Neutralizing IL-6 reduces human arterial allograft rejection by allowing emergence of CD161+ CD4+ regulatory T cells - PubMed (original) (raw)

Neutralizing IL-6 reduces human arterial allograft rejection by allowing emergence of CD161+ CD4+ regulatory T cells

Birgit Fogal et al. J Immunol. 2011.

Abstract

Perioperative injuries to an allograft exacerbate graft rejection, which in humans is primarily mediated by effector memory T cells. IL-6 transcripts in human coronary artery segments rapidly increase posttransplantation into immunodeficient mouse hosts compared with those of pretransplant specimens and fall dramatically by 30 d. Adoptive transfer of human PBMCs allogeneic to the artery 2 d postoperatively results in T cell infiltrates and intimal expansion 4 wk later. Ab neutralization of human IL-6 reduces the magnitude of intimal expansion and total T cell infiltration but increases the relative expression of CD161 while decreasing other Th17 markers. Coculture of MHC class II-expressing human endothelial cells (ECs) with allogeneic CD4(+) memory T cells results in T cell activation and EC secretion of IL-6. Neutralizing IL-6 in primary allogeneic T cell-EC cocultures results in enhanced T cell proliferation of CD161(+) CD4(+) T cells, reduces total T cell proliferation upon restimulation in secondary cultures (an effect dependent on CD161(+) T cells), increases expression of FOXP3 in CD161(+) T cells, and generates T cells that suppress proliferation of freshly isolated T cells. These data suggest that IL-6 released from injured allograft vessels enhances allogeneic T cell infiltration and intimal expansion in a model of human allograft rejection by inhibiting an increase in CD161(+) regulatory T cells.

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Figures

Figure 1. Time course of IL-6 expression after arterial transplantation in vivo

Segments of three independent human coronary arteries were collected prior to transplantation (0h) or at various times after transplantation into immunodeficient mouse hosts and analyzed for expression of IL-6 mRNA by qRT-PCR as described in the Methods. The bars represent the mean values normalized to GAPDH mRNA +/- SEM (n = 3); note that the 6h time point lacks SEM, since only two samples were analyzed at this time point.

Figure 2. IL-6 neutralization reduces T cell-mediated injury in human artery allografts

(A) Immunohistochemical detection of human α-smooth muscle actin (SMA) to define the media in representative human artery interposition grafts (5µm transverse sections) from animals treated with IgG Control (top) or anti-IL6 mAb (bottom) for 28 d (bars: 100µm). The areas of the lumen, total vessel, and intima are reported as the mean area in mm2 + SEM. Statistical significance was assessed via paired t test analysis (n=8 pairs of animals from 4 independent experiments, p < 0.05). (B) Immunohistochemical detection of human CD45RO(+) T cells in representative human artery interposition grafts (5µm transverse sections). The numbers of CD45RO+ cells in the intima are reported as the mean number of CD45RO+ T cells/intima + SEM. Paired t-test analysis was performed to assess statistical significance (n=8 pairs of animals from 4 independent experiments, p < 0.05).

Figure 3. IL-6 release from EC is increased by co-culture with allogeneic T cells

(A) Co-culture of CD4(+) T cells with allogeneic IFN-γ-pretreated MHC class II(+) HUVECs. Media was taken for ELISA from either HUVEC cultures or HUVECs co-cultured with CD4(+) T cells for 24 h (n=12, pooled from three independent experiments). (B) IL-6 levels in cultures after 24 h were compared between HUVECs and HUVEEC/CD4(+) T cell co-cultures using HUVECs either transduced with CIITA (gray) or treated with IFNγ for 3 d (black) to induce MHC class II expression (n = 12 from three independent experiments). (C) IL-6 levels in lysates of CD4(+) T cells (black) or HUVECs (gray) separated after 24h in co-culture as determined by ELISA (n=6, from two independent experiments). (D) IL-6 release in allogeneic MHC class II(+) HUVEC/CD4(+) T cell co-cultures after 24 h in the presence of an anti-TNF neutralizing or control antibody (n=12 from three independent experiments). An asterisk (*) indicates a statistically significant difference from control cells stimulated in presence of MHC-class II(-) HUVECs, at the same concentration of anti TNF antibody, as assessed by two-way ANOVA, followed by Bonferroni’s post hoc analysis (p < 0.05). (E) Flow cytometric analysis of intracellular TNF in permeabilized CD4(+) T cells after 24 h of co-culture with allogeneic MHC class II(-) or (+) HUVECs (n = xxx) (F) IL-6 release in HUVECs after 24 h in presence of increasing concentrations of recombinant human TNFα (n = 6 pooled from two independent experiments). An asterisk (*) indicates a statistically significant difference from 0 ng/ml TNF, as assessed by one-way ANOVA, followed by Student’s post hoc analysis (p < 0.05).

Figure 4. IL-6 neutralization increases proliferation of CD4(+) CD161(+) memory T cells during the anti-EC allogeneic response in vitro

(A) Effects of supplementing MHC class II(+) HUVEC/CD4(+) T cell co-cultures with an anti-human IL-6 neutralizing antibody (30µg/ml) vs. control IgG on proliferation after 7 days in culture as assessed via BrdU incorporation (n = 3 independent experiments), and on release of IFNγ or IL-2 after 24h as assessed by ELISA (n = 3 independent experiments). Paired t-test analysis was performed to assess statistical significance (p < 0.05). (B) Effect of supplementing MHC class II(+) HUVEC/CD4(+) T cell memory co-cultures with an anti-human IL-6 neutralizing antibody (30µg/ml) vs. control IgG on proliferation after 7 days in culture as assessed via BrdU incorporation. Paired t-test analysis was performed to assess statistical significance (p < 0.05; n = 3 independent experiments). (C) Effects of supplementing MHC class II(+) HUVEC/CD4(+) CD161(+) or CD161(-) memory T cells with an anti-human IL-6 neutralizing antibody (30µg/ml) vs. control IgG on proliferation after 7 days in culture as assessed via CFSE dilution. Statistical significance was assessed via two way ANOVA followed by Bonferroni’s post hoc test (p < 0.05).

Figure 5. IL-6 neutralization decreases T cell proliferation and cytokine production upon restimulation by allogeneic HUVEC

CIITA transduced HUVECs were co-cultured with CD4(+) memory T cells for 3 d in presence of either control IgG or an anti-IL6 neutralizing mAb. CD4(+) memory T cells were then rested for 3 d and restimulated with allogeneic CIITA transduced HUVECs from the same donor as in the primary culture. (A) ELISA analyses of media collected from co-cultures 24 h after restimulation (top, n=12 from three independent experiments). (B) CD4(+) memory T cells were labeled with CFSE after rest, and proliferation assessed 4 d after restimulation with allogeneic ECs via CFSE dilution. Shown are representative FACS blots from one experiment (left) and quantitative data pooled from three independent experiments (right). Paired t test analysis was performed to assess statistical significance (p < 0.05). (C) Cells were treated as in (B), but proliferation compared between complete population of CD4(+) memory T cells and CD161(+) T cell-depleted CD4(+) memory T cells. Statistical significance was assessed by two way ANOVA followed by Bonferroni’s post hoc test (p < 0.05).

Figure 6. IL-6 neutralization results in induction of a regulatory T cell population in EC/CD4(+) T cell co-cultures

(A) To test for generation of “suppressor T cells” in EC/T cell co-cultures CIITA-transduced HUVECs were co-cultured with allogeneic CD4(+) memory T cells for 3 d in presence of either control IgG or an anti-IL6 neutralizing mAb, at which time T cells were collected, rested for 3 d, and then added to co-cultures of CFSE-labeled freshly isolated CD4(+) memory T cells (“Responder T cells”) from the same blood donor and cultured with allogeneic CIITA-transduced HUVECs from the same endothelial cell donor as used in the primary stimulation. T cells were added at the following ratios (responder T cells: suppressor T cells) 1:0; 1:0.5, 1:1, 1:3. Proliferation of CFSE-labeled responder T cells was assessed after 7 days in culture. (B) Percent suppression was calculated for three independent experiments performed as in Panel A. Bars represent mean of suppression from the different experiments +/- SEM and asterisks (*) indicate a significant difference from the null hypothesis. (C) CD4(+) T cells were separated into CD161(+) and CD161(-) subsets and each subset was cultured with CIITA-transduced HUVECs in presence of an irrelevant isotype control Ab or anti-IL6 antibody for 7 days at which time T cells were recovered and analyzed for mRNA species normalized to CD3ε mRNA by qRT-PCR as described in the Methods. Note that wile RORc mRNA was higher in the CD161(+) subset, its expression was unchanged by co-culture in the presence of anti-IL6 whereas FoxP3 was increased by this treatment but only in the CD161(+) population. This change was statistically significant (n=5 independent experiments); a sixth experiment, which showed a much larger change in FoxP3 expression and much higher basal FoxP3 expression, appeared as an outlier and has been excluded so as to not skew the analysis.

Figure 7. IL-6 neutralization results in induction of CD161(+) FoxP3(+) CD127(-) regulatory T cells in EC/CD4(+) T cell co-cultures

CD4+CD45RA- memory T cells were co-cultured for 7 days with allogeneic HLA-DR+ ECs in the presence of an IL-6 neutralizing antibody or isotype control. Multiparameter flow cytometric analysis was performed for CD161, CD25, FoxP3, CD127, and Helios in freshly isolated cells or after co-culture for 7 days. A. Flow cytometric analysis for CD25 and FoxP3 expression on CD161(+) gated CD4(+) CD45RA(+) memory T cells. B. Flow cytometric analysis for CD127 and Helios expression on CD161(+) CD25(+) FoxP3(+)-gated cells.

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Neutralizing IL-6 reduces human arterial allograft rejection by allowing emergence of CD161+ CD4+ regulatory T cells - PubMed (original) (raw)