CTLA4-CD28 chimera gene modification of T cells enhances the therapeutic efficacy of donor lymphocyte infusion for hematological malignancy

Abstract

Donor lymphocyte infusion (DLI) followed by hematopoietic stem cell transplantation has served as an effective prevention/treatment modality against the relapse of some hematologic tumors, such as chronic myeloid leukemia (CML). However, the therapeutic efficacies of DLI for other types of leukemia, including acute lymphocytic leukemia (ALL), have been limited thus far. Therefore, we examined whether increasing the reactivity of donor T cells by gene modification could enhance the therapeutic efficacy of DLI in a murine model of ALL. When a CTLA4-CD28 chimera gene (CTC28) in which the intracellular signaling domain of CTLA4 was replaced with the CD28 signaling domain was introduced into CD4 and CD8 T cells in DLI, the graft-versus-tumor (GVT) effect was significantly increased. This effect was correlated with an increased expansion of donor CD8 T cells in vivo, and the depletion of CD8 T cells abolished this effect. The CD8 T cell expansion and the enhanced GVT effect were dependent on the transduction of both CD4 and CD8 T cells with CTC28, which emphasizes the role of dual modification in this therapeutic effect. The CTC28-transduced T cells that expanded in vivo also exhibited enhanced functionality. Although the potentiation of the GVT effect mediated by the CTC28 gene modification of T cells was accompanied by an increase of graft-versus-host disease (GVHD), the GVHD was not lethal and was mitigated by treatment with IL-10 gene-modified third-party mesenchymal stem cells. Thus, the combined genetic modification of CD4 and CD8 donor T cells with CTC28 could be a promising strategy for enhancing the therapeutic efficacy of DLI.

Figure 1

CTC28 gene modification of T cells increases the GVT effect in a therapeutic model of delayed DLI. Lethally irradiated B6 mice were reconstituted with a mixture of T cell-depleted BM cells from B10.A and B6 mice and left unmanipulated for at least 8 weeks (mixed chimera mice) before i.v. EL4 injection. (a) B10.A splenocytes were i.v. administered to the mice 7 days before EL4 injection (naive DLI » EL4) or 4–6 h after EL4 injection (EL4+ naive DLI). A group of the mice was injected with EL4 cells without DLI (EL4 only). The survival of the mice was monitored (P-value: naive DLI >> EL4 vs EL4 only). (b) B10.A splenocytes that were activated as described in the Materials and Methods section were i.v. administered to the mice 4–6 h after EL4 injection (EL4+activated DLI). (c and d) CD4 and CD8 T cells purified from B10.A mice were activated with anti-CD3 and anti-CD28 and subsequently transduced with CTC28 retrovirus respectively. After resting, both cells types were analyzed for transduction efficiency (GFP-positivity) and cell surface expression of CTC28 via flow cytometry (c) or i.v. administered to the mice as a mixed population at a 1:1 ratio 4–6 h after EL4 injection (EL4+CTC28-DLI) (d). The mixed CD4 and CD8 cells that were transduced with the empty vector (EV) retrovirus were used as a negative control (EL4+EV-DLI) (P-value: EL4+CTC28-DLI vs EL4+EV-DLI). The data were pooled from two independent experiments using five mice per group (a, b and d). P-values from log-rank (Mantel–Cox) tests.

Figure 2

Dual modification of CD4 and CD8 T cells is necessary for the in vivo expansion of CD8 T cells. CD4 and CD8 T cells that were purified from B10.A mice were stimulated with anti-CD3 and anti-CD28 and transduced with either EV or CTC28 retroviruses. After resting, various combinations of EV− or CTC28-transduced CD4 and CD8 T cells were mixed at a 1:1 ratio and i.v. injected into the mixed chimera mice 4–6 h after EL4 injection. (a) The survival of the mice was compared between the groups (P-value: EL4+CTC28-CD4+CTC28-CD8 vs EL4+EV-CD4+EV-CD8, log-rank (Mantel–Cox) test). Pooled data from three independent experiments using five mice per group are shown. (b–d) Peripheral blood samples were collected at 7, 14 or 21 days after DLI, and the in vivo expansions of the transduced CD4 T cells (CD4⁺GFP⁺) (b and c) and CD8 T cells (CD8⁺GFP⁺) (d and e) were analyzed by flow cytometry. Representative flow cytometry profiles are presented in (b and d). The percentages of the populations were calculated from the pooled data of two independent experiments using five mice per group and are presented in (c and e). P-values: each group vs EV-CD4+EV-CD8, Student’s t-test; n.s., not significant; error bars, s.e.m.

Figure 3

CD8 T cells are responsible for the GVT effect in the therapeutic model of delayed DLI. The combined CTC28-transduced CD4 and CD8 T cells were i.v. administered to the mixed chimera mice 4–6 h after EL4 injection. CD8 T cell depletion was performed by i.v. injecting anti-CD8 antibody into the mice periodically (P-value: compared with EL4 only). A group of mice was injected with normal rat IgG as a negative control (n.s. in comparison with EL4 only). The data were pooled from two independent experiments using five mice per group. P-values, log-rank (Mantel–Cox) test.

Figure 4

The functional activity of the CTC28-transduced T cells is enhanced after in vivo expansion. CTC28-transduced or EV-transduced CD4 and CD8 T cells were generated and introduced into EL4 tumor-bearing mixed chimera mice as in Figure 1d. (a and b) The transduced T cells prior to DLI (a) and splenocytes isolated 10 days after DLI (b) were stimulated with PMA and ionomycin for 4 h, and IFN-γ production was analyzed by intracellular staining via flow cytometry. Representative flow cytometry profiles of GFP-positive cells (the transduced T cells) are shown. (c–f) For the GFP-positive T cells that were isolated and stimulated as in (b), the percentages of IFN-γ producing cells (c and d) and the mean fluorescent intensities (MFIs) of the produced IFN-γ (e and f) were quantified from pooled data from four independent experiments using three to five mice per group. P-values, Student’s t-test.

Figure 5

Mixed chimerism in the peripheral blood shifts to the donor type following CTC28-transduced DLI. The proportions of blood cells that originated from the donor and host hematopoietic cells (mixed chimerism) were assessed periodically after CTC28− or EV-transduced DLI into EL4-tumor bearing mixed chimera mice as in Figure 1d. The percentages of H-2K^b-positive (host-derived) and H-2K^b-negative (donor-derived) cells in the CD4− (a and b), CD8− (c and d) and B220-positive cells (e and f) were analyzed via flow cytometry. Representative flow cytometry profiles 14 and 21 days after DLI are shown in (a, c and e). The percentages of donor-derived cells were analyzed using pooled data from two independent experiments using five mice per group in (b, d and f). P-values, Student’s t-test.

Figure 6

CTC28-transduced DLI induces late-onset and non-lethal GVHD. CTC28− and EV-transduced DLI were performed in the mixed chimera mice without EL4 tumor injection. A group of the mice did not receive donor lymphocytes (No DLI) as a negative control. (a and b) The degree of GVHD was assessed periodically by weight loss (a) and clinical scoring for GVHD (b) over 70 days. (c) The main tissues involved in GVHD were isolated 28 days after DLI, and tissue sections were stained with hematoxylin and eosin for histological examination. The results are representative of two independent experiments. The error bars indicate the s.e.m.

Figure 7

IL-10-transduced third-party MSCs mitigate DLI-induced GVHD and enhance mouse survival in the tumor-bearing mixed chimeras. (a) IL-10− and EV-transduced D3116 BALB/c MSCs (IL-10-MSC; EV-MSC) were generated by retroviral transduction. The amounts of IL-10 produced by various numbers of MSCs were measured in the 48 h culture supernatant by ELISA. (b–d) CTC28− and EV-transduced DLI were performed in the tumor-bearing mixed chimera mice as in Figure 1d. IL-10-MSCs were i.p. administered three times at 7-day intervals beginning on the day of tumor injection/DLI. The degree of GVHD was assessed by weight loss (b) and clinical scoring (c). The survival of the mice was periodically monitored (d). *, for the groups treated with tumor alone and with tumor plus DLI, the GVHD assessments were halted at ~28–35 days after DLI due to the significant deaths of the tumor-bearing mice. The data were pooled from three independent experiments using five mice per group. P-values, log-rank (Mantel–Cox) test.