. In this study we assessed the efficacy of the {Delta}CD34-tk suicide gene in the absence of extended ex vivo manipulation by generating transgenic animals that express {Delta}CD34-tk in the peripheral and thymic T cell compartments using the CD2 locus control region. We found that {Delta}CD34-tk-expressing T cells could be purified to near homogeneity by CD34 immunoselection and selectively eliminated ex vivo and in vivo when exposed to low concentrations of GCV. The optimal time to administer GCV after allogeneic BMT with {Delta}CD34-tk-expressing transgenic T cells was dependent on the intensity of the conditioning regimen, the leukemic status of the recipient, and the dose and timing of T cell infusion. Importantly, we used a controlled graft-vs-host reaction to promote alloengraftment in sublethally irradiated mice and provide a graft-vs-leukemia effect in recipients administered a delayed infusion of {Delta}CD34-tk-expressing T cells. This murine model demonstrates the potential usefulness of {Delta}CD34-tk-expressing T cells to control GVHD, promote alloengraftment, and provide a graft-vs-leukemia effect in man.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Graft-vs-host disease (GVHD)3 remains a major cause of morbidity and mortality after allogeneic bone marrow (BM) transplantation (BMT) (1). Because GVHD is initiated by mature donor T cells present in the graft, it can be significantly reduced by pretransplant T cell depletion (TCD) (2). However, such depletion procedures fail to improve the overall survival of transplanted recipients because of increased rates of graft failure, opportunistic infections, EBV-associated lymphoproliferative disorders, and disease relapse (2). These complications arising from TCD have demonstrated the crucial role mature donor T cells have in facilitating engraftment and providing graft-vs-infection and graft-vs-leukemia (GVL) effects after BMT.

One approach to preserve the beneficial effects of allogeneic donor T cells in BMT is to genetically modify the T cells with a drug-inducible suicide gene that can be selectively activated should GVHD develop (3). Both preclinical (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and clinical (16, 17) studies have demonstrated that alloreactive T cells expressing a HSV thymidine kinase (HSV-tk) suicide gene can be eliminated by in vivo administration of the nucleoside analog ganciclovir (GCV). Recently, we (18) and others (19, 20) developed new chimeric suicide genes by fusing HSV-tk to the extracellular and transmembrane domains of human CD34 ({Delta}CD34-tk). The {Delta}CD34-tk chimeric suicide gene strategy offers two distinct advantages over previously used selection systems in HSV-tk/GCV suicide gene therapy of GVHD. First, {Delta}CD34-tk-modified cells can be rapidly and efficiently selected using a well-established and clinically approved CD34 immunoselection selection technique (21). Second, in contrast to the previously used dual promoter or internal ribosome entry sequence-based HSV-tk expression systems (16, 17, 22, 23), the {Delta}CD34-tk fusion gene strategy ensures the expression of the suicide gene in all CD34-selected cells.

Because the {Delta}CD34-tk chimeric suicide gene appears to be an attractive candidate as a cell surface marker/suicide gene in man, in this study we assessed the efficacy of the {Delta}CD34-tk suicide gene in a murine model of allogeneic BMT. Importantly, we found that murine GVHD could be mitigated by {Delta}CD34-tk/GCV suicide gene therapy. The optimal time to administer GCV was dependent on several variables, including the intensity of the conditioning regimen, the leukemic status of the recipient, and the dose and timing of T cell infusion. These observations provide an important proof of principle for the use of {Delta}CD34-tk-expressing T cells in allogeneic BMT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c (H-2d, CD45.2+) and C57BL/6 (B6; H-2b, CD45.2+) mice were obtained from Taconic Farms (Germantown, NY). Congenic B6 mice expressing the CD45.1 gene were purchased from The Jackson Laboratory (Bar Harbor, ME). Animal care and euthanasia were approved by the Washington University Medical School animal studies committee. For BMT, donors were 6–12 wk of age, and recipients were 6–8 wk of age.

Reagents

GCV (Cytovene; Roche, Nutley, NJ) was dissolved in double-distilled water and stored in 50 mg/ml aliquots at –20°C. Before use, GCV was thawed and diluted to 5 mg/ml in a 5% dextrose saline solution. GCV was administered i.p. at a dose of 50 mg/kg/day.

Generation of {Delta}CD34-tk transgenic mice

The plasmid pCR2.1-{Delta}CD34-tk(wt) has been described previously (18). The {Delta}CD34-tk gene was excised from pCR2.1-{Delta}CD34-tk(wt) with EcoRI and ligated to p{beta}G-CD2 (24) (provided Dr. T. Enver, Institute of Cancer Research, London, U.K.) to generate p{beta}G-CD2-{Delta}CD34-tk. A KpnI/NotI fragment of p{beta}G-CD2-{Delta}CD34-tk was injected into (B6xC3H)F1 blastocysts. Two positive founder lines were established, and the line expressing the highest level of the {Delta}CD34-tk transgene was backcrossed (F16-F18) onto B6 mice to obtain progeny for these studies.

Immunomagnetic selection of T cells

Splenocytes were obtained by disrupting splenic capsules with the blunt end of a syringe, and erythrocytes were removed by hypotonic lysis with 154 mmol/L ammonium chloride, 10 mmol/L potassium bicarbonate, and 0.1 mmol/L EDTA. Transgenic (Tg) T cells expressing {Delta}CD34-tk were positively selected as previously described (18). Non-Tg T cells were negatively selected using FITC-conjugated mAbs to CD11b, B220, and GR1 (BD Pharmingen, San Diego, CA) and anti-FITC microbeads (Miltenyi Biotech, Auburn, CA) according to the manufacturers’ instructions.

In vitro sensitivity to GCV

Flat-bottom, non-tissue culture-treated, 96-well plates (Falcon; BD Biosciences, Franklin Lakes, NJ) were precoated with 0.67 µg of anti-CD3 mAb and 0.08 µg of anti-CD28 mAb (BD Pharmingen). Cells (5 x 104) were cultured in triplicate at 37°C for 5 days in phenol red-free RPMI 1640 medium (Invitrogen Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 10 mmol/L nonessential amino acids, 10 mmol/L sodium pyruvate, 20 mmol/L L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 50 µmol/L 2-ME, IL-2 (50 U/ml), and increasing GCV concentrations. Cell viability was determined using an XTT kit (Sigma-Aldrich, St. Louis, MO) as previously described (18).

Immunogenicity of {Delta}CD34-tk

Immunoselected Tg (CD45.2+) and non-Tg (CD45.1+) T cells from B6 mice were labeled with CFSE as described by others (25). Unconditioned B6 recipient mice (CD45.1+) were immunized by three injections of a 1/1 mixture of the CFSE-labeled T cells (1.2–2 x 107 total cells/injection) on days 0, 57, and 154. The persistence of transferred cells in peripheral blood was examined by flow cytometry.

BMT

BM from B6 (CD45.1+) mice was flushed from femurs and tibias with PBS and passed through sterile mesh filters, and TCD was performed with mouse CD90 microbeads (Miltenyi Biotech). BALB/c recipient mice were conditioned with total body irradiation (TBI) administered as a single exposure (500, 700, or 900 cGy) using a Mark I cesium irradiator (J. L. Shepherd and Associates, San Fernando, CA). Irradiated recipients received a single i.v. injection of TCD BM (4 x 106 cells) with or without added T cells from Tg and non-Tg B6 (CD45.2+) donors within 24 h. Delayed donor lymphocyte infusions (DLI) were injected i.v. at the indicated times post-BMT. Recipients were monitored daily for survival, and total body weights were recorded every 3–4 days.

Construction of luc-egfp (luciferase-enhanced GFP) fusion gene

Luciferase was modified from pFR-Luc (Stratagene, La Jolla, CA) by QuikChange site-directed mutagenesis (Stratagene) using primers (forward, 5'-GGCGGAAAGTCCAAATTGGATCCAAAATGTAACTGTATTCAGCG-3' and a complimentary antisense primer) that eliminated the stop codon of the luc gene and introduced a BamHI restriction site. The modified firefly luciferase pFR-Luc was then digested with KpnI and BamHI, and the insert was ligated to the pEGFP-N1 vector (BD Clontech, Palo Alto, CA) to generate the pLuc-EGFP-N1 vector.

Leukemia model

BALB/c-derived A20 B cell lymphoma cells (American Type Culture Collection, Manassas, VA) were transfected with the pLuc-EGFP-N1 vector using the Lipofectamine Plus protocol (Invitrogen Life Technologies) and selected with 1 mg/ml G418. Stable transfectants expressing EGFP were sterile-sorted using a MoFlo cell sorter (DakoCytomation, Fort Collins, CO), and a single high expressing clone (A20-luc/egfp) was expanded. BALB/c recipients were conditioned with 900 cGy of TBI and reconstituted with TCD BM (4 x 106 cells) with or without added A20-luc/egfp cells (1–2 x 104 cells) within 24 h. Donor lymphocytes (2 x 106 Tg T cells) were administered day 10 post-BMT. Mice were considered leukemic if they exhibited luciferase activity significantly greater than background (see below), lower limb paralysis, or macroscopic signs of tumor at autopsy.

Bioluminescence imaging (BLI) of animals

A20-luc/egfp tumor growth and distribution were assessed noninvasively by BLI with an IVIS CCD camera (Xenogen, Alameda, CA) as previously described (26, 27). Briefly, mice were injected i.p. with D-luciferin (150 µg/g in PBS) and imaged 10 min later with the IVIS (1- to 60-s exposure; binning 8; f-stop 1; field of view, 15 cm). Anesthesia was induced and maintained during imaging by vaporizer delivery of 2–2.5% isoflurane. Total photon flux (photons per second) was quantified on images using a rectangular region of interest encompassing the entire abdomen and thorax. The leukemic status of all surviving animals was evaluated 30–50 days after DLI by BLI, and mice were considered leukemic if they exhibited luciferase activity greater than TCD BM-only controls.

FACS

Single-cell suspensions from peripheral blood or spleen were stained on ice for 30 min with FITC-, PE-, or PerCP-conjugated mAb against CD3, CD4, CD8, B220, CD11b, Gr-1, pan-NK, H-2Kb, H-2Kd, or CD45.2 (BD Pharmingen). Cell surface expression of the {Delta}CD34-tk chimeric suicide gene was evaluated using PE-conjugated anti-human CD34 Abs (CD34 Pool-PE; Immunotech, Marseilles, France). Control staining was performed using a PE-conjugated isotype control Ab. Cells were analyzed on a FACScan cytometer (BD Biosciences, Mountain View, CA).

Statistics

Kaplan-Meier survival curves were compared using the log-rank test. Comparison between groups was performed using unpaired Student’s t test. All statistical analyses were performed using StatView software (Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of {Delta}CD34-tk transgenic mice

Previous studies by others have shown that ex vivo manipulation of T cells compromises their in vivo function (12, 28, 29, 30). Therefore, to assess the efficacy of the {Delta}CD34-tk suicide gene in the absence of extended ex vivo manipulation, we generated Tg animals in which {Delta}CD34-tk was targeted to the peripheral and thymic T cell compartment using the CD2 locus control region. Greater than 95% of peripheral blood CD3+ T cells expressed the {Delta}CD34-tk transgene, with equal expression in both the CD4+ and CD8+ subsets (Fig. 1A). In contrast, no {Delta}CD34-tk expression was observed in B220+ B cells, Mac-1+ macrophages, Gr-1+ myeloid cells, Pan-NK+ NK cells, or c-Kit+, lineage marker–/low, Sca-1+ hemopoietic stem cells (Fig. 1A and data not shown). Similar to Tg mice expressing human CD34 under the control of the CD2 locus control region (24), we observed no alterations in the development, maturation, CD4 and CD8 subset composition, or distribution of Tg T cells. In addition, the Tg mice exhibited normal life spans, with no evidence of leukemia in >500 evaluable animals. Importantly, the {Delta}CD34-tk Tg T cells could be efficiently purified to >97% using a human CD34 cell isolation kit (Fig. 1B). This observation further demonstrates that murine T cells process the human CD34 molecule similarly to human hemopoietic cells.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 1. Characterization of Tg mice expressing {Delta}CD34-tk. A, Peripheral blood from {Delta}CD34-tk Tg (upper panels) and non-Tg (lower panels) B6 mice were labeled with a PE-conjugated Ab to human CD34. Samples were then split and labeled with FITC-conjugated mAbs specific for T cells (CD3, CD4, and CD8), B cells (B220), macrophages (Mac-1), granulocytes (Gr-1), or NK cells (Pan-NK) and analyzed by FACS. The percentage of cells in each quadrant is indicated. B, {Delta}CD34-tk-expressing T cells from the spleen of a Tg mouse were isolated by CD34 immunoselection using a VarioMACS magnetic cell separator (Miltenyi Biotech). The percentage of CD34-expressing cells in the isolated Tg T cells (positive sort) was determined by FACS. C, Unconditioned {Delta}CD34-tk Tg ({blacktriangledown} and •) and non-Tg ({triangledown} and {circ}) mice were injected i.p. with a solution of either dextrose ({blacktriangledown} and {triangledown}) or GCV (50 mg/kg/day; • and {circ}) for 7 days. The percentage of T cells in the spleens of mice was determined by FACS on the indicated days post-GCV treatment (n = 2–3/day). D, Immunoselected {Delta}CD34-tk Tg (•) and non-Tg ({circ}) T cells were stimulated with immobilized anti-CD3 and anti-CD28 mAbs for 7 days in the presence of GCV. Cell viability was determined using an XTT kit, and the absorbance for each well was recorded and expressed as a percentage of the value for control wells with no GCV added. Each point represents the mean ± SD of a single experiment, in which each sample was assayed in triplicate.

 
GCV sensitivity of {Delta}CD34-tk Tg T cells

To evaluate the GCV sensitivity of the Tg T cells in vivo under steady state conditions, we treated 12- to 16-wk-old Tg and non-Tg mice with GCV for 7 days and determined the percentage of T cells in the spleen 1–3 wk post-GCV administration by FACS. As shown in Fig. 1C, {Delta}CD34-tk Tg T cells were largely unaffected by in vivo exposure to GCV. Therefore, similar to normal mice, the majority of mature T cells in Tg mice are in a quiescent state. The nonspecific 10–15% reduction in the percentage of splenic T cells 3 wk post-GCV treatment of Tg and non-Tg mice was most likely caused by the immunosuppressive effects of GCV (31).

To confirm HSV-tk activity in the Tg T cells, {Delta}CD34-tk-selected spleen cells were activated in vitro with immobilized anti-CD3 and anti-CD28 mAb for 7 days in the presence of IL-2 and increasing GCV concentrations. Non-Tg T cells were largely unaffected by GCV exposure, except at high concentrations, at which GCV is nonspecifically toxic (Fig. 1D). In contrast, treatment of Tg T cells with GCV resulted in a progressive and selective reduction in the number of viable cells, with >90% of the killing occurring in the presence of 0.1 µM GCV. This observation demonstrates that cycling {Delta}CD34-tk Tg T cells are highly sensitive to GCV in vitro.

Evaluation of {Delta}CD34-tk immunogenicity

To evaluate whether {Delta}CD34-tk was immunogenic, we immunized unconditioned B6 recipient mice with three injections of a 1/1 mixture of CFSE-labeled T cells from Tg and non-Tg B6 mice. Peripheral blood samples were collected at different time points after immunization, and the percentage of CFSE-labeled cells was assessed by FACS (Fig. 2A). We observed no difference between the in vivo persistence of the Tg and non-Tg T cells in two of the three immunized mice (Fig. 2B, mice 1 and 2). In contrast, Tg cells disappeared rapidly after the second infusion of cells in mouse 3. However, these cells were not rapidly eliminated after the third injection of cells into mouse 3 (Fig. 2B). Therefore, this mouse must not have generated an immune response to the {Delta}CD34-tk protein. Furthermore, splenocytes obtained from the vaccinated mice did not show any {Delta}CD34-tk-specific cytotoxicity, indicating the absence of cytotoxic T cell responses to the {Delta}CD34-tk protein (data not shown). These results suggest that the {Delta}CD34-tk protein is not immunogenic in B6 mice.



View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 2. Evaluation of {Delta}CD34-tk immunogenicity. Unconditioned B6 recipient mice (CD45.1+) were immunized by three injections of a 1/1 mixture of CFSE-labeled T cells (1.2–2 x 107 total cells/injection) from non-Tg (CD45.1+) and {Delta}CD34-tk Tg (CD45.2+) B6 mice on days 0, 57, and 154. A, Peripheral blood from untreated (control) and immunized mice (mice 1–3) were collected at the indicated time points postinjection. Samples were split, labeled with mAbs specific for CD34 or CD45.2 and CD3, and analyzed by FACS. The percentage of CD3+ cells in each quadrant is indicated. CFSE-labeled {Delta}CD34-tk Tg CD3+ T cells are located in the upper right quadrants. B, Peripheral blood from the immunized mice were collected at different time points after injection, and the percentages of CFSE-labeled {Delta}CD34-tk Tg (•) and non-Tg ({circ}) CD3+ cells were assessed by FACS.

 
{Delta}CD34-tk Tg T cells induce lethal GVHD

To determine whether {Delta}CD34-tk Tg T cells retain their GVHD potential, BALB/c recipients were lethally irradiated (900 cGy) and given TCD BM supplemented with either Tg or non-Tg purified T cells. Untreated animals receiving 5 x 105 or 2 x 106 Tg T cells died of GVHD in a T cell dose-dependent manner, with median survivals of 77 days (Fig. 3A) and 23 days (Fig. 3B), respectively. Consistent with GVHD, untreated mice lost >30% of their pretransplant body weight (Fig. 3, C and D), were complete donor chimeras (Fig. 3, E and F), and exhibited impaired lymphoid reconstitution (Fig. 3G and data not shown). Importantly, mice transplanted with 2 x 106 non-Tg T cells exhibited weight loss and overall survival similar to the Tg T cells (Fig. 3, B and D). These data indicate that expression of the {Delta}CD34-tk suicide gene in murine T cells does not alter their ability to induce lethal GVHD in a fully MHC-mismatched BMT model.



View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 3. GCV treatment mitigates GVHD in lethally irradiated mice transplanted with {Delta}CD34-tk Tg T cells. BALB/c mice were conditioned with 900 cGy of TBI and reconstituted with TCD B6 BM (CD45.1+) supplemented with 5 x 105 (upper panels; n = 8/group) or 2 x 106 (lower panels; BMT only, n = 25; no GCV, n = 20; days 1–7, n = 20; days 1–5, n = 6; days 1–3, n = 13; days 4–10, n = 16; days 10–16, n = 6; non-Tg, n = 18; non-Tg and GCV, n = 7; irradiation (XRT) only, n = 6) purified B6 {Delta}CD34-tk Tg or non-Tg T cells (CD45.2+). Animals receiving {Delta}CD34-tk Tg T cells were then either left untreated (no GCV) or were treated with GCV (50 mg/kg/day i.p.) as indicated. Animals receiving non-Tg T cells were either left untreated (Non-Tg) or treated with GCV (50 mg/kg/day i.p.) from days 1–7 post-BMT (Non-Tg + GCV). Surviving animals and age-matched unconditioned (No TBI) BALB/c mice were bled at 30 and 100 days post-BMT and analyzed by FACS. A and B, Kaplan-Meier survival curves. C and D, Percent change in pretransplant body weight. E and F, Overall engraftment 30 days post-BMT. G and H, Lymphoid chimerism 100 days post-BMT. Results are pooled from three experiments. The Non-Tg + GCV control was performed in only a single experiment in the absence of a Non-Tg only control. Survival and weight change for irradiation controls (XRT only) are shown in A–D. *, p < 0.01 compared with mice that received {Delta}CD34-tk Tg T cells but were not treated with GCV (No GCV control). **, p < 0.01 compared with BMT only control. Values of p in C and D were determined at 30, 60, and 90 days post-BMT.

 
Increased survival of 900 cGy-conditioned mice treated with GCV after BMT with {Delta}CD34-tk Tg T cells

To evaluate the ability of GCV to prevent GVHD after BMT with {Delta}CD34-tk-expressing T cells, we administered GCV beginning 1, 4, or 10 days after transplantation. GCV treatment significantly prolonged the survival of all groups compared with untreated mice (Fig. 3, A and B). Animals treated from days 1–7 had an overall survival rate of ~85% (Fig. 3, A and B), gained weight at a rate comparable to controls receiving BM alone (Fig. 3, C and D), remained mixed chimeras (Fig. 3, E and F), and exhibited normal lymphoid recovery (Fig. 3, G and H). We observed a similar prevention of GVHD when GCV was administered from days 1–3 or 1–5 post-BMT (Fig. 3, B, D, F, and H). Importantly, mice transplanted with non-Tg T cells and treated with GCV from days 1–7 post-BMT were not protected from GVHD. Taken together, these results indicate that GVHD initiated by allogeneic T cells expressing the {Delta}CD34-tk transgene is prevented by the early addition of GCV.

Although there were no statistically significant differences in survival between the different GCV administration schedules after conditioning with 900 cGy (Fig. 3, A and B), delaying GCV administration until day 4 or 10 post-BMT increased the severity of GVHD in a T cell dose- and GCV schedule-dependent manner. Mice receiving the lower dose of Tg T cells (5 x 105 cells) and GCV from days 4–10 exhibited normal body weight recovery (Fig. 3C) and lymphoid reconstitution (Fig. 3G). In contrast, day 4–10 treated mice receiving the higher T cell dose (2 x 106 cells) exhibited signs of ongoing GVHD, as evidenced by significantly impaired body weight (Fig. 3D; p < 0.05 from day 20 post-BMT onward compared with BMT-only control). Interestingly, these mice initially exhibited significant weight gain after GCV administration. However, the protective effect of GCV on weight loss was not sustained, as indicated by the >10% loss of pretransplant body weight between days 20 and 35 post-BMT (Fig. 3D). A similar pattern of weight loss was observed in animals treated with GCV from days 10–16 regardless of the T cell dose (Fig. 3, C and D).

At 30 days post-BMT, the percentage of donor T cells that were CD45.2+ ({Delta}CD34-tk+) in untreated mice transplanted with 2 x 106 Tg T cells ranged from 0.6–15.8% and averaged 4.8% (data not shown). Although treatment with GCV decreased this percentage to <1.5%, the numbers of circulating Tg T cells at 30 days post-BMT were too small to accurately evaluate the effectiveness of the different GCV administration schedules to eliminate these cells. Therefore, we killed mice 1 day after the final dose of GCV was administered and determined the percentage of CD45.2+ donor T cells in the spleen by flow cytometry. As shown in Fig. 4, the percentage of Tg T cells in the spleen of untreated mice decreased from 50 to 24% between days 8 and 17 post-BMT. Treatment with a 7-day course of GCV beginning 1, 4, or 10 days post-BMT reduced the percentage of CD45.2+ donor T cells 30-, 4.6-, and 2.4-fold, respectively (Fig. 4). These results confirm that GCV administration can selectively eliminate {Delta}CD34-tk Tg T cells mediating a GVH reaction and that early administration of GCV is more effective in eliminating cells that cause GVHD.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 4. Early GCV administration is more effective in eliminating cells that cause GVHD. Lethally irradiated (900 cGy) BALB/c mice were transplanted with TCD B6 BM supplemented with 2 x 106 purified {Delta}CD34-tk Tg T cells. Animals receiving T cells were then either left untreated (No GCV) or were treated with GCV (50 mg/kg/day i.p.) as indicated. Animals were killed 1 day after the final dose of GCV was administered, and the percentage of {Delta}CD34-tk Tg T cells in the spleen was analyzed by FACS (n = 1 for untreated; n = 2 for GCV treated).

 
Early GCV administration prevents donor engraftment in reduced intensity conditioning regimens

Because the intensity of the conditioning regimen affects the severity of GVHD (32), we reduced the TBI dose by increments of 200 cGy and evaluated the abilities of various GCV administration schedules to prevent GVHD and graft rejection after BMT with {Delta}CD34-tk-expressing Tg T cells. Eight of 14 recipients conditioned with 700 cGy of TBI and transplanted with TCD BM completely rejected the grafts, whereas the six remaining mice all exhibited <25% donor cell engraftment (Fig. 5E). Supplementing the TCD BM with 2 x 106 Tg T cells facilitated complete donor cell engraftment (Fig. 5E), but all mice died from GVHD (Fig. 5A). Although treatment with GCV significantly improved the overall survival of mice transplanted with Tg T cells compared with untreated animals (Fig. 5A; p < 0.01 for all GCV-treated groups compared with untreated mice), not all mice engrafted. As shown in Fig. 5E, eight of 11 mice transplanted with Tg T cells and treated with GCV from days 1–7 failed to engraft. This failure to engraft was dependent upon the expression of {Delta}CD34-tk, because similarly treated mice transplanted with non-Tg T cells fully engrafted and developed lethal GVHD.



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 5. Early GCV administration prevents alloengraftment in reduced intensity conditioning regimens. BALB/c mice were conditioned with 700 cGy (upper panels; BM only, n = 16; no GCV, n = 16; days 1–7, n = 17; days 4–10, n = 8; days 10–16, n = 8; days 20–26, n = 8; non-Tg and GCV, n = 8; irradiation (XRT) only, n = 3) or 500 cGy (lower panels; BM only, n = 16; no GCV, n = 17; days 1–7, n = 16; days 4–10, n = 8; days 10–16, n = 8; days 20–26, n = 8; non-Tg and GCV, n = 7; XRT only, n = 3) TBI and reconstituted with TCD B6 BM (CD45.1+) supplemented with 2 x 106 purified B6 {Delta}CD34-tk Tg or non-Tg T cells (CD45.2+). Animals receiving {Delta}CD34-tk Tg T cells were then either left untreated (No GCV) or were treated with GCV (50 mg/kg/day i.p.) as indicated. Animals receiving non-Tg T cells were treated with GCV (50 mg/kg/day i.p.) from days 1–7 post-BMT (Non-Tg + GCV). Surviving animals and age-matched unconditioned (No TBI) BALB/c mice were bled at 30 and 100 days post-BMT and analyzed by FACS. A and B, Kaplan-Meier survival curves. C and D, Percent change in pretransplant body weight. E and F, Overall engraftment 30 days post-BMT. G and H, Lymphoid chimerism 100 days post-BMT. Results are pooled from two experiments. Data from the irradiation controls (XRT only) are shown in A–F. *, p < 0.01 compared with mice that received {Delta}CD34-tk Tg T cells but were not treated with GCV (No GCV control). **, p < 0.01 compared with BMT only control. Values of p in C and D were determined at 30, 60, and 90 days post-BMT.

 
Although delayed administration of GCV facilitated engraftment and improved overall survival, mice conditioned with 700 cGy of TBI and treated with GCV from days 4–10, 10–16, or 20–26 exhibited significant weight loss compared with animals receiving BM alone (Fig. 5C; p < 0.01 from days 23–72 post-BMT for day 10–16 GCV-treated group compared with BMT-only control). Similar to mice conditioned with 900 cGy of TBI, this weight loss was progressively more severe as GCV treatment was delayed. It should be noted that the >30% increase in body weight of day 10–16 treated animals between days 50 and 120 post-BMT was primarily due to the death of three mice with severe GVHD. In addition to the weight loss, day 20–26 GCV-treated mice exhibited significantly impaired lymphoid reconstitution (Fig. 5G). These results further demonstrate that GVHD is not completely prevented after delayed administration of GCV.

Animals conditioned with 500 cGy of TBI failed to engraft unless the TCD BM was supplemented with 2 x 106 Tg T cells (Fig. 5, F and H). Reducing the donor T cell dose 4-fold, to 5 x 105 cells, resulted in graft failure (data not shown). Interestingly, not all untreated mice receiving 500 cGy of TBI and 2 x 106 Tg T cells died from GVHD (Fig. 6B). In fact, compared with mice conditioned with 900 or 700 cGy of TBI, untreated animals irradiated with 500 cGy of TBI exhibited significantly delayed mortality (Fig. 5B; p < 0.0001 compared with other TBI doses) and enhanced body weights (Fig. 5D) after {Delta}CD34-tk Tg T cell transfer.



View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 6. Effect of GCV administration on prevention of GVHD after delayed DLI of {Delta}CD34-tk Tg T cells. Lethally irradiated (900 cGy) BALB/c recipients were given TCD B6 BM (CD45.1+) and DLI of 2 x 106 (2e6) or 107 (1e7) purified B6 {Delta}CD34-tk Tg T cells (CD45.2+) on day 10 (upper panels; BMT only, n = 24; 2e6, no GCV, n = 20; 2e6, days 4–10, n = 13; 2e6, days 10–16, n = 8; 1e7, no GCV, n = 17; 1e7, days 4–10, n = 8; 1e7, days 10–16, n = 7) or day 20 (lower panels; BMT only, n = 21; 2e6, no GCV, n = 15; 2e6, days 4–10, n = 8; 2e6, days 10–16, n = 8; 1e7, no GCV, n = 12; 1e7, days 4–10, n = 8; 1e7, days 10–16, n = 8) post-BMT. Animals receiving T cells were then either left untreated (No GCV) or were treated with GCV (50 mg/kg/day i.p.) as indicated. Surviving animals and age-matched unconditioned (No TBI) BALB/c mice were bled at 30 and 100 days post-BMT and analyzed by FACS. A and B, Kaplan-Meier survival curves. C and D, Percent change in pretransplant body weight. E and F, Overall engraftment 30 days post-BMT. G and H, Lymphoid chimerism 100 days post-BMT. Results are pooled from three experiments. *, p < 0.01 compared with mice that received {Delta}CD34-tk Tg T cells but were not treated with GCV (No GCV control). **, p < 0.01 compared with BMT only control. Values of p in C and D were determined at 30, 60, and 90 days post-BMT.

 
The schedule of GCV administration also affected donor cell engraftment in mice conditioned with 500 cGy of TBI. Mice transplanted with {Delta}CD34-tk Tg T cells and treated with GCV from days 1–7 or 4–10 failed to engraft, whereas all the day 10–16 and day 20–26 GCV-treated mice had >85% donor cell engraftment (Fig. 5F). Again, the failure of mice to engraft after early GCV treatment was dependent upon the expression of {Delta}CD34-tk, because all but one mouse transplanted with non-Tg T cells and treated with GCV from days 1–7 post-BMT exhibited complete donor engraftment (Fig. 5F). It is important to note that the predominantly host lymphoid chimerism displayed in Fig. 5H for the non-Tg group treated with GCV is caused by the survival of the single mouse that failed to engraft.

Because reducing the irradiation dose to 500 cGy significantly reduced the severity of GVHD, we failed to observe a significant difference in survival upon treatment of engrafted recipients with GCV (Fig. 5B). However, both day 10–16 and day 20–26 GCV-treated mice exhibited significantly improved body weight recovery compared with the untreated mice (Fig. 5D; p < 0.05 from days 27–64 post-BMT for day 10–16 and day 20–26 GCV-treated groups compared with no GCV control). These results indicate that the severity of GVHD is reduced after delayed administration of GCV to 500 cGy TBI-conditioned mice.

Prevention of GVHD after delayed DLI of {Delta}CD34-tk Tg T cells

Delayed DLI are used to enhance GVL activity and increase the level of donor chimerism after allogeneic BMT (33). To evaluate the effectiveness of various GCV administration schedules to prevent GVHD after delayed DLI, lethally irradiated (900 cGy) BALB/c recipients were given TCD BM and DLI of 2 x 106 or 107 purified Tg T cells on day 10, 20, or 70 post-BMT. Similar to findings reported by others (34, 35, 36, 37, 38), we observed that the development of GVHD was dependent upon the timing of the DLI. Untreated animals receiving a DLI of 2 x 106 or 107 T cells on day 10 post-BMT died of GVHD, with median survivals of 21 and 15 days, respectively (Fig. 6A), similar to mice who received T cells the day of BMT (Fig. 3B). Consistent with GVHD, day 10 DLI mice lost >20% of their pretransplant body weight (Fig. 6C; p < 0.0001 for both untreated groups compared with BMT-only control) and had impaired lymphoid reconstitution (Fig. 6G). In contrast, the development of GVHD in untreated day 20 DLI recipients was less severe and T cell dose dependent. Mice receiving the higher dose of cells (107) on day 20 post-BMT exhibited a median survival of 24 days, similar to day 10 DLI recipients, and lost >20% of their pretransplant body weight (Fig. 6D). However, mice receiving the lower dose of cells (2 x 106) on day 20 post-BMT exhibited an overall survival (Fig. 6B) and lymphoid reconstitution (Fig. 6H) similar to those in the TCD BMT-only controls (p > 0.9 compared with BMT-only control). All untreated day 10 and day 20 DLI recipients converted from mixed T cell chimerism to full donor chimerism within 30 days post-DLI (Fig. 6, E and F). In contrast, we observed mixed T cell chimerism and no GVHD in recipients that received DLI 70 days post-BMT (data not shown).

To evaluate the effectiveness of GCV in mitigating delayed DLI-induced GVHD, we treated BMT recipients with a 7-day course of GCV beginning 4 or 10 days post-DLI. Day 10 DLI recipients (2 x 106 or 107 T cells) treated with GCV from days 4–10 post-DLI exhibited overall survival (Fig. 6A), weight gain (Fig. 6C), and lymphoid reconstitution (Fig. 6G) that were similar to those of the TCD BMT-only controls. Importantly, the level of donor chimerism increased in these mice, indicating that a controlled GVH reaction was achieved (Fig. 6E; p < 0.01 for both T cell doses compared with BMT-only control). Delaying GCV administration for 10 days after the day 10 DLI resulted in mortality (Fig. 6A; p < 0.0001 for both T cell doses compared with BMT-only control), weight loss (Fig. 6C; p < 0.01 from day 20 post-BMT onward for both T cell doses compared with BMT-only control), and impaired lymphoid reconstitution (Fig. 6G) similar to those in untreated mice.

In contrast to the day 10 DLI recipients, both GCV administration schedules reduced the severity of GVHD in day 20 DLI recipients infused with 107 T cells (Fig. 6, B and D). However, early administration of GCV (days 4–10 post-DLI) to day 20 DLI recipients prevented the conversion to full donor chimerism (Fig. 6F). Taken together, these observations suggest that the optimal GCV administration schedule in a DLI setting using HSV-tk-modified T cells is dependent upon the T cell dose and timing of DLI.

Delayed administration of GCV is required for maintenance of a GVL effect after suicide gene therapy of GVHD

We next evaluated whether delayed DLI of {Delta}CD34-tk-expressing T cells and treatment with GCV could provide a GVL effect in the absence of GVHD. To induce leukemia, BALB/c mice were lethally irradiated and reconstituted with TCD BM and A20-luc/egfp cells. Ten days after BMT, we administered a DLI of 2 x 106 Tg T cells and assessed tumor growth at various time intervals by BLI. Eight of 10 recipients who received TCD BM and A20-luc/egfp cells exhibited tumor engraftment and growth (data not shown). Importantly, GCV had no inhibitory effect on the growth of A20-luc/egfp cells in vivo (data not shown). As previously described by others (39, 40), we observed homing of the A20 cells to the BM, spleen, liver, mesenteric lymph nodes, and spinal cord, with no evidence of luciferase or EGFP immunogenicity (Fig. 7A and data not shown).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 7. GVL effect obtained by delaying GCV administration after day 10 DLI of {Delta}CD34-tk Tg T cells. Lethally irradiated (900 cGy) BALB/c recipients were given TCD B6 BM (CD45.1+) with (A20; n = 10) or without (BMT only; n = 6) added A20-luc/egfp cells (1–2 x 104 cells). Donor lymphocytes (CD45.2+; 2 x 106 {Delta}CD34-tk Tg T cells) were administered 10 days post-BMT. Animals receiving T cells were then either left untreated (A20 + DLI; n = 10) or were treated with GCV (50 mg/kg/day i.p.) as indicated (days 1–7, n = 10; days 4–10, n = 12; days 10–16, n = 10). Surviving animals were bled at 30 and 100 days post-BMT and analyzed by FACS. Tumor growth was assessed at various time intervals by BLI using a cooled CCD optical system (Xenogen IVIS). A, BLI of Luc activity. Representative images of mice transplanted with BM only or treated with GCV from days 10–16 post-DLI. Photon flux is indicated in the color scale bars. Mice 1 and 2 in the day 10–16 GCV treatment group had tumor signal above background. Mouse 3 did not exhibit significant tumor signal and is representative of the remaining seven mice in the day 10–16 GCV treatment group. B, Kaplan-Meier survival curve. C, Percent change in pretransplant body weight. D, Overall engraftment 30 days post-BMT. E, Lymphoid chimerism 100 days post-BMT. Results are pooled from two experiments. *, p < 0.01 compared with A20 and DLI control. **, p < 0.01 compared with BMT only control. Values of p in C were determined at 30, 60, and 990 days post-BMT.

 
In contrast to leukemic recipients who received only TCD BM, we found no evidence of leukemia in untreated mice that received a DLI 10 days after BMT. However, these animals developed GVHD with high mortality (Fig. 7B; p = 0.033 compared with BMT-only control) and significant weight loss (Fig. 7C; p < 0.05 from days 8–65 post-BMT compared with BMT-only control). To evaluate the ability of GCV to prevent this DLI-induced GVHD and maintain a GVL effect, we administered GCV beginning 1, 4, or 10 days post-DLI. As expected, both day 1–7 and day 4–10 treated leukemic animals were protected from GVHD (Fig. 7, C–E). However, 60% of the day 1–7 treated mice and 58% of the day 4–10 treated animals developed leukemia, with an overall survival rate not significantly different than the leukemia controls (Fig. 7B). In contrast, only two of 10 mice developed leukemia if GCV treatment was delayed until day 10 post-DLI (Fig. 7, A and B). Importantly, this GVL effect was obtained in the absence of GVHD, as evidenced by the improved survival (Fig. 7B) and weight gain (Fig. 7B) of the day 10–16 GCV-treated mice compared with the untreated controls (A20+ DLI).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we demonstrated, for the first time, that murine GVHD could be mitigated by {Delta}CD34-tk/GCV suicide gene therapy. To date, clinical trials using HSV-tk-modified T cells to control GVHD in allogeneic BMT have selected gene-modified cells using either the neomycin phosphostransferase gene and G418, or {Delta}LNGFR and immunomagnetic selection (16, 17, 41). Unfortunately, both these selection strategies have disadvantages that limit their clinical usefulness. G418 selection of neomycin phosphotransferase-transduced T cells requires prolonged culture periods and impairs T cell alloreactivity (16, 17, 41, 42, 43). Although both these limitations were overcome with the development of the {Delta}LNGFR selection marker (23), this approach is still limited by the independent expression of {Delta}LNGFR and HSV-tk and the lack of a clinically approved isolation system for {Delta}LNGFR-modified cells. As mentioned previously, the {Delta}CD34-tk chimeric suicide gene strategy offers two main advantages over the neomycin phosphotransferase and {Delta}LNGFR selection approaches. First, fusing HSV-tk to {Delta}CD34 ensures the expression of the suicide gene in all selected cells. Second, {Delta}CD34-tk-modified cells can be rapidly and efficiently selected using a well-established and clinically approved CD34 immunoselection selection technique (21).

Multiple preclinical studies by others have demonstrated that GCV administration can prevent acute GVHD induced by allogeneic HSV-tk-expressing T cells after myeloablative BMT (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). The level of protection from GVHD in these studies has been primarily dependent upon the degree of MHC incompatibility between the donor and the recipient and the GCV administration schedule. In general, early administration of GCV (before day 3) prevented GVHD, whereas delayed GCV treatment reduced the severity of the disease. We observed a similar trend in GVHD protection in this study. Lethally irradiated (900 cGy) mice transplanted with {Delta}CD34-tk Tg T cells and treated with GCV beginning 4 or 10 days post-BMT exhibited significant and prolonged weight loss and impaired lymphoid reconstitution. This failure of delayed GCV treatment to prevent GVHD is most likely caused by multiple factors, including the inability of GCV to 1) cure recipient tissue (skin, intestine, and liver) damage that occurred before prodrug administration, 2) kill mature donor effector T cells that are not dividing or expressing sufficient levels of HSV-tk at the time of GCV administration, and 3) prevent ongoing tissue damage caused by inflammatory cytokines (IFN, TNF, IL-1, and IL-2) and secondary immune effector cells (i.e., mononuclear phagocytes and NK cells). Whatever the explanation for the failure of delayed GCV treatment to prevent GVHD, the most important observation in this study is that murine GVHD could be mitigated by {Delta}CD34-tk/GCV suicide gene therapy.

One potential limitation with the use of {Delta}CD34-tk-modified T cells in allogeneic BMT may be the development of a host immune response to the {Delta}CD34-tk chimeric protein. Because of its human origin, the {Delta}CD34 Ag should not be immunogenic. However, some patients have developed CD8-mediated immune responses to HSV-tk (16, 23, 44, 45). Interestingly, the development of immunity to HSV-tk-modified donor T cells has been inconsistent in allogeneic transplant recipients. In one study, eight of 24 patients developed HSV-tk-specific immunity after delayed infusion of genetically modified T cells (45). In contrast, Tiberghein et al. (17) found no evidence of HSV-tk immunity when genetically modified T cells were administered at the time of BMT. In this study we observed no immune response against the {Delta}CD34-tk suicide gene upon repeated infusion of Tg T cells into immunocompetent B6 mice. Although the exact mechanism responsible for the induction of tolerance to HSV-tk-modified T cells in our studies and some human allogeneic recipients remains unknown, initial results from the two clinical studies (16, 17) have clearly demonstrated that any concerns associated with the risk of HSV-tk immunogenicity are diminished by the ability to control GVHD.

A recent advancement in BMT has been the development of reduced intensity conditioning regimens (46). Although these regimens have demonstrated clinical benefits, GVHD remains a significant problem (47, 48, 49, 50, 51, 52, 53). Similar to a recent report by Drobyski et al. (8), we observed that a controlled GVH reaction could facilitate alloengraftment in the absence of lethal GVHD after nonmyeloablative BMT. At a TBI exposure of 700 cGy, GCV treatment had to be delayed 4 days to achieve engraftment and initiated before day 20 post-BMT to protect recipients from GVHD. In contrast, mice conditioned with 500 cGy of TBI required a longer GVH reaction for engraftment to be achieved. All 500 cGy conditioned recipients treated with GCV beginning 4 days post-BMT failed to engraft, whereas mice treated with GCV beginning 10 or 20 days post-BMT engrafted and were protected from GVHD. These data further support the need for clinical evaluation of HSV-tk-expressing allogeneic T cells in reduced intensity conditioning regimens.

In contrast to preclinical studies in mice (34, 35, 36, 37, 38), GVHD remains the most significant complication after delayed administration of donor lymphocytes in humans (33, 53, 54). Although initial clinical data from the study by Bonini et al. (16) demonstrated the ability of the HSV-tk/GCV suicide gene system to control GVHD after delayed DLI, no preclinical studies have evaluated the effectiveness of various GCV administration schedules to prevent GVHD and preserve a GVL effect after delayed administration of HSV-tk-expressing donor lymphocytes. In this study we found that the optimal GCV administration schedule for prevention of GVHD after delayed DLI was dependent upon the timing of the DLI and the leukemic status of the recipient. In nonleukemic mice, treatment with GCV from days 4–10 post-DLI protected both day 10 and day 20 DLI recipients from lethal GVHD, whereas day 10–16 GCV administration failed to prevent GVHD in day 10 DLI recipients. In contrast, we observed no GVHD in leukemic recipients who were treated with GCV from days 10–16 after a day 10 DLI. Why donor T cells preferentially targeted the A20 cells as opposed to host tissues in leukemic recipients is an interesting question that requires further investigation.

Two recent reports demonstrated that HSV-tk/GCV suicide gene therapy could be used to dissociate GVL activity from GVHD after allogeneic BMT (14, 15). Although the kinetics of GCV treatment required to obtain a GVL effect were slightly different between the two studies, both reports showed that the GVL effect was lost if GCV treatment was initiated at or close to the time of BMT. Our results have extended these observations by demonstrating that appropriately timed administration of GCV after delayed DLI of HSV-tk-modified T cells retains a GVL effect while controlling GVHD. Importantly, this was the first study to use in vivo BLI to assess disease burden after HSV-tk/GCV suicide gene therapy. Using BLI, we found that eight of 10 mice treated with GCV from days 10–16 post-DLI were free of disease. In contrast, ~60% of mice treated with GCV from days 1–7 and 4–10 developed leukemia. This ability to noninvasively and repetitively image residual disease using each animal as its own control should facilitate the development of therapeutic interventions to treat relapse after suicide gene therapy of GVHD.

In summary, this study demonstrated that {Delta}CD34-tk-expressing Tg T cells function similar to HSV-tk-expressing Tg T cells after allogeneic BMT. This demonstration that the {Delta}CD34-tk suicide gene is functional in vivo represents an important proof-of principle for the development of clinical trials evaluating {Delta}CD34-tk-expressing T cells in allogeneic BMT.


   Acknowledgments
 
We thank Bill Eades (High Speed Cell Sorter Core, Washington University) for assistance with cell sorting.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants RO1CA83845 (to J.F.D.) and P50CA94056 (to D.P.-W.). Back

2 Address correspondence and reprint requests to Dr. John F. DiPersio, Washington University School of Medicine, Box 8007, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: jdipersi{at}im.wustl.edu Back

3 Abbreviations used in this paper: GVHD, graft-vs-host disease; BLI, bioluminescence imaging; BM, bone marrow; BMT, BM transplantation; DLI, donor lymphocyte infusion; EGFP/egfp, enhanced GFP; GCV, ganciclovir; GVL, graft-vs-leukemia; HSV-tk, HSV thymidine kinase; {Delta}LNGFR, truncated human low affinity receptor for nerve growth factor; LUC/luc, luciferase; TBI, total body irradiation; TCD, T cell depleted; Tg, transgenic. Back

Received for publication December 4, 2003. Accepted for publication July 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Appelbaum, F. R.. 2003. The current status of hematopoietic cell transplantation. Annu. Rev. Med. 54:491.[Medline]
  2. Ho, V. T., R. J. Soiffer. 2001. The history and future of T-cell depletion as graft-versus-host disease prophylaxis for allogeneic hematopoietic stem cell transplantation. Blood 98:3192.[Free Full Text]
  3. Springer, C. J., I. Niculescu-Duvaz. 2000. Prodrug-activating systems in suicide gene therapy. J. Clin. Invest. 105:1161.[Medline]
  4. Cohen, J. L., O. Boyer, B. Salomon, R. Onclercq, F. Charlotte, S. Bruel, G. Boisserie, D. Klatzmann. 1997. Prevention of graft-versus-host disease in mice using a suicide gene expressed in T lymphocytes. Blood 89:4636.[Abstract/Free Full Text]
  5. Cohen, J. L., S. Lacroix-Desmazes, F. Charlotte, L. Lejeune, P. J. Martin, D. Klatzmann, O. Boyer. 1999. Immunological defects after suicide gene therapy of experimental graft-versus-host disease. Hum. Gene Ther. 10:2701.[Medline]
  6. Cohen, J. L., M. F. Saron, O. Boyer, V. Thomas-Vaslin, B. Bellier, L. Lejeune, F. Charlotte, D. Klatzmann. 2000. Preservation of graft-versus-infection effects after suicide gene therapy for prevention of graft-versus-host disease. Hum. Gene Ther. 11:2473.[Medline]
  7. Cohen, J. L., O. Boyer, D. Klatzmann. 2001. Suicide gene therapy of graft-versus-host disease: immune reconstitution with transplanted mature T cells. Blood 98:2071.[Abstract/Free Full Text]
  8. Drobyski, W. R., H. C. Morse, III, W. H. Burns, J. T. Casper, G. Sandford. 2001. Protection from lethal murine graft-versus-host disease without compromise of alloengraftment using transgenic donor T cells expressing a thymidine kinase suicide gene. Blood 97:2506.[Abstract/Free Full Text]
  9. Helene, M., V. Lake-Bullock, J. S. Bryson, C. D. Jennings, A. M. Kaplan. 1997. Inhibition of graft-versus-host disease: use of a T cell-controlled suicide gene. J. Immunol. 158:5079.[Abstract]
  10. Kornblau, S. M., I. Stiouf, V. Snell, D. Przepiorka, L. C. Stephens, R. Champlin, F. C. Marini, III. 2001. Preemptive control of graft-versus-host disease in a murine allogeneic transplant model using retrovirally transduced murine suicidal lymphocytes. Cancer Res. 61:3355.[Abstract/Free Full Text]
  11. Liu, J., B. E. Anderson, M. E. Robert, J. M. McNiff, S. G. Emerson, W. D. Shlomchik, M. J. Shlomchik. 2001. Selective T-cell subset ablation demonstrates a role for T1 and T2 cells in ongoing acute graft-versus-host disease: a model system for the reversal of disease. Blood 98:3367.[Abstract/Free Full Text]
  12. Weijtens, M., A. van Spronsen, A. Hagenbeek, E. Braakman, A. Martens. 2002. Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats. Hum. Gene Ther. 13:187.[Medline]
  13. Contassot, E., C. Ferrand, R. Angonin, J. L. Cohen, M. de Carvalho Bittencourt, F. Lorchel, V. Laithier, J. Y. Cahn, D. Klatzmann, et al 2000. Ganciclovir-sensitive acute graft-versus-host disease in mice receiving herpes simplex virus-thymidine kinase-expressing donor T cells in a bone marrow transplantation setting. Transplantation 69:503.[Medline]
  14. Litvinova, E., S. Maury, O. Boyer, S. Bruel, L. Benard, G. Boisserie, D. Klatzmann, J. L. Cohen. 2002. Graft-versus-leukemia effect after suicide-gene-mediated control of graft-versus-host disease. Blood 100:2020.[Abstract/Free Full Text]
  15. Drobyski, W. R., M. Gendelman, S. Vodanovic-Jankovic, J. Gorski. 2003. Elimination of leukemia in the absence of lethal graft-versus-host disease after allogenic bone marrow transplantation. J. Immunol. 170:3046.[Abstract/Free Full Text]
  16. Bonini, C., G. Ferrari, S. Verzeletti, P. Servida, E. Zappone, L. Ruggieri, M. Ponzoni, S. Rossini, F. Mavilio, C. Traversari, et al 1997. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276:1719.[Abstract/Free Full Text]
  17. Tiberghien, P., C. Ferrand, B. Lioure, N. Milpied, R. Angonin, E. Deconinck, J. M. Certoux, E. Robinet, P. Saas, B. Petracca, et al 2001. Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft. Blood 97:63.[Abstract/Free Full Text]
  18. Rettig, M. P., J. K. Ritchey, T. E. Meyerrose, J. S. Haug, J. F. DiPersio. 2003. Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease. Mol. Ther. 8:29.[Medline]
  19. Fehse, B., O. S. Kustikova, Z. Li, A. Wahlers, W. Bohn, W. R. Beyer, D. Chalmers, P. Tiberghien, K. Kuhlcke, A. R. Zander, et al 2002. A novel ‘sort-suicide’ fusion gene vector for T cell manipulation. Gene Ther. 9:1633.[Medline]
  20. Junker, K., U. Koehl, S. Zimmerman, S. Stein, D. Schwabe, T. Klingebiel, M. Grez. 2003. Kinetics of cell death in T lymphocytes genetically modified with two novel suicide fusion genes. Gene Ther. 10:1189.[Medline]
  21. Schumm, M., P. Lang, G. Taylor, S. Kuci, T. Klingebiel, H. J. Buhring, A. Geiselhart, D. Niethammer, R. Handgretinger. 1999. Isolation of highly purified autologous and allogeneic peripheral CD34+ cells using the CliniMACS device. J. Hematother. 8:209.[Medline]
  22. Gallardo, H. F., C. Tan, M. Sadelain. 1997. The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes. Gene Ther. 4:1115.[Medline]
  23. Verzeletti, S., C. Bonini, S. Marktel, N. Nobili, F. Ciceri, C. Traversari, C. Bordignon. 1998. Herpes simplex virus thymidine kinase gene transfer for controlled graft-versus-host disease and graft-versus-leukemia: clinical follow-up and improved new vectors. Hum. Gene Ther. 9:2243.[Medline]
  24. Healy, L., G. May, K. Gale, F. Grosveld, M. Greaves, T. Enver. 1995. The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion. Proc. Natl. Acad. Sci. USA 92:12240.[Abstract/Free Full Text]
  25. Wells, A. D., H. Gudmundsdottir, L. A. Turka. 1997. Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J. Clin. Invest. 100:3173.[Medline]
  26. Luker, G. D., J. P. Bardill, J. L. Prior, C. M. Pica, D. Piwnica-Worms, D. A. Leib. 2002. Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice. J. Virol. 76:12149.[Abstract/Free Full Text]
  27. Luker, G. D., C. M. Pica, J. Song, K. E. Luker, D. Piwnica-Worms. 2003. Imaging 26S proteasome activity and inhibition in living mice. Nat. Med. 9:969.[Medline]
  28. Contassot, E., W. Murphy, R. Angonin, J. J. Pavy, M. C. Bittencourt, E. Robinet, C. W. Reynolds, J. Y. Cahn, P. Herve, P. Tiberghien. 1998. In vivo alloreactive potential of ex vivo-expanded primary T lymphocytes. Transplantation 65:1365.[Medline]
  29. Drobyski, W. R., D. Majewski, K. Ozker, G. Hanson. 1998. 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. 161:2610.[Abstract/Free Full Text]
  30. Tan, R., X. Xu, G. S. Ogg, P. Hansasuta, T. Dong, T. Rostron, G. Luzzi, C. P. Conlon, G. R. Screaton, A. J. McMichael, et al 1999. Rapid death of adoptively transferred T cells in acquired immunodeficiency syndrome. Blood 93:1506.[Abstract/Free Full Text]
  31. Faulds, D., R. C. Heel. 1990. Ganciclovir: a review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections. Drugs 39:597.[Medline]
  32. Hill, G. R., J. L. M. Ferrara. 2000. The primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation. Blood 95:2754.[Abstract/Free Full Text]
  33. Luznik, L., E. J. Fuchs. 2002. Donor lymphocyte infusions to treat hematologic malignancies in relapse after allogeneic blood or marrow transplantation. Cancer Control 9:123.[Medline]
  34. Billiau, A. D., S. Fevery, O. Rutgeerts, W. Landuyt, M. Waer. 2002. Crucial role of timing of donor lymphocyte infusion in generating dissociated graft-versus-host and graft-versus-leukemia responses in mice receiving allogeneic bone marrow transplants. Blood 100:1894.[Abstract/Free Full Text]
  35. Blazar, B. R., C. J. Lees, P. J. Martin, R. J. Noelle, B. Kwon, W. Murphy, P. A. Taylor. 2000. Host T cells resist graft-versus-host disease mediated by donor leukocyte infusions. J. Immunol. 165:4901.[Abstract/Free Full Text]
  36. Johnson, B. D., R. L. Truitt. 1995. Delayed infusion of immunocompetent donor cells after bone marrow transplantation breaks graft-host tolerance allows for persistent antileukemic reactivity without severe graft-versus-host disease. Blood 85:3302.[Abstract/Free Full Text]
  37. Pelot, M. R., D. A. Pearson, K. Swenson, G. Zhao, J. Sachs, Y. G. Yang, M. Sykes. 1999. Lymphohematopoietic graft-vs.-host reactions can be induced without graft-vs.-host disease in murine mixed chimeras established with a cyclophosphamide-based nonmyeloablative conditioning regimen. Biol. Blood Marrow Transplant. 5:133.[Medline]
  38. Sykes, M., M. A. Sheard, D. H. Sachs. 1988. Graft-versus-host-related immunosuppression is induced in mixed chimeras by alloresponses against either host or donor lymphohematopoietic cells. J. Exp. Med. 168:2391.[Abstract/Free Full Text]
  39. Edinger, M., Y. A. Cao, M. R. Verneris, M. H. Bachmann, C. H. Contag, R. S. Negrin. 2003. Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging. Blood 101:640.[Abstract/Free Full Text]
  40. Levitsky, H. I., J. Montgomery, M. Ahmadzadeh, K. Staveley-O’Carroll, F. Guarnieri, D. L. Longo, L. W. Kwak. 1996. Immunization with granulocyte-macrophage colony-stimulating factor-transduced, but not B7-1-transduced, lymphoma cells primes idiotype-specific T cells and generates potent systemic antitumor immunity. J. Immunol. 156:3858.[Abstract]
  41. Link, C. J., Jr, R. K. Burt, A. E. Traynor, W. R. Drobyski, T. Seregina, J. P. Levy, L. Gordon, S. T. Rosen, W. H. Burns, B. Camitta, et al 1998. Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene for remission induction. HGTRI 0103. Hum. Gene Ther. 9:115.[Medline]
  42. Di Ianni, M., S. Di Florio, G. Venditti, C. Liberatore, F. Lucheroni, F. Falzetti, A. Terenzi, C. C. Stella, F. Spinozzi, P. Mannoni, et al 2000. T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection. Cancer Gene Ther. 7:920.[Medline]
  43. Sauce, D., M. Bodinier, M. Garin, B. Petracca, N. Tonnelier, A. Duperrier, J. V. Melo, J. F. Apperley, C. Ferrand, P. Herve, et al 2002. Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms. Blood 99:1165.[Abstract/Free Full Text]
  44. Riddell, S. R., M. Elliott, D. A. Lewinsohn, M. J. Gilbert, L. Wilson, S. A. Manley, S. D. Lupton, R. W. Overell, T. C. Reynolds, L. Corey, et al 1996. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat. Med. 2:216.[Medline]
  45. Roncarolo, M. G., M. K. Levings, P. Mangia, C. Bordignon, S. Marktel, C. Bonini, C. Traversari. 2000. Characterization and modulation of the undesirable T cell-mediated responses to transgenes. Mol. Ther. 5:1228a.
  46. Georges, G. E., R. Storb. 2003. Review of "minitransplantation:" nonmyeloablative allogeneic hematopoietic stem cell transplantation. Int. J. Hematol. 77:3.[Medline]
  47. Childs, R., E. Clave, N. Contentin, D. Jayasekera, N. Hensel, S. Leitman, E. J. Read, C. Carter, E. Bahceci, N. S. Young, et al 1999. Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses. Blood 94:3234.[Abstract/Free Full Text]
  48. Giralt, S., P. F. Thall, I. Khouri, X. Wang, I. Braunschweig, C. Ippolitti, D. Claxton, M. Donato, J. Bruton, A. Cohen, et al 2001. Melphalan and purine analog-containing preparative regimens: reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation. Blood 97:631.[Abstract/Free Full Text]
  49. McSweeney, P. A., D. Niederwieser, J. A. Shizuru, B. M. Sandmaier, A. J. Molina, D. G. Maloney, T. R. Chauncey, T. A. Gooley, U. Hegenbart, R. A. Nash, et al 2001. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects. Blood 97:3390.[Abstract/Free Full Text]
  50. Mielcarek, M., P. J. Martin, W. Leisenring, M. E. Flowers, D. G. Maloney, B. M. Sandmaier, M. B. Maris, R. Storb. 2003. Graft-versus-host disease after nonmyeloablative versus conventional hematopoietic stem cell transplantation. Blood 102:756.[Abstract/Free Full Text]
  51. Mineishi, S., Y. Kanda, T. Saito, K. Nakai, A. Makimoto, M. Kami, R. Tanosaki, H. Wakasugi, K. Tobinai, Y. Takaue. 2003. Impact of graft-versus-host disease in reduced-intensity stem cell transplantation (RIST) for patients with haematological malignancies. Br. J. Haematol. 121:296.[Medline]
  52. Slavin, S., A. Nagler, E. Naparstek, Y. Kapelushnik, M. Aker, G. Cividalli, G. Varadi, M. Kirschbaum, A. Ackerstein, S. Samuel, et al 1998. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 91:756.[Abstract/Free Full Text]
  53. Marks, D. I., R. Lush, J. Cavenagh, D. W. Milligan, S. Schey, A. Parker, F. J. Clark, L. Hunt, J. Yin, S. Fuller, et al 2002. The toxicity and efficacy of donor lymphocyte infusions given after reduced-intensity conditioning allogeneic stem cell transplantation. Blood 100:3108.[Abstract/Free Full Text]
  54. Collins, R., Jr, O. Shpilberg, W. Drobyski, D. Porter, S. Giralt, R. Champlin, S. Goodman, S. Wolff, W. Hu, C. Verfaillie, et al 1997. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J. Clin. Oncol. 15:433.