Population analysis of antiviral T cell responses using MHC class I-peptide tetramers

Abstract

MHC class I-peptide tetrameric complexes ('tetramers') have revolutionized the study of antiviral CD8+ T cell responses. They allow accurate quantification of immune responses ex vivo independent of function, with high levels of sensitivity. They have revealed unexpectedly large frequencies of 'memory' T cell responses against viruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), and provided information about their phenotypic and functional variation. However, such studies have generally concentrated on limited numbers of individuals analysed in detail. To allow larger population-based studies, we devised a method for tetramer analysis using 50-100 microlitre blood volumes in a 96-well plate format. We adapted this method to study the effect of age on responses in a cohort of nearly 600 individuals to an immunodominant HLA-A2 restricted response to CMV pp65 (NLVPMVATV). We observed the phenomenon of steady 'memory inflation' with age, similar to recently observed longitudinal data from murine studies. These data show that tetramers can be used as population screening tools and could be used to study age-related, geographical or seasonal effects in a number of other viral infections.

Fig. 1

Tetramer staining for population studies. (a) Examples of staining from HLA-A2-positive subjects. The FACS plots are gated upon live lymphocyte populations from two donors, one negative (left) and one positive (right), for CMV responses. Proportions of positive CD8⁺ lymphocytes are shown in the upper right quadrant. (b) Effect of storage on tetramer staining. Samples were taken into EDTA and stored at room temperature. On day 0 and after 1, 4 and 7 days the blood was stained for CMV tetramer-positive cells as described in Methods. The proportions of positive lymphocytes are shown in the upper right quadrants. A day 10 sample showed much weaker staining intensity and lower frequency (0·14%) of positive cells.

Fig. 2

CMV-specific T cell populations as a function of age. Mean frequencies of tetramer positive cells as a fraction of total CD8⁺ cells in individuals of different ages. Of 581 individuals screened, 275 were HLA-A2-positive. Only tetramer positive samples are included in the analysis (mean ± s.e.m. displayed in 10-year age groups). Staining using matched controls established a threshold of detection of 0·05%. Frequencies of those HLA-A2-positive individuals scoring tetramer-positive in each 10-year age group are shown in Table 1 − the highest and lowest 10-year age groups are not included due to lack of positive samples. The correlations shown are for the 10-year age groups included. Values for breakdown by individual year age groups showed P= 0·0014, r= 0·32.