Suppressing T cell motility induced by anti-CTLA-4 monotherapy improves antitumor effects

Abstract

A promising strategy for cancer immunotherapy is to disrupt key pathways regulating immune tolerance, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4). However, the determinants of response to anti-CTLA-4 mAb treatment remain incompletely understood. In murine models, anti-CTLA-4 mAbs alone fail to induce effective immune responses to poorly immunogenic tumors but are successful when combined with additional interventions, including local ionizing radiation (IR) therapy. We employed an established model based on control of a mouse carcinoma cell line to study endogenous tumor-infiltrating CD8+ T lymphocytes (TILs) following treatment with the anti-CTLA-4 mAb 9H10. Alone, 9H10 monotherapy reversed the arrest of TILs with carcinoma cells in vivo. In contrast, the combination of 9H10 and IR restored MHC class I-dependent arrest. After implantation, the carcinoma cells had reduced expression of retinoic acid early inducible-1 (RAE-1), a ligand for natural killer cell group 2D (NKG2D) receptor. We found that RAE-1 expression was induced by IR in vivo and that anti-NKG2D mAb blocked the TIL arrest induced by IR/9H10 combination therapy. These results demonstrate that anti-CTLA-4 mAb therapy induces motility of TIL and that NKG2D ligation offsets this effect to enhance TILs arrest and antitumor activity.

Figures

Figure 1. Characterization of 4T1-CFP tumor model for imaging CD8+ TILs in Cxcr6+/gfp mice treated with local radiotherapy and anti–CTLA-4 mAb.

(A) Treatment schedule. (B) Growth of 4T1-CFP tumors of untreated and IR+9H10-treated mice (n = 6/group). Treated mice showed significant tumor inhibition (P = 0.0006). Vertical dashed lines indicate the days at which in vivo TPLSM was performed. (C–E) Ex vivo characterization of CD8 TILs on day 21. Samples were gated on CD8+ T cells. (C) Most GFP+ TILs express CD69, and represent a greater proportion of CD8+ cells in tumors treated with IR+9H10 than controls. (D) IR+9H10 treatment increases the density of CD8+ TILs about 1.8-fold, but the density of CD8 GFP+ TILs is increased by more than 4-fold. (E) The majority CD8+ TILs producing IFN-γ are GFP+, as determined by intracellular staining. Error bars are absent because pooling of tumors within each group was necessary to obtain sufficient number of cells for analysis. Results are from 10 mice/group and are representative of 2 experiments. (F) In vivo TPLSM images of GFP+ TILs in 4T1-CFP tumors on day 22 (Supplemental Videos 1 and 2). Mice were mock treated (Control) or treated as indicated. Images are representative of 6–8 independent experiments for each treatment. Scale bars: 58 μm. T cells are green (GFP), 4T1 cells are blue (CFP), blood vessels are red (quantum dots). (G) Quantification of GFP+ TILs on day 22. Results are the mean ± SD of 9 fields (9 × 104 μm2) from 3 mice per group. *P < 0.05, **P < 0.005.

Figure 2. Treatment with IR and 9H10 alters the migratory behavior of TILs.

Cxcr6+/gfp mice injected with 4T1-CFP cells and treated as described in Figure 1A were imaged on day 16. Movement of individual cells was tracked in the xy plane through stacks of 3D time-lapse images. Data are derived from 4 mice for each treatment. Each time lapse lasted 15 minutes and was acquired as a z-stack of 30 μm between 60 and 90 μm of depth below the capsule. (A) Trajectories of individual GFP+ TILs in CFP+ cell–rich areas (Supplemental Videos 3 and 4). The color scale shows time length of each path. Both IR and 9H10 as single treatment led to increased path lengths compared with controls. In contrast, when given in combination, IR+9H10 led to reduced migration of GFP+ TILs. Scale bar: 30 μm. Scatter plots of mean velocity (B), arrest coefficient (C), and confinement index (D) of GFP+ TILs in tumor cell–rich areas. The arrest coefficient was defined as the percentage of time a cell was moving at a speed less than 1.5 μm/min. Each data point represents a single cell, and red bars indicate mean values. *P < 0.05, **P < 0.005. (E) Random walk analysis. Mean displacement plotted as a function of the square root of time. The slope of each line represents the motility coefficient M and indicates the area an average cell scans per unit time. A liner curve indicates a random walk, a plateau indicates confinement, and a higher slope indicates directed motion.

Figure 3. T cell arrest requires MHC-I recognition.

Cxcr6+/gfp mice injected with 4T1-CFP cells and treated as described in Figure 1A were imaged on day 16. Migratory behavior of GFP+ TILs was analyzed 18 hours after injection of MHC-I blocking mAbs or irrelevant isotype mAb in untreated control mice and mice treated with IR+9H10, as indicated. Data were derived from 4 mice for each treatment. (A) Trajectories of individual GFP+ TILs, and scatter plots of mean velocity (B) and arrest coefficient (C). MHC blockade resulted in increased T cell migration in both treated and untreated mice. Each data point represents a single cell, and red bars indicate mean values. **P < 0.005.

Figure 4. Upregulation of ICAM-1 and RAE-1γ on 4T1 cells after in vivo irradiation.

Cxcr6+/gfp mice were injected s.c. with CFP+ 4T1 cells on day 0 and mock treated (Control) (n = 9) or treated with local IR in 2 doses of 12 Gy on days 13 and 14 (n = 9). Tumors were analyzed on day 16. To obtain sufficient material, we pooled tumors from 3 mice in each treatment group to obtain 3 independent samples. Data are representative of 3 experiments. (A) 4T1 tumor cells were identified as CFP+CD45–. Histograms show the expression of H2-Kd, ICAM-1, and RAE-1γ on the gated CFP+CD45– mock-treated (thin lines) and irradiated (bold lines) cells. Cells stained with isotype control (dashed lines). Bar graphs show MFI after subtraction of background of 3 samples ± SD. In vivo irradiation significantly increased ICAM-1 but not H2-Kd and induced the expression of RAE-1γ on 4T1 cells. (B) Expression of H2-Kd, ICAM-1, RAE-1γ, and H60 was compared in CFP+ 4T1 tumor cells cultured in vitro (blue lines) and CFP+ 4T1 tumor cells isolated from tumors implanted in mice (red lines). Gray lines, isotype control. H60 and RAE-1 molecules were equally undetectable on 4T1 cells from in vivo growing tumors digested with liberase or dissociated with EDTA, as described in Methods. Data are representative of 3 experiments.

Figure 5. RAE-1/NKG2D interactions promote T cell arrest in the presence of TCR ligation and 9H10.

(A and B) In vitro analysis of T cell motility. Migration of preactivated CD8+ T cells over an ICAM-1–coated glass surface. Trajectories (A) and mean velocity (B) of individual CD8+ T cells stimulated with anti-CD3 and/or 9H10 in the absence or presence of RAE-1β, as indicated. Paths were tracked over time from confocal 2D time lapses. Each line corresponds to the path of an individual cell. Each dot represents the average speed of a cell tracked over 15 minutes. Data are representative of 3 independent experiments. Scale bar: 10 εm. (C and D) In vivo analysis of T cell motility. Cxcr6+/gfp mice were injected with 4T1-CFP cells on day 0 and left untreated or treated with IR+9H10 as described in Figure 2, and tumors were imaged by TPLSM on day 16 (Supplemental Videos 5 and 6). All mice received an i.p. injection of NKG2D blocking mAb CX5 30 minutes before imaging. (C) Trajectories of individual GFP+ TILs show that CX5 treatment led to increased migratory activity in mice treated with IR+9H10 but not in control mice. Scale bar: 30 εm. (D) Scatter plots showed increased speed and decreased arrest coefficient of TILs in the presence of NKG2D blockade in tumors of mice treated with IR+9H10. Data are derived from analysis of 3 mice per treatment group. Each data point represents a single cell, and red bars indicate mean values. **P < 0.005.

Figure 6. Blocking NKG2D abrogates immune-mediated inhibition of the irradiated primary tumor in mice treated with IR+9H10 and reduces metastasis inhibition.

WT mice inoculated with parental 4T1 cells on day 0 (n = 7–13/group) were randomly assigned to different treatments, as indicated. IR was delivered in 2 fractions of 12 Gy to the s.c. tumors on day 12 and 13. Anti–CTLA-4 mAb 9H10 was given i.p. on days 15, 18, and 21. Anti-NKG2D mAb CX5 was given i.p. on days 15, 18, 21, 25, and 28. (A) Tumor volume is shown as the mean ± SEM at each time within each treatment group. CX5 by itself did not have any effect on tumor growth (P > 0.05, CX5 versus control). IR by itself caused significant tumor growth inhibition (P < 0.0005, IR versus control) that was not altered by CX5 (P = 0.4349, CX5+IR versus IR) but was significantly enhanced in the presence of 9H10 (P < 0.0005, IR+9H10 versus IR). The latter effect was completely abrogated by addition of CX5 (P = 0.1643, IR+9H10+CX5 versus IR). (B) The number of surface lung metastases was not significantly altered by IR or CX5 used alone (P > 0.05 versus control) or by IR in combination with CX5 (P > 0.05 versus control). IR+9H10 caused a significant inhibition of lung metastases (P < 0.005, IR+9H10 versus all other groups) that was partially abrogated by addition of CX5 (P < 0.05, IR+9H10 versus IR+9H10+CX5). Data shown are from 2 independent experiments combined. Bars indicate the mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005. (C) Images of lungs from representative animals in each treatment group, as indicated.