Conventional dendritic cells (cDCs) include functionally and phenotypically diverse populations, such as cDC1s and cDC2s. The latter population has been variously subdivided into Notch-dependent cDC2s, KLF4-dependent cDC2s, T-bet + cDC2As and T-bet − cDC2Bs, but it is unclear how all these subtypes are interrelated and to what degree they represent cell states or cell subsets. All cDCs are derived from bone marrow progenitors called pre-cDCs, which circulate through the blood to colonize peripheral tissues. Here, we identified distinct mouse pre-cDC2 subsets biased to give rise to cDC2As or cDC2Bs. We showed that a Siglec-H + pre-cDC2A population in the bone marrow preferentially gave rise to Siglec-H − CD8α + pre-cDC2As in tissues, which differentiated into T-bet + cDC2As. In contrast, a Siglec-H − fraction of pre-cDCs in the bone marrow and periphery mostly generated T-bet − cDC2Bs, a lineage marked by the expression of LysM. Our results showed that cDC2A versus cDC2B fate specification starts in the bone marrow and suggest that cDC2 subsets are ontogenetically determined lineages, rather than cell states imposed by the peripheral tissue environment.

Plasmacytoid dendritic cells (pDCs) are the main source of type I interferon (IFN-I) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DCs (tDCs), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. In the present report, we present a reporter mouse, pDC-Tom, designed through intersectional genetics based on unique Siglech and Pacsin1 coexpression in pDCs. The pDC-Tom mice specifically tagged pDCs and, on breeding with Zbtb46GFP mice, enabled transcriptomic profiling of all splenic DC types, unraveling diverging activation of pDC-like cells versus tDCs during a viral infection. The pDC-Tom mice also revealed initially similar but later divergent microanatomical relocation of splenic IFN+ versus IFN- pDCs during infection. The mouse models and specific gene modules we report here will be useful to delineate the physiological functions of pDCs versus other DC types.

In the thymus, T cell progenitors differentiate in order to generate naive T lymphocytes which migrate in the periphery where they will fulfill their function in the adaptive immune response. During thymopoiesis, genomic alterations in thymocytes can promote leukemia development. Among recurrent alteration is PTEN inactivation, which is associated to MYC overexpression. Herein, we used conditional Pten and Myc knockout mouse models and single-cell RNA-sequencing approach, to investigate the impact of MYC loss on physio-pathological development of PTEN-proficient or PTEN-deficient T lymphocytes. First, our results confirm that MYC is mandatory for PTEN loss-mediated leukemogenesis, while it is not required for terminal steps of thymopoiesis. In contrast, we uncovered that Myc ablation in CD4+CD8+ thymocytes disrupts T lymphocytes homeostasis in the spleen, notably by drastically reducing the number of MYC-deficient effector/memory T cells. Collectively, our data show that besides naive T cells proliferation, MYC is essential for effector/memory differentiation.

Whooping cough is a severe, highly contagious disease of the human respiratory tract, caused by Bordetellapertussis. The pathogenicity requires several virulence factors, including pertussis toxin (PTX), a key component of current available vaccines. Current vaccines do not induce mucosal immunity. Tissue-resident memory T cells (Trm) are among the first lines of defense against invading pathogens and are involved in long-term protection. However, the factors involved in Trm establishment remain unknown. Comparing two B.pertussis strains expressing PTX (WT) or not (ΔPTX), we show that the toxin is required to generate both lung CD4+ and CD8+ Trm. Co-administering purified PTX with ΔPTX is sufficient to generate these Trm subsets. Importantly, adoptive transfer of lung CD4+ or CD8+ Trm conferred protection against B. pertussis in naïve mice. Taken together, our data demonstrate for the first time a critical role for PTX in the induction of mucosal long-term protection against B. pertussis.

Specific antigen recognition by T cell receptor (TCR) activates TCR signaling pathway, leading to T cell proliferation and differentiation into effector and memory cells. Herein, we describe protocols for TCR stimulation assays, including procedures for the isolation and enrichment of mouse splenic T cells for ex vivo TCR stimulation with anti-CD3/CD28 antibodies, and the use of ovalbumin-OT-II mouse model for in vivo TCR stimulation. We applied this protocol to show that MYC protein is essential for T cell proliferation and differentiation.For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).

Atopic dermatitis (AD) is a chronic allergic dermatosis characterized by epidermal thickening and dermal inflammatory infiltrates with a dominant Th2 profile during the acute phase, whereas a Th1 profile is characteristic of the chronic stage. Among chemokines and chemokine receptors associated with inflammation, increased levels of CX(3)CL1 (fractalkine) and its unique receptor, CX(3)CR1, have been observed in human AD. We have thus investigated their role and mechanism of action in experimental models of AD and psoriasis. AD pathology and immune responses, but not psoriasis, were profoundly decreased in CX(3)CR1 deficient mice and upon blocking CX(3)CL1-CX(3)CR1 interactions in wild- type mice. CX(3)CR1 deficiency affected neither antigen presentation nor T cell proliferation in vivo upon skin sensitization, but CX(3)CR1 expression by both Th2 and Th1 cells was required to induce AD. Surprisingly, unlike in allergic asthma, where CX(3)CL1 and CX(3)CR1 regulate the pathology by controlling effector CD4(+) T cell survival within inflamed tissues, adoptive transfer experiments established CX(3)CR1 as a key regulator of CD4+ T cell retention in inflamed skin, indicating a new function for this chemokine receptor. Therefore, although CX(3)CR1 and CX3CL1 act through distinct mechanisms in different pathologies, our results further indicate their interest as promising therapeutic targets in allergic diseases.

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-β deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.

Millions of lymphocytes enter and exit mammal lymph nodes (LNs) each day, accessing the parenchyma via high endothelial venules (HEVs) and egressing via lymphatics. Despite this high rate of cellular flux and the many entry and exit sites within a given LN, the number of lymphocytes present in a resting LN is extraordinary stable over time, raising the question of how this steady-state is maintained. Here we have examined the anatomic details of lymphocyte movement in HEVs, finding that HEVs create pockets within which lymphocytes reside for several minutes before entering the LN proper. The function of these pockets was revealed in experiments performed under conditions in which lymphocyte egress from the LN was compromised by any of several approaches. Under such conditions, the HEVs pockets behaved as “waiting areas” in which lymphocytes were held until space was made available to them for entry into the parenchyma. Thus, rather than being simple entry ports, HEVs act as gatekeepers able to stack, hold and grant lymphocytes access to LN parenchyma in proportion to the rate of lymphocyte egress from the LN, enabling the LN to maintain a constant steady-state cellularity while supporting the extensive cellular trafficking necessary for repertoire scanning.

Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4+ T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.

Allergic asthma is a T cell-dependent inflammatory lung disease that results from complex interactions between genetic predisposition and environmental factors, including exposure to lipopolysaccharide (LPS). In this study, we have shown that airway LPS exposure was sufficient to induce airway hyperreactivity (AHR) and eosinophil recruitment in mice that had previously experienced an acute episode of allergic asthma. LPS-induced disease reactivation depended on the activation of allergen-specific CD4(+) T cells by a subset of lung langerin(+) dendritic cells (DCs) that retained the allergen. Upon LPS exposure, migration of langerin(+) DCs from lungs to draining lymph nodes increased and LPS-exposed langerin(+) DCs instructed CD4(+) T cells toward a T helper (Th) 2 response. Selective depletion of langerin(+) DCs prevented LPS-induced eosinophil recruitment and T-cell activation, further demonstrating a critical role for langerin + DCs in disease reactivation. This finding provides a possible explanation for the subclinical worsening of asthmatics following exposure to low-dose LPS.

Guanine nucleotide exchange factor activation of Rho G-proteins is critical for cytoskeletal reorganization. In the yeast Saccharomyces cerevisiae, the sole guanine nucleotide exchange factor for the Rho G-protein Cdc42p, Cdc24p, is essential for its site-specific activation. Several mammalian exchange factors have been shown to oligomerize; however, the function of this homotypic interaction is unclear. Here we show that Cdc24p forms oligomers in yeast via its catalytic Dbl homology domain. Mutation of residues critical for Cdc24p oligomerization also perturbs the localization of this exchange factor yet does not alter its catalytic activity in vitro. Chemically induced oligomerization of one of these oligomerization-defective mutants partially restored its localization to the bud tip and nucleus. Furthermore, chemically induced oligomerization of wild-type Cdc24p does not affect in vitro exchange factor activity, yet it results in a decrease of activated Cdc42p in vivo and the presence of Cdc24p in the nucleus at all cell cycle stages. Together, our results suggest that Cdc24p oligomerization regulates Cdc42p activation via its localization.