Quantitation of CMV Specific T-Cell Expansion Using T Cell Receptor Beta Locus Deep Sequencing to Identify Patients at Risk of Viral Complications

Background/Aim An effective T cell response is necessary for the control of cytomegalovirus (CMV) reactivation after allogeneic stem cell transplant (alloBMT) - CMV is associated with significant morbidity, especially in patients (pts) who require recurrent episodes of antiviral therapy u0026 those who develop associated end organ dysfunction (CMV disease). We sought to determine whether deep sequencing of T cell receptor beta (TRB) loci could identify u0026 quantitate T cell clonotypes with specificity for CMV epitopes in the early post-alloBMT period and assess association with virus related outcomes. Methods TRB deep sequencing was performed using LymphoTrack TRB (Invivoscribe) on stored DNA samples, extracted from CD3+ selected peripheral blood samples taken at Day+30 u0026 Day+60 from 65 consecutive pts receiving alloBMT at our institution, who were donor (D) u0026/or recipient (R) CMV seropositive. Sequence assembly, annotation u0026 error correction was performed by MiXCR/VDJtools. Epitope specificity was assessed using a curated database of T cell clonotype/epitope specificity (VDJdb). Statistical analysis was performed using the Kaplan-Meier method for time to event endpoints, Fisheru0027s exact test for discrete variables u0026 the Mann Whitney U test for continuous variables. Results CMV specific T cells were identifiable in 97% samples at D+30 u0026 100% samples at D+60 - the proportion of the T cell repertoire comprising CMV specific T cells (CMV-Trep%) was median (IQR) 0.15% (0.04-0.55%) u0026 0.12% (0.05-0.66%) respectively. Antecedent viremia was associated with a higher CMV-Trep% at both time points (D+30: 0.45% vs, 0.095%, p=0.01; D+60: 0.20% vs. 0.07%, p=0.02), as was D+R+ serostatus at D+60 (0.28%, p=0.003). Pts who developed detectible viremia between the two sample timepoints had a median 2.1-fold increase in CMV-Trep% (vs. 0.1-fold decrease for those without viremia, p=0.03). 45% of pts had at least 1 episode of anti-CMV therapy, 52% of whom required u003e1 episodes of treatment. CMV disease developed in 11% of pts. Time to first antiviral treatment was associated with ATG (HR 2.5, p Conversely, following initial reactivation requiring antiviral therapy, pts with CMV-Trep u003e0.1% at D+60 were significantly less likely to require additional episodes of antiviral therapy (HR 0.32, p=0.05) or develop CMV related organ dysfunction (HR 0.15, p=0.02). No clinical factor was associated with these outcomes. Conclusion The degree of post-reactivation expansion of CMV specific T cells can be quantified using TRB deep sequencing. Identification of pts who fail to mount a T cell response to initial reactivation may be predictive of those at high risk for recurrent viremia u0026 CMV disease, in whom secondary prophylactic interventions (e.g. antiviral or cellular therapy) could be used.