Compensatory upregulation of PD-1, LAG-3, and CTLA-4 limits the efficacy of single-agent checkpoint blockade in metastatic ovarian cancer - PubMed (original) (raw)

Compensatory upregulation of PD-1, LAG-3, and CTLA-4 limits the efficacy of single-agent checkpoint blockade in metastatic ovarian cancer

Ruea-Yea Huang et al. Oncoimmunology. 2016.

Abstract

Tumor-associated or -infiltrating lymphocytes (TALs or TILs) co-express multiple immune inhibitory receptors that contribute to immune suppression in the ovarian tumor microenvironment (TME). Dual blockade of PD-1 along with LAG-3 or CTLA-4 has been shown to synergistically enhance T-cell effector function, resulting in a delay in murine ovarian tumor growth. However, the mechanisms underlying this synergy and the relative contribution of other inhibitory receptors to immune suppression in the ovarian TME are unknown. Here, we report that multiple immune checkpoints are expressed in TALs and TILs isolated from ovarian tumor-bearing mice. Importantly, blockade of PD-1, LAG-3, or CTLA-4 alone using genetic ablation or blocking antibodies conferred a compensatory upregulation of the other checkpoint pathways, potentiating their capacity for local T-cell suppression that, in turn, could be overcome through combinatorial blockade strategies. Whereas single-agent blockade led to tumor outgrowth in all animals, dual antibody blockade against PD-1/CTLA-4 or triple blockade against PD-1/LAG-3/CTLA-4 resulted in tumor-free survival in 20% of treated mice. In contrast, dual blockade of LAG-3 and CTLA-4 pathways using PD-1 knockout mice led to tumor-free survival in 40% of treated mice, suggesting a hierarchical ordering of checkpoint function. Durable antitumor immunity was most strongly associated with increased numbers of CD8+ T cells, the frequency of cytokine-producing effector T cells, reduced frequency of Tregs and arginine-expressing monocytic myeloid-derived suppressor cells in the peritoneal TME. These data provide a basis for combinatorial checkpoint blockade in clinical intervention for ovarian cancer.

Keywords: Antibody blockade; CTLA-4; LAG-3; PD-1; immune inhibitory checkpoint; ovarian cancer.

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Figures

Figure 1.

Figure 1.

Multiple immune checkpoints are expressed on TALs of murine ovarian cancer microenvironment. Pooled data of eight inhibitory receptor expression on CD8+ (A) and CD4+ (B) TALs from tumor-bearing mice. IE9mp1 tumor cells (1×107) were injected intraperitoneally. TALs were isolated at day 25 (Materials and Methods) and stained for surface expression of the checkpoints and analyzed with flow cytometry. Expression of eight checkpoints in T cells from splenocytes is shown for comparison. Data were obtained from 10 animals and are representative of two independent experiments. Data were analyzed using GraphPad Prism 6. Error bars represent SD. Statistical significance was determined by Student's _t_-test. *p < 0.05; **p < 0.01; ***p < 0.001. Examples of CD8+ (C) and CD4+ (D) TALs coexpressing two checkpoint molecules are shown. Representative flow cytometry analysis of TALs stained with Live/Dead dye, mAbs to CD4+, CD8+, PD-1, LAG-3, TIM-3, and CTLA-4. Dot plot analyses were gated on live cells, then on CD8+ or CD4+ and showed percentages of single and double stained PD-1, LAG-3, CTLA-4, and TIM-3-positive cells.

Figure 2.

Figure 2.

Expression of four immune checkpoints on TALs from the human ovarian cancer microenvironment. (A) Pooled data of the PD-1, LAG-3, TIM-3, and CTLA-4 expression on CD8+ (left panel) and CD4+ (right panel) TALs from 10 human ovarian cancer patients. (B, C) Examples of flow cytometry analysis displaying co-expression of dual immune checkpoints. TALs were isolated as described in Materials and Methods, stained with antibodies against CD8+, CD4+, and the above mentioned checkpoints and analyzed by flow cytometry. Numbers represent the percentage of each population.

Figure 3.

Figure 3.

Compensatory upregulation of immune checkpoints in murine OVC after blockade of single checkpoint pathway. (A, B) Elevated expression of other checkpoints in tumor-bearing PD-1KO or LAG-3KO mice. The levels of LAG-3, CTLA-4, and TIM-3 are elevated in the TALs from the tumor-bearing PD-1KO mice, whereas PD-1, TIM-3, and 2B4 are increased in tumor-bearing LAG-3KO mice. Tumor implantation was performed as described in Fig. 1. TALs and TILs (data not shown) were collected from tumor-bearing C57BL6 (wild-type), PD-1KO, and LAG-3KO mice (five animals per group) at early time points and stained for the antibody against the indicated checkpoints. Data are representative of two independent experiments. (C, D) Individual blockade with immune checkpoint antibody anti-PD-1, or anti-LAG-3, or anti-CTLA-4 in the wild-type mice results in elevated expression of the other immune inhibitory checkpoints. Ten days after tumor implantation, mice were treated intraperitoneally with antibody (200 μg per mouse) every other day for four times. T cells were analyzed 7 d after the end of the treatment. Data were analyzed using GraphPad Prism 6. Error bars represent SD. Statistical significance was determined by Student's _t_-test. *p < 0.05; **p < 0.01; ***p < 0.001. Data were obtained from five animals and are representative of three independent experiments.

Figure 4.

Figure 4.

Combinatorial and triple blockade of PD-1, CTLA-4, and LAG-3 pathways exerts differential antitumor immunity. (A) Experimental scheme and antibody blockade treatment. C57BL/6 mice were implanted with IE9mp1 tumors as described in Fig. 3, randomized and treated with control IgG, single antibody, and combination of two or three antibodies were administered every other day for six times (Materials and Methods). Tumor progression was monitored by measuring the abdominal circumference due to accumulation of ascites. Survival was determined when the abdominal circumference reached 12 cm or moribund. (B) Combinatorial blockade of PD-1/LAG-3 or CTLA-4/LAG-3 pathways mildly improve antitumor immunity. Antibody treatment was as described above. Survival data were analyzed with the Mantel–Cox log-rank test. Data were obtained from 14 to 15 mice per group and were representative of two independent experiments. (C) Triple blockade of three checkpoints and dual antibody blockade of PD-1 and CTLA-4 significantly enhanced the survival of ovarian tumor-bearing wild-type mice. Antibody treatment and data analysis were performed at the same time as that described in (B).

Figure 5.

Figure 5.

Combinatorial blockade of PD-1, CTLA-4, and LAG-3 significantly enhance lymphocyte infiltration and function at early time point. (A) Frequency of CD8+ and CD4+ TALs (ascites) and TILs (tumor) from ovarian tumor-bearing mice after checkpoint blockade at early time point. Both CD8+ and CD4+ TALs were significantly elevated in triple antibody-treated group. (B) Frequency of cytokine-producing CD8+ TALs is enhanced after antibody blockade treatment. T effector function was assessed by percentage of cytokine-producing cells (IFNγ, TNFα, and IL2). Dual IFNγ- and TNFα-producing cells represent polyfunctionality of the population. Mice were treated every other day with total six doses of antibodies on days 10–20. TALs and TILs were isolated at 25 d posttumor implantation. For cytokine production TALs were stimulated with PMA/Ionomycin in the presence of BFA for 5 h. Cytokine producing cells were analyzed as described in Material and Methods. Data are representative of two independent experiments with 3–5 animals per group. Error bars represent SD. Statistical significance was determined by Student's _t_-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6.

Figure 6.

Effects of combinatorial blockade of PD-1, CTLA-4, and LAG-3 on Treg and MDSC population in ovarian TME. (A) Dual and triple antibody blockade treatment decreased the frequency of CD4+CD25+FoxP3+ cells at early time point of ovarian tumor progression. TALs and TILs were isolated as described above and stained for the expression of CD4+, CD25, and FoxP3 protein and analyzed using flow cytometry. (B) Arginase 1 (ARG1)-expressing population was increased in the ovarian TME as compared with that in spleen. Splenocytes and TALs were isolated as described in Fig. 5. Frequency of ARG1+ population in the CD45+CD11b+ cells was determined based on the gating shown in Fig. S3. (C–E) Various checkpoint blockade combinations reduced the frequencies of ARG1+-MDSCs. The frequency of ARG1+-Ly6G+6C+ cells was significantly reduced by PD-1/CTLA-4 dual blockade (C). All three dual blockades of PD-1/LAG-3, PD-1/CTLA-4, and LAG-3/CTLA-4 significantly reduced the frequency of ARG1+-Ly6G−6C+ (D) and ARG1+-F4/80+ cells (E). Data were obtained from 5 mice per group, analyzed using GraphPad Prism 6, and are representative of two independent experiments. Error bars represent SD. Statistical significance was determined by Student's _t_-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7.

Figure 7.

Genetic knockout of PD-1 or LAG-3 enhances the impact of combination antibody treatment. (A) Combinatorial blockade of LAG-3 and CTLA-4 significantly enhanced the survival of ovarian tumor-bearing PD-1KO mice. Antibody treatment and data analysis were performed as described in Fig. 4. Survival data were derived from 10 to 12 mice per group and are representative of two independent experiments. (B) Combinatorial blockade of PD-1 and CTLA-4 significantly enhanced survival of LAG-3KO mice bearing ovarian tumors. Survival data were derived from 8 to 10 mice per group and are representative of two independent experiments.

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