Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry
HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their...
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MDPI AG
2021-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/1/263 |
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| author | Amélie Rousselière Laurence Delbos Céline Bressollette Maïlys Berthaume Béatrice Charreau |
| author_facet | Amélie Rousselière Laurence Delbos Céline Bressollette Maïlys Berthaume Béatrice Charreau |
| author_sort | Amélie Rousselière |
| collection | DOAJ |
| description | HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA<sub>peptide</sub> tetramer complexes (HLA-E<sub>VMAPRTLIL</sub>, HLA-E<sub>VMAPRSLLL</sub>, and HLA-A2<sub>NLVPMVATV</sub>) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV<sup>−</sup> and HCMV<sup>+</sup> individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E<sub>UL40</sub> and HLA-A2<sub>pp65</sub>CD8 T cells, and expansion of γδT and NK subsets including δ2<sup>−</sup>γT and memory-like NKG2C<sup>+</sup>CD57<sup>+</sup> NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines. |
| first_indexed | 2024-03-10T03:40:07Z |
| format | Article |
| id | doaj.art-f41d1eb2cbec439bbdfe6305c5ee0a05 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T03:40:07Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-f41d1eb2cbec439bbdfe6305c5ee0a052023-11-23T11:37:18ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-12-0123126310.3390/ijms23010263Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow CytometryAmélie Rousselière0Laurence Delbos1Céline Bressollette2Maïlys Berthaume3Béatrice Charreau4INSERM, Center for Research in Transplantation and Translational Immunology, Nantes Université, UMR 1064, CHU Nantes, F-44000 Nantes, FranceINSERM, Center for Research in Transplantation and Translational Immunology, Nantes Université, UMR 1064, CHU Nantes, F-44000 Nantes, FranceINSERM, Center for Research in Transplantation and Translational Immunology, Nantes Université, UMR 1064, CHU Nantes, F-44000 Nantes, FranceINSERM, Center for Research in Transplantation and Translational Immunology, Nantes Université, UMR 1064, CHU Nantes, F-44000 Nantes, FranceINSERM, Center for Research in Transplantation and Translational Immunology, Nantes Université, UMR 1064, CHU Nantes, F-44000 Nantes, FranceHCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA<sub>peptide</sub> tetramer complexes (HLA-E<sub>VMAPRTLIL</sub>, HLA-E<sub>VMAPRSLLL</sub>, and HLA-A2<sub>NLVPMVATV</sub>) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV<sup>−</sup> and HCMV<sup>+</sup> individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E<sub>UL40</sub> and HLA-A2<sub>pp65</sub>CD8 T cells, and expansion of γδT and NK subsets including δ2<sup>−</sup>γT and memory-like NKG2C<sup>+</sup>CD57<sup>+</sup> NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.https://www.mdpi.com/1422-0067/23/1/263HLA-ECD8 T cellsNKγδTHCMVpHLA tetramers |
| spellingShingle | Amélie Rousselière Laurence Delbos Céline Bressollette Maïlys Berthaume Béatrice Charreau Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry International Journal of Molecular Sciences HLA-E CD8 T cells NK γδT HCMV pHLA tetramers |
| title | Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry |
| title_full | Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry |
| title_fullStr | Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry |
| title_full_unstemmed | Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry |
| title_short | Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry |
| title_sort | mapping and characterization of hcmv specific unconventional hla e restricted cd8 t cell populations and associated nk and t cell responses using hla peptide tetramers and spectral flow cytometry |
| topic | HLA-E CD8 T cells NK γδT HCMV pHLA tetramers |
| url | https://www.mdpi.com/1422-0067/23/1/263 |
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