Dendritic cell-targeted therapy expands CD8 T cell responses to bona-fide neoantigens in lung tumors.

Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage.

FLT3L-dependent dendritic cells control tumor immunity by modulating Treg and NK cell homeostasis.

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector T cells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.

Breast cancer remotely imposes a myeloid bias on haematopoietic stem cells by reprogramming the bone marrow niche.

Myeloid cell infiltration of solid tumours generally associates with poor patient prognosis and disease severity1-13. Therefore, understanding the regulation of myeloid cell differentiation during cancer is crucial to counteract their pro-tumourigenic role. Bone marrow (BM) haematopoiesis is a tightly regulated process for the production of all immune cells in accordance to tissue needs14. Myeloid cells differentiate during haematopoiesis from multipotent haematopoietic stem and progenitor cells (HSPCs)15-17. HSPCs can sense inflammatory signals from the periphery during infections18-21 or inflammatory disorders22-27. In these settings, HSPC expansion is associated with increased myeloid differentiation28,29. During carcinogenesis, the elevation of haematopoietic growth factors supports the expansion and differentiation of committed myeloid progenitors5,30. However, it is unclear whether cancer-related inflammation also triggers demand-adapted haematopoiesis at the level of multipotent HSPCs. In the BM, HSPCs reside within the haematopoietic niche which delivers HSC maintenance and differentiation cues31-35. Mesenchymal stem cells (MSCs) are a major cellular component of the BM niche and contribute to HSC homeostasis36-41. Modifications of MSCs in systemic disorders have been associated with HSC differentiation towards myeloid cells22,42. It is unknown if MSCs are regulated in the context of solid tumours and if their myeloid supportive activity is impacted by cancer-induced systemic changes. Here, using unbiased transcriptomic analysis and in situ imaging of HSCs and the BM niche during breast cancer, we show that both HSCs and MSCs are transcriptionally and spatially modified. We demonstrate that breast tumour can distantly remodel the cellular cross-talks in the BM niche leading to increased myelopoiesis.

Dendritic cell type 3 arises from Ly6C+ monocyte-dendritic cell progenitors.

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.

In Vitro Generation of Murine Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) are mononuclear phagocytes of hematopoietic origin residing in lymphoid and nonlymphoid tissues. DCs are often referred as the sentinels of the immune system as they can sense pathogens and danger signals. Upon activation, DCs migrate to the draining lymph nodes and present antigens to naïve T cells to trigger adaptive immunity. Hematopoietic progenitors for DCs reside in the adult bone marrow (BM). Therefore, BM cell culture systems have been developed to generate large amounts of primary DCs in vitro conveniently enabling to analyze their developmental and functional features. Here, we review various protocols enabling to generate DCs in vitro from murine BM cells and discuss the cellular heterogeneity of each culture system.

In Vitro Generation of Human Dendritic Cell Subsets from CD34+ Cord Blood Progenitors.

Dendritic cells (DCs) are professional antigen-presenting cells controlling the activation of T cells and thus regulating adaptive immune response against pathogens or tumors. Modeling human DC differentiation and function is crucial for our understanding of immune response and the development of new therapies. Considering DC rarity in human blood, in vitro systems allowing their faithful generation are needed. This chapter will describe a DC differentiation method based on the co-culture of CD34+ cord blood progenitors together with mesenchymal stromal cells (eMSCs) engineered to deliver growth factors and chemokines.

Tissue-resident FOLR2+ macrophages associate with CD8+ T cell infiltration in human breast cancer.

Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2+ tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2+ macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8+ T cells. FOLR2+ macrophages efficiently prime effector CD8+ T cells ex vivo. The density of FOLR2+ macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies.

Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

Harnessing Mesenchymal Stromal Cells for the Engineering of Human Hematopoietic Niches.

Tissue engineering opens multiple opportunities in regenerative medicine, drug testing, and modeling of the hematopoiesis in health and disease. Recapitulating the organization of physiological microenvironments supporting leukocyte development is essential to model faithfully the development of immune cells. Hematopoietic organs are shaped by spatially organized niches defined by multiple cellular contributions. A shared feature of immune niches is the presence of mesenchymal stromal cells endowed with unique roles in organizing niche development, maintenance, and function. Here, we review challenges and opportunities in harnessing stromal cells for the engineering of artificial immune niches and hematopoietic organoids recapitulating leukocyte ontogeny both in vitro and in vivo.

TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor responses.

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8+ T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8+ T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors.

Development and function of human dendritic cells in humanized mice models.

Dendritic cells (DCs) are sentinel cells of the immune system arising from hematopoietic stem cells. DCs play a key role in the regulation of both adaptive and innate lymphocyte responses. As such, experimental models enabling a thorough analysis of human DCs development and function are needed. Humanized mice models (termed collectively as HIS mice, or human immune system mice models) provide unique opportunities to model human hematopoiesis and tackle the function of human immune cell types in vivo. Here, we review experimental approaches enabling to recapitulate the ontogeny of DC subsets in HIS mice and discuss studies addressing the biology of human DC subsets implementing HIS mice models.

Transcriptional and Functional Analysis of CD1c+ Human Dendritic Cells Identifies a CD163+ Subset Priming CD8+CD103+ T Cells.

Dendritic cells (DCs) are antigen-presenting cells controlling T cell activation. In humans, the diversity, ontogeny, and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DCs (called DC3s) as immediate precursors of inflammatory CD88-CD14+CD1c+CD163+FcεRI+ DCs. DC3s develop via a specific pathway activated by GM-CSF, independent of cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors. Like classical DCs but unlike monocytes, DC3s drove activation of naive T cells. In vitro, DC3s displayed a distinctive ability to prime CD8+ T cells expressing a tissue homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor ß (TGF-ß) signaling. In vivo, DC3s infiltrated luminal breast cancer primary tumors, and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Together, these findings define DC3s as a lineage of inflammatory DCs endowed with a strong potential to regulate tumor immunity.

IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses.

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation.

Engineered niches support the development of human dendritic cells in humanized mice.

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.

Phosphatase PTPN22 Regulates Dendritic Cell Homeostasis and cDC2 Dependent T Cell Responses.

Dendritic cells (DCs) are specialized antigen presenting cells that instruct T cell responses through sensing environmental and inflammatory danger signals. Maintaining the homeostasis of the multiple functionally distinct conventional dendritic cells (cDC) subsets that exist in vivo is crucial for regulating immune responses, with changes in numbers sufficient to break immune tolerance. Using Ptpn22-/- mice we demonstrate that the phosphatase PTPN22 is a highly selective, negative regulator of cDC2 homeostasis, preventing excessive population expansion from as early as 3 weeks of age. Mechanistically, PTPN22 mediates cDC2 homeostasis in a cell intrinsic manner by restricting cDC2 proliferation. A single nucleotide polymorphism, PTPN22R620W, is one of the strongest genetic risk factors for multiple autoantibody associated human autoimmune diseases. We demonstrate that cDC2 are also expanded in mice carrying the orthologous PTPN22619W mutation. As a consequence, cDC2 dependent CD4+ T cell proliferation and T follicular helper cell responses are increased. Collectively, our data demonstrate that PTPN22 controls cDC2 homeostasis, which in turn ensures appropriate cDC2-dependent T cell responses under antigenic challenge. Our findings provide a link between perturbations in DC development and susceptibility to a broad spectrum of PTPN22R620W associated human autoimmune diseases.

Origin and development of classical dendritic cells.

Classical dendritic cells (cDCs) are mononuclear phagocytes of hematopoietic origin specialized in the induction and regulation of adaptive immunity. Initially defined by their unique T cell activation potential, it became quickly apparent that cDCs would be difficult to distinguish from other phagocyte lineages, by solely relying on marker-based approaches. Today, cDCs definition increasingly embed their unique ontogenetic features. A growing consensus defines cDCs on multiple criteria including: (1) dependency on the fms-like tyrosine kinase 3 ligand hematopoietic growth factor, (2) development from the common DC bone marrow progenitor, (3) constitutive expression of the transcription factor ZBTB46 and (4) the ability to induce, after adequate stimulation, the activation of naïve T lymphocytes. cDCs are a heterogeneous cell population that contains two main subsets, named type 1 and type 2 cDCs, arising from divergent ontogenetic pathways and populating multiple lymphoid and non-lymphoid tissues. Here, we present recent knowledge on the cellular and molecular pathways controlling the specification and commitment of cDC subsets from murine and human hematopoietic stem cells.

The protein tyrosine phosphatase PTPN22 negatively regulates presentation of immune complex derived antigens.

A C1858T single nucleotide polymorphism within PTPN22 (which encodes PTPN22R620W) is associated with an enhanced susceptibility to multiple autoimmune diseases including type 1 diabetes and rheumatoid arthritis. Many of the associated autoimmune diseases have an autoantibody component to their pathology. Fc receptors (FcRs) recognise autoantibodies when they bind to autoantigens and form immune complexes. After immune complex binding and receptor crosslinking, FcRs signal via Src and Syk family kinases, leading to antigen uptake, presentation and cytokine secretion. Ptpn22 encodes a protein tyrosine phosphatase that negatively regulates Src and Syk family kinases proximal to immunoreceptor signalling cascades. We therefore hypothesised that PTPN22 regulates immune complex stimulated FcR responses in dendritic cells (DCs). Bone marrow derived DCs (BMDCs) from wild type (WT) or Ptpn22-/- mice were pulsed with ovalbumin:anti-ovalbumin immune complexes (ova ICs). Co-culture with WT OT-II T cells revealed that ova IC pulsed Ptpn22-/- BMDCs have an enhanced capability to induce T cell proliferation. This was associated with an increased capability of Ptpn22-/- BMDCs to present immune complex derived antigens and to form ova IC dependent DC-T cell conjugates. These findings highlight PTPN22 as a regulator of FcR mediated responses and provide a link between the association of PTPN22R620W with autoantibody associated autoimmune diseases.

Protein tyrosine phosphatase PTPN22 is dispensable for dendritic cell antigen processing and promotion of T-cell activation by dendritic cells.

The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice. Results indicated no differential ability of DCs to uptake antigen via macropinocytosis or receptor-mediated endocytosis. Antigen degradation and presentation was also equal as was WT T-cell conjugate formation and subsequent T-cell proliferation. Despite the likely presence of multiple phosphatase-regulated pathways in the antigen uptake, processing and presentation pathways that we investigated, we observed that Ptpn22 and the R619W autoimmune associated variant were dispensable. These important findings indicate that under non-inflammatory conditions there is no requirement for Ptpn22 in DC dependent antigen uptake and T-cell activation. Our findings reveal that perturbations in antigen uptake and processing, a fundamental pathway determining adaptive immune responses, are unlikely to provide a mechanism for the risk associated with the Ptpn22 autoimmune associated polymorphism.