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CD45RC isoform expression identifies functionally distinct T cell subsets differentially distributed between healthy individuals and AAV patients.

Ordonez L, Bernard I, L'faqihi-Olive FE, Tervaert JW, Damoiseaux J, Saoudi A - PLoS ONE (2009)

Bottom Line: Interestingly, AAV patients exhibit an increased proportion of CD45RC(low) CD4 T cells as compared to HC and SLE patients.In conclusion, we have shown that CD45RC expression divides human T cells in functionally distinct subsets that are imbalanced in AAV.Since this imbalance is stable over time and independent of several disease parameters, we hypothesize that this is a pre-existing immune abnormality involved in the etiology of AAV.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U563, Institut Fédératif de Recherche (IFR) 30, Hôpital Purpan and Université Paul Sabatier, Toulouse, France.

ABSTRACT
In animal models of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), the proportion of CD45RC T cell subsets is important for disease susceptibility. Their human counterparts are, however, functionally ill defined. In this report, we studied their distribution in healthy controls (HC), AAV patients and in Systemic lupus erythematous (SLE) patients as disease controls. We showed that CD45RC expression level on human CD4 and CD8 T cells identifies subsets that are highly variable among individuals. Interestingly, AAV patients exhibit an increased proportion of CD45RC(low) CD4 T cells as compared to HC and SLE patients. This increase is stable over time and independent of AAV subtype, ANCA specificity, disease duration, or number of relapses. We also analyzed the cytokine profile of purified CD4 and CD8 CD45RC T cell subsets from HC, after stimulation with anti-CD3 and anti-CD28 mAbs. The CD45RC subsets exhibit different cytokine profiles. Type-1 cytokines (IL-2, IFN-gamma and TNF-alpha) were produced by all CD45RC T cell subsets, while the production of IL-17, type-2 (IL-4, IL-5) and regulatory (IL-10) cytokines was restricted to the CD45RC(low) subset. In conclusion, we have shown that CD45RC expression divides human T cells in functionally distinct subsets that are imbalanced in AAV. Since this imbalance is stable over time and independent of several disease parameters, we hypothesize that this is a pre-existing immune abnormality involved in the etiology of AAV.

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Cytokine profile of human CD45RC CD4 T cell subsets.(A) Representative example of the purification of CD4 CD45RC T cell subsets. Results are shown as histograms for CD45RC expression on CD4 T cells before (left histogram) and after CD45RC subsets purification (right histograms). The values within the histograms represent the percentage of CD45RC T cell subsets. (B) Purified CD45RChigh (High) and CD45RClow (Low) CD4 T cell subsets, were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 mAbs. The supernatants were collected at 72 h of culture and analyzed for the presence of cytokines using the CBA kit and Elisa. The results obtained in 20 healthy individuals are presented as box plot diagrams. The p-values were calculated using the Wilcoxon matched-pairs test; *, p<0.05; **, p<0.02; ***, p<0.002. (C) For intracellular measurement of cytokines, purified CD4 CD45RChigh and CD45RClow T cells were stimulated and stained using FITC-labeled anti-IFN-γ mAb and PE-labeled anti-IL-4 or anti-IL-10 mAbs. The results are expressed as dot plot representing IFN-γ/IL-4 or IFN-γ/IL-10 production by CD4 T cell subsets. The values within the plots represent the fraction of CD4 T cells producing the indicated cytokine. The results are representative of three independent experiments.
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pone-0005287-g004: Cytokine profile of human CD45RC CD4 T cell subsets.(A) Representative example of the purification of CD4 CD45RC T cell subsets. Results are shown as histograms for CD45RC expression on CD4 T cells before (left histogram) and after CD45RC subsets purification (right histograms). The values within the histograms represent the percentage of CD45RC T cell subsets. (B) Purified CD45RChigh (High) and CD45RClow (Low) CD4 T cell subsets, were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 mAbs. The supernatants were collected at 72 h of culture and analyzed for the presence of cytokines using the CBA kit and Elisa. The results obtained in 20 healthy individuals are presented as box plot diagrams. The p-values were calculated using the Wilcoxon matched-pairs test; *, p<0.05; **, p<0.02; ***, p<0.002. (C) For intracellular measurement of cytokines, purified CD4 CD45RChigh and CD45RClow T cells were stimulated and stained using FITC-labeled anti-IFN-γ mAb and PE-labeled anti-IL-4 or anti-IL-10 mAbs. The results are expressed as dot plot representing IFN-γ/IL-4 or IFN-γ/IL-10 production by CD4 T cell subsets. The values within the plots represent the fraction of CD4 T cells producing the indicated cytokine. The results are representative of three independent experiments.

Mentions: In order to characterize the function of CD45RChigh and CD45RClow CD4 T cell subsets, we determined their cytokine profile. For this purpose, we purified these sub-populations from peripheral blood of 20 healthy individuals using magnetic beads. The purity was always higher than 92% (Fig. 4A). Purified CD45RC CD4 T cell subsets were then stimulated in vitro in an antigen-presenting cell independent system using plate bound anti-CD3 mAb in the presence of soluble anti-CD28 mAb. Initial experiments showed that the peak of cytokine production was reached after 3 days of stimulation (data not shown). Upon this in vitro stimulation, both T cell subpopulations proliferated equally well, but produced different cytokines (Fig. 4B). The type-1 cytokines, IL-2, TNF-α and IFN-γ, were produced by both subsets, but the CD45RChigh population produced more IL-2 (Fig. 4B). In contrast, IL-17, IL-10 and the type-2 cytokines (IL-4, IL-5) were mainly produced by the CD45RClow CD4 T cells (Fig. 4B). Similar results were also obtained when CD45RC subsets were highly purified by flow cytometry (>99%), thus excluding a possible contribution of contaminating cells in these differences (data not shown). Intracellular staining confirmed the above results and showed that IFN-γ was produced by both the CD45RChigh and CD45RClow subsets, while IL-4 and IL-10 producing cells were mainly contained within the CD45RClow subset (Fig. 4C). In addition, these experiments showed that the majority of IL-4 or IL-10 producing cells did not produce IFN-γ (Fig. 4C). We also analysed CD4 T cells according to the expression of CD45RA isoform. We showed that the majority of CD45RChigh subset expresses also high levels of CD45RA isoform. In contrast, the CD45RClow population is heterogeneous and contains both CD45RAhigh and CD45RAlow subsets (Fig. S1A, S1B). After stimulation with anti-CD3 and anti-CD28 mAbs, we showed that purified CD45RClowCD45RAhigh CD4 T cells and CD45RClowCD45RAlow CD4 T cells exhibited a similar pattern of cytokine production as total CD45RClow CD4 T cell subsets (Fig. S1C and S1D). Similar phenotypic and functional studies were obtained when CD45RB was used instead of CD45RA (data not shown). Since the CD45RAhigh (and CD45RBhigh) cells within the CD45RClow subset produced IL-4, IL-5 IL-10 and IL-17, we conclude that CD45RC expression is more reliable than CD45RA (and CD45RB) expression to identify human CD4 T cells that are responsible for type-2, IL-17 and regulatory cytokine production. Altogether, these data demonstrate that CD45RC expression identifies two human CD4 T cell subsets with different cytokine profiles. The CD45RChigh subset produce mainly type-1 cytokines while T cells responsible for IL-17, IL-10 and type-2 cytokine production are mainly contained within the CD45RClow subset.


CD45RC isoform expression identifies functionally distinct T cell subsets differentially distributed between healthy individuals and AAV patients.

Ordonez L, Bernard I, L'faqihi-Olive FE, Tervaert JW, Damoiseaux J, Saoudi A - PLoS ONE (2009)

Cytokine profile of human CD45RC CD4 T cell subsets.(A) Representative example of the purification of CD4 CD45RC T cell subsets. Results are shown as histograms for CD45RC expression on CD4 T cells before (left histogram) and after CD45RC subsets purification (right histograms). The values within the histograms represent the percentage of CD45RC T cell subsets. (B) Purified CD45RChigh (High) and CD45RClow (Low) CD4 T cell subsets, were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 mAbs. The supernatants were collected at 72 h of culture and analyzed for the presence of cytokines using the CBA kit and Elisa. The results obtained in 20 healthy individuals are presented as box plot diagrams. The p-values were calculated using the Wilcoxon matched-pairs test; *, p<0.05; **, p<0.02; ***, p<0.002. (C) For intracellular measurement of cytokines, purified CD4 CD45RChigh and CD45RClow T cells were stimulated and stained using FITC-labeled anti-IFN-γ mAb and PE-labeled anti-IL-4 or anti-IL-10 mAbs. The results are expressed as dot plot representing IFN-γ/IL-4 or IFN-γ/IL-10 production by CD4 T cell subsets. The values within the plots represent the fraction of CD4 T cells producing the indicated cytokine. The results are representative of three independent experiments.
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Related In: Results  -  Collection

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pone-0005287-g004: Cytokine profile of human CD45RC CD4 T cell subsets.(A) Representative example of the purification of CD4 CD45RC T cell subsets. Results are shown as histograms for CD45RC expression on CD4 T cells before (left histogram) and after CD45RC subsets purification (right histograms). The values within the histograms represent the percentage of CD45RC T cell subsets. (B) Purified CD45RChigh (High) and CD45RClow (Low) CD4 T cell subsets, were stimulated in vitro with plate-bound anti-CD3 and anti-CD28 mAbs. The supernatants were collected at 72 h of culture and analyzed for the presence of cytokines using the CBA kit and Elisa. The results obtained in 20 healthy individuals are presented as box plot diagrams. The p-values were calculated using the Wilcoxon matched-pairs test; *, p<0.05; **, p<0.02; ***, p<0.002. (C) For intracellular measurement of cytokines, purified CD4 CD45RChigh and CD45RClow T cells were stimulated and stained using FITC-labeled anti-IFN-γ mAb and PE-labeled anti-IL-4 or anti-IL-10 mAbs. The results are expressed as dot plot representing IFN-γ/IL-4 or IFN-γ/IL-10 production by CD4 T cell subsets. The values within the plots represent the fraction of CD4 T cells producing the indicated cytokine. The results are representative of three independent experiments.
Mentions: In order to characterize the function of CD45RChigh and CD45RClow CD4 T cell subsets, we determined their cytokine profile. For this purpose, we purified these sub-populations from peripheral blood of 20 healthy individuals using magnetic beads. The purity was always higher than 92% (Fig. 4A). Purified CD45RC CD4 T cell subsets were then stimulated in vitro in an antigen-presenting cell independent system using plate bound anti-CD3 mAb in the presence of soluble anti-CD28 mAb. Initial experiments showed that the peak of cytokine production was reached after 3 days of stimulation (data not shown). Upon this in vitro stimulation, both T cell subpopulations proliferated equally well, but produced different cytokines (Fig. 4B). The type-1 cytokines, IL-2, TNF-α and IFN-γ, were produced by both subsets, but the CD45RChigh population produced more IL-2 (Fig. 4B). In contrast, IL-17, IL-10 and the type-2 cytokines (IL-4, IL-5) were mainly produced by the CD45RClow CD4 T cells (Fig. 4B). Similar results were also obtained when CD45RC subsets were highly purified by flow cytometry (>99%), thus excluding a possible contribution of contaminating cells in these differences (data not shown). Intracellular staining confirmed the above results and showed that IFN-γ was produced by both the CD45RChigh and CD45RClow subsets, while IL-4 and IL-10 producing cells were mainly contained within the CD45RClow subset (Fig. 4C). In addition, these experiments showed that the majority of IL-4 or IL-10 producing cells did not produce IFN-γ (Fig. 4C). We also analysed CD4 T cells according to the expression of CD45RA isoform. We showed that the majority of CD45RChigh subset expresses also high levels of CD45RA isoform. In contrast, the CD45RClow population is heterogeneous and contains both CD45RAhigh and CD45RAlow subsets (Fig. S1A, S1B). After stimulation with anti-CD3 and anti-CD28 mAbs, we showed that purified CD45RClowCD45RAhigh CD4 T cells and CD45RClowCD45RAlow CD4 T cells exhibited a similar pattern of cytokine production as total CD45RClow CD4 T cell subsets (Fig. S1C and S1D). Similar phenotypic and functional studies were obtained when CD45RB was used instead of CD45RA (data not shown). Since the CD45RAhigh (and CD45RBhigh) cells within the CD45RClow subset produced IL-4, IL-5 IL-10 and IL-17, we conclude that CD45RC expression is more reliable than CD45RA (and CD45RB) expression to identify human CD4 T cells that are responsible for type-2, IL-17 and regulatory cytokine production. Altogether, these data demonstrate that CD45RC expression identifies two human CD4 T cell subsets with different cytokine profiles. The CD45RChigh subset produce mainly type-1 cytokines while T cells responsible for IL-17, IL-10 and type-2 cytokine production are mainly contained within the CD45RClow subset.

Bottom Line: Interestingly, AAV patients exhibit an increased proportion of CD45RC(low) CD4 T cells as compared to HC and SLE patients.In conclusion, we have shown that CD45RC expression divides human T cells in functionally distinct subsets that are imbalanced in AAV.Since this imbalance is stable over time and independent of several disease parameters, we hypothesize that this is a pre-existing immune abnormality involved in the etiology of AAV.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U563, Institut Fédératif de Recherche (IFR) 30, Hôpital Purpan and Université Paul Sabatier, Toulouse, France.

ABSTRACT
In animal models of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), the proportion of CD45RC T cell subsets is important for disease susceptibility. Their human counterparts are, however, functionally ill defined. In this report, we studied their distribution in healthy controls (HC), AAV patients and in Systemic lupus erythematous (SLE) patients as disease controls. We showed that CD45RC expression level on human CD4 and CD8 T cells identifies subsets that are highly variable among individuals. Interestingly, AAV patients exhibit an increased proportion of CD45RC(low) CD4 T cells as compared to HC and SLE patients. This increase is stable over time and independent of AAV subtype, ANCA specificity, disease duration, or number of relapses. We also analyzed the cytokine profile of purified CD4 and CD8 CD45RC T cell subsets from HC, after stimulation with anti-CD3 and anti-CD28 mAbs. The CD45RC subsets exhibit different cytokine profiles. Type-1 cytokines (IL-2, IFN-gamma and TNF-alpha) were produced by all CD45RC T cell subsets, while the production of IL-17, type-2 (IL-4, IL-5) and regulatory (IL-10) cytokines was restricted to the CD45RC(low) subset. In conclusion, we have shown that CD45RC expression divides human T cells in functionally distinct subsets that are imbalanced in AAV. Since this imbalance is stable over time and independent of several disease parameters, we hypothesize that this is a pre-existing immune abnormality involved in the etiology of AAV.

Show MeSH
Related in: MedlinePlus