Global Foxp3⁺ T cell numbers are reduced in HD patients.

<p>(A) Representative FACS analysis of CD4⁺CD25⁺Foxp3⁺T cells and their distinct subsets on PBMC from control (top) and HD patients (bottom). (B) CD4⁺CD25⁺Foxp3⁺T cell numbers are reduced in HD patients. (C to E) Among these gated cells, cytokine-secreting CD45RA⁻Foxp3^lo nonsuppressive (C), resting suppressive CD45RA⁺Foxp3^lo (D) and activated suppressive CD45RA⁻Foxp3^hi (E) cells, all these subsets are reduced in HD patients compared to controls. Bars represent the median. *, P<0.01; ****, P<0.0001.</p

Patient demographic characteristics.

<p>Two hemodialysed patients had glomerulonephritis and vascular nephropathy at the same time.</p

Both ILT and conventional T cells are equally deficient in HD and KD patients.

<p>(A) HD patients had no significant differences in their number of iNKT, MAIT, CD4⁺ and CD8⁺ T cells when compared to kidney transplanted (KT) patients. Bars represent the median. (B) CD4⁺CD25⁺Foxp3⁺T cell numbers are similarly reduced in HD as in KD patients. Among these gated cells, cytokine-secreting CD45RA⁻Foxp3^lo nonsuppressive, resting suppressive CD45RA⁺Foxp3^lo and activated suppressive CD45RA⁻Foxp3^hi cell levels, are also similar in HD and KD patients. (C and D) The percentage of IFN<b>γ</b>⁺, IL-4⁺ or IL-17⁺ cells among gated CD4⁺ (C) or CD8⁺ (D) T lymphocytes was assessed after 6 h stimulation with PMA and ionomycin. Box-and-whisker plots are used to represent the distributions. The bottom and the top of a box represent the 5th and 95th percentiles, and the bar in the box shows the median. (A and B) Bars represent the median. *, P<0.01.</p

CD4⁺ and CD8⁺ T cell numbers are reduced in HD patients.

<p>(A) CD4⁺ and CD8⁺T cell numbers are significant decreased in the peripheral blood of HD patients compared to healthy donors. Bars represent the median. (B) The percentage of IFN<b>γ</b>⁺ cells among gated CD8⁺ or CD4⁺ T lymphocytes or (C) the percentage of IL-4⁺ and IL-17⁺ cells among gated CD4⁺ T lymphocytes was assessed after 6 h stimulation with PMA and ionomycin. Box-and-whisker plots are used to represent the distributions. The bottom and the top of a box represent the 5th and 95th percentiles, and the bar in the box shows the median. *, P<0.01; **, P<0.001; ***, P<0.0005 versus controls. Representative FACS analysis of IL-4, IL-17 and IFN<b>γ</b> production by gated CD4⁺ (E) or CD8⁺ (F) T cells from health donor controls (top) or HD patients (bottom) are represented.</p

iNKT cell deficit in HD patients.

<p>(A) iNKT cells were double stained by anti-CD3 and the PBS57-loaded CD1d-tetramer. Representative FACS profile showing the percentage of iNKT and MAIT cells in control versus HD patients. (B and C) HD patients had significant low numbers (B) and percentage (C) of peripheral blood iNKT and MAIT cells when compared to healthy donors. Bars represent the median. (D) The frequency of CD4⁺ and CD8⁻ subsets respectively among gated iNKT and MAIT cells in control versus HD patients is represented. (E) The percentage of IL-4⁺ or IFN<b>γ</b>⁺ cells among gated iNKT lymphocytes was assessed after 5 h stimulation with PMA and ionomycin. Box-and-whisker plots are used to represent the distributions. The bottom and the top of a box represent the 5th and 95th percentiles, and the bar in the box shows the median. **, P<0.001; ***, P<0.0005; ****, P<0.0001 versus controls.</p

Activation of iNKT cells inhibits the VP2_122–130-specific CD8 T cell response in TMEV infected C57Bl/6 mice.

<p>Frequency (left) and absolute number (right) of the VP2_122–130-specific CD8 T cells in the spleen (A) and CNS (B) of C57Bl/6 mice 8 or 9 days after infection with 10⁴ pfu of TMEV. On the day of infection mice received a single injection of 4 µg of α-GalCer, OCH, or vehicle <i>i.p</i>. The magnitude of the TMEV-specific CD8 T cell response was determined by <i>ex vivo</i> IFN-γ ELISpot. To this end, mononuclear cells were restimulated or not with 10 µM of the H2-D^b-restricted VP2_122–130 epitope and IFN-γ spot-forming cells (SFC) were enumerated. (A) Data represents two individual experiments in which the organs of 4 mice/group were analysed individually. The mean of 8 individual mice ± SEM is presented. (B) Represents 2 independent experiments on pooled mononuclear cells purified from the CNS of 4 mice/group. Data represent the mean ± SEM.</p

Increased mortality and viral load in TCR Vα14 Tg mice infected with TMEV.

<p>(A) Kaplan-Meyer survival curve of Vα14 Tg C57Bl/6 mice (n = 21) and C57Bl/6 littermates (n = 19) infected with 10⁴ pfu of TMEV. (B) TMEV viral load in the spinal cord 8 days after infection of Vα14 Tg C57Bl/6 mice (n = 13) and C57Bl/6 littermates (n = 19). (C) TMEV viral load in the spinal cord 45 days after infection of Vα14 Tg C57Bl/6 mice (n = 19) and C57Bl/6 littermates (n = 8). Data represent the pooled observations of 2–3 independent experiments.</p

iNKT cells reside in the CNS in the absence of inflammation.

<p>(A) Mononuclear cells were isolated from the brain and spinal cord (left), spleen (middle), and liver (right) of unmanipulated adult C57Bl/6 (top) or Jα18^-/- C57Bl/6 mice (bottom) by percoll gradient. A representative CD1d:α-GalCer tetramer staining on mononuclear cells is presented. (B) Frequency of CD1d:α-GalCer tetramer positive cells among αβ T cells in the liver (n = 6), spleen (n = 7), peripheral blood (n = 5), CNS (n = 7, 3–16 pooled tissues/observation). (C) Absolute numbers of CD1d:α-GalCer tetramer positive αβ T cells in the liver (n = 5) and CNS (n = 7, 3–16 pooled tissues/observation) among percoll purified mononuclear cells. Data represent the mean ± SEM of n individual mice per group.</p

iNKT cells and CD8 T cells in the CNS of TMEV-infected mice express a cytotoxic phenotype.

<p>CD1d:α-GalCer tetramer⁺ TCRβ⁺ iNKT cells (A) and TCRβ⁺ CD8⁺CD4⁻ (B) T cells were FACS purified from the CNS of Vα14 Tg mice 8 days after TMEV infection. Real-time PCR analysis was performed on mRNA from purified iNKT cells and CD8 T cells from the spleen (white bars) and CNS (black bars). Cells were purified (black bars) from CNS-infiltrating mononuclear cells of 6–8 pooled Vα14 Tg mice or (white bars) from individual spleens. Data represent the mean ± SD of 2 individual experiments (CNS) or the mean of 4 individual mice ± SD (spleen).</p