Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. Using two newly generated transgenic mice that report and trace the expression of terminal deoxynucleotidyl transferase (TdT), transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset that lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction on MPPs reflected a transcriptionally dynamic but uncommitted stage, characterized by low expression of lineage-associated genes. Single-cell CITE-seq indicated that multipotency in the TdT+ MPPs is associated with expression of the endothelial cell adhesion molecule ESAM. Stable and progressive upregulation of TdT defined the lymphoid developmental trajectory. Collectively, we here identify a new multipotent progenitor within the MPP4 compartment. Specification and commitment are defined by downregulation of ESAM which marks the progressive loss of alternative fates along all lineages.

Progenitors of distinct lineages shape the diversity of mature type 2 conventional dendritic cells.

Conventional dendritic cells (cDC) are antigen-presenting cells comprising cDC1 and cDC2, responsible for priming naive CD8+ and CD4+ T cells, respectively. Recent studies have unveiled cDC2 heterogeneity and identified various cDC2 progenitors beyond the common DC progenitor (CDP), hinting at distinct cDC2 lineages. By generating Cd300ciCre-hCD2R26tdTomato reporter mice, we identified a bone marrow pro-cDC2 progenitor exclusively generating cDC2 in vitro and in vivo. Single-cell analyses and multiparametric flow cytometry demonstrated that pro-cDC2 encompasses myeloid-derived pre-cDC2 and lymphoid-derived plasmacytoid DC (pDC)-like precursors differentiating into a transcriptionally convergent cDC2 phenotype. Cd300c-traced cDC2 had distinct transcriptomic profiles, phenotypes, and tissue distributions compared with Ms4a3CreR26tdTomato lineage-traced DC3, a monocyte-DC progenitor (MDP)-derived subset that bypasses CDP. Mice with reduced Cd300c-traced cDC2 showed impaired humoral responses to T cell-dependent antigens. We conclude that progenitors of distinct lineages shape the diversity of mature cDC2 across tissues. Thus, ontogenesis may impact tissue immune responses.

Malt1 Protease Deficiency in Mice Disrupts Immune Homeostasis at Environmental Barriers and Drives Systemic T Cell-Mediated Autoimmunity.

The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ-producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.

pDC-like cells are pre-DC2 and require KLF4 to control homeostatic CD4 T cells.

Plasmacytoid dendritic cells (pDCs) have been shown to play an important role during immune responses, ranging from initial viral control through the production of type I interferons to antigen presentation. However, recent studies uncovered unexpected heterogeneity among pDCs. We identified a previously uncharacterized immune subset, referred to as pDC-like cells, that not only resembles pDCs but also shares conventional DC (cDC) features. We show that this subset is a circulating precursor distinct from common DC progenitors, with prominent cDC2 potential. Our findings from human CD2-iCre and CD300c-iCre lineage tracing mouse models suggest that a substantial fraction of cDC2s originates from pDC-like cells, which can therefore be referred to as pre-DC2. This precursor subset responds to homeostatic cytokines, such as macrophage colony stimulating factor, by expanding and differentiating into cDC2 that efficiently prime T helper 17 (TH17) cells. Development of pre-DC2 into CX3CR1+ ESAM- cDC2b but not CX3CR1- ESAM+ cDC2a requires the transcription factor KLF4. Last, we show that, under homeostatic conditions, this developmental pathway regulates the immune threshold at barrier sites by controlling the pool of TH17 cells within skin-draining lymph nodes.

Requirement of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Protease Activity for Fcγ Receptor-Induced Arthritis, but Not Fcγ Receptor-Mediated Platelet Elimination, in Mice.

OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.

Accumulation of Multipotent Hematopoietic Progenitors in Peripheral Lymphoid Organs of Mice Over-expressing Interleukin-7 and Flt3-Ligand.

Interleukin-7 (IL-7) and Flt3-ligand (FL) are two cytokines important for the generation of B cells, as manifested by the impaired B cell development in mice deficient for either cytokine or their respective receptors and by the complete block in B cell differentiation in the absence of both cytokines. IL-7 is an important survival and proliferation factor for B cell progenitors, whereas FL acts on several early developmental stages, prior to B cell commitment. We have generated mice constitutively over-expressing both IL-7 and FL. These double transgenic mice develop splenomegaly and lymphadenopathy characterized by tremendously enlarged lymph nodes even in young animals. Lymphoid, myeloid and dendritic cell numbers are increased compared to mice over-expressing either of the two cytokines alone and the effect on their expansion is synergistic, rather than additive. B cell progenitors, early progenitors with myeloid and lymphoid potential (EPLM), common lymphoid progenitors (CLP) and lineage-, Sca1+, kit+ (LSK) cells are all increased not only in the bone marrow but also in peripheral blood, spleen and even lymph nodes. When transplanted into irradiated wild-type mice, lymph node cells show long-term multilineage reconstitution, further confirming the presence of functional hematopoietic progenitors therein. Our double transgenic mouse model shows that sustained and combined over-expression of IL-7 and FL leads to a massive expansion of most bone marrow hematopoietic progenitors and to their associated presence in peripheral lymphoid organs where they reside and potentially differentiate further, thus leading to the synergistic increase in mature lymphoid and myeloid cell numbers. The present study provides further in vivo evidence for the concerted action of IL-7 and FL on lymphopoiesis and suggests that extramedullary niches, including those in lymph nodes, can support the survival and maintenance of hematopoietic progenitors that under physiological conditions develop exclusively in the bone marrow.