OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

Abstract

Plasmacytoid DCs (pDCs), the major producers of type I interferon, are principally recognized as key mediators of antiviral immunity. However, their role in tumor immunity is less clear. Depending on the context, pDCs can promote or suppress antitumor immune responses. In this study, we identified a naturally occurring pDC subset expressing high levels of OX40 (OX40+ pDC) enriched in the tumor microenvironment (TME) of head and neck squamous cell carcinoma. OX40+ pDCs were distinguished by a distinct immunostimulatory phenotype, cytolytic function, and ability to synergize with conventional DCs (cDCs) in generating potent tumor antigen-specific CD8+ T cell responses. Transcriptomically, we found that they selectively utilized EIF2 signaling and oxidative phosphorylation pathways. Moreover, depletion of pDCs in the murine OX40+ pDC-rich tumor model accelerated tumor growth. Collectively, we present evidence of a pDC subset in the TME that favors antitumor immunity.

Keywords: Antigen presenting cells; Dendritic cells; Head and neck cancer; Immunology; Oncology.

Figure 1. OX40 expression on pDCs in the TME of HNSCC.

(A) OX40 expression in the TME (measured by flow cytometry) of HNSCC patients on different immune cell subsets — pDCs (n = 89), cDCs (n = 53), CD8⁺ T cells (n = 16), CD4⁺ T cells (n = 17), CD4⁺ Th1 T cells (n = 12), and CD4⁺ Treg cells (n = 14). T cell subsets were gated from live CD45⁺CD3⁺ cells. Th1 cells were defined as CD4⁺Tbet⁺ T cells and Treg cells were defined as CD4⁺Foxp3⁺ cells. (B) Gating strategy for FACS analysis and sorting of OX40⁺ and OX40^lo/– pDCs from patient specimens. After selecting for singlets and live cells, pDCs were gated from HLA-DR^hiLineage^– cells, followed by CD11c^–CD123⁺ cells. pDCs were further confirmed by expression of CD303 (BDCA-2). OX40 expression on pDCs was determined using internal negative controls. (C) Immunofluorescence of pDCs in the TME demonstrating OX40 and CD123 coexpression. n = 4, with 4 patient repeats. Original magnification, ×63. Scale bar: 5 μm. Red, OX40; green, CD123; blue, DAPI. (D) OX40 expression on pDCs from different anatomic sites: PBMC (n = 17), dLN^– (n = 50) or dLN⁺ (n = 59), and primary tumor (n = 53). (E) Correlation (Pearson, with a line of best fit) between OX40 and ICOSL expression on matched patient TME pDCs (n = 28). One-way ANOVA followed by Tukey’s post hoc test (A and D). **P < 0.01; ***P < 0.001; ****P < 0.0001. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (A, D, and E).

Figure 2. OX40⁺ pDCs have a distinct immunostimulatory phenotype.

(A) After overnight incubation, pDCs from the dLNs^– of HNSCC patients (n = 7) were harvested and measured by flow cytometry for expression of different surface markers. Single gradient mean values are shown. (B) Representative histograms of OX40⁺ and OX40^lo/– pDC surface marker expression. (C) Expression of IL-12p70⁺CD86⁺ populations on sorted cDCs and pDCs, either unstimulated (controls; bottom) or in the presence of Resiquimod (top). n = 2; 2 experimental patient repeats. (D) Percentages (by flow cytometry) of dLN^– pDCs positive for TRAIL (n = 5) and GzB (n = 4) after overnight stimulation with CpG or Resiquimod. (E) The concentration (pg/mL) of IFN-α (n = 10) and TRAIL (n = 6) in the supernatant from sorted OX40⁺ and OX40^lo/– pDCs from the TME and non-TME stimulated with either CpG or Resiquimod. Data normalized to 2 × 10³ pDCs per sample. (F) May-Grunwald staining of OX40⁺ and OX40^lo/– pDC subsets stimulated with Resiquimod. Scale bar: 5 μm. n = 4, 4 experimental patient repeats. Two-way ANOVA with Sidak’s test for multiple comparisons (A). Two-tailed paired t test (D and E). *P < 0.05. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points.

Figure 3. OX40⁺ pDCs promote antigen-specific CD8⁺ T cell responses.

(A) Illustration of the antigen-specific in vitro coculture model, in which autologous OX40⁺/OX40^lo/– pDCs from the TME/non-TME of HNSCC patients (n = 9) were cocultured with autologous TAA peptide-loaded mDCs and CD8⁺ T cells for 5 to 6 days, at which point antigen-specific CD8⁺ T cell responses were measured, (B) including for proliferation (eFluor 450–low) and IFN-γ production as demonstrated in flow plots of a patient’s CD8⁺ T cells cocultured with OX40⁺ or OX40^lo/– pDCs sorted from their tumors. CD8⁺ T cell positivity was also measured for (C) Tbet and (D) eFluor 450–low in these coculture experiments. (E) CD8⁺ T cell positivity for CD69 after coculture with TAA peptide–loaded mDCs without pDCs (control) or with OX40⁺ or OX40^lo/– pDCs from the TME versus non-TME (dLN^–) (n = 5). (F) Illustration depicting the Transwell coculture assay in which OX40⁺ or OX40^lo/– pDCs in the top chamber were separated from autologous CD8⁺ T cells and peptide-loaded mDCs in the bottom chamber. (G) Percentage of proliferating (eFluor 450–low) and GzB⁺ CD8⁺ T cells in Transwell versus contact coculture (n = 3). Representative flow plots show GzB production by CD8⁺ T cells cocultured with E7-loaded mDCs and OX40⁺ or OX40^lo/– pDCs in coculture contact or separated by Transwell. (H) Flow plots comparing antigen presentation capacities of autologous OX40⁺ and OX40^lo/– pDCs with mDCs, based on cytolytic CD8⁺ T cell responses (no peptide controls for these plots are shown in Supplemental Figure 2B). Shown is GzB production by CD8⁺ T cells in the presence or absence of OX40⁺/OX40^lo/– pDCs (top) and IL-12p70 production by mDC/pDC subsets (bottom). n = 2; 2 experimental repeats. One-way ANOVA followed by Tukey’s post hoc test (C–E and G). Bar graph data are mean ± SEM; *P < 0.05. NS, not significant. Middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (C–E and G).

Figure 4. The OX40-OX40L axis is utilized by pDCs in the TME.

(A) OX40L expression measured by flow cytometry on OX40⁺ and OX40^lo/– pDCs (n = 7). Correlation (Pearson, with line of best fit) of OX40 and OX40L expression intensities (per-cell normalized counts, total weighting) on pDCs measured on an HNSCC TME tissue section. n = 4; 4 patient repeats. (B) Immunofluorescence images from the TME showing a cell’s coexpression of OX40 (red) with CD123 (green), sitting adjacent to CD8-expressing (magenta) cells. Gallery view of Z-stacks (collected at 0.29-μm intervals). Original magnification, ×63. Scale bar: 5 μm. n = 4; 4 patient repeats. (C) Processed multispectral image (steps outlined in Supplemental Figure 2E for same example image) of the TME. Original magnification, ×40. Inset: a cluster CD123⁺ (green) cells, including one with OX40 expression (orange), next to a CD8⁺ (magenta) cell. (D) Representative image file written using Phenoptr to calculate touching pairs of phenotyped OX40L⁺CD68⁺ macrophages with phenotyped OX40⁺ pDCs. (E and F) Percentage of phenotyped OX40⁺/OX40^lo/– pDCs touching other OX40L⁺ cells, including pDCs, macrophages (Mφ), and “other cells” (Methods), in the TME and non-TME of patients (n = 3). (G) Ratios of CD8⁺ T cell counts within 30 μm of pDC subsets to total counts of CD123⁺ pDC subsets (OX40⁺/OX40^lo/–) in the TME and non-TME of patients (n = 5). (H) Intercellular distances of phenotyped pDC subsets to the closest tumor margin (μm). Original magnification, ×40. n = 3; 3 patient repeats. One-way ANOVA followed by Tukey’s post hoc test (F) and unpaired (E), and paired (A, G, and H) t tests. *P < 0.05; **P < 0.01; NS, not significant. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points.

Figure 5. OX40⁺ pDCs harbor a unique transcriptome.

(A–F and I) Bulk RNA sequencing was performed on cell-sorted pDCs from HNSCC patients (n = 7) (Supplemental Table 2). DEGs were determined using the criterion of fold changes (FC) greater than or equal to 1.5 (see Methods). (A) Principal component analysis showing the clustering of transcriptional profiles of TME and non-TME pDCs (top), including pDCs from HPV⁺ patients (bottom). (B) Top: log₂ expression of TNF-receptor genes upregulated in the TME of pDC samples. Bottom: expression of TNFSF4 in the TME versus non-TME pDCs. (C) Volcano plot of gene expression in OX40⁺ pDCs relative to their expression in OX40^lo/– pDCs (up- and downregulated in OX40⁺ pDCs colored as magenta and orange, respectively) against the FDR. (D) Top pathways (–log[P value] > 2.0) generated from IPA enriched in OX40⁺ and OX40^lo/– pDCs, respectively. Corresponding ratios for each enriched pathway are measured on the second y axis. Full pathway title: Nitrous Oxide and Reactive Oxygen Species Production in Macrophages. (E) Expression of genes involved in MHC I antigen presentation enriched in HPV⁺ HNSCC OX40⁺ pDCs. (F) Heat map reporting relative expression of 183 DEGs in OX40⁺/OX40^lo/– pDCs. Gene pathway clusters are demarcated by boxes. (G) Functional correlative data for cluster II (OXPHOS) in OX40⁺ pDCs. Left: Mitochondrial mass measurements in pDC subsets from the TME were calculated using Imaris (see Methods). Original magnification, ×100. Scale bar: 5 μm. n = 3; 3 experimental repeats. Right: Real-time analysis of OXPHOS (oxygen consumption ratios measured by Seahorse assay) in sorted pDCs from the TME. n = 2; 2 experimental repeats. (H) Functional correlative data for cluster III (detoxification of ROS): total ROS volumes and ROS colocalized to mitochondria volumes in pDCs from the TME. n = 2; 2 patient experimental repeats. (I) Expression of genes involved in cluster VI (type I IFN signaling) upregulated in OX40⁺ pDCs. Unpaired Student’s t test (B and F–I) and right-tailed Fisher’s exact test (E). *P < 0.05; **P < 0.01, ***P < 0.001; NS, not significant. Error bars represent mean ± SEM of technical duplicates. Middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Z scores and genes for IPA in Source Data 1.

Figure 6. OX40⁺ pDCs correlate to survival in cancer patients and suppress tumor growth.

(A) Prospective recurrence-free survival (log-rank, Mantel-Cox test) of HNSCC cohort (n = 80), stratified by median (45%) intratumoral pDC OX40 expression, as measured by flow cytometry. (B) Overall survival (log-rank, Mantel-Cox test) of HNSCC patients (n = 500) from the GDC data portal, stratified first by median pDC gene signature Z scores followed by stratification of mean TNFRSF4 (encodes OX40) mRNA levels. (C) Correlation (Pearson, with line of best fit) of TNFRSF4 log₂ mRNA levels (among cases with pDC_hi gene signatures) with CD8⁺ T effector scores in HNSCC (n = 172). (D) OX40 expression on intratumoral pDCs from different murine tumor models. n =4; 4 experimental replicates. (E) gp100-specific Pmel-1 CD8⁺ T cell IFN-γ production by proliferating (eFluor 450–low) CD8⁺ T cells, measured in the presence or absence of pDCs from the dLNs of B16-F10– and B16CCR7-bearing mice. n = 2; 2 experimental repeats. (F) gp100-specific proliferating (eFluor 450–low) Pmel-1 CD8⁺ T cells in the presence or absence of B16CCR7 pDCs prestimulated with Resiquimod and OX86. n = 2; 2 experimental repeats. (G) Effect of pDC depletion (anti-PDCA1) in B16-F10– and B16CCR7-bearing mice compared with controls (anti-polyclonal IgG). Data are pooled from at least 2 independent experiments with 3 to 5 mice per group. (H) Quantification (by flow cytometry) of conventional cDCs (CD11c⁺CD11b^–) and CD8a⁺ cDCs from B16CCR7-bearing mice treated with anti-PDCA1 or anti-polyclonal IgG. Data are pooled from individual experiments and normalized to 5 × 10⁵ live cells. One-way ANOVA followed by Tukey’s post hoc test (D), 2-way ANOVA with Sidak’s test for multiple comparisons (H), and unpaired Student’s t test (G). **P < 0.01; ***P < 0.001. Tumor burden data and bar graph data are mean ± SEM.