Analysis of transgene-specific immune responses that limit the in vivo persistence of adoptively transferred HSV-TK-modified donor T cells after allogeneic hematopoietic cell transplantation

Abstract

The introduction of an inducible suicide gene such as the herpes simplex virus thymidine kinase (HSV-TK) might allow exploitation of the antitumor activity of donor T cells after allogeneic hematopoietic cell transplantation (HCT) without graft versus host disease. However, HSV-TK is foreign, and immune responses to gene-modified T cells could lead to their premature elimination. We show that after the infusion of HSV-TK-modified donor T cells to HCT recipients, CD8+ and CD4+ T-cell responses to HSV-TK are rapidly induced and coincide with the disappearance of transferred cells. Cytokine flow cytometry using an overlapping panel of HSV-TK peptides allowed rapid detection and quantitation of HSV-TK-specific T cells in the blood and identified multiple immunogenic epitopes. Repeated infusion of modified T cells boosted the induced HSV-TK-specific T cells, which persisted as memory cells. These studies demonstrate the need for nonimmunogenic suicide genes and identify a strategy for detection of CD4+ and CD8+ T-cell responses to transgene products that should be generally applicable to monitoring patients on gene therapy trials. The potency of gene-modified T cells to elicit robust and durable immune responses imply this approach might be used for vaccination to elicit T-cell responses to viral or tumor antigens.

Figure 1.

Adoptively transferred HyTK-modified T cells. (A-C) Persistence of adoptively transferred HyTK-modified T cells in vivo. PBMCs from each of the 3 patients were obtained at various time points before and after infusion of HyTK-positive T cells and evaluated for the presence of HyTK-transduced cells by quantitative PCR as described in “Patients, materials, and methods.” (D-F) Rapid induction of a HyTK-specific CTL response after adoptive transfer of HyTK-modified T cells. PBMCs from each of the 3 patients were obtained prior to and at various times after infusion and stimulated in vitro with γ-irradiated HyTK-positive donor T cells. Aliquots of these cultures were then evaluated in a chromium release assay for recognition of HyTK-positive target cells (▪) or unmodified controls (□). The killing of HyTK-positive LCLs is shown at an effector-target ratio of 10:1. The arrows indicate the day of the HyTK-positive T-cell infusion.

Figure 2.

HyTK-specific immunity is mediated by HyTK-specific CD8⁺ cytotoxic T cells. Postinfusion PBMCs were obtained from UPN 1574, UPN 4103, and UPN 7826 after the HyTK-positive T-cell infusion and stimulated in vitro with γ-irradiated HyTK-expressing T cells twice 1 week apart. Aliquots of these cultures were depleted of CD8⁺ T cells as described in “Patients, materials, and methods” and then evaluated in a chromium release assay for recognition of HyTK-positive target cells (▪) or untransduced LCLs (□). Aliquots of the undepleted cultures () were assayed in a similar fashion for HyTK-positive target cell lysis. Data are shown at an effector-target ratio of 10:1.

Figure 3.

HyTK-specific CD8⁺ CTL recognize epitopes derived from both the Hy and TK domain of the HyTK fusion gene and are restricted by multiple class I MHC alleles. Samples of PBMCs were obtained from UPN 1574, UPN 4103, and UPN 7826 after the HyTK-positive T-cell infusion and stimulated twice 1 week apart with γ-irradiated HyTK-expressing donor T cells (A-C) or with HSV-TK–expressing donor T cells (UPN 7826 [D]). To map the HLA-restricting alleles, EBV-transformed LCL lines that matched at all 4 class I MHC alleles (Auto), allogeneic LCL lines that matched only at a single class I MHC allele, and mismatched LCLs (MM) were modified to express either the Hy () or the HSV-TK (▪) domain of the HyTK fusion protein. Aliquots of the CD8⁺ CTL lines were then examined for recognition of nontransduced LCL- (□), Hy- (), or HSV-TK– (▪) modified target cells. Data are shown at an effector-target ratio of 5:1.

Figure 4.

HSV-TK–specific CD8⁺ CTLs can be visualized by CFC and recognize multiple epitopes within the HSV-TK protein. (A) Design of peptide pools. The numbers of the pools (left column and top row) are shown in bold. Individual 15-mer peptides overlapping by 11 aa (n = 95) in these 20 pools correspond to the numbers in the respective columns and rows. Peptides 1 to 3 comprise the C-terminal aa sequence of the Hy protein (Hy_300-324), peptides 4 to 7 encompass the HyTK fusion site, and peptides 8 to 95 span the aa sequence of the HSV-TK protein (HSV-TK_10-376). The gray shading illustrates how a candidate peptide was chosen. (B) Identification of an immunogenic peptide from the peptide pools that induced IFN-γ production by CD8⁺ T cells. Samples of postinfusion PBMCs were obtained from UPN 4103 stimulated in vitro with γ-irradiated HyTK-positive donor T cells twice 1 week apart. Aliquots of the cultures were incubated for 6 hours with medium alone or with each 1 of the 20 peptide pools or the individual 15-mer peptides. Cells were then stained with FITC-coupled anti–IFN-γ and PE-coupled anti-CD8β mAbs and examined by flow cytometry. Cells were gated to identify CD8⁺ T cells and stained for intracellular IFN-γ. Values indicate the percentage of cells in the culture that produced IFN-γ in response to peptide stimulation. (C) Multiple 15-mer peptides throughout the sequence of the HSV-TK protein (boldface) are immunogenic. The underlined sequences indicate the 15-mer candidate peptides within the HSV-TK protein that were identified to induce IFN-γ production by CD8⁺ T cells in aliquots of the T-cell lines or postinfusion PBMCs from the 3 patients. Immunogenic peptides within the adjacent Hy protein (italics) or fusion site were not detected in these patients.

Figure 5.

HSV-TK–specific CD8⁺ and CD4⁺ T cells can be directly visualized by CFC in samples of peripheral blood after HyTK-positive DLI. Samples of PBMCs obtained from the 3 patients after the first T-cell infusion were incubated for 6 hours with medium alone, the candidate 15-mer peptides, or with peptide pools that elicited responses from the HSV-TK–reactive T-cell lines. Cells were then stained with FITC-coupled anti–IFN-γ, PE-coupled anti-CD8β, and peridinin chlorophyll protein (PerCP)-Cy5.5–coupled anti-CD4 mAbs, respectively, and examined by flow cytometry. (A) Gated on CD8⁺ T cells. (B) Gated on CD4⁺ T cells. Values indicate the percentage of cells producing IFN-γ. Data are shown for representative HSV-TK peptides.

Figure 6.

The HSV-TK–specific CD8⁺ T cells are boosted after reinfusion of HyTK-positive donor T cells. Samples of PBMCs were obtained from UPN 1574 before and at various time points after the first and second infusion of HyTK-modified donor T cells. Aliquots of these cells were incubated for 6 hours with medium alone or with all 6 candidate 15-mer peptides that were identified to induce IFN-γ production in samples of PBMCs obtained from this patient. After 6 hours of stimulation, cells were permeabilized, stained with anti–IFN-γ and anti-CD8 mAbs, and examined by flow cytometry. Values indicate the percentage of CD8⁺ T cells producing IFN-γ.

Figure 7.

Adoptive transfer of HSV-TK–modified T cells results in the induction of a durable memory T-cell response. Samples of PBMCs were obtained from UPN 1574 more than 7 years after HSV-TK–modified DLI, stained with anti-CD8 and anti-CD62L mAbs, and separated by fluorescence-activated cell sorting (FACS) in a CD8⁺CD62L⁺ and CD8⁺CD62L^– fraction. These fractions were then incubated with medium alone or stimulated for 6 hours with a peptide mix consisting of the 6 candidate 15-mer peptides as described in “Patients, materials, and methods.” After 6 hours of stimulation, cells were then stained with FITC-coupled anti–IFN-γ and PE-Cy7–coupled anti-CD8 mAbs and examined by flow cytometry. Cells were gated to identify CD8⁺ T cells and assessed for cytokine production.