Nrp-1 expression defines a highly activated CD8⁺ T cell phenotype.

Splenic CD8+ T cells from naïve C57BL/6 mice were stimulated with αCD3 and αCD28 in vitro. (A) Nrp-1, CD69 and PD-1 expression of CD8+ T cells was analyzed by flow cytometry after 0, 24, 48, 72 or 96 hours of in vitro stimulation with 1 μg/ml αCD3/αCD28. (B) Nrp-1 expression on gated CD8+ T cells (left) and the expression of activation-associated molecules was analyzed by flow cytometry on Nrp-1+ (black bars) and Nrp-1- CD8+ T cells (white bars) 48 hours after in vitro stimulation with 1 μg/ml, 0.5 μg/ml or 0.1 μg/ml αCD3/αCD28 and is summarized as mean ± SD. Experiments have been performed as technical duplicates of stimulated T cells from one mouse. Mean values of duplicates have been included as one data point that represents one biological replicate (cells from one mouse). (C) The stability of Nrp-1 expression after in vitro induction was analyzed by isolating Nrp-1+CD8+ T cells using FACS after 48 hours of stimulation. The cells were re-cultured with IL-2 and Nrp-1 expression was analyzed at 0, 24, 48, 72 and 96 hours after re-cultivation by flow cytometry as shown by exemplary contour plots. Data from one to two independent experiments with (A) n = 8 mice in total and (B) n = 3–8 mice in total and (C) one experiment with n = 3 mice in total are presented as mean values with SD. (A, B) Ordinary one-way ANOVA with Holm-Sidak’s or Tukey’s multiple comparisons test or (C) Kruskal-Wallis test with Dunn’s multiple comparisons test were used to test significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

T cell-specific ablation of Nrp-1 expression in Nrp-1KO mice.

Splenocytes from Nrp-1fl/fl x CD4crewt (WT, black bars) and Nrp-1fl/fl x CD4cretg (KO, white bars) littermates were cultured in vitro (unstimulated) and stimulated with αCD3 and αCD28 (stimulated) for 48 hours. Successful ablation of Nrp-1 expression in (A) Foxp3-CD4+ conventional T cells, (B) Foxp3+CD4+ Tregs and (C) CD8+ T cells was verified by flow cytometry and is shown as mean ± SD. Representative FACS plots including FMO controls are shown in the upper panels. Data from one experiment with n = 2 mice is depicted. (D) The percentages of CD8+ and CD4+ T cells in spleen of naïve Nrp-1fl/fl x CD4crewt (WT) and Nrp-1fl/fl x CD4cretg (KO) littermates were determined by flow cytometry. Data from 2–3 independent experiments with n = 9–12 mice in total are shown as mean ± SD. Each dot represents one animal. (TIF)</p

Cytokine secretion of sorted Nrp-1⁺CD8⁺ and Nrp-1^-CD8⁺ T cells re-stimulated with low concentrations of αCD3/αCD28.

MACS-sorted CD8+ T cells from spleen of C57BL/6 mice were stimulated in vitro with 1 μg/ml αCD3/αCD28 for 48h. Nrp-1+CD8+ and Nrp-1-CD8+ T cells were sorted by FACS and re-stimulated with 0.5 μg/ml or 0.1 μg/ml αCD3 plate-bound/αCD28 soluble for another 48h. The concentration of cytokines in the supernatant was determined by Luminex technology. Data from 2 independent experiments with n = 3 mice per experiment are summarized as mean ± SD. Statistical analysis was performed with One-Way ANOVA. *, p (TIF)</p

<i>Plasmodium berghei ANKA</i> infection induces Nrp-1 expression on CD8⁺ T cells that correlates with disease severity.

Nrp-1 expression on CD8+ T cells was analyzed by flow cytometry in naïve (0 days post infection) and PbA-infected C57BL/6 mice at day 0, 3, 4, 5 and 6 post infection. (A) A representative gating strategy, including fluorescence minus one (FMO) control, is shown for immune cells in the brain 5 days after PbA infection or from non-infected (d0) and PbA-infected mice (d6). The frequency of Nrp-1-expressing CD8+ T cells was analyzed in (B) spleen, (C) blood and (D, left) brain. (D, right) Absolute numbers of Nrp-1+CD8+ T cells are shown for the brain. (E) The percentages of Nrp-1 expressing cells among gated CD8+CD11+ antigen-experienced T cells were analyzed in spleen, blood and brain 6 days post PbA infection by flow cytometry. (F) Correlation was calculated between the frequency of cerebral CD8+ T cells (left) or Nrp-1+CD8+ T cells (right) during the course of PbA infection and the severity of experimental cerebral malaria (ECM) pathology assessed by the Rapid-Murine-Coma-and-Behavior-Scale (RMCBS) score. ECM pathology characterized by neurologic deficits results in a decline in score with increasing severity. (B-D) Results from one (day 3, 4, 5) or two (day 0 and 6) experiments with n = 5–11 mice per time point are summarized as mean (± SD). (E) Results from two experiments with n = 8–14 mice in total are shown as mean. Each dot represents one animal. Statistical analysis was performed with (C, D, E) nonparametric Kruskal-Wallis test and Dunn’s multiple comparisons test and with (B) ordinary one-way ANOVA and Holm-Sidak’s multiple comparisons test. *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001. (F) Correlation is shown based on n = 26 mice and statistical significance was calculated using nonparametric Spearman correlation test. P and r values are displayed in the graphs.</p

Antigen-specific Nrp-1⁺CD8⁺ T cells exhibit a more activated phenotype than Nrp-1^-CD8⁺ counterparts.

MACS-sorted CD8+ T cells isolated from spleen of OT-I mice were stimulated with indicated concentration of OVA in the presence of irradiated splenocytes as APCs for 48h. The expression of (A) Nrp-1 and (B) CD69, PD1 and GzmB on gated Nrp-1+CD8+ T cells and Nrp-1-CD8+ T cells was analyzed by flow cytometry. Results from three independent experiments with cells from n = 6–8 mice in total are summarized as mean ± SD. Statistical significance was calculated with One-Way ANOVA and Tukey`s multiple comparisons test. **, p (TIF)</p

Re-stimulated Nrp-1⁺CD8⁺ T cells maintained their high level of T cell activation.

CD8+ T cells from Nrp-1tdTomato reporter mice or from C57BL/6 mice were separated into Nrp-1tdtomato+ or Nrp-1+ fluorochrome-labeled antibody-stained (represented as black bars) and Nrp-1- not endogenously expressing tdTomato or negative for Nrp-1 fluorochrome-labeled antibody staining (white bars) by FACS sorting 48 hours after in vitro stimulation. Cells were then stimulated again with αCD3 and αCD28 for 48 hours and analyzed by flow cytometry. (A) Nrp-1 expression of Nrp-1+ or Nrp-1- CD8+ T cells after sorting (post-sort) and re-stimulation with indicated concentrations of αCD3 and αCD28 was measured by flow cytometry. (B and C) The activation state (CD44, PD-1), effector function (GzmB) and proliferation (Ki-67) of CD8+ T cells that were either Nrp-1+ or Nrp-1- prior to re-stimulation was analyzed 48 hours after re-stimulation with (B) 1 μg/ml, (C) 0.5 μg/ml or 0.1 μg/ml αCD3/αCD28 on gated CD8+ T cells by flow cytometry. (D) The supernatants of this re-cultivation (with 1 ug/ml αCD3/αCD28) were collected and the concentrations of cytokines were determined using Luminex technology. Data from two to three independent experiments with n = 5–8 mice in total are shown as mean ± SD. Depending on the sorted cell number of Nrp-1+ or Nrp-1- CD8+ T cells, some of the experiments have been performed as technical duplicates. In this case, the mean was calculated and plotted as one dot. (A, C) ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test, (B) Student’s t-test or (D) Mann-Whitney test were used to test significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

<i>Plasmodium berghei ANKA</i> infection is associated with neurological deficits and activation of T cells.

C57BL/6 mice were infected i.v. with 105 Plasmodium berghei ANKA (PbA) GFP+-infected erythrocytes. (A) The severity of experimental cerebral malaria (ECM) was assessed by the RMCBS score, which quantifies neurological deficits during development of ECM. Mice with a RMCBS score below 12 are considered to have ECM. (B) Parasitemia was determined by flow cytometry and calculated as the proportion of GFP+PbA-infected RBCs of total Ter119+ erythrocytes on day 3, 5 and 6 after PbA infection. (C) The frequency of PD-1-, GzmB- and IFN-γ-expressing CD8+ T cells was measured by flow cytometry in the spleen. Results from (A, B) n = 5 mice (d0, 3, 4, 5) from one experiment, n = 11 mice in total (d6) from two experiments, (C) n = 3–5 mice (d3, 4, 5, 6) from one experiment and n = 9–11 mice (d0) from three experiments are shown as mean + SD. Each dot represents one animal. Statistical significance was calculated with ordinary one-way ANOVA and Dunett`s multiple comparisons test. ***, p (TIF)</p

T cell numbers and T cell activation in spleen and blood are not altered in Nrp-1KO mice upon PbA infection.

Nrp-1fl/fl x CD4crewt (WT, black bars) and Nrp-1fl/fl x CD4cretg (KO, white bars) littermates were infected i.v. with 105 PbA-infected red blood cells (iRBCs) at day 0. Absolute numbers of CD8+ T cells, CD11a+CD8+ T cells and GzmB+CD8+ T cells in (A) spleen and (B) blood were determined at day 6 or 7 after infection by flow cytometry. Data from 2–4 independent experiments with n = 13–22 mice in total (A) or from 2 experiments with n = 7–8 mice in total (B) are depicted as mean ± SD. Each dot represents one animal. (TIF)</p

CD8⁺ T cells upregulate Nrp-1 expression during LCMV infection and T cell-specific Nrp-1 ablation ameliorates immunopathology.

C57BL/6 mice (A-D) or Nrp-1fl/fl x CD4cretg (KO, white bars) and Nrp-1fl/fl x CD4crewt littermates (WT, black bars) (E) were infected with 200 PFU LCMV-WE i.v. and T cell phenotypes were analyzed in the spleen 8 days post infection. (A) Nrp-1 expression on CD8+ T cells was measured by flow cytometry in the spleen and liver of naïve (white bar, grey dots) and LCMV-infected C57BL/6 mice (black bars, white dots). (B) The correlation of the frequency of Nrp-1+CD8+ T cells in the spleen with serum LDH and AST concentrations was determined in C57BL/6 mice infected with 200 PFU LCMV-WE, 200 PFU LCMV-docile and 2x106 PFU LCMV-docile on day 8 post infection. (C and D) Expression of CD69, Lag-3, PD-1, GzmB and TNF-α was determined on Nrp-1+ (black bars) and Nrp-1- CD8+ T cells (white bars) (C) or Tet+CD8+T cells (D). (E and F) Nrp-1, GzmB, TNF-α or IFN-γ expression was determined on antigen-specific Tet+CD8+ T cells in the spleen (E) and in the liver (F) of infected Nrp-1WT or Nrp-1KO mice by flow cytometry. LDH concentrations were measured in the serum at day 8 post LCMV infection. Data from two independent experiments with n = 5–8 mice per group are shown as mean values with SD. Statistical significance was calculated with (A) ordinary one-way ANOVA and Tukey’s multiple comparisons test. (B) Correlation is based on two independent experiments with n = 18 mice and statistics were analyzed using Pearson correlation coefficients. P and r values are displayed in the graphs. (C-F) Significance was tested using unpaired Student’s t test. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Differential gene expression analysis reveals differences in T cell activation.

For analysis of differentially expressed genes, Nrp-1+ and Nrp-1-CD8+ T cells were sorted from spleens of PbA-infected C57BL/6 mice at day 6 post infection (n = 2–3), processed and analyzed by Clariom S microarray analysis. (A) The volcano plot summarizes 173 genes significantly downregulated (colored green) and 280 genes significantly upregulated (colored red) in Nrp-1+ compared to Nrp-1- CD8+ T cells. (B) Differential gene expression of selected genes of Nrp-1+CD8+ vs. Nrp-1-CD8+ T cells. (C) mRNA expression of selected genes was analyzed in sorted Nrp-1+CD8+ vs. Nrp-1-CD8+ T cells from spleens of C57BL/6 mice 6 days post PbA infection by qRT-PCR (n = 5 mice, each dot represents the mean of technical duplicates from one mouse). Data are summarized as mean ± SD and Student`s t-test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. (D) Enriched Gene Ontology Analysis was performed with GO-Term “Biological Process”. The gene ratio increased with perimeter and the adjusted p-value is shown as high in blue and low in red color of the dots.</p