Priming of naive CD8+ T cells in the presence of IL-12 selectively enhances the survival of CD8+CD62Lhi cells and results in superior anti-tumor activity in a tolerogenic murine model

Abstract

During the antigen-dependant activation process several subsets CD8+ T cells appear with different phenotypic and functional characteristics. Recent studies indicate that the state of T cell differentiation radically affects their ability to effectively respond to tumor challenge, with early effector CD8+ T (CD62Lhigh) cells having better anti-tumor activity. Thus strategies aimed at optimizing the generation of such subpopulations could significantly enhance the effectiveness of adoptive cell therapy (ACT) for cancer. In this study, we show that priming of naïve CD8+ T cells in the presence of IL-12 selectively rescued early CD8+ CD62L(hi) from activation induced cell death and resulted in the increased accumulation of this subset of CD8+ T cells. Furthermore, we demonstrated that IL-12 directly modulated the expression of CD62L on activated CD8+ T cells. When used for ACT, naïve CD8+ T cells primed in vitro in the presence of IL-12 showed superior anti-tumor activity toward B16 melanoma. Importantly, using the Pmel-1 model, priming pmel-1 cells in vitro with IL-12 reduced the state of functional tolerance associated with the non-mutated "self" tumor antigen gp100, as demonstrated by significant tumor responses in the absence of vaccination. Together, our results suggest that in vitro conditioning of naïve CD8+ T cells with IL-12 prior to ACT could significantly enhance their anti-tumor activity.

Fig. 1

Expression of CD62L on CD8⁺ T cells primed in the presence of IL-12. Spleen and LN suspensions (1 × 10⁶ cells/ml) from OT-1 mice were primed with 1 ug/ml SIINFEKL. Cell suspensions were either: (1) pretreated overnight with 10 ng/ml of IL-12 and then washed and primed with antigen (IL-12→antigen), (2) primed with antigen and IL-12 simultaneously (IL-12 + antigen), (3) primed with antigen overnight and then treated with IL-12 (antigen→IL-12), or (4) primed with antigen alone. Three days after priming cells were re-cultured at a density of 1 × 10⁶ cells/ml. Cells were harvested and expression of CD62L was determined by flow cytometry on Vα2 (antigen specific transgenic cells) on days 4, 5, and 6 after priming (a). Absolute numbers were calculated for each experimental group (b) and were expressed as total cell number

Fig. 2

In vitro anti-apoptotic effect of IL-12 on CD8⁺ CD62L^hi cells. OT-1 cells were primed with or without 10 ng/ml of IL-12. Cells were harvested at day 5 and 7 after priming and stained with a mixture of mAbs against Vα2, CD62L and Annexin-V. Cells positive for Vα2 were assayed for CD62L and annexin-V binding (a). Expression of caspase-3, caspase-8, Bax, and Bad was determined on sorted OT-1⁺CD62L^lo/IL-12 or OT-1⁺CD62L^hi/IL-12 at day-5 after priming by real-time PCR (b). Results are presented as the mean normalized expression which is the number of transcripts per 10³ copies of β-actin ± SEM

Fig. 3

IL-12 modulated the expression of CD62L on in vitro activated OT-1 cells. OT-1 cells primed with or without 10 ng/ml of IL-12 were cultured for 3 days and sorted into CD8⁺ CD62L^lo/sham, CD8⁺ CD62L^hi/sham, CD8⁺ CD62L^lo/IL-12 or CD8⁺ CD62L^hi/IL-12 (a). Sham-treated (b) or IL-12-treated (c) cells were re-cultured with or without IL-12 at a cell density of 1 × 10⁶/ml. Expression of CD62L was analyzed by flow cytometry at days 5 and 7 after priming (days 2 and 4 after reculture)

Fig. 4

In vitro IL-12 conditioning induces “Early effector/T_CM-like” phenotype of OT-1⁺CD62L^hi/IL-12 cells. OT-1 cells were primed in vitro with 1 μg/ml SIINFEKL. Ten ng/ml of IL-2 or IL-12 was added at the time of priming. Cells were cultured for 3 days, harvested, washed and recultured at a density of 1 × 10⁶ cells/ml. At day-7 after priming cells were harvested and stained with mAbs against Vα2, CD62L, CD127 and CD44. Percentages (a) and absolute number (b) of CD62L^hiCD127⁺ and CD62L^hiCD44⁺ cells were determined

Fig. 5

In vivo survival, recall potential and anti-tumor activity of OT-1 cells primed in vitro in the presence of IL-12. OT-1⁺Ly5.1⁺ cells were primed with 1 μg/ml of SIINFEKL with or without 10 ng/ml of IL-12 and cultured for 3 days. After culture cells were sorted into CD62L^hi/sham or CD62L^hi/IL-12 cells and adoptively transferred (1 × 10⁶ cells/mice) into recipient wild type Ly5.2 mice (n = 4 mice) previously treated with CTX. The percentage of Ly5.1⁺ cells in the peripheral blood was determined at the indicated time points (a). The same mice were vaccinated at day-22 post-transfer and frequency of donor cells (Ly5.1⁺) in the periphery was determined 3 days after vaccination (b). Wild type C57BL/6 mice were injected s.c with 5 × 10⁵ B16-OVA cells. Tumors were allowed to grow for 7 days. Mice received sorted CD8⁺CD62L^hi/IL-12, CD8⁺CD62L^lo/IL-12, CD8⁺CD62L^hi/sham, CD8⁺CD62L^lo/sham or saline. Tumor size was determined at days 7 (C) and 14 (D) after transfer. Tumor size is shown as the surface area in mm²

Fig. 6

Anti-tumor activity of Pmel-1^IL-12 cells in the absence of vaccination. Spleen and LN suspensions from Pmel-1 mice were cultured for 3 days with 1 μg/ml gp100_25–33 peptide with or without 10 ng/ml of IL-12. After culture cells were washed and recultured at a density of 1 × 10⁶ cells/ml (a) or transfused i.v. (1 or 5 × 10⁶ cells/mouse) to mice bearing 10-day old B16 tumors (b). Recultured cells were harvested at day 5 after priming and stained with antibodies against Vβ13 and CD62L and Annexin-V. Annexin binding and CD62L expression of Vβ13⁺ cells was analyzed by flow cytometry. Percentages and absolute number of non-apoptotic cells expressing high levels of CD62L was determined (a). B16-bearing mice were adoptively transferred (7 days after tumor challenge) with 1 × 10⁶ of Pmel-1^IL12 cells and the tumor size was determined at the indicated time points, and it is shown as the surface area in mm² (b)