Universal CD8+ T cell epitopes across IAV, IBV and ICV

To investigate the breadth of CD8+ T cell cross-reactivity across IAVs, IBVs and ICVs, we first assessed the conservation of previously identified IAV-specific CD8+ T cell epitopes across IAVs, IBVs and ICVs (Fig. 1a and Supplementary Fig. 1), as IAV-specific CD8+ T cells have been our the research focus so far. Our conservation analysis of >67,000 influenza segment sequences identified 31 conserved epitopes (with >70% amino acid identity) across IAV and IBV, and 8 epitopes across IAV, IBV and ICV types (Supplementary Table 1). Based on the prevalence of HLA-restricting molecules in the population and the nature of alterations within the peptide variants, we selected nine epitopes across both HLA-A (HLA-A*01:01, HLA-A*02:01 and HLA-A*03:01/A*11:01/*31:01/A*68:02) and HLA-B (HLA-B*07:02, HLA-B*44:02 and HLA-B*37:01) alleles (Table 1) for further investigation.

Fig. 1: CD8+ T cell cross-reactivity across influenza A, B and C viruses. a, Conservation of known IAV epitopes in IBV and ICV. Bars indicate percentage conservation (average amino acid identity) of each peptide across the three types of viruses in the indicated number of sequences. IEDB, Immune Epitope Database. b, Immunogenicity of memory CD8+ T cells directed at conserved peptides in healthy adults. PBMCs were cultured with the peptides (as outlined in B) for ~10 d and responses were assessed in an IFN-γ ICS. Frequency of IFN-γ+CD8+ T cells after subtracting ‘no peptide’ control and responding donors are shown. Dots indicate individual donors (n = 3, except for A1-PB1 591 , n = 5, A2-PB1 407 , n = 4 and A2-PB1 413 , n = 6; different donors were assessed over at least two independent experiments), median and interquartile range (IQR) shown; ND, not detected. c, Conservation of PB1 413–421 in orthomyxoviruses. Alignment of PB1 sequences derived from viruses representing each genus is shown. Box indicates the PB1 413–421 peptide. d, A2-PB1 413–421 -mediated cross-reactivity across IAV, IBV and ICV. PBMCs were stimulated with one of the viruses for ~10 d and responses to the peptide were assessed in an ICS. A2-PB1 413–421 +CD8+ T cell responses measured directly ex vivo by ICS are shown for comparison. Representative concatenated FACS plots for IFN-γ production are shown. Data points indicate individual donors, median and IQR (n = 6 from two independent experiments). Statistical significance was determined using two-tailed Wilcoxon matched-pairs signed-rank test, *P < 0.05, **P < 0.005. e,f, B37-NP 338–345 -mediated (e) and A1-PB1 591–599 -mediated (f) cross-reactivity across IAV and IBV. On about day 10 of peptide culture, CD8+ T cell responses to either IAV or IBV variants were assessed by ICS. Dots indicate individual donors (n = 3 for B37-NP 338 and n = 4 for A1-PB1 591 from one experiment). d–f, ‘No peptide’ control was subtracted. d, *P = 0.049, **P = 0.0099. Full size image

Table 1 Highly conserved peptides across IAV, IBV and ICV types selected for dissection of cross-reactive CD8+ T cell responses Full size table

To determine CD8+ T cell immunogenicity towards these epitopes, we probed memory CD8+ T cells within peripheral blood mononuclear cells (PBMCs) obtained from healthy adults using in vitro peptide expansion and measured IFN-γ after peptide re-stimulation. Three (A1-PB1 591 , n = 3; A2-PB1 413 , n = 5; B37-NP 338 , n = 3) of nine conserved CD8+ T cell epitopes recalled memory responses across multiple donors (Fig. 1b). These conserved CD8+ T cell peptides (PB1 591–599, PB1 413–421 and NP 338–345 ) are restricted by three prominent HLA molecules (HLA-A*01:01, HLA-A*02:01 and HLA-B*37:01, respectively), providing broad global coverage as ~54% of the population carry at least one of these alleles.

The NMLSTVLGV PB1 413–421 peptide in IAV (positioned as PB1 414–422 in IBV and ICV; referred to as PB1 413 hereafter) was universally (>98% of sequences) conserved (>99.9%) across IAV, IBV and ICV, but not in influenza D viruses, where a L7F alteration was found, or in other genera of the Orthomyxoviridae family (Fig. 1c). The PB1 413–421 peptide has been reported as an IAV epitope22,23 shared in sequence with IBV24, however CD8+ T cell cross-reactivity has not been shown. To demonstrate the ability of HLA-A2-PB1 413 -specific CD8+ T cells to confer cross-reactivity across IAV, IBV and ICV, PBMCs obtained from HLA-A*02:01-expressing donors were stimulated in vitro with autologous PBMCs infected with IAV, IBV or ICV, followed by measurement of A2-PB1 413 +CD8+ T cells by IFN-γ on day 10 (n = 5) (Fig. 1d). In contrast to minimal IFN-γ production toward PB1 413 directly ex vivo (Fig. 1d), ten-day culture with IAV-, IBV- or ICV-infected targets markedly increased the magnitude of A2-PB1 413 -specific CD8+ T cells (Fig. 1d), owing to expansion of A2-PB1 413 +CD8+ T cells towards all IAV, IBV and ICV. Our data thus provide evidence that memory A2-PB1 413 +CD8+ T cells are activated after stimulation with IAV-, IBV- or ICV-infected targets, suggesting that CD8+ T cells can exhibit universal cross-reactivity across IAV, IBV and ICV, and hence have a much broader cross-reactivity potential than has been previously thought.

Analysis of the remaining two conserved and immunogenic peptides (PB1 591–599 and NP 338–345 in IAV; BPB1 590–598 and BNP 394–401 in IBV) revealed variations at 1–2 amino acids (S2A and L8I for PB1 591 and F1Y within NP 338 ) between IAV and IBV, and a lack of conservation in ICV (Table 1). In vitro expansion with either IAV- or IBV-derived peptides showed unidirectional cross-reactivity, with IAV-expanded CD8+ T cells recognizing both IAV- and IBV-derived peptides. However, the IBV variants could not expand the number of CD8+ T cells directed at the cognate peptides, suggesting lower immunogenicity of these variants (Fig. 1e,f).

Collectively, our data demonstrate that human CD8+ T cells can confer heterotypic cross-reactivity across IAV, IBV and ICV. As the findings are based on the currently known IAV-derived epitopes and thus limited to IAV peptides, such universal cross-reactivity might be broader than defined here. Furthermore, our data suggest a need for identification of novel CD8+ T cell epitopes recognizing both IAV- and IBV-derived peptides that are restricted by a broad range of HLAs represented across different ethnicities.

Identification of novel HLA-A*02:01-restricted IBV epitopes

As there is a general lack of CD8+ T cell epitopes for clinically relevant and understudied IBVs, we identified novel CD8+ T cell epitopes derived from IBV viruses and presented by HLA-A*02:01, owing to the high global prevalence of this allele. We used immunopeptidomics to define peptides naturally processed and presented on the surface of IBV-infected cells. Epstein Barr virus-transformed B lymphoblastoid class I–reduced (C1R) cell lines stably expressing high levels of HLA-A*02:01 were used, together with parental C1R cells expressing background levels of HLA-B*35:01 and HLA-C*04:01 (ref. 25), to exclude peptides derived from these HLAs. Infection of C1R cells with the B/Malaysia strain resulted in high infection rates (~70% BNP+ cells) and cell viability (~93%) (Supplementary Fig. 2a). Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of peptides isolated from HLA-A*02:01 revealed predominantly 9-mer (n = 1,490) peptides, followed by 11-mer (n = 695) and 10-mer (n = 589) peptides (Fig. 2a,b and Supplementary Fig. 2b). These peptides exhibited canonical anchor residues of HLA-A*02:01 ligands (leucine at P2 and leucine or valine at the C terminus26; Fig. 2b). Length distributions were similar for human peptides from uninfected cells and human or viral peptides from infected cells (Fig. 2c and Supplementary Fig. 2c). Analyses from two experiments yielded 73 potential HLA-A*02:01-presented IBV-derived peptides, with ~64% overlap between experiments (Supplementary Table 2). The IBV-derived peptides originated from hemagglutinin (BHA) (22.3%), followed by BNP (16.4%) and BM1 (11.9%), with all IBV proteins contributing to the HLA-A*02:01 immunopeptidome, except BM2 and NB (Fig. 2d). In contrast, peptides from BM2 were identified with high confidence as HLA-II ligands (Supplementary Fig. 2d and Supplementary Table 2). Of 73 HLA-A2-binding IBV peptides, 67 were synthesized for further investigation.

Fig. 2: Identification of novel protective IBV CD8+ T cell epitopes by immunopeptidomics. a, Immunopeptidomics outline. b, Peptide-binding motifs for host and IBV HLA-A*02:01 ligands generated from combined nonredundant lists of 9-mer, 10-mer and 11-mer, using Icelogo by the static reference method against the SWISS-PROT human proteome. c, Length distribution of filtered HLA-A*02:01 ligands (nonredundant by sequence) from uninfected (single experiment) and B/Malaysia-infected (two independent experiments) C1R.A*02:01 cells. Numbers of peptides of each length identified from the human proteome (5% FDR cutoff) and B/Malaysia proteome (all confidences) are shown. d, Distribution of IBV-derived HLA ligands (nonredundant by sequence) across the B/Malaysia proteome identified as probable HLA-A*02:01 ligands. Pooled data from two independent experiments. e–g, In vitro screening of novel peptides in human HLA-A*02:01-expressing PBMCs. e, Representative concatenated FACS plots for each peptide pool are shown from one donor, with a representative mock (unstimulated) control of a day 9–10 T cell line outlined for comparison. Frequency of IFN-γ+TNF+CD8+ T cells for each pool. Dots indicate individual donors; median and IQR are shown (n = 11, from at least two independent experiments). f, Frequency of responding donors for each pool (n = 11). g, Frequency of IFN-γ+TNF+CD8+ T cells directed toward individual peptides from pool 2 (n = 6 from two independent experiments); median and IQR are shown. h, Representative FACS plots for a positive CD8+ T cell response directed toward each peptide. Frequency of IFN-γ+TNF+CD8+ T cells. Donors are color coded; medians and IQRs are shown (n = 6 from one experiment). i,j, Immunodominance of universal CD8+ T cells during in vitro IAV or IBV infection. Responses during IAV infection against A2-M1 58 , A2-PA 46 and A2-PB1 413 (i) and during IBV infection against A2-BHA 543 , A2-BNS1 266 and A2-PB1 413 (j). Bar charts show the contribution of each peptide to the total measured (sum of tetramer+ (Tet+)) response. n = 11 from one experiment. **P = 0.002, ***P = 0.001 (i); *P = 0.0352, #P = 0.0312, **P = 0.0098 (j). Full size image

Novel IBV BHA 543 + and BNS1 266 +CD8+ T cell epitopes in humans

To dissect IBV-specific CD8+ T cells toward the 67 LC–MS/MS-identified IBV peptides, we probed memory CD8+ T cell pools in HLA-A*02:01-expressing individuals. We assigned peptides to six pools of 10–12 peptides, avoiding overlapping peptides in the same pool. We established CD8+ T cell lines for each of the six peptide pools, and then re-stimulated each T cell line with the cognate pool in an IFN-γ and ΤΝF intracellular cytokine staining (ICS) assay (Fig. 2e,f). CD8+ T cell responses were predominantly targeted towards pool 2 (80% of donors, n = 11), with smaller responses detected for pools 1, 3, 4 and 6 (Fig. 2e and Supplementary Fig. 3a). Dissection of pool 2 identified A2-BHA 543–551 as the prominent epitope among HLA-A*02:01+ donors (n = 6) (Fig. 2g). Smaller responses towards A2-BHA 538–551 , A2-NS1 266–274 , A2-BNS1 264–274 and BM1 132–140 were detected in some donors (Supplementary Fig. 3b). To validate these responses independently, we established CD8+ T cell lines towards individual immunogenic peptides. CD8+ T cell responses to A2-BHA 543–551 were of the greatest magnitude (median 7.35%; n = 6) and more frequent among donors (6 of 6) than A2-NS1 266–274 and A2-BM1 132–140 (0.035% and 0.025%, respectively) (Fig. 2h and Supplementary Fig. 3b). Thus, our analysis identified five novel peptides recognized by CD8+ T cells in complex with HLA-A*02:01, with BHA 543–551 being most prominent.

Having identified novel IBV CD8+ T cell epitopes, we determined the conservation of the most prominent peptides, BHA 543–551 and BNS1 266–274 , across IBVs. Both peptides were highly conserved at >98% in >14,000 sequences per segment, spanning both lineages and 77 years (1940–2017) (Supplementary Fig. 3c). Although peptides identified by immunopeptidomics were highly conserved (>70%) in IAV (n = 6 peptides) or in ICV (n = 1) (Supplementary Fig. 3d), these were not immunogenic.

Overall, immunopeptidomics identified 73 novel IBV-derived HLA-A*02:01 peptide ligands. We tested 67 for immunogenicity; CD8+ T cell responses were targeted predominantly to BHA 543–551 , and highly conserved across IBV, but not IAV or ICV.

Universal PB1 413 +CD8+ dominate over BHA 543 +CD8+ T cells in IBV infection

Our data described so far identified three conserved HLA-A*02:01-restricted epitopes for IBV: the universal A2-PB1 413 and two IBV-specific epitopes (A2-BHA 543–551 and A2-NS1 266–274 , hereafter called A2-BHA 543 and A2-BNS1 266 ). To understand the immunodominance hierarchy of the universal A2-PB1 413 +CD8+ T cells after IAV or IBV infection, we established IAV- or IBV-specific CD8+ T cell lines in vitro from PBMCs of healthy adults (n = 11) and assessed tetramer-specific CD8+ T cells against IAV epitopes (A2-M1 58–66 (A2-M1 58 ) , A2-PA 46–54 (A2-PA 64 ) and A2-PB1 413 ) and IBV epitopes (A2-BHA 543 , A2-BNS1 266 and A2-PB1 413 ). Consistent with IFN-γ staining (Fig. 1d), A2-PB1 413 -tetramer detected universal A2-PB1 413 +CD8+ T cells within both IAV- or IBV-specific CD8+ T cell lines, although these cells displayed differential immunodominance hierarchies after either IAV or IBV infection (Fig. 2i,j). Within IAV-specific CD8+ T cell lines, A2-M1 58 -tetramer+CD8+ T cells were significantly dominant (median 3.9% tetramer+CD8+ T cells; detected in all 11 donors) over universal A2-PB1 413 + (0.12%; detected in 10 of 11 donors) and subdominant A2-PA 46 +CD8+ T cells (0.05%) (Fig. 2i). Conversely, the universal A2-PB1 413 epitope within IBV-specific lines was immunodominant (0.3%; detected in 8 of 11 donors) over the IBV-specific A2-BHA 543 (0.11%; detected in 10 of 11 donors) and A2-BNS1 266 epitopes (0.01%) (Fig. 2j). These data demonstrate that (1) the universal A2-PB1 413 and the newly identified IBV-specific A2-BHA 543 and A2-BNS1 266 CD8+ T cells can be expanded in number after virus stimulation in vitro, and (2) immunodominance of the universal A2-PB1 413 epitope depends on influenza type.

Universal PB1 413 +CD8+ T cells recruited during human IAV and IBV infection

To evaluate the recruitment and activation of universal A2-PB1 413–421 +CD8+ T cells during influenza virus infection, we analyzed PBMCs from three clinical cohorts of PCR-confirmed IAV- or IBV-infected pediatric and adult individuals (Fig. 3). Using tetramer-associated magnetic enrichment (TAME), we detected influenza-specific CD8+ T cells directly ex vivo in IAV- and IBV-infected pediatric and adult subjects (Fig. 3a,b)27,28,29. A healthy adult cohort and HLA-A*02:01-positive subjects hospitalized with a noninfluenza respiratory illness (influenza-PCR negative) were analyzed for comparison (Fig. 3b). A2-M1 58 - and A2-PB1 413 -specific CD8+ T cells were detected in 100% and 50% of IAV+ individuals (n = 16), respectively, whereas A2-BHA 543 - and A2-PB1 413 -specific CD8+ T cells were detected in 75% and 87.5% of IBV+ individuals, respectively (n = 8). The frequency of A2-M1 58 - and A2-PB1 413 -specific CD8+ T cells in the blood was significantly increased (4.3- and 6-fold increase, respectively) in IAV-infected subjects as compared to memory CD8+ T cells in healthy donors (Fig. 3c). The numbers of A2-BHA 543 - and A2-PB1 413 -specific CD8+ T cells in IBV-infected subjects increased 2.2- and 2.6-fold, respectively, above the healthy donors, however these increases did not reach statistical significance, most probably owing to the differential age distribution in IBV-infected, but not IAV-infected, subjects compared to healthy controls. In influenza-negative hospitalized subjects, CD8+ T cells for three specificities were in the same range as for the healthy donors (Fig. 3c). Notably, tetramer+CD8+ T cells for all specificities were detected across all age groups (Fig. 3d).

Fig. 3: Prominence of memory and effector pools of universal CD8+ T cells in healthy adults, influenza-infected individuals and human tissues. a–d, Tetramer-specific CD8+ T cells in healthy and influenza-infected individuals. a, Ex vivo TAME on PBMCs from healthy and infected donors. Representative FACS plots are shown. b, Characteristics of healthy and influenza-infected or influenza-negative influenza-like illness (ILI) cohorts used in this study. c, Precursor frequency of tetramer+ cells in healthy controls (HC), influenza-infected individuals and influenza-negative ILI subjects (n = 6 for flu-negative (flu-neg.) ILI, n = 8 for IBV and n = 24 for IAV, assessed over at least two independent experiments). Data points for flu-negative ILI were pooled across CD8+ T cell specificities owing to their varying detection levels across the six donors (5 of 6 for A2-M1 58 , 2 of 6 for A2-PB1 413 and 1 of 6 for A2-BHA 543 ). Statistical significance was determined using a two-tailed Mann-Whitney test, *P = 0.03, **P = 0.0037. Median and IQR are shown. d, Precursor frequency of tetramer+ CD8+ T cells in healthy and influenza-infected individuals across age. e, Expression profiles of tetramer+CD8+ T cells for activation markers CD38 and Ki-67. Representative FACS plots are shown. Frequency of CD38+Ki-67+ tetramer+ CD8+ T cells from healthy controls (n = 3–5) and influenza-infected donors (n = 6 for flu-negative ILI, n = 8 for IBV and n = 24 for IAV, assessed over at least two independent experiments). Statistical significance for changes in the frequency of CD38–Ki-67– cells was determined using a two-tailed Mann-Whitney test A, *P = 0.017, **P = 0.0025, ##P = 0.0095. Full size image

Tetramer-positive A2-PB1 413 +CD8+, IBV-A2-BHA 543 + and IAV-A2-M1 58 + CD8+ T cells detected in IAV- or IBV-infected subjects displayed increased CD38+ and Ki-67+ expression (Fig. 3e and Supplementary Fig. 4), representing an activated phenotype during human viral infections30,31,32. This suggests that they are recruited during influenza virus infection. This was not observed in the influenza-negative cohort, indicating that this activation is influenza-specific. Upregulation of additional activation markers, HLA-DR and PD-1, was also increased on tetramer+CD8+ T cells (Supplementary Fig. 5). The observed variability in numbers and phenotype is probably due to (1) the donors’ age range and exposure history and (2) varying times of sampling after influenza virus infection (Fig. 3b). Indeed, CD8+ T cell responses after human influenza A pandemic H1N1 infection peak within 7 d and then contract rapidly33. Additionally, the magnitude and activation status of peripheral CD8+ T cells can underrepresent virus-specific cells at the site of respiratory infections31.

These data show that A2-PB1 413 +CD8+ T cells are universal and can be detected with an activated phenotype in HLA-A*02:01-expressing influenza-infected subjects following either IAV or IBV. Additionally, activated CD8+ T cells specific for A2-BHA 543–551 , the epitope identified by immunopeptidomics, can be detected during IBV infection, illustrating the ability of mass spectrometry to identify novel peptide ligands.

Tissue-resident memory universal PB1 413 +CD8+ T cells in human lungs

As human memory CD8+ T cells also reside outside the blood circulation34,35,36, we used rare human lung samples from deceased HLA-A*02:01-expressing organ donors (n = 5) to assess the presence of universal A2-PB1 413 +CD8+ T cells at the site of infection. We also used human spleens (n = 11), tonsils (n = 4) and lymph nodes (n = 4) to assess the presence of influenza-specific CD8+ T cells in the secondary lymphoid organs (SLOs), where memory CD8+ T cells are enriched. CD8+ T cells specific for A2-M1 58 (4 of 5), A2-PB1 413 (2 of 5), and A2-BHA 543 (1 of 5) were detected within human lung (Fig. 4a). Similarly, CD8+ T cells directed at A2-M1 58 (17 of 19), A2-PB1 413 (6 of 19) and A2-BHA 543 (4 of 19) were detected within human SLOs. Importantly, the majority of A2-PB1 413 + and A2-BHA 543 CD8+ T cells exhibited a tissue-resident memory (T RM ) CD69+CD103+CD45RO+ phenotype in human lung but not in SLOs (Fig. 4b), with central (CD27+CD45RA–) or effector (CD27–CD45RA–) memory-like phenotype dominating in SLOs (Fig. 4c). This indicates the presence of universal A2-PB1 413 + tissue-resident memory CD8+ T cells in human lung and memory pools in human SLOs.

Fig. 4: Universal CD8+ T cells with a tissue-resident phenotype in the human lung. a, Ex vivo detection of universal CD8+ T cells in human lung and secondary lymphoid organ (SLO; spleen, tonsils and lymph nodes) samples. Frequency of tetramer+CD8+ T cells (n = 8, lungs; n = 11, spleens; n = 4, tonsils; n = 4, lymph nodes); bars indicate the median. b, Phenotype of tetramer+CD8+ T cells was based on CD103 and CD69 expression within CD45RO+tetramer+CD8+ T cells. c, Phenotype of tetramer+ CD8+ T cells based on CD27 and CD45RA expression. Representative FACS plots are shown. Mean and s.e.m. are shown. Full size image

Overall, effector and memory IAV-A2-M1 58 +, IBV-A2-BHA 543 + and universal A2-PB1 413 + CD8+ T cells can be detected directly ex vivo in blood and SLOs of healthy individuals, and tissue-resident IAV-A2-M1 58 -specific and universal A2-PB1 413 -specific memory CD8+ T cells can be detected in human lung.

Single-cell RNA analysis of universal and IBV CD8+ T cells

To further understand the recruitment and activation of universal and novel IBV-specific CD8+ T cells during influenza virus infection, we used single-cell RNA sequencing (scRNA-seq) to assess the transcriptome of ex vivo–isolated tetramer+CD8+ T cells from longitudinal PBMC samples obtained from an IBV-infected HLA-A*02:01-expressing individual. Infection with a B/Victoria strain was confirmed by PCR37 and serological analysis (Supplementary Fig. 6a,b). Blood samples were obtained at baseline (~3 months before infection), and on 14 days, 3 months and 1.5 years after IBV infection (Fig. 5a). A2-PB1 413 +CD8+ T cells were detected at baseline at 19 tetramer+ per 106 CD8+ T cells, then increased 19-fold to 367 tetramer+ per 106 CD8+ T cells on day 14 and remained at a similar level (327 tetramer+ per 106 CD8+ T cells) for up to 1.5 years after infection (Fig. 5b). Conversely, A2-BHA 543 +CD8+ T cells were undetectable at baseline, suggesting that this was the first IBV infection for this donor, despite a previous immunization against the B/Yamagata strain with inactivated vaccine not eliciting CD8+ T cells19. A2-BHA 543 +CD8+ T cells increased to 73.4 tetramer+ per 106 CD8+ T cells on day 14 after infection; this was five-fold lower than for universal A2-PB1 413 +CD8+ T cells, and close to the detection level at 1.5 years. Thus, A2-PB1 413 +CD8+ T cells were assessed at all time points, whereas IBV-specific A2-BHA 543 +CD8+ T cells were analyzed on day 14.

Fig. 5: Single-cell RNA sequencing of universal CD8+ T cells in an IBV-infected individual. a, Timeline of infection and number of tetramer+CD8+ T cells isolated from each sample. b, FACS plots and precursor frequency of tetramer+CD8+ T cells before, during and after IBV infection. c, Principal component analysis of tetramer+CD8+ T cells sequenced. Time points are distinguished by color and specificity by shape. d, Heatmap illustrating expression of differentially expressed genes identified across all the time points compared to the baseline as reference using MAST. Cells grouped by epitope and time point. e, Heatmap representing gene set enrichment of upregulated (pink) and downregulated (green) genes of tetramer+CD8+ T cells sorted at day 14 compared to baseline. f, Heatmap representing gene set enrichment of upregulated (pink) and downregulated (green) genes of tetramer+CD8+ T cells sorted at day 14 compared to 1.5-year time point. Full size image

A total of 209 tetramer+CD8+ T cells were analyzed using scRNA-seq, with ~1,201 expressed genes identified per cell. Principal component analysis revealed clear segregation of A2-PB1 413 +CD8+ T cells by time point but no segregation between the two antigenic specificities on day 14 (Fig. 5c). Notably, differential expression analysis identified distinct gene expression signatures across time points (Fig. 5d). Gene-set enrichment analysis showed that signatures of T cell activation and differentiation, cell division, immune cell migration and chemotaxis were enriched in day 14 cells as compared to those from baseline or 1.5 years (Fig. 5e,f).

We next analyzed the expression of specific genes associated with T cell differentiation, activation, cytotoxicity and effector function (Supplementary Fig. 6c,d). Importantly, effector CD8+ T cells across both IBV specificities isolated from day-14 upregulated genes associated with activation, cytotoxic molecules, cytotoxic receptors and effector cytokines. The expression profiles for selected genes associated with differentiation and activation were confirmed by flow cytometry (Supplementary Fig. 6c,d).

Although single-cell RNA-seq data were obtained from one subject naturally infected with IBV, this experiment provided a rare opportunity to examine baseline PBMC samples from a HLA-A*0201-expressing subject before natural IBV infection, at the acute (day 14), short-term memory (3 months) and long-term memory (1.5 years) time points after IBV. Our results provide evidence of transcriptome changes associated with differentiation and activation of A2-PB1 413 +CD8+ and A2-BHA 543 +CD8+ T cells during IBV infection. To the best of our knowledge, these are the first data on transcriptome changes within tetramer-specific CD8+ T cells at the single-cell level from the baseline to long-term memory CD8+ T cells in humans. Thus, through the flow cytometric analysis of IBV-infected subjects and longitudinal scRNA-seq analysis of a naturally infected individual, we demonstrate that A2-PB1 413 +CD8+ and A2-BHA 543 +CD8+ T cells are recruited to the immune response during IBV infection.

BHA 543 + and BNS1 266 +CD8+ T cells in IBV-infected mice

Having shown recruitment of activated CD8+ T cells against universal (A2-PB1 413 ) and novel IBV-specific (A2-BHA 543 ) epitopes during influenza in humans, we subsequently investigated the protective efficacy of these cells, especially as the role of CD8+ T cells in IBV infection remains unclear. To achieve this, we used HLA-A2.1-expressing transgenic (HHD-A2) mice previously used for IAV infection38, and established a HHD-A2 mouse model of IBV and ICV infection. HHD-A2 mice express a chimeric MHC-I monochain comprising human β2-microglobulin covalently linked to the HLA-A*02:01 α1 and α2 domains, and murine α3 and transmembrane domains39, and thus can respond to many human HLA-A*02:01-restricted epitopes, including IAV-derived A2-M 1 58 (ref. 38) and cancer-derived A2-WT1A neoantigen40. These mice are not confounded by infection history or coexpression of other MHC-I molecules and provide a tool for understanding influenza-specific CD8+ T cells in vivo and determining their protective role in influenza.

To verify the immunogenicity of novel IBV-derived peptides, we infected HHD-A2 mice intranasally with B/Malaysia. On day 10 after infection, we stimulated splenocytes with the 67 novel IBV peptides individually and measured IFN-γ and ΤΝF production. As in humans (Fig. 2h), CD8+ T cell responses were targeted towards A2-BHA 543–551 (mean 5% of CD8+ T cells) and A2-BNS1 266–274 (mean 1.8%), with smaller responses observed for A2-BHA 538–551 and A2-BNS1 264–274 (mean <0.5%), which overlap with A2-BHA 543–551 and A2-BNS1 266–274 , respectively (Fig. 6a,b). We also assayed six pools of 10–12 peptides, as for humans (Supplementary Table 2), and assessed CD8+ T cells to those at the site of infection (bronchoalveolar lavage, BAL). CD8+ T cell responses were targeted to pools 2 and 3, which contained the BHA 543–551 , BNS1 266–274 and BNS1 264–274 peptides, as confirmed separately (Fig. 6c,d).

Fig. 6: In vivo CD8+ T cell responses to novel IBV peptides in HHD (A2+) mice. a, Representative FACS plots for immunogenic peptides. b, Frequency of IFN-γ+TNF+CD8+ T cells of total CD8+ T cells in the spleen of IBV-infected mice toward each peptide. Mean and s.e.m. are shown (n = 8 from at least two independent experiments). c,d, CD8+ T cell responses in the BAL. c, Cytokine responses to each peptide pool. Data from two independent experiments in which the BAL of multiple (n = 3 or 5 per experiment) mice were pooled. d, Cytokine responses to individual immunogenic peptides in the BAL (n = 4 from one experiment). Mean and s.e.m. are shown. Full size image

To compare primary CD8+ T cells directed at BHA 543–551 and BNS1 266–274 epitopes with secondary responses, we primed HHD-A2 mice intranasally with B/Malaysia, and then intranasally infected them with the heterologous strain B/Phuket 6 weeks later (Supplementary Fig. 7b,c). Assessment of CD8+ T cell responses against the main A2-BHA 543–551 and A2-BNS1 266–274 epitopes on day 8 after challenge showed that the number of secondary IFN-γ+TNF+CD8+ T cells in the spleen was ~27-fold higher than that after primary infection (Supplementary Fig. 7a). Additionally, CD8+ T cells for both specificities showed increased polyfunctionality (IFN-γ+TNF+IL-2+) after challenge (0.14 and 2.14%, respectively, for BHA 543 ) (Supplementary Fig. 7d). Thus, using our model of IBV in HHD-A2 mice, we verified the novel (identified by immunopeptidomics) immunodominant IBV-specific A2-BHA 543–551 and A2-BNS1 266–274 epitopes in both primary and secondary IBV infections.

Lack of A2-PB1 413 +CD8+ T cells in HHD-A2 mice

As universal A2-PB1 413 +CD8+ T cells can be detected in both IAV- and IBV-infected subjects, we assessed A2-PB1 413 +CD8+ T cells after IAV (A/X31), IBV (B/Malaysia) or ICV (C/Perth) infection of HHD-A2 mice. To our surprise, CD8+ T cells specific for the A2-PB1 413 epitope were not detected after (1) primary IAV, IBV or ICV infection (Supplementary Fig. 8a), (2) secondary infection with a heterologous virus (for example, A/X31→A/PR8) or a heterotypic virus (for example, A/X31→B/Mal) in all four possible combinations (A→A, A→B, B→B and B→A) (Supplementary Fig. 8b) or (3) tertiary (A→B→A, B→A→B) (Supplementary Fig. 8c) influenza infections (Supplementary Results). Additionally, A2-PB1 413 +CD8+ T cells in HHD-A2 mice were not detected after lipopeptide (Supplementary Fig. 8d) or peptide (Supplementary Fig. 8f) vaccination or with tetramer enrichment in naive mice. Thus, the above experiments (Supplementary Fig. 8) provide strong evidence for a lack of naive A2-PB1 413 -specific precursors in HHD-A2 mice, probably owing to a T cell antigen receptor (TCR) repertoire hole in HHD-A2 mice toward the A2-PB1 413 epitope. Hence, the protective role of universal A2-PB1 413 +CD8+ T cells toward IAV, IBV and ICV infection could not be assessed in HHD-A2 mice (Supplementary Note).

Protective capacity of BHA 543 + and BNS1 266 +CD8+ T cells in HHD-A2 mice

Previous studies using CD8+ T cell depletion in mice lacking antibodies have demonstrated a role for CD8+ T cells during IBV infection41. To determine the protective capacity of novel IBV-derived CD8+ T cell epitopes in HHD-A2 mice, we vaccinated mice with the BHA 543 and BNS1 266 peptides using a prime-boost approach, then infected mice intranasally with 5 × 103 plaque-forming units (p.f.u.) B/Malaysia (Fig. 7a). On day 6 after boosting and before vaccination, numbers of innate cells (neutrophils, macrophages and γδ T cells) were comparable between mock (adjuvant alone) and peptide-adjuvant-vaccinated groups in blood (data not shown). Thus, any nonspecific inflammatory or innate effects of vaccination are controlled for in mock animals.

Fig. 7: CD8+ T cells against novel epitopes mediated protection from IBV challenge. a, Detailed experimental plan of vaccination. b–d, Tetramer-specific CD8+ T cells responses on days 6 and 7 after infection in BAL and spleen. b, Representative FACS plots. c, Number of total (A2-BHA 543 and A2-BNS1 266 ) tetramer+ CD8 T cells in the spleen on day 7 after IBV infection. d, Number of individual A2-BHA 543 + and A2-BNS1 266 + tetramer+ CD8+ T cells in the spleen and BAL on days 6 (D6) and 7 (D7) after IBV infection. e, Viral titers in lungs and nose of peptide-vaccinated and mock-vaccinated mice after IBV infection. Days 5 (n = 5) and 6 (n = 5) were assessed in an independent experiment to day 7 (n = 4–5). f,g, Cytokine responses in the BAL on day 7 after IBV challenge (n = 4–5). Throughout the figure, means and s.e.m. are shown (n = 5 mice per group, except from mock group day 7 where n = 4). Data from days 6 and 7 are from two independent experiments. For c,d, statistical significance was determined using a two-way analysis of variance (Sidak’s multiple comparisons). c, **P = 0.002; d, **P = 0.0014, *P = 0.016. For e–g, statistical significance was determined using an unpaired two-tailed t-test. e, Lung, *P = 0.016, **P = 0.005; nose, **P = 0.0076; g,*P = 0.0038, #P = 0.024, **P = 0.0075. Full size image