Skewed Repertoire Reconstitution and Gene Expression of HLA-A2402 CMV-CTLs after Allogeneic HCT

Abstract

<h3>Background</h3> Cellular immunity is important for the control of Cytomegalovirus (CMV) reactivation after allogeneic hematopoietic cell transplantation (HCT). However, the actual <i>in vivo</i> dynamics of CMV-specific cytotoxic T-cell (CMV-CTL) clones are still unclear. Thus, we investigated the T-cell receptor (TCR)-beta repertoire and gene expressions of HLA-A*2402-restricted CMV-CTLs in allo-HCT, using next-generation sequence methods (NGS). <h3>Patients</h3> The clone repertoire and dynamics of HLA-A*2402-restricted CMV-CTL were analyzed in 20 cases (30-1000 cells in each). First, we focused on the clone diversity in the group who had never experienced CMV reactivation after HCT from CMV-positive donors (the transferred-immunity group without CMV reactivation, n=7 cases). Next, we focused on the group who received HCT from CMV-negative donors (the naively-introduced immunity group after CMV reactivation, n=13 cases). Patients who received HCT from CMV-positive donors but experienced CMV reactivation were excluded, since there might simultaneously exist the transferred and introduced immunities. <h3>Results</h3> First, we focused on the clone diversity in the transferred immunity group (n = 7) and identified 78 clones. The top 2 clones in each case at one month after HCT exclusively accounted for >75% (range: 78-100%) of CMV-CTLs in all cases (Fig.1). In addition, these top 2 clones were also dominant in their corresponding donors (n = 3), suggesting that the transferred CMV immunity from donors would play a critical role for the prevention of CMV reactivation. Next, looking at the naively-introduced immunity group (n = 13) where 139 clones were identified, the similar skewed CMV-CTL repertoire reconstitution was also observed, and the top 2 clones accounted for >75% in 10 of the 13 cases (range: 61-100%) at 3 months after HCT (Fig.1). Focusing on the diversity of CMV-CTLs in the late phase after HCT (range: 6 -24 months, n=16), some novel clones appeared and became one of dominant clones in several cases, but the top 1 dominant clones at the early phase in each case stayed at top in 13 of the 16 cases. TRBV7 and BJ1-2 were frequently used in their CDR3 of CMV-CTLs (Fig. 2). In terms of specific amino acid sequences of CDR3 of TCR-ƒÀ, no common motif was found through all cases. However, [TSG, Q(G)GG, DPG, and NQG] motifs were frequently observed 3 months after HCT in the naively-introduced group, while [G(Q)GG or DPG] was observed even among the different cases of the transferred-immunity group. Single-cell RNA sequences demonstrated relatively uniform population in terms of gene expression even if different CMV-CTL clones existed in a patient (Fig.3). <h3>Conclusion</h3> <i>In vivo</i> clone-monitoring and gene expression of CMV-CTLs using NGS could provide an insight into the mechanism of immunological reconstitution following HCT.