T cell epitope immunotherapy induces a CD4+ T cell population with regulatory activity

Abstract

Background: Synthetic peptides, representing CD4(+) T cell epitopes, derived from the primary sequence of allergen molecules have been used to down-regulate allergic inflammation in sensitised individuals. Treatment of allergic diseases with peptides may offer substantial advantages over treatment with native allergen molecules because of the reduced potential for cross-linking IgE bound to the surface of mast cells and basophils.

Methods and findings: In this study we address the mechanism of action of peptide immunotherapy (PIT) in cat-allergic, asthmatic patients. Cell-division-tracking dyes, cell-mixing experiments, surface phenotyping, and cytokine measurements were used to investigate immunomodulation in peripheral blood mononuclear cells (PBMCs) after therapy. Proliferative responses of PBMCs to allergen extract were significantly reduced after PIT. This was associated with modified cytokine profiles generally characterised by an increase in interleukin-10 and a decrease in interleukin-5 production. CD4(+) cells isolated after PIT were able to actively suppress allergen-specific proliferative responses of pretreatment CD4(neg) PBMCs in co-culture experiments. PIT was associated with a significant increase in surface expression of CD5 on both CD4(+) and CD8(+) PBMCs.

Conclusion: This study provides evidence for the induction of a population of CD4(+) T cells with suppressor/regulatory activity following PIT. Furthermore, up-regulation of cell surface levels of CD5 may contribute to reduced reactivity to allergen.

Figure 1. PIT Reduces Antigen-Specific Proliferation of Memory T Cells

(A–D) PBMCs taken before and after PIT were labelled with CFSE to track cell division after antigen stimulaton. Proliferation of cat-allergen-specific CD45RO⁺ lymphocytes was reduced following PIT (A) and (B). (C) and (D) represent CD45RO⁺ T cells as shown in panels (A) and (B), respectively, analysed with ModFit software. The right-hand peaks represent the parental population, and generations of dividing cells are depicted leftwards along the x-axis. (E) Summary of the percentage of proliferating CD45RO⁺ T cells pre- and post-PIT (percent proliferating cells is defined as the fraction of the starting population that has proliferated during the course of the experiment, determined with Modfit) for all nine patients tested. Open symbols represent patients enrolled in treatment Group 1, while solid symbols depict patients from treatment Group 2. Horizontal solid bars show mean levels of proliferation. Background proliferation (in the absence of a stimulus) was less than 2% and was subtracted. The Wilcoxon signed rank test was used for statistical analysis.

Figure 2. PIT Reduces Antigen-Specific Proliferation of CD4⁺ and CD8⁺ T Cells

CD4⁺ and CD8⁺ proliferation data were obtained and interpreted as for Figure 1. (A) and (B) represent percentage proliferation of PBMCs to cat allergen for each patient as determined with ModFit. Open symbols represent patients from treatment Group 1, while solid symbols depict patients from treatment Group 2. Horizontal solid bars indicate means. Background proliferation has been subtracted. The Wilcoxon signed rank test was used for statistical analysis.

Figure 3. CD4⁺ Cells Isolated after PIT Suppress the Proliferative Response of Baseline CD4^neg Cells

PBMCs taken before and after PIT were separated into CD4⁺ and CD4^neg populations by immunomagnetic separation. CD4^neg cells were labelled with CFSE and served as target cells. CD4⁺ cells were labelled with PKH-26 and were evaluated for suppressor/regulator function by co-culture with CD4^neg cells. (A) and (B) show antigen-stimulated proliferation of CD4^neg target cells before and after PIT. Proliferation of CD4^neg target cells was reduced after PIT (B). In (C) and (E), pre-PIT CD4^neg cells were employed as target cells. The addition of post-PIT (E), but not pre-PIT (C) CD4⁺ cells inhibited proliferation. In (D) and (F), post-PIT CD4^neg cells were employed as target cells. Addition of either pre-PIT (D) or post-PIT (F) CD4⁺ cells had no further effect on proliferation. Proliferation in the absence of a stimulus was less than 2% in all experiments. Representative data for one patient are shown. Data for an additional four patients were obtained using the same protocol. A data summary of percentage proliferation of pre-PIT CD4^neg PBMCs in the presence of pre-PIT or post-PIT CD4⁺ T cells for five patients from treatment Group 2 is shown in (G). The paired t-test was used for statistical analysis.

Figure 4. PIT Enhances CD5 Expression on Resting CD4⁺ and CD8⁺ PBMCs

(A and B) Box-and-whiskers plots representing changes in MFI of CD5 expression levels on unstimulated CD4⁺ (A) and CD8⁺ (B) pre- and post-PIT PBMCs from seven patients in treatment Group 2. Isotype control MFI values have been subtracted. (C and D) CD5 levels on pre-PIT (heavy black line) and post-PIT (grey, filled) CD4⁺ and CD8⁺ PBMCs of one representative patient. M1 marks the CD5^low population, with a pre- to post-PIT decrease in CD5^lowCD4⁺ PBMCs from 6.6% (in black) to 1.5% (in grey), and a decrease in CD5^lowCD8⁺ PBMCs from 32.3% to 13.9%. M2 indicates the concomitant increases in CD5⁺CD4⁺ and CD5⁺CD8⁺ cells post-PIT. Changes in MFI values for the total pre- and post-PIT CD4⁺ or CD8⁺ populations of the single representative patient are shown in the upper right-hand corner of each histogram. The Wilcoxon signed rank test was used for statistical analysis.