Six to eight-week-old C57BL/6 Ly5.1 (CD45.1), Ly5.2 (CD45.2), WT mice were purchased from Charles River or Jackson Laboratory. CCR2^-/-, IL-10^-/-, H–2K^bm1, OTI, OTII, MHCII^-/- and IL-10 GFP (Tiger) mice were purchased from Jackson Laboratory. PDL1/2^-/- mice were kindly provided by Dr. Daniel Barber and Keith Kauffman. OTII mice were crossed with IL-10 GFP to generate OTIl IL-10 GFP reporter mice. All mice were genotyped upon arrival and prior to their use in experiments. Mice were housed in a specific pathogen-free environment at National Jewish Health and Dartmouth Hitchcock Medical College, an AAALAC accredited institution, and used in accordance with protocols approved by the Institutional Animal Care and Utilization Committee.

Eight-week-old CD45.1 or CD45.2 C57BL/6 mice were lethally irradiated with 900 rads and reconstituted intravenously at a bone marrow (BM) cell ratio of 80:20 from CCR2^-/- and either WT, H–2K^bm1, PDL1/2^-/-, or IL-10^-/- mice. Six to eight weeks post-reconstitution, mice were assessed for chimerism prior to use.

Lung-draining LNs were teased with 26G needles and digested in 1ml of 2.5 mg ml⁻¹ collagenase D (Roche) solution in 1X RPMI at 37°C for 30 min. After incubation, 100 μl of 100 mM EDTA was added to stop the enzymatic reaction. The cells were pipetted up and down with glass Pasteur pipets and then passed through a 70 μm nylon filter. Single cell suspensions were collected and centrifuged at 300 g for 5 min. Cells were stained with the following monoclonal Abs: Phycoerythrin (PE)-conjugated to Vα2, CD4 and CD64; PerCP-Cy5.5- conjugated to CD11b and CD4; PE-Cy7-conjugated to CD11c and CD44; BUV395-conjugated to CD11b; BUV805-conjugated to CD8a, fluorescein isothiocyanate (FITC)-conjugated to CD4, CD45.1 and CD103; allophycocyanin (APC)-conjugated to Vβ51, CD45.2, CD45 and CD103; APC-Cy7-conjugated to CD45.2 and MHCII; and BV510 conjugated to Ly6C and MHCII. The PE-conjugated MHC Tetramer H-2kb OVA SIINFEKL was purchased from MBL international. The viability dye 4’,6-diamidino-2-phenylindole (DAPI) (#D9542, Sigma) was added immediately before each sample acquisition on a BD Symphony A3 analyzer (BD Biosciences). Data were analyzed using FlowJo (Tree Star, Ashland, OR). Antigen-specific antibodies and isotype controls were obtained from BioLegend, eBioscience and BD Biosciences.

Spleen cells from OTI or OTII GFP mice, in which the TCR of CD8⁺ or CD4⁺ T cells are restricted to an OVA peptide, were labeled with 10 μM carboxyfluorescein succinimidyl ester (CFSE) for 10 mins. Half a million of these cells were transferred intravenously into recipient mice 1 day before intranasal delivery of 2 μg soluble OVA and 20 μg Poly I:C. Mice were sacrificed 3 days after intranasal deliveries for analysis of T cell proliferation (CFSE dye dilution) in the LLN. For OTI: Isolated cells were cultured 5 hours in RPMI 10% FCS containing 10 μM OVA peptide (257-264) and Brefeldin A 10 μg/ml. Following surface staining, BD Foxp3 intracellular staining kit was used. Cells were stained with APC-conjugated to IFNγ, PerCP-Cy5.5-TNFα, or isotype controls (BD Pharmingen). For OTII: Isolated cells were cultured 5 hours in RPMI 10% FCS containing 10 μM OVA peptide (323-339) and Brefeldin A 10 μg/ml. Following stimulation, cells were stained with PE-conjugated to CD4, APC-conjugated to Vβ51, PE-Cy7-conjugated to Vα2, APC-Cy7-conjugated to CD44, BUV805-conjugated to CD8 (BD Pharmingen, eBioscience or Biolegend).

Monocytes from CD11b enriched cell suspension (Miltenyi) were sorted (FACSAriaIII sorter). Total RNA was extracted using the RNeasy Minikit (Qiagen). Equal amounts of total RNA from each sample were reverse transcribed into cDNA using SuperScript™ VILO™ Master Mix (Invitrogen). qPCR was set up using PowerUp SYBR Green PCR Master Mix (Applied Biosystems) and analyzed on Bio-Rad C-1000 CFX96. RT-PCR primers for IL-10: Forward-GGTTGCCAAGCCTTATCGGA; Reverse-ACCTGCTCCACTGCCTTGCT. Relative quantification of gene expression was performed by normalizing expression levels of genes of interest over housekeeping (Thoc1) gene within each sample followed by relative expression to control (mean of triplicate).

B16F10 melanoma cells were purchased from ATCC (CRL-6475) and maintained in RPMI with 10% FCS, 1% Pen/Strep/L-glutamine (Sigma), 1% non-essential amino acids (Sigma), 1% sodium pyruvate (Sigma), 10 mM HEPES (Sigma) and 0.1 mM β-mercaptoethanol.

We received de-identified non-diseased human lung with attached LLNs from the International Institute for the Advancement of Medicine (Edison, NJ, USA). We selected donors without a history of chronic lung disease and with reasonable lung function with a P_aO₂/F_iO₂ ratio of >225, a clinical history and X-ray that did not indicate infection, and limited time on a ventilator. Lungs were removed en bloc in the operating room and included the trachea, LNs and pulmonary vessels. Pulmonary arteries were perfused in the operating room with cold histidine-tryptophan-ketoglutarate (HTK) solution to preserve endothelial cell function and prevent intravascular clot formation. The lungs were submerged in HTK, and immediately shipped on ice. All lungs were processed within 24 hours of removal. The lungs were visually inspected for lesions or masses and were eliminated from the study if grossly abnormal. Peribronchial LNs were identified and removed. Procedure additionally described in (35, 36). The Committee for the Protection of Human Subjects at National Jewish Health approved this research. RNA sequencing was completed as previously described (11).

Statistical analysis was conducted using Prism software. All bar graphs are expressed as the mean ± SEM. Statistical tests were performed using two-tailed Student’s t-test and ANOVA. P < 0.05 was considered statistically significant.

Monocytes require CCR2 to exit the bone marrow and enter tissue under steady-state and inflammatory conditions (7, 37). As anticipated, 24 hours post intranasal delivery of antigen and Poly I:C, the total number of migratory DCs in the LLN was similar between WT and CCR2^-/- mice ( Figure 1A ). In contrast, monocytes are substantially deficient in the CCR2^-/- mice compared to WT mice (17, 23, 37).

To examine the role of LN monocytes in a Poly I:C-induced antigen-specific CTL response, CFSE labeled naïve OVA-specific CD8⁺ T cells (OTI) were adoptively transferred into WT and CCR2^-/- mice. Three days post inoculation of antigen and Poly I:C, LLN OTI T cells were analyzed for the production of CTL cytokines, IFNγ and TNFα. Although we observed a relatively similar number of proliferating CFSE⁺ OTI T cells between WT and CCR2^-/- mice ( Figure 1B ), the frequency of IFNγ and TNFα producing OTI T cell was significantly greater in CCR2^-/- mice compared to WT mice ( Figure 1C ), showing that monocytes downregulate the intensity of cytokine production in proliferated OTI T cells.

To support the findings in the CCR2^-/- mice, where LN monocytes are continually deficient, we examined whether temporary depletion of monocytes would also result in an increased Poly I:C induced CTL response. Using anti-Gr1, monocytes were temporarily depleted in WT mice on the same day as the inoculation of antigen and Poly I:C. A caveat to note is that in addition to monocytes, anti-Gr1 depletes neutrophils, plasmacytoid DCs and Ly6C⁺ T cells but not DCs for up to 2-3 days (6, 38). As observed in CCR2^-/- mice, WT mice with transiently depleted monocytes displayed a significant increase in IFNγ and TNFα producing antigen-specific CD8⁺ T cells compared to mice treated with control antibody ( Supplementary Figure 1 ). Collectively, data from mice either constitutively (CCR2^-/-) or temporarily (via anti-Gr1 treatment) depleted of LN monocytes suggest that the role of monocytes is to restrict an exaggerated CTL response in the context of antigen and Poly I:C.

We investigated three possible mechanisms by which monocytes could dampen a Poly I:C induced CTL response. First, we hypothesized that monocytes directly interfere with the cross-presentation and cross-priming of CTLs by TLR3 stimulated DC1s. To address this hypothesis, we took advantage of the H–2K^bm1 mice which have mutations to the antigen binding site of MHCI, rendering antigen presenting cells incapable of inducing proliferation of OTI T cells (39). Lethally irradiated CD45.1 C57BL/6 mice were reconstituted with a mixture of 80% CCR2^-/- and 20% H–2K^bm1 bone marrow (BM) cells or with 20% CD45.1 WT BM in control mice. In this competitive setting, CCR2-sufficient BM cells will outcompete CCR2-deficent cells in the reconstitution of CCR2 dependent cell types, resulting in the complete repopulation of LN monocytes by H–2K^bm1-derived BM cells (or CD45.1 WT BM cells in control mice). Importantly DCs, which do not depend on CCR2 for their migration into tissue, are found at a ratio of 80:20 CD45.2:CD45.1 in the LNs of control mice, reflecting the ratio of transferred BM cells ( Figure 2A ). Thus, in chimeric mice, 100% of LN monocytes are incapable of directly presenting antigen to OTI T cells while antigen presentation by DCs is minimally affected.

In BM chimeras where the LN monocytes were derived from WT or H–2K^bm1 BM, we observed no difference in the cytokine production in Poly I:C induced CTLs ( Figure 2C ). Therefore, we examined our second hypothesis that PDL1 expression on monocytes directly dampen the CTL response. In the LLN, PDL1 is highly expressed in steady-state and inflamed monocytes ( Figure 2B ). However, in the lung, monocytes and not DCs upregulate PDL1 after Poly I:C exposure ( Figure 2B ) (7). When we created BM chimeric mice where monocytes lacked PDL1/2 expression, there was a slight decrease in cytokine production from proliferating antigen-specific T cells compared to PDL1/2 expressing monocytes ( Figure 2D ). In summary, the mechanism of action that LN monocytes use to regulate a Poly I:C-induced antigen-specific CTL response is not mediated via cross-presentation or PDL1 expression.

Thirdly, we hypothesized that the production of IL-10, a well-described anti-inflammatory cytokine secreted by monocytes and macrophages in response to PAMP stimulation, might provide the mechanism by which monocytes dampen a Poly I:C-induced CTL response. First, we confirmed that monocytes produce IL-10 after an intranasal bolus of antigen and Poly I:C using IL-10 reporter mice ( Figure 3A ). Among extravascular CD11b⁺ cells, CD64⁺Ly6C⁺ monocytes uniquely expressed IL-10 after exposure to Poly I:C; intravascular cells were excluded by labeling with injection of anti-CD45 antibody given 5 minutes prior to harvest. Moreover, in the LLN, monocytes increased their mRNA expression of IL-10 after nasal inoculation of antigen and Poly I:C ( Figure 3B ). Using the same bone marrow chimera approach described previously ( Figure 2 ), we created mice with IL-10 deficient LN monocytes. Compared to IL-10 sufficient LN monocytes, IL-10 deficient LN monocytes were unable to dampen the CTL response induced by antigen and Poly I:C ( Figure 3C ). In conclusion, these data suggest that monocytes require IL-10 production to dampen a CTL response. However, whether this effect is direct or indirect is unclear.

Next, we investigated whether other immune cell populations were involved in the modulation of adaptive immune response promoted by monocytes. Suppressor T cells (Treg) are known for their critical role in controlling autoimmunity and suppressing inflammatory responses. They also promote tumor development and progression by dampening the anti-tumor immune response. Therefore, we examined whether there was an increase in suppressor T cells in WT mice compared to CCR2^-/- mice. Suppressor T cells can be either Foxp3⁺ or Foxp3^- (35). We observed no difference in the frequency of Foxp3⁺ regulatory T cells between WT and CCR2^-/- mice ( Supplementary Figure 2 ). Then, we examined the frequency of endogenous CD44⁺CD4⁺ IL-10 producing T cells post antigen and Poly I:C exposure and observed an increase in IL-10 producing CD4⁺ T cells in WT mice compared to CCR2^-/- mice ( Supplementary Figure 2 ). Since these data do not convey antigen-specificity, OTII OVA-specific CD4 T cells were crossed with an IL-10 reporter and transferred these into WT or CCR2^-/- mice. After delivery of antigen with Poly I:C, WT mice displayed a significant increase in IL-10 producing antigen-specific CD4⁺ T cells compared to CCR2^-/- monocyte deficient mice ( Figure 4A ).

Based on this finding, we examined ex vivo whether endogenous CD4⁺ T cells from antigen and Poly I:C treated WT mice are more suppressive than those from CCR2^-/- mice. We isolated CD4⁺ T cells from the LLNs of WT and CCR2^-/- mice treated with antigen and Poly I:C and co-cultured them with antigen-specific CD8 T cells and stimulants (splenic DCs, antigen and Poly I:C). After five days in culture, antigen-specific CD8⁺ T cells co-cultured with CD4⁺ T cells from WT mice compared to CCR2^-/- mice produced significantly less IFNγ ( Figure 4B ). This suggests that LN monocytes are required for the induction of an antigen-specific CD4⁺ T cell that suppresses the Poly I:C induced CTL response.

To clearly demonstrate that antigen presentation by monocytes is required for the induction of suppressor T cells, we created BM chimeric mice where monocytes lacked MHCII expression (80:20, CCR2^-/-MHCII^-/- BM). First, we examined whether endogenous Poly I:C-induced antigen-specific CD8⁺ T cells were significantly increased when monocytes lacked the ability to differentiate suppressor CD4 T cells. Indeed, when monocytes lacked MHCII expression, tetramer staining illustrated a significant increase in endogenous Poly I:C induced antigen-specific CD8⁺ T cells ( Figure 5A ). When CCR2^-/-:MHCII^-/- BM chimeras were challenged with antigen, Poly I:C, and antigen-specific CD8⁺ T cells, as in Figure 1 , mice lacking MHCII expression on monocytes, displayed significantly enhanced cytokine production in Poly I:C-induced antigen-specific T cells ( Figure 5B ). Finally, we demonstrated in MHCII sufficient monocyte BM chimeras that the differentiation of IL-10 producing antigen-specific CD4⁺ T cells was significantly greater than in MHCII deficient monocyte BM chimeras ( Figure 5C ). Overall, these data suggest that MHCII expression on monocytes and not DCs is required for the differentiation of IL-10 suppressor antigen-specific CD4⁺ T cells that regulate the Poly I:C induced CTL response.

Next, the relevance of these findings was demonstrated in a prophylactic metastatic melanoma model. Treatment with anti-IL-10 was performed instead of monocyte depletion, as there are currently no human monocyte specific depleting antibodies. A triple combination therapy of anti-IL-10, tumor peptides and Poly I:C, was compared to tumor peptides +/- Poly I:C, types of therapy already known to promote anti-tumor immunity. Although the tumor peptides with Poly I:C alone displayed a robust anti-tumor immune response, the triple combination therapy of melanoma challenged mice displayed the greatest anti-tumor immunity ( Figure 6 and Supplementary Figure 3 ). This finding demonstrates that an anti-tumor immune response can be significantly enhanced in combination with IL-10 neutralization.

Finally, to translate our findings to clinical practice, we examined whether human LN monocytes, like mice, express IL-10. Indeed, like mice, human LLNs contained a similar number of CD14⁺CD11c^hiHLADR⁺ monocytes as CD14^-CD11c^hiHLADR⁺⁺ DCs ( Figure 7A ) (36, 40), and expressed significantly more IL-10. Transcriptionally, the lung and LLN monocytes expressed ~10 to 100-fold greater amounts of IL-10 compared to LN DCs and immature blood monocytes ( Figure 7B ). This demonstrates that human monocytes produce IL-10 after extravasation into tissue and LN and perhaps, play a continuous role of regulating immunity.