Chain-specific remodeling of the γδ T-cell TCR repertoire in preterm infants with cCMV: a gestational-age–stratified case–control study

Background: Congenital cytomegalovirus (cCMV) is a major cause of intellectual disability. γδ T-cells contribute to early-life antiviral defense, yet their T-cell receptor (TCR) repertoire in preterm cCMV remains incompletely defined. Methods: We profiled the γδ T-cell TCR repertoire in preterm infants allocated to three non-overlapping groups: Congenital CMV infection (cCMV) group (n=9), Acquired CMV infection (aCMV) group (n=10), and Preterm controls (CMV-negative) (n=7). RNA was extracted from RNAlater-stabilized heel-prick whole blood. Libraries were generated using constant-region-anchored primers for TRD/TRG, multiplex target enrichment, and index PCR, and sequenced on an Illumina platform (paired-end 150 bp). Repertoire features included CDR3 length distributions, V(D)J usage, VJ pairing, and clonal diversity. Nine lineage-associated rearrangements were quantified by intercalating dye-based qPCR. Results: TRD: the 25-aa CDR3 bin was less frequent in the cCMV group versus controls, and the cCMV group showed a lower Gini index, indicating less clonal dominance (greater evenness) than controls. TRG: cCMV and aCMV differed in a length-interval-dependent manner. V-gene usage shifted by group (TRAV29/DV5 and TRAV38-2/DV8 in TRD; TRGV2/4/8/9 in TRG), and VJ pairings were more frequent in cCMV than in aCMV. The cCMV group harbored more "specific" clonotypes than controls (P = 0.020). qPCR showed higher expression of Dδ2-Dδ3, Vδ2-Jδ1, Vγ-Jγ1.3/2.3 in cCMV; the mixed Vγ9+Vγ11-Jγ1.1/2.1 signal was also elevated but requires deconvolution. Conclusions: cCMV is associated with chain-specific remodeling of the γδ T-cell repertoire in preterm infants. These data may inform molecular risk stratification in neonatal CMV. Findings should be interpreted in light of small sample size and RNA-level profiling.