Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

Dendritic cells (DCs) are key antigen-presenting cells that control the induction of both tolerance and immunity. Understanding the molecular mechanisms regulating DCs commitment toward a regulatory- or effector-inducing profile is critical for better designing prophylactic and therapeutic approaches. Initially identified in dexamethasone-treated thymocytes, the glucocorticoid-induced leucine zipper (GILZ) protein has emerged as a critical factor mediating most, but not all, glucocorticoids effects in both non-immune and immune cells. This intracellular protein exerts pleiotropic effects through interactions with transcription factors and signaling proteins, thus modulating signal transduction and gene expression. GILZ has been reported to control the proliferation, survival, and differentiation of lymphocytes, while its expression confers anti-inflammatory phenotype to monocytes and macrophages. In the past twelve years, a growing set of data has also established that GILZ expression in DCs is a molecular switch controlling their T-cell-priming capacity. Here, after a brief presentation of GILZ isoforms and functions, we summarize current knowledge regarding GILZ expression and regulation in DCs, in both health and disease. We further present the functional consequences of GILZ expression on DCs capacity to prime effector or regulatory T-cell responses and highlight recent findings pointing to a broader role of GILZ in the fine tuning of antigen capture, processing, and presentation by DCs. Finally, we discuss future prospects regarding the possible roles for GILZ in the control of DCs function in the steady state and in the context of infections and chronic pathologies.

Ag sampling is a key process in dendritic cell (DC) biology. DCs use constitutive macropinocytosis, receptor-mediated endocytosis, and phagocytosis to capture exogenous Ags for presentation to T cells. We investigated the mechanisms that regulate Ag uptake by DCs in the steady-state and after a short-term LPS exposure in vitro and in vivo. We show that the glucocorticoid-induced leucine zipper protein (GILZ), already known to regulate effector versus regulatory T cell activation by DCs, selectively limits macropinocytosis, but not receptor-mediated phagocytosis, in immature and recently activated DCs. In vivo, the GILZ-mediated inhibition of Ag uptake is restricted to the CD8α⁺ DC subset, which expresses the highest GILZ level among splenic DC subsets. In recently activated DCs, we further establish that GILZ limits p38 MAPK phosphorylation, providing a possible mechanism for GILZ-mediated macropinocytosis control. Finally, our results demonstrate that the modulation of Ag uptake by GILZ does not result in altered Ag presentation to CD4 T cells but impacts the efficiency of cross-presentation to CD8 T cells. Altogether, our results identify GILZ as an endogenous inhibitor of macropinocytosis in DCs, the action of which contributes to the fine-tuning of Ag cross-presentation.

Dendritic cells (DCs) are key players in the control of tolerance and immunity. Glucocorticoids (GCs) are known to regulate DC function by promoting their tolerogenic differentiation through the induction of inhibitory ligands, cytokines, and enzymes. The GC-induced effects in DCs were shown to critically depend on increased expression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ). GILZ expression levels were further shown to control antigen-presenting cell function, as well as T-cell priming capacity of DCs. However, the pattern of GILZ expression in DC subsets across tissues remains poorly described, as well as the modulation of its expression levels in different pathological settings. To fill in this knowledge gap, we conducted an exhaustive analysis of GILZ relative expression levels in DC subsets from various tissues using multiparametric flow cytometry. This study was performed at steady state, in the context of acute as well as chronic skin inflammation, and in a model of cancer. Our results show the heterogeneity of GILZ expression among DC subsets as well as the complexity of its modulation, that varies in a cell subset- and context-specific manner. Considering the contribution of GILZ in the control of DC functions and its potential as an immune checkpoint in cancer settings, these results are of high relevance for optimal GILZ targeting in therapeutic strategies.

Tumor infiltration by dendritic cells (DCs) has been identified as a biomarker associated with favorable outcomes and improved responses to immunotherapies by T cell immune checkpoint blockade. However, tumor induced immunosuppressive pathways contrive efficient DC infiltration. This calls for the development of DC-targeted interventions purposed to stimulate DC infiltration and local expansion. Here we deliver a proof-of-concept for a cell-based immunotherapeutic approach promoting circulating DC progenitors infiltration in the tumor together with their local expansion. We report that engraftment of autologous mesenchymal stromal cells engineered to express the membrane bound form of FLT3L (eMSC-FLT3L) uniquely induce DC infiltration, T cells activation and anti-tumor immunity, when combined with an adjuvant like the poly(I:C) double stranded RNA mimic. Mechanistic experiments demonstrate that poly(I:C)-induced CXCR3 and CCR5 ligands (CXCL9, CCL5) are required for intra-tumoral DC and T cell recruitment and for the therapeutic effect of eMSC-FLT3L + poly(I:C) immunotherapy. Stromal cell-based delivery of CXCL9 and CCL5, together with FLT3L, by-passes the requirement for poly(I:C) agonist and is sufficient to induce T cell and DC recruitment within tumors. These findings are transferrable to human settings as we show that eMSCs-FLT3L-CXCL9-CCL5 stimulate tissue infiltration by human XCR1⁺ DC1s in mice harboring a ‘Human Immune System’ (HIS mice). Lastly, we report that engraftment of eMSC-FLT3L-CXCL9-CCL5 in the tumor induces anti-tumor immunity and partially overcomes resistance to immune checkpoint blockade. Altogether, these data support the therapeutic potential of expanding intra-tumoral DC1s using a continuous and local source of FLT3L, CXCL9 and CCL5. One sentence summary Intra-tumoral delivery of FLT3L synergizes with CXCL9 and CCL5 to activate the expansion of intra-tumoral DCs.

Receptors for the Fc fragment of immunoglobulin G (FcyRs) are critical in the defense against pathogens and in monoclonal antibody-based therapies. When activated by immune complexes or opsonized particles, FcyRs are endocytosed. Components of the endocytosis machinery are used during autophagy, a process which is triggered by starvation or by activation of specific receptors. In this work, we demonstrate that activation of FcyRs initiates autophagy, characterized by formation of p62 protein puncta and activation of ULK1, a major component of the autophagy initiation complex. Autophagy induction downstream of FcyRs activation involves the protein phosphatase Pp2a and its enzymatic activity, as demonstrated by in situ protein labeling. In animal models in which autophagy was inactivated or enhanced in myeloid cells, autophagy negatively regulates pro-inflammatory cytokine production downstream of FcyRs receptors, while being required for FcyRs -mediated antibody-induced cell phagocytosis and myeloid cell survival. Our results suggest that, for antibody-based therapeutic strategies that target the activation of FcyRs, an additional level of control can be obtained by manipulation of autophagy.