Identification and Characterization of Circulating Naïve CD4+ and CD8+ T Cells Recognizing Nickel

Abstract

Allergic contact dermatitis caused by contact sensitizers is a T-cell-mediated inflammatory skin disease. The most prevalent contact allergens is nickel. Whereas, memory T cells from nickel-allergic patients are well-characterized, little is known concerning nickel-specific naïve T-cell repertoire. The purpose of this study was to identify and quantify naïve CD4+ and CD8+ T cells recognizing nickel in the general population. Using a T-cell priming in vitro assay based on autologous co-cultures between naïve T cells and dendritic cells loaded with nickel, we were able to detect a naïve CD4+ and CD8+ T-cell repertoire for nickel in 10/11 and 7/8 of the tested donors. We calculated a mean frequency of 0.49 nickel-specific naïve CD4+ T cells and 0.37 nickel-specific naïve CD8+ T cells per million of circulating naïve T cells. The activation of these specific T cells requires MHC molecules and alongside IFN-γ production, some nickel-specific T-cells were able to produce granzyme-B. Interestingly, nickel-specific naïve CD4+ and CD8+ T cells showed a low rate of cross-reactivity with cobalt, another metallic hapten, frequently mixed with nickel in many alloys. Moreover, naïve CD4+ T cells showed a polyclonal TCRβ composition and the presence of highly expanded clones with an enrichment and/or preferentially expansion of some TRBV genes that was donor and T-cell specific. Our results contribute to a better understanding of the mechanism of immunization to nickel and propose the T-cell priming assay as a useful tool to identify antigen-specific naïve T cells.

Keywords: T-cell assay; allergic contact dermatitis; allergy and immunology; in vitro tests; metals hapten; naive T cells; repertoire.

Figure 1

Naïve CD4+ and CD8+ T-cell lines can recognize nickel. (A) IFN-γ ELISpot response of naïve CD4+ T cells from donor PR4 stimulated with unloaded DC or DC loaded with NiSO_4. (B) Spots count for naïve CD4+ T cells specific to nickel from donor PR4. Dashed line represents the minimum required spots count (count = 30) for the analysis to be considered acceptable. (C) IFN-γ ELISpot response of naïve CD8 + T cells from donor PR13 stimulated with unloaded DC or DC loaded with NiSO₄. (D) Spots count for naïve CD8 + T cells specific to nickel from donor PR13. Dashed line represents the minimum required spots count (count = 30) for the analysis to be considered acceptable. PHA: Phytohemagglutinin.

Figure 2

Frequency of human naive CD4 + and CD8 + T cells recognizing nickel. Frequency of human naïve CD4+ (A) and CD8+ (B) T cells specific to nickel. Frequency was calculated for each donor using the Poisson distribution law. On the x-axis is represented the donor's number. For the different donors tested, the number of T-cell lines found specific for nickel out of the number of T-cell lines tested is indicated in brackets. (C,D) Comparison of the nickel-specific T-cell frequency to the KLH-specific T-cell frequency measured on different donors.

Figure 3

Granzyme B production from CD4 + and CD8 + T cells recognizing nickel. (A) Granzyme-B (Grz-B)/ IFN-γ spots count for nickel-specific CD4+ T-cells, from donor PN1, stimulated with unloaded DC or DC loaded with NiSO₄. (B) Representative figure from donor PN1 (CD4+; well number 14) and PN2 (CD8+; well number 17) performed using an IFN-γ/Grz-B fluorospot assay.

Figure 4

Implication of MHC class II and I molecules in nickel-specific T-cell response. (A) IFN-γ ELISpot response for naïve CD4+ T cells from donors PR20, PR29, and PR43 stimulated with unloaded DC or DC loaded with NiSO₄ or DC loaded with MHC blocking antibodies prior to nickel treatment. (B) Spots count for naïve CD4+ T cells specific to nickel from donors PR20, PR29, and PR43 stimulated with unloaded DC or DC loaded with NiSO₄ or DC loaded with MHC blocking antibodies prior to nickel treatment. Dashed line represents the minimum required spots count (count = 30) for the analysis to be considered acceptable. (C) IFN-γ ELISpot response of naïve CD8+ T cells from donors PR42 and PR44 stimulated with unloaded DC or DC loaded with NiSO₄ or DC loaded with MHC blocking antibodies prior to nickel treatment. (D) Spots count for naïve CD8+ T cells specific for nickel from donors PR42 and PR44 stimulated with unloaded DC or DC loaded with NiSO₄ or DC loaded with MHC blocking antibodies prior to nickel treatment. Dashed line represents the minimum required spots count (count = 30) for the analysis to be considered acceptable.

Figure 5

Cobalt cross-reactivity of nickel-recognizing naïve CD4+ and CD8+ T cells. (A) Number of cross-reactive T-cell lines among nickel-specific T cells from different donors. (B) Spots count for naïve CD4+ T cells specific to nickel from donors PR5, PR6, and PR20 stimulated with unloaded DC or DC loaded with NiSO₄ or DC loaded with CoCl₂. (C) Spots count for naïve CD8+ T cells specific to nickel from donors PR18, PR31, and PR44 stimulated with unloaded DC or DC loaded with NiSO₄ or DC loaded with CoCl_2. Dashed line represents the minimum required spots count (count = 30) for the analysis be considered acceptable. w, well-number.

Figure 6

TCRβ repertoire analysis of nickel-recognizing naïve CD4+ T-cell lines. TCRβ variable gene usage of the highly expanded clones (HECs) detected in the six nickel-specific T-cell lines analyzed (A) and in the sorted naïve CD4+ T-cells from donors PR19 and PR20 (B). TCRβ variable gene usage in the total repertoire pre- and post-stimulation in donors PR19 (C) and PR20 (D). The percentage of clones (or HECs) carrying a particular TCRβ variable gene (TRBV) is depicted on the y-axis. All the genes names follow the IMGT nomenclature.