Infiltration of Tumors Is Regulated by T cell-Intrinsic Nitric Oxide Synthesis

Abstract

Nitric oxide (NO) is a signaling molecule produced by NO synthases (NOS1-3) to control processes such as neurotransmission, vascular permeability, and immune function. Although myeloid cell-derived NO has been shown to suppress T-cell responses, the role of NO synthesis in T cells themselves is not well understood. Here, we showed that significant amounts of NO were synthesized in human and murine CD8+ T cells following activation. Tumor growth was significantly accelerated in a T cell-specific, Nos2-null mouse model. Genetic deletion of Nos2 expression in murine T cells altered effector differentiation, reduced tumor infiltration, and inhibited recall responses and adoptive cell transfer function. These data show that endogenous NO production plays a critical role in T cell-mediated tumor immunity.

Figure 1.

NO production and NOS expression in CD8⁺ T cells. A, CD8⁺ T cells were activated with anti-CD3/CD28 Dynabeads (or SIINFEKL peptide, when using OT-I CD8⁺ T cells) for 1 to 4 days in 21%, 5%, and 1% O₂. After activation, NO production and NOS expression were analyzed. B, NO production determined by extracellular quantification of nitrites (NO₂⁻, a NO byproduct) in mouse CD8⁺ T cells activated for 3 days (N = 11–13). C, Western blot analysis of NOS using a panNOS antibody (all isoforms detected) in lysates of mouse CD8⁺ T cell activated for 3 days. Results normalized to total protein stain (TPS, top) and representative blot (bottom; N = 3). D, Time course qRT-PCR analysis of Nos2 and Nos3 mRNA expression in activated mouse CD8⁺ T cells (N = 3–9). E, Western blot analysis of NOS2 protein levels in mouse OT-I CD8⁺ T cells treated or untreated with 50 μmol/L FG-4592 and activated for 3 days in 1% O₂ with increasing amounts of SIINFEKL peptide (0.001, 0.1, 1, and 1000 ng/mL). Quantification normalized to TPS (top) and representative blot (bottom). BMDMs polarized to M1 with 100 U/mL LPS were used as positive control for NOS2 expression (N = 3). F, NO production as determined by extracellular quantification of nitrites in human T cells cultured for 1 day in 21% and 1% O₂ with or without anti-CD3/CD28 beads (N = 4). G, Time course qRT-PCR analysis of NOS2 and NOS3 mRNA levels in activated human CD8⁺ T cells. NOS1 mRNA levels were under the detection limit (N = 5–8). H, Western blot analysis of panNOS (antibody detecting all NOS isoforms) and NOS3 in human CD8+ T cells. HUVEC cells were used as positive control and PPIB was used as loading control (representative of N = 3). Apart from panel D and G, each data point represents an independent animal and results are shown as median ± interquartile range (IQR). *, P < 0.05; Wilcoxon matched-pairs signed-rank test.

Figure 2.

Effect of pharmacologic inhibition of NO production by human CD8⁺ T cells. A, Human CD8⁺ T cells were activated with anti-CD3/CD28 Dynabeads in 21% O₂ and cultured for 3 days in the presence of L-NAME (NOS inhibitor) or NOC-18 (NO donor). Flow cytometry analysis was used to assess the effects on T-cell differentiation and expansion. B–D, Cell number determined with counting beads (B), cell division determined with CTV staining (C), and expression of CD45RO in cells treated with increasing concentrations of L-NAME and NOC-18. Horizontal grey line represents the DMSO control cells (CT). Cell division shown as division index and CD45RO expression shown as log₂ fold-change (FC) in mean fluorescence intensity (MFI) relative to CT (N = 3). E, NO production determined by quantification of nitrites in lysates of human CD8⁺ T cells; N = 3. F, Proportion of cells positive for phospho-S6 Ribosomal Protein (Ser235/236, pS6; N = 3). G, Expression of differentiation markers shown as log₂ FC in MFI relative to DMSO CT (horizontal grey line) following treatment with 200 μmol/L L-NAME (N = 4–10). Results are shown as median ± interquartile range (IQR). *, P < 0.05; *, P < 0.01; Wilcoxon matched-pairs signed-rank test.

Figure 3.

In vitro characterization of Nos2 KO CD8⁺ T cells. A, After activation in 1% O₂ with anti-CD3/CD28 beads, NOS2 expression was determined by Western blot in Nos2^fl/fl (WT, gray) and Nos2^fl/fldLck^Cre (NOS2^KO, orange) cells. Representative blot (top) and quantification normalized to total protein stain (bottom; N = 2). B, WT and NOS2^KO CD8⁺ T cells were activated for 3 days in 21% or 1% O₂, and NO production was determined by the extracellular nitrite concentration (N = 5–8). C, WT and NOS2^KO mouse CD8⁺ T cells were activated for 72 hours in 21%, 5%, or 1% O₂. Viable CD8⁺ T-cell number was determined by flow cytometry using count beads (left); cell proliferation assessed with CTV staining and expressed as division index (right; N = 8–18). D, Proportion of CD62L⁻CD44⁺ in cells activated as in C (left) and representative FACS plots for 1% O₂ activated cells (right; N = 11–18). E, Heat map illustrating expression of markers of differentiation determined by flow cytometry in CD8⁺ T cells activated for 72 hours in 21%, 5%, or 1% O₂. Increased and reduced expression of proteins are shown in gray and orange, respectively. Rows represent averaged z-scores (N = 11–18). F, Seahorse metabolic analysis of mouse T cells activated for 3 days in 1% O₂, as determined by OCR and ECAR after injection of anti-CD3/CD28 beads or antibodies, oligomycin (O), FCCP (F), or rotenone+antimycin A (R+A; left). Effect of T-cell activation on T-cell OCR and ECAR was determined by % change from baseline following injection of anti-CD3/CD28 beads or antibodies. Seahorse analysis was conducted in a hypoxia chamber set to 3%O₂ (N = 8). G, OT-I CD8⁺ T cells activated for 3 days in 1% O₂ were cocultured with 10,000 OVA-expressing B16-F10 tumor cells at different effector:target (E:T) ratios. Cytotoxicity was assessed with Alamar blue assay after 14 to 18 hours of coculture at 21% O₂. A nonlinear regression [(agonist) vs. normalized response] was used to determine dose–response curves (shaded areas: 95% confidence intervals; N = 4–6). H, CD8⁺ T cells were activated for 6 days and incubated for 48 hours in 1% O₂ before being loaded with calcein-AM and cocultured with mouse endothelial cells in a transwell system. mCCL19 and mCCL21 were added to the lower chamber as chemoattractant. Calcein signal corresponding to T cells migrating through the endothelial barrier was assessed after 3 hours of coculture in a plate reader (N = 7–9). All results [median ± interquartile range (IQR)] are pooled from a minimum of two independent experiments, and each data point from panels B, F (right), and G and H represent an independent animal. ns, P > 0.05; *, P < 0.05; **, P < 0.01; Mann–Whitney test relative to respective WT control.

Figure 4.

Tumor growth in animals lacking Nos2 expression in T cells. A, Tumor growth model. 5×10⁵ MC38 or B16-F10-OVA were subcutaneously injected in Nos2^fl/fl (WT) Nos2^fl/fldlck^Cre (NOS2^KO) animals. On day 10 after tumor inoculation, peripheral blood and tumors were processed to single-cell suspensions and analyzed by flow cytometry. Tumor growth was monitored until day 30. B, MC38 (top) and B16-F10-OVA (bottom) tumor growth data. Tumor growth curves in WT and NOS2^KO animals; thin lines represent individual animals and thick line represents an exponential (Malthusian) growth curve (left). Survival curves using 500 mm³ as threshold (right). Results pooled from two independent experiments (N = 18–26 animals per group). C, Immune composition was analyzed by flow cytometry on peripheral blood of animals bearing MC38 (top; N = 6–8) and B16-F10-OVA (bottom; N = 13–18) tumors for 10 days. Results expressed as cells per milliliter of blood; median ± interquartile range (IQR). D, Representative flow cytometry plots from CD4⁺ and CD8⁺ T-cell infiltration in B16-F10-OVA tumors collected on day 10 following inoculation in WT and NOS2^KO animals (top). Immune cell infiltration in B16-F10-OVA analyzed by flow cytometry and expressed as counts per million CD45⁺ cells (bottom). Cells pregated on live, singlet, CD45⁺ events (N = 9, median ± IQR). Each data point represents an individual animal. ns, P > 0.05; *, P < 0.05; **, P < 0.05; log-rank (Mantel–Cox) test relative to WT animals (B) and Mann–Whitney test relative to WT control (C and D).

Figure 5.

Antitumor function and tissue infiltration capacity of NOS2^KO OT-I T cells. A, ACT model. C57BL/6j mice were injected subcutaneously with 1×10⁶ OVA-expressing B16-F10 tumor cells, and 4 days later were lymphodepleted with 300 mg/kg CPA. Mice bearing tumors for 7 days were then intraperitoneally injected with 1×10⁶ of 4 days activated WT or NOS2^KO OT-I cells. Tumor growth was monitored every 2 to 3 days until day 60. B, B16-F10-OVA tumor growth after ACT. Tumor growth curves after No ACT or ACT with VC or NOS2^KO OT-I cells; vertical dotted lines represent day of ACT, thin lines represent individual animals, and thick lines represent an exponential (Malthusian) growth curve (left). Survival curves using 500 mm³ as threshold (right; N = 9–20 animals per group). C, Tumor infiltration model. C57BL/6j mice were injected subcutaneously with 1×10⁶ OVA-expressing B16-F10 tumor cells, and 11 days later were lymphodepleted with CPA. Mice bearing tumors for 14 days were then intraperitoneally injected with Nos2^fl/fl (WT) and Nos2^fl/fldlck^Cre (NOS2^KO) OT-I CD8⁺ T cells (1×10⁶ each in 1:1 NOS2^KO:WT ratio). Spleen, peripheral blood, liver, and tumor were harvested on day 19 and processed to single-cell suspensions for flow cytometric analysis. Endogenous and adoptive populations were distinguished by the allelic variants of CD45. D, Total OT-I T-cell expansion in all analyzed tissues expressed as a ratio between NOS2^KO and WT cell counts (gray horizontal line represents the NOS2^KO/WT ratio at the time of injection; N = 22, median ± interquartile range [IQR]). E, Percentage of cells expressing granzyme B (GZMB) within CD8⁺ T cells in all tissues analyzed by flow cytometry on day 19 (bottom; N = 19–22, median ± IQR). F, Representative FACS plots (left) and flow cytometry analysis (right) of percentage of CD44⁺CD8⁺ T cells in peripheral blood on day 19 (N = 19–22, median ± IQR). Results are pooled from at least two independent experiments, and each data point represents an independent animal. *, P < 0.05; **, P < 0.01; ***, P < 0.001; log-rank (Mantel–Cox) test relative to WT animals (B), one sample t test relative to 1 (D) and Wilcoxon matched-pairs signed-rank test relative to WT control (E and F).

Figure 6.

In vivo activation and recall response of NOS2^KO OT-I CD8⁺ T cells. A, Scheme of in vivo T-cell activation and recall response model. C57BL/6j mice were injected intraperitoneally with 1×10⁶ naïve Nos2^fl/fl (WT) and Nos2^fl/fldlck^Cre (NOS2^KO) OT-I CD8⁺T (1:1 NOS2^KO:WT ratio). The next day, mouse BMDMs differentiated for 7 days and polarized with LPS for 24 hours were pulsed with SIINFEKL peptide for 4 hours prior to intraperitoneal injection. Peripheral blood was sampled at days 7 and 10 after BMDM transfer and analyzed by flow cytometry. SIINFEKL-pulsed BMDMs (or PBS controls) were administered again on day 30. At day 37, the spleen, inguinal lymph nodes, and a liver portion were harvested and analyzed by flow cytometry. Endogenous and adoptive populations were distinguished by the allelic variants of CD45. B, OT-I T-cell expansion in peripheral blood expressed as the relative ratio between KO and WT cell counts on days 7 and 10 after BMDM injections (horizontal gray line represents the range of the initial NOS2^KO/WT ratio; N = 32–36). C, Percentage of CD62L-CD44+ cells (left) and CD8 and CD127 MFI (right) of WT, KO, and endogenous CD8⁺ T cells harvested from peripheral blood on day 7 (N = 31). D, Recall response as determined by the ratio between WT and KO OT-I CD8⁺T-cell infiltration in the spleen, lymph node, and liver 7 days after recall with SIINFEKL-pulsed BMDMs (+) or with PBS control (−). Horizontal gray line represents the range of the initial NOS2^KO/WT ratio (N = 17–21). E, Recall response as determined by amount of CD44⁺ WT and NOS2^KO OT-I T cells per million CD45⁺ cells infiltrated in the spleen, lymph node, and liver 7 days after recall with BMDMs or PBS control (N = 9–18). F, Flow cytometry analysis of CD44 in CD8⁺ T cells infiltrating the spleen, lymph node, and liver on day 37. All results [median ± interquartile range (IQR)] are pooled from three independent experiments and each data point represents an independent animal. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One sample t test relative to 1 (B and D), Tukey multiple comparisons paired test (C–F). #, P < 0.05; ##, P < 0.01; ###, P < 0.001; Unpaired t test.