Transcriptional profiles reveal a stepwise developmental program of memory CD8(+) T cell differentiation

Abstract

The generation of CD8(+) T-cell memory is a major aim of vaccination. While distinct subsets of CD8(+) T-cells are generated following immunization that differ in their ability to confer long-term immunity against infection, the transcriptional profiles of these subsets within endogenous vaccine-induced CD8(+) T cell responses have not been resolved. Here, we measure global transcriptional profiles of endogenous effector (TEFF), effector memory (TEM) and central memory (TCM) CD8(+) T-cells arising from immunization with three distinct prime-boost vaccine regimens. While a proportion of transcripts were uniquely regulated within distinct CD8(+) T cell populations, we observed progressive up- or down-regulation in the expression of a majority of differentially expressed transcripts when subsets were compared in the order TN>TCM>TEM>TEFF. Strikingly, when we compared global differences in gene expression between TN, TCM, TEM and TEFF cells with known transcriptional changes that result when CD8(+) T cells repetitively encounter antigen, our analysis overwhelmingly favored a model whereby cumulative antigen stimulation drives differentiation specifically from TN>TCM>TEM>TEFF and this was common to all vaccines tested. These findings provide insight into the molecular basis of immunological memory and identify potential biomarkers for characterization of vaccine-induced responses and prediction of vaccine efficacy.

Keywords: Adenovirus vector; CD8; LCMV vector; Memory T cells; Prime-boost vaccination; T cell memory.

Fig. 1.

Immunization schema and characterization of endogenous immune reponses. (A) Animals were immunized intramuscularly with DNA-rAd, rAd-rAd or rAd-LCMV vaccine regimens encoding the HIV-1 Env gene insert. H2-Dd/PA9 tetramer staining of splenocytes isolated at indicated time points following primary immunization showing the magnitude of the endogenous immune responses against the PA9 epitope generated by the three vaccine regimens. (B) Phenotypic characterization of splenic Env-specific immune responses (CM: CD62L⁺ IL7R⁺; EM: CD62L⁻ IL7R⁺; EFF: CD62L⁻ IL7R⁻) at the indicated time point. No significant differences in the frequency of T_CM, T_EM and T_EFF cells were observed between immunization groups at the indicated time point (at p < 0.05; Student’s t-test). Data are representative of two repeated experiments with five mice per group per time point.

Fig. 2.

Dynamic changes in gene expression between endogenous T_N, T _CM, T_EM and T_EFF Env-specific CD8+ T cell responses to vaccination. Expression of selected mRNA transcripts in indicated CD8⁺ T-cell subsets arising endogenously following immunization with given vaccines. Expression values for each transcript are provided as a relative ratio to expression in naïve cells. Gene expression measurements represent pooled results from two independently conducted experiments and results from ≥4 mice per vaccine group are shown. Asterisks p-values; * p < 0.05; ^** p < 0.01; ^*** p < 0.005; ^****p < 0.001; two-tailed t-test.

Fig. 3.

T_CM cells exhibit greater transcriptional relatedness to T_N cells than either T_EM or T_EFF cells. (A) Number of differentially expressed genes between the antigen-experienced CD8⁺ T-cell subsets (T_CM, T_EM and T_EFF) and T_N cells from the core signature. (B) Multidimensional scaling analysis of global gene expression data from the CD8⁺ T-cell subsets separated by vaccine protocol indicating transcriptional proximity of endogenously derived CD8⁺ T-cell subsets. (C) Venn diagram indicating uniquely up- or down-regulated transcripts within distinct CD8⁺ T cell subsets as compared with T_N. Members of the Venn diagram are provided in Supplemental Tables 1–7. (D–F) Uniquely regulated transcripts in T_CM (D), T_EM (E) and T_EFF (F) cells; selected transcripts and the direction in which they are either up- or down-regulated are identified.

Fig. 4.

Progressive up- or down-regulation of gene expression accounts for the majority of transcriptional changes occurring in the order T_N > T_CM > T_EM > T_EFF. (A) Hierarchical clustering analysis of differentially expressed genes between subsets. Clusters [A-F] of genes exhibiting distinct patterns of regulation between subsets are highlighted along the right hand side corresponding to the dendrogram on the left side of the heatmap. Clustering of CD8⁺ T-cell subsets based on global transcriptional proximity is highlighted above the heatmap and corresponds to the dendrogram. (B) Average and individual patterns of gene expression within the six identified clusters. Samples are distributed along the x-axis of plots based upon their position in the hierarchical clustering analysis in A and y-axes represent standardized gene expression levels. The top four ranked members of each cluster are displayed to the right of the average gene expression plot for each cluster.

Fig. 5.

Differences in gene expression between CD8⁺ T-cell subsets isolated at a single time point following immunization resemble known transcriptional changes that occur when T cells repetitively encounter antigen. (A) Differences in gene expression between subject and comparator CD8⁺ T cell subsets were evaluated in the context of known transcriptional changes that occur when CD8⁺ T cells repetitively encounter antigen [21]. Pearson’s correlation coefficient between the fold-changes (log2) of a given pairwise CD8⁺ T cell subset comparison (subject and comparator subsets are indicated as column and row titles) and the fold-changes of a given repetitive antigen stimulation (red boxes) comparison are shown. Statistically significant values (p < 0.05) are indicated in bold text. Naïve to primary (top left), primary to secondary (bottom left), secondary to tertiary (top right) and tertiary to quaternary (bottom right) gene expression changes from the dataset of Wirth et al. were evaluated independently. Only genes that reached statistical significance in the subset comparisons were considered for the correlation calculations. (B) Deconvolution analysis showing relative similarity of T_N, T_CM, T_EM and T_EFF cell fractions within cell populations generated following repetitive antigen stimulations of Wirth et al. (naïve, primary (I), secondary (II), tertiary (III) and quaternary (IV)). Colored asterisks represent p-values (* p <0.05; Wilcoxon rank sum test) and indicate statistical significance of enrichment of the indicated CD8⁺ T-cell subset (as indicated by the color of the asterisk) in populations resulting from indicated repetitive antigen stimulation in vivo.