CD85j (Leukocyte Ig-Like Receptor-1/Ig-Like Transcript 2) Inhibits Human Osteoclast-Associated Receptor-Mediated Activation of Human Dendritic Cells

Abstract Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated γ-chain, induced Ca2+ flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs

Redundant Notch1 and Notch2 Signaling Is Necessary for IFNγ Secretion by T Helper 1 Cells During Infection with Leishmania major

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4+ T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4+ T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4+ T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2ΔCD4Cre) were infected with the protozoan parasite Leishmania major. N1N2ΔCD4Cre mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4+ T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4+ T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection

Caractérisation moléculaire et fonctionnelle de hOSCAR, nouveau récepteur associé à la chaîne FcRg (rôle dans la présentation antigénique et l'activation des cellules myéloïdes)

Le système immunitaire est régulé par des récepteurs activateurs et inhibiteurs, dont ceux signalant respectivement par des motifs à tyrosine, les ITAM et ITIM. hOSCAR est un nouveau récepteur exprimé uniquement par les cellules myéloïdes et associé à la chaîne à ITAM, FcRg. Dans les cellules dendritiques (DC), l'engagement d'hOSCAR permet l'endocytose et la présentation antigénique en CMH de classe II. De plus, hOSCAR est capable de déclencher un programme de maturation des DC (marqueurs d'activation, production de cytokines/chimiokines). L'engagement d'hOSCAR en combinaison avec des ligands de TLR augmente la capacité des DC à stimuler les cellules T naïves, module le profil des cytokines produites par les DC et peut influencer l'orientation Th1. hOSCAR est également capable d'activer la réponse pro-inflammatoire des monocytes et neutrophiles. hOSCAR est donc un nouveau récepteur activateur impliqué dans l'initiation et la modulation des réponses des immunités innée et adaptativeLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Ly49D-mediated ITAM signaling in immature thymocytes impairs development by bypassing the pre-TCR checkpoint.

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRβ-chain. High levels of CD5 were expressed on ic TCRβ(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRβ(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint

N1 and N2 alone can drive CD4⁺ Th1 differentiation.

<p>(A) N1^ΔCD4Cre, N2^ΔCD4Cre, N1N2^ΔCD4Cre, and control mice were infected with 3×10⁶<i>L. major</i> promastigotes and lesion size measured weekly. Data are represented as the mean of lesion size ± SEM with n≥3 mice per group. (B) Parasite load in the lesion was assessed by LDA 6 weeks after infection. Mean parasite number is given ± SEM (n≥3 mice per group) (C, D) Six weeks after infection, IFNγ (C), IL-4 and IL-13 (D) secretion was assessed in supernatant of dLN cells restimulated or not with UV-irradiated <i>L. major</i> for 72 h. Histograms show the mean cytokine secretion ± SEM (n≥3 mice per group). n.d. not-detectable, n.s. not significant. * p-value<0.05 versus control mice.</p

N1N2^ΔCD4Cre mice transcribe T-bet and IFNγ in dLN CD4⁺ T cells but do not secrete it.

<p>(A) Proliferation of CD4⁺ T cells was assessed by FACS. Draining LN cells of <i>L. major</i>-infected mice were isolated 6 weeks after infection, stained with CFSE and restimulated with UV-treated <i>L. major</i> for 72 h. Representative flow cytometry plots gated on CD4⁺ T cells are shown. Numbers in plots represent the mean percentage of proliferating cells ± SEM for 5 mice. (B) Intracellular levels of IFNγ were analysed by FACS in <i>L. major</i>-infected dLN cells restimulated for 72 h with UV-treated <i>L. major</i>. Representative flow cytometry plots are given. Numbers in plots represent the mean percentage of IFNγ⁺ cells within CD4⁺ T cells ± SEM for 5 mice. (C) Draining LN CD4⁺ T cells from N1N2^ΔCD4Cre and N1N2<i>^lox/lox</i> mice were sorted by FACS 21 days post <i>L. major</i> infection, T-bet and IFNγ mRNA levels were analyzed by quantitative RT-PCR. Data are represented as the mean ± SEM mRNA transcript levels normalized to HPRT mRNA levels (n≥3 mice per group). (D) Phosphorylation of STAT1 was assessed by FACS on dLN cells of N1N2^ΔCD4Cre and N1N2<i>^lox/lox</i> mice 3 weeks post infection. Naive mice were used as control. Representative flow cytometry plots gated on CD4⁺ T cells are shown. Numbers in quadrants indicate the mean frequency of pSTAT1⁺CD4⁺ T cells ± SEM. pSTAT1 mean fluorescence intensity MFI ± SEM is shown (n≥3 mice per group). (E) Draining LN cells of <i>L. major</i>-infected mice were restimulated <i>ex vivo</i> with PMA/ionomcyin for 4 h and level of intracellular IFNγ was assessed by FACS. The frequency of CD4⁺IFNγ⁺ T cells is given ± SEM for n≥3 mice per group. (F) mRNA expression of IL-13 and IL-5 was analyzed by quantitative real-time PCR in dLN cells isolated from N1N2^ΔCD4Cre and N1N2<i>^lox/lox</i> mice 6 weeks post <i>L. major</i> infection. Results are given as mean mRNA expression relative to HPRT ± SEM for n≥3 mice per group. Data are representative of 2–3 individual experiments. * p-value<0.05 versus control mice.</p