Crucial role of dendritic cells in the generation of anti-tumor T-cell responses and immunogenic tumor microenvironment to suppress tumor development

Dendritic cells (DCs) are known as unique professional antigen (Ag)-presenting cells (APCs) to prime naïve T cells for the initiation of adaptive immunity. While DCs are believed to play a pivotal role in generating anti-tumor T-cell responses, the importance of DCs in the protection from the progression of tumors remains elusive. Here, we show how the constitutive deficiency of CD11chi DCs influences the progression of tumors with the use of binary transgenic mice with constitutive loss of CD11chi DCs. Constitutive loss of CD11chi DCs not only enhances the progression of tumors but also reduces the responses of Ag-specific T cells. Furthermore, the congenital deficiency of CD11chi DCs generates the immunosuppressive tumor microenvironment (TME) that correlates with the marked accumulation of myeloid-derived suppressor cells (MDSCs) and the prominent productions of immunosuppressive mediators. Thus, our findings suggest that CD11chi DCs are crucial for generating anti-tumor T-cell responses and immunogenic TME to suppress the development of tumors.


Introduction
Dendritic cells (DCs) are regarded as antigen (Ag)-presenting cells (APCs) that play a pivotal role in linking innate and adaptive immunity (1)(2)(3).DCs are divided into functionally distinguishable two subsets, conventional DCs (cDCs) and plasmacytoid DCs (pDCs) (1)(2)(3).DCs recognize and process intracellular or extracellular Ags for the presentation of the complexes of the antigenic peptides and major histocompatibility complex (MHC) class I (MHC I) or MHC class II (MHC II), together with costimulatory molecules and cytokines, for differentiation of naïve T cells into effector T (T eff ) cells (1)(2)(3).cDCs are subdivided into type 1 cDC (cDC1) and type 2 cDC (cDC2) with different developmental pathways and functions (1)(2)(3).The differentiation of cDC1 depends on interferon (IFN) regulatory factor (IRF) 8 and basic leucine zipper ATF-like transcription factor 3 (Batf3), whereas the development of cDC2 requires IRF4, Notch2, and/or kruppel-like factor (KLF) 4 (1)(2)(3).Upon sensing foreign Ags, cDC1 mainly generates type 1 helper T (T H 1) cells or cytotoxic T lymphocytes (CTLs) through an MHC class IImediated pathway or MHC I-dependent pathway known as cross-presentation (3).However, cDC2 preferentially induces T H 2 and T H 17 cells via the MHC class II-mediated pathway of exogenous Ags (3).Conversely, DCs are suggested to be essential in maintaining immune homeostasis under steady-state and certain environmental conditions by generating immune tolerance through mechanisms involving clonal deletion and anergy of Ag-specific T cells as well as the generation of CD4 + Foxp3 + regulatory T (T reg ) cells (3).
Accumulating lines of evidence suggest that DCs are crucial for the induction of anti-tumor immunity (4,5).Tumorinfiltrating DCs are thought to initiate anti-tumor immune responses in tumor-draining lymph nodes (TdLNs) that migrated from tumor tissues after capturing and processing tumor Ags (6)(7)(8).A recent analysis of cDC1-deficient mice revealed that cDC1 acts as a distinctive subset capable of crosspresenting tumor Ags to naïve CD8 + T cells in TdLNs, resulting in the generation of tumor-specific CTLs for the elimination of tumor (4)(5)(6)(8)(9)(10). cDC1 is also known as a major producer of interleukin (IL)-12, which elicits T H 1 responses to participate in the induction of anti-tumor immunity (11).Furthermore, analysis of cDC2-deficient mice showed that cDC2 derived from tumor tissues enhances the ability of cDC1 to induce tumor-specific CTLs through the activation of CD4 + T cells in TdLNs (4)(5)(6)12).However, it has been reported that pDCs enhance tumor-specific CTL response generated by cDC1 through the secretion of type I IFN (7,8).Although the importance of DC subsets in the induction of anti-tumor immunity has been highly appreciated, their role in the progression of tumors remains elusive.
In this study, we examine the impact of the constitutive deficiency of CD11c hi DCs on tumor progression with the use of binary transgenic (Tg) mice that constitutively lacked CD11c hi DCs (13,14).

Flow cytometry
Cells were stained with fluorescein-conjugated monoclonal antibodies (mAbs) after Fc blocking with mAb to CD16/CD32 (clone 2.4G2, BD Biosciences) listed in Supplementary Table 1 in the Supplementary Material.Intracellular staining was performed using the Fixation and Permeabilization kit (eBioscience, San Diego, CA, USA).Analysis of fluorescence staining was performed using FACSVerse flow cytometer (BD Biosciences), CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA), and FlowJo software (version 10.9.0; Tree Star, Ashland, OR, USA) according to the gating strategy to identify the leukocytes in LNs, Spl, and tumor tissues as described in Supplementary Figures 1-3 in the Supplementary Material.

Quantitative reverse transcriptionpolymerase chain reaction
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed as described previously (14,21,22).Total RNA was extracted from cells by RNeasy plus micro kit (Qiagen, Valencia, CA, USA), and the first-strand cDNA was synthesized with oligo(dT)20 primer using the PrimeScript RT Master Mix (Takara, Shiga, Japan) from total RNA.Transcriptional expression levels were analyzed by using SYBR ® Premix Ex Taq II on Thermal Cycler Dice (Takara) with specific primer pairs listed in Supplementary Table 2 in the Supplementary Material after normalization for Gapdh expression.

Treatment with Flt3 inhibitor
Mice were treated daily with or without gilteritinib, an inhibitor of Fms-like tyrosine kinase 3 (Flt3), (SelleckChem, Houston, TX, USA; 200 mg/mouse) (23) by oral gavage using 20-gauge, 1.5-inch feeding needles (Natume, Tokyo, Japan) for 20 days.Spl was obtained from the mice on day 21 after the start of administration.

Tumor growth assay and treatment
WT mice or CD11c:DTA mice were inoculated subcutaneously (s.c.) with B16-OVA (1 × 10 5 ) or MC38 (5 × 10 5 ) in the right back.Tumor size was measured every day after the inoculation for 8 to 18 days using a digital caliper (CP-15CP; Mitsutoyo, Kanagawa, Japan), and tumor volumes were estimated using the following ellipsoidal formula: length × width × height ×0.52.Spl, TdLNs, and tumor tissues were obtained from mice on day 18 after tumor inoculation.In some experiments, Spl, TdLNs, and tumor tissues were obtained from tumor-bearing WT mice or tumor-bearing CD11c:DTA mice with delayed tumor growth on days 19-21 after tumor inoculation when the values of the tumor volumes of tumor-bearing WT mice as controls were in the range of their mean ± standard deviation (s.d.).For the depletion of Gr-1 + cells (24, 25), B16-OVA-bearing mice received anti-Gr-1 mAb (clone RB6-8C5, Bio X Cell, Lebanon, NH, USA; 200 µg/mouse) or control Ab (rat IgG Wako; 200 µg/mouse) by i.p. injections every 3 days for 15 days starting from the day of tumor inoculation until the end of the experiment.

Statistical analysis
Data are expressed as the mean ± s.d.from three to eight individual samples in a single experiment of at least three independent experiments.Statistical analysis was performed using Prism 9 (GraphPad Software, Boston, MA, USA) using the two-sided unpaired Student's t-test.A P-value for significance was indicated with <0.05 or 0.01.

Constitutive deficiency of CD11c hi DCs causes abnormal composition of leukocytes
To clarify the contribution of CD11c hi DCs to the protection from the development of tumors, we generated a mouse model with the constitutive deficiency of CD11c hi DCs by cross-mating CD11c-Cre mice (15) and R-DTA mice harboring the diphtheria toxin (DT) a chain (DTA) under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus (16) for producing CD11c-Cre: R-DTA double-Tg mice (referred to as CD11c:DTA mice) (14).CD11c:DTA mice showed almost complete depletion of CD11c hi DCs in LNs and Spl under homeostatic conditions when compared with WT mice (Supplementary Figures 4, 5 in the Supplementary Material).Similar to the published report (14), the partial reduction of the frequency of pDCs was observed in LNs and Spl of CD11c:DTA mice as compared with WT mice (Supplementary Figures 4, 5 in the Supplementary Material).
We addressed the influence of the constitutive ablation of CD11c hi DCs on the constituency of leukocytes in lymphoid tissues under steady-state conditions.CD11c:DTA mice showed reduced frequencies of CD4 + T cells and CD8 + T cells, whereas they exhibited the enhanced proportions of Gr-1 + CD11b + F4/80 − polymorphonuclear leukocytes (PMNs) and Gr-1 + CD11b + F4/80 + monocytes/macrophages in LNs and Spl when compared with WT mice (Supplementary Figures 4, 5 in the Supplementary Material).In addition, CD11c:DTA mice showed the enhanced frequency of B220 + B cells in LNs (Supplementary Figure 4 in the Supplementary Material).
Taken together, these results indicate that the constitutive deficiency of CD11c hi DCs leads to abnormal composition of leukocytes in lymphoid tissues.
Given that Flt3 ligand (Flt3L) is crucial for the development of various types of leukocytes (26-28), we have previously reported the linking of the enhanced generation of myeloid-lineage cells with massive serum production of Flt3L in CD11c:DTA mice (14).To clarify the role of Flt3L on the enhanced development of Gr-1 + CD11b + F4/80 − PMNs and Gr-1 + CD11b + F4/80 + monocytes/ macrophages under the constitutive absence of CD11c hi DCs, we examined the influence of gilteritinib, an inhibitor of Flt3 ( 23), on their generation in CD11c:DTA mice.Treatment of CD11c:DTA mice with gilteritinib inhibited the enhanced generation of Gr-1 + CD11b + F4/ 80 − PMNs and Gr-1 + CD11b + F4/80 + monocytes/macrophages in LNs and Spl under homeostatic conditions (Supplementary Figure 6 in the Supplementary Material).
Collectively, these results indicate that Flt3L is required for the enhanced generation of PMNs and monocytes/macrophages under the constitutive loss of CD11c hi DCs.
Taken together, these results indicate that CD11c hi DC subsets are mainly ablated in lymphoid tissues in CD11c:DTA mice.

Congenital deficiency of CD11c hi DCs enhances tumor development and accumulation of MDSCs in tumor tissues
We utilized the experimental model of a murine melanoma tumor cell line with poor immunogenicity that was engineered to express OVA (B16-OVA) (19).When compared with WT mice, CD11c:DTA mice displayed a significant progression of tumor growth for the duration of the experiment (Figures 1A-D).We  1E-H).Therefore, these results indicate that the congenital deficiency of CD11c hi DCs causes the enhanced progression of tumors.We also compared the cellularity of tumor-infiltrating leukocytes between WT mice and CD11c:DTA mice under tumor-bearing conditions.When compared with tumor-bearing WT mice, tumor-bearing CD11c:DTA mice exhibited the marked infiltrations of Gr-1 + CD11b + F4/80 − PMNs, Gr-1 + CD11b + F4/80 + monocytes/macrophages, and B220 + B cells, whereas they displayed the significant reduction in the accumulations of CD11c hi cDCs, CD11c + Siglec-H + pDCs, and CD3 + CD8 + T cells (Figures 1I-K).Similar differences in the cellular compositions in TdLN and Spl between WT mice and CD11c:DTA mice were observed under tumor-bearing conditions when compared with those under steadystate conditions (Supplementary Figures 10, 11 in the Supplementary Material).
Taken together, these results indicate that the constitutive loss of CD11c hi DCs enhances the accumulation of MDSCs in tumor tissues under tumor-bearing conditions.
It has been shown that MDSCs exhibited an immunosuppressive potential to support tumor progression (40)(41)(42).To clarify the contribution of MDSCs to the development of tumors, we addressed the effect of the depletion of MDSCs by anti-Gr-1 mAb (24, 25) on the progression of tumor growth in CD11c:DTA mice.The elimination of MDSCs by anti-Gr-1 mAb exhibited a more dramatic reduction of tumor growth in CD11c:DTA mice than tumor-bearing WT mice (Supplementary Figures 15C-F in the Supplementary Material), probably due to a more prominent accumulation of MDSCs in tumor tissues (Figures 1I-K).
Taken together, these results indicate that MDSCs are involved in the enhanced development of tumors under the constitutive loss of CD11c hi DCs.

Constitutive loss of CD11c hi DCs affects the activation status of T cells
We compared the activation status of T cells in lymphoid tissues between WT mice and CD11c:DTA mice.CD11c:DTA mice had reduced fractions of CD4 + T cells and CD8 + T cells expressing CD44 as a marker for T-cell activation (43) in LNs and Spl as compared with WT mice in the homeostatic conditions (Supplementary Figures 16, 17 in the Supplementary Material).Furthermore, CD4 + T cells and CD8 + T cells in LNs and Spl did not express any immune checkpoint molecules including programmed death (PD)-1, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and lymphocyte activation gene (LAG)-3 in both WT mice and CD11c: DTA mice under steady-state conditions (Supplementary Figures 16, 17 in the Supplementary Material).However, tumor-infiltrating CD4 + tumor-infiltrating lymphocytes (TILs) and CD8 + TILs obtained from tumor-bearing WT mice not only had the marked proportions of CD4 + CD44 + CD62L − T cell subset and CD8 + CD44 + CD62L − T cell subset, which function as effector memory T (T EM ) cells (44), but also expressed PD-1, TIM-3, and LAG-3 (Figures 2A-D; Supplementary Figures 18A, B in the Supplementary Material).In contrast, CD11c: DTA mice had a higher proportion of CD4 + CD44 + CD62L − T cells in tumor tissues than WT mice (Figures 2A, B; Supplementary Figure 18A in the Supplementary Material).Interestingly, CD4 + TILs, but not CD8 + TILs, obtained from CD11c:DTA mice showed lower expressions of PD-1 and TIM-3 than those obtained from WT mice (Figures 2A-D; Supplementary Figures 18A, B in the Supplementary Material).
CD4 + T cells and CD8 + T cells displayed an enhanced expression of CD44 in TdLNs and Spl in WT mice during tumor progression (Figures 2E-L; Supplementary Figure 18C-F in the Supplementary Material).When compared with tumor-bearing WT mice, CD4 + T cells, but not CD8 + T cells, obtained from tumor-bearing CD11c:DTA mice exhibited enhanced expressions of PD-1 and TIM-3 in TdLNs ( Figures 2E-H; Supplementary Figures 18C, D in the Supplementary Material).Similar to the homeostatic conditions, CD11c:DTA mice showed reduced or enhanced fractions of CD4 + CD44 + T cells and CD8 + CD44 + T cells or CD8 + CD62L − T cells in Spl as compared with WT mice under tumor-bearing conditions (Figures 2I-L; Supplementary Figures 18E, F in the Supplementary Material).Furthermore, tumor-bearing CD11c:DTA mice showed an enhanced expression of TIM-3 on CD4 + T cells and PD-1 and TIM-3 on CD8 + T cells in Spl as compared with those obtained from tumor-bearing WT mice (Figures 2I-L; Supplementary Figures 18E, F in the Supplementary Material).Collectively, these results indicate that the constitutive loss of CD11c hi DCs alters the activation status of T cells under tumor-bearing conditions.

Constitutive absence of CD11c hi DCs affects Ag-specific responses of T cells
To clarify the role of CD11c hi DCs in the Ag-specific T-cell priming, WT mice and CD11c:DTA mice were adoptively transferred with eFluor ™ 670-labeled OT-II CD4 + T cells or OT-I CD8 + T cells expressing the OVA-specific TCR (14,17,18) before systemic injection of OVA protein to analyze their Ag-specific division in LNs.In contrast to the prominent division of OT-II CD4 + T cells or OT-I CD8 + T cells in LNs in WT mice after systemic administration of OVA protein, their responses were significantly reduced in CD11c:DTA mice (Supplementary Figure 19 in the Supplementary Material).
To determine the influence of the constitutive elimination of tissues and TdLNs as well as OT-II CD4 + T cells in TdLNs, they did not show the apparent responses of OT-II CD4 + T cells in tumor tissues and Spl as well as OT-I CD8 + T cells in Spl (Figures 3A-L).However, tumor-bearing CD11c:DTA mice displayed the profound reduction of Ag-specific divisions of OT-I CD8 + T cells in tumor tissues and TdLNs as well as OT-II CD4 + T cells in TdLNs as compared with tumor-bearing WT mice (Figures 3C, D, G-J).
To examine the effect of the constitutive elimination of CD11c hi DCs on the generation of Ag-specific CTLs, we evaluated the occurrence of Ag-specific CD8 + T cells based on binding with the MHC I-OVA pentamer in tumor-bearing mice.MHC I-OVA pentamer + CD44 high CD8 + T cells were detected in tumor tissues, TdLNs, and Spl in tumor-bearing WT mice (Figures 3M-U).However, tumor-bearing CD11c:DTA mice showed a marked reduction of MHC I-OVA pentamer + CD44 high CD8 + T cells in tumor tissues, TdLNs, and Spl as compared with tumor-bearing WT mice (Figures 3M-U).Similarly, tumor-bearing CD11c:DTA mice exhibited a lower freque ncy of MHC I-gp100 tetramer + CD44 high CD8 + T cells in tumor tissues than tumorbearing WT mice (Supplementary Figure 20 in the Supplementary Material).Taken together, these results indicate that the congenital deficiency of CD11c hi DCs diminishes Agspecific responses of T cells under tumor-bearing conditions.

Constitutive absence of CD11c hi DCs affects the appearance of CD4 + Foxp3 + T reg cells
To address the effect of the constitutive absence of CD11c hi DCs on the composition of CD4 + Foxp3 + T reg cells, we compared their proportion between WT mice and CD11c:DTA mice.CD11c:DTA mice displayed a reduced frequency of CD4 + Foxp3 + T reg cells, whereas they exhibited the enhanced frequency of CD4 + Foxp3 + retinoic acid-related orphan receptor gt (RORgt) + T reg cells (45,46), in LNs and Spl when compared with WT mice in homeostatic conditions (Supplementary Figure 21 in the Supplementary Material).However, tumor-bearing WT mice and CD11c:DTA mice showed comparable accumulations of CD4 + Foxp3 + T reg cells and CD4 + Foxp3 + RORgt + T reg cells in tumor tissues (Figures 4A-C).Similar to the homeostatic conditions, tumor-bearing CD11c:DTA mice showed a reduced proportion of CD4 + Foxp3 + T reg cells and an enhanced proportion of CD4 + Foxp3 + RORgt + T reg cells in TdLNs and Spl as compared with tumor-bearing WT mice (Figures 4D-I).Taken together, these results suggest that the constitutive elimination of CD11c hi DCs alters the composition of CD4 + Foxp3 + T reg cells in lymphoid tissues under homeostatic and tumor-bearing conditions.

Constitutive loss of CD11c hi DCs enhances the production of immunosuppressive mediators in tumor tissues
Tumor cells and tumor-infiltrating immune cells secrete several soluble factors that are thought to generate the immunosuppressive tumor microenvironment (TME), which attenuates the anti-tumor immune responses, leading to progressive tumor growth (47-49).We therefore compared the transcriptional expressions of the immunosuppressive cytokines and metabolite-generating enzymes in tumor tissues between tumor-bearing WT mice and CD11c:DTA mice (Figure 5).Tumor tissues obtained from tumor-bearing CD11c:DTA mice exhibited higher transcriptional expressions of IL-10, transforming growth factor (TGF)-b, indoleamine 2,3dioxygenase (IDO), vascular endothelial growth factor (VEGF), arginase, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and membrane-associated prostaglandin E2 synthase (mPGES) than tumor-bearing WT mice.Collectively, these results indicate that the congenital deficiency of CD11c hi DCs promotes the production of immunosuppressive mediators in tumor tissues.

Discussion
While DCs are reportedly critical for the control of anti-tumor immunity, the contribution of DCs to the protection from the development of tumors remains elusive.In the present study, we demonstrated that the congenital deficiency of CD11c hi DCs promoted the progression of tumors associated with the impaired responses of T cells as well as the prominent accumulation of MDSCs and the marked productions of immunosuppressive mediators in tumor tissues.Thus, our results suggest that CD11c hi DCs are required for the initiation and regulation of anti-tumor T-cell responses as well as the formation of immunogenic TME for the inhibition of the progression of tumors.
Analysis of CD11c:DTA mice revealed that the congenital deficiency of CD11c hi DCs caused the abnormal composition of leukocytes in lymphoid tissues correlated with the enhanced proportions of B cells, PMNs, and monocytes/macrophages under homeostatic and tumor-bearing conditions.Flt3L is reportedly important for the regulation of the development of leukocytes (26-28).We have previously reported that the serum production of Flt3L was enhanced in CD11c:DTA mice under homeostatic conditions, suggesting that cDC lineage is responsible for the major consumption of Flt3L (14).However, we showed that the inhibitor of Flt3 suppressed the enhanced proportions of PMNs and monocytes/ macrophages in lymphoid tissues in CD11c:DTA mice.Thus, the secretion of an excess amount of Flt3L in the periphery under the constitutive loss of CD11c hi DCs could lead to the abnormal composition of leukocytes in lymphoid tissues, and that may affect the formation of the pathophysiological immune responses.
Given the decreased proportion of NK cells under the transient depletion of CD11c + DCs (50), CD11c + DCs could be required to maintain NK cell homeostasis through the production of IL-15 (51, 52).However, it has been also shown that IL-2 favors the rapid proliferation of short-lived effector phenotypes of CD8 + T cells and NK cells, while IL-15 has its own unique effects on supporting the maintenance of their long-lived memory phenotypes (53).We showed that CD11c:DTA mice exhibited normal proportions of CD3 − CD19 − NK1.1 + NKp46 + CD49 a − C D49b + NK cells.Furthermore, they exhibited the enhanced or reduced productions of serum IL-2 or IL-15.Different from the direct linking of the CD11c:DTA mice showed a reduced constituency of CD3 − CD19 − CD127 + RORgt + ILC3s in LNs and Spl, although they did not show an expression of CD11c.Furthermore, they exhibited a reduced production of serum IL-1b.Given that IL-1b produced by DCs plays a relevant role in the proliferation of ILC3s (54), a reduced constituency of CD3 − CD19 − CD127 + RORgt + ILC3s in lymphoid tissues may be due to the constitutive loss of CD11c hi DCs as sources of IL-1b rather than the direct toxicity of DTA under the control of the expression of CD11c.
While the transient depletion of cDC1 and cDC2 in zDC (Zbtb46)-DTR mice or CD11c + DCs in CD11c-DTR mice reportedly reduced the effect of tumor vaccination to enhance the survival rates of tumorbearing mice and to inhibit the development of tumor (55, 56), their transient depletion had little or no effect on the tumor growth (56).In contrast, the constitutive absence of cDC1 in Batf3-deficient mice not only enhanced the progression of tumors but also abrogated the effect of tumor vaccination to inhibit the growth of tumors (9, 57).We showed that the congenital deficiency of CD11c hi DCs in CD11c:DTA mice caused the marked progression of tumor.The reason why the constitutive absence of CD11c hi DCs in CD11c:DTA mice or cDC1 in Batf3-deficient mice leads to the enhanced progression of tumor may be due to the failure of the establishment of the tumor immunosurveillance associated with the occurrence of anti-tumorspecific T eff cells.Indeed, we showed that the constitutive absence of CD11c hi DCs showed a marked reduction of MHC I-OVA pentamer + CD44 high CD8 + T cells in tumor tissues and lymphoid tissues.However, CD11c-DTR mice or zDC (Zbtb46)-DTR mice reportedly exhibited the short-term ablation of CD11c + DCs or cDC1 and cDC2 and their rapid recovery following DT injection.Therefore, the transient depletion of CD11c + DCs in CD11c-DTR mice or cDC1 and cDC2 in zDC (Zbtb46)-DTR mice may be insufficient for the induction of the breakdown of the tumor immunosurveillance to enhance the development of tumor, whereas their transient depletion could be sufficient for impairment of the enhanced anti-tumor immune response triggered by tumor vaccination.
In contrast to the published reports (55, 56), analysis of tumorbearing zDC-DTR mice showed that the transient depletion of cDC1 and cDC2 resulted in the delayed growth of tumors and the enhanced survival rates possibly due to the drastic reduction of CD4 + Foxp3 + T reg cells and maintenance of T H cells in TdLNs (50).The constitutive elimination of CD11c hi DCs exhibited a more potent enhancement of the growth of the immunogenic tumor than that of the poor immunogenic tumor.Therefore, the impact of the immunogenic function of CD11c hi DCs on adaptive anti-tumor immunity could be linked with the immunogenicity of tumors.However, the TME-mediated modulation of tumor-infiltrated DCs is suggested to impair their function in enhancing the adaptive antitumor immunity, thereby promoting the development of tumors (4,58).While DCs could have the potential to initiate anti-tumor immune responses by recruiting and activating various types of immune cells in TdLNs, the TME could secrete immunosuppressive mediators that limit the immunogenic function of DCs and instead skew DCs to a tolerogenic function.
Previous studies have shown that CD11c:DTA mice had an unimpaired compartment of T cells in peripheral lymphoid tissues, while they exhibited myeloproliferative disorder (MPD) with lymphadenopathies (13).However, we showed that CD11c:DTA mice displayed a reduced constituency of T cells in LNs and Spl, whereas they did not develop MPD with normal sizes of LNs and Spl.Although the precise reason for these apparent discrepancies remains unclear, the different environmental conditions may affect the immune cell homeostasis.
The congenital deficiency of CD11c hi DCs not only reduced the expression of CD44 on CD4 + T cells and CD8 + T cells in lymphoid tissues under homeostatic and tumor-bearing conditions but also enhanced their expressions of several immune checkpoint molecules under tumor-bearing conditions.Taken together, these results suggest that CD11c hi DCs are required to control the activation status of T cells in lymphoid tissues under homeostatic and tumor-bearing conditions.
We showed that the congenital deficiency of CD11c hi DCs enhanced the proportion of tumor-infiltrating CD4 + T EM cells, while it reduced their expressions of certain immune checkpoint molecules.However, the constitutive ablation of CD11c hi DCs had little or no effect on the proportion of tumor-infiltrating CD8 + T EM cells and their expressions of the immune checkpoint molecules.Thus, these results suggest that CD11c hi DCs control the manifestation of the effector memory phenotype of tumor-infiltrating CD4 + T cells and their expressions of the immune checkpoint molecules.
We showed that the constitutive deficiency of CD11c hi DCs caused the almost complete inhibition of the priming of Ag-specific CD4 + T cells in LNs and TdLNs under homeostatic conditions with soluble antigenic immunization and tumor-bearing conditions.Therefore, these results suggest that CD11c hi DCs are critical APCs to present soluble and tumor cell-associated Ags to CD4 + T cells for their Ag-specific priming.The constitutive absence of CD11c hi DCs partially inhibited the Ag-specific priming of CD8 + T cells in LNs under homeostatic conditions with soluble antigenic immunization.It has been reported that pDCs could prime Ag-specific CD8 + T cells through cross-presentation (59-61).Since pDCs were partially depleted in lymphoid tissues of CD11c:DTA mice, it is possible that residual pDCs as well as other APCs, including monocytes/macrophages, participate in cross-priming of CD8 + T cells with soluble Ag.
We showed that the congenital deficiency of CD11c hi DCs not only suppressed Ag-specific priming of CD8 + T cells in TdLNs and tumor tissues but also abolished the generation of Ag-specific CTLs in lymphoid and tumor tissues under tumor-bearing conditions.Thus, our findings suggest that CD11c hi DCs are the most potent APCs to present tumor cell-associated Ag to CD8 + T cells through cross-presentation for their Ag-specific priming to generate tumorspecific CTLs.Previous studies have shown that the transient depletion of CD11c + DCs, including cDC1, cDC2, and Mo-DCs/ DC3, inhibited Ag-specific priming of CD8 + T cells in tumor tissues (62).However, it has been shown that Ag-specific priming of CD8 + T cells was reduced in TdLNs, whereas their response was not affected in tumor tissues under the transient depletion of cDC1 and cDC2, but not Mo-DCs/DC3 (30).Furthermore, Mo-DCs/DC3 have been shown to cross-prime Ag-specific CD8 + T cells (30).Collectively, these phenomena imply that Mo-DCs/DC3 play a key role in the cross-priming of CD8 + T cells in tumor tissues, while cDC1 and cDC2 are essential for this response in TdLNs.
Similar to the published reports (14, 63), the congenital deficiency of CD11c h i DCs reduced the proportion of CD4 + Foxp3 + T reg cells in lymphoid tissues under homeostatic and tumor-bearing conditions, suggesting the importance of CD11c hi DCs in the maintenance of the population size of CD4 + Foxp3 + T reg cells.Conversely, the constitutive absence of CD11c hi DCs enhanced the proportion of CD4 + Foxp3 + RORgt + T reg cells in lymphoid tissues under homeostatic and tumorbearing conditions.It has been reported that CD4 + Foxp3 + RORgt + T reg cells, possibly derived from CD4 + Foxp3 + RORgt − T reg cells, had a prominent suppressive activity against the responses of CD4 + T eff cells (45,46).Furthermore, the intestinal CD4 + Foxp3 + RORgt + T reg cells induced by gut microbiota have been shown to contribute to the suppression of gut inflammation (64, 65), the inhibition of T H 2driven defense against helminths and T H 2-associated colitis (66), and the inhibition of intestinal T H 2-associated allergy (67).While the development of CD4 + Foxp3 + RORgt + T reg cells and their role in the regulation of anti-tumor immunity as well as their relation to the intestinal CD4 + Foxp3 + RORgt − T reg cells remain unclear, it is possible that CD4 + Foxp3 + RORgt − T reg cells convert into CD4 + Foxp3 + RORgt + T reg cells in the milieu of the abundance of various proinflammatory cytokines under the constitutive absence of CD11c hi DCs (14), and they suppress anti-tumor functions of CD4 + T eff cells and CTLs in lymphoid tissues.
Previous studies have reported that tumor cells and immune cells as well as fibroblasts in the tumor stroma could produce a variety of inflammatory mediators, including myeloid growth factors and proinflammatory cytokines, leading to the creation of autocrine and paracrine loops for the generation of the chronic inflammation in the TME (41,68).Furthermore, the chronic inflammation in the TME could contribute to the generation of MDSCs, possibly due to the occurrence of the aberrant myelopoiesis to skew the differentiation of PMNs and monocytes/macrophages in favor of MDSCs (41,68).However, we have previously reported that the congenital deficiency of CD11c hi DCs triggers spontaneous systemic inflammation with the massive productions of various proinflammatory cytokines and Flt3L, accompanied by the abnormal composition of leukocytes, including the enhanced generation of PMNs, in lymphoid tissues (14).Furthermore, we showed that the inhibitor of Flt3 suppressed the enhanced generation of PMNs and monocytes/macrophages in lymphoid tissues in CD11c:DTA mice.Thus, these phenomena imply that the constitutive loss of CD11c hi DCs triggers the massive secretion of Flt3L in the periphery and the chronic inflammation in the TME, which enhances the generation of MDSCs from myeloid cells in the context of the aberrant myelopoiesis during the development of tumor.
Accumulating lines of evidence suggest that MDSCs play a critical role in the development of tumors (40)(41)(42).We showed that the elimination of MDSCs exhibited a more potent reduction of tumor growth in tumor-bearing CD11c:DTA mice than in tumorbearing WT mice.Thus, these results suggest that the promoted generation of MDSCs and their accumulation in the TME contribute to the enhanced progression of tumors under the constitutive loss of CD11c hi DCs.
Having demonstrated the linking of the constitutive deficiency of CD11c hi DCs with the marked accumulation of MDSCs in tumor tissues, tumor-infiltrating MDSCs could contribute to the enhanced secretions of several immunosuppressive cytokines and metabolitegenerating enzymes in tumor tissues under the constitutive ablation of CD11c hi DCs.Thus, our findings suggest that the congenital deficiency of CD11c hi DCs promotes the emergence of MDSCs in tumor tissues and facilitates the formation of the immunosuppressive TME through the production of immunosuppressive mediators.Taken together, CD11c hi DCs could be crucial for generating the immunogenic TME by attenuating the generation of MDSCs and their accumulation in tumor tissues for the suppression of the development of tumors.
In conclusion, our results suggest that the congenital deficiency of CD11c hi DCs leads to the impaired responses of tumor-specific T eff cells and the occurrence of the immunosuppressive TME, possibly mediated through the marked accumulation of MDSCs and their prominent productions of immunosuppressive mediators, and that results in the acceleration of the development of tumor.Thus, our findings unravel the importance of CD11c hi DCs in the protection from the progression of tumors by generating anti-tumor T eff -cell responses and immunogenic TME.A better understanding of the nature of CD11c hi DCs and the manipulation to enforce their function may open new avenues for exploring novel tumor-specific immunotherapeutic treatments.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material.Further inquiries can be directed to the corresponding author.

1
FIGURE 1 Deficiency of CD11c hi DCs promotes the progression of tumors.(A-D) WT mice and CD11c:DTA mice were inoculated with B16-OVA, and tumor growth was monitored.(A-C) Average tumor volume curves of WT mice and CD11c:DTA mice (A) or tumor volume curves of individual WT mice (B) and CD11c:DTA mice (C) for 18 days.(D) Tumor volume on 17 days; n = 7 per group.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.(E-H) WT mice and CD11c:DTA mice were inoculated with MC38, and tumor growth was monitored.(E-G) Average tumor volume curves of WT mice and CD11c:DTA mice (E) or tumor volume curves of individual WT mice (F) and CD11c:DTA mice (G) for 18 days.(H) Tumor volume on 17 days; n = 7 per group.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.(I-K) WT mice and CD11c:DTA mice were inoculated with B16-OVA.Cell surface expression profile (I), proportion (J), and absolute number (K) of leukocytes in tumor tissues on days 18-21 after tumor inoculation.Data are obtained from five to six individual samples in a single experiment.Numbers in the dot plot represent the proportion of the indicated cell populations among leukocytes.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.All data are representative of at least three independent experiments.
a n d I -A / I -E hi CD11c + XCR1 − SIRPa + CD64 + Mo-DCs/DC3 in TdLNs as well as I -A / I -E + C D 1 1 c + X C R 1 + S I R P a − c D C 1 a n d I -A / I -E + CD11c + XCR1 − SIRPa + cDC2 in Spl compared with those in tumor-bearing WT mice (Supplementary Figures

2
FIGURE 2 Deficiency of CD11c hi DCs affects the activation status of T cells under tumor-bearing conditions.WT mice and CD11c:DTA mice were inoculated with B16-OVA.Cell surface expression profile (A, C, E, G, I, K) and proportion (B, D, F, H, J, L) of CD4 + T cells (A, B, E, F, I, J) and CD8 + T cells (C, D, G, H, K, L) in tumor tissues (A-D), TdLNs (E-H), and Spl (I-L) on days 18-21 after tumor inoculation.Data are obtained from five to eight individual samples in a single experiment.Numbers in the histogram represent the proportion of the indicated cell populations.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.All data are representative of at least three independent experiments.

3
FIGURE 3 Deficiency of CD11c hi DCs decreases the generation of Ag-specific T eff cells under tumor-bearing conditions.(A-L) WT mice and CD11c:DTA mice that had been inoculated with or without B16-OVA were adoptively transferred i.t.(A, B, G, H) or i.v.(C-F, I-L) with eFluor ™ 670-labeled CD45.1 + OT-II CD4 + T cells (A-F) or CD45.1 + OT-I CD8 + T cells (G-L).Cell dividing profile (A, C, E, G, I, K) and proportion (B, D, F, H, J, L) in tumor tissues (A, B, G, H), TdLNs (C, D, I, J), and Spl (E, F, K, L) at 2 days after the administration.Data are obtained from four to six individual samples in a single experiment.Numbers in the histogram represent the proportion of the dividing cells.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.(M-U) WT mice and CD11c:DTA mice were inoculated with or without B16-OVA.Cell surface expression profile (M, P, S), proportion (N, Q, T), and absolute number (O, R, U) of MHC I-OVA pentamer + CD44 high CD8 + T cells among CD8 + T cells in tumor tissues (M-O), TdLNs (P-R), and Spl (S-U) on days 18-21 after tumor inoculation.Data are obtained from four to six individual samples in a single experiment.Numbers in the dot plot represent the proportion of the indicated cell populations.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.All data are representative of at least three independent experiments.

4
FIGURE 4 Deficiency of CD11c hi DCs affects the proportion of CD4 + Foxp3 + T reg cells under tumor-bearing conditions.WT mice and CD11c:DTA mice were inoculated with B16-OVA.Cell surface expression profile (A, D, G), proportion (B, E, H), and absolute number (C, F, I) of CD4 + Foxp3 + T cells and CD4 + Foxp3 + RORgt + T cells among CD4 + T cells in tumor tissues (A-C), TdLNs (D-F), and Spl (G-I) on days 18-21 after tumor inoculation.Data are obtained from five to seven individual samples in a single experiment.Numbers in the dot plot represent the proportion of the indicated cell populations.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.All data are representative of at least three independent experiments.

FIGURE 5
FIGURE 5 Deficiency of CD11c hi DCs affects the formation of the TME.WT mice and CD11c:DTA mice were inoculated with B16-OVA.Transcriptional expressions of the immunosuppressive molecules in tumor tissues on days 18-21 after tumor inoculation.Data are obtained from five individual samples in a single experiment.*P < 0.05 compared with WT mice by two-sided unpaired Student's t-test.All data are representative of at least three independent experiments.