siRNA silencing of PD-1 ligands on dendritic cell vaccines boosts the expansion of minor histocompatibility antigen-specific CD8(+) T cells in NOD/SCID/IL2Rg(null) mice

Abstract

Allogeneic stem cell transplantation (allo-SCT) can be a curative therapy for patients suffering from hematological malignancies. The therapeutic efficacy is based on donor-derived CD8(+) T cells that recognize minor histocompatibility antigens (MiHAs) expressed by patient's tumor cells. However, these responses are not always sufficient, and persistence and recurrence of the malignant disease are often observed. Therefore, application of additive therapy targeting hematopoietic-restricted MiHAs is essential. Adoptive transfer of MiHA-specific CD8(+) T cells in combination with dendritic cell (DC) vaccination could be a promising strategy. Though effects of DC vaccination in anti-cancer therapy have been demonstrated, improvement in DC vaccination therapy is needed, as clinical responses are limited. In this study, we investigated the potency of program death ligand (PD-L) 1 and 2 silenced DC vaccines for ex vivo priming and in vivo boosting of MiHA-specific CD8(+) T cell responses. Co-culturing CD8(+) T cells with MiHA-loaded DCs resulted in priming and expansion of functional MiHA-specific CD8(+) T cells from the naive repertoire, which was augmented upon silencing of PD-L1 and PD-L2. Furthermore, DC vaccination supported and expanded adoptively transferred antigen-specific CD8(+) T cells in vivo. Importantly, the use of PD-L silenced DCs improved boosting and further expansion of ex vivo primed MiHA-specific CD8(+) T cells in immunodeficient mice. In conclusion, adoptive transfer of ex vivo primed MiHA-specific CD8(+) T cells in combination with PD-L silenced DC vaccination, targeting MiHAs restricted to the hematopoietic system, is an interesting approach to boost GVT immunity in allo-SCT patients and thereby prevent relapse.

Fig. 1

Priming of functional MiHA-specific CD8⁺ T cells is enhanced by PD-L silenced DCs. CD8⁺ T cells were co-cultured with MiHA peptide-loaded allogeneic DCs and analyzed at day 7 by flow cytometry for the presence of MiHA-specific CD8⁺ T cells. a Representative FACS plot of two independent cultures. The numbers in the FACS plots represent the percentage of MiHA-specific CD8⁺ T cells within the total CD3⁺CD8⁺ T cell population. b Combined data of nine independent cultures. c Representative FACS plot of CD28 expression (black) on MiHA-specific CD8⁺ T cells at day 7 of culture, isotype control in gray. The numbers in the FACS plots represent mean fluorescence intensity (MFI). One out of four independent cultures is shown. d Representative FACS plot of cultured cells at day 7, which were overnight re-stimulated with peptide followed by intracellular staining for IFNγ and CD137. The number in the FACS plots represents the percentage of IFNγ⁺ cells within CD137^hi CD8⁺ T cells. e–f CD8⁺ T cells were cultured for two consecutive weeks with HA-1 peptide-loaded PD-L silenced or control DCs (relative expression: PD-L1 6 %, PD-L2 23 %), and analyzed at day 14 by flow cytometry for the presence of MiHA-specific CD8⁺ T cells. FACS plot (e) and absolute cell numbers (f) of cultures with control or PD-L silenced DCs, n = 1

Fig. 2

MiHA-specific CD8⁺ T cell priming and expansion from the naive repertoire is augmented by PD-L silenced DCs. Naïve (N, CD45RA⁺CCR7⁺) and effector memory (EM, CD45RA⁻CCR7⁻) CD8⁺ T cells were sorted and cultured for two consecutive weeks with HA-1 peptide-loaded PD-L silenced or control DCs (relative expression: PD-L1 7 %, PD-L2 12 %). The percentage (a) and cumulative numbers (b) of HA-1-specific CD8⁺ T cells obtained after stimulation with PD-L silenced or control DCs

Fig. 3

CMV-specific CD8⁺ T cell expansion by DC vaccination in vivo. Peripheral blood lymphocytes containing 2000 CMV-specific CD8⁺ T cells were injected in NSG mice and stimulated with no (ctrl), one, two or three peptide-loaded (5 µM) DC vaccinations at weekly intervals. Mice were killed 7 days after their last DC vaccination or at the end of the experiment (ctrl). Peripheral blood and spleen were analyzed by flow cytometry for human CMV-specific CD8⁺ T cells. Engraftment of human cells (a) and CD8⁺ T cell (b). Statistical analysis was performed using one-way ANOVA followed by a Bonferroni post hoc test. c Representative FACS plot of CMV-specific CD8⁺ T cells after three DC vaccinations. The numbers in the FACS plots represent the percentage of CMV-specific CD8⁺ T cells within the total CD3⁺CD8⁺ T cell population. d Percentage and absolute cell numbers of CMV-specific CD8⁺ T cells of mice non-vaccinated, or vaccinated three times. Statistical analysis was performed using a one-tailed Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

PD-L silenced DCs enhanced MiHA-specific CD8⁺ T cell expansion in vivo. a HA1-primed PBLs containing 2900 HA1-specific T cells were injected in NSG mice and vaccinated three times with control or PD-L silenced (relative expression: PD-L1 13.1 %, PD-L2 10.5 %) DCs at weekly intervals. Mice were killed at day 21, and peripheral blood and spleen were analyzed by flow cytometry for human HA-1-specific CD8⁺ T cells. b Representative FACS plot of HA-1-specific CD8⁺ T cells in spleen after control or PD-L silenced DC vaccinations. The numbers in the FACS plots represent the percentage of HA-1-specific CD8⁺ T cells within the CD3⁺CD8⁺ T cell population. c Percentages within CD8⁺ T cells, and absolute levels of HA-1-specific CD8⁺ T cells in peripheral blood and spleen. Each dot represents a single mouse, n = 6 mice per group. Statistical analysis was performed using a one-tailed Student’s t test