1. Identification of three immunodominant spike-specific CD4 + T cell responses with effector function in individuals recovered from COVID-19
We previously identified three dominant SARS-CoV-2 spike protein (S) CD4+ T cell epitopes: S166−180 (CTFEYVSQPFLMDLE), S751−765 (NLLLQYGSFCTQLNR) and S866−880 (TDEMIAQYTSALLAG)18. The HLA-restriction of these epitopes was defined using interferon (IFN)-γ -ELISPOT or peptide-MHC-Class II tetramer staining (Extended Data Fig. 1). S166−180-specific T cells are restricted by HLA-DPB1*04:01 and are also detected in vaccinated healthy donors19. The remaining two spike epitopes are presented by HLA-DRB1*15:01 as defined by peptide-MHC-Class II tetramer staining. Our cohort includes 45 individuals that had recovered from COVID-19, comprising 26 mild cases and 19 severe cases (including 6 critical cases) based on oxygen requirements during the acute illness (Supplementary Table 1 and Extended Data Fig. 2). Among them, 26 (26/41, 63.41%) were HLA-DPB1*04:01 positive (13 mild and 13 severe cases) and 17 (17/45, 37.78%) carried HLA-DRB1*15:01 (7 mild and 10 severe cases) (Fig. 1a). Our ex vivo IFN-γ ELISpot assays with convalescent samples collected 1-3 months post-infection revealed that 68% (17/25) of DPB1*04:01 individuals responded to S166−180, while 85.71% (12/14) and 71.43% (10/14) of DRB1*15:01 positive patients showed responses to S751−765 and S866−880 respectively (Fig. 1b). This further confirmed the immunodominance of these three epitopes. Among S166−180 responders, a high proportion of individuals (64.7%, 11/17) had recovered from severe disease (Fig. 1c) and) with significantly stronger responses compared to individuals who had recovered from mild symptoms (P = 0.031; Fig. 1d). Although our data does not provide statistical evidence for an association of S751−765- and S866−880- specific T cell responses with disease severity, we observed a higher proportion (60%, 6/10) of S866−880 responders who had recovered from mild disease (Fig. 1c and d).
To further characterize these three dominant T cell responses, we generated 50 S166−180-specific T cell clones, 54 S751−765-specific T cell clones and 49 S866−880-specific T cell clones from convalescent samples and evaluated their functionality. T cell receptors (TCRs) of each clone were also sequenced (Supplementary Table 2). All clones expressed cytokines including TNF-α, IFN-γ and IL-2 upon antigen activation (Fig. 1e). S866−880-specific T cell clones displayed the highest antigen-sensitivity, with the lowest EC50 calculated from TNF-α, IFN-γ and IL-2 production. Interestingly, in addition to cytokine expression, we observed a substantial proportion of T cell clones (4 from S166−180, 8 from S751−765 and 22 from S866−880) capable of killing target cells by more than 10%, with the highest level of killing being 60% for S866−880 (Fig. 1f). This highlights the existence of SARS-CoV-2-specific CD4+ CTLs following SARS-CoV-2 infection. We defined CD4+ killer cells as clones with a killing capacity of > 10%. Among these spike-specific T cell clones, S866−880-specific T cells showed the strongest cytotoxic and killing capacity (Fig. 1f, 61% P < 0.001) and proportion of killer cells (Fig. 1g, 60% P < 0.001) whereas S166−180 T cells showed the least cytotoxic killing potential. The highest effector function and antiviral efficacy was seen for S866−880, suggesting significant effector function of cytotoxic CD4+ T cell acting on virus infected cells and controlling virus replication.
2. Spike-specific CD4 + T cell antiviral activity is associated with cytotoxic activity, cytokine production and antigen load
Taking advantage of our in vitro SARS-CoV-2 virus infection system5, we assessed the antiviral activity of these spike-specific CD4+ T cells. In brief, Epstein Barr virus (EBV)-transformed B cell lines (BCLs) ectopically expressing ACE2 were infected with SARS-CoV-2 (Fig. 2a) then cocultured with spike-specific CD4+ T cells. T cell recognition of virus-infected cells was examined by intracellular cytokine staining (ICS), and the suppression of SARS-CoV-2 replication by T cells was assessed by quantifying the number of viral copies in the infected cells after 48hrs of co-culturing. Our data showed that CD4+ T cells targeting these three dominant spike epitopes can recognise virus-infected cells and produce cytokines after activation (Fig. 2b), with S866−880−specific-T cells having the highest proportion of TNF-α (P < 0.001), IFN-γ (P < 0.001) and IL-2 (P < 0.001) producing CD4+ T cells after encountering virus-infected BCLs (Fig. 2c). More importantly, CD4+ T cell clones targeting each of the three epitopes exhibited, to varying extents, direct effector function against the virus, capable of suppressing virus replication (Fig. 2d). In particular, T cells targeting S866−880 showed significantly better antiviral efficacy compared to T cells targeting epitope S751−765 restricted by the same HLA-DRB1*15:01 (P = 0.002). We sought to investigate whether this strong antiviral activity was a result of high effector function or exposure to high antigen loads by examining single cell gene expression from tetramer-sorted short term cultured T cell lines. First, we compared single cell gene expression profiles of T cells targeting S751−765 (n = 1629) and S866−880 cells (n = 2233) (Fig. 2e). We observed significant upregulation of genes encoding effector molecules, such as cytotoxic molecules KLRK1 (P = 1.37 x 10−44), GZMB (P = 6.13 x 10−4) and GZMK (P = 5.13 x 10−10), and cytokines CCL3 (P = 2.79 x 10−6), CCL4 (P = 4.4 x 10−8), TNF (P = 9.08 x 10−12) in S866−800-specific T cells compared to S751−765 -specific T cells (Fig. 2f). This further confirms the cytotoxic potential of S866−880-specific T cells.
To estimate the antigen load of each epitope on virus-infected cells, we cultured T cells with the same number of target cells either infected with SARS-CoV-2 or loaded with variable amounts of peptide, then assessed T cell responses by ICS (Fig. 2g). The antigen-load in virus-infected cells was equivalent to the peptide concentration that elicited a similar level of response to virus-infected cells. Surprisingly, much lower concentrations (equivalent to 0.06µM) of S866−880 peptide were presented on virus-infected cells, when compared to S166−180 (about 0.11µM) and S751−765 (equivalent to 2.57µM) (Fig. 2h). Our data suggests that the higher antiviral activity of S866−880-specific T-cells is likely to result from cytotoxicity and high antigen sensitivity even when antigen load on the surface of infected cells was relatively low.
Next, we compared the cytotoxic activity of S751−765 and S866−880 tetramer-sorted short term cultured single cells isolated from those who had recovered from mild or severe acute COVID-19 (n = 2 mild, 2728 cells; n = 2 severe, 1823 cells; Fig. 2i). We observed that T cells isolated from patients who had recovered from severe disease were more activated and expressed higher levels of T cell effector function genes CD69 (P = 2.75 x 10−5), CCL5 (P = 1.61 x 10−22), IL2RG (P = 3.27 x 10−35), MX1 (P = 3.76 x 10−13), in particular cytotoxic molecules such as GZMB (P = 6 x 10−17) and GZMM (P = 3.87 x 10−3), compared to cells from mild cases (Fig. 2j). Collectively, these data suggest that cytotoxic CD4+ T cells may play a role in the immunopathogenesis of the severe disease.
3. Diverse TCR usage and public TCR clonotypes are commonly observed among immunodominant spike T cells
Single cell TCR sequences of ex vivo stimulated and cytokine sorted S166−180 (n = 152 cells from 5 patients), tetramer-sorted S751−765 (n = 77 cells from 4 patients) and tetramer-sorted S866−880 (n = 100 cells from 4 patients) -specific T cells from 1-3 months convalescent patients were analysed to assess TCR diversity. Interestingly, each epitope displays a different dominant αV gene usage, with TRAV35, TRAV12-1 and TRAV26-1 being dominant for S166−180-, S751−765-, and S866−880-specific T cells respectively, where each dominant αV gene pairs with multiple different βV genes (Fig. 3a). This highlighted the importance of the α chain in these spike epitope TCRs; hence we decided to focus on the αV chain when investigating TCR clonotypes further. Single cells from epitope specific T cells sampled at different timepoints (S166−180 ex vivo acute, ex vivo 1-3 months, short term culture 9 months convalescence from total 5 patients; S751−765 and S866−880 ex vivo acute, ex vivo 1-3 months, short term culture 6 months and 9 months convalescence from total 6 patients) were analysed together to identify public clonotypes (CDR3 amino acid and V gene usage), which are unique clonotypes shared among more than one unrelated patient. We found that public α clonotypes (CDR3α and TRAV) were shared by many patients whereas β clonotypes (CDR3β and TRBV) were shared by a smaller number of patients. For example, the maximum number of patients with one particular α clonotype (CAGTGNNRKLIW, TRAV 25) from S866−880-specific T cells could be 6/6 whereas the highest number of patients sharing any β clonotype from the same epitope was 3/6 (Table 1 and Supplementary Table 3). Examination of paired αβ public clonotypes revealed that the β public clonotypes were more diverse than α public clonotypes, with no clear dominant Vβ gene usage for any epitope. By focusing on α clonotypes, we reasoned that we would be better able to study the dominant α V genes for each epitope, which should also capture the diversity of β clonotypes.
Next, we sought to compare the proportion of public and private clonotypes from each epitope. S166−180-specific T cells have higher proportions of TCRs matching public clonotypes compared to the other two epitopes (n = 21 S166−180 public α clonotypes, n = 16 S751−765 public, n = 19 S866−880 public; Fig. 3b). We also investigated if there were any differences between public and private clonotype expansion for each epitope at different timepoints (acute, 1-3 months and 6-9 months convalescence) and found that T cells with public clonotypes were present at higher frequencies compared to cells with private clonotypes, with the exception of S866−880 at 6-9 months convalescence (acute: S166−180 P = 1.7 x 10−6, S751−765 P = 0.04, S866−880 P = 0.0098; 1-3 months: S166−180 P = 1.4 x 10−15, S751−765 P = 6.3 x 10−8, S866−880 P = 3.8 x 10−6; 6-9 months: S166−180 P = 3.1 x 10−9, S751−765 P = 0.026, S866−880 P = 0.15; Fig. 3c). To further investigate differences between T cells with public and those with private TCRs, we looked at single cell gene expression between these two groups. Ex vivo stimulated and cytokine sorted S166−180-specific single cells with public TCRs (n =128 cells) had higher expression of activation markers [IFNG (P =2.92 x 10−5), FASLG (P = 9 x 10−8), CD40LG (P = 4.86 x 10−16), BTLA (P = 1.48 x 10−3), PDCD1 (P = 1.61 x 10−3)], Th1 activators [ID2 (P = 1.07 x 10−5)] and Th2 suppressors [NR4A2 (P = 1.94 x 10−14) and IRF4 (P = 1.18 x 10−6), Fig. 3d] compared with T cells with private TCRs (n = 285 cells). This may indicate that high expansion of T cells with public clonotypes might drive a Th1 response in convalescent COVID-19 patients. Conversely, tetramer-sorted S866−880-specific T cells from short term culture with private TCRs (n = 1325 cells) display higher expression of cytotoxic genes [KLRK1 (P = 5.65 x 10−8), GZMA (P = 1.22 x 10−3), GZMK (P = 2.95 x 10−5), NKG7 (P = 3.92 x 10−4), CCL5 (P = 1.19 x 10−8), Fig. 3e] compared with T cells with public TCRs (n = 194 cells). Similar results were observed with S751−765-specific T cells (Extended Data Fig. 3). In summary, these results highlight the potential different functional activities between epitope-specific T cells.
4. Cytotoxicity and function of spike-specific CD4 + T cells are regulated by more than TCR usage alone.
We next investigated whether the effector function of spike-specific CD4+ T cells was due to their broad TCR usage by comparing antigen sensitivity and killing capacity of T cell clones bearing the same TCR. We found that T cells with the same TCRs had very different antigen sensitivities, reflected in a wide range of EC50 values for IFN-γ, TNF-α and IL-2 production (Fig. 4a). These T cell clones also produced different cytokine profiles upon antigen stimulation (Extended Data Fig. 4). Moreover, S866−880-specific T cell clones with shared TCRs had distinct killing capacities (Fig. 4b), for example, clone 2 had 40% killing capacity while clone 3 showed minimal killing despite sharing the same TCR (TRAV12-1/TRBV5-1). This suggested that the cytolytic activity of these spike-specific CD4+ T cells was due to factors beyond TCR usage. This observation was further confirmed by variable expression levels of cytotoxic molecule genes, such as PRF1, GZMA and GZMB, in tetramer-sorted S866−880 single cells (n = 399 cells) with the same TCR usage (Fig. 4c). Indeed, we noted a positive association between killing ability with degranulation activity, as measured by CD107a expression, upon antigen stimulation (R = 0.413, P = 0.001, Fig. 4d). This suggests that high expression of CD107a can act as a potential marker for CD4+ CTLs.
5. Antiviral activity of spike-specific CD4 + T cells strongly correlates with their killing capacity and IL-2 production
As previously highlighted, a number of spike-specific CD4+ T cell clones were capable of suppressing virus replication (Fig. 2d). We further examined whether this antiviral effector function was mediated via direct killing of virus-infected cells or by the expression of soluble inhibitory factors. Firstly, our data demonstrated that the antiviral activity of the CD4+ T cell clones strongly correlated to the proportion of cells producing IL-2 upon stimulation with virus-infected cells (R = 0.226, P = 0.030, Fig. 5a), but did not correlate with the proportion of cells producing IFN-γ or TNF-α (Extended Data Fig. 5). Secondly, we found a significant association between the killing capacity of T cells and their potential to control virus replication. Indeed, we observed that killer cells (more than 10% killing capacity) could suppress virus replication more efficiently than non-killers (less than 10% of killing) (Fig. 5b R = 0.390, P < 0.001), suggesting the importance of direct killing of virus-infected cells in viral control by CD4+ CTLs. Strikingly, some non-killer cells were capable of inhibiting viral replication as efficiently as killer cells, indicating the potential contribution of soluble factors to viral control (Fig. 5c). Subsequently we compared cytokine and chemokine production of these viral suppressing non-killer CD4+ T cells with other non-killer cells (Fig. 5d). We noticed that non-killer CD4+ T cells capable of suppressing virus replication (suppressor clone) produced significantly higher concentrations of IL-2 (P = 0.040) and IFN-γ (P = 0.040) than non-killer CD4+ T cells incapable of viral suppression (non-suppressor clone), highlighting a role for IL-2 and possibly IFN-γ in CD4+ T cell control of SARS-CoV-2 infection.
6. Cytotoxic spike-specific CD4 + CTLs utilise distinct cytolytic pathways with increased migration potential
Activated CD8+ CTLs carry out their killing function primarily by releasing cytotoxic granules such as perforin and granzymes, which subsequently induce apoptosis of target cells. To determine whether the killing of target cells by spike-specific CD4+ CTLs was also mediated through the perforin-dependent pathway, S866−880−specific CD4+ T cell clones were treated with concanamycin A (CMA), an inhibitor of perforin, prior to adding to target cells loaded with peptide. The cytolytic activity mediated by CD4+ T cell clones was completely blocked by CMA, resulting in decreased or ablated killing capacity (Fig. 6a), and reduced or no viral suppression (Fig. 6b). To identify other factors involved with this perforin-mediated effective viral control, we grouped tetramer-sorted S866−880−specific single cells from short term cultured lines into perforin-high (n = 693 cells) and perforin-low (n = 724 cells) subsets according to their perforin module scores (Supplementary Table 4) and compared their gene expression profiles (Fig. 6c). Perforin-high T cells not only upregulate cytotoxic associated genes such as GZMA (P = 4.43 x 10−144), NKG7 (P = 1.03 x 10−139), GZMK (P = 4.34 x 10−39), KLRD1 (P = 2.01 x 10−2) and CTSW (P = 6.54 x 10−35) but also genes associated with migration for example: chemokines such as CCL3 (P = 8.48 x 10−16), CCL4 (P = 2.26 x 10−59), CCL5 (P = 9.29 x 10−82), chemokine receptors CCR3 (P = 4.6 x 10−3) and IL2RG (P = 3.17 x 10−24), tissue homing receptors ITGB1 (P = 1.19 x 10−8), ITGA4 (P = 1.26 x 10−3), ITGAL (P = 3.78 x 10−4) and inhibitory receptors such as TIGIT (P = 2.82 x 10−4) and KLRG1 (P = 1.94 x 10−2) (Fig. 6d). This suggests there is increased migration potential of activated cytotoxic CD4+ T cells to infected tissue.
To understand whether these spike-specific CD4+ CTLs utilise similar cytolytic pathways as CD8+ CTLs, we compared single cell gene expression, in particular the expression of effector molecules, between perforin-high CD4+ cytotoxic T cells (tetramer-sorted S866−880 specific, n = 693) and CD8+ cytotoxic T cells (Pentamer-sorted HLA-B*0702/NP105−113-specific, n = 1041) from short term cultured lines5 (Fig. 6e). We found that CD8+ CTLs expressed significantly higher levels of granzyme B and H (GZMB, average log2 fold change = -2.68, P = 2.24 x 10−25; GZMH, average log2 fold change = -0.57, P = 2.09 x 10−5), classic cytolytic molecules secreted by CD8+ T cells and induce apoptosis in target cells (Fig. 6f). Compared to CD8+ CTLs, CD4+ CTLs displayed upregulated expression of other cytolytic molecules such as GZMM (P = 1.91 x 10−10), GZMK (P = 1.78 x 10−10), NKG7 (P = 9.51 x 10−12), KLRK1 (P = 8.3 x10−6) and CTSW (P = 2.86 x 10−11), suggesting the involvement of different cytolytic machineries in CD4+ CTL killing.
7. Dominant spike-specific CD4 + T cells are maintained nine months after infection with diverse TCR repertoire and preserved antiviral activity.
To examine whether memory T cells established following natural infection could provide sufficient protection against secondary viral infection, we collected peripheral blood mononuclear cells (PBMCs) from the same patients 6-9 months after infection. Although T cell responses to these three spike epitopes (S166−180, S751−765 and S866−880) significantly declined six months after infection (Fig. 7a), we were able to sort, sequence, and expand these spike-specific CD4+ T cells after in vitro antigen stimulation. We discovered that the diversity of the TCR repertoire of these CD4+ T cells were maintained 6-9 months after infection (Fig. 7b). We then assessed the antiviral efficacy of these bulk spike-specific T cell lines using our in vitro SARS-CoV-2 infection assays. The bulk lines targeting all three epitopes elicited strong responses against BCLs infected with SARS-CoV-2 (Victoria strain) and variants of concerns (VOCs; Delta and Omicron). The CD4+ T cell lines produced profoundly elevated level of IFN-γ, TNF-α and IL-2 (Fig. 7c and Extended Fig. 6) and showed significant cytotoxic potential with upregulated CD107a expression (Fig. 7d). In addition, we found that these antigen-specific CD4+ bulk cell lines are capable of suppressing SARS-CoV-2 replication and showed strong inhibition against VOCs (Fig. 7e). Our data highlight the protective role of these dominant spike-specific CD4+ T cells in secondary infection against different SARS-CoV-2 variants.