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Experimental Therapeutics |
Section of Molecular Hematology and Therapy, Department of Blood and Marrow Transplantation [S. M. K., I. S., V. S., R. C., F. C. M.] and Department of Veterinary Medicine and Surgery [L. C. S.], the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and the Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 [D. P.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AKR/J) was adapted. B10.BR splenic lymphocytes stimulated with concanavalin A and interleukin 2 were infected with a retrovirus containing the low-affinity nerve growth factor receptor (LNGFR) and the HSV-TK gene and immunomagnetically selected; these LNGFR+/TK+ allogeneic lymphocytes were then cotransplanted with 1 x 107 bone marrow cells into lethally irradiated AKR/J recipients. The LNGFR+/TK+ donor lymphocytes persisted in the peripheral circulation for 6 months in both syngeneic and allogeneic settings. Doses of 2 x 106 TK+ allogeneic lymphocytes produced GVHD with a severity and time course similar to that induced by naive lymphocytes. Survival of TK+ allogeneic lymphocyte-bearing mice was significantly improved (P = 0.01) when ganciclovir (GCV; 2 mg/day) was administered on days 7–13 post transplant by i.p. injection, demonstrating that GVHD could be prevented. Fluorescence-activated cell sorting analysis demonstrated 4-fold reduction but persistent circulation of LNGFR+ lymphocytes in mice treated with GCV at various time points 1–3 months after transplantation, demonstrating selective killing of GVHD-reactive cells. We conclude that retrovirally transduced LNGFR+/TK+ murine lymphocytes can be produced, persist after transplant, remain alloreactive, and can be killed by GCV administration, resulting in reduced GVHD. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability to selectively remove the GVHD-initiating T cell after it has been activated might permit control of GVHD with less toxicity than current immunosuppressive GVHD therapies. A promising strategy to achieve this relies on the insertion of a suicide gene into T lymphocytes prior to transplant, with the administration of a prodrug to induce T-lymphocyte suicide if GVHD arises. Numerous groups, including ours, have demonstrated the ability to use retroviruses to introduce the stable transduction of the HSV-TK gene into T lymphocytes ex vivo (6, 7, 8, 9) . The HSV-TK-transduced lymphocytes will convert the prodrug GCV to the toxic metabolite GCV-triphosphate (10) , which interferes with DNA and RNA transcription, culminating in cell death (11) . Several small pilot trials using suicidal lymphocytes clinically, either in the setting of donor lymphocyte infusion (12, 13, 14, 15) or as a T-cell add back to T-cell-depleted marrow at the time of the initial transplant (16) , have been performed. These studies have demonstrated proof of concept with reduction of GVHD after the administration of GCV (12 , 16) . However, the incidence of GVHD and GVL in these studies has been lower than expected, possibly because of decreased alloreactivity arising from alterations in the lymphocyte subset composition (9) . Consequently, the optimal dose of suicidal lymphocytes to use, optimal timing of suicide induction relative to GVHD development, completeness of suicide required to suppress GVHD, and the ability of these heavily manipulated cells to deliver a GVL effect remain unknown. Therefore, an animal model could provide experimental direction toward the effective clinical application of suicidal lymphocytes in humans.
Here we report the development of a murine model that exactly duplicates the process used to generate retrovirally transduced HSV-TK+ lymphocytes for human use. Additionally, we demonstrate that these gene-modified lymphocytes can be produced in sufficient numbers, survive transplantation into mice, and produce GVHD if left unchecked and that GVHD can be decreased by the administration of GCV.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20–25% of the total spleen cells). An alternative stimulation strategy using 500 units/ml IL-2 and irradiated (40 Gy) allogeneic splenic lymphocytes was also investigated. The stimulated lymphocytes resulting from this were used for the following retroviral infections.
Retroviral Production and Infection Protocol.
A retroviral vector (plasmid pLNGFR-Tkneo) and a transduced amphotropic producer cell line containing the LNGFR-TK/neo construct (generous gift from Dr. C. Bordignon; Refs. 17
, 18
) was used to create a stable murine virus-producing cell line. Briefly, GP+Am-12 packaging cells (provided by D. Markowitz and A. Banks; Ref. 19
) were electroporated with pLNGFR-Tkneo. After 48 h, the supernatant was collected and passed on to GP+E 86 cells. Infected cells were selected with 1 mg/ml G418 for 14 days (20)
. Neo-positive colonies were screened for LNGFR expression by FACS using an anti-LNGFR antibody (LN-12; Boehringer Mannheim, Indianapolis, IN). After ping/pong amplification (21)
, LNGFR+ colonies were selected and tested for high titer viral production and viral rearrangement. Clone 2 was selected for intact viral DNA; titers of 3 x 106 LNGFR+ colonies/ml were achieved, and a master cell stock was established. For T-lymphocyte transduction, irradiated (40 Gy) clone 2 cells were plated at 75% confluence in T-150 flasks (Falcon), and 0.5 x 106/ml stimulated lymphocytes were cocultured in the presence of 3 µg/ml polybrene for 5 days, according to the method of Baum and Ostertag (22)
. Alternatively, static retroviral infection of these stimulated murine lymphocytes (1 x 106 cells/ml) was performed using medium harvested from one 5-liter 24-h collection from clone 2 at an multiplicity of infection of 3–5 for 6 h in the presence of 10 µg/ml polybrene, added daily for 5 days. After the 5 days, the lymphocytes were collected in AIM-V and softly pelleted (800 rpm for 5 min at 4°C) before isolation of LNGFR+ cells. Of a critical nature for the success of either method of infection was that the viral supernatant must contain a very high serum concentration (25%) and 500 units/ml IL-2. Significant variance from the optimum lymphocyte medium routinely resulted in the lymphocyte population dying after 4–7 days.
Selection of LNGFR+ Murine Lymphocytes (TK+ T Cells).
Forty-eight h after the final retroviral infection, viable murine lymphocytes were isolated using a Ficoll-Hypaque gradient, washed twice, pelleted, and reacted with anti-LNGFR antibody at a concentration of 5 µg/107 cells. This reaction was performed in AIM-V (no serum) at room temperature. After 30 min, the cells were washed once with ice-cold PBS, reacted with an antimouse IgG microbead (Miltenyi Biotech, Auburn, Ca), and selected on Miltenyi Vario MACS columns according to the manufacturer’s protocol. Transduced LNGFR+/TK+ lymphocyte populations were >90% pure cells, and yields were typically three to four LNGFR+/TK+ lymphocytes for each starting T cell.
Mouse Model.
To demonstrate the use and application of suicidal lymphocytes, an existing murine model created by Truitt and Atasoylu was adapted (23
, 24
, 25)
. This model system uses MHC-identical mouse strains, the AKR/J (female) and the congenic B10.BR (male) strain, in a recipient-donor arrangement. Splenic lymphocytes taken from B10.BR males will cause GVDH in the recipient AKR/J females within 10–15 days post transplant in a dose-dependent manner. These mice are MHC-H-2 identical, and differ by only two minor antigens. Additionally, lymphocytes taken from AKR/J animals express the THY-1.1 antigen, whereas B10.BR lymphocytes express the THY-1.2 surface antigen, allowing monitoring of engraftment and subsequent chimerism.
BMT and Experimental GVHD.
Recipient AKR/J mice were irradiated on day 0 with 60Co (9 Gy) 4–6 h prior to transplantation. Donor B10.BR bone marrow cells were isolated fresh as described in Truitt and Atasoylu (23)
On day 0, AKR/J mice received injections, via the tail vein, of 1 x 107 B10.BR bone marrow cells and varying amounts of either fresh control or LNGFR+/TK+ cultured splenic lymphocytes. Some transplanted animals received injections of saline or GCV (Cytovene; Syntex, Boulder, CO) at 2 mg/kg i.p. daily starting on day 7 post transplant. These injections continued for 7 days.
Monitoring for GVHD.
Mice were monitored daily, and weights were recorded. Additionally, signs of morbidity and clinical signs of GVHD, i.e., weight loss, hunched appearance, skin rashes, erythematous tails/ears, or diarrhea, and mortality were noted. Kaplan-Meier survival curves were established for each group (26)
. Mice suffering from advanced disease were sacrificed following institutional animal care and use policies, and were considered a death in the analysis.
Histology.
Ear punch biopsies were performed when GVHD was suspected, and tissue samples from target GVHD organs (gut, skin, heart, lungs) were taken from moribund mice sacrificed at various days post transplant or at time of death. Fixed tissues were embedded in paraffin and 4–6-µm tissue sections were mounted on glass slides, stained, and evaluated by an in-house veterinary pathologist (L. C. S.) for clinical sign of engraftment and GVHD.
Engraftment.
Engraftment of donor cells following induction of GVHD was assessed by flow cytometric analysis of peripheral blood taken from tail vein bleeds (200 µl) at various days post transplant. Three-color flow cytometry was performed by staining the peripheral lymphocytes with a FITC-labeled monoclonal antibody against CD3 (145-2C11; PharMingen, San Diego, CA,), a phycoerythrin-labeled monoclonal antibody against Thy-1.1 (OX-7; PharMingen), and a Cy-5-labeled antibody against Thy-1.2. (53-2.1; PharMingen). Normal AKR/J (Thy-1.1) and B10.BR (Thy-1.2) splenic lymphocytes were used as controls.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 1
, the number of the murine lymphocytes grew slowly over the first 4 days, at which point the lymphocytes that received 500 units/ml IL-2 grew the fastest. Lymphocyte expansion was higher and more rapid when ConA was combined with IL-2, although there was no apparent difference between 3 and 25 µg/ml ConA in growth or expansion rate with either 50 or 500 units/ml IL-2. The higher dose of ConA was detrimental compared with the lower dose when combined with 100 units/ml IL-2. Cell growth ceased after 15–16 days, suggesting that these lymphocytes had a limited expansion potential ex vivo.
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, coincubation of murine lymphocytes with irradiated allogeneic stimulators on days 2 and 8 of culture produced expansion similar to that observed with 3 µg/ml ConA and 500 units/ml IL-2, (900 x 104 cells/ml versus 825 x 104 cells/ml versus 550 x 104 cells/ml). However, this growth was also short lived because these lymphocytes started to die off very quickly. Furthermore, expansion was slightly higher among those cells that underwent retroviral infection, suggesting that infection was not detrimental to cell growth and expansion. This suggests that stimulation with allogeneic cells is also a potent stimulus for expansion.
Retrovirally Transduced Lymphocytes Survive Transplantation in Mice.
To demonstrate that these retrovirally transduced LNGFR+/TK+ murine lymphocytes would survive after reinfusion, two scenarios favorable to long-term survival were selected: infusion into the host strain (nonirradiated B10.BR), and delayed infusion (shown to have a decreased risk of GVHD development; Ref. 27
) into an allogeneic AKR/J mouse 49 days after BMT. Mice from each group received injections of 1 x 106 LNGFR+/TK+ lymphocytes because this dose is below that associated with rapid death from GVHD in this model.
As shown in Fig. 2
, after injection, circulating LNGFR+/TK+ lymphocytes were detectable by FACS in both mouse backgrounds. In the syngeneic animal, we found LNGFR+ cells as long as 186 days after injection, at which point these cells composed 66% of the splenic lymphocytes. In the allogeneic animal receiving LNGRF+/TK+ lymphocytes 49 days post allogeneic BMT, 27% of the circulating lymphocytes detected on day 63 days were transduced. Interestingly, we were unable to detect these positive lymphocytes during the first few days after injection.
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, analysis of CD3-positive lymphocytes revealed similar proportions of residual host cells containing Thy-1.1 and donor-derived Thy-1.2 antigen-positive cells in mice transplanted with naive or LNGFR+/TK+ T lymphocytes, respectively. This suggests a similar ability to induce chimerism.
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, a surrogate marker for gut GVHD (28)
. Marrow control mice initially manifested a 10% weight reduction but quickly recovered to near their original weight. Mice receiving naive or LNGFR+/TK+ lymphocytes demonstrated similar patterns of continued weight loss that continued until death from GVHD and had identical survival outcomes (Fig. 5A
). Stigmata of GVHD, including ruffled fur, perianal staining of fur with diarrhea, and hunched posture, were noted at similar times and with similar severity in mice receiving LNGFR+/TK+ and naive lymphocytes. Histological examination of skin and gut biopsies obtained at the time of necropsy revealed identical changes consistent with GVHD from both naive and LNGFR+/TK+ bearing mice (Fig. 5B
; Refs. 29, 30, 31
). Skin from naive and LNGFR+/TK+-bearing mice both had epidermal hyperplasia and epidermolysis accompanied by diffuse mixed inflammation. Similarly, the intestines of both naive and LNGFR+/TK+-bearing mice demonstrated epidermal hyperplasia and diffuse mixed inflammation. By all measurements used, weight loss, clinical stigmata, survival, and histology, the TK+ lymphocytes behaved similarly to fresh naive lymphocytes and were able to cause GVHD.
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and survival data (Fig. 5)
from five separate experiments yielded similar results and were pooled.
As shown in Fig. 5
, radiation control mice not rescued by B10.BR bone marrow faired the worst, with only one animal remaining alive until day 42. In contrast, mice that received only B10.BR bone marrow survived the longest, with 60% of the starting population alive after 10 weeks. Mice that received the naive lymphocytes (negative control for LNGFR+/TK+ T cells) and GCV, as well as mice that received LNGFR+/TK+ T cells with no GCV faired poorly, with almost all of the animals deceased by day 42. In contrast, mice that received the LNGFR+/TK+ T cells plus 7 days of injection of GCV performed well, with >40% of the starting population remaining alive after 10 weeks. The difference in survival between the bone marrow-only control group and the LNGFR+/TK+ T cells plus GCV group was not statistically significant (P = 0.47). However, the survival of mice receiving LNGFR+/TK+ lymphocytes plus GCV was significantly greater compared with mice receiving naive or LNGFR+/TK+ lymphocytes without GCV (P = 0.006–0.02 for the four comparisons). Administration of GCV to transplanted mice receiving normal lymphocytes offered no protection against death from GVHD.
Effects of Therapy with GCV on the Level of Circulating LNGFR+/TK+ Lymphocytes.
We evaluated the completeness of suicide induction in vivo in mice transplanted as described above. Peripheral blood obtained on day 35 was analyzed for circulating LNGFR+ cells via FACS. As shown in Fig. 6
, 65% of the circulating CD3-positive cells from the mouse that received LNGFR+/TK+ lymphocytes but no GCV expressed LNGFR. However, in the three mice that received 7 days of GCV injections, on days 7–13, the percentage of CD3-positive cells that expressed LNGFR was markedly reduced (to 19.2, 14.6, and 18.8% in mice 1, 2, and 3, respectively), but transduced cells were still readily detectable in the circulation. Interestingly, the mouse that did not receive GCV had clinical evidence of GVHD at the time of sampling, whereas the other three mice that received GCV did not.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The lower than expected rates of GVHD in human trials (13 , 14) of suicidal lymphocytes raises questions about the immunological function of these cells after the production process. Contassot et al. (4) demonstrated that ex vivo culture can diminish the alloreactivity of murine lymphocytes in a GVHD model. The TK+ lymphocytes produced in these experiments caused GVHD of similar magnitude and with a similar time course compared with fresh, naive lymphocytes when transplanted into MHC-matched donors. This suggests retention of immunological function despite the production process. The administration of GCV to mice that received the TK+/LNGFR+ T cells reduced the severity of GVHD and resulted in significantly longer survival compared with mice that did not receive GCV, demonstrating the ability of GCV to prevent the emergence of GVHD. The optimal time to initiate suicide induction relative to the development of GVHD is unknown at present. Because control mice began dying of GVHD by day 12, it is reasonable to believe that the GVHD process was already initiated by day 7 when GCV therapy was started. Whether GVHD can also be suppressed if GCV therapy is initiated later in the time course of GVHD can be evaluated. A leukemia cell line for the AKR/J mouse M1 exists, and in future experiments it may be possible to determine whether GVL function is retained as well.
Four groups have reported data on murine suicidal lymphocyte models that used mice transgenic for the HSV-TK gene (32, 33, 34, 35) . However, the conditions used in these studies were very different from those used in the clinical setting. Transgenic human donors are not available, so TK+ lymphocytes must be created for each patient in an ex vivo process that requires (a) stimulation (36) to permit retroviral transduction of the HSV-TK gene and a second selection gene; (b) separation from untransduced, and hence uncontrollable, lymphocytes; (c) expansion prior to clinical use. In contrast, because transgenic T lymphocytes need only one gene and do not require ex vivo culture, stimulation, selection, or expansion procedures, it is reasonable to question whether transgenic TK+ lymphocytes will behave similar to retrovirally transduced TK+ lymphocytes. In this study, the GCV was given on days 7–13 as opposed to starting therapy on day 0, as was done in many of the studies using transgenic mice (32 , 34) . If this was tried in a patient who just received a dose of already activated cultured lymphocytes, nearly all of the infused cells would be eradicated. Therefore, early GCV administration could not be used in humans in the context of current HSV-TK+ lymphocyte production procedures. Hence, differences in transgenic models may limit the applicability of information gathered from these models to the use of HSV-TK lymphocytes in humans. In contrast, the production process used in this model closely mimics the process used in several human trials (13, 14, 15, 16) and may therefore produce more representative results.
In the trial by Bonini et al. (12) , circulating LNGFR+ lymphocytes were not observed after the administration of GCV. In contrast, in all of the mice studied, we were able to detect TK+ lymphocytes at day 35 and beyond after GCV administration. Because GCV will kill only actively dividing cells, it makes sense that only some TK+ lymphocytes would die because not all circulating lymphocytes are expected to be activated. Our ability to detect TK+ cells after GCV administration suggests that the killing was more selective for activated GVHD-producing lymphocytes and that this strategy may actually spare other nonactivated lymphocytes. Because cyclosporine, FK-506, steroids, or methotrexate affect all lymphocytes, this strategy may provide a less immunosuppressive means of treating GVHD. This could reduce the rate of severe or fatal infections arising after therapy for active acute GVHD.
In summary, we have developed a new model for testing and optimizing the use of suicidal lymphocytes that more closely approximates the current use of suicidal lymphocytes in humans than prior transgenic models. Further refinement and use of this model will permit testing of the timing of suicide induction, optimal cell dose, optimal GCV administration, the GVL function of these cells, and the ability to control GVHD by this method. The importance of lymphocyte subset composition on GHVD and GVL activity can also be determined (9) . This model could be further adapted to haploidentical transplant settings to determine whether GVHD can be reliably controlled by this strategy when there is greater disparity between host and donor. If GVHD can be controlled reliably, then the barrier to transplantation imposed by GVHD can be eliminated and a higher percentage of patients can be offered the potential for cure that comes from the GVL effect associated with allogeneic transplantation. This strategy might also find use in other solid tumors that express HLA class II markers, such as breast cancer and melanoma. The immunospecificity may be further enhanced by making dendritically primed and stimulated allogeneic TK+ suicidal lymphocytes. For all of these potential applications, a murine model will speed the evaluations of these strategies.
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FOOTNOTES |
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1 Supported by a grant from the Adler Foundation and Grant PO1 CA49639 (PP-4)-9A1. 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Section of Molecular Hematology and Therapy, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 448, Houston, TX 77030-4009. E-mail: skornblau{at}mdacc.tmc.edu
4 The abbreviations used are: GVHD, graft-versus-host disease; BMT, bone marrow transplantation; HSV-TK, herpes simplex virus thymidine kinase; GCV, ganciclovir; GVL, graft-versus leukemia; conA, concanavalin A; IL-2, interleukin 2; LNGFR, low-affinity nerve growth factor receptor; TK, thymidine kinase; FACS, fluorescence-activated cell sorting.
Received 11/22/00. Accepted 2/12/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation. J. Immunol., 154: 5554 1995.
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