Fig 1: NRP1 is expressed in activated CD8+ T cells and controls their antitumoral function in mice(A) NRP1 expression and cell trace intensity analyzed by flow cytometry in OT1 murine CD8+ T cells activated during 24, 48, 72, and 96 h with OVA257 peptide pulsed on dendritic cells (SIINFEKL, 10-9 mg/mL). Data are representative of 5 independent experiments.(B) Flow cytometry analysis of NRP1 expression in OT1 murine CD8+ T cells, 72 h after activation with OVA peptide (SIINFEKL, 10-9 mg/mL). Expression is shown according to different effector CD8+ T cell / antigen-presenting cell (DC) ratios (E/A ratio: 1/1, 2/1, 4/1, and 8/1). p value (p = 0.0006) was determined by one-way ANOVA. Data are representative of 2 independent experiments. Expression was assessed by flow cytometry and data are presented as the mean ± SEM of percentage of NRP1 on CD8+ T cells (c) Expression of NRP1 in CD8+ T cells from B6 mice, after intramuscular immunization with AAV-OVA vector. Expression was assessed by flow cytometry and data are presented as the mean ± SEM of percentage of NRP1 on iTAg Tetramer/PE - H-2 Kb OVA, at day 7, 14, 21, 28, and 35 after immunization. Data are representative of 5 independent experiments.(D) NRP1 expression profiles in H2-Db GP33-specific CD8+ T cells according to in vivo infection in mice with LCMV Armstrong (n = 16), LCMV clone 13 (n = 16), or naïve CD44low CD8+ T cells from controls (n = 4) at days 6, 8, 15, and 30. Raw transcriptomic data were from Doering et al. (2012) microarray experiments [32]. Data are presented as the mean ± SEM p value was determined by two-way ANOVA(p = 0.0008).(E) Flow cytometry analysis of NRP1 expression (blue line curve) on iTAg Tetramer/PE - H-2 Kb OVA CD8+ TILs collected from C57BL/6 mice bearing a B16-OVA tumor at day 14 post-immunization with ovalbumin and poly-IC. Data are representative of 3 independent experiments.(F) Flow cytometry analysis of NRP1 and PD1 expression in OT1 CD8+ T cells activated with OVA257 peptide (SIINFEKL, 10-9M) at 24, 48, 72, and 96 h post-activation. Data are representative of 3 independent experiments.(H) Mice were pre-immunized (immunized) or not pre-immunized (control) with ovalbumin and poly-IC. B16-OVA tumor volume was assessed at day 0, 8, 11, 14, and 18 post-immunization in CD8Nrp1KO (KO) and control C8Cre (WT) mice. Data are presented as mean ± SEM. p values were determined by using student t-test ***p < 0.001, *p < 0.05. Data are representative of 3 independent experiments.(G) CD8Nrp1KO (KO) and control (WT) mice were injected in the right flank with 1 × 105 TC1 lung tumor cells subcutaneously. Data are presented as mean ± SEM p values was determined by using student t-test **p < 0.01, *p < 0.05. Data are representative of 3 independent experiments (I) CD8Nrp1KO (KO) and control (WT) mice were pre-immunized with ovalbumin and poly-IC. Tumor volume was assessed 21 days after immunization. Data are presented as mean ± SEM p values was determined by using student t-test (p = 0.0012). Data are representative of 3 independent experiments.(J) Number of CD8+ TILs per fields with highest CD8+ T cells infiltration from CD8Nrp1KO mice (KO) or controls (WT) assessed by confocal microscopy at day 21 post immunization with ovalbumin and poly-IC. Data are presented as mean ± SEM. p values (p < 0.0001) was determined by using student t-test. Data are representative of 3 tumors per group.(K) Percentages of Tetramer/PE - H-2 Kb OVA CD8+ TILs in B16-OVA tumors of four different mice group assessed at day 14 post-immunization by flow cytometry from CD8Nrp1KO (KO) and control (WT) mice immunized or not immunized (control) with ovalbumin and poly-IC. Data are presented as the mean percentage of CD8+ TILs Tetramer positive ± SEM. p values were determined by using student T test **p < 0.01, *p < 0.05. Data are representative of 2 independent experiments.(L) Ex vivo TILs proliferation was analyzed by flow cytometry 72 h post-activation with anti-CD3 and anti-CD28. TILs were collected from B16-OVA tumors at day 21 post-immunization from 3 mice. Data are presented as the mean percentage of divided CD8+ T cells ± SEM. p values was determined by using student t-test ***p < 0.001. Data are representative of 2 independent experiments.
Fig 2: The distribution of immune cells in the spleens, lymph nodes, and tumors was confirmed by flow cytometry. The presentation of activated effector CD8 and CD4 T cells, such as activated CD8+CD25+, CD8+CD69+, and CD8+CD44+ or CD4+CD25+, CD4+CD69+, and CD4+CD44+ in spleens (n = 6 for each treatment group), lymph nodes (n = 7 for each treatment group), and tumors (n = 6 for each treatment group) were also measured (A-D). Central memory and effector memory CD8+ and CD4+ T cells in spleens (CD44highCD62L- and CD44highCD62L+) (n = 6 for each treatment group), lymph nodes (CD44highCD62L- and CD44highCD62L+) (n = 7 for each treatment group), and tumors (CD44+CD62L- and CD44+CD62L+) (n = 6 for each treatment group) were also measured (E, F). The percentages of regulatory T cells (Tregs) (CD4+CD25+Foxp3+) in spleens (n = 7 for each treatment group), lymph nodes (n = 8 for each treatment group), and tumors (n = 6 for each treatment group) were determined (G, H). All data is shown as the mean ± standard deviation (SD). No treatment: no treatment group; Long pep: long multi-epitope peptide; Long pep+Lena: long multi-epitope peptide plus lenalidomide; Lena+anti-PD1: Lenalidomide plus anti-PD1; Long pep+Lena+anti-PD1: long multi-epitope peptide plus lenalidomide and anti-PD1; Cocktail pep+Lena+anti-PD1: cocktail of multi-epitope peptide plus lenalidomide and anti-PD1. p< 0.05 (*), p< 0.001 (**), p< 0.0001 (***).
Fig 3: NRP1 modulates PD1 activity at the synapse between CD8+ T cells and tumor cells(A) Illustrative image of phalloidin (yellow), CD8 (pink), CFP from EL4 (purple), and NRP1 (red) labeling in the synapse model between activated OT1 CD8+ T cells and EL4-CFP tumor cells bearing OVA257 (SIINFEKL), observed by ImageStream. The synapse is the high phalloidin labeling zone. Bright field image is in white (scale bar = 7µm). Data are representative of 4 independent experiments from 2 synapse models.(B) Quantification by ImageStream of NRP1 expression (mean pixel intensity/MPI) in an allogeneic synapse model between activated CD8+ T cells and cell tracer violet labeled A20 cells. NRP1 expression was analyzed in activated CD8+ T cells at the synapse junction (high phalloidin labeling zone). Data are presented as the mean MPI ± SEM. p value (p < 0.0001) was determined by Wilcoxon matched pairs test. Data are representative of 4 independent experiments from 2 synapse models.(C) Illustrative image of phalloidin (yellow), phospho-ZAP70 (green), CD8 (red), cell tracer violet labeled A20 tumor cells (purple), and NRP1 (white) between activated NRP1high or NRP1low CD8+ T cells and cell tracer violet labeled A20 tumor cells observed by ImageStream. The synapse is the high phalloidin labeling zone. Bright field image is in white (scale bar = 7µm). Data are representative of 2 independent experiments.(D) Quantification by Imagestream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in the synapse junction (high phalloidin labeling zone) between activated NRP1high or NRP1low CD8+ T cells and cell tracer violet labeled A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.(E) Left panel: CD8 (green) NRP1 (red), PD1 (blue), and NRP1/PD1 merge (purple) expression observed by confocal microscopy in CD8+ TILs from control mice (WT) at day 21 post-activation (x63 oil objective, scale bar = 10 µm). Data are representative of 3 tumors. Right panel: Colocalization of NRP1 and PD1 was assessed by the calculation of Pearson coefficient. Data from 10 CD8+ TILs analyzed are presented as mean ± SEM.(F) Phospho-ZAP70 signal according to its localization within the synapse between the CD8+ T cells and the tumor cells. Illustrative image of phalloidin (yellow), phospho-ZAP70 (green), CD8 (red), and Cell Tracer (A20 cells, purple) labeling in the synapse model between activated CD8+ T cells from CD8Nrp1KO mice (KO) or controls (WT) and allogeneic A20 tumor cells, by ImageStream. Bright field image is in white (scale bar = 7µm). Data are representative of 2 independent experiments.(G) Phospho-ZAP70 signal according to its localization within the synapse between the CD8+ T cells and the tumor cells. Quantification by Image stream of phospho-ZAP70 amounts (mean pixel intensity/MPI) in the synapse junction (high phalloidin labeling zone) between activated CD8+ T cells from CD8Nrp1KO mice (KO) or control mice (WT), and cell tracer violet labeled A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.(H) Proximity of NRP1 and PD1 proteins demonstrated by Duolink assay on in vitro activated CD8+ T cells from C57BL/6J mice. Upper panel: Left: Negative control experiments performed using anti-IRAP and anti-NRP1 antibodies (PLA-Duolink). Right: NRP1/PD1 complexes (anti-NRP1 and anti-PD1 antibodies with PLA-Duolink). The red spots indicate less than 40nm proximity between cellular-bound antibodies. Nuclei are stained with DAPI (blue). Images have been observed by confocal microscopy (x63 oil objective, scale bar = 10µm). Data are representative of 5 independent experiments. Lower panel: Comparison of number of PLA plots per cell. Data are presented as mean ± SEM.(I) NRP1 and PD1 interaction was demonstrated by CoIP experiments performed in splenocytes from C57BL/6J mice activated with anti-CD3 and anti-CD28 antibodies. NRP1 and PD1 immunoblot (IB) detection is shown in total lysate (TL) as control, in eluate from IgG Control (ctl) IP, and from NRP1 IP (N = 1 experiment). NRP1/PD1 Co-IP was also observed after PD1 IP (N = 2 experiment). Data are representative of 3 independent experiments.(J) Quantification by Imagestream of PD1 expression (MPI) in the synapse junction (high phalloidin labeling zone) between activated CD8+ T cells from CD8Nrp1KO mice (KO) or controls (WT), and allogeneic A20 tumor cells. Data are presented as mean MPI ± SEM. p value (p < 0.0001) was determined by Mann Whitney test. Data are representative of 2 independent experiments.
Fig 4: Adoptive Cell Transfer of Nr2f6-Deficient OT-II T Cells into Wild-Type Hosts Results in Increased Host CD4 Tfh, Th1, and Th2 Subsets(A) Experimental scheme showing adoptive transfer of 3 × 106 OT-II CD4+CD45.2+Nr2f6+/+ (black) or Nr2f6-/- (green) into Nr2f6+/+ CD45.1/CD45.2 congenic mice with OVA-alum immunization (n = 7 per group).(B) Total splenocytes, with Nr2f6+/+ or Nr2f6-/- OT-II adoptive cell transfer.(C) Total CD4 T cell counts, including both host and transferred OT-II cells.(D and E) Transferred OT-II T cell counts (D) and total host CD4 T cell counts (E).(F) Frequency of Th1 cells from the indicated OT-II populations or host cells (left and middle panels). Total host Th1 cells in the spleen with indicated OT-II T cell transfer (right panel).(G) Th2 as a frequency of OT-II or host CD4 T cell populations and total Th2 cells from the host CD4 T cells.(H) Frequency and total cell count of Th17 cells.(I) Treg subset defined as CXCR5- PD-1- CD4+ FoxP3+ from both OT-II transferred cells and host cells.(J) Tfh cells defined as CXCR5+ PD-1+ Foxp3- CD4+ from OT-II and host cells.Results shown are derived from three independent experiments (n = 7). The middle bar shows an average of each dataset. Error bars represent SD, and an asterisk indicates statistically significant differences between genotypes calculated using Student’s t test or Mann-Whitney U test. A p value of <0.05 was considered statistically significant. *p < 0.05; **p < 0.01.
Fig 5: Expression Analysis of TfH Markers by NKT Cells and Strategy for the Generation of CD1d-Floxed ESCs, Related to Figure 2(A) Flow cytometry gating strategy for the analysis of NKT cells (TCRß+ CD1d tetramer+), CD4+ T cells (TCRß+ CD1d tetramer- CD4+) and TfH cells (TCRß+ CD1d tetramer- CD4+ CXCR5+ PD-1+) in mediastinal lymph nodes at day 9 of influenza infection.(B) RT-qPCR analysis of IL-21 expression at day 3 and 9 of influenza infection by sorted NKT and TfH cells compared to CD4+ cells.(C and D) Representative contour plots show the expression of (C) Bcl-6 and (D) SLAM by NKT, TfH and CD4+ T cells at day 3 and 9 of infection. Quantification on the right charts shows the mean fluorescence intensity for (C) Bcl-6 and (D) SLAM.(E) Scheme showing the wild type and targeted CD1d locus containing two loxP sites flanking the exon 3 of CD1d as well as two FRT sites for the removal of the Neomycin resistance cassette.(F) Specific fragments amplified by long-range PCR for the 5' end (7.1kb) and 3' end (6.6kb) of the clones that correctly performed homologous recombination.(G) DNA from three positive clones was digested with SacI restriction enzyme and Southern blot performed using a labeled Southern probe. The upper band corresponds to the wild type locus (10.6kb) and the lower band to the targeted locus (7.3kb).(H) Southern blot of the three positive clones performed with a labeled Southern probe against the Neomycin cassette. Orange arrow indicates the single 5.2kb band recognized by this probe, indicating single insertion of the construct. The clone 2, in red, was chosen for injections.In all quantification charts, each dot represents one mouse. Data are representative from three experiments. Statistical analysis two-tailed Student’s t test; **p < 0.01, ***p < 0.001 and ****p < 0.0001.
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