Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment - PubMed (original) (raw)

Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment

Stefani Spranger et al. J Immunother Cancer. 2014.

Abstract

Background: Blockade of immune inhibitory pathways is emerging as an important therapeutic modality for the treatment of cancer. Single agent treatments have partial anti-tumor activity in preclinical models and in human cancer patients. Inasmuch as the tumor microenvironment shows evidence of multiple immune inhibitory mechanisms present concurrently, it has been reasoned that combination therapies may be required for optimal therapeutic effect.

Methods: To test this notion, we utilized permutations of anti-CTLA-4 mAb, anti-PD-L1 mAb, and/or the IDO inhibitor INCB23843 in the murine B16.SIY melanoma model.

Results: All three combinations showed markedly improved tumor control over single treatments, with many mice achieving complete tumor rejection. This effect was seen in the absence of vaccination or adoptive T cell therapy. The mechanism of synergy was investigated to examine the priming versus effector phase of the anti-tumor immune response. Only a minimal increase in priming of anti-tumor T cells was observed at early time points in the tumor-draining lymph nodes (TdLN). In contrast, as early as three days after therapy initiation, a marked increase in the capacity of tumor-infiltrating CD8(+) T cells to produce IL-2 and to proliferate was found in all groups treated with the effective combinations. Treatment of mice with FTY720 to block new T cell trafficking from secondary lymphoid structures still enabled restoration of IL-2 production and proliferation by intratumoral T cells, and also retained most of the tumor growth control.

Conclusions: Our data suggest that the therapeutic effect of these immunotherapies was mainly mediated through direct reactivation of T cells in situ. These three combinations are attractive to pursue clinically, and the ability of intratumoral CD8(+) T cells to produce IL-2 and to proliferate could be an important biomarker to integrate into clinical studies.

Keywords: Anti-CLTA-4; Combinatorial immunotherapy; IDO inhibitor; Immune inhibitory pathways; PD-1/PD-L1; T cell anergy/exhaustion; Tumor microenvironment; Tumor-infiltrating lymphocytes.

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Figures

Figure 1

Figure 1

Pairwise combinations of αCTLA-4, αPD-L1 or IDOi blockade results in retarded tumor outgrowth. A. Schema of experimental design illustrating the time points of drug administration throughout the experiment. αCTLA-4 was administered on day 4, 7, 10 i.p., αPD-L1 on days 4, 6, 8, 10, 12, 14, 16 i.p. and IDOi was given on a 5 day/2 day off regimen via oral gavage Monday-Friday. B-D. Tumor outgrowth measured in mm2 comparing the single treatment to the respective combined double treatment of B. αCTLA-4 and αPD-L1 C. αCTLA-4 and IDOi and D. αPD-L1 and IDOi (pool of 3 experiments). Depicted are means +/− SEM of 6 mice from one representative experiment. All experiments were at least done three times with the same overall result. Numbers next to curves indicate # of survivors/total number of mice for all experiments done. Significance to the single treatments was tested using a two-way-Anova with Bonferroni post-test and is shown in the figure while all treatments regimens were significantly different to the no treatment control (**** <0.0001, ** <0.01).

Figure 2

Figure 2

Double regimen therapy does not result in substantially increased frequency of tumor-reactive T cells at early time points in the periphery. A-B. Peptide/Kb pentamer staining was performed on gated CD3+CD8+ T cells, isolated from TdLN (A) or spleen (B) on day 7. Shown are means +/− SEM of a total of 10 mice collected from two experiments. For statistical analysis, Mann–Whitney-U test was performed comparing single treatments to double treatments (* = 0.0317 αCTLA-4 to αCTLA-4 + αPD-L1 in spleen). None of the other treatments was significantly different to the no treatment control. C. IFN-γ ELISpot was performed on splenocytes collected on day 7. Data are shown as mean +/− SEM from 10 mice out of two experiments with no stimulation as open bar and SIY-specific stimulation as filled bar. The Mann–Whitney-U test was used to assess significant differences between the no treatment group and treatment regimens with * in the figure indicating significant difference compared to no treatment. Results with αCTLA-4 were significantly different to αCTLA-4 + IDOi (p = 0.0185) and αPD-L1 was significantly different to αCTLA-4 + αPD-L1 (p = 0.0172).

Figure 3

Figure 3

Double treatments restore capacity of lymphocytes within the tumor to produce IL-2 and proliferate. Tumors were harvested on day 7 and single cell suspensions were prepared. Pools of cells from 3–5 mice were combined and subsequently stained with cell trace. Cells were cultured with or without plate-bound anti-CD3 for 48 h then treated with anti-CD3/anti-CD28-stimulation in the presence of Brefeldin A for 6 h. Cells were then stained for production of IL-2, IFN-γ and TNF-α by intracellular flow cytometry. A. A representative FACS plot showing proliferation via cell trace dilution on the x-axis and intracellular IL-2 staining on the y-axis. A pool of five mice was analyzed. B. Statistical analysis of the amount of proliferating CD3+CD8+ cells in the non-stimulated (left) and stimulated (right) group. Only double treatments show a significant increase in proliferation compared to non-stimulated and single treatments when tested with a one-way Anova. Bars represent mean +/− SEM of a total of 4 pools collected out of 2 experiments. C. The percentages of IFN-γ+ (open bar), IFN-γ+ and IL-2+ (gray bar), and proliferating IFN-γ+IL-2+ cells (filled bar) were calculated within the CD3+CD8+ cell population. D. The percentages of polyfunctional (TNF-α+IFN-γ+IL-2+ proliferated) CD8+ T cells (black) compared to other less functional subsets is shown. Dark gray represents no proliferation, light gray represents no IL-2 production, white represents no IFN-γ production for each treatment group. C and D were calculated based on the results shown in A and B.

Figure 4

Figure 4

Restoration of IL-2 production and proliferation of tumor-infiltrating lymphocytes in the absence of new T cell migration. B16.SIY-bearing mice were either treated with FTY720 or control vehicle prior to initiation of therapy, to prevent migration of new lymphocytes into the tumor. A. Peripheral blood T cell numbers following FTY720 treatment on the day of tumor harvest for analysis. Open bars depict the number of CD45+CD3+ T cells detected in 200ul peripheral blood of vehicle treated mice set to 100%. Filled bars represent the number found in FTY720-treated mice, relative to the vehicle-treated group. B &C. Single cell suspensions from tumor were labeled with cell trace and stimulated with plate-bound anti-CD3 antibody for 48 h prior then with anti-CD3 and anti-CD28 in the presence of Brefeldin A. Cells were then analyzed for proliferation by cell trace dilution and production of IL-2 via intracellular staining. Depicted are the percentages of proliferating cells (B) or proliferating and IL-2 producing cells (C) comparing vehicle-treated groups (open bar) to FTY720-treated groups (filled bar). Results are shown as the mean +/− SEM combining two experiments with each having 2 pools of 3 mice. Significance was tested using Mann–Whitney-U test but no significant change between FTY720 and vehicle control could be detected except for increased IL-2 production in the αPD-L1 + IDOi treatment group (p = 0.02).

Figure 5

Figure 5

Tumor control is largely preserved despite FTY720 administration at later time points. B16.SIY-bearing mice were either treated with FTY720 or control vehicle prior to tumor inoculation (B), prior to initiation of therapy (C) or with the last dose of αCTLA-4 mAb on day 10 (D). A. Schematic overview of the experimental design bold arrow heads indicate initiation of FTY720 treatment, which was always carried out through the entire experiment. B-D. Tumor outgrowth measured in mm2 comparing vehicle control (circle) to FTY720 treatment (square) on no treatment (open) vs. αCTLA-4 + αPD-L1 (filled). B. FTY720 treatment was initiated 24 h prior to tumor inoculation. C. FTY720 treatment was given on day 4, 2.5 h before therapy was initiated. D. FTY720 was given at the same time point as the last dose of αCTLA-4 mAb treatment. Depicted is a representative experiment, showing mean of 5 mice +/− SEM. Significance was determined using a two-way ANOVA with Bonferroni post-test.

Figure 6

Figure 6

Immunotherapy doublets result in increased BrdU incorporation by CD8 + and CD4 + tumor-infiltrating T cells in vivo . TILs were harvested on day 7, 24 h after a single BrdU pulse in vivo, and cells were stained for BrdU along with anti-CD3, anti-CD4, and anti-CD8. Depicted are percentages of BrdU+ cells that were CD3+ CD8+(A) and CD3+ CD4+(B). Data shown present the mean of a total of 5 mice from one experiment and are representative of two independent experiments. Differences were assessed using a two-way Anova test and taking proliferation values from spleen and TdLN into account. * indicates significantly different to no treatment and all double treatments were significantly different to their corresponding single treatments with the exception of αCTLA-4 to both double treatments for CD4 T cell proliferation.

Figure 7

Figure 7

Immunotherapy doublets result in increased frequencies of tumor antigen-specific T cells at later time points in the periphery. Depicted is an IFN-γ ELISpot of splenocytes harvested on day 14 with open bars being the un-stimulated control and filled bars representing SIY-stimulation. Data are shown as the mean +/− SEM from 10 mice pooled from two experiments. Statistical analysis was done using Mann–Whitney-U test comparing all groups to no treatment. All double treatment groups were significantly different to their respective single treatment and when comparing double treatments within each other a significant difference between αCTLA4 + αPD-L1 and αPD-L1 + IDOi was observed.

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