Major TCR Repertoire Perturbation by Immunodominant HLA-B*44:03-Restricted CMV-Specific T Cells

Lack of disease during chronic human cytomegalovirus (CMV) infection depends on the maintenance of a high-frequency CMV-specific T cell response. The composition of the T cell receptor (TCR) repertoire underlying this response remains poorly characterised, especially within African populations in which CMV is endemic from infancy. Here we focus on the immunodominant CD8+ T cell response to the immediate-early 2 (IE-2)-derived epitope NEGVKAAW (NW8) restricted by HLA-B*44:03, a highly prevalent response in African populations, which in some subjects represents >10% of the circulating CD8+ T cells. Using pMHC multimer staining and sorting of NW8-specific T cells, the TCR repertoire raised against NW8 was characterised here using high-throughput sequencing in 20 HLA-B*44:03 subjects. We found that the CD8+ T cell repertoire raised in response to NW8 was highly skewed and featured preferential use of a restricted set of V and J gene segments. Furthermore, as often seen in immunity against ancient viruses like CMV and Epstein-Barr virus (EBV), the response was strongly dominated by identical TCR sequences shared by multiple individuals, or “public” TCRs. Finally, we describe a pair “superdominant” TCR clonotypes, which were germline or nearly germline-encoded and produced at remarkably high frequencies in certain individuals, with a single CMV-specific clonotype representing up to 17% of all CD8+ T cells. Given the magnitude of the NW8 response, we propose that this major skewing of CMV-specific immunity leads to massive perturbations in the overall TCR repertoire in HLA-B*44:03 individuals

Cohort characteristics.

<p>Cohort characteristics.</p

Cohort characteristics.

<p>Cohort characteristics.</p

Immunodominant CMV-specific CD8+ T-cell responses within pp65, IE-1 and IE-2 measured by IFN-g ELISpot assay for CMV+ subjects.

<p>(A) Percentage responders to each protein-covering peptide pool. (B) Percentage responders among 152 CMV+ individuals to pp65 15mer overlapping peptides that were targeted by >4% of the study population. (C) Percentage responders among 95 CMV+ individuals to IE-1 15mer overlapping peptides that were targeted by >3% of the study population. (D) Percentage responders among 92 CMV+ individuals to IE-2 15mer overlapping peptides that were targeted by >3% of the study population.</p

HLA associations with the remaining 15-mer OLP’s in CMV pp65, IE-1 and IE-2 that were targeted by ≥4% of the study cohort.

<p>HLA associations with the remaining 15-mer OLP’s in CMV pp65, IE-1 and IE-2 that were targeted by ≥4% of the study cohort.</p

Validation of novel CD8+ T-cell epitopes in CMV using HLA-Class I tetramers.

<p>(A) Flow cytometry plot of CD8+ T-cell responses from a Durban cohort subject SK-251 (HLA-A*02:05, -A*30:01, -B*42:01, -B*58:01, -C*07:01, -C*017:01) to HLA-A*30:01-restricted IE-2-342/343 epitope-KK9 and an HLA-mismatched tetramer stain as a control. (B) Flow cytometry plot from a Durban cohort subject SK-331 (HLA-A*30:01, -A*66:01, -B*42:01, -B*58:02, -C*06:02, -C*017:01) to B*42:01-restricted pp65-66/67 epitope RL11 and an HLA-mismatched tetramer stain as a control. The plots show gated live CD3+ T-cells; the number shown above each gate is the percentage of live CD3+ CD8+ cells that are tetramer-specific.</p

Individuals making CD8+ T cell responses against CMV IE-2 epitope HLA-B*44:03-NW8.

<p>(A) Representative FACS plot of a Durban cohort subject 202-30-0064 (expressing HLA-B*44:03/B*58:01) to HLA-B*44:03-restricted NW8 tetramer. (B) FACS plot of subject R048 (HLA-A*29:02, -A*6802, -B*15:03, -B*44:03, -C*02:10, -C07:01) with NW8 tetramer and HLA-mismatched tetramer. (C) FACS plot of a TVC subject R112 (HLA-A*23:01, -A*3002, -B*18:01, -B*18:01, -C*02:02, -C07:04) to HLA-B*44:03-restricted NW8 tetramer, as B*44:03-negative control. The FACS plots show tetramer-specific cells expressed as the percentage of live CD3+ CD8+ T-cells. (D) Percentage of live CD3+ CD8+ tetramer-specific T-cells in individuals expressing HLA-B*44:03 measured using the B*44:03-NW8 tetramer.</p

Phenotypic frequencies of closely related HLA I molecules and their impact on CMV specific CD8+ T-cell hierarchies.

<p>(A) The phenotypic frequency of some of the HLA class I molecules in A2 superfamily in white populations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189612#pone.0189612.ref028" target="_blank">28</a>] and our African cohort. (B) The percentage of subjects with HLA alleles A*02:01 (n = 23), A*02:02 (n = 10) and A*02:05 (n = 16), responding to epitope (NLVPMVATV) in pp65-123/124 peptide and predicted epitope (FVFPTKDV) in pp65-46/47 peptide. (C) The phenotypic frequency of the HLA class I molecules in B07 superfamily in white populations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189612#pone.0189612.ref028" target="_blank">28</a>] and our African cohort. (D) The percentage of subjects with B*07:02 (n = 21), B*35:01 (n = 10), B*39:10 (n = 6), B*42:01 (n = 26) and B*81:01(n = 12) alleles in our African cohort responding to predicted epitopes in pp65, GPISGHVL (OLP-4), RPHERNGFTVL (OLP-66/67) and TPRVTGGGAM (OLP-104/105). The percentage of subjects in our African cohort responding to predicted epitope in IE-1, TMYGGISLL (OLP-212/213) with HLA-B*07:02 (n = 11), B*35:01 (n = 6) B*39:10 (n = 5), B*42:01 (n = 19) and B*81:01(n = 1) alleles.</p