Depleting tumor-specific Tregs at a single site eradicates disseminated tumors

Abstract

Activation of TLR9 by direct injection of unmethylated CpG nucleotides into a tumor can induce a therapeutic immune response; however, Tregs eventually inhibit the antitumor immune response and thereby limit the power of cancer immunotherapies. In tumor-bearing mice, we found that Tregs within the tumor preferentially express the cell surface markers CTLA-4 and OX40. We show that intratumoral coinjection of anti-CTLA-4 and anti-OX40 together with CpG depleted tumor-infiltrating Tregs. This in situ immunomodulation, which was performed with low doses of antibodies in a single tumor, generated a systemic antitumor immune response that eradicated disseminated disease in mice. Further, this treatment modality was effective against established CNS lymphoma with leptomeningeal metastases, sites that are usually considered to be tumor cell sanctuaries in the context of conventional systemic therapy. These results demonstrate that antitumor immune effectors elicited by local immunomodulation can eradicate tumor cells at distant sites. We propose that, rather than using mAbs to target cancer cells systemically, mAbs could be used to target the tumor infiltrative immune cells locally, thereby eliciting a systemic immune response.

Figure 1. OX40 and CTLA-4 are highly expressed at the tumor site.

(A) Surface expression of OX40 and CTLA-4 on T and B cells (mean ± SEM) in tumor-bearing mice (n = 3) challenged with 5 × 10⁶ A20 lymphoma tumor cells s.c. On day 7, cells from blood, bone marrow, spleen, draining lymph nodes (DLN), and tumors were analyzed by flow cytometry. Proportions of OX40- and CTLA-4–positive cells within B220⁺CD3^– (B cells), CD3⁺CD4^–, and CD3⁺CD4⁺ (T cells) are plotted (isotype background ~0.5%). The proportion of positive cells in i.t. CD4⁺ T cells was significantly higher (*P < 0.05) than in any other site or cell subset, except for the expression of CTLA-4 in blood cells. (B–F) Surface expression of OX40 and CTLA-4 (mean ± SEM) (B) within FOXP3⁺ and FOXP3^– CD3⁺CD4⁺ T cells collected from tumors, draining lymph nodes, and spleens of mice bearing tumors established for 7 days (FACS analysis, n = 5; *P < 0.001); (C) within CD3⁺CD4⁺ T cells collected from tumors (quadrant values are mean percentages (± SEM) obtained from 3 tumor-bearing mice); (D) on i.t. Tregs (n = 3, *P = 0.003); (E) within tumor-infiltrating lymphocytes of patients with mantle cell lymphoma (n = 5) and follicular lymphoma (n = 9) tumors (FACS analysis; *P < 0.05); and (F) within tumor-infiltrating CD4⁺ T cells of human mantle cell lymphoma (n = 5) and follicular lymphoma (n = 9) tumors (FACS analysis; *P < 0.05).

Figure 2. OX40 and CTLA-4 are highly expressed at the tumor site, especially by tumor-specific Tregs.

(A) DO11.10 mice were transplanted s.c. with A20 lymphoma tumor cells at one site and with A20-OVA at another (5 × 10⁶ cells per site). About 70% of CD4⁺ T cells from DO11.10 mice are directed against the OVA peptide. They can be identified by the KJ1-26 clonotypic mAb, which specifically binds their OVA-specific TCR. (B) 10 days after challenge, tumors were collected and tumor-infiltrating OVA-specific T cells (KJ1-26⁺) were analyzed by FACS (pool of tumors from 5 mice). (C) The proportion of FOXP3⁺ cells within the OVA-specific CD4⁺ T cells (gated on KJ1-26⁺) and the proportion of expression of OX40 and CTLA-4 within the OVA-specific Tregs (gated on CD4⁺KJ1-26⁺FOXP3⁺) (mean ± SEM, n = 5, *P < 0.05).

Figure 3. Local immunomodulation does better than systemic immunomodulation for the efficacy of antitumor immune responses.

(A) Mice were challenged s.c. with 5 × 10⁶ A20 tumor cells on the right and left flanks. Therapy was started when tumors reached 0.5–0.7 cm in diameter (usually between day 5 and 8). Treated mice received CpG i.t. only in their right tumor for 5 consecutive days. On day 1 and 5 of CpG therapy, αOX40 and αCTLA4 mAbs were either injected i.p. or i.t. (together with CpG in the right tumor). The systemic antitumor immune response generated by these systemic (i.p.) and local (i.t.) maneuvers was assessed by measuring the size of the contralateral (noninjected) left tumor and mouse disease-free survival. Results were pooled from 2 distinct experiments (n = 10 mice per group). (B) Tumor growth of the distant tumors (nontreated left tumors) when mAbs were injected systemically (i.p.) or locally (i.t. into the right tumor). Black arrows indicate day 1 of therapy. Both strategies (mAbs injected i.p. or i.t.) result in disappearance of the distant (left) tumors. (C) Relapse-free survival of mice treated with either local or systemic immunomodulation. Most of the mice treated systemically (i.p.) with αOX40/CTLA4 relapsed in the left tumor-draining lymph nodes, whereas almost all the mice who received αOX40 and αCTLA4 locally (i.t.) had a long-term survival (*P = 0.002). The number of mice per group is shown into parenthesis. (D) Therapeutic efficacy of 1:100 and 1:1,000 doses of αOX40 and αCTLA4 either injected i.p. or i.t. together with CpG and the resulting long-term disease-free survival. ttt, treatment.

Figure 4. Combination therapy of i.t. CpG and low-dose immunomodulatory antibodies is specifically required to trigger an efficient antitumor immune response.

Mice were treated as in Figure 3A, and. i.t. injections of therapy were done in right (local) tumors (red), and systemic antitumor effect was assessed by measuring growth of left (distant) tumors (blue). CpG was injected at 100 μg daily for 5 consecutive days. Low doses of mAbs (4 μg αOX40 or rat isotype or/and 1 μg αCTLA4 or hamster isotype) were injected on day 1 and 5 of CpG therapy into the same tumor. (A) Growth of distant tumors without therapy. (B–F) Systemic antitumor effect of CpG injections (B) alone on injected and distant tumors; (C) in combination with rat and hamster isotypes of αOX40 and αCTLA4 mAbs, respectively; (D) in combination with αOX40; (E) in combination with αCTLA4; and (F) in combination with αOX40 and αCTLA4. Previous curves pooled from at least 2 different experiments per group. (A–F) The number of surviving mice at day 60 is shown in parenthesis. (G) Survival of mice bearing 2 s.c. tumors (right and left flanks) that received CpG plus rat/hamster isotypes, CpG plus αOX40, CpG plus αCTLA4, or CpG plus αOX40/CTLA4 in right tumors. Survival with CpG plus αOX40/CTLA4 was significantly higher than with CpG plus αOX40 (P = 0.004) or CpG plus αCTLA4 (P = 0.03). Data are pooled from at least 2 different experiments per group; the number of mice per group is shown into parenthesis (*P < 0.05). Systemic antitumor effect of (H) αOX40 plus αCTLA4 local low-dose therapy without CpG (n = 4) and (I) s.c. CpG and i.t. αOX40 plus αCTLA4. (J) Survival of tumor-bearing mice treated with i.t. CpG and low-dose αOX40 plus αCTLA4 in the context of CD4 or CD8 T cell depletion (*P < 0.05).

Figure 5. Depletion of tumor-specific Tregs at the injected site.

(A) OVA-specific CD4⁺ T cells negatively selected from DO11.10 mice were stained with violet dye and injected into 7-day-old A20-OVA tumors of WT mice. Four days later, the tumors were treated or not with CpG plus low-dose αOX40/CTLA4. Tumor-infiltrating OVA-specific CD4⁺ T cells were analyzed 4 days after beginning therapy. Data are representative of cohorts of 3 mice. (B) Proportions of CD25⁺ and FOXP3⁺ cells within OVA-specific (KJ1-26⁺) CD4⁺ cells from untreated and treated A20-OVA tumors. Seven days after A20-OVA tumor challenge, 1 site was injected with 1.8 × 10⁶ OVA-specific CD4 cells purified from DO11.10 mice. Four days later, 1 group of mice received 1 day of CpG and low-dose αOX40/CTLA4 in these tumors, followed by 2 days of i.t. CpG alone; the phenotype of OVA-specific CD4 cells was analyzed by FACS on day 4. Quadrants values are mean percentages (± SEM) obtained in each group (3 mice per group). (C) Activated OVA-specific CD4⁺ T cells infiltrating A20-OVA tumors at day 0 and 4 days later without therapy (no treatment) or after 4 days of i.t. low-dose immunomodulation (CpG plus αOX40/CTLA4). Results are presented as proportions of KJ1-26⁺CD25⁺ cells among total CD4⁺ T cells and absolute numbers of cells per 1,000 live cells (n = 3, *P < 0.05 at day 4). (D) DO11.10 mice (n = 5 per group) were challenged with 5 × 10⁶ A20-OVA tumor cells s.c. Seven days later, one group received i.t. CpG plus αOX40/CTLA4 as described before. Infiltrating CD4⁺ T cells of A20-OVA tumors were analyzed by FACS for CD25 surface expression and FOXP3 intracellular expression. *P < 0.05. (E) Tumor-infiltrating CD4⁺FOXP3⁺ T cells within injected or uninjected A20-OVA tumors in WT mice on day 4 of therapy (n = 3 per group, *P < 0.05). (F) Effect of therapy on tumor-infiltrating tumor-specific Tregs (as defined by CD3⁺CD4⁺CD25⁺OX40⁺FOXP3⁺ cells) in injected A20 tumors of WT mice on day 4 of therapy (*P < 0.0001). The ratio of Teffs (CD3⁺CD4⁺CD25⁺OX40^–FOXP3^–) over tumor-specific Tregs (CD3⁺CD4⁺CD25⁺OX40⁺FOXP3⁺) is also displayed (^†P = 0.01). Mean ± SEM.

Figure 6. In situ immunization with CpG plus αOX40/CTLA4 induces depletion of i.t. tumor-specific Tregs.

After a CD4-negative selection, OVA-specific FOXP3-GFP Tregs were FACS sorted from splenocytes of Thy1.2 DO11.10 FOXP3-GFP mice. 2.5 × 10⁵ cells were injected into 5-day-old A20-OVA tumors on the right flank of Thy1.1 BALB/c mice. On day 8, right tumors were treated with i.t. CpG plus low-dose αOX40/CTLA4. On day 4 of therapy, the number of Thy1.2⁺ donor Tregs was counted by FACS in the injected (right) and distant (left) A20-OVA tumors, the right draining lymph nodes (DLN R), the spleen, and the blood of Thy1.1⁺ recipients.

Figure 7. i.t. CpG plus low-dose αOX40/CTLA4 immunotherapy is efficient against aggressive, disseminated tumor models.

(A) Mice bioluminescence on day 1 (d 1) after i.v. challenge with 2 × 10⁶ of A20-Luc tumors cells. Mice were concomitantly injected s.c. with 10 × 10⁶ A20 tumor cells. i.t. immunomodulation with CpG plus low-dose αOX40/CTLA4 was started on day 7 in the s.c. tumors. On day 31, only mice treated locally with CpG plus low-dose αOX40/CTLA4 in their A20 s.c. tumors showed disappearance of their systemic disease. (B) Ascites (black arrows) and metastases (white arrows) in internal organs of 1 representative mouse 32 days after an i.v. tumor challenge with 2 × 10⁶ A20 tumor cells (inset image illustrates predominant metastatic disease of the liver). (C) Long-term survival of mice concomitantly challenged on day 1 with 10 × 10⁶ s.c. and 2 × 10⁶ i.v. A20 tumor cells. On day 7, most of the mice treated with CpG plus low-dose αOX40/CTLA4 in their s.c. tumor were cured (*P < 0.0001). (D) Growth of the distant (untreated) 2F3 leukemia tumors (*P < 0.0001) and survival (^†P = 0.0018) after a single course of in situ immunomodulation with CpG plus low-dose αOX40/CTLA4 in mice (n = 5) challenged with 5 × 10⁴ 2F3 leukemia cells into 2 different s.c. sites (black arrows indicate day 1 of therapy). (E) Growth of the distant (untreated) 4T1 breast carcinoma tumors (*P < 0.05) and number of lung metastases on day 22 after a single course of i.t. immunomodulation with CpG plus low-dose αOX40/CTLA4 (mean ± SEM). Mice (n = 5) were challenged with 1 × 10⁴ 4T1 cells in 2 different s.c. sites; only 1 tumor site was treated (black arrows indicate day 1 of therapy). The number of mice per group is shown into parenthesis.

Figure 8. Two courses of i.t. CpG plus low-dose αOX40/CTLA4 immunotherapy enhances the antitumor immune response.

(A) Tumor growth and viability of distant tumors after 2 courses of i.t. CpG plus low-dose αOX40/CTLA4. Mice (n = 5) were challenged with 1 × 10⁴ 4T1 and 1 × 10⁴ 4T1-Luc tumor cells in their right and left flank, respectively, followed by 2 courses of i.t. CpG plus low-dose αOX40/CTLA4 in their right tumor (black arrows indicate day 1 of each course). The viability of the distant (untreated) 4T1-Luc breast carcinoma tumors was assessed by bioluminescence. *P < 0.05. (B) Spontaneous lung metastases of 4T1 tumors on day 29 after s.c. tumor inoculation in untreated mice and after 2 courses of i.t. CpG plus low-dose αOX40/CTLA4 (mean ± SEM). (C) OX40 and CTLA-4 surface expression on CD4⁺ T cells infiltrating 4T1 breast carcinoma tumors and their draining lymph nodes (FACS analysis; 5 mice per group, *P < 0.0001 for both OX40 and CTLA-4; mean ± SEM).

Figure 9. CNS lymphoma development after i.c. tumor challenge.

(A) CNS disease-free survival after i.c. challenge. CNS protection of mice previously cured from s.c. lymphoma with i.t. CpG plus low-dose αOX40/CTLA4. 150 days later, these mice and naive mice were challenged i.c. with 0.5 × 10⁶ A20 tumor cells. Mice were sacrificed when presenting with neurological symptoms. *P < 0.05. (B) CNS disease-free survival in the same experimental settings but with mice depleted of CD4⁺ or CD8⁺ T cells a few days prior the i.c. challenge. (C) CNS lymphoma mouse model. 0.5 × 10⁶ to 1 × 10⁶ A20 or A20-Luc lymphoma tumor cells were stereotactically injected into the brain parenchyma of syngeneic BALB/c mice. Tumor development was monitored longitudinally using bioluminescence signal of A20-Luc tumor cells. i.c. tumor engraftment rate was higher than 95%. The bioluminescence signal after A20-Luc injections revealed spontaneous leptomeningeal metastases in 20% to 40% of the cases. The correlation between brain and spinal cord bioluminescent signals and pathological infiltration by tumor cells was confirmed by histology (Supplemental Figures 2 and 3). White arrows indicate the localization of the small blue tumor cells within the brain or the spinal cord after H&E staining. (D) CNS lymphoma treatment groups. Mice were challenged s.c. with 10 × 10⁶ A20 and i.c. with 1 × 10⁶ A20-Luc tumors cells. Once the tumors were established (day 5 after i.c. tumor inoculation), these mice received either systemic conventional therapies (chemotherapies or passive immunotherapy) or local in situ active immunomodulation.

Figure 10. i.t. low-dose immunomodulation cures established CNS lymphoma.

The CNS lymphoma burden assessed over time by bioluminescence for 1 representative mouse of each treatment group and the survival of the whole group (5 mice per group). MTX, 400 mg/kg s.c. MTX, followed by 12 mg/kg s.c. calcium leucovorin rescue started 16 hours later and given once every 2 hours for a total of 5 doses; CTX, 100 mg/kg i.p CTX for 2 subsequent days; αId, 100 mg i.p.; i.t. CpG and 1/10 or 1/100 doses of αOX40/CTLA4.

Figure 11. i.t. low-dose immunomodulation conveys a sustainable cytotoxic antitumor immune response, even in an immune sanctuary site.

(A) Mice were challenged with 1 × 10⁶ A20 tumor cells i.c. and 10 × 10⁶ A20 tumor cells s.c. and treated with CpG plus low-dose aOX40/CTLA4 as previously described (see Figure 9D). On day 6 after the beginning of therapy, brains were collected and digested, and mononuclear cells were separated by Percoll. These cell suspensions were analyzed by FACS for T cell (CD3⁺) infiltration (gate on all viable cells, *P < 0.05). (B) Brains of mice bearing CNS lymphoma were extracted on day 7 of therapy and stained by IF for CD8⁺ and CD4⁺ T cells (red). Tumor cells were identified by tumor idiotype staining (green). Original magnification, ×20. (C) Ratio of number of brain-infiltrating CD8⁺ T cells over CD4⁺ T cells upon therapy. *P < 0.05. (D) Proportion of activated (CD69⁺) CD8⁺ T cells upon therapy. *P < 0.05. (E) Brains from CNS lymphoma-bearing mice were collected on day 8 of s.c. therapy and reexposed overnight to irradiated A20 tumor cells. T cells were subsequently stained for surface CD44 and intracellular IFN-γ. (F) As in E, but median values from 5 mice (*P < 0.0001 for both CD8⁺ and CD4⁺ cells). (G) Mice cured from CNS lymphoma were rechallenged i.c. 140 days later in the contralateral hemisphere with 0.5 × 10⁶ A20 lymphoma tumor cells as were naive mice. Survival after rechallenge of i.c. cured is shown (6 mice per group, P < 0.001).