Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides

Abstract

The frequencies of human cytomegalovirus (HCMV) protein‐specific CD8 T cells, identified by the presence of intracellular IFN‐γ, were measured by flow cytometry following stimulation of freshly isolated peripheral blood mononuclear cells (PBMC) with comprehensive peptide pools. These pools spanned the entire amino acid sequences of the HCMV pp65 and major immediate early (IE‐1) proteins and consisted of 15‐amino acid peptides with at least nine overlaps between neighboring peptides. As a result all potential CD8 T cell epitopes contained in these proteins were provided by the complete pools and, therefore, unlike with single epitopes, testing was independent of donor HLA type. Individual stimulating peptides from the same pools were identified in parallel experiments. Thus we found that our results with the complete pools using PBMC from 26 healthy HCMV‐seropositive donors were 100 % sensitive and specific with respect to predicting the presence of recognized epitopes in the respective proteins. In addition, cells from 15 renal transplant patients were tested with complete pools alone. While our results confirmed our previous contention that HCMV IE‐1 is an important CD8 T cell target, the technical improvement we made in order to address this question has clearly wider implications. Similar pools may be applied to examine the role of proteins from other pathogens, in autoimmune disease or following vaccination.

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

“…Until recently, monitoring of HCMV-specific CD8 + T cell response remained elusive and difficult. To overcome this limitation, some authors [12,13] proposed the use of peptide pools covering the entire sequence of the two most immunogenic HCMV proteins (pp65 and IE-1 proteins). Using 15-mer peptide pools, both CD4 + and CD8 + IFN-c-producing T cell responses can be elicited, as with 8-12-mer mixes [12].…”

“…To overcome this limitation, some authors [12,13] proposed the use of peptide pools covering the entire sequence of the two most immunogenic HCMV proteins (pp65 and IE-1 proteins). Using 15-mer peptide pools, both CD4 + and CD8 + IFN-c-producing T cell responses can be elicited, as with 8-12-mer mixes [12]. HCMVspecific CD8 + T cell frequency of healthy subjects determined by the method herein reported is similar to those previously reported for IE-or pp65-specific CD8 + T cells [12,16].…”

“…Using 15-mer peptide pools, both CD4 + and CD8 + IFN-c-producing T cell responses can be elicited, as with 8-12-mer mixes [12]. HCMVspecific CD8 + T cell frequency of healthy subjects determined by the method herein reported is similar to those previously reported for IE-or pp65-specific CD8 + T cells [12,16]. Although pp65 and IE proteins are currently considered the predominant viral stimuli of the HCMV-specific cell-mediated immune response, other viral proteins are involved in this process [17].…”

“…Peptides used in this study are pools of 15-mers [12,13] with an overlap of 11 amino acids spanning the entire pp65 or IE-1 proteins of HCMV (Jerini AG, Berlin, Germany). Peptides were stored according to the manufacturer's instructions and used at a final concentration of 1 lg/ml.…”

“…Recently some authors [12,13] developed a method involving the activation of both CD4 + and CD8 + T cell subsets, irrespective of the HLA type and known epitopes, using whole-protein-spanning peptide pools. This method is useful to detect protein-specific CD4 + or CD8 + T cell reactivity, but does not enable simultaneous quantification of HCMV-specific CD4 + and CD8 + T cell responses to multiple viral antigens.…”