Diagnosing Viral Infections Through T-Cell Receptor Sequencing of Activated CD8+ T Cells

Abstract

T-cell-based diagnostic tools identify pathogen exposure but lack differentiation between recent and historical exposures in acute infectious diseases. Here, T-cell receptor (TCR) RNA sequencing was performed on HLA-DR+/CD38+CD8+ T-cell subsets of hospitalized coronavirus disease 2019 (COVID-19) patients (n = 30) and healthy controls (n = 30; 10 of whom had previously been exposed to severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]). CDR3α and CDR3β TCR regions were clustered separately before epitope specificity annotation using a database of SARS-CoV-2-associated CDR3α and CDR3β sequences corresponding to >1000 SARS-CoV-2 epitopes. The depth of the SARS-CoV-2-associated CDR3α/β sequences differentiated COVID-19 patients from the healthy controls with a receiver operating characteristic area under the curve of 0.84 ± 0.10. Hence, annotating TCR sequences of activated CD8+ T cells can be used to diagnose an acute viral infection and discriminate it from historical exposure. In essence, this work presents a new paradigm for applying the T-cell repertoire to accomplish TCR-based diagnostics.

Keywords: COVID-19; NGS-based diagnostics; T cells; TCR sequencing; immunoinformatics; immunology.

Figure 1.

Characterization of sorted T-cell fractions of coronavirus disease 2019 patient and healthy controls. A, Patient overview. One patient represents 1 line where severity, sex, symptomatic prehospitalization days, days in the hospital, and time points of sampling are shown. Patients are ordered according to duration of symptomatic period. Of 5 patients, a postrecovery sample was analyzed. B. Laboratory method workflow. Study volunteer blood samples were collected from which peripheral blood mononuclear cells were isolated and sorted to obtain T-cell subsets: “activated T cells” (CD3⁺/CD8⁺/CD38⁺/HLA-DR⁺) and “nonactivated T cells” (CD3⁺/CD8⁺/CD38⁻/HLA-DR^–). RNA from the sorted cells underwent high-throughput T-cell receptor VDJ sequencing. Abbreviations: COVID-19, coronavirus disease 2019; F, female; FACS, fluorescence-activated cell sorting; M, male; PBMC, peripheral blood mononuclear cell; TCR, T-cell receptor; VDJ, variable (V), joining (J), and diversity (D) gene segments.

Figure 2.

Increased presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–associated T-cell receptors (TCRs) in the skewed activated T-cell subset during acute coronavirus disease 2019 (COVID-19). The figure depicts only those patient samples in which both the activated T-cell subset and the nonactivated T-cell subset were analyzed (left: COVID-19 patients, n = 26; right: previously SARS-CoV-2–exposed individuals, n = 10) A, Chao1 diversity (sum of Chao1 index of the CDR3α and CDR3β chains). B, Clonal expansion of SARS-CoV-2–associated T cells expressed as SARS-CoV-2–associated TCR depth (defined as the sum of SARS-CoV-2–associated TCR clones divided by the repertoire size). P values denote paired Student t test. **P ≤ .01; ****P ≤ .0001; ns, not significant (P > .05).

Figure 3.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–associated clonotype frequency in CD8⁺/CD38⁺/HLA-DR⁺ populations detected during active disease and after recovery (rec) in the same individuals (n = 5). A, SARS-CoV-2 CDR3α matches. B, SARS-CoV-2 CDR3β matches. Depicted are the top 20 SARS-CoV-2–associated T-cell receptors after coronavirus disease 2019 (COVID-19) recovery compared to the acute COVID-19 phase.

Figure 4.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) predictive performance of CD8⁺/CD38⁺/HLA-DR⁺ populations. A, Depth of the SARS-CoV-2–associated immune response. Of the 30 included patients, 20 had moderate, 5 severe, and 5 critical coronavirus disease 2019. In the control group, 10 individuals had past SARS-CoV-2 infection (exposed [exp]), 10 had no evidence of a previous SARS-CoV-2 infection although being sampled during the pandemic (nonexposed-pandemic [non-exp-p]), and 10 were non-exposed controls sampled before the pandemic (nonexposed-prepandemic [non-exp-pp]). P value denotes Student t test. ***P ≤ .001. B, Receiver operating characteristic curve using the SARS-CoV-2–associated depth in a logistic regression classifier. Abbreviations: AUC, area under the curve; COVID-19, coronavirus disease 2019; ROC, receiver operating characteristic; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.