Negative Regulation of NKG2D Expression by IL-4 in Memory CD8 T Cells

International audienceIL-4 is one of the main cytokines produced during Th2-inducing pathologies. This cytokine has been shown to affect a number of immune processes such as Th differentiation and innate immune responses. However, the impact of IL-4 on CD8 T cell responses remains unclear. In this study, we analyzed the effects of IL-4 on global gene expression profiles of Ag-induced memory CD8 T cells in the mouse. Gene ontology analysis of this signature revealed that IL-4 regulated most importantly genes associated with immune responses. Moreover, this IL-4 signature overlapped with the set of genes preferentially expressed by memory CD8 T cells over naive CD8 T cells. In particular, IL-4 downregulated in vitro and in vivo in a STAT6-dependent manner the memory-specific expression of NKG2D, thereby increasing the activation threshold of memory CD8 T cells. Furthermore, IL-4 impaired activation of memory cells as well as their differentiation into effector cells. This phenomenon could have an important clinical relevance as patients affected by Th2 pathologies such as parasitic infections or atopic dermatitis often suffer from viral-induced complications possibly linked to inefficient CD8 T cell responses

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8α+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8α+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1−/− mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria

NOD1 Cooperates with TLR2 to Enhance T Cell Receptor-Mediated Activation in CD8 T Cells

<div><p>Pattern recognition receptors (PRR), like Toll-like receptors (TLR) and NOD-like receptors (NLR), are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR). This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions.</p> </div

NOD1 is expressed by CD8 T cells.

<p>NLR mRNA expression assessed by quantitative RT-PCR in murine CD8 T cells (black bars), macrophages (white bars) and splenocytes (grey bars) (nd: not detected). Results are the mean expression of NLR relative to HPRT ± SD of 3 independent experiments.</p

NOD1 cooperates with TLR2 to enhance TCR-mediated CD8 T cell activation.

<p>(A–C) Flow cytometry assessment of the proliferation (the percentage of proliferating cells is indicated within the histograms) (A), cell numbers (B) and CD25 expression (the mean fluorescence intensity of CD8 T cells is indicated within the histograms) (C) of CFSE stained murine CD8 T cells cultured for 48 h with anti-CD3, in the absence or presence of C12, Pam, or both C12 and Pam. (D–F) Determination of IL-2 (D), IFN-γ (E) and TNF-α (F) concentrations in the supernatants of CD8 T cells cultured for 48 h with anti-CD3, in the absence or presence of C12, Pam, or both C12 and Pam. (G) Determination by western blotting of IκBα, β-actin, Phospho-ERK (P-ERK), total ERK, Phospho-JNK (P-JNK), total JNK, Phospho-p38 (P-p38) and total p38 protein levels in F5 CD8 lymphoblasts cultured for 30 minutes in medium alone or with 1 nM of NP68, in the absence or presence of C12, Pam, or both C12 and Pam. (B) Cell number values are the mean fold increases of anti-CD3 stimulated CD8 T cell numbers in the different conditions, in comparison to the control condition anti-CD3 alone, ± SD from 4 independent experiments (** = p<0.01; Student <i>t</i> test). The other results are representative of 4 (A and C) or 3 (D, E, F and G) independent experiments.</p

NOD1 ligand directly increases TCR-activated CD8 T cell proliferation and effector functions.

<p>(A) Flow cytometry assessment of the proliferation of CFSE stained murine CD8 T cells cultured for 72 h with or without anti-CD3 antibody, in the absence or presence of a dose range of C12, anti-CD28 or TLR2 ligand Pam. The percentage of proliferating cells is indicated within the histograms. The column graph represents the mean fold increase of anti-CD3 stimulated CD8 T cell proliferation in the different conditions, in comparison to the control condition anti-CD3 alone, ± SD from 5 independent experiments. (B–E) Flow cytometry assessment of CD69 (B) expression by CD8 T cell after 20 h of culture and of CD25 (C), CD44 (D) and CD62L (E) expression after 48 h of culture in medium containing or not anti-CD3 antibody, in the absence (solid grey) or presence of C12, anti-CD28 or Pam (black lines). The column graphs represent the mean fold increase of anti-CD3 stimulated CD8 T cell expression level of the different activation markers in the different conditions, in comparison to the control condition anti-CD3 alone, ± SD from 3 independent experiments. (F–H) Determination of IL-2, IFN-γ and TNF-α concentrations in CD8 T cells supernatants following 48 h of activation with anti-CD3, in the absence or presence of C12, anti-CD28 or Pam. Results are the mean concentrations of cytokines determined ± SD from 3 independent experiments. (I) Flow cytometry assessment of the surface expression of CD107a by CD8 T cells activated for 72 h with anti-CD3 in the absence or presence of C12, anti-CD28 or Pam, and restimulated for 4 h with anti-CD3. The column graph represents the mean fold increase of CD8 T cell surface expression level of CD107a in the different conditions, in comparison to the control condition anti-CD3 alone, ± SD from 3 independent experiments. (* = p<0.05 and ** = p<0.01; Student <i>t</i> test).</p

C12 effect on activated CD8 T cells is NOD1- and RIP2- dependent, and is associated with activation of the NF-κB, JNK and p38 signaling pathways.

<p>(A–B) Flow cytometry assessment of the proliferation of CFSE stained WT, NOD1^−/−, RIP2^−/−, MyD88^−/− and TRIF^−/− CD8 T cells activated for 72 h with anti-CD3, in the absence or presence of C12 or Pam. (A) The percentage of proliferating cells is indicated within the histograms. (B) The column graph represents the mean fold increase of anti-CD3 stimulated CD8 T cell proliferation in the different conditions, in comparison to the control condition anti-CD3 alone, ± SD from 3 independent experiments. (C) Determination by western blotting of IκBα, β-actin, Phospho-ERK (P-ERK), total ERK, Phospho-JNK (P-JNK), total JNK, Phospho-p38 (P-p38) and total p38 protein levels in F5 CD8 lymphoblasts cultured for 30 minutes in medium alone or with 1 nM of their specific antigenic peptide, NP68, in the absence or presence of C12, anti-CD28 or Pam. Results are representative of 3 independent experiments.</p

NOD1 cooperates with TLR2 to enhance TCR-mediated activation in human CD8 T cells.

<p>Flow cytometry assessment of the (A) proliferation (the percentage of proliferating cells is indicated), (B) CD25 expression (the mean fluorescence intensity of CD8 T cells is indicated) and (C) cell numbers of CFSE stained human CD8 T cells activated for 72 h with anti-CD3 in the absence or presence of C12, Pam, or both C12 and Pam. Results are representative of 3 independent experiments. CD8 T cell numbers are the mean cell number ± SD of triplicates.</p

T inflammatory memory CD8 T cells participate to antiviral response and generate secondary memory cells with an advantage in XCL1 production

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