CD4+ regulatory T cell responses induced by T cell vaccination in patients with multiple sclerosis - PubMed (original) (raw)

Clinical Trial

. 2006 Mar 28;103(13):5024-9.

doi: 10.1073/pnas.0508784103. Epub 2006 Mar 17.

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Clinical Trial

CD4+ regulatory T cell responses induced by T cell vaccination in patients with multiple sclerosis

Jian Hong et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Immunization with irradiated autologous T cells (T cell vaccination) is shown to induce regulatory T cell responses that are poorly understood. In this study, CD4(+) regulatory T cell lines were generated from patients with multiple sclerosis that received immunization with irradiated autologous myelin basic protein-reactive T cells. The resulting CD4(+) regulatory T cell lines had marked inhibition on autologous myelin basic protein-reactive T cells and displayed two distinctive patterns distinguishable by the expression of transcription factor Foxp3 and cytokine profile. The majority of the T cell lines had high Foxp3 expression and secreted both IFN-gamma and IL-10 as compared with the other pattern characteristic of low Foxp3 expression and predominant production of IL-10 but not IFN-gamma. CD4(+) regulatory T cell lines of both patterns expressed CD25 and reacted with activated autologous T cells but not resting T cells, irrespective of antigen specificity of the target T cells. It was evident that they recognized preferentially a synthetic peptide corresponding to residues 61-73 of the IL-2 receptor alpha chain. T cell vaccination correlated with increased Foxp3 expression and T cell reactivity to peptide 61-73. The findings have important implications in the understanding of the role of CD4(+) regulatory T cell response induced by T cell vaccination.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Expression of Foxp3 mRNA in CD4+ regulatory T cell lines derived from MS patients immunized with irradiated autologous MBP-reactive T cells. (A) Two representative patterns of CD4+ regulatory T cell lines by flow cytometry. Representative T cell lines (lines B9 and F8) were analyzed for the expression of CD4 paired with that of CD25, HLA-DR, CD71, and Foxp3. Open curves in the histograms represent isotype control. Staining with specific antibody to CD4 or Foxp3 is indicated by gray or solid curves, respectively. (B) Immunoblot analysis of the same T cell lines for protein expression of transcription factor Foxp3. (C) Foxp3 mRNA expression by real-time PCR in all 30 CD4+ regulatory T cell lines and six reference MBP-reactive T cell clones derived from the same patients.

Fig. 2.

Fig. 2.

Expression of Foxp3 in paired PBMCs obtained from MS patients before and after T cell vaccination. Purified CD4+CD25+ T cells were obtained from MS patients (n = 20) before and after T cell vaccination. The resulting T cells were subject to real-time PCR analysis for mRNA expression (A) and inhibition rate on the proliferation of autologous T cells stimulated by anti-CD3 antibody (B). Percent inhibition is calculated as [1 − (experimental cpm per control cpm)] × 100%. ∗, statistical differences between the groups (P < 0.05).

Fig. 3.

Fig. 3.

Reactivity of CD4+ regulatory T cell lines to autologous T cell targets. CD4+ regulatory T cell lines were examined for reactivity to resting T cells, activated MBP-reactive T cells used for vaccination, and activated T cells raised by anti-CD3/CD28 mAbs. All target T cells were of the autologous origin and irradiated before use. T cell reactivity to target cells was determined in proliferation assays by measuring [3H]-thymidine incorporation.

Fig. 4.

Fig. 4.

Reactivity of CD4+ regulatory T cells to peptides derived from IL-2R α chain. The reactivity of CD4+ regulatory T cells to three selected peptides derived from IL-2 receptor α chain was measured by ELISPOT. An irrelevant T cell antigen receptor peptide was used as a control. CD4+ regulatory T cell lines were cultured at 200,000 cells per well in triplicate in the presence of irradiated antigen-presenting cells and the indicated synthetic peptides at a concentration of 20 μg/ml in precoated ELISPOT plates. The results were presented as IL-10 secreting T cells per 200,000 CD4+ regulatory T cells. Among the T cell lines examined, E11, G4, F4, D4, F9, F5, F8, G5, G6, and B6 are considered as CD4+CD25+Foxp3− T cell lines.

Fig. 5.

Fig. 5.

The frequency of CD4+ regulatory T cells in response to peptide 61–73 of IL-2R α chain in patients before and after T cell vaccination. The frequency of T cells reactive to peptide 61–73 was measured in PBMCs obtained from MS patients (n = 20) before and after T cell vaccination. PBMCs were cultured in triplicate at 200,000 cells per well in the presence of peptide 61–73 at a concentration of 20 μg/ml in precoated ELISPOT plates. The data are presented as IL-10 secreting T cells in 1 million PBMCs. ∗, statistical difference between the groups (P < 0.05).

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