Adoptive immunotherapy of disseminated leukemia with TCR-transduced, CD8+ T cells expressing a known endogenous TCR

Abstract

Adoptive T-cell immunotherapy has shown promise in the treatment of human malignancies, but the challenge of isolating T cells with high avidity for tumor antigens in each patient has limited application of this approach. The transfer into T cells of T-cell receptor (TCR) genes encoding high-affinity TCRs recognizing defined tumor-associated antigens can potentially circumvent this obstacle. Using a well-characterized murine model of adoptive T-cell immunotherapy for widely disseminated leukemia, we demonstrate that TCR gene-modified T cells can cure mice of disseminated tumor. One goal of such adoptive therapy is to establish a persistent memory response to prevent recurrence; however, long-term function of transferred TCR-transduced T cells is limited due to reduced expression of the introduced TCR in vivo in quiescent resting T cells. However, by introducing the TCR into a cell with a known endogenous specificity, activation of these T cells by stimulation through the endogenous TCR can be used to increase expression of the introduced TCR, potentially providing a strategy to increase the total number of tumor-reactive T cells in the host and restore more potent antitumor activity.

Figure 1

Design of retroviral constructs. The gag-specific Vα3 and Vβ12 TCR chains were cloned into pUC6S³⁶ and separated by either an IRES element, murine phosphoglycerate kinase (pgk) promoter, or the 2A peptide sequence derived from the foot-and-mouth disease virus (F2A), and each entire cassette cloned into the BamHI and NotI sites of the retroviral vector pLZRSpBMN-Z, replacing the lacZ gene. Only the retrovirus portion of the vector is shown. IRES, internal ribosomal entry site; LTR, long terminal repeat; TCR, T-cell receptor.

Figure 2

Transduction efficiency and T-cell receptor α and β chain expression in P14 T cells. (a) P14 T cells were transduced with one of the three retroviral constructs shown in Figure 1 and incubated with antibodies to CD8α, Vα3, and Vβ12 2 days post-transduction. Dot plots were gated on CD8⁺ lymphocytes, and histograms were gated on CD8⁺, Vα3⁺ lymphocytes to illustrate relative levels of Vβ12 expression in transduced T cells compared to mock-transduced controls. (b) Histograms were analyzed as in a above to compare Vβ12 expression in three separate experiments. Means and standard deviations for the mean fluorescence intensity (MFI) of Vβ12 expression for the transduced T cells gated on CD8⁺, Vα3⁺ lymphocytes from the three experiments were calculated (right, upper bar graph). The means and standard deviations for the MFI of Vβ12 expression in transduced T cells were also calculated after eliminating T cells expressing low or absent levels of Vβ12 by gating on CD8⁺, Vα3⁺, Vβ12⁺ lymphocytes (right, lower bar graph). F2A, foot and mouth disease virus–derived 2A peptide; IRES, internal ribosomal entry site; pgk, phosphoglycerate kinase.

Figure 3

Maintenance of transduced T-cell receptor (TCR) expression with repetitive in vitro stimulation. P14 T cells transduced with the TCR_αgag receptor chains were incubated with antibodies to CD8α and either the introduced, gag-specific TCR chains (Vα3 and Vβ12) or the endogenous gp33-specific TCR chains (Vα2 and Vβ8). (a) Expression of the gag-specific TCR on day 9 following the 8th in vitro stimulation when cells had been in culture for ~2.5 months. (b) Expression of the gp33-specific TCR at the same time point as in a. All plots shown are gated on CD8⁺ lymphocytes and the numbers in the histograms indicate the mean fluorescence intensity of α- and β−chain expression. F2A, foot and mouth disease virus–derived 2A peptide; IRES, internal ribosomal entry site.

Figure 4

Therapy of mice bearing disseminated FBL leukemia with P14 T cells transduced with a gag-specific TCR. B6 mice were injected with 2 × 10⁶ live FBL tumor cells intraperitoneal on day 0 and cyclophosphamide (Cy) on day 5. Six hours following Cy treatment, mice received no T cells (dashed black line) or 6 × 10⁶ mock-transduced P14 T cells (solid gray line), P14 T cells transduced with a Vα3F2AVβ12 retrovirus (dotted gray line), P14 T cells transduced with a Vα3IRESVβ12 retrovirus (dashed gray line), or TCR_αgag transgenic T cells (solid black line). The transduced T cells had been expanded in vitro for nine stimulation cycles and were compared for in vivo efficacy to similarly expanded transgenic T cells expressing the same TCR. Mice were euthanized if progressive tumor growth became evident (mortality anticipated in 48 h). F2A, foot and mouth disease virus–derived 2A peptide; IRES, internal ribosomal entry site; TCR, T-cell receptor.

Figure 5

Analysis of long-term persistence and function of transferred cells in treated mice. On day 214 following tumor challenge, splenocytes from surviving treated mice from Figure 4 were stimulated in vitro with irradiated FBL or gp33 peptide. Six days later, cells were restimulated with no peptide (φ), gag peptide, or gp33 peptide for 6 h and analyzed for production of IFN-γ and expression of Thy1.1. All transduced and adoptively transferred P14 T cells were derived from T cells expressing the Thy1.1 congenic marker. (a–d) Mice received Vα3IRESVβ12-transduced P14 T cells and (e,f) mice received Vα3F2AVβ12-transduced T cells. All plots are gated on CD8⁺ lymphocytes. IFN-γ, interferon-γ.