Neutralization of CXCR3 ligands does not block preferential migration of CXCR3⁺ CD4⁺ T cells to the PerC.

<p>CXCR3⁺ and CXCR3⁻ CD4⁺ T cells isolated from donor animals were differentially labeled with CFSE or CMTMR, and mixtures containing equal numbers of cells were injected intravenously into naïve recipients pretreated with a cocktail of anti-CXCL9, anti-CXCL10 and anti-CXCL11 antibody or an appropriate isotype control. Eighteen hours after transfer, the ratio of transferred CXCR3⁺ cells to total transferred cells was determined in different organs by flow cytometry. Dots represent the ratio for individual animals. Horizontal lines indicate the mean for the group, and error bars correspond to the SEM. n.s. – not significant. Sp, spleen; PerC, peritoneal cavity.</p

The percentage of CXCR3⁺ CD4⁺ T cells is higher in PerC than in other organs.

<p>The percentage of CXCR3⁺ CD4⁺ T cells in different organs was determined by flow cytometry. (A) Cells isolated from naïve Balb/c animals were analyzed (n = 5). Each point represents an individual animal. (B) Cells isolated from 10 to 20 naïve Balb/c animals were pooled. Results from 8 independent experiments are presented. Error bars indicate SEM. cLN, cervical lymph node; mLN, mesenteric lymph node; PP, Peyer's patches; Sp, spleen; PerC, peritoneal cavity. (*) Statistically significant at P<0.05.</p

The PerC environment results in increased expression of CXCR3 on CD4⁺ T cells.

<p>(A) The expression level of CXCR3 on CD4⁺ cells from spleen (black line) or PerC (grey area) of BALB/c mice was determined by flow cytometry. (B) CXCR3⁺ and CXCR3⁻ CD4⁺ T cells were sorted from Sp of donor BALB/c mice and stained with CFSE. Cells were then adoptively transferred to naïve recipient animals by i.v or i.p. route. After 24 h, cells were re-isolated from spleens or peritoneal cavities and re-stained with an anti-CXCR3 antibody. The expression level of CXCR3 on CFSE⁺ cells was then analyzed by flow cytometry. The differences were statistically significant at P<0.001 (*), n.s. – not significant.</p

Peritoneal memory CD4⁺ T cells produce more cytokines than Sp derived cells.

<p>CXCR3⁺ CD62L^low CD44^high, CXCR3⁻ CD62L^low CD44^high and CXCR3⁻ CD62L^high CD44^low CD4⁺ T cells were sorted from Sp or PerC of C57BL/6 naïve (specific pathogen free) mice. Then, cells were re-stimulated with ionomycin and PMA and the expression of (A) IFNγ, (B) IL-4 and (C) IL-17 was measured by ELISA. Similar results were obtained in 2 independent experiments.</p

CXCR3⁺ CD4⁺ T cells preferentially migrate to the PerC.

<p>CXCR3⁺ and CXCR3⁻ CD4⁺ T isolated from donor animals were differentially labeled with CFSE or CMTMR, and mixtures containing equal numbers of cells were injected intravenously into naïve recipients. Eighteen hours after transfer, the ratio of transferred CXCR3⁺ cells to total transferred (CXCR3⁺+CXCR3⁻) cells was determined in different organs by flow cytometry. (A) CD4⁺ CXCR3⁺ and CD4⁺CXCR3⁻ T cells isolated from Sp of donor BALB/c mice. (B) CD4⁺ CXCR3⁺ and CD4⁺ CXCR3⁻ T cells isolated from PerC of donor BALB/c mice. (C) CD4⁺ CXCR3⁺ CD62L^low CD44^high and CD4⁺ CXCR3⁻ CD62L^low CD44^high T cells isolated from Sp of C57BL/6 donor mice. Dots represent the ratio for the individual animals analyzed. Similar results were obtained in three independent experiments. Horizontal lines indicate the mean for the group, error bars correspond to the SEM. (*) Statistical significance at P<0.001. cLN cervical lymph node; mLN, mesenteric lymph node; PP Peyer's patches; Sp, spleen; PerC, peritoneal cavity.</p

Th cell subsets are differentially distributed in different anatomic niches.

<p>The percentage of different Th cell subsets in CD4⁺ cell population were determined by FACS analysis in different organs (n = 5). (A) Th1 cells (IFNγ secreting cells), (B) Th2 cells (IL-4 secreting cells) and (C) Th17 cells (IL-17 secreting cells). The differences were statistically significant at P<0.001 (*). Similar results were obtained in 2 independent experiments. NALT, nasal associated lymphoid tissues; cLN, cervical lymph node; brLN, bronchial lymph nodes; mLN, mesenteric lymph node; PP, Peyer's patches; Sp, spleen; PerC, peritoneal cavity.</p

The environment of the PerC results in increased expression of IFNγ by CD4⁺ T cells.

<p>CXCR3⁺ CD62L^low CD44^high, CXCR3⁻ CD62L^low CD44^high and CXCR3⁻ CD62L^high CD44^low CD4⁺ T cells were sorted from Sp of naïve C57BL/6 mice and stained with CFSE. Then, cells were transferred to naïve recipient animals by i.v. or i.p. rout and re-isolated after 24 h. Cells were re-stimulated with ionomycin, PMA and brefeldinA, stained for (A) IFNγ, (B) IL-4 and (C) IL-17 production and further analyzed by flow cytometry. The percentage of cells producing the different cytokines was subsequently determined. Similar results were obtained in 2 independent experiments. The differences were statistically significant at P<0.01 (*).</p

NK1.1⁻ NKT cells also block Th17 differentiation.

<p>Naïve CD4⁺ cells were sorted from spleen of OTII animals and stained with CFSE. Cells were then co-cultured with DC under Th17 inducing conditions with the specific peptide (OVA323–339) and αGCPEG in the absence (left panels) or in the presence of either NK1.1⁺ (central panels) or NK1.1⁻ (right panels) NKT cells. Some cultures also received blocking antibodies against IL-4, IL-10 or IFNγ. After 4 days in culture, cells were stained for IL-17A production and analyzed by FACS. Results belong to one representative out of 3 independent experiments.</p

αGCPEG efficiently modulates the effect of other adjuvants.

<p>C57BL/6 mice were immunized with OVA co-administered with different combinations of LPS, Curdlan and αGCPEG. Control animals received PBS or OVA alone. After 2 boosts splenocytes were isolated from animals and cells (5×10⁵/well) were then cultured in ELISpot plates coated with anti-IL-17A antibody for 48 h. Subsequently, plates were incubated with detection antibody, developed and the numbers of spots were counted. From the presented data the background was subtracted. *, statistically significant (p<0.05). Results belong to one representative out of 3 independent experiments.</p

NKT cells block Th17 differentiation <i>in vitro</i> by soluble factors.

<p>Naïve CD4⁺ cells were sorted from spleen of OTII animals and stained with CFSE. Cells were then co-cultured with DC under Th17 inducing conditions together with a peptide encompassing the specifically recognized epitope (OVA323–339) in the presence of either αGCPEG or BPPcysPEG. After 4 days in culture the cells were stained for IL-17A production and analyzed by FACS. (A) To some cultures NKT cells were added (right panels). (B) To some cultures were added supernatants collected from 24 h cultures of DC in presence of αGCPEG (left panel) or co-cultures of DC and NKT cells in the presence of αGCPEG (right panel). Results belong to one representative out of at least 3 independent experiments.</p