Nonmyeloablative immunosuppressive regimen prolongs In vivo persistence of gene-modified autologous T cells in a nonhuman primate model - PubMed (original) (raw)

Nonmyeloablative immunosuppressive regimen prolongs In vivo persistence of gene-modified autologous T cells in a nonhuman primate model

C Berger et al. J Virol. 2001 Jan.

Abstract

The in vivo persistence of gene-modified cells can be limited by host immune responses to transgene-encoded proteins. In this study we evaluated in a nonhuman primate model whether the administration of a nonmyeloablative regimen consisting of low-dose total-body irradiation with 200 cGy followed by immunosuppression with mycophenolate mofetil and cyclosporin A for 28 and 35 days, respectively, could be used to facilitate persistence of autologous gene-modified T cells when a transgene-specific immune response had already been established or to induce long-lasting tolerance in unprimed recipients. Two macaques (Macaca nemestrina) received infusions of T cells transduced to express either the enhanced green fluorescent protein and neomycin phosphotransferase genes or the hygromycin phosphotransferase and herpes simplex virus thymidine kinase genes. In the absence of immunosuppression, both macaques developed potent class I major histocompatibility complex-restricted CD8(+) cytotoxic T-lymphocyte (CTL) responses that rapidly eliminated the gene-modified T cells and that persisted long term as memory CTL. Treatment with the nonmyeloablative regimen failed to abrogate preexisting memory CTL responses but interfered with the induction of transgene-specific CTL and facilitated in vivo persistence of gene-modified cells in an unprimed host. However, sustained tolerance to gene-modified T cells was not achieved with this regimen, indicating that further modifications will be required to permit sustained persistence of gene-modified T cells.

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Figures

FIG. 1

FIG. 1

Schematic diagram of the experimental design. Each arrow indicates an infusion of 109 autologous gene-modified T cells/m2. For immunosuppression, macaque 90152 was given a nonmyeloablative dose (200 cGy) of TBI prior to the infusion of both EGFP/_neo_- and HyTK-modified T cells (day 140). CSP (1.5 mg/kg b.i.d. i.v.) was administered on days 137 to 175, and MMF (10 to 15 mg/kg b.i.d. i.v.) was given on days 140 to 168.

FIG. 2

FIG. 2

(A) Successful gene transfer of M. nemestrina T lymphocytes, demonstrated by representative flow cytometry analysis of EGFP expression in T cells after transduction with the LGSN (PG13) retroviral vector. Gene transfer was accomplished using an optimized transduction protocol including anti-CD3 and anti-CD28 stimulation of T cells. Viable cells were stained with anti-CD3 MAb and analyzed by flow cytometry. Percentages of cells positive for both EGFP and CD3 are as indicated. (B) Transferred EGFP-expressing T cells are detectable by flow cytometry in vivo. PBMC from a control animal (left) and PBMC collected from macaque 90152 at 30 min postinfusion (day 410) (right) were analyzed for expression of both EGFP and CD3. Percentages of cells positive for both EGFP and CD3 are as indicated.

FIG. 3

FIG. 3

Analysis of PBMC for the in vivo persistence of gene-modified T lymphocytes following in vivo priming. (A) Frequency of EGFP/_neo_-modified T cells transferred to macaque 90152 in peripheral blood. PBMC collected before and at the indicated days after infusion were analyzed by the quantitative real-time PCR assay for the presence of the neo sequence or by flow cytometry for EGFP-expressing cells. For enumeration of transferred EGFP-expressing T cells in PBMC, >100,000 viable cells were analyzed for each assay. Negative fluorescence gates were defined using PBMC from control animals. (B) Detection of HyTK-modified T cells transferred to macaque 97140 in circulating PBMC by the real-time PCR assay. Arrows indicate the days of T-cell infusions.

FIG. 4

FIG. 4

CTL responses specific for the transgene products are elicited following transfer of gene-modified T cells. (A) Analysis of PBMC collected from macaque 90152 prior to the infusion of EGFP/_neo_-modified T cells for the presence of EGFP/_neo_-specific CTL. (B and C) Analysis of PBMC collected from macaque 90152 on day 7 following infusion of EGFP/_neo_-modified T cells for the presence of EGFP-specific CTL (B) and _neo_-specific CTL (C). Aliquots of PBMC were analyzed following stimulation with autologous T cells expressing EGFP and/or neo in a chromium release assay for recognition of target cells, either parental (◊) or transduced to express the EGFP (▵) and neo (●) genes alone and together (▴). (D to F) PBMC from macaque 97140 obtained before (D) and on days 8 (E) and 10 (F) following the first cell dose were cocultured with autologous HyTK-modified T cells and then assayed for HyTK-specific cytolytic activity against target cells, either parental (◊) or transduced to express the HyTK gene (■).

FIG. 5

FIG. 5

Transferred gene-modified T cells induce potent memory T-cell responses specific for the transgene products. (A and B) Aliquots of PBMC from macaque 90152 were collected on day 70 prior to the readministration of EGFP/_neo_-modified T cells and cocultured with autologous T cells either expressing both EGFP and neo (A) or neo alone (B). Cells from these cultures were then assayed in a chromium release assay for recognition of target cells, either parental (◊) or expressing both the EGFP and neo genes (▴) or the neo (●) gene alone. (C) Frequency of EGFP/_neo_-modified T cells readministered to macaque 90152 in peripheral blood. PBMC collected before and at the indicated days postinfusion were analyzed by real-time PCR for the presence of the neo sequence as described in the text or by flow cytometry to enumerate EGFP-expressing cells. Arrows indicate the days of T-cell infusions.

FIG. 6

FIG. 6

(A) Analysis of PBMC for the presence of both EGFP/_neo_- and HyTK-modified T cells transferred with a transient nonmyeloablative regimen to macaque 90152. The frequency of neo or HyTK sequences was evaluated in samples of DNA derived from PBMC collected before and on the indicated days after infusion, using the real-time PCR assay. The frequency of transferred EGFP-expressing T cells in the same samples of PBMC was assessed by flow cytometry as described in the text. Arrows indicate the days of T-cell infusions. (B) EGFP/_neo_- and HyTK-specific CTL responses before and after immunosuppressive treatment. Aliquots of PBMC obtained from macaque 90152 at the days indicated on the horizontal axis were stimulated twice with autologous T cells expressing either both the EGFP and neo genes or the HyTK gene and then assayed in a chromium release assay at various E/T ratios for recognition of target cells, either parental (white bars) or transduced to express either both the EGFP and neo genes (hatched bars) or the HyTK gene (black bars). The percent specific lysis of the target cells indicated on the vertical axis is shown for an E/T ratio of 20:1. The presence of EGFP-specific CTL was evaluated in samples of PBMC collected on day 371 instead of day 389. The duration of the treatment is indicated by the horizontal bar, and arrows indicate the days of T-cell infusions.

FIG. 7

FIG. 7

HyTK-specific CTL are elicited following readministration of HyTK-modified T cells to macaque 90152. (A) In vivo persistence of HyTK-modified T cells. Samples of PBMC collected before and on the indicated days after infusion were evaluated by real-time PCR for the frequency of HyTK sequences. Arrows indicate the days of T-cell infusions, and the star denotes an inguinal lymph node biopsy. (B and C) CTL responses specific for target antigens derived from HyTK in samples of PBMC collected before (study day 389) (B) and 7 days after (study day 417) (C) infusion. PBMC were cocultured with autologous γ-irradiated HyTK-expressing T cells and then assayed for HyTK-specific cytolytic activity against target cells, either parental (◊) or transduced to express the HyTK gene (■).

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