Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation

Abstract

Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting.

Keywords: CMV; T cell receptor alpha; T cell receptor repertoire; allogeneic transplantation; next-generation sequencing.

Figure 1

Time–course of the cytomegalovirus (CMV) virus load and CMV_pp65‐specific CD8⁺ T cells, patient 1. The patient developed a CMV‐specific immunity, as seen by a high frequency of detectable CMV_pp65‐specific CD8⁺ T cells. At day 74 after transplantation the patient was diagnosed with a graft‐versus‐host disease (GVHD) requiring prednisolone (2 mg/kgKG). Following steroid exposure whole lymphocytes (not shown) and CMV_pp65‐specific CD8⁺ T cells decreased and the patient developed a CMV reactivation with CMV enteritis requiring anti‐viral treatment. Samples for next‐generation sequencing (NGS) of CMV_pp65‐specific CD8⁺ T cells were taken at days 74, 173, 404, 525 and 547 following transplantation. The patients showed a donor chimerism >98% from day 27 and >99% from day 62 following transplantation throughout the observed period. Total CMV_pp65‐specific CD8⁺ T cells (solid line) were calculated based on total lymphocyte count in peripheral blood (10⁹/l).

Figure 2

Distribution of T cell receptor alpha (TCR‐α) clonotypes in patient 1. (a) Cytomegalovirus (CMV)‐specific CD8⁺ T cells were sequenced at four time‐points. One dominant clonotype was detected in all samples (75·0–90·1% of reads). (b) CMV_IE‐specific CD8⁺ T cells were sequenced at day 547 following transplantation, resulting in two dominant clonotypes. (c) The three dominant clonotypes from CMV_pp65‐specific and CMV_IE‐specific cells accounted for approximately 50% of the whole CD8⁺ TCR‐α repertoire. Clonotypes with a frequency >1% of reads in the CMV samples are shown in colour. Previously published public CMV motifs are underlined. Sequences that were also identified by single‐cell sequencing are marked by *.

Figure 3

Sorting, phenotype and T cell receptor alpha (TCR‐α) sequences of cytomegalovirus (CMV)_pp65‐specific CD8⁺ T cells (day 547 following transplantation). (a) CD3⁺CD8⁺ T cells (52·0% of lymphocytes) and CMV_pp65‐specific cells (11·4% of CD3⁺CD8⁺ T cells) were sorted for next‐generation sequencing (NGS) analyses of the TCR‐α. Additionally, single‐cell sequencing with the amplification of TCR‐α and TCR‐β was performed on sorted single cells of the two CMV_pp65‐specific populations (pop I: 10·3% of CD3⁺CD8⁺ T cells and pop II: 0·7% of CD3⁺CD8⁺ T cells). One single clonotype was identified in pop I cells, whereas clonotypes of pop II cells were heterogeneous. (b) Phenotyping of pop I and pop II cells of CD3⁺CD8 ⁺ CMV_pp65‐specific T cells. Pop I cells were mainly CCR7^–CD45RA^– effector memory (EM) cells (84·3% of CD3⁺CD8⁺ CMV_pp65‐specific T cells) and for pop II, 77·2% were CCR7^–CD45RA⁺ terminally differentiated effector memory (TEMRA) cells.

Figure 4

Gene expression analyses of single cytomegalovirus (CMV)_pp65‐specific CD8⁺ T cells (day 547 following transplantation). Heatmap for selected genes. Pop I of CMV_pp65‐specific cells (blue, n = 84) and pop II of CMV_pp65‐specific cells (green, n = 84) showed comparable expression patterns. Gene expression profiles of influenza‐specific CD8⁺ T cell clones (FluMP, red, n = 36) and CD8⁺ T cells from a healthy donor (purple, n = 80) are shown for comparison. Whole gene names are provided in Supporting information, Table S3.

Figure 5

Time course of the cytomegalovirus (CMV) virus load and cytomegalovirus (CMV)_pp65‐specific CD8⁺ T cells, patients 2–4. The date of sampling for each patient is implemented as vertical arrows. Patients showed a full donor chimerism starting from day 29 (for patient 2) and day 14 (for patients 3 and 4). There were no cases of mixed chimerism over the whole time. Total CMV_pp65‐specific CD8⁺ T cells (solid line) were calculated based on total lymphocyte count in peripheral blood [10⁹/l]. GVHD = graft‐versus‐host disease; p = prednisolone.

Figure 6

Distribution of T cell receptor alpha (TCR‐α) clonotypes in patient 2. Cytomegalovirus (CMV)_pp65‐specific CD8⁺ T cells and CD8⁺ T cells from day 71 following transplantation were sequenced by next‐generation sequencing (NGS) for their TCR‐α. Clonotypes with a read frequency >1% in the CMV_pp65‐specific cells are shown in colour and mapped in the CD8⁺ repertoire. Additionally, frequent clonotypes containing a public CMV motif are shown in yellow. Previously published public CMV motifs are underlined. Sequences that were also identified by single‐cell sequencing are marked by *.

Figure 7

Shared clonotypes of patients 1 and 2. Comparison of clonotypes from cytomegalovirus (CMV)_pp65 samples of patients 1 and 2. Shared clonotypes are shown in black. Non‐shared clonotypes are shown in white.