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 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

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

The Bfl-1-α9 peptide induces mitochondrial permeabilization through a membrane-destabilizing mechanism.

<p>Bfl-1-α9 peptide was incubated for the indicated times with mitochondria at lower concentrations (0.5 µM and 2.5 µM, top panels) and previously used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a> (10 µM and 25 µM, bottom panels). The release of cytochrome c and the expression of MitoHsp70 were monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a>, combined with the detection of the external membrane associated protein hexokinase 1 (HK1) and the matrix-contained protein MnSOD.</p

Subcellular localization of the GFP-tagged, Bfl-1/Bax-derived (poly)peptides.

<p>MEF (left panels) and MEF DKO (right panels) cells were co-transfected with mitoDsRed plasmid (encoding DsRed2 fused to the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase) and the GFP-tagged constructs. Subcellular distribution was analyzed by confocal microscopy 24 h after transfection. Confocal images showing GFP (green) and MitoDsRed (red) fluorescence. The DNA staining dye Topro-3 (blue) was used to visualize the nuclei. In merged images, the yellow color shows the co-localization of GFP and MitoDsRed at mitochondria. Scale bar, 10 µm.</p

μ-calpain cleaves Bfl-1 at two major sites in its N-terminus and releases a large C-terminal fragment with cytotoxic activity.

<p>(A) GST-Bfl-1(1–151) was digested with recombinant µ-calpain <i>in vitro</i> and the fragments were separated by SDS-PAGE (left panel). Bands corresponding to cleaved products (arrows) were analyzed by mass spectrometry (MS). A higher concentration of recombinant GST-Bfl-1(1–151) was treated with µ-calpain, products were separated by SDS-PAGE and blotted to PVDF (right panel). A sub-band (asterisk) was excised and subjected to Edman degradation, which indicated that Bfl-1 had been N-terminally cleaved between residues F71 and N72. (B) Schematic representation of the wild type Bfl-1 protein showing the location of the identified μ-calpain cleavage sites (asterisks, upper sequence), of mutant Bfl-1 protein with a 6 aminoacids deletion surrounding the two cleaved residues (Bfl-1DD, middle sequence), and of mutant Bfl-1 in which the region overlapping the first cleavage site was swapped with a structurally homologous region in Bcl2L10 (Bfl-1SD, bottom sequence) (C) Confirmation of the two calpain cleavage sites identified in Bfl-1 using noncleavable mutants. 293T cell lysates expressing GFP-tagged Bfl-1 constructs were exogenously treated with μ-calpain. Lysates containing equal amount of GFP-tagged Bfl-1 proteins were separated by SDS-PAGE and analyzed by western blot with an anti-GFP antibody to detect the full length protein and N-terminal truncated fragments and with a polyclonal anti-Bfl-1 antibody to detect C-terminal truncated fragments. Upper and lower panels represent two independent experiments with different time of exposure. (D) BJAB cells were cultured with or without treatment with TNF/CHX in the presence or absence of the calpain inhibitor ALLN. Lysates were separated by SDS-PAGE and the presence of a cleaved fragment was assayed by western blot using an anti-Bfl-1 antibody. (E) Secondary structure of Bfl-1 in which the nine helices of the protein are represented by boxes along with the different BH domains (left panel).The two cleavage sites mapped in (A) are indicated. A comparison with the previously published µ-calpain cleavage site in Bax is also shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Wood1" target="_blank">[26]</a> (bottom sequence). 3D structure of Bfl-1 (2VM6) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a> and position of the different cleaved sites (red circles) are indicated. The different Bcl-2 Homology domains are colored in yellow (putative BH4), red (BH3), green (BH1) and blue (BH2). (F) FACS assays of Annexin V staining in HT1080 cells. Chimeric GFP constructs encoding GFP alone, or fusions of GFP with full-length Bfl-1 or Bax or with the various membrane-active α-helices corresponding to the C-terminal part of Bfl-1 or Bax, i.e. α5, α6 (PFD, pore forming domain) and α9 (FE, final exon) are represented. The α-helical topology of Bax and Bfl-1 corresponds to the structures solved in aqueous environment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Suzuki1" target="_blank">[50]</a>. Transfected cells were stained for phosphatidylserine exposure using Cy3-conjugated Annexin V and the percentage of apoptotic GFP-expressing cells was determined by FACS 24 hours post transfection (right panel). Death of GFP-expressing and staurosporine (STS)-treated cells were also monitored as controls. Graphs shown are representative of three independent experiments.</p

Bfl-1-derived peptides have different abilities to permeabilize the MOM of mitochondria isolated from cultured cells.

<p>Peptides were incubated at different concentrations (10 µM and 25 µM) with isolated mitochondria for the indicated times (5, 15, 30 and 60 min) and the release of cytochrome c was monitored by immunoblot (IB). MitoHsp70 (mHsp70) was used as control indicative of equal-loading and proper isolation of the pellet fraction containing mitochondria (Mito) in comparison to the supernatant fraction (SN). Cytochrome c release assays were performed using iBMKW2 (wild type) and iBMKD3 (double KO Bax/Bak) for all tested peptides. For Bfl-1-α5, wild type MEF and MEF DKO (Bax/Bak −/− double KO) cells were used in parallel.</p