High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4+ T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19

Abstract

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4⁺ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4⁺ T-cell response in both patients, with higher frequencies of virus-specific CD4⁺ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4⁺ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4⁺ T-cells with CD45RA^- CXCR5⁺ PD-1⁺ circulating T follicular helper cell (cT_FH) phenotype. Furthermore, we observed a delayed contraction of CD127^- virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4⁺ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell-T-cell interaction, a robust virus-specific CD4⁺ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection.

Keywords: CD39; CD4+ T-cells; CD73; COVID-19; MHC class II Tetramer; PD-1; SARS-CoV-2; T-cell memory; TIGIT; anti-CD20 therapy.

Figure 1

Clinical course and kinetics of Tetramer⁺ SARS-CoV-2-specific CD4⁺ T-cells. (A) PCR results for SARS-CoV-2 from nasopharyngeal swabs and sputum are shown as “+” for a positive and “−“ for a negative test result. Overview of the lymphocytes (B- and T-cells) and inflammatory blood markers CRP [mg/L] and IL-6 [ng/L] during the infection of the index patient are depicted. (B) Representative flow cytometry plot of DRB1*11 Tetramer staining of PBMC from the index patient on day 112 after the onset of symptoms. Shown are living CD3⁺ T-cells. (C) The frequencies of Tetramer⁺ CD4⁺ T-cells in the peripheral blood of the index patient (red) and a reference patient (blue) are depicted longitudinally (left), and pooled for each patient (right). (D) Comparison of the proportions of naïve (T_n; CCR7⁺ CD45RA⁺), central memory (T_cm; CCR7⁺ CD45RA⁻), effector memory (T_em; CCR7⁻ CD45RA⁻), and late effector memory (T_emRA; CCR7⁻ CD45RA⁺) T-cells between the index patient (red) and the reference patient (blue) among the SARS-CoV-2-specific T-cells. (E) The index patient showed cumulatively increased frequencies of SARS-CoV-2-specific CD4⁺ T-cells with a T_em phenotype. (F) Development of IL7Rα (CD127) negative SARS-CoV-2-specific effector CD4⁺ T-cells of the index (red) and the reference patient (blue). Dotted lines indicate the last positive PCR result. (G) The index patient showed cumulatively reduced frequencies of SARS-CoV-2-specific CD4⁺ T-cells with a circulating T follicular helper cell (cT_FH) phenotype. Shown are representative flow cytometry plots for both patients from day 32 (index patient; red) or day 34 (reference patient; blue). In cumulative analyses, data are depicted as mean with SD, and for statistical testing, a Mann–Whitney test was performed. Results were considered statistically significant if p < 0.05. Levels of significance are translated to asterisks as follows: * p < 0.05; ** p < 0.01.

Figure 2

Co-inhibitory receptors on SARS-CoV-2-specific CD4⁺ T-cells. Expression frequencies of PD-1, LAG-3, and TIGIT on SARS-CoV-2-specific CD4⁺ T-cells of the index patient (red) and the reference patient (blue), as well as the respective relevant correlations, are depicted. (A) PD-1 expression was elevated on virus-specific CD4⁺ T-cells of the index patient, and correlated with CD127 expression. (B) LAG-3 expression on virus-specific CD4⁺ T-cells was elevated in early infection, and was associated with plasma CRP levels in the index patient (r = 0.945; p = 0.055). (C) TIGIT expression was elevated on virus-specific CD4⁺ T-cells of the reference patient, and significantly correlated with plasma IL-6 levels in the index patient (r = 0.997; p = 0.003). In correlation analyses, each dot represents an individual time point, and values are given as Pearsons’s r. Results were considered statistically significant if p < 0.05. Levels of significance are translated to asterisks as follows: * p < 0.05; ** p < 0.01.

Figure 3

Ectonucleotidases CD39 and CD73 on SARS-CoV-2-specific CD4⁺ T-cells. Exemplary flow cytometry plots of the index patient (day 112) for expression of CD39 (A) and CD73 (B) on bulk and virus-specific CD4⁺ T-cells. Shown are living CD3⁺ CD4⁺ T-cells. (C) Longitudinal assessment of CD39 expression on bulk and virus-specific CD4⁺ T-cells of the index patient (left) and the reference patient (right). (D) Longitudinal assessment of CD73 expression on bulk and virus-specific CD4⁺ T-cells of the index patient (left) and the reference patient (right). (E) Representative flow cytometry plot of the reference patient, with backgating of the virus-specific CD4⁺ T-cells illustrating the CD39/CD37 expression pattern. (F) Correlation of CD39⁺ virus-specific T-cells with overall Tetramer⁺ CD4⁺ T-cells (r = 0.695; p = 0.018) and PD-1 expression frequency (r = 0.725; p = 0.012). In correlation analyses, each dot represents an individual time point, and values are given as Pearsons’s r. Results were considered statistically significant if p < 0.05. Levels of significance are translated to asterisks as follows: * p < 0.05; ** p < 0.01.