Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells

Abstract

Tumor escape from immune-mediated destruction has been associated with immunosuppressive mechanisms that inhibit T cell activation. Although evidence for an active immune response, including infiltration with CD8(+) T cells, can be found in a subset of patients, those tumors are nonetheless not immunologically rejected. In the current report, we show that it is the subset of T cell-inflamed tumors that showed high expression of three defined immunosuppressive mechanisms: indoleamine-2,3-dioxygenase (IDO), PD-L1/B7-H1, and FoxP3(+) regulatory T cells (T(regs)), suggesting that these inhibitory pathways might serve as negative feedback mechanisms that followed, rather than preceded, CD8(+) T cell infiltration. Mechanistic studies in mice revealed that up-regulated expression of IDO and PD-L1, as well as recruitment of T(regs), in the tumor microenvironment depended on the presence of CD8(+) T cells. The former was driven by interferon-γ and the latter by a production of CCR4-binding chemokines along with a component of induced proliferation. Our results argue that these major immunosuppressive pathways are intrinsically driven by the immune system rather than being orchestrated by cancer cells, and imply that cancer immunotherapy approaches targeting negative regulatory immune checkpoints might be preferentially beneficial for patients with a preexisting T cell-inflamed tumor microenvironment.

Fig. 1. Correlation of FoxP3 expression with CD8⁺ cell infiltration and IDO and PD-L1 expression in melanomas

(A) From a series of metastatic melanoma patient tumor biopsies, FoxP3, PD-L1, and IDO mRNA expression levels were determined using real-time RT-PCR. 18S was used as an internal control. (B) IHC analysis was performed on a representative subset of tumors. CD8 was developed using Vulcan Red, and FoxP3 was developed using horseradish peroxidase. Similar results were observed for three independent tumors of each category. Scale bars, 20 μm. (C) Number of CD8⁺ and FoxP3⁺ cells was assessed in three visual fields (depicted as area). Correlation studies were performed with R² = 0.6689 (Pearson correlation; total of 16 patients). (D and E) Tumors were grouped in CD8^high and CD8^low and analyzed for the amount of PD-L1 (D) or IDO (E) staining. The number of CD8⁺ T cells correlated significantly with the level of PD-L1 (p = 0.035) and IDO (p = 0.002) expression when tested with a two-sided χ² in combination with Fisher's exact test.

Fig. 2. Dependence of IDO, PD-L1, and FoxP3⁺ T_regs in the B16 tumor microenvironment on host CD8⁺ T cells and IFN-γ

B16.SIY melanoma cells were implanted subcutaneously in wild-type (WT) C57BL/6 mice treated with isotype control antibody (n = 2; gray) or CD8-depleting antibody (n = 3; dashed line) or in IFN-γ^−/− mice (n = 2; solid line). (A to D) At day 7, the tumors were subjected to ex vivo RT-PCR and fluorescence-activated cell sorting (FACS) analysis for IDO (A), PD-L1 (B), and FoxP3 (C and D). FACS data for PD-L1 are shown as geometric mean within the GFP⁺CD45⁻ population, and the T_regs were identified as GFP⁻CD8⁻CD4⁺CD25⁺FoxP3⁺ cells. Shown is the mean ± SEM and tested for significance using one-way analysis of variance (ANOVA) (Kruskal-Wallis) test (WT = 5; IFN-γ = 6; CD8-depleted = 5) with P = 0.0016 for IDO expression, P =0.01 for PD-L1 expression, and P =0.0003 for FoxP3 expression (qRT-PCR and FACS). The results shown are representative of two independent experiments, which were combined for statistical purposes. KO, knockout; n.s., not significant.

Fig. 3. Lack of detectable conversion of FoxP3⁻ CD4⁺ T cells to induced T_regs in B16 melanoma in vivo

After tumor inoculation of B16 cells, Rag2^−/− mice were injected with either 3 × 10⁶ total splenocytes or GFP-depleted splenocytes of a FoxP3-GFP reporter mouse. On day 7 tumor, tumor-draining lymph node (TdLN) and spleen were analyzed for the amount of GFP⁺/CD25⁺/FoxP3⁺ cells within their CD4⁺ T cell compartment. (A) Representative example for results obtained with total splenocytes (top row) or GFP-depleted splenocytes injected in vivo (lower row). Cells were pregated on living cells and CD3⁺CD4⁺ cells. (B) Statistical analysis of six mice shown as mean ± SEM and two-sided Mann-Whitney U test to determine significance. ****P < 0.0001; ***P < 0.001).

Fig. 4. CD8⁺ T cells contribute to T_reg proliferation in the tumor site in vivo

B16 cells were injected subcutaneously in WT mice treated with CD8-depleting antibody or isotype control. Twenty-four hours after BrdU pulse, given on day 7 after tumor inoculation, tumor and tumor-draining lymph node were analyzed for the amount of proliferated CD4⁺FoxP3⁺ T_regs. (A) PercentageofFoxP3⁺ cells detected within the CD4⁺ T cell fraction either BrdU⁻ (black) or BrdU⁺ (gray) (n = 6 out of two independent experiments; shown as mean ± SEM). (B) Fraction of proliferated (BrdU⁺) cells detected within the total T_reg fraction (CD3⁺CD4⁺FoxP3⁺) in tumor (left) and tumor-draining lymph node (right). Using a two-sided Mann-Whitney U test to compare the two proliferated fractions, we confirmed a significantly reduced proliferation rate within the tumor-infiltrating T_regs under CD8-depleting conditions. *P =0.002; **P = 0.0094.

Fig. 5. Chemokine-mediated recruitment of T_regs in the tumor micro-environment supported by CD8⁺ T cells in vivo

(A and B) B16 cells were engrafted into Rag2^−/− (subcutaneously) followed, 24 hours later, by injection of 3 × 10⁶ CD8⁺ T cells (intravenously) (if indicated), and on day 10 after tumor inoculation, 1 × 10⁶ T_regs (CD4⁺ CD25⁺) were given (intravenously). PTX T_regs were treated with pertussis toxin for 1.5 hours in vitro before injection, and C021 T_regs were treated with the CCR4 antagonist C021 for 2 hours before injection. Forty-eight hours after T_reg injection, tumor and spleen were analyzed for the number of infiltrating T_regs (CD3⁺, CD4⁺,FoxP3⁺). T_regs could only be detected in the tumor site if nontreated and given in combination with pre-engrafted CD8⁺ T cells (A), whereas chemokine receptor inhibitory treatment did not alter homing to the spleen (B). Shown are means ± SEM of n = 6 out of two independent experiments; significance was tested using two-sided Mann-Whitney U test (**P = 0.0024; ****P < 0.0001). (C) Naïve (CD62L⁺) CD8 T cells were cultured for 6 hours or 7 days ± 6 hours or assayed ex vivo. After activation, mRNA expression levels of CCL22 were assessed by qRT-PCR, normalized to 18S RNA, and were relative to the ex vivo expression level of CCL22 (*) (left panel). Tumor-infiltrating CD8 T cells were sorted out of B16 tumors 12 days after engraftment and analyzed either ex vivo or after activation for 6 hours (right panel). Shown are means ± SEM of n = 3, with numbers indicating the fold change compared to ex vivo naïve or tumor-infiltrating CD8⁺ T cells.

Fig. 6. Human CD8⁺ T cells support the recruitment of T_regs in a xenograft model in vivo

(A) Purified CD8⁺ T cells were stimulated with anti-CD3/anti-CD28 mAb-coated beads for different terms, and supernatants were collected and analyzed using CCL22 ELISA. Data represent means ± SEM and are representative of two independent experiments done with two normal donors each. (B) Supernatants from activated human CD8⁺ effector cells or control culture medium were pretreated with anti-CCL22 mAb (aCCL22) or mouse IgG for 30 min and then placed in the bottom wells of a Transwell system. Flow cytometrically sorted CD4⁺CD25^hi cells were added to the top wells, and 2 hours later, the cells that migrated to the bottom well were counted. Data represent means ± SEM and are representative of three independent experiments done in duplicate. PTX, pertussis toxin–treated T_regs. (C) The indicated cell subsets were injected into mice bearing human melanoma xenografts by tail vein injection. Forty-eight hours later, tumors were collected, made into single-cell suspensions, and stained for human CD8, CD4, and FoxP3. Representative FACS plots are shown for two independent experiments done with five mice in each group. P < 0.01, T_reg cell injection followed by CD8⁺ T cell injections versus T_regs injected alone.