In Vivo comparison of 3 Suicide Gene-Prodrug Combinations in a Mouse Graft-Versus-Host-Disease Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3121-3121
Author(s):  
Jae H. Park ◽  
Raymond Yeh ◽  
Isabelle Riviere ◽  
Michel Sadelain ◽  
Renier Brentjens
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Normal Saline ◽  
Genetically Modified ◽  
Suicide Gene ◽  
Suicide Genes ◽  
Graft Versus Host

Abstract Abstract 3121 Cellular therapies through allogeneic hematopoietic stem cell transplants (allo-HSCT) and infusion of genetically modified T cells targeted against tumor associated antigens hold great promise for cancer therapy. However, significant risk of graft-versus-host-disease (GvHD) with allo-HSCT and potential toxicities from the transgene products in the case of genetically modified T cells have generated considerable interest in developing safeguards in the form of suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events. We have previously reported the comparable in vitro function of 3 different suicide gene-prodrug combinations (Park et al. ASH 2010 abstract #3771). The three suicide genes examined included herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and a novel suicide gene nitroreductase (NTR) derived from E.coli combined with the prodrug metronidazole. Here, we report the comparison of in vivo efficacy of these 3 suicide gene-prodrug combinations using our previously published mouse GvHD model (Santos et al. Nat Med 2009). SCID-Beige mice were injected i.v. with 1×106 FACS-sorted major histocompatibility complex mismatched C57/BL6 T cells transduced to express each suicide gene (HSV-TK SR39, iCasp9 or NTR) cloned in frame with external Gaussia Luciferase (extGLuc), separated by the 2A peptide in an SFG-based vector. Seven days after intravenous injection of modified T cells, SCID-Beige mice developed GvHD as evidenced by in vivo expansion of the transferred T cells detected by bioluminescent imaging (BLI) after a bolus injection of coelenterazine. Subsequently, the mice injected with the HSV-TK SR39-transduced T cells were treated intraperitoneally (i.p.) twice daily for 5 days with ganciclovir at 25mg/kg; the mice with the NTR-transduced T cells were treated i.p. three times daily for 5 days with metronidazole at 200μg; the mice with the iCasp9-transduced T cells were treated i.p. daily for 3 days with CID at 50μg; and the mice with the HSV-TK SR39-transduced T cells treated i.p. twice daily for 5 days with normal saline served as a control group. All treated mice were imaged by BLI on days 3, 7, 11, 18, 25, 32, and weekly thereafter for a total of 3 months following the first dose of each prodrug. All of the control mice treated with normal saline developed rapidly progressing GvHD both clinically and by BLI, meeting the criteria for euthanasia by day 18. In contrast, the administration of prodrugs successfully eradicated the modified T cells expressing respective suicide genes by day 3 and no evidence of GvHD was detected. However, a long-term follow-up of these mice revealed the re-expansion of the transferred T cells or relapse of GvHD in all mice in the NTR group (by day 39) and in 2 out of 3 mice in the HSV-TK SR39 group (by day 53), but none in the mice in the iCasp9 group. The relapsed mice in the NTR and HSV-TK SR groups were treated again with the prodrugs, but none responded to the re-treatment. At the end of the 3 month follow-up, all mice in the NTR group and 2 out of 3 mice from the HSV-TK SR39 group died from progressive GvHD, while all of the mice in the iCasp9 group survived with no evidence of GvHD. In conclusion, our results suggest that the long-term safety and efficacy data of iCasp9, together with its lack of immunogenicity and the absence of reported side effects of CID (Di Stasi et al. ASH 2010 abstract #559), might be advantageous as a promising suicide gene-based strategy in ensuring the rapid and sustained eradication of the adoptively transferred T cells in case of unanticipated severe toxicities of cellular therapies. Based on this in vivo data, we are planning to incorporate the iCasp9 suicide gene in our future clinical trials with T cells genetically modified to express a chimeric antigen receptor targeted against a tumor specific antigen. Disclosures: No relevant conflicts of interest to declare.

Long-Term Immunological Profile of Patients Treated with Haploidentical HSCT and TK-Cells to Study the Requirements of Memory T Cell Persistence

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4793-4793
Author(s):  
Giacomo Oliveira ◽  
Maria Teresa Lupo Stanghellini ◽  
Eliana Ruggiero ◽  
Nicoletta Cieri ◽  
Mattia D'Agostino ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Suicide Gene ◽  
Suicide Gene Therapy ◽  
Cell Compartment

Abstract BACKGROUND: Suicide gene therapy applied to haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. RESULTS: We studied 9 adult patients who underwent haplo-HSCT and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-39.5x106) for high-risk hematologic malignancies between 1995 and 2010 (TK patients). At a median follow-up of 7,4 years (range 3.2-12.3), all patients are in complete remission. Two out of 9 patients (22%) experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. A complete recovery of NK cells, B lymphocytes and αβ or γδ T cells was observed. The CD8+ and CD4+ T cell compartment of TK patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. The quantification of CD4+ CD31+ CD62L+ CD45RA+ CD95- recent thymic emigrants and measure of single joint T-cell receptor excision circles demonstrated that the normalization of the T cell compartment was supported by a completely recovered thymic output. TK-cells were detected in all patients (100%), at low levels (median=4cells/uL). Ex vivo selection of pure TK-cells after polyclonal stimulation and LNGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Of notice TK-cells could be retrieved also in patients successfully treated with GCV for GvHD, thus confirming the selective action of GCV only on proliferating TK-cells. Accordingly, GCV sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. TK-cells circulating in patients displayed a memory phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells and exhibited a low level of Ki-67 positivity, thus suggesting the maintenance of a pool of gene modified memory cells through homeostatic proliferation. The number of TK-cells circulating at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. We evaluated whether the phenotype of infused TK-cells was able to affect the long-term fate of gene-modified memory T cells. We observed that the number of infused TSCM cells positively correlated with early TK-cell expansion and with their long-term persistence, suggesting that TSCMmight play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: These data show that a complete and physiological donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 12 years in treated patients. This setting can be exploited to investigate the requirements at the basis of the generation of a long-lasting immunological memory in vivo. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Chairman and CEO Other. Bonini:MolMed S.p.A: Consultancy.

Tracking Genetically Engineered Lymphocytes Long-Term Reveals the Dynamics of T-Cell Immunological Memory

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 263-263
Author(s):  
Giacomo Oliveira ◽  
Eliana Ruggiero ◽  
Maria Teresa Lupo Stanghellini ◽  
Nicoletta Cieri ◽  
Mattia D'Agostino ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Suicide Gene ◽  
T Cell Subsets ◽  
Memory T Cell ◽  
Donor T Cells

Abstract BACKGROUND: Long-term T-cell survival is pivotal for the development of effective therapeutic approaches against pathogens and cancer, since the success of immunotherapy requires the generation of a robust, safe but also durable immune response. Even if it is established that memory cells can survive and persist for years, little is known about the requirements for their long-term persistence. Suicide gene therapy after T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) provides a unique model to study memory T cells. In this setting, patients receive the post-transplant infusion of donor-derived gene-modified memory T lymphocytes retrovirally transduced to express the Herpes Simples Virus Thymidine Kinase (TK) suicide gene and the DLNGFR selection marker. The presence of a safety switch allows the infusion into patients of a broad T-cell repertoire in the absence of immune suppression, while the surface marker enables unambiguous detection and close monitoring of gene-modified cells circulating in treated patients. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the fate of persisting TK cells to shed light on memory T cell dynamics in vivo and to unravel the requirements for long-term persistence directly in humans. RESULTS: We studied 10 adult patients who underwent haplo-HSCT and infusion of suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:1-39.5x106) for high-risk hematologic malignancies between 1995 and 2012. Three out of 10 patients (33%) experienced GvHD early after HSCT; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. At a median follow-up of 7 years (range 2-14), all patients were in complete remission and free of GvHD, and displayed a complete and broad donor-derived immune system characterized by physiological counts of NK cells, B lymphocytes, γδ T cells and naïve and memory CD4+ or CD8+ T cells. TK cells were detected in all patients, at low levels (median=4cells/uL), even in patients treated with GCV. Ex vivo selection of pure TK-cells confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Importantly, GCV sensitivity was preserved in long-term persisting TK cells, independently from their differentiation phenotype. Longitudinal follow up revealed that TK cells circulated in patients at stable levels and displayed a conserved phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells. The low level of Ki-67 positivity suggested the maintenance of a pool of gene-modified memory cells through homeostatic proliferation. Polyclonality was demonstrated by sequencing among TK cells of thousands of diverse TCRs with a broad usage of V and J alpha and beta genes. The number of TK cells persisting at the longest follow-up did not correlate with the amount of infused cells, but instead with the peak of TK cells measured within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Accordingly, we documented the persistence of CMV and Flu-specific TK cells only after post-transplant CMV reactivation or after Flu infection. Characterization of TK cell products infused to patients showed that the amount of infused TSCM cells positively correlates with early expansion and long-term persistence of gene-marked cells. By combining sorting of memory T-cell subsets with sequencing of integration sites, TCRα and TCRβ clonal markers, we longitudinally traced T-cell clones from infused products to late follow-up time-points. We showed that although T cells retrieved long-term are enriched in clones originally shared in different memory T-cell subsets, dominant long-term clonotypes preferentially originate from infused TSCM clones, suggesting that TSCM might play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: In a completely restored immune system, suicide gene-modified donor T cells persist for up to 14 years in treated patients. Long-term persistence of memory T cells is determined by antigen exposure, and by the original phenotype of infused cells, according to a hierarchical model in which TSCM are superior to TCM and TEM/EFF. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Membership on an entity's Board of Directors or advisory committees. Bonini:MolMed S.p.A: Consultancy.

In Vivo Suicide Gene Therapy of Human T Lymphocytes To Prevent Graft Versus Host Disease in a Murine Xenograft Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4979-4979
Author(s):  
Bruno Nervi ◽  
Michael Rettig ◽  
Julie Ritchey ◽  
Jon Walker ◽  
Gerhard Bauer ◽  
...  
Keyword(s):  
T Cells ◽  
Body Weight ◽  
Graft Versus Host Disease ◽  
Genetically Modify ◽  
Xenograft Model ◽  
Suicide Gene ◽  
Host Disease ◽  
Graft Versus Host ◽  
Post Infusion

Abstract Graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality following allogeneic stem cell transplantation. One approach to preserve the beneficial graft-versus-leukemia while minimizing GvHD is to genetically modify donor T cells with a herpes simplex virus thymidine kinase (HSV-tk) suicide gene that can selectively phosphorylate the prodrug ganciclovir (GCV) resulting in T cell suicide should GvHD develop. We recently developed a new chimeric suicide gene by fusing HSV-tk to the extracellular and transmembrane domains of human CD34 (ΔCD34-tk; Mol. Ther.2003; 8:29–41). ΔCD34-tk-modified cells can be rapidly and efficiently selected using a well-established and clinically approved CD34 immunoselection technique ensuring coordinated expression of the CD34 epitope and tk suicide gene in transduced affinity purified human T cells (HuT). We have developed a new model for xenogeneic GvHD induced by HuT in NOD-SCID-β2M null mice (β2 mice). β2 mice lack macrophage activity, T, B and NK cells. In this model 79% (11/14) of the β2 mice conditioned with 250 cGy and injected retro-orbitaly (ro) with 107 HuT developed lethal GvHD, with loss of pretransplant body weight (>20%), decreased activity, hunched posture, ruffled fur, accompanied by HuT expansion and infiltration of blood and multiple tissues. In this study, we evaluated the ability of GCV to prevent GvHD after infusion of CD34-tk-modified HuT in this xenogeneic model. Human PBMCs cells were activated with anti-CD3 and anti-CD28 mAbs immobilized on magnetic beads (CD3/CD28 beads) in the presence of IL-2 (50 U/mL). Two days post-activation, cells were incubated with the 293 GPG-derived VSV-G pseudotyped CD34-tk oncoretroviral supernatants for 6 h at 37°C. Transduced (Td) cells were then expanded in media containing IL-2 and CD3/CD28 beads and isolated by MACS (Miltineyi Biotech) on day 4 postactivation (transduction efficiency >60%). Td cells were purified to >94% by CD34 immunomagnetic selection using a VarioMACS magnetic cell separator. Naive (unmanipulated) T cells (n=2), naive PBMC (n=3), activated non Td T cells (Activated; n=3) and CD34-tk transduced T cells (Td; n=6) were then injected (107/mouse) ro into 250 cGy conditioned β2 mice. Animals receiving Td cells were then either left untreated or treated with GCV (1 mg/day, intraperitoneal) from days 1–7 post- HuT cell injection. Mice that received naive T cells died of lethal GvHD on days 14 and 16. Although we observed an average of 15.5% and 33.6% HuT engraftment in the Activated and Td groups respectively, and increased expression of CD25, CD30, and CD69 in the peripheral blood of mice 4 weeks post-infusion, we observed no GvHD (mice maintained their pretransplant body weight and did not developed any signs of GvHD). However, we were able to demonstrate that Td T cells could be efficiently eliminated in vivo by treatment with ganciclovir. Animals treated with ganciclovir from days 1–7 had less than 1% engraftment at 4 weeks post-infusion. This selective elimination of the Td T cells was not sustained however, as evidenced by the 3.5% HuT engraftment on blood (5.4% of these HuT were CD34+) and 18% HuT in the spleen (24% CD34+) at week 10 post-infusion. In conclusion, this xenograft model provides a unique opportunity for preclinical testing of the CD34-tk/GCV suicide gene system as well as other methods of GvHD control.

Suicide gene therapy of graft-versus-host disease: immune reconstitution with transplanted mature T cells

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2071-2076 ◽  
Author(s):  
José L. Cohen ◽  
Olivier Boyer ◽  
David Klatzmann
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Immune System ◽  
Graft Versus Host Disease ◽  
Immune Reconstitution ◽  
Suicide Gene ◽  
Suicide Gene Therapy ◽  
Allogeneic Hsct ◽  
Graft Versus Host ◽  
Donor T Cells

After allogeneic hematopoietic stem cell transplantation (HSCT), mature transplanted T cells play a major role in restoration of the immune system. However, they can also induce a life-threatening complication: graft-versus-host disease (GVHD). Suicide gene therapy of GVHD aims to selectively eliminate alloreactive T cells mediating GVHD while sparing nonalloreactive T cells that should contribute to immune reconstitution. It was demonstrated previously that treatment with ganciclovir (GCV) can control GVHD in mice by killing donor T cells engineered to express the thymidine kinase (TK) suicide gene. TK allows phosphorylation of nontoxic GCV into triphosphate GCV, which is selectively toxic for dividing cells. Thus, in the TK-GCV system, the specificity of cell killing depends on the cycling status of TK T cells rather than allogeneic recognition. This is a potential drawback because in recipients of lymphopenic allogeneic HSCT, alloreactive and homeostatic signals drive the proliferation of donor T cells. It is shown here that the onset of alloreactive T-cell division occurs earlier than that of nonalloreactive T cells, thus establishing a time frame for GCV administration. A 7-day GCV treatment initiated at the time of HSCT allowed efficient prevention of GVHD, while sparing a pool of nondividing donor TK T cells. These cells later expanded and contributed to the replenishment of the recipient immune system with a diversified T-cell receptor repertoire. These results provide a rationale for designing the therapeutic scheme when using TK-GCV suicide gene therapy in allogeneic HSCT.

Prevention of Transfusion-Associated Graft-Versus-Host Disease by Photochemical Treatment

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3140-3147 ◽  
Author(s):  
Joshua A. Grass ◽  
Tamim Wafa ◽  
Aaron Reames ◽  
David Wages ◽  
Laurence Corash ◽  
...  
Keyword(s):  
T Cells ◽  
Gamma Radiation ◽  
Graft Versus Host Disease ◽  
Clinical Signs ◽  
Peripheral Blood Cell ◽  
Control Group ◽  
Host Disease ◽  
Graft Versus Host

Abstract Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model. Recipient mice in four treatment groups were transfused with 108 splenic leukocytes. (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 μmol/L S-59 and 2.1 J/cm2UVA. Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin. Mice in the control group remained healthy and free of detectable disease. Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia. In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 μmol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD. Using an in vitro T-cell proliferation assay, greater than 105.1 murine T cells were inactivated by PCT. Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.

Thymic atrophy in murine acute graft-versus-host disease is effected by impaired cell cycle progression of host pro-T and pre-T cells

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 347-354 ◽  
Author(s):  
Werner Krenger ◽  
Simona Rossi ◽  
Luca Piali ◽  
Georg A. Holländer
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
Graft Versus Host Disease ◽  
Cell Cycle Progression ◽  
Acute Gvhd ◽  
Chronic Gvhd ◽  
Cycle Progression ◽  
Thymic Atrophy ◽  
Graft Versus Host

Abstract Reconstitution of the peripheral T-cell compartment is a critical aspect for the success of bone marrow transplantation and is also dependent on the reestablishment of normal thymic structure and function. Graft-versus-host disease (GVHD), however, exacerbates posttransplant immunodeficiency through a deleterious effect on thymic function. To investigate the mechanisms of GVHD-mediated thymic disease, 2 murine parent→F1transplantation models of acute and chronic GVHD, respectively, were studied. Acute GVHD was associated with changes in thymic architecture and a reduction in cellularity mainly because of the decrease in CD4+CD8+, or double-positive (DP) thymocytes, to less than 15% of values found in mice without GVHD. Simultaneously, mature donor-derived T cells expanded in the confines of the allogeneic thymic microenvironment, leading to local inflammation. Through analysis of in vivo cell proliferation, we demonstrated that the ensuing depletion of DP thymocytes was secondary to a decreased commitment of resident pro-T and pre-T cells to enter the cell cycle. Moreover, DP cells themselves showed altered proliferative capacities in the presence of acute GVHD. These findings suggested that thymic atrophy in acute GVHD is effected by impaired cellular proliferation of immature host thymocytes and that the failure of these cells to enter the cell cycle is dependent on an interferon (IFN)-γ–driven immune response. In contrast, interleukin-4–driven chronic GVHD was not accompanied by a sustained thymic infiltration of donor T cells. Consequently, there was a lack of apparent structural changes, a restricted in situ transcription of inflammatory cytokines, and a virtually unchanged cell cycle progression in vivo.

scholarly journals Prevention of Graft-Versus-Host Disease in Mice Using a Suicide Gene Expressed in T Lymphocytes

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4636-4645 ◽  
Author(s):  
José L. Cohen ◽  
Olivier Boyer ◽  
Benoı̂t Salomon ◽  
Rosine Onclercq ◽  
Frédéric Charlotte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Therapeutic Strategy ◽  
Suicide Gene ◽  
Graft Versus Host ◽  
Alloreactive T Cells ◽  
Cell Pool ◽  
Ganciclovir Treatment

Abstract Alloreactive T cells present in a bone marrow transplant are responsible for graft-versus-host disease (GVHD), but their depletion is associated with impaired engraftment, immunosuppression, and loss of the graft-versus-leukemia effect. We developed a therapeutic strategy against GVHD based on the selective destruction of these alloreactive T cells, while preserving a competent T-cell pool of donor origin. We generated transgenic mice expressing in their T lymphocytes the Herpes simplex type 1 thymidine kinase (TK) suicide gene that allows the destruction of dividing T cells by a ganciclovir treatment. T cells expressing the TK transgene were used to generate GVHD in irradiated bone marrow grafted mice. We show that a short 7-day ganciclovir treatment, initiated at the time of bone marrow transplantation, efficiently prevented GVHD in mice receiving TK-expressing T cells. These mice were healthy and had a normal survival. They maintained a T-cell pool of donor origin that responded normally to in vitro stimulation with mitogens or third party alloantigens, but were tolerant to recipient alloantigens. Our experimental system provides the proof of concept for a therapeutic strategy of GVHD prevention using genetically engineered T cells.

Characterization of in vitro antimurine thymocyte globulin–induced regulatory T cells that inhibit graft-versus-host disease in vivo

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1726-1734 ◽  
Author(s):  
Melanie C. Ruzek ◽  
James S. Waire ◽  
Deborah Hopkins ◽  
Gina LaCorcia ◽  
Jennifer Sullivan ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Graft Versus Host Disease ◽  
Antithymocyte Globulin ◽  
Mrna Level ◽  
Regulatory Cells ◽  
Host Disease ◽  
Graft Versus Host

Abstract Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin–induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4+CD25+ T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

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