Cytomegalovirus-Mediated T Cell Receptor Repertoire Perturbation Is Present in Early Life

Abstract

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus, particularly in sub-Saharan Africa, where it is endemic from infancy. The T cell response against CMV is important in keeping the virus in check, with CD8 T cells playing a major role in the control of CMV viraemia. Human leukocyte antigen (HLA) B^*44:03-positive individuals raise a robust response against the NEGVKAAW (NW8) epitope, derived from the immediate-early-2 (IE-2) protein. We previously showed that the T cell receptor (TCR) repertoire raised against the NW8-HLA-B^*44:03 complex was oligoclonal and characterised by superdominant clones, which were shared amongst unrelated individuals (i.e., "public"). Here, we address the question of how stable the CMV-specific TCR repertoire is over the course of infection, and whether substantial differences are evident in TCR repertoires in children, compared with adults. We present a longitudinal study of four HIV/CMV co-infected mother-child pairs, who in each case express HLA-B^*44:03 and make responses to the NW8 epitope, and analyse their TCR repertoire over a period spanning more than 10 years. Using high-throughput sequencing, the paediatric CMV-specific repertoire was found to be highly diverse. In addition, paediatric repertoires were remarkably similar to adults, with public TCR responses being shared amongst children and adults alike. The CMV-specific repertoire in both adults and children displayed strong fluctuations in TCR clonality and repertoire architecture over time. Previously characterised superdominant clonotypes were readily identifiable in the children at high frequency, suggesting that the distortion of the CMV-specific repertoire is incurred as a direct result of CMV infection rather than a product of age-related "memory inflation." Early distortion of the TCR repertoire was particularly apparent in the case of the TCR-β chain, where oligoclonality was low in children and positively correlated with age, a feature we did not observe for TCR-α. This discrepancy between TCR-α and -β chain repertoire may reflect differential contribution to NW8 recognition. Altogether, the results of the present study provide insight into the formation of the TCR repertoire in early life and pave the way to better understanding of CD8 T cell responses to CMV at the molecular level.

Keywords: HLA-B*44:03; T cell receptor; T cell receptor repertoire; cytomegalovirus; memory inflation; paediatric repertoire; repertoire dynamics; superdominance.

Figure 1

High magnitude HLA-B*44:03-restricted, NW8-specific T cell responses are detectable over 12+ years in four mother-child pairs. Clinical data and frequency of tet+ CD8 T cells are shown for four mother-child pairs. (A) 64C and 64M, as well as 64S, an HIV-uninfected sibling of 64C. (B) 76C and 76M. (C) 35C and 35M. (D) 21C and 21M. For 21M, only one timepoint was sampled and clinical data was not available. In all panels, data from the child is displayed on the left and data from the mother on the right side. Clinical data (top) includes the CD4 count in blue, HIV viral load in red, and CMV viral load in purple. The shaded areas indicate the periods when the subjects were on antiretroviral therapy. Longitudinal tetramer data (bottom) is shown as green symbols for children, and black symbols for mothers.

Figure 2

TCR-α and -β chain bias in NW8-specific CD8 T cell responses is manifest in children and adults alike. CD8 T cells were stained with HLA-B*44:03/NW8 tetramer and sorted for TCR-α and -β sequencing, as described in Materials and methods. TRAV-TRAJ (left) and TRBV-TRBJ (right) gene co-occurrence wheels are shown for CMV/HIV infected children, and their mothers. A single time point is shown for every individual, chosen so that the mothers and the children would be of similar age in their respective group. The size of the arcs is proportional to V or J frequency. The area joining any V-J pair is proportional to the co-occurrence frequency of that pair. Co-occurrence (“circos”) wheels were generated by VDJviz (40). Gene segments encoding dominant clonotypes are highlighted in colours matching the corresponding section in the wheel. Gene segment co-occurrence is shown for (A) the 64 family: 64M, the mother; 64C, the child. (B) the 76 family: 76M, the mother; 76C, the child. (C) the 35 family: 35M, the mother; 35C, the child. (D) The 21 family: 21M, the mother; 21C, the child.

Figure 3

CMV-associated TCR repertoire perturbation arises in early life and is maintained over time. Oligoclonality of the TCR-α (A) and -β chain (B) repertoire was calculated as outlined in Materials and methods for each time point. J′ values of 0 have been used to represent monoclonal samples. Spearman's r was computed for every parameter and shown above the corresponding graph. P-values < 0.05 were considered significant (**p < 0.01, NS, not significant).

Figure 4

Clonotypic TCR superdominance is maintained in children and adults over time. The frequency of TCR-α (left) and TCR-β (right) is shown for three mother-child pairs monitored between 2005 and 2017. Superdominant clonotypes for each subject are shown in solid coloured lines. All other clonotypes are represented by solid black lines. A clonotype is considered superdominant if its frequency exceeds >30% at any point.