Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium

<div><p>Cytomegalovirus (CMV) infection elicits a very strong and sustained intravascular T cell immune response which may contribute towards development of accelerated immune senescence and vascular disease in older people. Virus-specific CD8+ T cell responses have been investigated extensively through the use of HLA-peptide tetramers but much less is known regarding CMV-specific CD4+ T cells. We used a range of HLA class II-peptide tetramers to investigate the phenotypic and transcriptional profile of CMV-specific CD4+ T cells within healthy donors. We show that such cells comprise an average of 0.45% of the CD4+ T cell pool and can reach up to 24% in some individuals (range 0.01–24%). CMV-specific CD4+ T cells display a highly differentiated effector memory phenotype and express a range of cytokines, dominated by dual TNF-α and IFN-γ expression, although substantial populations which express IL-4 were seen in some donors. Microarray analysis and phenotypic expression revealed a profile of unique features. These include the expression of CX3CR1, which would direct cells towards fractalkine on activated endothelium, and the β2-adrenergic receptor, which could permit rapid response to stress. CMV-specific CD4+ T cells display an intense cytotoxic profile with high level expression of granzyme B and perforin, a pattern which increases further during aging. In addition CMV-specific CD4+ T cells demonstrate strong cytotoxic activity against antigen-loaded target cells when isolated directly <i>ex vivo</i>. PD-1 expression is present on 47% of cells but both the intensity and distribution of the inhibitory receptor is reduced in older people. These findings reveal the marked accumulation and unique phenotype of CMV-specific CD4+ T cells and indicate how such T cells may contribute to the vascular complications associated with CMV in older people.</p></div

Analysis of inhibitory molecule PD-1 on CMV-specific T cells.

<p>PBMCs were stained with LIVE/DEAD fixeable dye, followed by HLA class II tetramer, before staining to detect surface molecules. (A) Percentage of PD-1+ cells within T cell memory subsets defined by co-expression of CCR7 and CD45RA for the total CD4+ population (n = 53). (B) Percentage of PD-1+ cells within those subsets for the TM+ cells (n = 53). Columns represent medians with IQR. (C) The frequency of PD-1+ cells within the CD4+EM T cell subset, LLQ-, AGI- and DYS-specific T cell populations. (D) MFI of PD-1 expression on CD4+EM T cells as well as LLQ-, AGI- and DYS-specific T cells. Error bars indicate medians and IQR. p-values: * p = 0.01–0.05, ** p = 0.001–0.01. Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values (C and D). (E) The proportion of TM+ cells and CD4+EM cells expressing PD-1 in relation to donor age. (F) Median fluorescence intensity (MFI) of PD-1 expression on TM+ cells and CD4+EM cells correlated with donor age. Spearman‘s rank correlation was used to analyse the strength of associations between variables (D and E).</p

Phenotype of CMV-specific CD4+ T cells.

<p>(A) The memory phenotype of virus-specific T cells was determined initially using co-expression patterns of CCR7 and CD45RA (n = 56). Bars represent median with interquartile range. The cells were then further characterised by staining with CCR7, CD27, CD28, CD57, CD45RA and CD45RO. (B) Using Boolean gating all possible combinations of the six surface markers were determined and cells were categorised according to differentiation status, with the least differentiated subset on the left. The heatmap represents data for each individual response, showing the proportion of tetramer positive (TM+) cells within each subset (% of TM+ cells, scale bar at the top). Labelled to the right are the number of the donor and in brackets the peptide epitope and frequency of TM+ cells. (C) We then examined and compared expression patterns between T cells specific for all three CMV-derived epitopes. Each pie chart summarises data for one of the epitopes studied, arcs representing the proportion of TM+ cells expressing any particular marker. To compare T cells specific for the different epitopes (pie charts) permutation analysis (non-parametric test) was performed in SPICE. (D) Proposed stages of differentiation of CMV-specific CD4+ T cells.</p

Transcriptional analysis of CMV-specific T cells.

<p>(A) CD4+ T cells specific for gB-derived epitope DYS and pp65-derived epitope LLQ were sorted as well as EM CD4+ T cells (CCR7-CD45RA-) of CMV-seronegative individuals and the mRNA transcriptional profile determined using microarray analysis. Transcription of 55 genes was at least 2-fold up or downregulated in CMV-specific T cells compared to the EM cells, which is graphically presented here. (B) The Venn diagram summarises differences observed between EM cells and T cells specific for the two CMV-derived epitopes. (C) Relative transcript levels of aquantile normalised data are depicted for selected genes, comparing EM, LLQ- and DYS-specific T cells. (D) qPCR analysis was performed for genes identified as showing transcriptional upregulation in the microarray analysis. Gene expression levels in TM+ cells were related to those in CD4+ EM cells. Analysis was performed in 2–3 individuals in duplicate. Bars represent means with SEM, Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values.</p

Functional characterisation of CMV-specific CD4+ T cells.

<p>Following peptide stimulation of PBMCs the cytokine profile of activated CD4+ T cells was analysed using multicolour flow cytometry. Pie charts in the top summarise the data for gB (DYS)- and pp65-specific T cells (AGI and LLQ), each arc representing one of the cytokines studied, and showing the proportion of responding cells making them. The bottom graph shows individual responses and the proportion of cells detected in relevant different functional subsets.</p

CMV-specific CD4+ T cells are highly cytotoxic directly <i>ex vivo</i>.

<p>PBMCs were stained with LIVE/DEAD fixable dye, before staining with HLA class II tetramer followed by anti-CD3, CD4 and CX3CR1. After fixation and permeabilisation Granzyme B and Perforin were stained intracellularly. (A) Pie charts represent the proportion of CD4+ EM T cells (in CMV seronegative individuals) or the TM+ population expressing the indicated markers and combinations thereof. To compare pie charts permutation analysis was performed in SPICE. (B) Expression of Granzyme B, perforin and CX3CR1 on pp65-specific (on the left) and gB-specific (on the right) T cells in relation to donor age. Spearman‘s rank correlation was used to analyse the strength of associations between variables. (C) Percentage killing of peptide-loaded target cells (HLA-matched LCLs) mediated by <i>ex vivo</i> separated CD4+TM+ or CD4+EM T cells following over night co-culture at effector:target (E:T) ratios indicated. All conditions were performed in triplicate; bars represent means with SEM. (D) Proportion of cells expressing NKG2D within the CD4+ EM or the CMV-specific T cell population in young, middle aged or older adults. Error bars represent medians and IQR. p-values: ** p = 0.001–0.01, *** p = 0.001–0.0001. Kruskal-Wallis test was performed with Dunn‘s multiple comparison in GraphPad Prism5 to derive p-values.</p

Detection of CMV-specific CD4+ T cells in healthy virus carriers using HLA class II tetramers.

<p>(A) Specificity of HLA class II tetramers was tested by staining HLA-matched PBMCs of a CMV seronegative donor and an epitope-specific T cell clone (representative example shown for DYS:DR7). At the same time sensitivity of the tetramer reagents was tested by mixing decreasing numbers of the clonal T cells with PBMCs of the CMV-seronegative donor. Plots are representative of two independent experiments. Similar results were obtained for all three HLA class II tetramers tested. (B) Representative examples of healthy donor PBMCs stained with each of the three HLA-peptide tetramers used in this study. Background staining without tetramer is very low and examples are shown of epitope-specific T cells at low, intermediate and high frequencies. Numbers indicate the proportion of tetramer+ (TM+) cells within the total CD4+ T cell population. (C) Summary of all data for all individuals tested in this study depicting frequency of TM+ cells within the total CD4+ T cell population for each epitope and (D) the correlation between frequency of TM+ cells and donor age (n = 56). (E) The frequency of cells detected by tetramer staining correlates strongly with the proportion of IFN-γ producing cells detected by intracellular cytokine staining following stimulation with the cognate peptide. Spearman‘s rank correlations were performed to measure the strength of associations between variables (D and E).</p

Potential mechanism of virus-directed vascular injury in response to stress and endothelial damage.

<p>CMV-specific CD4+ T cells expressing β2-adrenergic receptor would be mobilised into the bloodstream in response to epinephrine released during stress. Epinephrine also leads to reactivation of virus from latently infected endothelial cells. Furthermore, activation/inflammation of endothelium induces expression of membrane-bound fractalkine. CX3CR1+ CMV-specific CD4+ T cells would adhere to endothelium, antigen recognition leading to activation of these highly cytotoxic effector cells and direct killing of virus-infected endothelial cells. Release of cytokines and chemokines from CMV-specific CD4+ T cells potentially attracts further immune cells to such sites of viral reactivation and inflammation which may enhance vascular injury.</p