Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy

Abstract

PD-1/PD-L1 checkpoint therapy for cancer is commonly considered to act by reactivating T cells in the tumor microenvironment. Here, we present data from 2 mouse tumor models demonstrating an essential involvement of tumor-draining lymph nodes in PD-1 and PD-L1 therapeutic efficacy. Immune activation induced by checkpoint treatment was predominantly observed in the tumor-draining, but not nondraining, lymph nodes and was reflected in local accumulation of CD8+ T cells. Surgical resection of these lymph nodes, but not contralateral lymph nodes, abolished therapy-induced tumor regressions and was associated with decreased immune infiltrate in the tumor microenvironment. Moreover, inhibitor FTY720, which locks lymphocytes in lymph organs, also abrogated checkpoint therapy, suggesting that the tumor-draining lymph nodes function as sites of T cell invigoration required for checkpoint blockade therapy. Now that PD-1/PD-L1 checkpoint treatment is applied in earlier clinical stages of cancer, our preclinical data advocate for enrolling patients with their tumor-draining lymph nodes still in place, to optimally engage the antitumor immune response and thereby enhance clinical benefit.

Keywords: Cancer immunotherapy; Immunology; Mouse models; Oncology.

Figure 1. Immune activation takes place in tumor-draining, but not in nondraining, lymph nodes.

(A) TDLNs (inguinal and axillary) and NDLNs (opposite flank) were isolated when subcutaneous MC38 tumors reached an average size of 100 mm³ and were analyzed with flow cytometry. Mean fluorescence expression of PD-L1 on myeloid cells (CD19^–CD11b⁺) is shown. Statistical difference was calculated with a paired 2-tailed t test. (B and C) Three days after PD-1 Ab treatment, lymph nodes were analyzed for (B) immune cell counts and numbers of (C, left) CD8⁺ T cells, (C, middle) proliferating CD8⁺ T cells, and (C, right) T-bet⁺ CD8⁺ T cells. Statistical differences were analyzed with 2-way ANOVA. All data represent mean ± SEM from 1 experiment (n = 4 per group) out of 3 independent experiments with similar outcome (*P < 0.05, **P < 0.01).

Figure 2. Essential role of TDLNs in checkpoint therapy.

Mice bearing subcutaneous tumors in the right flank were treated with checkpoint blocker therapy immediately following lymph node resection surgery or mock surgery. (A–D) C57BL/6 mice bearing MC38 tumors were treated with PD-1–blocking Ab immediately following lymph node resection surgery. (A) Average outgrowth of mice treated with PD-1 Ab systemically (2 × 100 πg i.p.). Average ± SEM are depicted. Statistical analysis of average tumor outgrowth difference on day 14 was calculated by 1-way ANOVA, 10 mice per group. Res: resection. (B) Survival of mice treated with PD-1 Ab systemically (2 × 100 πg i.p.). Statistical difference was analyzed with log-rank test, 10 mice per group. (C) Survival of mice treated with PD-1 Ab systemically (2 × 100 πg i.p.). TDLNs were resected 7 days after (“res after”) or 1 day before (“res before”) tumor inoculation. Pooled data of 2 independent experiments, with 16 mice per group. Statistical difference was analyzed with log-rank test. (D) Average tumor outgrowth of mice treated with local injection of low-dose PD-1 Ab (1 × 50 πg s.c.), with TDLNs or NDLNs resected before PD-1 treatment, 8 mice per group. Average ± SEM are depicted. Statistical difference in average tumor size on day 20 was calculated by 1-way ANOVA. (E) BALB/c mice bearing subcutaneous CT26 tumors in the right flank were treated with PD-L1 Ab systemically. Average tumor outgrowth ± SEM are depicted. One representative experiment is shown from 2 performed, with 8 mice per group. Statistical analysis of average tumor size on day 20 was calculated with 1-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.005, NS: nonsignificant).

Figure 3. Increase of intratumoral CD8⁺ T cell numbers after PD-1 treatment is abrogated in the absence of TDLNs.

(A) CD3⁺CD8⁺ content in peripheral blood of mice treated with FTY720. Mean ± SEM are depicted. Statistical differences were calculated with 1-way ANOVA. (B) Survival of mice treated with PD-1–blocking Ab with or without FTY720. Pooled data of 2 comparable experiments with 16 mice per group are shown. Statistical analysis was done by log-rank test. (C–E) Analysis of subcutaneous MC38 tumors at day 13 after PD-1 treatment, with or without TDLN resection immediately before PD-1 treatment. (C) Percentage of CD45⁺ cells out of live gate. (D) Percentage of CD8⁺ cells out of CD45⁺ gate. (E) Percentage of Ki67⁺ cells out of CD8⁺ cells. Statistical analysis was performed with 1-way ANOVA, 5–8 mice per group (*P < 0.05, **P < 0.01, ***P < 0.005); 1 representative experiment is shown out of 2 performed.