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

Abstract

<h3>Background/Aim</h3> 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 & 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 & quantitate T cell clonotypes with specificity for CMV epitopes in the early post-alloBMT period and assess association with virus related outcomes. <h3>Methods</h3> TRB deep sequencing was performed using LymphoTrack TRB (Invivoscribe) on stored DNA samples, extracted from CD3+ selected peripheral blood samples taken at Day+30 & Day+60 from 65 consecutive pts receiving alloBMT at our institution, who were donor (D) &/or recipient (R) CMV seropositive. Sequence assembly, annotation & 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, Fisher's exact test for discrete variables & the Mann Whitney U test for continuous variables. <h3>Results</h3> CMV specific T cells were identifiable in 97% samples at D+30 & 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%) & 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 >1 episodes of treatment. CMV disease developed in 11% of pts. Time to first antiviral treatment was associated with ATG (HR 2.5, p<0.01), unrelated donor (HR 2.0, p=0.04) & non D+R- serostatus (HR 6.0, p<0.01), and was not predicted by CMV-Trep% at either timepoint. Conversely, following initial reactivation requiring antiviral therapy, pts with CMV-Trep >0.1% 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. <h3>Conclusion</h3> 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 & CMV disease, in whom secondary prophylactic interventions (e.g. antiviral or cellular therapy) could be used.