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Abstract
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 HLApeptide tetramer complexes (HLA-EVMAPRTLIL, HLA-EVMAPRSLLL, and HLA-A2NLVPMVATV) 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− and HCMV+ 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-EUL40 and HLA-A2pp65CD8 T cells, and expansion of γδT and NK subsets including δ2−γT and memory-like NKG2C+CD57+ 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.
Highlights
Human cytomegalovirus (HCMV; human herpesvirus 5, HHV5) is the prototype member of β-herpesvirus family and a widespread opportunistic pathogen
We have shown that HLA-EUL40 CD8 T cell responses may represent up to 30% of total blood CD8 T cells in a host post-infection [11]
To define our ability to map the various anti-HCMV cellular responses postinfection, we used a combination of three pHLA tetramer complexes (HLA-EVMAPRTLIL, HLA-EVMAPRSLLL, HLA-A2NLVPMVATV ) 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 multiparameter flow cytome
Summary
Human cytomegalovirus (HCMV; human herpesvirus 5, HHV5) is the prototype member of β-herpesvirus family and a widespread opportunistic pathogen. HCMV is a major cause of morbidity and mortality in immunocompromised individuals such as transplant recipients and patients with HIV infection. Immune response against HCMV is complex, multifactorial, and includes a set of persistent and virus-specific effector NK and CD8 αβT and γδT cell populations [2,3,4]. These effector cells display cytotoxic functions devoted to eliminating infected cells and preventing further HCMV reactivation [5,6].
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