Clonal analysis of immunodominance and cross-reactivity of the CD4 T cell response to SARS-CoV-2

1The identification of T cell epitopes in disease causing organisms is a challenge in view of the polymorphism of HLA class II molecules and the variability of rapidly mutating pathogens. In the context of the COVID-19 pandemic, bioinformatic analysis has been used to predict T cell epitopes in SARS-CoV-2 proteins and to produce peptide pools to stimulate peripheral blood mononuclear cells (PBMCs) and enumerate antigen-specific T cells. These studies revealed a robust CD4+ and CD8+ T cell response against SARS-CoV-2 proteins in recovered patients and a level of cross-reactivity with endemic coronaviruses in pre-pandemic samples.

2A limitation of bioinformatic predictions is the difficulty in identifying immunodominant epitopes, since immunodominance is determined by multiple factors such as antigen processing, T cell repertoire, HLA alleles, and preexisting cross-reactive immunity. To identify naturally processed immunodominant CD4+ T cell epitopes, we took the unbiased approach of stimulating memory T cells with protein-pulsed antigen presenting cells (APCs), followed by the isolation of T cell clones to precisely map the epitope recognized.

3PBMCs from a first cohort of 14 patients who had recovered from mild-to-severe COVID-19 (table S1) were used to isolate total CD4+ T memory cells or T central memory (Tcm), T effector memory (Tem), and circulating T follicular helper (cTfh) cells (fig. S1A). The cells were labeled with CFSE and stimulated with autologous monocytes in the presence of recombinant SARS-CoV-2 spike (S) or nucleoprotein (N). In all individuals, we observed a strong response to both antigens in terms of proliferation and IFN-γ production (Fig. 1, A and B, and fig. S1, B and C). Proliferating T cells were detected at different levels in Tcm, Tem, and cTfh, consistent with a recent report, and over a one-year period (fig. S1D). By contrast, the CD4+ memory T cell response to SARS-CoV-2 proteins in unexposed individuals was low or undetectable (Fig. 1B and fig. S1C), consistent with the presence of a few cross-reactive T cells primed by endemic coronaviruses.

4The clonal composition of SARS-CoV-2-reactive T cells and the relationship between different memory subsets was studied in three individuals (P28, P31, and P33) by TCR Vβ sequencing. Tcm, Tem, and cTfh cell lines comprised on average 908, 480, and 697 S-reactive clonotypes and 1452, 623, and 908 N-reactive clonotypes, respectively (Fig. 1C and fig. S2). Interestingly, several of the most expanded clonotypes were shared between two and even among all three subsets (Fig. 1, C and D), indicating a polyfunctional response consistent with previous studies on intraclonal diversification of antigen-primed CD4+ T cells.

5In view of the interest for the design of a subunit vaccine, we analyzed in depth the CD4+ T cell response to the S protein and in particular to the RBD, which is the main target of neutralizing antibodies. CD4+ T cells from a larger cohort of 34 COVID-19 individuals (table S1) were stimulated with S protein-pulsed monocytes and proliferating T cells were cloned by limiting dilution. We obtained 2943 T cell clones and mapped their specificity using three pools of peptides spanning S1ΔRBD, RBD, and S2 (Fig. 2, A and B). RBD-specific T cell clones were found in 32 out of 34 donors, accounting on average for 20% of the response to the S protein (Fig. 2B). Using a matrix-based approach, we mapped the epitope specificity of 1254 RBD-reactive CD4+ T cell clones (Fig. 2C) and found that in each individual the clones recognize multiple sites that collectively spanned almost all the RBD sequence. However, certain regions emerged as immunodominant, such as those spanning residues S346-385 and S446-485. Strikingly, a 20-amino-acid region (S346-365) was recognized by 94% of the individuals (30 out of 32) and by 33% of the clones (408 out of 1254) (Fig. 2D). This region is highly conserved among human Sarbecoviruses, including the recently emerged variants of concern (VOC), and zoonotic Sarbecoviruses (Fig. 2E). RBD- and S346-365-specific T cell clones were found in different memory subsets of COVID-19 individuals and were also isolated from individuals following SARS-CoV-2 mRNA vaccination (fig. S3). Thus, RBD is highly immunogenic in vivo and contains a large number of naturally processed T cell epitopes including a conserved immunodominant region.

6To dissect the CD4+ T cell response to the immunodominant S346-365 region, we sequenced TCR Vβ chains of 329 specific T cell clones. The 206 clonotypes identified used a broad spectrum of TCR Vβ genes and, even in the same individual, carried different CDR3 sequences (Fig. 3, A and B, and table S2). In P31 and P33, certain S346-365 clonotypes were detected in ex vivo memory T cells among the top 5% (Fig. 3C). Using blocking antibodies we determined that most of the T cell clones analyzed (n=247 from 22 individuals) were HLA-DR-restricted, whereas the remaining (n=50 from five individuals) were HLA-DP- and one was HLA-DQ-restricted (Fig. 3, D and E). Using truncated peptides and T cell clones from individuals with different HLA type (table S3), we defined two HLA-DR-restricted epitopes (VYAWNRKRIS and RFASVYAWNRKR), and one HLA-DP-restricted epitope (NRKRISNCVAD) (Fig. 3F). Thus, the S346-365 region comprises at least three nested epitopes recognized in association with different allelic forms of HLA-DR or HLA-DP by T cell clones that use a large set of TCR Vβ genes and CDR3 of different sequence and length.

7To address the extent of T cell cross-reactivity between different S proteins, SARS-CoV-2 S-specific T cell lines from P28 and P33 were relabeled with CFSE and stimulated with S proteins from endemic human coronaviruses. In the secondary cultures, a robust proliferation was observed in response to SARS-CoV and HKU1 (Fig. 4A). Interestingly, a sizeable fraction of SARS-CoV-2 clonotypes (ranging from 7 to 25%) were found in SARS-CoV and/or HKU1 secondary cultures, consistent with a substantial degree of T cell crossreactivity (fig. S4). To corroborate this finding, we isolated from secondary cultures several clones that proliferated in response to two or even three different naturally processed S proteins (Fig. 4B and table S4).

8Crossreactive T cells may derive from preexisting memory cells or from the priming of naïve T cells. We therefore analyzed a COVID-19-recovered individual from whom we had previously cryopreserved PBMCs. A robust memory CD4+ T cell proliferation in the pre-COVID-19 sample was detected against NL63 and 229E S proteins, whereas the response to HKU1 and OC43 was limited and the response to SARS-CoV and SARS-CoV-2 undetectable (Fig. 4C). Conversely, in the post-COVID-19 sample, strong T cell proliferation was observed not only in response to SARS-CoV-2, but also in response to all other alpha and beta coronavirus S proteins (Fig. 4, C and D) and shared clonotypes were detected between SARS-CoV-2 and endemic coronavirus S protein-stimulated cultures (fig. S5A). Furthermore, T cell clones isolated from cultures stimulated by SARS-CoV, OC43, or NL63 proliferated in response to the SARS-CoV-2 S peptide pool and their specificity was mapped primarily to the S2 region (Fig. 4, E and F), consistent with its high degree of sequence conservation. Notably, T cell clones that fully cross-reacted with all S proteins mapped to the highly conserved fusion peptide (Fig. 4G).

9To ask whether S-reactive T cells in the post-COVID-19 sample could be detected in pre-pandemic samples, we performed clonotypic analysis of total memory T cells on the post-COVID-19 sample and on samples collected in 2014 and 2017. Most of the SARS-CoV-2 reactive clonotypes identified above were found only in the post-COVID-19 sample, consistent with priming of naïve T cells (fig. S5B). By contrast, clonotypes reactive to endemic coronaviruses were found at comparable number at all time points. Notably, T cell clonotypes against the highly conserved fusion peptide could be tracked back to the 2014 sample and found to be expanded in the post-COVID-19 sample (fig. S5C). These findings demonstrate that pre-existing crossreactive memory T cells are recalled and expanded upon SARS-CoV-2 infection.

10The robust CD4+ T cell response to the RBD and the identification of the S346-365 immunodominant region conserved in the emerging SARS-CoV-2 VOC provide the rationale for the development of a subunit vaccine based on RBD, since this is the target of most neutralizing antibodies. These findings were not anticipated in previous studies based on bioinformatics predictions and short-term peptide stimulation of PBMCs, highlighting the value of combining T cell stimulation with protein antigens with cloning and TCR sequencing for the dissection of antigen-specific T cell repertoires.

11The immunodominance of RBD S346-365 at the individual and at the population level may be due to the presence of three nested T cell epitopes presented by HLA-DR and HLA-DP and to the relative abundance of naturally processed peptides, as recently reported by immunopeptidomics. Interestingly, the S346-365 region is also a contact site for the broadly reactive neutralizing antibody S309, providing a striking example of convergence of B and T cells around a conserved epitope.

12Our study provides also evidence for the recall of preexisting cross-reactive memory T cells upon SARS-CoV-2 infection. However, this phenomenon reminiscent of the “original antigenic sin” does not prevent a robust and persistent primary response to new epitopes of SARS-CoV-2 that is characterized by extensive intraclonal diversification into Tem, cTfh and Tcm, which represent inflammatory, helper and long-lived memory T cells. The availability of a large number of cross-reactive T cell clones is not only instrumental to defining target sites in relevant pathogens but also to understanding whether cross-reactivity is due to epitope structural similarities or to TCR binding degeneracy.

13The possibility of leveraging a robust cross-reactive T helper cell function against conserved sites will be instrumental to drive neutralizing antibody responses to adaptive vaccines that incorporate escape mutations found in emerging SARS-CoV-2 variants.