Immune Netw.  2013 Jun;13(3):86-93. 10.4110/in.2013.13.3.86.

Priming of Autoreactive CD8+ T Cells Is Inhibited by Immunogenic Peptides Which Are Competitive for Major Histocompatibility Complex Class I Binding

• You S
• Choi YS
• Hong S
• Shin EC

• KMID: 2150771
• DOI: http://doi.org/10.4110/in.2013.13.3.86

Abstract

In the present study, we investigated if priming of autoreactive CD8+ T cells would be inhibited by competitive peptides for major histocompatibility complex (MHC) class I binding. We used a mouse model of vitiligo which is induced by immunization of Kb-binding tyrosinase-related protein 2 (TRP2)-180 peptide. Competitive peptides for Kb binding inhibited IFN-gamma production and proliferation of TRP2-180-specific CD8+ T cells upon ex vivo peptide restimulation, while other MHC class I-binding peptides did not. In mice, the capability of inhibition was influenced by T-cell immunogenicity of the competitive peptides. The competitive peptide with a high T-cell immunogenicity efficiently inhibited priming of TRP2-180-specific CD8+ T cells in vivo, whereas the competitive peptide with a low T-cell immunogenicity did not. Taken together, the inhibition of priming of autoreactive CD8+ T cells depends on not only competition of peptides for MHC class I binding but also competitive peptide-specific CD8+ T cells, suggesting that clonal expansion of autoreactive T cells would be affected by expansion of competitive peptide-specific T cells. This result provides new insights into the development of competitive peptides-based therapy for the treatment of autoimmune diseases.

Keyword

Autoimmune disease; Autoreactive CD8+ T cell; Competitive peptide; Major histocompatibility complex class I

MeSH Terms

Animals
Autoimmune Diseases
Immunization
Major Histocompatibility Complex
Mice
Peptides
T-Lymphocytes
Vitiligo
Peptides

Figure

• 

  Figure 1 Relative affinity of T cell epitope peptides for Kb binding. Splenocytes of naive C57BL/6 mice were incubated with peptides. Peptide-pulsed splenocytes were fixed with 4% paraformaldehyde, and the density of SIINFEKL-binding Kb molecules on lymphocytes was analyzed by flow cytometry using antibody specific to SIINFEKL-binding Kb molecule (clone 25-D1.16). (A) A dashed line represents the mean fluorescence intensity (MFI) of 25-D1.16-stained lymphocytes pulsed with the indicated concentration of SIINFEKL without additional peptides. Each solid line represents MFI of 25-D1.16-stained lymphocytes pulsed with SIINFEKL at 0.5µM in combination with the indicated concentration of additional peptide. Each dot represents the mean+SEM from 3 independent experiments. (B) The percentage of decreased MFI of SIINFEKL-binding Kb molecules by additional peptides (SIINFEKL at 0.5µM and additional peptide at 5µM) was calculated as follows: [100-MFI of SIINFEKL-binding Kb in SIINFEKL at 0.5µM with additional peptides at 5µM/MFI of SIINFEKL-binding Kb in SIINFEKL at 0.5µM×100]. Each bar graph represents the mean+SEM from 3 independent experiments.

• 

  Figure 2 Inhibition of TRP2-180-specific IFN-γ production of CD8+ T cells by competitive peptides for Kb binding in vitro. C57BL/6 mice were immunized with TRP2-180, and splenocytes were harvested and stimulated ex vivo with TRP2-180 peptide alone or in combination with competitive peptides. Then, IFN-γ ICS was performed and analyzed by flow cytometry. (A) The splenocytes were stimulated ex vivo with 0.1µM of TRP2-180 peptide alone or in combination with competitive peptides at 10µM. Representative FACS dot plots for IFN-γ and CD44 are shown in the gate of the CD8+ T cell population. (B) The splenocytes were stimulated ex vivo with TRP2-180 peptide at 0.1µM alone or in combination with the indicated concentration of competitive peptides, and the percentages of TRP2-180-specific IFN-γ+ cells in the CD8+ T-cell population are presented as bar graphs. Each bar graph represents the mean+SEM from 6 independent experiments (**p<0.01).

• 

  Figure 3 Inhibition of TRP2-180-specific CD8+ T-cell proliferation by competitive peptides for Kb binding in vitro. A TRP2-180-specific T-cell line (CD45.2) labeled with CFSE at 10µM was cocultured with splenocytes (CD45.1) pulsed with TRP2-180 peptide alone or in combination with competitive peptides. Four days after coculture, cocultured cells were stained with anti-CD45.2-APC-H7 and analyzed by flow cytometry. (A) CFSE-labeled T-cell line was cocultured with splenocytes pulsed with the indicated concentration of TRP2-180. The fluorescence intensity of CFSE was analyzed in the gate of the CD45.2+-cell population. (B) CFSE labeled T cell line was cocultured with splenocytes pulsed with TRP2-180 at 0.1µM in combination with additional peptides at 10µM. The fluorescence intensity of CFSE was analyzed in the gate of the CD45.2+-cell population. (C) The percentage of proliferation inhibition by additional peptide was calculated as follows: [100-(% of proliferation in TRP2-180 at 0.1µM with additional peptides-% of proliferation in the absence of TRP2-180)/(% of proliferation in TRP2-180 at 0.1µM-% of proliferation in the absence of TRP2-180)×100], and presented by bar graphs.

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  Figure 4 Inhibition of TRP2-180-specific CD8+ T-cell priming by competitive peptides in vivo. C57BL/6 mice were subcutaneously immunized in the footpad with TRP2-180 (5 nmole), LPS, and CpG ODN. One day after the immunization, competitive peptides at 50 nmole were injected into the same footpad without adjuvants twice at 1-day intervals. One week after TRP2-180 immunization, splenocytes were harvested. (A) TRP2-180-specific IFN-γ+ CD8+ T cells were analyzed by ICS and flow cytometry in each group. (B) Competitive peptide-specific IFN-γ+ CD8+ T cells were analyzed by ICS and flow cytometry in each group. Each bar graph represents the mean+SEM from 3 independent experiments with 3 mice per group (*p<0.05).

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  Figure 5 Competitive peptides do not prevent progression of depigmented skin lesions in a mouse model of vitiligo. C57BL/6 mice were subcutaneously immunized in the footpad with TRP2-180 (5 nmole), LPS, and CpG ODN twice at 1-week intervals. One week after the second immunization, TRP2-180 (5 nmole) with the same adjuvants was injected into the tail dermis for the disease induction twice at 1-week intervals. Fifty nmole competitive peptides were injected into the tail dermis 1 day after third and fourth TRP2-180 immunization without adjuvants twice at 1-day intervals. Five weeks after the last TRP2-180 immunization, splenocytes were harvested. (A) TRP2-180-specific IFN-γ+ CD8+ T cells were analyzed by ICS and flow cytometry in each group. (B) Competitive peptide-specific IFN-γ+ CD8+ T cells were analyzed by ICS and flow cytometry in each group. (C) Disease progression was evaluated by the percentage of depigmented skin lesions in the tail in each group. Each bar graph represents the mean+SEM from 3 independent experiments with 3 mice per group (n.s., not significant).

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