Ex vivo conditioning with IL-12 protects tumor-infiltrating CD8+ T cells from negative regulation by local IFN-γ

Abstract

Optimal ex vivo expansion protocols for adoptive cell therapy (ACT) must yield T cells able to effectively home to tumors and survive the inhospitable conditions of the tumor microenvironment (TME), while simultaneously exerting persistent anti-tumor effector functions. Our previous work has shown that ex vivo activation in the presence of IL-12 can induce optimal expansion of murine CD8⁺ T cells, thus resulting in significant tumor regression after ACT mostly via sustained secretion of IFN-γ. In this report, we further elucidate the mechanism of this potency, showing that IL-12 additionally counteracts the negative regulatory effects of autocrine IFN-γ. IL-12 not only downregulates PD-1 expression by T cells, thus minimizing the effects of IFN-γ-induced PD-L1 upregulation by tumor stromal cells, but also inhibits IFNγR2 expression, thereby protecting T cells from IFN-γ-induced cell death. Thus, the enhanced anti-tumor activity of CD8⁺ T cells expanded ex vivo in the presence of IL-12 is due not only to the ability of IL-12-stimulated cells to secrete sustained levels of IFN-γ, but also to the additional capacity of IL-12 to counter the negative regulatory effects of autocrine IFN-γ.

Keywords: Adoptive T cells transfer; Melanoma/skin cancers; Models of host–tumor interactions; PD-1; Tumor microenvironment; Tumor promotion and progression.

Fig. 1

Anti-tumor activity of Pmel^Ag+IL-12 cells associates with sustained levels of IFN-γ. a Adoptive cell therapy approach involving the infusion of Pmel cells activated in the presence of antigen with or without IL-12 into a lymphopenic host. This approach mediates the regression of pre-established melanoma lesions which results in improved survival. b Tumors from mice treated with Pmel^Ag or Pmel^Ag+IL-12 were harvested 3 and 9 days after adoptive transfer. Expression of IFN-γ in RNA isolated from tumors was measured using SYBR green quantitative real-time PCR. The relative quantification of the target transcripts normalized to the endogenous control (actin) was determined by the comparative Ct method. Data are presented as average fold change ± SD (n = 3) relative to tumors from control mice that did not receive adoptive transfer. Two-tailed Student’s t test was used, *p < 0.05, **p < 0.005, and ***p < 0.0001. c Tumors were digested to single cell suspensions and analyzed for frequency of infiltrating Pmel cells (Thy1.1⁺) by flow cytometry. Data shown are representative of at least three independent experiments

Fig. 2

IL-12 downregulates PD-1 expression on adoptively transferred TILs independently of antigen. a Expression of PD-1 among Pmel cells activated in the presence of antigen plus or minus IL-12 prior to and 7 days after adoptive transfer in cell suspensions from spleen and tumor was determined by flow cytometry. Decreased levels of PD-1 expression among tumor-infiltrating Pmel^Ag+IL-12 was confirmed by quantitative real-time PCR. b Mice bearing B16 melanoma tumors treated with Pmel^Ag, Pmel^Ag+IL-12, or CTX or PBS alone were injected i.p with 50 µg of cognate peptide every other day for 10 days. At day 11, blood was collected and expression of PD-1 was assessed on circulating Pmel (Thy1.1⁺) cells. c 10 days after transfer, mice bearing B16 melanoma tumors untreated or treated with Pmel^Ag or Pmel^Ag+IL-12 cells were injected i.v. with 5 × 10⁴ B16 melanoma cells. 25 days after transfer mice were killed and lungs excised and examined for the presence of B16 nodules. d At the same time, primary tumors were excised and weighed. Tumor size is presented as the average weight ± SD (n = 3). Data shown are representative of at least three independent experiments

Fig. 3

IL-12 can directly downregulate the expression of PD-1 on human TILs. a Tumor cell suspensions from human melanoma tumors were incubated with 10 ng/ml human IL-12 for 72 h, and the expression of PD-1 among CD8⁺ T cells was determined by flow cytometry before and after culture. (n = 3). b The same tumor cell suspensions were cultured in the presence of anti-CD3/CD28 beads with or without IL-12, and production of IFN-γ among CD8⁺ T cells was determined by flow cytometry (n = 5). One-tailed paired Student’s t test was used, *p < 0.05, **p < 0.005, and ***p < 0.0001. c Cell suspensions from a B16 melanoma tumor were incubated with 10 ng/ml murine IL-12 for 72 h, and the expression of PD-1 among CD8⁺ T cells was determined by flow cytometry

Fig. 4

Lack of IFN-γ sensitivity protects activated CD8 + T cells from IFN-γ-induced apoptosis. a Adoptively transferred Thy1.1⁺ Pmel cells were sorted from cell suspensions prepared from similar size tumors (n = 3) 5 days after treatment with Pmel^Ag or Pmel^Ag+IL-12 cells, and analyzed for IFNγR1 and IFNγR2 expression by qPCR. Data are expressed as fold expression of Pmel^Ag+IL-12 over Pmel^Ag ±SD (p = 0.07). 1 × 10⁶ ex vivo expanded wild-type Pmel^Ag or Pmel^Ag+IL-12, and IFNγR^−/− Pmel^Ag cell were recultured in fresh media. b Cell viability was determined after 24, 48, and 72 h of culture using the LIVE/DEAD^® assay. c Apoptosis was measured by annexin V stain 24 h after culture. Data are presented as the average ratio of CD8⁺ annexin V⁺ over CD8⁺ annexin V⁻ cells ± SD (n = 3). d RNA isolated from cells after 24 h of culture was analyzed by qPCR for expression of Bcl-2 and Bax. These values were used to calculate Bax/Bcl-2 ratio which directly associates with apoptosis [20]. Data are presented as the average change in Bax/Bcl-2 ratio relative to their respective baseline measurement ± SD (n = 3). Two-tailed Student’s t test was used, *p < 0.05, **p < 0.005, and ***p < 0.0001. Data shown are representative of at least three independent experiments

Fig. 5

Adoptively transferred ex vivo expanded Pmel cells unable to respond to IFN-γ stimuli have longer in vivo survival and superior anti-tumor activity. a Circulating levels of adoptively transferred Pmel^Ag or IFNγR^−/− Pmel^Ag ex vivo expanded in the presence of antigen plus or minus IL-12 at days 5 and 13 after infusion into wild-type mice bearing B16 melanoma tumors. Data are presented as the average percent of CD8⁺/Thy1.1⁺ cells of total lymphocytes ± SD. (n = 4–5). b Tumor progression and c overall survival of mice treated with CTX alone or with either Pmel^Ag, Pmel^Ag+12 or IFNγR^−/− Pmel^Ag cells. Two-tailed Student’s t test was used, *p < 0.05, **p < 0.005, and ***p < 0.0001. Cumulative survival was calculated using a Kaplan–Meier curve using GraphPad Prism (n = 5). Data shown are representative of at least three independent experiments

Fig. 6

IL-12-induced mechanisms to counter negative feedback by IFN-γ Exposure to IL-12 during ex vivo expansion protects tumor-infiltrating transferred CD8⁺ T cells from negative feedback by IFN-γ, directly by downregulating IFNγR2 which results in reduced apoptosis, and indirectly by downregulating PD-1 hence circumventing the impact of IFN-γ-induced PD-L1 upregulation on tumor stromal cells