PD-L1 surface expression can regulate the DC-T time of contact.

<p>(<b>A</b>) Cell surface expression of PD-1 (left panel) and CD40L (right panel) was analysed by FACS on OT1 T cells activated with coated anti-CD3 and soluble anti-CD28 at 1, 2 and 3 days post-activation. (<b>B</b>) Time of contact between DCs and T cells. DCs from wildtype or PD-L1 KO mice were pre-treated with nothing (control), polyI:C (25 μg/mL) or LPS (10 ng/ml) and co-cultured with <i>Ubi</i>-GFP OT-I T cells pre-activated for 2 days with anti-CD3/CD28. The percentage of T cells forming contacts of <600 mins is plotted for each condition. More than 50 contacts were measured for each condition, pValues determined by Z-test (<b>C</b>) The cumulative distribution of the T-DC measured contact times. Only contact with duration <600 min are shown in the graph. ****: p<0.0001 and *: p <0.05. n.s., non-significant. Data is representative of 2 independent experiments.</p

TLR3-Induced Maturation of Murine Dendritic Cells Regulates CTL Responses by Modulating PD-L1 Trafficking

<div><p>Targeting TLR3 through formulations of polyI:C is widely studied as an adjuvant in cancer immunotherapy. The efficacy of such targeting has been shown to increase in combination with anti-PD-L1 treatment. Nevertheless, the mechanistic details of the effect of polyI:C on DC maturation and the impact on T-DC interactions upon PD-L1 blockade is largely unknown. Here we found that although DC treatment with polyI:C induced a potent inflammatory response including the production of type I interferon, polyI:C treatment of DCs impaired activation of peptide specific CD8⁺ T cells mainly due to PD-L1. Interestingly, we found that PD-L1 trafficking to the cell surface is regulated in two waves in polyI:C-treated DCs. One induced upon overnight treatment and a second more rapid one, specific to polyI:C treatment, was induced upon CD40 signaling leading to a further increase in surface PD-L1 in DCs. The polyI:C-induced cell surface PD-L1 reduced the times of contact between DCs and T cells, potentially accounting for limited T cell activation. Our results reveal a novel CD40-dependent regulation of PD-L1 trafficking induced upon TLR3 signaling that dictates its inhibitory activity. These results provide a mechanistic framework to understand the efficacy of anti-PD-L1 cancer immunotherapy combined with TLR agonists.</p></div

PolyI:C pre-treatment impairs the capacity of DCs to cross-present antigen.

<p>(<b>A</b>) Degradation of bead-bound OVA was measured in DCs treated with nothing (Control) or polyI:C (25 μg/mL). Pre-treated DCs were pulsed with bead-bound OVA for 30 min at 18°C and chased for 0, 60, 120 or 240 min. The amount of OVA present on internalised beads was evaluated using an anti-OVA polyclonal antibody by FACS. (<b>B</b>) Cross-presentation of bead-bound OVA by DCs pre-treated with different concentrations of polyI:C for 20 h. After incubation with bead-bound OVA, cells were washed and incubated with B3Z T cells at a 1:1 ratio for 20 h. Proliferation was measured by detection of ß-galactosidase activity in the cells. (<b>C</b>) Presentation of the soluble OVA peptide. DCs were treated with nothing (Ctrl) or polyI:C (25 μg/mL) for 20 h and then loaded with different concentrations of the SIINFEKL peptide for 3 h, washed and incubated with B3Z T cells at a 1:1 ratio for 20 h. Proliferation of B3Z T cells was evaluated by detection of ß-galactosidase activity in the cells. Error bars represent SEM. ** p value < 0.001. Data are representative of 4 independent experiments.</p

PolyI:C pre-treatment impedes the capacity of DCs to elicit an antigen-specific cytotoxic T cell response.

<p>(<b>A-B</b>) DCs were either left non-treated (Ctrl) or treated with different concentrations of polyI:C or LPS as indicated for 20 h, washed, loaded with different doses of SIINFEKL peptide and incubated with CFSE-labelled OT1 CD8⁺ T cells at a 1:1 ratio. Three days later, the number of total CFSE-high and low positive T cells were enumerated by FACS-based cell counting. Data are presented as an absolute number of OT1 T cells present in each well. (<b>C</b>) OT1 T cells co-cultured for 3 days with polyI:C (25 μg/mL) or LPS (10 ng/ml) pre-treated DCs loaded with 4 pg/mL of SIINFEKL peptide were permeabilized and stained for IFNγ and Granzyme B. Numbers in the windows indicate the percentage of positive cells among total T cells in the well. The figures are representative of 2 independent experiments.</p

CD40L signalling induces the re-distribution of PD-L1 to the cell surface in polyI:C-treated DCs.

<p>(<b>A</b>) PD-L1 expression on treated DCs. DCs were treated with nothing (Control), polyI:C (25 μg/mL) or LPS (10 ng/ml) for 20 h followed by incubation with medium alone (no CD40L) or with rCD40L-Fc (500 ng/mL) for 90 min. PD-L1 expression was analysed by confocal microscopy. Scale bar is 10μm. Images are a single confocal plane. (<b>B</b>) Translocation of PD-L1 to the surface. DCs treated as above were incubated with rCD40L-Fc for the indicated times. Surface expression of PD-L1 (left panel) and CD86 (right panel) was evaluated by flow cytometry. Data is represented as a fold change over expression values at time 0 of non-treated (Ctrl) DCs. (<b>C</b>) DCs stimulated as above were incubated with celastrol (3 μM) for 1hr before addition of rCD40L-Fc for the indicated times. Surface expression of PD-L1 (left panel) and CD86 (right panel) were evaluated by FACS. Data in B and C are a compilation of 5 and 3 independent experiments respectively. Significance is calculated for each time point as compared to time 0 of CD40L treatment. *** represents p<0.001, * p<0,05, * p<0,05 by one-way anova test. Error bars represent SEM.</p

CD8+ T cells from PCC immunized mice protect naïve mice from EAE.

<p>A. Naïve C57BL/6 mice were immunized with PBS or 100µg of 50V PCC in CFA. Three weeks later, CD8+ T cells purified from the draining lymph nodes and spleen of the PBS immunized (CD8 PBS, squares) or PCC immunized (CD8 PCC, triangles) mice were adoptively transferred into naïve C57BL/6 recipients (CD8 PBS n = 10, CD8 PCC n = 10). Sixteen hours later, EAE was induced in the recipients upon subcutaneous immunization with MOG peptide accompanied by intravenous pertussis toxin on the day of EAE induction and 2 days later. B. Similar protocol was carried out with a second transfer of CD8+ T cells 9 days after EAE induction. The clinical course of EAE was analyzed using a paralysis grading score. Mean ± SEM. * indicates that the p-value<0.05 as assessed using ANOVA Fisher's PLSD. Arrows indicate the time points of CD8+ T cell transfer.</p

Regulatory CD8+ T cells prevent lymphocyte infiltration and CNS inflammation.

<p>Naïve mice were adoptively transferred with CD8+ T cells from PBS-immunized (CD8 PBS, n = 4) or PCC-immunized (CD8 PCC, n = 4) mice followed by EAE induction 16 hours later. Thirteen days after EAE induction, the mice were sacrificed and the lymphocyte populations in the brain and spinal cord were examined by flow cytometry. A. Absolute numbers of lymphocytes in the CNS gated according to their forward and side-scatter characteristics. B. MOG-specific CD4+ T cells were stained using MOG_38–49-I-Ab tetramers. Represented is the percentage of tetramer-positive cells among total CD4+ T cells in the CNS. C. Absolute numbers of CD4+ T cells that produce IL-17 in the CNS. D. Absolute numbers of IFNγ-producing CD4+ T cells in the CNS. Mean ± SEM. * indicates that the p-value<0.05 as assessed using Mann-Whitney non-parametric analysis.</p

Qa-1-binding molecular pattern.

<p>A. The alignment of previously described 9 amino acid-long Qa-1-binding peptides (Qdm, pre-proinsulin, HSP60 signal sequence derived peptide) using the Geneious software (ClustalW analysis). B. A consensus sequence with conserved amino acids in position (P) 1, 2, 5, 7 and 9 was revealed from this alignement. C. The search of such a consensus sequence in mouse Vß chains derived from the IMGT database revealed the presence of consenting 9 amino acid long peptides in the leader sequences of all mouse Vß chains. * Described as being a Qa-1-binding peptide in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021628#pone.0021628-Panoutsakopoulou1" target="_blank">[12]</a>.</p

Regulatory CD8+ T cells are restricted to Qa-1.

<p>Naïve C57BL/6 mice were immunized with PBS or 100µg of 50V PCC in CFA. Three weeks later, CD8+ T cells purified from the draining lymph nodes and spleen of PBS-immunized (CD8 PBS) and PCC-immunized (CD8 PCC) mice were adoptively transferred into either wildtype (WT, n = 7 for ‘CD8 PBS in WT’ and ‘CD8 PCC in WT’) or Qa-1-deficient mice (Qa-1°, n = 5 for ‘CD8 PBS in Qa-1°’ and n = 6 for ‘CD8 PCC in Qa-1°’). EAE was induced in the recipients 16 hours later. * indicates that the p-value<0.05 as assessed using ANOVA Fisher's PLSD.</p

Phenotypic and functional features of M1 and M2 macrophages.

<p><b>A</b>: The effect of an overnight IFNγ priming step was tested on C57Bl/6 mouse bone marrow-derived MØ subjected to M1- or M2-polarizing conditions. The expression of iNOS, ArgI, ArgII, and Ym1/2 were determined by real time RT-PCR on RNA extracted 10 hours after the induction of polarization. Data were calculated using the 2^−ΔΔCt Pfaffl formula <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008852#pone.0008852-Pfaffl1" target="_blank">[30]</a> in which experimental conditions (M1 and M2) are compared to Ct values obtained in M0 MØ and normalized to the Ct values of the HPRT house-keeping gene. <b>B</b>: Primary aortic vascular smooth muscle cells (VSMCs) from C57Bl/6 mice were cultured for 48 hours in the presence of media conditioned by C57Bl/6 M1 or M2 MØ which were polarized in the presence of the PPARγ agonist pioglitazone (Pio), the PPARγ antagonist GW9662 (GW), or the arginase inhibitor Nor-NOHA. As controls, VSMCs were also cultured with the same concentration of the polarizing agents, of the PPARγ agonists and antagonists, or of the arginase inhibitor. At the end of the assay, the number of viable cells in each condition was evaluated by the MTT assay and by using a standard curve established with known numbers of cells. *; **: p<0.05; p<0.01 vs matched medium conditioned by MØ polarized in the standard way (−). Note that the number of cells obtained with the M2-conditioned medium were significantly greater than with M1-conditioned medium (p<0.01 vs matched condition).</p