T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells

Abstract

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

Figure 1. NOS2 is essential for effective ACT

(A) Representative images of CD11b⁺ cells sorted from spleens or tumors of EG7 tumor-bearing WT mice stained for NOS2, ARG1 and DAPI. Scale bar, 25 μm. (B, C) CD11b⁺ cells sorted from spleens or tumors of EG7 tumor-bearing WT mice were quantified for the percentage of ARG1⁺, NOS2⁺, and ARG1⁺NOS2⁺ cells (B) or for fluorescence intensity of NOS2 expression (C, Median [50^th percentile] represented as a line inside the box. Lines at the bottom and top of the box represent, respectively, the 25^th and the 75^th quartile, the whiskers represent 10^th and 90^th percentile. Outliers beyond the whiskers are individually plotted as black dots; *** p ≤ 0.001, unpaired Student t-test analysis). n=357 cells from spleen or from tumor were evaluated. (D) CD11b⁺ cells from tumors of WT, ARG KO, NOS KO or ARG NOS KO mice were sorted and co-cultured with OVA-specific, CFSE-labeled, CD8⁺CD45.1⁺ T lymphocytes in the presence of OVA peptide. Percent of proliferating T cells within a given number of cell divisions (from 0 to 7 as indicated) is shown in bar graph format relative to positive control (mixed lymphocyte peptide culture [MLPC], lymphocytes stimulated with OVA peptide) or negative controls without OVA (NS, not stimulated). (E, F) WT and EG7 tumor-bearing WT and NOS KO mice were adoptively transferred with OVA-specific CD8⁺CD45.1⁺ T lymphocytes. Cells derived from spleens (E) or tumors (F) were stimulated with OVA peptide and percent of CD45.1⁺CD8⁺ cells and IFN-γ⁺ cells shown in CD45.1⁺CD8⁺ gate. Mean ± s.d.; n=4, representative of 3 independent experiments. ***p ≤ 0.001, **p ≤ 0.01 and *p ≤ 0.05, by using One Way ANOVA. (G) Survival percentages of EG7 tumor bearing WT and KO mice either untreated (n=5, for each group) or treated with ACT (WT n=20; ARG KO n= 15; NOS KO n=18; ARG NOS KO n=17). *p ≤ 0.05, logrank test. See also figure S1.

Figure 2. Adoptive transfer of tumor-specific CD8⁺ T cells induces Tip-DC expansion

(A) Dot plots indicating the overall gating strategy to define the myeloid sub-populations in EG7 tumor mass. (B) Quantification of tumor-infiltrating myeloid subpopulations in the CD11b⁺ gate in mice treated (CD8) or untreated (w/o CD8) with ACT, n=18, pooled from 3 experiments (C, D) Frequency (C, n=18 pooled from 3 experiments) and representative flow cytometry plots (D, percentages of different populations are indicated in each quadrant.) for tumor infiltrating myeloid populations in CD11b⁺NOS2⁺ gate (red boxes) in WT mice untreated (w/o CD8) or treated with (CD8) ACT. (E) Frequency of different myeloid subpopulations in tumor cell suspensions stimulated or not with LPS in CD11b⁺ gate, n=6 representative experiment of 3. (F) Sorted myeloid subpopulations were cultured with naive CD4⁺ lymphocytes to evaluate IFN-γ released in supernatants by ELISA, representative experiment of 3. (B, C, E, F) Mean ± s.d., ***p ≤ 0.001, **p ≤ 0.01, by using One Way ANOVA. See also figure S2.

Figure 3. Tip-DCs are functionally active in tumor environment

(A) A schematic representation of the experiment. (B) Survival of EG7-tumor-bearing NOS KO mice treated with ACT and with sorted Tip-DCs from WT or NOS KO mice; n=6. **p ≤ 0.01, Log-rank test. (C) In vitro generated human Tip-DCs were analyzed by flow cytometry for the indicated molecules. (D) NOG mice were injected s.c. with MDA-MB-231 and intratumorally with CD14 monocytes, Tip-DCs, or PBS and either treated or untreated i.v. with anti-hTERT specific CD8⁺ T cells. Survival percentages of n=15, pooled from 3 independent experiments, are reported. ***p ≤ 0.001, logrank test. See also figure S3.

Figure 4. Affinity between TCR and MHC-peptide complexes regulates Tip-DC generation and ACT effectiveness

(A) Percentage of survival (*p ≤ 0.05, logrank test) and percent of different tumor infiltrating myeloid subpopulations in CD11b⁺NOS2⁺ gate (Mean ± s.d., representative of 3 independent experiments, **p ≤ 0.01, by using One Way ANOVA) for WT mice bearing B16-OVA^high (n=7) or B16-OVA^low (n=6) tumor treated or not with ACT. (B) Splenic CD11b⁺ cells were cultured in the presence of cell culture supernatant of antigen-stimulated OT-I, TRP2^high or TRP2^low CD8⁺ T cells and anti-CD40 antibody for 48 hr then analyzed for Tip-DC differentiation. Representative dot plots are shown. (C) Percentage of CD11b⁺Ly6C⁺MHCII⁺ cells generated in vitro by splenic CD11b⁺ cells in different culture conditions is shown. (D) As in D showing MFI of NOS2 and TNF for CD11b⁺Ly6C⁺MHCII⁺ cells. (C, D) Mean ± s.d.; n=4 representative experiment of 3, ***p ≤ 0.001, **p ≤ 0.01 and *p ≤ 0.05, by using One Way ANOVA. (E) B16 tumor-bearing mice were adoptively transferred with CD8⁺ T cells from mice expressing the TCR specific for TRP-2 antigen either with TRP-2^high or TRP-2^low. As negative control, anti-OVA CD8⁺ T cells were transferred. Survival of mice untreated or treated with ACT is reported; n=10 mice for group, 3 independent experiments. **p ≤ 0.01, logrank test. See also figure S4.

Figure 5. CD40-CD40L is required for ACT effectiveness

(A) CFSE-labeled, CD8⁺ T cells specific for either OVA or gp100 antigens were incubated with EG7 tumor slices from either WT or different KO mice as indicated. NO was detected by confocal microscopy of slices loaded with DAR-4M AM. NO-release levels were measured as mean of fluorescence and expressed as fold induction over control (without CD8). Mean ± s.e.m.; n=12 slices pooled from 3 independent experiments, ***p ≤0.001, **p ≤0.01, by using One Way ANOVA. (B) CFSE-labeled, polyclonal CD8⁺ T cells specific for OVA were derived from immunized WT or E8I^cre x Cd40lg^flox/flox mice and were incubated with EG7 tumor slices from either WT or CD40L KO mice, as indicated. NO was detected as in A. Error bars, mean ± s.e.m.; n=12 slices pooled from 3 independent experiments, ***p ≤ 0.001, **p ≤ 0.01, by using One Way ANOVA and the Holm–Sidak method of correction for all pairwise multiple comparison. (C) Survival percentages of WT, CD40 KO, and CD40L KO EG7 tumor-bearing mice untreated or treated with ACT (n=10). *p ≤ 0.05 *** p ≤ 0.001, logrank test. (D) EG7 tumor-bearing RAG-deficient mice were reconstituted with CD8⁺ T lymphocytes isolated from spleens and lymph nodes of WT and CD40L KO, either EG7 tumor-bearing or tumor-free, mice. After 2 days, ACT with OVA-specific CD8⁺CD45.1⁺ T lymphocytes was performed. Tumor area at days 9, and 13 following ACT are reported. Horizontal lines represent means of n=7. **p ≤ 0.01, unpaired Student t-test. See also figure S5.

Figure 6. Improving ACT effectiveness by favoring intra-tumoral myeloid balance towards Tip-DC accumulation

(A) EG7 tumor-bearing WT mice were treated with either anti-CSF-1R or Ctrl antibody, followed or not by ACT with OVA-specific CD8⁺CD45.1⁺ T lymphocytes. Percent of different myeloid subpopulations in CD11b⁺NOS2⁺ gate within the tumor mass is shown. n=12, pooled from 2 independent experiments. (B–C) WT (B) or NOS KO (C) MCA203 tumor-bearing mice were treated weekly with either anti-CSF-1R or Ctrl antibody and injected or not with mTERT-specific CD8⁺ T lymphocytes. Tumor volume at indicated days is shown as mean ± s.d., (n=15). (D) Percent of different immune subpopulations in tumors from mice treated with anti-CSF-1R or Ctrl antibody with or without ACT, (n=8). (A, D) Error bars indicate mean ± s.d., ***p ≤ 0.001, **p ≤ 0.01 and *p ≤ 0.05, by using One Way ANOVA. See also figure S6.

Figure 7. CD40LG, NOS2 and TNF correlate with survival in human CRC

(A) Correlations between CD40LG, NOS2 and TNF expression (qPCR) in CRC (n=125 patients) and immunome markers visualized in a ClueGO-CluePedia network. Markers associated to specific immune cell populations share the color of the node. The lines between nodes (edges) represent Spearman’s rank correlation values and are colored in red (CD40LG, ρ >0.5), blue (NOS2, ρ >0.2) and green (TNF, ρ >0.5), respectively. Negative correlation is shown with a sinusoidal line. The lower graphs show pair-wise correlation plots corresponding to the network edges. (B) Density of immune cells (number of positive cells per mm² of tissue surface area) infiltrating the center of the tumor (CT) in patients with (HiHiHi), heterogeneous (Htg) or low (LoLoLo) expression of CD40LG, NOS2 and TNF. A parametric or nonparametric test was applied. Error bars, mean ± s.e.m.; n=107. The median cell count/mm² is shown in blue, ** p ≤0.01, and * p ≤0.05. (C) Disease free survival (DFS) for patients having high, heterogeneous, and low expression of CD40LG, NOS2 and TNF is shown. See also figure S7.