T cell receptor sequencing reveals reduced clonal breadth of T-cell responses against SARS-CoV-2 after natural infection and vaccination in allogeneic hematopoietic stem cell transplant recipients.

Allogeneic hematopoietic stem cell transplantation (HSCT) recipients have a higher risk to develop severe COVID-19 and a higher mortality rate compared to the general population (Ljungman et al.) potentially also as a consequence of their reduced ability to respond to vaccination (Mamez et al. ; Redjoul et al. ; Einarsdottir et al.). To evaluate the magnitude and breadth of T cell responses against SARS-CoV-2 in allogeneic HSCT recipients, we performed high-throughput T cell receptor (TCR) repertoire profiling on cells recovered from allogeneic HSCT recipients or healthy controls after COVID-19 natural infection or mRNA-based vaccination.

Peripheral blood samples were obtained after COVID-19 infection from allogeneic HSCT recipients (n=11; Supplemental Table 1-2) or healthy controls (HC; n=10; Supplemental Materials). Six out of 11 patients were under immunosuppression for active (n=3) or resolved (n=3) GvHD. T-cell receptor (TCR) beta sequencing (Supplemental Materials) identified SARS-CoV-2 specific T cell clonotypes in both HC and HSCT recipients after COVID-19 infection (Figure 1 A). No difference was observed in the proportion of T cells specific for SARS-CoV-2 between HSCT recipients and HC (data not shown). However, the diversity of the SARS-CoV-2-specific T cell clonotypes, a measure previously shown to be inversely associated with severity of the disease (Elyanow et al. ), was significantly reduced in HSCT recipients compared with HC (Figure 1B). Enzyme-Linked ImmunoSpot (ELISpot) assay (Supplemental Materials) showed significantly lower numbers of IFN-γ spot forming units (SFU) after stimulation of PBMCs from HSCT recipients with peptides from both the SARS-CoV-2 Spike (S) protein (Figure 1B, upper panel) and the membrane glycoprotein (M) plus the nucleocapside phosphoprotein (N) proteins (data not shown) compared with HC. A significant positive correlation between SARS-CoV-2-specific T cell clonotypes and IFN-γ SFU was observed (Figure 1B, upper panel). Conversely, we detected no significant difference in anti-Spike IgG titers and no correlation between antibody titers and different clonotypes (Figure 1B, middle panel). HSCT recipients displayed a less diverse TCR repertoire compared with HC as revealed by higher Simpson clonality and the Simpson clonality negatively correlated with the number of different SARS-CoV-2-specific T cell clonotypes (Figure 1B, lower panel).

Figure 1

Reduced SARS-CoV2-specific T cell clonotypes after COVID-19 infection and vaccination in allogeneic HSCT recipients. (A, C) SARS-CoV-2 specific T cell clonotypes visualized based on the putative sequence of the SARS-CoV-2 genome recognized. (B, D) Scatter plots and marginal bar plots correlating and comparing the number of different SARS-CoV-2 specific T cell clonotypes/1000 T cells, the anti-S IFN-γ SFU, the anti-S IgG titers and the Simpson clonality index in HC and HSCT. Differences between groups were assessed using the Mann–Whitney U test. Correlations were evaluated using a Spearman rank correlation coefficient test.

We next performed the same analysis on samples recovered from allogeneic HSCT recipients (n=11; Supplemental Table 1-2) or from vaccinated healthy controls (n=10) after vaccination with 3 doses of mRNA-based SARS-CoV-2 vaccines (Supplemental Materials). We observed a significant reduction in different S-protein-specific T cell clonotypes in allogeneic HSCT recipient compared to HC (Figure 1C-D). ELISpot analysis revealed significantly lower numbers of IFN-γ SFU in HSCT recipients compared with HC and a slightly significant positive correlation between the ELISpot and the TCR-seq results (Figure 1D, upper panel). We observed slightly reduced anti-S titers in HSCT recipient compared to HC and a trend toward a positive correlation between S-specific clonotypes and anti-S titers (Figure 1D, middle panels). We detected a negative correlation between the Simpson clonality and the number of different S-protein specific T cell clonotypes after vaccination (r2=0.55, p=7.8e-05; Figure 1F).

Our results indicate that allogeneic HSCT recipients display reduced breadth of SARS-CoV-2-specific T cell clonotypes after COVID-19 infection and vaccination. No clear correlation was detected between TCR clonal breadth and anti-S IgG titers. The clonal breadth defect was associated with increased T-cell clonality after HSCT, pointing to the reduced diversity of the TCR repertoire as a mechanism leading to impaired cellular responses against SARS-CoV-2 in HSCT recipients.