Visual Abstract

Induction of lung mucosal immune responses is highly desirable for vaccines against respiratory infections. We recently showed that monocyte-derived dendritic cells (moDCs) are responsible for lung IgA induction. However, the dendritic cell subset inducing lung memory TH cells is unknown. In this study, using conditional knockout mice and adoptive cell transfer, we found that moDCs are essential for lung mucosal responses but are dispensable for systemic vaccine responses. Next, we showed that mucosal adjuvant cyclic di-GMP differentiated lung moDCs into Bcl6+ mature moDCs promoting lung memory TH cells, but they are dispensable for lung IgA production. Mechanistically, soluble TNF mediates the induction of lung Bcl6+ moDCs. Our study reveals the functional heterogeneity of lung moDCs during vaccination and paves the way for an moDC-targeting vaccine strategy to enhance immune responses on lung mucosa.

Vaccination is the most cost-effective approach to fight infectious diseases. Approved vaccines mainly elicit Ab-mediated protection and do not generate strong mucosal responses. Vaccines that induce T cell–mediated protection in lung mucosa would provide major health and economic benefits. Memory T cells include central memory T cells, effector memory T cells, and tissue-resident memory T (TRM) cells. TRM cells comprise a majority of memory T cells in the lung and play a crucial role in maintaining long-term protective immunity in the lung mucosa (1, 2). Lung TRM cells include CD4+ TRM and CD8+ TRM cells.

Lung CD4+ TRM cells are crucial for protection against influenza virus and Streptococcus pneumoniae infection (3, 4). Swain’s group showed that lung memory CD4+ T cells protect against influenza through multiple synergizing mechanisms (5). Memory CD4+ T cells can accelerate primary CD8+ T cell responses (6), activate innate immune cells to combat infections (7), and secret TH cytokines (8). The dendritic cell (DC) subset that promotes the induction of lung CD4+ TRM cells is unknown.

Lung TRM cells (both CD4+ and CD8+ TRM cells) require local Ag presentation (912). In contrast, TRM cells in the intestine, genital tract, or skin do not require tissue Ag recognition (13, 14). Swain’s group (11) first reported that CD4+ T cell memory formation depends on the re-engagement of CD4+ T cells with APCs at their effector stage (“Signal 4”), day 5–8 of their response (9, 11). Lung CD8+ TRM cell establishment also requires cognate Ag recognition in the lung (10). It is unclear which lung APCs provide the “Signal 4” for lung CD4+ TRM cell induction at this stage of vaccination or infection.

As professional APCs, DCs comprise developmental and functional diverse populations. Common DC progenitors generate preconventional DCs (precDCs) (15). In the lung, precDCs develop into conventional DC (cDC)–1 and cDC2. cDC1 expresses CD103, whereas cDC2 expresses CD11b and CD24 but are CD64 negative. cDC1 are specialized in Ag cross-presentation and priming cytotoxic CD8+ T cells (16). cCD2 prime CD4+ T cells and generate TH responses (17). Circulating monocytes also give rise to lung DCs (18). Upon infection or inhalation of allergens, lung epithelial cells produce chemokines CCL2 that recruit CCR2hi monocytes (19, 20). In the lung, these infiltrating CCR2hi monocytes upregulate MHC class II (MHCII) and CD11c and become monocyte-derived DCs (moDCs). moDCs are found virtually in any disease with substantial inflammation (21).

Bona fide moDCs are highly capable of Ag uptake, processing, presentation, and priming CD4+ and CD8+ T cells (21). For example, tumor-infiltrating moDCs prime CD8+ T cells and induce antitumor immunity (22). CCR2+ moDCs are critical for TH17 induction and the development of experimental autoimmune encephalomyelitis (23). During cutaneous Listeria major infection, moDCs could induce TH1 polarization (20). moDCs were also sufficient to induce TH2 immunity by high-dose house dust mite asthma (24). Last, we recently showed that moDCs induce T follicular helper (TFH) cells in the lung by mucosal adjuvant cyclic di-GMP (CDG) (25). Thus, there is a significant degree of plasticity and heterogeneity in moDCs in vivo. The underlying mechanism for moDCs plasticity and heterogeneity is unknown (21).

In this report, we study the role of lung moDCs in generating lung mucosal memory TH cells. We found that moDCs are specifically required for the generation of memory TH cells in lung mucosa but not in the systemic compartments. Furthermore, we identified a new, to our knowledge, differentiated moDCs subpopulation expressing the transcriptional factor (transcriptional repressor B cell chronic lymphocytic leukemia/lymphoma 6) Bcl6 that is responsible for memory TH cells generation in lung mucosa.

Age- and sex-matched mice (2–3 mo old) were used for immunization. C57BL/6J, B6.CD45.1 (no. 002014), Bcl6fl/fl (no. 023727), CCR2–/– (no. 004999), RelAfl/fl (no. 024342), IL-21–VFP (no. 030295), CD11ccre (no. 008068), and LysMcre (no. 004781) mice were purchased from The Jackson Laboratory. Mice were housed and bred under pathogen-free conditions in the Animal Research Facility at the University of Florida. Cre-negative littermates were used as controls for experiments. All mouse experiments were performed by the regulations and approval of the Institutional Animal Care and Use Committee at the University of Florida, Institutional Animal Care and Use Committee number 201909362.

Eα-OVA (ASFEAQGALANIAVDKA-OVA) fusion protein was produced by GeneCust. TNF-IgG2a-Fc fusion proteins were produced by Creative Biolabs. Wild-type (WT) mouse TNF (aa80–aa235) or mutant mouse TNF (TNFD221N/A223R) was fused with the Fc portion of the mouse IgG2A. The following reagents were from Invivogen: endotoxin-free OVA (vac-pova) and vaccine-grade CDG (vac-nacdg). The following reagent was from eBioscience: YAE mAb (14-5741-82). The following reagent was from Biosearch Technologies: NP6CGG (N-5055A).

The following reagents were obtained from BioLegend: allophycocyanin-Mouse IgG2A (clone: MOPC173, catalog no. 981906), anti-mouse CD19–PerCP/Cy5.5 (clone: 1D3/CD19, catalog no. 152405), anti-mouse LAP (TGF-β1) (clone: TW7-16B4, catalog no. 141407, 141413), anti-mouse CD45.1-allophycocyanin(clone: A20, catalog no. 110713), anti-mouse CD69–allophycocyanin (clone: H1.2F3, catalog no. 104578), anti-mouse CD80-FITC (clone: 16-10A1,catalog no. 104705), anti-mouse CD86–allophycocyanin-Cy7 (clone: GL-1, catalog no. 105029), anti-mouse Bcl6–allophycocyanin (clone: 7D1, catalog no. 358505), anti-mouse Bcl6–PE (clone: IG191E/A8, catalog no. 648304), anti-mouse CD4-PE/Cy7 (clone: GK1.5, catalog no. 100422), anti-mouse IL-4–allophycocyanin (clone: 11B11, catalog no. 504106), anti-mouse IL-17a–PE (clone: TC11-1810.1, catalog no. 506903), anti-mouse IL-4–allophycocyanin (clone: 11B11, catalog no. 504105), anti-mouse CD45-PercP/Cy5.5 (clone: 30-F11, catalog no. 103131), anti-mouse MHCII(I-A/I-E)–Brilliant Violet 421 (clone: M5/114.15.2, catalog no. 107636), anti-mouse MHCII(I-A/I-E)–Alexa Fluor (clone: M5/114.15.2, catalog no. 107622), anti-mouse CD11c-allophycocyanin/Cy7 (clone: N418, catalog no. 117323), anti-mouse/human CD11b-PE/Cy7 (clone: M1/70, catalog no. 101216), anti-mouse/human CD11b–Brilliant Violet 605 (clone: M1/70, catalog no. 101237), anti-mouse CD64-PerCP/Cy5.5 (clone: X54-5/7.1, catalog no. 139307), anti-mouse TNFR2-PE (clone: TR75-89, catalog no. 113405), anti-mouse PD1 (clone: 29F.1A12, catalog no. 135214, no. 135223), anti-mouse IL-10–allophycocyanin (clone: JESS-16E3, catalog no. 505016), anti-mouse CXCR–allophycocyanin (clone: L138D7, catalog no. 145506), anti-mouse CD49A–PE (clone: HMa1, catalog no. 142604).

Additional reagents were from Invitrogen (anti-mouse IL-12p35–PE [clone: 27537, catalog no. MA5-23559], anti-mouse IL-23–FITC [clone: fc23cpg, catalog no. 53-7023-80]), Miltenyi Biotec (anti-mouse TNFR2-allophycocyanin [clone: REA228, catalog no. 130-104-698]), R&D Systems (anti-mouse CXCL13 [catalog no. AF470]), anti-mouse CCL20–allophycocyanin-Cy7 [clone: 114906, catalog no. IC760N]), Cell Signaling Technologies (anti-mouse/human pRelB-PE [clone: D41B9, catalog no. 13567], anti-mouse pRelA–allophycocyanin [clone: 93H1, catalog no. 4887S]).

The following reagents were obtained through Biological and Emerging Infections Resources, National Institute of Allergy and Infectious Diseases, and the National Institutes of Health: S. pneumoniae Family 2, Clade 3 Pneumococcal Surface Protein A (PspA UAB099) with C-Terminal Histidine Tag, Recombinant from Escherichia coli, NR-33179.

Groups of mice were intranasally (i.n.) vaccinated with CDG (5 µg), adjuvanted Ag (2 µg), or Ag alone (26). For i.n. vaccination, animals were anesthetized using isoflurane in an E-Z Anesthesia system (Euthanex, Palmer, PA). Ag with or without CDG was administered in 30 µl saline. Sera were collected at the indicated time points after the last immunization. The Ag-specific Abs were determined by ELISA. The secondary Abs used were anti-mouse IgG-HRP (1033– 05; SouthernBiotech), and anti-mouse IgA-HRP (1040–05; SouthernBiotech). To determine Ag-specific TH response, splenocytes and lung cells from Ag or CDG + Ag-immunized mice were stimulated with 5 µg/ml Ag for 4 d in culture. TH cytokines were measured in the supernatant by ELISA.

Cells were isolated from the lung, as previously described (25). The lungs were perfused with ice-cold PBS and removed. Lungs were digested in DMEM containing 200 μg/ml Dnase I (10104159001; Roche Diagnostics), 25 μg/ml liberase (05401119001; Roche Diagnostics) at 37°C for 2 h. RBCs were then lysed and a single-cell suspension was prepared by filtering through a 70-µm cell strainer.

For transcription factor Bcl6 staining of murine and human cells, cells were fixed and permeabilized with the Foxp3-staining buffer set (catalog no 00-5523-00; eBioscience). The intracellular cytokine staining was performed using the Cytofix/Cytoperm Kit from BD Biosciences (catalog no. 555028). The single lung cell suspension was fixed in Cytofix/perm buffer (BD Biosciences) in the dark for 20 min at room temperature. Fixed cells were then washed and kept in the Perm/Wash buffer at 4°C. Golgi-plug was present during every step before fixation.

Mouse Ly6Chi monocytes were purified from the bone marrow of I mice (C57BL/6J or CD45.1) following the protocol according to the manufacturer (19861; StemCell Technologies). Mice were i.n. vaccinated with CDG (5 µg) and Ag. Ly6Chi monocytes (1.5 million per mouse) were administered i.n. into immunized mice at 30 mins, 2 h, and 4 h postimmunization.

Single-cell suspensions were stained with fluorescent dye–conjugated Abs in PBS containing 2% FBS and 1 mM EDTA. Surface stains were performed at 4°C for 20 min. Cells were washed and stained with surface markers. Cells were then fixed and permeabilized (catalog no. 00-5523-00; eBioscience) for intracellular cytokine stain. Data were acquired on a BD LSRFortessa and analyzed using the FlowJo software package (FlowJo). Cell sorting was performed on the BD FACSAriaIII Flow Cytometer and Cell Sorter.

Data exclusion was justified when the positive or negative control did not work. All experiments will be repeated at least two times. All repeats are biological replications that involve the same experimental procedures on different mice. Experiments comparing different genotypes and adjuvant responses are designed with individual treatments being assigned randomly. When possible, treatments will be assigned blindly to the experimenter by another individual in the laboratory. When comparing samples from different groups, samples from each group will be analyzed in concert, thereby preventing any biases that may arise from analyzing individual treatments on different days.

All data are expressed as mean ± SEM. Statistical significance was evaluated using Prism 6.0 software. One-way ANOVA was performed with post hoc Tukey multiple comparison test or Student t test applied, as appropriate for comparisons between groups. A p value <0.05 was considered significant.

Mucosal immunization with the adjuvant CDG elicits protective immunity in the systemic and mucosal compartments (2530). However, how CDG promotes mucosal immunity is unknown. We set out to define the mechanisms by which CDG targets pulmonary DCs to generate lung mucosal immunity. Pulmonary DCs comprise three unique subsets: cDC1, cDC2, and moDCs (Fig. 1A). We have previously shown that moDCs promote lung mucosal TFH cells and IgA responses (25, 31). To further evaluate the role of moDCs on lung mucosal immunity, we used CCR2–/– mice, as CCR2-deficient mice have reduced numbers of moDCs in the lung at a steady state (Fig. 1B). Mice were immunized i.n. twice with CDG and the Ag (pneumococcal surface protein A) PspA at 2-wk intervals, and the immune responses were examined on day 28 (Fig. 1C). We found that CCR2–/– mice had unaltered anti-PspA IgG in the serum but lacked anti-PspA IgA in the bronchoalveolar lavage fluid (BALF) (Fig. 1D). We then examined memory TH1/2/17 responses in the spleen and lung from immunized CCR2–/– mice by the ex vivo recall. Again, CCR2–/– mice retained memory TH responses in the spleen but not in the lung (Fig. 1E, 1F). Thus, CDG-induced lung mucosal adjuvant responses can be uncoupled from systemic responses. moDCs may be specifically needed for promoting adjuvant responses in lung mucosa.

moDC activation is required for efficient CDG-induced immune responses (22). Lung moDCs require the NF-κB transcription factor RelA for optimal moDC activation (Supplemental Fig. 1A) (25). We generated the RelAfl/flCD11ccre mice to examine the role of moDCs activation in CDG adjuvanticity in the lung mucosa. RelAfl/flCD11ccre mice had no Ag-specific IgA in the BALF despite maintaining Ag-specific IgG production in the serum (Supplemental Fig. 1B, 1C). Similar to the CCR2–/– mice, RelAfl/flCD11ccre mice did not generate memory TH responses in the lung, despite having normal memory TH responses in the spleen (Supplemental Fig. 1D, 1E).

To further establish that moDCs were responsible for the lack of mucosal immune responses, we performed a monocyte adoptive cell transfer experiment. moDCs differentiate from Ly6Chi monocytes (32). We isolated bone marrow Ly6Chi monocytes from C57BL/6J mice and i.n. transferred the cells into RelAfl/flCD11ccre mice. The recipient RelAfl/flCD11ccre mice were then immunized with CDG/OVA. On day 14, the recipient RelAfl/flCD11ccre mice received a second dose of Ly6Chi monocytes from C57BL/6J mice and were subsequently immunized with CDG/OVA (Supplemental Fig. 1F). Lung mucosal immune responses were analyzed on day 28. RelAfl/flCD11ccre mice that received WT monocytes restored the lung memory TH responses and IgA production in the BALF (Supplemental Fig. 1G, 1H). Together, the data indicate that lung moDCs mediate CDG-induced lung mucosal–specific IgA and memory TH cells.

How do moDCs promote mucosal IgA and memory TH responses in the lung but is dispensable in the spleen? We hypothesized that moDCs might differentiate into a lung-specific subpopulation to induce lung mucosal vaccine responses.

We first examined moDCs in the lungs and mediastinal lymph nodes (medLNs) of WT mice following CDG/PspA immunization (Fig. 2A). We noticed that on day 14 postimmunization, the lungs contained a population of moDCs that expressed the transcription factor Bcl6 (Fig. 2B). This population was unique to the lung, as moDCs in the medLNs of CDG-immunized mice did not express Bcl6 (Fig. 2C). Bcl6 is upregulated in CXCR5+PD1+ TFH cell as well as in B cells prior to entry into the germinal center (33, 34). Bcl6 expression in lung moDCs is lower than those observed in lung Bcl6+ B cells and CXCR5+PD1+ T cells (Fig. 2D). Bcl6 controls the chemokine receptor CXCR5 expression. The new Bcl6+ moDCs express CXCR5+, but the expression is lower than those observed in lung Bcl6+ B cells and CXCR5+PD1+ T cells (Fig. 2D).

FIGURE 1.

CCR2–/– mice selectively lose CDG adjuvant responses in lung mucosa but not in the systemic compartments. (A) Gating strategy for lung DCs. Lung cDC1s are gated as MHCIIhiCD11c+CD11bCD64, moDCs are MHCIIhiCD11c+CD11b+CD64+, and cDC2 are MHCIIhiCD11c+CD11b+CD64. (B) Frequency of lung DCs at steady state in WT and CCR2–/– mice (n = 3 mice per group). Data are representative of four independent experiments. (C) Experimental design for the immunization of WT and CCR2–/– mice. Mice were immunized i.n. with two doses of PspA (1 µg) or PspA (1 µg) plus CDG (5 μg) at 2-wk intervals. (D) Anti-PspA IgG in serum (left) and IgA in BALF (right) were determined by ELISA 28 d postimmunization (n = 3 mice per group). Data are representative of three independent experiments. (E and F) Lung cells (E) or splenocytes (F) from immunized mice were stimulated with 5 μg/ml PspA for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. Graphs represent the mean with error bars indicating SEM. The p values are determined by unpaired Student t test (B) or one-way ANOVA Tukey multiple comparison test (E and F). *p < 0.05.

FIGURE 1.

CCR2–/– mice selectively lose CDG adjuvant responses in lung mucosa but not in the systemic compartments. (A) Gating strategy for lung DCs. Lung cDC1s are gated as MHCIIhiCD11c+CD11bCD64, moDCs are MHCIIhiCD11c+CD11b+CD64+, and cDC2 are MHCIIhiCD11c+CD11b+CD64. (B) Frequency of lung DCs at steady state in WT and CCR2–/– mice (n = 3 mice per group). Data are representative of four independent experiments. (C) Experimental design for the immunization of WT and CCR2–/– mice. Mice were immunized i.n. with two doses of PspA (1 µg) or PspA (1 µg) plus CDG (5 μg) at 2-wk intervals. (D) Anti-PspA IgG in serum (left) and IgA in BALF (right) were determined by ELISA 28 d postimmunization (n = 3 mice per group). Data are representative of three independent experiments. (E and F) Lung cells (E) or splenocytes (F) from immunized mice were stimulated with 5 μg/ml PspA for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. Graphs represent the mean with error bars indicating SEM. The p values are determined by unpaired Student t test (B) or one-way ANOVA Tukey multiple comparison test (E and F). *p < 0.05.

Close modal
FIGURE 2.

moDCs differentiate into lung-specific Bcl6+ moDCs on day 14 post–CDG immunization. (A) Experimental design for moDC characterization. C57BL/6 mice were immunized i.n. with CDG (5 µg) and OVA (2 µg). Lung moDCs were characterized by flow cytometry on day 14. (B) Flow cytometry analysis of Bcl6 expressing cells in the lung of mice immunized with CDG/OVA as in (A) on day 14. (n = 3 mice per group). Data are representative of two independent experiments. (C) Flow cytometry analysis of Bcl6 expressing DCs in the medLNs and lungs of WT mice immunized with CDG/OVA as in (A). Lungs were harvested on day 14. Data are representative of two independent experiments. (D) Bcl6 and CXCR5 expression on indicated cell populations in the lung of C57BL/6J mice on day 14 post–CDG/NP6CGG immunization. Non-TFH cells were gated as CD4+CXCR5PD1. TFH cells were gated as CD4+CXCR5+PD1+. Bcl6+ B cells were gated as CD19+Bcl6+ (n = 3 mice per group). Data are representative of three independent experiments. (E) Experimental design for monocyte adoptive transfer. Ly6chi monocytes were isolated from the bone marrow of CD45.1 mice. CD45.1+ monocytes (1.5 million cells) were i.n. transferred into C57BL/6J mice. Recipient WT mice were immunized with CDG (5 µg) and PspA (1 µg). Mice were harvested on day 14. (F) Flow cytometry analysis of CD45.1+ cells following monocyte adoptive transfer. On day 14, CD45.1+Bcl6+ moDCs were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. (G) Flow cytometry analysis of Bcl6 expression in AMs in the BALF of WT mice on day 14 post–CDG/OVA immunization (n = 3 mice per group). Data are representative of three independent experiments. (H–J) Flow cytometry analysis of the kinetics of pRelA+ (H), CD80+ (I), and Bcl6+ moDCs (J) from WT mice immunized with CDG (5 µg) and OVA (2 µg). Lungs were harvested at different time points (n = 3 mice per group). Data are representative of two independent experiments.

FIGURE 2.

moDCs differentiate into lung-specific Bcl6+ moDCs on day 14 post–CDG immunization. (A) Experimental design for moDC characterization. C57BL/6 mice were immunized i.n. with CDG (5 µg) and OVA (2 µg). Lung moDCs were characterized by flow cytometry on day 14. (B) Flow cytometry analysis of Bcl6 expressing cells in the lung of mice immunized with CDG/OVA as in (A) on day 14. (n = 3 mice per group). Data are representative of two independent experiments. (C) Flow cytometry analysis of Bcl6 expressing DCs in the medLNs and lungs of WT mice immunized with CDG/OVA as in (A). Lungs were harvested on day 14. Data are representative of two independent experiments. (D) Bcl6 and CXCR5 expression on indicated cell populations in the lung of C57BL/6J mice on day 14 post–CDG/NP6CGG immunization. Non-TFH cells were gated as CD4+CXCR5PD1. TFH cells were gated as CD4+CXCR5+PD1+. Bcl6+ B cells were gated as CD19+Bcl6+ (n = 3 mice per group). Data are representative of three independent experiments. (E) Experimental design for monocyte adoptive transfer. Ly6chi monocytes were isolated from the bone marrow of CD45.1 mice. CD45.1+ monocytes (1.5 million cells) were i.n. transferred into C57BL/6J mice. Recipient WT mice were immunized with CDG (5 µg) and PspA (1 µg). Mice were harvested on day 14. (F) Flow cytometry analysis of CD45.1+ cells following monocyte adoptive transfer. On day 14, CD45.1+Bcl6+ moDCs were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. (G) Flow cytometry analysis of Bcl6 expression in AMs in the BALF of WT mice on day 14 post–CDG/OVA immunization (n = 3 mice per group). Data are representative of three independent experiments. (H–J) Flow cytometry analysis of the kinetics of pRelA+ (H), CD80+ (I), and Bcl6+ moDCs (J) from WT mice immunized with CDG (5 µg) and OVA (2 µg). Lungs were harvested at different time points (n = 3 mice per group). Data are representative of two independent experiments.

Close modal

To confirm that the Bcl6+ moDCs were indeed monocyte-derived, we isolated Ly6Chi monocyte from CD45.1 mice. The monocytes were then i.n. transferred into C57BL/6J mouse. The recipient WT mice were immunized with CDG/PspA (Fig. 2E). On day 14 postimmunization, we identified CD45.1+ Bcl6+ moDCs (Fig. 2F). Thus, these Bcl6+ moDCs are indeed monocyte-derived. As alveolar macrophages (AMs) are also monocyte-derived, we examined AMs from the BALF of immunized mice. AMs did not express Bcl6 (Fig. 2G). Thus, the Bcl6+ monocyte-derived cells were not AMs.

Next, we examined the kinetics of moDCs differentiation in the lung during CDG immunization. The activation of moDCs, indicated by pRelA and CD80, peaked on days 6 and 9, respectively, postimmunization (Fig. 2H, 2I). Intriguingly, Bcl6+ moDCs differentiation had different kinetics from moDCs activation. Bcl6+ moDCs appeared early on day 2 and peaked on day 14, even when moDCs activation was waning (Fig. 2J). On day 14 postimmunization, ∼40% of lung moDCs expressed Bcl6 (Fig. 2J).

To understand the functional significance of Bcl6 expression in moDCs populations in vivo, we generated Bcl6fl/flLysMcre and Bcl6fl/flCD11ccre mice. Bcl6fl/flLysMcre mice delete Bcl6 in myeloid cells. The cDC population in the Bcl6fl/flLysMcre mice were unaltered (Fig. 3A, 3B). Bcl6fl/flLysMcre mice had expanded lung neutrophils and Ly6Chi monocytes populations (Fig. 3C), as previously reported (35). However, Bcl6fl/flLysMcre mice had reduced lung moDCs (Fig. 3A, 3B), similar to the CCR2–/– mice (Fig. 1B), suggesting that Bcl6 expression in myeloid cells is crucial for the differentiation of lung moDCs.

Next, Bcl6fl/flLysMcre mice were immunized with two doses of CDG/NP6CGG at 2-wk intervals. Immune memory responses were determined on day 120 (Fig. 3D). Bcl6fl/flLysMcre mice did not produce IgA in the BALF (Fig. 3E) or lung memory TH responses (Fig. 3G). Similar to the CCR2–/– mice, spleen memory TH responses and serum Ag-specific IgG in Bcl6fl/flLysMcre mice were comparable to the Bcl6fl/fl mice 4 mo post–CDG immunization (Fig. 3F, 3H).

FIGURE 3.

Bcl6 expression in LysM+ cells is required for lung moDCs development. (A and B) Flow cytometry analysis (A) and absolute number (B) of pulmonary DCs subsets in Bcl6fl/fl and Bcl6fl/flLysMcre mice at steady state (n = 3 mice per group). Data are representative of two independent experiments. (C) Flow cytometry analysis lung Ly6Chi monocytes and neutrophils in Bcl6fl/fl and Bcl6fl/flLysMcre mice at steady state (n = 3 mice per group). Data are representative of two independent experiments. (D) Experimental design for the immunization of Bcl6fl/fl and Bcl6fl/flLysMcre mice. Mice were immunized i.n. with two doses of NP6CGG (1 μg) or NP6CGG (1 μg) plus CDG (5 μg) at 2-wk intervals. Mice were then harvested on day 120 postimmunization. (E and F) Anti-NP6CGG IgG in serum (E) and BALF IgA (F) were determined by ELISA on day 120 postimmunization (n = 3 mice per group). Data are representative of three independent experiments. (G and H) Lung cells (G) and splenocytes (H) from immunized Bcl6fl/fl and Bcl6fl/flLysMcre mice were recalled with 5 μg/ml NP6CGG for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments (n = 3 mice per group). Graphs represent the mean with error bars indicating the SEM. The significance is determined by one-way ANOVA Tukey multiple comparison test (G) or unpaired Student t test (B). *p < 0.05, **p < 0.001.

FIGURE 3.

Bcl6 expression in LysM+ cells is required for lung moDCs development. (A and B) Flow cytometry analysis (A) and absolute number (B) of pulmonary DCs subsets in Bcl6fl/fl and Bcl6fl/flLysMcre mice at steady state (n = 3 mice per group). Data are representative of two independent experiments. (C) Flow cytometry analysis lung Ly6Chi monocytes and neutrophils in Bcl6fl/fl and Bcl6fl/flLysMcre mice at steady state (n = 3 mice per group). Data are representative of two independent experiments. (D) Experimental design for the immunization of Bcl6fl/fl and Bcl6fl/flLysMcre mice. Mice were immunized i.n. with two doses of NP6CGG (1 μg) or NP6CGG (1 μg) plus CDG (5 μg) at 2-wk intervals. Mice were then harvested on day 120 postimmunization. (E and F) Anti-NP6CGG IgG in serum (E) and BALF IgA (F) were determined by ELISA on day 120 postimmunization (n = 3 mice per group). Data are representative of three independent experiments. (G and H) Lung cells (G) and splenocytes (H) from immunized Bcl6fl/fl and Bcl6fl/flLysMcre mice were recalled with 5 μg/ml NP6CGG for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments (n = 3 mice per group). Graphs represent the mean with error bars indicating the SEM. The significance is determined by one-way ANOVA Tukey multiple comparison test (G) or unpaired Student t test (B). *p < 0.05, **p < 0.001.

Close modal

As monocytes develop into moDCs, the expression of CD11c is increased. To bypass the influence of Bcl6 on lung moDCs development, we generated Bcl6fl/flCD11ccre mice to ablate Bcl6 after moDCs have developed. Different from the Bcl6fl/flLysMcre mice, Bcl6fl/flCD11ccre mice had increased numbers of lung moDCs at steady state (Fig. 4A, 4B).

Bcl6fl/flCD11ccre mice will delete Bcl6 gene in CD11c+ cells, including cDCs and AMs. Watchmaker, et al. (36) first showed that CD103+/CD8+ cDC1, not Sirpα+IRF4+ cDC2, from gastrointestinal tract and spleen specifically express Bcl6, which is important for cDC1 development. Later, Zhang, et.al., found that Bcl6 protein expression is elevated in peripheral precDCs, especially cDC1 (37). We observed that Bcl6fl/flCD11ccre mice lack cDC1s in the lung (Fig. 4A, 4B), confirming that Bcl6 expression in DCs is required for lung cDC1 development. The numbers of lung cDC2 also reduced in Bcl6fl/flCD11ccre mice, although was not statistically significant (Fig. 4B).

FIGURE 4.

Bcl6 expression in CD11c+ cells is dispensable for CDG-induced systemic vaccine responses. (A and B) Flow cytometry analysis (A) and absolute number (B) of pulmonary DCs subsets in Bcl6fl/fl and Bcl6fl/flCD11ccre mice at steady state (n = 3 mice per group). Data are representative of three independent experiments. (C and D) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were i.n. treated with CDG (5 μg) or PBS for 16 h. CD86 and CCR7 expression on lung cDC2 were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. (E) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG/H1N1-NP twice at the 2-wk interval. Serum anti–H1N1-NP IgG was determined by ELISA on day 90 postimmunization (n = 3 mice per group). Data are representative of two independent experiments. (F) Lung cells from immunized Bcl6fl/fl and Bcl6fl/flCD11ccre mice were recalled with 5 μg/ml H1N1-NP for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of two independent experiments. (G) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG/OVA or OVA. Bcl6 expression in lung moDCs were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. Graphs represent mean ± SE. The significance is determined by unpaired Student t test (B) or one-way ANOVA Tukey multiple comparisons (F). *p < 0.05, **p < 0.001.

FIGURE 4.

Bcl6 expression in CD11c+ cells is dispensable for CDG-induced systemic vaccine responses. (A and B) Flow cytometry analysis (A) and absolute number (B) of pulmonary DCs subsets in Bcl6fl/fl and Bcl6fl/flCD11ccre mice at steady state (n = 3 mice per group). Data are representative of three independent experiments. (C and D) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were i.n. treated with CDG (5 μg) or PBS for 16 h. CD86 and CCR7 expression on lung cDC2 were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. (E) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG/H1N1-NP twice at the 2-wk interval. Serum anti–H1N1-NP IgG was determined by ELISA on day 90 postimmunization (n = 3 mice per group). Data are representative of two independent experiments. (F) Lung cells from immunized Bcl6fl/fl and Bcl6fl/flCD11ccre mice were recalled with 5 μg/ml H1N1-NP for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of two independent experiments. (G) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG/OVA or OVA. Bcl6 expression in lung moDCs were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. Graphs represent mean ± SE. The significance is determined by unpaired Student t test (B) or one-way ANOVA Tukey multiple comparisons (F). *p < 0.05, **p < 0.001.

Close modal
FIGURE 5.

Bcl6 expression in moDCs is specifically required for CDG-induced lung memory TH response. (A) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG (5 µg) and PspA (2 µg). BALF anti-PspA IgA was determined on day 14 by ELISA (n = 3 mice per group). Data are representative of three independent experiments. (B) Lung cells from immunized mice (A) were recalled with 5 μg/ml PspA for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. (C) Flow cytometry plots (left) and frequency (right) of CD4+CD69+CD49a+ TRM cells in immunized Bcl6fl/fl and Bcl6fl/flCD11ccre mice from (A). Data are representative of three independent experiments. (D) Experimental design for moDC adoptive transfer. moDCs were isolated from the lungs of naive WT mice. moDCs were (∼35,000 cells) were i.n. transferred into Bcl6fl/flCD11ccre mice. Recipient Bcl6fl/flCD11ccre mice were immunized with CDG (5 µg) and NP6CGG (1 µg). Mice were harvested on day 14. (E) Lung cells from immunized mice (D) were recalled with 5 μg/ml NP6CGG for 4 d in culture. Cytokines were measured in the supernatant by ELISA (n = 3 mice per group). (F) Flow cytometry analysis (left) and frequency (right) of CD4+CD69+CD49a+ TRM cells following adoptive transfer in (E). Data are representative of two independent experiments. Graphs represent means ± SEM. The significance is determined by unpaired Student t test (B and C) or one-way ANOVA Tukey multiple comparison test (E and F). *p < 0.05, **p < 0.001.

FIGURE 5.

Bcl6 expression in moDCs is specifically required for CDG-induced lung memory TH response. (A) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG (5 µg) and PspA (2 µg). BALF anti-PspA IgA was determined on day 14 by ELISA (n = 3 mice per group). Data are representative of three independent experiments. (B) Lung cells from immunized mice (A) were recalled with 5 μg/ml PspA for 4 d in culture. Cytokines were measured in the supernatant by ELISA. Data are representative of three independent experiments. (C) Flow cytometry plots (left) and frequency (right) of CD4+CD69+CD49a+ TRM cells in immunized Bcl6fl/fl and Bcl6fl/flCD11ccre mice from (A). Data are representative of three independent experiments. (D) Experimental design for moDC adoptive transfer. moDCs were isolated from the lungs of naive WT mice. moDCs were (∼35,000 cells) were i.n. transferred into Bcl6fl/flCD11ccre mice. Recipient Bcl6fl/flCD11ccre mice were immunized with CDG (5 µg) and NP6CGG (1 µg). Mice were harvested on day 14. (E) Lung cells from immunized mice (D) were recalled with 5 μg/ml NP6CGG for 4 d in culture. Cytokines were measured in the supernatant by ELISA (n = 3 mice per group). (F) Flow cytometry analysis (left) and frequency (right) of CD4+CD69+CD49a+ TRM cells following adoptive transfer in (E). Data are representative of two independent experiments. Graphs represent means ± SEM. The significance is determined by unpaired Student t test (B and C) or one-way ANOVA Tukey multiple comparison test (E and F). *p < 0.05, **p < 0.001.

Close modal

cDC2 are essential for CDG mucosal adjuvanticity whereas cDC1 are dispensable (25). We examined the function of lung cDC2 in the Bcl6fl/flCD11ccre mice. Mice were treated (i.n.) with CDG for 16 h. Lung cDC2 activation was determined by CD86 and CCR7 expression. CD86 and CCR7 expression on CDG-activated lung cDC2 were comparable between Bcl6fl/fl and Bcl6fl/flCD11ccre mice (Fig. 4C, 4D). However, cDC2 from the Bcl6fl/flCD11ccre mice had elevated basal CD86 and CCR7 expression (Fig. 4C, 4D). To further determine the function of cDC2 in Bcl6fl/flCD11ccre mice, we measured CDG adjuvanticity in the systemic compartments. Three months after CDG immunization, Bcl6fl/flCD11ccre mice had unaltered Ag-specific serum IgG (Fig. 4E) and memory TH responses in the spleen (Fig. 4F). Intracellular Bcl6 stain confirmed the lack of Bcl6 upregulation in lung moDCs from Bcl6fl/flCD11ccre (Fig. 4G). The phenotypes of the conditional Bcl6 mice were summarized in Table I.

Table I.

Lung DC subsets and CDG vaccine responses in various genetically modified mouse strains

Mouse StrainsLung moDCsLung cDC1Lung cDC2Lung Mucosal CDG ResponseSystemic CDG Response
CCR2–/– Reduced in numbers Normal Normal No IgANo memory TH cells Normal 
Batf3–/– 25 Normal Depleted Normal Normal Normal 
IRF4fl/flCD11ccre 25 Normal Normal Depleted No IgANo Memory TH cells No serum IgG No spleen
memory TH cells 
RelAfl/flCD11ccre No activation by CDG Normal Normal No IgANo memory TH cells Normal 
Bcl6fl/flCD11ccre Increased in numbers Depleted Constitutively activated Has IgANo memory TH cells Normal 
Bcl6fl/flLysMcre Reduced in numbers Normal Normal No IgANo memory TH cells Normal 
Mouse StrainsLung moDCsLung cDC1Lung cDC2Lung Mucosal CDG ResponseSystemic CDG Response
CCR2–/– Reduced in numbers Normal Normal No IgANo memory TH cells Normal 
Batf3–/– 25 Normal Depleted Normal Normal Normal 
IRF4fl/flCD11ccre 25 Normal Normal Depleted No IgANo Memory TH cells No serum IgG No spleen
memory TH cells 
RelAfl/flCD11ccre No activation by CDG Normal Normal No IgANo memory TH cells Normal 
Bcl6fl/flCD11ccre Increased in numbers Depleted Constitutively activated Has IgANo memory TH cells Normal 
Bcl6fl/flLysMcre Reduced in numbers Normal Normal No IgANo memory TH cells Normal 

Examining lung moDCs, cDC1, and cDC2 populations and their responses to CDG adjuvant in vivo.

Next, Bcl6fl/flCD11ccre mice were immunized with CDG/PspA and the vaccine responses were examined 14 d later. Unlike Bcl6fl/flLysMcre mice, the Bcl6fl/flCD11ccre mice had unaltered lung IgA (Fig. 5A). However, Bcl6fl/flCD11ccre mice did not generate lung memory lung TH responses (Fig. 5B). We examined CD4 TRM cells, as these cells are a unique population of lung-specific memory T cells. CD4+ TRM cells were characterized based on the expression of CD69 and CD49a. Bcl6fl/flCD11ccre mice did not generate CD4+ TRM cells compared with the Bcl6f/f mice following immunization (Fig. 5C). Thus, CDG induces lung memory TH responses but not IgA in Bcl6fl/flCD11ccre mice.

To further demonstrate that Bcl6 expression in moDCs, not other CD11c+ cells, are responsible for the loss of lung memory TH responses in Bcl6fl/flCD11ccre mice, we adoptively transferred WT moDCs into Bcl6fl/flCD11ccre mice (Fig. 5D). We sorted out lung moDCs frInaive C57BL/6 mouse and i.n. transferred the moDCs into Bcl6fl/flCD11ccre mice. Recipient Bcl6fl/flCD11ccre mice were immunized with CDG/NP6CGG. We examined lung memory TH responses on day 14 postimmunization. WT moDCs restored memory TH1 responses and lung CD4+ TRM cells in the immunized Bcl6fl/flCD11ccre (Fig. 5E, 5F). Together, the data indicate that Bcl6 expression in moDCs is required for generating lung memory TH responses, but not IgA.

To further understand the role of Bcl6 expression in moDCs, we examined the ability of Bcl6+ moDCs to stimulate T cells. We first asked if lung Bcl6+ DCs were able to present Ag at the effector stage (day 14) in vivo. We immunized C57BL/6J mice with CDG plus a fusion protein of Eα peptide (aa52–68) with OVA (Eα-OVA) (Fig. 6A). The YAE mAb will detect cells that express the Eα peptide on I-Ab of MHCII. On day 14 postimmunization, Bcl6+ moDCs were able to present the Eα peptide on MHCII (Fig. 6B). Furthermore, the YAE+Bcl6+ moDCs were activated and marked by CD86 expression (Fig. 6B). Thus, Bcl6+ moDCs were mature APCs and were capable of activating T cells.

We next examined the ability of Bcl6+ moDCs to produce T cell–promoting cytokines (the Signal 3). We immunized C57BL/6J mice with CDG/OVA and characterized the moDCs on day 14. We measured the production of prototypic TH1 (IL-12p70)-, TH2 (IL-4)-, and TH17 (IL-23)-promoting cytokines by moDCs (Fig. 6C–E). Bcl6+ moDCs were proficient at producing all these cytokines. Besides, Bcl6+ moDCs were capable of producing IL-10 and TGF-β1, two cytokines that are critical for the induction of TRM cells (Fig. 6F, 6G) (38, 39). Together, the data indicate that the lung-specific Bcl6+ moDCs are mature on day 14 postimmunization and may promote TH responses in the lung.

FIGURE 6.

Lung Bcl6+ moDCs are mature DCs producing T cell–promoting cytokines on day 14 post–CDG immunization. (A) Experimental design for moDC characterization. C57BL/6 mice were immunized i.n. with CDG (5 µg) and Eα-OVA (1 µg). Control C57BL/6 mice were immunized i.n. with Eα-OVA (1 µg). Lung moDCs were characterized by flow cytometry on day 14. (B) WT mice were immunized with Eα-OVA or Eα-OVA/CDG (5 µg). Cells that presented Eα on I-Ab of MHCII were determined with the YAE mAb by flow cytometry in the lung on day 14 (n = 3 mice per group). Data are representative of three independent experiments. (C–G) Flow cytometry analysis of IL-12p70 (C), IL-4 (D), IL-23 (E), IL-10 (F), and TGF-β1 (G) production by lung moDCs in C57BL/6J mice on day 14 post–CDG/OVA immunization (n = 3 mice per group). Data are representative of two independent experiments. (H) Analysis of IL-21+ cells in the lungs of IL-21–VFP reporter mice on day 14 postimmunization with CDG/OVA (i.n.) (n = 3 mice per group). Data are representative of two independent experiments. (I) Flow cytometry analysis of CXCL13+ lung moDCs in C57BL/6J mice on day 14 postimmunization (i.n.) with CDG/OVA (n = 3 mice per group). Data are representative of two independent experiments.

FIGURE 6.

Lung Bcl6+ moDCs are mature DCs producing T cell–promoting cytokines on day 14 post–CDG immunization. (A) Experimental design for moDC characterization. C57BL/6 mice were immunized i.n. with CDG (5 µg) and Eα-OVA (1 µg). Control C57BL/6 mice were immunized i.n. with Eα-OVA (1 µg). Lung moDCs were characterized by flow cytometry on day 14. (B) WT mice were immunized with Eα-OVA or Eα-OVA/CDG (5 µg). Cells that presented Eα on I-Ab of MHCII were determined with the YAE mAb by flow cytometry in the lung on day 14 (n = 3 mice per group). Data are representative of three independent experiments. (C–G) Flow cytometry analysis of IL-12p70 (C), IL-4 (D), IL-23 (E), IL-10 (F), and TGF-β1 (G) production by lung moDCs in C57BL/6J mice on day 14 post–CDG/OVA immunization (n = 3 mice per group). Data are representative of two independent experiments. (H) Analysis of IL-21+ cells in the lungs of IL-21–VFP reporter mice on day 14 postimmunization with CDG/OVA (i.n.) (n = 3 mice per group). Data are representative of two independent experiments. (I) Flow cytometry analysis of CXCL13+ lung moDCs in C57BL/6J mice on day 14 postimmunization (i.n.) with CDG/OVA (n = 3 mice per group). Data are representative of two independent experiments.

Close modal
FIGURE 7.

Lung moDCs in Bcl6fl/flCD11ccre mice are defective in producing TH cell–polarizing cytokines and chemokines. (A–E) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG (5 µg) and NP6CGG (1 µg). Mice were harvested on day 14. Flow cytometry analysis and frequency of TGF-β1 (A), IL-12p70 (B), IL-4 (C), IL-23 (D), and CCL20 (E) production by lung moDCs from Bcl6fl/fl and Bcl6fl/flCD11ccre mice on day 14 post–CDG/NP6CGG immunization (n = 3 mice per group). Data are representative of two independent experiments. Graphs represent mean ± SEM. The significance is determined by the unpaired Student t test. * p < 0.05, **p < 0.001.

FIGURE 7.

Lung moDCs in Bcl6fl/flCD11ccre mice are defective in producing TH cell–polarizing cytokines and chemokines. (A–E) Bcl6fl/fl and Bcl6fl/flCD11ccre mice were immunized (i.n.) with CDG (5 µg) and NP6CGG (1 µg). Mice were harvested on day 14. Flow cytometry analysis and frequency of TGF-β1 (A), IL-12p70 (B), IL-4 (C), IL-23 (D), and CCL20 (E) production by lung moDCs from Bcl6fl/fl and Bcl6fl/flCD11ccre mice on day 14 post–CDG/NP6CGG immunization (n = 3 mice per group). Data are representative of two independent experiments. Graphs represent mean ± SEM. The significance is determined by the unpaired Student t test. * p < 0.05, **p < 0.001.

Close modal

IL-21 is a major TFH cell–inducing cytokine. Using an IL-21 reporter mouse, we found that lung moDCs were the main IL-21–producing lung DCs in the lung on day 14 postimmunization (Fig. 6H). However, the IL-21+ moDCs were Bcl6 moDCs (Fig. 6H). CXCL13 is essential for germinal center formation. We found that on day 14 postimmunization, moDCs produced CXCL13 (Fig. 6I). However, most CXCL13+ moDCs were Bcl6 moDCs (Fig. 6I). Thus, the lung-specific Bcl6+ moDCs do not produce IL-21 or CXCL13.

TGF-β1 are critical cytokines for the induction of TRM cells (38, 39). We found that on day 14 postimmunization, lung moDCs from immunized Bcl6fl/flCD11ccre mice had decreased production of TGF-β1 (Fig. 7A). Lung moDCs from immunized Bcl6fl/flCD11ccre mice also produced less TH-promoting cytokines IL-12p70, IL-4, and IL-23 (Fig. 7B–D). CCL20 (MIP3A), the CCR6 ligand, recruits lymphocytes and DCs to mucosal lymphoid tissues and is critical for inducing mucosal immune responses (40). We found that CCL20 production by lung moDCs was also reduced in CDG-immunized Bcl6fl/flCD11ccre mice on day 14 (Fig. 7E). Thus, Bcl6 expression in moDCs is required for the production of T cell–promoting cytokines and chemokines.

The Bcl6+ moDCs produce TH cell– and TRM cell–promoting cytokines, chemokines and are likely responsible for lung memory TH responses. How does CDG induce the differentiation of lung Bcl6+ moDCs in vivo? We previously showed that the lung TNFR2+ cDC2 population induced lung memory TH responses (Fig. 8A) (25). Furthermore, moDCs did not take up i.n. administered CDG (25). We hypothesized that the CDG directly activated TNFR2+ cDC2, which drives moDCs differentiation into Bcl6+ moDCs.

IRF4fl/flCD11ccre mice lack the cDC2 population and do not respond to CDG immunization (25). We performed an adoptive cell transfer to identify whether TNFR2+ cDC2 drive moDC differentiation in IRF4fl/flCD11ccre mice. Lung TNFR2+ cDC2 were sorted from WT mice and transferred i.n. into IRF4fl/flCD11ccre mice (Fig. 8B). Recipient IRF4fl/flCD11ccre mice were immunized with CDG/PspA. moDCs were characterized on day 14. IRF4fl/flCD11ccre that received no cells did not generate Bcl6+ moDCs (Fig. 8C). In contrast, IRF4fl/flCD11ccre received WT TNFR2+ cDC2 population had lung Bcl6+ moDCs (Fig. 8C).

FIGURE 8.

TNFR2+ cDC2 produce sTNF to generate lung Bcl6+ moDCs in vivo. (A) Gating strategy for the identification of TNFR2+ cDC2 in the lung of naive WT mice. (B) Experimental design for TNFR2+ cDC2 adoptive transfer. Lung TNFR2+ cDC2 were isolated from the lungs of naive WT mice. TNFR2+ cDC2 (∼50,000 cells) were i.n. transferred into IRF4fl/flCD11ccre mice. Recipient IRF4fl/flCD11ccre mice were immunized with CDG (5 µg) and PspA (1 µg). Mice were harvested on day 14. (C) Flow cytometry analysis of Bcl6 expression on moDCs in IRF4fl/flCD11ccre mice receiving TNFR2+ cDC2 (n = 3 mice per group). Data are representative of two independent experiments. (D) WT mice were treated (i.n.) with 200 ng of recombinant TNF and PspA or PspA alone. On day 14, Bcl6+ moDCs in the lung were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. (E) C57BL/6J mice were administered (i.n.) with CDG/APC-mouse IgG2a (clone: MOPC-173) or CDG/APC only. Flow cytometry analysis of APC+ cells were done in the lungs 16 h posttreatment (n = 3 mice per group). Data are representative of three independent experiments. (F) Cartoon illustrating the sTNF-Fc (IgG2A) and tmTNF-Fc (IgG2A) fusion proteins. (G) IRF4fl/flCD11ccre mice were immunized (i.n.) with CDG/NP6CGG and 100ng tmTNF-Fc (IgG2A) or 100ng sTNF-Fc (IgG2A). Lungs were harvested on day 14. (H) Flow cytometry analysis of Bcl6, CCL20, and CXCL13 expression by lung moDCs from immunized mice. (n = 3 mice per group). Data are representative of three independent experiments.

FIGURE 8.

TNFR2+ cDC2 produce sTNF to generate lung Bcl6+ moDCs in vivo. (A) Gating strategy for the identification of TNFR2+ cDC2 in the lung of naive WT mice. (B) Experimental design for TNFR2+ cDC2 adoptive transfer. Lung TNFR2+ cDC2 were isolated from the lungs of naive WT mice. TNFR2+ cDC2 (∼50,000 cells) were i.n. transferred into IRF4fl/flCD11ccre mice. Recipient IRF4fl/flCD11ccre mice were immunized with CDG (5 µg) and PspA (1 µg). Mice were harvested on day 14. (C) Flow cytometry analysis of Bcl6 expression on moDCs in IRF4fl/flCD11ccre mice receiving TNFR2+ cDC2 (n = 3 mice per group). Data are representative of two independent experiments. (D) WT mice were treated (i.n.) with 200 ng of recombinant TNF and PspA or PspA alone. On day 14, Bcl6+ moDCs in the lung were determined by flow cytometry (n = 3 mice per group). Data are representative of two independent experiments. (E) C57BL/6J mice were administered (i.n.) with CDG/APC-mouse IgG2a (clone: MOPC-173) or CDG/APC only. Flow cytometry analysis of APC+ cells were done in the lungs 16 h posttreatment (n = 3 mice per group). Data are representative of three independent experiments. (F) Cartoon illustrating the sTNF-Fc (IgG2A) and tmTNF-Fc (IgG2A) fusion proteins. (G) IRF4fl/flCD11ccre mice were immunized (i.n.) with CDG/NP6CGG and 100ng tmTNF-Fc (IgG2A) or 100ng sTNF-Fc (IgG2A). Lungs were harvested on day 14. (H) Flow cytometry analysis of Bcl6, CCL20, and CXCL13 expression by lung moDCs from immunized mice. (n = 3 mice per group). Data are representative of three independent experiments.

Close modal

How does the lung TNFR2+ cDC2 population stimulate the differentiation of Bcl6+ moDCs in the lung? We previously discovered that TNF is essential for CDG mucosal adjuvant activity (26). cDC2 is the main source for i.n. CDG-induced lung TNF (25). We hypothesize that TNFR2+ cDC2 cells promote the differentiation of Bcl6+ moDCs via TNF secretion. Indeed, the i.n. administration of recombinant soluble TNF (sTNF)/PspA-induced lung Bcl6+ moDCs in C57BL/6J mice on day 14 postimmunization (Fig. 8D).

To demonstrate that lung TNF acts on moDCs directly to promote the differentiation of Bcl6+ moDCs, we generated moDCs-targeting TNF fusion proteins. moDCs express the high-affinity FcR, FcγRI, also known as CD64 that is not found on cDCs or lymphocytes (41). FcγRI binds the Fc of IgG2a with the highest affinity (108 M–1), more than 1,000-fold higher than its next binding partner IgG2b-Fc (42). Indeed, i.n. administration of APC-conjugated mouse IgG2A was taken up exclusively by CD64+ lung cells, including MHCIIhi CD64+ moDCs (Fig. 8E). The CD64+ MHCIIlow/int macrophages were also targeted. However, macrophages are dispensable for CDG mucosal adjuvanticity in vivo (25, 29). We thus generated a fusion protein TNF-Fc (IgG2A) to target TNF to moDCs (Fig. 8F).

CDG immunization generates sTNF and transmembrane TNF (tmTNF) (25). We fused TNF with the Fc portion of IgG2A to generate TNF-Fc (IgG2A), which represents sTNF. We also made a TNFD221N/A223R-Fc (IgG2A) fusion protein that targets tmTNF to moDCs. The TNFD221N/A223R mutant mimics tmTNF that binds only to TNFR2, not TNFR1 (43).

We used IRF4fl/flCD11ccre as CDG does not generate lung TNF in the IRF4fl/flCD11ccre (25), which facilitates the TNF-Fc (IgG 2A) complement experiment. We immunized IRF4fl/flCD11ccre mice with CDG/NP6CGG with tmTNF or sTNF (Fig. 8G). On day 14, we examined lung moDCs (Fig. 8G). The addition of sTNF, not mTNF, generated Bcl6+ and CCL20+ moDCs in the lung (Fig. 8H). In contrast, the addition of mTNF generated CXCL13+ moDCs (Fig. 8H). We propose that sTNF directly induces moDCs differentiation into Bcl6+ moDCs.

moDCs have a universal presence at the site of inflammation and can promote CD4+ and CD8+ T cell responses in vivo. Yet, we know very little about how moDCs achieve this plasticity in vivo. Consequently, we have few approaches to control this common and versatile APC population in vivo during inflammation. The most exciting discoveries in this report were the identification of Bcl6+ moDCs responsible for lung memory TH in vivo and a potential method to induce Bcl6+ moDC in vivo. Indeed, we recently reported that moDCs-targeting TNF fusion proteins enhanced CDG adjuvanticity in 2-y-old mice (44).

We used the mucosal adjuvant CDG system to study moDCs in vivo. CDG activates mainly the STING pathway (26) and does not induce lung damage (29). Its simplicity uncouples the lung mucosal versus systemic, lung memory TH versus lung IgA vaccine responses, thus facilitates the extraction of the underlying mechanisms. Lung TNF generates Bcl6+ moDCs. TNF is a hallmark of inflammation. We propose that Bcl6+ moDCs exist at the site of all inflammation and may play a critical role in driving TH cell–mediated inflammatory responses.

The role of Bcl6 in DCs is not well understood. Ohtsuka et al. (45) first reported that cDCs in the spleen requires Bcl6 expression for development in total Bcl6 knockout mice. However, Watchmaker, et al. showed that Bcl6 was the transcriptional factor specifically controlling cDC1 development in the gut, lymph nodes, and spleen (36). Zhang, et al. also found that cDC1 have the highest Bcl6 expression (37). In this study, we found that the Bcl6f/fCD11ccre mice lack cDC1 in the lung but retain functional cDC2 confirming that Bcl6 is specifically required for cDC1 differentiation. We also found that Bcl6f/fLysMcre had reduced moDCs similar to the CCR2–/– mice. Future study is needed to understand the mechanism by which Bcl6 controls cDC1 and moDCs differentiation.

How do Bcl6+ moDCs promote the induction of lung CD4+ memory T cells? Lung CD4+ T cell memory formation depends on the re-engagement of CD4+ T cells with APC at their effector stage (Signal 4) (9, 11). The lung Bcl6+ moDCs are a differentiated moDCs population that appears in the lung after the initial priming and peaked on day 14. We propose that the differentiated Bcl6+ moDCs are the long-thought lung APCs that provide the “Signal 4” during the effector stage to induce lung memory CD4+ T cells. Indeed, lung Bcl6+ moDCs bore Ags and expressed coactivators on day 14 postimmunization.

The recall response is a hallmark of immune memory cells, which establishes Ag-specificity and distinguishes TH1, TH2, and TH17 memory CD4+ T cells. We used lung ex vivo recall assays to determine the total lung CD4+ memory T cells, including memory TH1, TH2, and TH17 cells induced by immunization. We reasoned that although the lung memory TH responses are mainly from lung CD4+ TRM cells, effector memory T cells, or other unknown memory CD4 T cells in the lung may also contribute to the total lung memory TH responses (46). Furthermore, growing evidence suggests that TRM cells, even at the same tissue site (e.g., lung), are heterogeneous (2). Our goal is to generate Ag-specific protection against lung infections for which all memory CD4+ T cells in the lung contribute. We believe measuring total lung memory TH1/2/17 cells by ex vivo lung recall is a better indicator for the vaccine efficacy in lung mucosa than enumerating total cell numbers of lung CD69+CD49a+ CD4 TRM cells.

In summary, moDCs can differentiate into the new Bcl6+ lung moDCs to promote memory CD4+ TH cells in the lung mucosa. Targeted activation of moDCs by sTNF fusion protein could be a valuable method to enhance vaccine responses in vulnerable populations (e.g., the elderly) (44).

We thank the Center for Immunology and Transplantation at the University of Florida for the assistance.

This work was supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health Grants AI110606. AI125999, and AI132865 (to L.J.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

AM

alveolar macrophage

BALF

bronchoalveolar lavage fluid

cDC

conventional DC

CDG

cyclic di-GMP

DC

dendritic cel

i.n.

intranasally

medLN

mediastinal lymph node

MHCII

MHC class II

moDC

monocyte-derived DC

precDC

preconventional DC

sTNF

soluble TNF

TFH

T follicular helper

tmTNF

transmembrane TNF

TRM

tissue-resident memory T.

1
Sathaliyawala
,
T.
,
M.
Kubota
,
N.
Yudanin
,
D.
Turner
,
P.
Camp
,
J. J.
Thome
,
K. L.
Bickham
,
H.
Lerner
,
M.
Goldstein
,
M.
Sykes
, et al
.
2013
.
Distribution and compartmentalization of human circulating and tissue-resident memory T cell subsets.
Immunity
38
:
187
197
.
2
Mueller
,
S. N.
,
L. K.
Mackay
.
2015
.
Tissue-resident memory T cells: local specialists in immune defence.
Nat. Rev. Immunol.
16
:
79
89
.
3
Teijaro
,
J. R.
,
D.
Turner
,
Q.
Pham
,
E. J.
Wherry
,
L.
Lefrançois
,
D. L.
Farber
.
2011
.
Cutting edge: Tissue-retentive lung memory CD4 T cells mediate optimal protection to respiratory virus infection.
J. Immunol.
187
:
5510
5514
.
4
Smith
,
N. M.
,
G. A.
Wasserman
,
F. T.
Coleman
,
K. L.
Hilliard
,
K.
Yamamoto
,
E.
Lipsitz
,
R.
Malley
,
H.
Dooms
,
M. R.
Jones
,
L. J.
Quinton
,
J. P.
Mizgerd
.
2017
.
Regionally compartmentalized resident memory T cells mediate naturally acquired protection against pneumococcal pneumonia.
Mucosal Immunol.
11
:
220
235
.
5
McKinstry
,
K. K.
,
T. M.
Strutt
,
Y.
Kuang
,
D. M.
Brown
,
S.
Sell
,
R. W.
Dutton
,
S. L.
Swain
.
2012
.
Memory CD4+ T cells protect against influenza through multiple synergizing mechanisms.
J. Clin. Invest.
122
:
2847
2856
.
6
Krawczyk
,
C. M.
,
H.
Shen
,
E. J.
Pearce
.
2007
.
Memory CD4 T cells enhance primary CD8 T-cell responses.
Infect. Immun.
75
:
3556
3560
.
7
Strutt
,
T. M.
,
K. K.
McKinstry
,
J. P.
Dibble
,
C.
Winchell
,
Y.
Kuang
,
J. D.
Curtis
,
G.
Huston
,
R. W.
Dutton
,
S. L.
Swain
.
2010
.
Memory CD4+ T cells induce innate responses independently of pathogen.
Nat. Med.
16
:
558
564
.
8
McKinstry
,
K. K.
,
T. M.
Strutt
,
S. L.
Swain
.
2010
.
The potential of CD4 T-cell memory.
Immunology
130
:
1
9
.
9
Bautista
,
B. L.
,
P.
Devarajan
,
K. K.
McKinstry
,
T. M.
Strutt
,
A. M.
Vong
,
M. C.
Jones
,
Y.
Kuang
,
D.
Mott
,
S. L.
Swain
.
2016
.
Short-lived antigen recognition but not viral infection at a defined checkpoint programs effector CD4 T cells to become protective memory.
J. Immunol.
197
:
3936
3949
.
10
McMaster
,
S. R.
,
A. N.
Wein
,
P. R.
Dunbar
,
S. L.
Hayward
,
E. K.
Cartwright
,
T. L.
Denning
,
J. E.
Kohlmeier
.
2018
.
Pulmonary antigen encounter regulates the establishment of tissue-resident CD8 memory T cells in the lung airways and parenchyma.
Mucosal Immunol.
11
:
1071
1078
.
11
McKinstry
,
K. K.
,
T. M.
Strutt
,
B.
Bautista
,
W.
Zhang
,
Y.
Kuang
,
A. M.
Cooper
,
S. L.
Swain
.
2014
.
Effector CD4 T-cell transition to memory requires late cognate interactions that induce autocrine IL-2.
Nat. Commun.
5
:
5377
.
12
Haddadi
,
S.
,
M.
Vaseghi-Shanjani
,
Y.
Yao
,
S.
Afkhami
,
M. R.
D’Agostino
,
A.
Zganiacz
,
M.
Jeyanathan
,
Z.
Xing
.
2019
.
Mucosal-pull induction of lung-resident memory CD8 T cells in parenteral TB vaccine-primed hosts requires cognate antigens and CD4 T cells.
Front. Immunol.
10
:
2075
.
13
Mackay
,
L. K.
,
A. T.
Stock
,
J. Z.
Ma
,
C. M.
Jones
,
S. J.
Kent
,
S. N.
Mueller
,
W. R.
Heath
,
F. R.
Carbone
,
T.
Gebhardt
.
2012
.
Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation.
Proc. Natl. Acad. Sci. USA
109
:
7037
7042
.
14
Casey
,
K. A.
,
K. A.
Fraser
,
J. M.
Schenkel
,
A.
Moran
,
M. C.
Abt
,
L. K.
Beura
,
P. J.
Lucas
,
D.
Artis
,
E. J.
Wherry
,
K.
Hogquist
, et al
.
2012
.
Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues.
J. Immunol.
188
:
4866
4875
.
15
Onai
,
N.
,
A.
Obata-Onai
,
M. A.
Schmid
,
M. G.
Manz
.
2007
.
Flt3 in regulation of type I interferon-producing cell and dendritic cell development.
Ann. N. Y. Acad. Sci.
1106
:
253
261
.
16
Desch
,
A. N.
,
G. J.
Randolph
,
K.
Murphy
,
E. L.
Gautier
,
R. M.
Kedl
,
M. H.
Lahoud
,
I.
Caminschi
,
K.
Shortman
,
P. M.
Henson
,
C. V.
Jakubzick
.
2011
.
CD103+ pulmonary dendritic cells preferentially acquire and present apoptotic cell-associated antigen.
J. Exp. Med.
208
:
1789
1797
.
17
Beaty
,
S. R.
,
C. E.
Rose
Jr.
,
S. S.
Sung
.
2007
.
Diverse and potent chemokine production by lung CD11bhigh dendritic cells in homeostasis and in allergic lung inflammation.
J. Immunol.
178
:
1882
1895
.
18
Jakubzick
,
C.
,
F.
Tacke
,
F.
Ginhoux
,
A. J.
Wagers
,
N.
van Rooijen
,
M.
Mack
,
M.
Merad
,
G. J.
Randolph
.
2008
.
Blood monocyte subsets differentially give rise to CD103+ and CD103- pulmonary dendritic cell populations.
J. Immunol.
180
:
3019
3027
.
19
Hammad
,
H.
,
M.
Chieppa
,
F.
Perros
,
M. A.
Willart
,
R. N.
Germain
,
B. N.
Lambrecht
.
2009
.
House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells.
Nat. Med.
15
:
410
416
.
20
León
,
B.
,
M.
López-Bravo
,
C.
Ardavín
.
2007
.
Monocyte-derived dendritic cells formed at the infection site control the induction of protective T helper 1 responses against Leishmania.
Immunity
26
:
519
531
.
21
Chow
,
K. V.
,
R. M.
Sutherland
,
Y.
Zhan
,
A. M.
Lew
.
2016
.
Heterogeneity, functional specialization and differentiation of monocyte-derived dendritic cells.
Immunol. Cell Biol.
95
:
244
251
22
Sharma
,
M. D.
,
P. C.
Rodriguez
,
B. H.
Koehn
,
B.
Baban
,
Y.
Cui
,
G.
Guo
,
M.
Shimoda
,
R.
Pacholczyk
,
H.
Shi
,
E. J.
Lee
, et al
.
2018
.
Activation of p53 in immature myeloid precursor cells controls differentiation into Ly6c+CD103+ monocytic antigen-presenting cells in tumors.
Immunity
48
:
91
106.e6
.
23
Ko
,
H. J.
,
J. L.
Brady
,
V.
Ryg-Cornejo
,
D. S.
Hansen
,
D.
Vremec
,
K.
Shortman
,
Y.
Zhan
,
A. M.
Lew
.
2014
.
GM-CSF-responsive monocyte-derived dendritic cells are pivotal in Th17 pathogenesis.
J. Immunol.
192
:
2202
2209
.
24
Plantinga
,
M.
,
M.
Guilliams
,
M.
Vanheerswynghels
,
K.
Deswarte
,
F.
Branco-Madeira
,
W.
Toussaint
,
L.
Vanhoutte
,
K.
Neyt
,
N.
Killeen
,
B.
Malissen
, et al
.
2013
.
Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen.
Immunity
38
:
322
335
.
25
Mansouri
,
S.
,
S.
Patel
,
D. S.
Katikaneni
,
S. M.
Blaauboer
,
W.
Wang
,
S.
Schattgen
,
K.
Fitzgerald
,
L.
Jin
.
2018
.
Immature lung TNFR2- conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo.
Mucosal Immunol.
12
:
277
289
.
26
Blaauboer
,
S. M.
,
V. D.
Gabrielle
,
L.
Jin
.
2013
.
MPYS/STING-mediated TNF-α, not type I IFN, is essential for the mucosal adjuvant activity of (3'-5')-cyclic-di-guanosine-monophosphate in vivo.
J. Immunol.
192
:
492
502
.
27
Ebensen
,
T.
,
K.
Schulze
,
P.
Riese
,
C.
Link
,
M.
Morr
,
C. A.
Guzmán
.
2007
.
The bacterial second messenger cyclic diGMP exhibits potent adjuvant properties.
Vaccine
25
:
1464
1469
.
28
Allen
,
A. C.
,
M. M.
Wilk
,
A.
Misiak
,
L.
Borkner
,
D.
Murphy
,
K. H. G.
Mills
.
2018
.
Sustained protective immunity against Bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces IL-17-secreting TRM cells.
Mucosal Immunol.
11
:
1763
1776
.
29
Blaauboer
,
S. M.
,
S.
Mansouri
,
H. R.
Tucker
,
H. L.
Wang
,
V. D.
Gabrielle
,
L.
Jin
.
2015
.
The mucosal adjuvant cyclic di-GMP enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo.
eLife
4
:
e06670
.
30
Gogoi
,
H.
,
S.
Mansouri
,
L.
Jin
.
2020
.
The age of cyclic dinucleotide vaccine adjuvants.
Vaccines (Basel)
8
:
453
.
31
Richmond
,
B. W.
,
S.
Mansouri
,
A.
Serezani
,
S.
Novitskiy
,
J. B.
Blackburn
,
R.-H.
Du
,
H.
Fuseini
,
S.
Gutor
,
W.
Han
,
J.
Schaff
, et al
.
2020
.
Monocyte-derived dendritic cells link localized secretory IgA deficiency to adaptive immune activation in COPD.
Mucosal Immunol.
14
:
431
442
.
32
Randolph
,
G. J.
,
S.
Beaulieu
,
S.
Lebecque
,
R. M.
Steinman
,
W. A.
Muller
.
1998
.
Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking.
Science
282
:
480
483
.
33
Kitano
,
M.
,
S.
Moriyama
,
Y.
Ando
,
M.
Hikida
,
Y.
Mori
,
T.
Kurosaki
,
T.
Okada
.
2011
.
Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.
Immunity
34
:
961
972
.
34
Denton
,
A. E.
,
S.
Innocentin
,
E. J.
Carr
,
B. M.
Bradford
,
F.
Lafouresse
,
N. A.
Mabbott
,
U.
Mörbe
,
B.
Ludewig
,
J. R.
Groom
,
K. L.
Good-Jacobson
,
M. A.
Linterman
.
2019
.
Type I interferon induces CXCL13 to support ectopic germinal center formation.
J. Exp. Med.
216
:
621
637
.
35
Zhu
,
B.
,
R.
Zhang
,
C.
Li
,
L.
Jiang
,
M.
Xiang
,
Z.
Ye
,
H.
Kita
,
A. M.
Melnick
,
A. L.
Dent
,
J.
Sun
.
2019
.
BCL6 modulates tissue neutrophil survival and exacerbates pulmonary inflammation following influenza virus infection.
Proc. Natl. Acad. Sci. USA
116
:
11888
11893
.
36
Watchmaker
,
P. B.
,
K.
Lahl
,
M.
Lee
,
D.
Baumjohann
,
J.
Morton
,
S. J.
Kim
,
R.
Zeng
,
A.
Dent
,
K. M.
Ansel
,
B.
Diamond
, et al
.
2013
.
Comparative transcriptional and functional profiling defines conserved programs of intestinal DC differentiation in humans and mice.
Nat. Immunol.
15
:
98
108
.
37
Zhang
,
T. T.
,
D.
Liu
,
S.
Calabro
,
S. C.
Eisenbarth
,
G.
Cattoretti
,
A. M.
Haberman
.
2014
.
Dynamic expression of BCL6 in murine conventional dendritic cells during in vivo development and activation.
PLoS One
9
:
e101208
.
38
Thompson
,
E. A.
,
P. A.
Darrah
,
K. E.
Foulds
,
E.
Hoffer
,
A.
Caffrey-Carr
,
S.
Norenstedt
,
L.
Perbeck
,
R. A.
Seder
,
R. M.
Kedl
,
K.
Loré
.
2019
.
Monocytes acquire the ability to prime tissue-resident T cells via IL-10-mediated TGF-β release.
Cell Rep.
28
:
1127
1135.e4
.
39
Nath
,
A. P.
,
A.
Braun
,
S. C.
Ritchie
,
F. R.
Carbone
,
L. K.
Mackay
,
T.
Gebhardt
,
M.
Inouye
.
2019
.
Comparative analysis reveals a role for TGF-β in shaping the residency-related transcriptional signature in tissue-resident memory CD8+ T cells.
PLoS One
14
:
e0210495
.
40
Lee
,
A. Y. S.
,
H.
Körner
.
2019
.
The CCR6-CCL20 axis in humoral immunity and T-B cell immunobiology.
Immunobiology
224
:
449
454
.
41
Langlet
,
C.
,
S.
Tamoutounour
,
S.
Henri
,
H.
Luche
,
L.
Ardouin
,
C.
Grégoire
,
B.
Malissen
,
M.
Guilliams
.
2012
.
CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization.
J. Immunol.
188
:
1751
1760
.
42
Guilliams
,
M.
,
P.
Bruhns
,
Y.
Saeys
,
H.
Hammad
,
B. N.
Lambrecht
.
2014
.
The function of Fcγ receptors in dendritic cells and macrophages. [Published erratum appears in 2014 Nat. Rev. Immunol. 14: 349.].
Nat. Rev. Immunol.
14
:
94
108
.
43
Loetscher
,
H.
,
D.
Stueber
,
D.
Banner
,
F.
Mackay
,
W.
Lesslauer
.
1993
.
Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors.
J. Biol. Chem.
268
:
26350
26357
.
44
Gogoi
,
H.
,
S.
Mansouri
,
D. S.
Katikaneni
,
L.
Jin
.
2020
.
New MoDC-targeting TNF fusion proteins enhance cyclic di-GMP vaccine adjuvanticity in middle-aged and aged mice.
Front. Immunol.
11
:
1674
.
45
Ohtsuka
,
H.
,
A.
Sakamoto
,
J.
Pan
,
S.
Inage
,
S.
Horigome
,
H.
Ichii
,
M.
Arima
,
M.
Hatano
,
S.
Okada
,
T.
Tokuhisa
.
2010
.
Bcl6 is required for the development of mouse CD4+ and CD8α+ dendritic cells.
J. Immunol.
186
:
255
263
.
46
Reagin
,
K. L.
,
K. D.
Klonowski
.
2018
.
Incomplete memories: the natural suppression of tissue-resident memory CD8 T cells in the lung.
Front. Immunol.
9
:
17
.

L.J. and S.M. are coinventors on a patent (PCT/US19/53548) on moDCs-targeting TNF fusion proteins. The other authors have no financial conflicts of interest.

Supplementary data