Lymphatic PD-L1 Expression Restricts Tumor-Specific CD8+ T-cell Responses

Abstract

Lymph node (LN)-resident lymphatic endothelial cells (LEC) mediate peripheral tolerance by self-antigen presentation on MHC-I and constitutive expression of T-cell inhibitory molecules, including PD-L1 (CD274). Tumor-associated LECs also upregulate PD-L1, but the specific role of lymphatic PD-L1 in tumor immunity is not well understood. In this study, we generated a mouse model lacking lymphatic PD-L1 expression and challenged these mice with two orthotopic tumor models, B16F10 melanoma and MC38 colorectal carcinoma. Lymphatic PD-L1 deficiency resulted in consistent expansion of tumor-specific CD8⁺ T cells in tumor-draining LNs in both tumor models, reduced primary tumor growth in the MC38 model, and increased efficacy of adoptive T-cell therapy in the B16F10 model. Strikingly, lymphatic PD-L1 acted primarily by inducing apoptosis in tumor-specific CD8⁺ central memory T cells. Overall, these findings demonstrate that LECs restrain tumor-specific immunity via PD-L1, which may explain why some patients with cancer without PD-L1 expression in the tumor microenvironment still respond to PD-L1/PD-1-targeted immunotherapy. SIGNIFICANCE: A new lymphatic-specific PD-L1 knockout mouse model reveals that lymphatic endothelial PD-L1 expression reduces tumor immunity, inducing apoptosis in tumor-specific CD8⁺ central memory cells in tumor-draining lymph nodes.

Figure 1.

PD-L1 impairs CD8⁺ T-cell priming by LECs in vitro. A and B, Example histograms (A) and quantification (B) of PD-L1 expression in wild-type (WT) and PD-L1^ko LECs determined by FACS (N = 3 independent experiments). IFNγ was used as positive control to further induce PD-L1 expression. C–G, WT and PD-L1^ko LECs were loaded with SIINFEKL peptide and cocultured with naive CD8⁺ OT-1 cells O/N. OT-1 expression of CD69 (C), IFNγ (D), Ki67 (E, left), proliferation (CFSE dilution; E, right), PD-L1 (F), and PD-1 (G) was determined by FACS. One representative of three independent experiments is shown (N = 5). *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA with paired Sidak post-test.

Figure 2.

Lymphatic PD-L1 reduces tumor-specific CD8⁺ T-cell responses in mice bearing orthotopically implanted B16-ova melanomas and MC38-ova colorectal carcinomas. A, Growth of B16-ova cells in Cre⁺ PD-L1^LECKO and Cre⁻ controls [one representative of three independent experiments is shown (N = 4 Cre⁻/5 Cre⁺ mice)]. B, Quantification of CD8⁺ T cells (expressed as the percentage of all living singlets) in tumor, draining LNs, and spleen on day 16 after inoculation of B16-ova cells. Data were pooled from three independent experiments (N = 15 Cre⁻/11 Cre⁺ mice). C–E, Representative FACS plots (pre-gated for CD8⁺ T cells) and quantification of CD8⁺ T cells specific for ova (ovalbumin) or pmel in tumors (C), draining LNs (D), and spleen (N = 15 Cre⁻/11 Cre⁺ mice for ova; N = 8 for pmel; E). F, Representative histogram (left) and quantification (right) of PD-L1 expression on LECs in normal colorectal mucosa (naive) compared with LECs in orthotopic MC38-ova tumors in Cre⁻ control mice on day 21 after tumor cell inoculation (N = 3 mice/group). Graph represents the fluorescence intensity of PD-L1 compared with the isotype control. G, Weight of orthotopic MC38-ova tumors in Cre⁺ PD-L1^LECKO mice and Cre⁻ controls on day 21 after inoculation (N = 27 Cre⁻/21 Cre⁺ mice). H, Quantification of CD8⁺ T cells in tumor, draining LNs, and spleen on day 21 after inoculation of MC38-ova cells (N = 4 Cre⁻/6 Cre⁺ mice in tumor; N = 7 Cre⁻/6 Cre⁺ mice in draining LN and spleen). I–K, Representative FACS plots (pre-gated for CD8⁺ T cells) and quantification of CD8⁺ T cells specific for ova and p15e in tumors (I), draining LNs (J), and spleen (N = 4 Cre⁻/6 Cre⁺ mice in tumor and N = 7 Cre⁻/6 Cre⁺ mice in draining LN and spleen for ova; N = 9 Cre⁻/6 Cre⁺ mice in tumor and spleen and N = 15 Cre⁻/12 Cre⁺ mice in draining LNs for p15e; K). *, P < 0.05, Student t test (D, E, F, and J) or Welch t test (G).

Figure 3.

Deletion of lymphatic PD-L1 increases the efficiency of adoptive T-cell transfer in the B16-ova melanoma model. A, Schematic representation of the adoptive T-cell therapy (ACT) approach in B16-ova–bearing mice. B, Primary tumor weight in Cre⁺ PD-L1^LECKO mice and Cre⁻ controls at the endpoint. C, Quantification of total CD8⁺ T cells in tumor, draining LNs, and spleen. D–F, Representative FACS plots (pre-gated for endogenous, CD45.1⁻ CD8⁺ T cells) and quantification of endogenous CD8⁺ T cells specific for ova (ovalbumin) or pmel in tumors (D), draining LNs (E), and spleen (F). G, Representative FACS plots (pre-gated for CD8⁺ T cells) and quantification of CD45.1⁻ endogenous and CD45.1⁺-transferred OT-1 CD8⁺ T cells in tumors, draining LNs, and spleens (N = 6 Cre⁻/7 Cre⁺ mice). *, P < 0.05, Student t test.

Figure 4.

Lymphatic PD-L1 induces apoptosis in tumor-specific CD8⁺ central memory T cells in tumor-draining LNs. A–C, Representative FACS plots [pre-gated for tetramer-specific (A), ova-specific T_CM and naive (B) or pmel-specific T_CM and naive (C) CD8⁺ T cells] and frequency of apoptotic cells [pooled early (Zombie⁻) and late (Zombie⁺) apoptotic] among all ova- and pmel-specific CD8⁺ T cells (A) or within T_EM, T_CM and naive ova-specific (B) and pmel-specific (C) CD8⁺ T cells in B16-ova–draining LNs (N = 10 Cre⁻/9 Cre⁺ mice). D and E, Representative FACS plots [pre-gated for all ova-specific (D) or ova-specific T_CM and naive (E) CD8⁺ T cells] and frequency of apoptotic cells among all ova-specific CD8⁺ T cells (D) or within T_EM, T_CM and naive ova-specific (E) CD8⁺ T cells in MC38-ova–draining LNs (N = 5 mice/group). F–G, Apoptosis among endogenous and transferred CD45.1⁺ OT-1 T cells (F) and among CD45.1⁺ T_EM, T_CM and naive T cells (G) in B16-ova–draining LNs after adoptive transfer of freshly isolated, unstimulated OT-1 cells (N = 3 mice/group). *, P < 0.05; **, P < 0.01, Student t test.

Figure 5.

Lymphatic PD-L1 deletion increases the functionality of tumor-specific memory T cells. A, C57Bl/6 wild-type mice received an adoptive transfer of CD8⁺ T cells from B16-ova–draining LNs of PD-L1^LECKO mice or Cre⁻ controls and were subsequently challenged by ovalbumin injection into the hind paw. B, Ratio of the absolute number of ova-specific CD8⁺ T cells in challenged (draining) popliteal LNs compared with contralateral (non-draining) popliteal LNs. C and D, Representative FACS plots (pre-gated for ova-specific CD8⁺ T cells) and ratio of ova-specific CD8⁺ T_EM, T_CM, and naive (F) or activated (G) T cells in challenged (draining) versus contralateral (non-draining) popliteal LNs (N = 3 Cre⁻/4 Cre⁺ mice). E and F, C67Bl/6 wild-type mice received T cells as in A and were subsequently challenged with B16-ova tumor cells. Graphs show survival (E) and tumor volume (F) for each individual mouse (N = 4 Cre⁻/5 Cre⁺ mice). *, P < 0.05; Student t test.

Figure 6.

Schematic representation of the proposed role of lymphatic PD-L1 in tumor immunity. Within LNs, LECs lining the floor of the subcapsular sinus as well as medullary sinuses express high levels of PD-L1. Tumor-specific T_CM cells may enter tumor-draining LNs either via high endothelial venules (HEV) or through afferent lymphatic vessels (LV), and will leave the LNs again via cortical and medullary lymphatic sinuses. Thus, T_CM cells may receive apoptosis-inducing signals from PD-L1⁺ floor LECs as they enter the LN or from PD-L1⁺ medullary LECs as they leave the LN.