Conceived and designed the experiments: SAV SG NLLG AWP JR PCD SJT KK. Performed the experiments: SAV SG CG NLLG KK. Analyzed the data: SAV SG CG PGT KK. Contributed reagents/materials/analysis tools: AWP JR KK. Wrote the paper: SAV SG CG NLLG PGT AWP JR PCD SJT KK.
The authors have declared that no competing interests exist.
Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8+ T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP336–374 peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8+ T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vβ regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2DbNPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2Db, including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8+ responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8+ T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected
Introduction of a new influenza strain into human circulation leads to a rapid global spread of the virus due to minimal antibody immunity. Established T-cell immunity towards conserved viral regions provides some protection against influenza and promotes rapid recovery. However, influenza viruses mutate to escape the protective immunity. We found that established T cell immunity can recognise influenza mutants with variations at positions that are partially involved in T cell recognition. However, an initial priming with the mutated variant decreases recognition of the original parental virus. This finding results from a markedly lower functional quality and limited structural interactions of the mutant. In terms of possible vaccination strategies for rapidly changing viruses or tumours, it appears that priming with cross-reactive mutants that display such characteristics would be of no benefit as the same level of T cell immunity against such mutants can be elicited by exposure to the original virus.
Virus-specific CD8+ T cells play a critical role in host defence via the production of antiviral cytokines, the direct killing of virus-infected cells and the establishment of immunological memory
Virus escape mutants are well documented for persistent infections and constitute a major problem for CD8+ T cell-mediated control and vaccine design
Using influenza A virus infection of B6 mice
Earlier analysis
NP366–374 peptide binds to the H-2Db in an extended conformation. The P3-Asp, P5-Asn and P9-Met are the anchor residues, whereas the P4-Glu, P6-Met, P7-Glu and P8-Thr are solvent exposed and available for contact by the TCR.
The NPN3A mutation was engineered into PR8 (H1N1) and HKx31 (H3N2) influenza viruses (PR8NPN3A, HKNPN3A) to allow cross-challenge experiments in the absence of antibody neutralisation. The B6 mice were immunised i.p. with the virulent PR8 mutant and wt viruses, while the HK viruses were used for primary (1o) i.n. infection of naïve mice or secondary (2o) i.n. challenge of PR8-immune (>30d previously) mice. Naïve (primary) or PR8-immune (PR8, or PR8NPN3A) mice were challenged i.n. with the homologous virus (HK, or HKNPN3A) and CD8+ T cell responses were measured using the DbNP366 and DbNPN3A tetramers (
The magnitude of CD8+ T cell responses at the peak (d10 1o A,B; d8 2o D,E) or memory (d28, CF) phases following 1o (A–C) or 2o (D–F) infection. Cells were stained with the DbNP366-PE, DbNPN3A-PE or DbPA224-PE tetramers and anti-CD8-PerCPCy5.5 mAb. The numbers of epitope specific CD8+ T cells were calculated from the % cells staining and the total cell counts. The wt HK or HK-NPN3A viruses were used for 1o i.n. infection and 2o i.n. challenge of i.p.-primed (PR8 or PR8-NPN3A i.p. >30d previously) B6 mice. Data are mean±SD n = 5 mice per group, * = p<0.01. Memory T cell numbers were also analysed on d60, and the tetramer analysis was replicated using the ICS assay (data not shown). (G, H) Representative dot plots are shown for (G) DbNP366 or NPN3A tetramer staining and (H) intracellular cytokine assay after infection with either HK or HK-NPN3A for the same individual mouse.
A high proportion of the wt DbNP366+CD8+ T cells in BAL (94.0% (10, d10); 93.2 (20, d8);
Despite the decreased DbNPN3A+CD8+ T cell numbers generated following primary infection (
Given that the NPN3A mutation was associated with a numerically diminished response following infection with either the wt or NPN3A influenza A viruses (
Can the smaller response to DbNPN3A be correlated with structural constraints or any decrease in stability for the pMHC-I complex? The DbNPN3A crystal structure containing the heavy chain of H-2Db (residues 1–275), the β2 microglobulin (residues 1–99) and the 9 residues of the NPN3A peptide was determined to a 2.6 Å resolution (
The H2Db molecule is in a cartoon representation with the α1-helix on the back and the α2-helix removed for better clarity. The peptide is represented in stick conformation with the C terminus on the right. (A) The NPN3A is in purple and (B) the NP366 in blue, with the p3 mutation in yellow. (B) Overlay of the peptide-binding cleft for H2DbNP366 and H2DbNPN3A with the α1-helix on top and the α2-helix on the bottom. (C) Contacts with the H-2Db molecule by (C) P3N in NP366 and (D) P3A in NPN3A. The Asn3 mutation to alanine abolishes contacts between the P3 residue of the peptide with His155 and the Tyr156 of the H-2Db.
Data Collection Statistics | H2D |
Temperature | 100K |
Space group | |
Cell Dimensions (a,b,c) (Å) | 93.64, 94.98, 132.24 |
Resolution (Å) | 50.00 - 2.60 (2.70-2.60) |
Total number of observations | 131799 (13218) |
Number of unique observations | 18241 (1905) |
Multiplicity | 7.2 (6.9) |
Data completeness (%) | 98.6 (97.7) |
I/σI | 27.55 (6.37) |
Rmerge |
6 (30.3) |
Refinement Statistics | |
Non-hydrogen atoms | |
Protein | 3040 |
Water | 46 |
Resolution (Å) | 2.60 |
22.1 | |
30.4 | |
Rms deviations from ideality | |
Bond lengths (Å) | 0.012 |
Bond angles (°) | 1.468 |
Ramachandran plot (%) | |
Most Favoured Region | 85.5 |
Allowed Region | 12.5 |
Generously allowed region | 1.7 |
B-factors (Å2) | |
Average main chain | 40.806 |
Average side chain | 40.807 |
Rmerge = Σ|Ihkl−<Ihkl>|/ΣIhkl.
Rfactor = Σhkl| |Fo|−|Fc| |/Σhkl|Fo| for all data except ≈5% which were used for Rfree calculation. Values in parentheses are for the bin of highest resolution (approximate interval = 0.5 Å).
However, the mutation of P3-Asn to Ala leads to a loss of 35 contacts between the peptide and the MHC molecule. In comparison to the wt NP366 that makes 636 contacts with the H-2Db molecule, the NPN3A peptide achieves only 601 contacts. Interestingly, the Asn3 Ala mutation abolishes contacts between the P3 residue of the peptide with His155 and the Tyr156 of the MHC and eliminates one hydrogen bond between the P3-AsnNδ2 and the P4-GluO (
The loss of contacts between the peptide and the MHC molecule could lead to decreased stability of the peptide and subsequent changes in NPN3A presentation. To test this hypothesis, we performed a thermostability assay on both NP366 and NPN3A bound to the H-2Db molecule. The NP366 and NPN3A peptides are equally effective at stabilising H-2Db. The pMHC-I complex with the NP366 wt peptide had a Tm of 51.8±0.7°C and DbNPN3A showed a comparable level of thermostability (Tm = 51.4±1°C), irrespective of the concentrations of the complex used for the assay. This suggests that the NPN3A mutation does not modify the stability of the pMHC-I complex when compared to the cognate epitope.
Is the smaller DbNPN3A+CD8+ T cell response a consequence of diminished naïve CTL
Effects of the N3A substitution within NP366–374 on (A) naïve T cell precursor frequency, (B) Vβ8.3 bias and (C) naïve TCRβ repertoire within the DbNPN3A+Vβ8.3+CD8+ set. (A) Naïve DbNPN3A+ and DbNP366+CD8+ T cell precursors within lymph nodes and spleens were identified with the DbNPN3A-PE and DbNP366-PE tetramers using an established tetramer enrichment protocol
The next step was to dissect the immune DbNPN3A+CD8+ CTL repertoire to determine how TCR diversity relates to the size of the immune DbNPN3A+CD8+ T cell response. The DbNPN3A-specific CD8+ T cells were first analysed for Vβ usage by staining with a panel of anti-TCRVβ mAbs and the DbNPN3A tetramer. After infection with the NPN3A viruses, the strong Vβ8.3 bias characteristic of responding DbNP366+CD8+ T cells
We analysed TCRβ clonotypes within the Vβ8.3 as (i) it is still a preferred region (29.8%±20.2) of NPN3A+CD8+ T cell response) and (ii) clonotypes within this region could be highly relevant for cross-reactive CD8+ T cell responses between NP366 and NPN3A as they are prominent in both populations. Overall, the mutant and the wt immune Vβ8.3 repertoires appear different. Single-cell RT-PCR and sequencing of the CDR3β region of tetramer+Vβ8.3+CD8+ T cells following either HK or HK-NPN3A infections showed that (
HK-NPN3A infection | 10 response | 20 response | ||||||||||||||
M1 | M2 | M3 | M4 | M5 | M6 | M7 | ||||||||||
CDR3β | Jβ | aa | NP | N3A | NP | N3A | NP | N3A | NP | N3A | NP | N3A | NP | N3A | NP | N3A |
SGGANTGQL | 2S2 | 9 | 3 | 9 | 2 | 5 | ||||||||||
SGGSNTGQL | 2S2 | 9 | 11 | 1 | 2 | 3 | ||||||||||
SGGGSTGQF | 2S2 | 9 | 11 | |||||||||||||
SGGGNTGQL | 2S2 | 9 | 12 | 1.5 | 99 | 3 | ||||||||||
SGGGNNGHL | 2S2 | 9 | 2 | |||||||||||||
KGGGNTGQL | 2S2 | 9 | 100 | 80 | ||||||||||||
SDAASTEV | 1S1 | 8 | 84 | 88 | ||||||||||||
SDAANTEV | 1S1 | 8 | 16 | 12 | 19 | 11 | ||||||||||
SDAVATEV | 1S1 | 8 | 62 | 82 | ||||||||||||
SDASSTEV | 1S1 | 8 | 98 | 92 | ||||||||||||
SDSANTEV | 1S1 | 8 | 3 | 4.5 | ||||||||||||
RRDRGGNTL | 1S3 | 9 | 95 | 100 | ||||||||||||
SGGTENSPL | 1S6 | 9 | 95 | |||||||||||||
SDAQLYAEQ | 2S1 | 9 | 2 | |||||||||||||
SVGGRAEQ | 2S1 | 8 | 1.5 | |||||||||||||
SDWGSQNTL | 2S4 | 9 | 87 | 100 | ||||||||||||
SDGGGTYEQ | 2S5 | 9 | 2 | |||||||||||||
M: individual mouse; NP: DbNP366 tetramer; N3A: DbNPN3A tetramer.
10 responses were generated by i.n. HK-NPN3A infection of mice; 20 responses were generated by priming mice with i.p. PR8-NPN3A virus then challenging with i.n. HK-NPN3A virus; DbNP366: complex of H2Db and NP366–374 peptide; DbNPN3A: complex of H2Db and NPN3A366–374 peptide.
These results also establish that the naïve DbNPN3A+ CD8+ TCR repertoire (
Similarly, when the DbNP366+Vβ8.3+CD8+ T cells induced by wt HK infection were sequenced, the majority of the TCRβ clonotypes were detected with both the DbNP366 and DbNPN3A tetramers (
HK infection | 10 response | 20 response | ||||||||||||
M8 | M9 | M10 | M11 | M12 | M13 | |||||||||
CDR3β | Jβ | aa | NP | N3A | NP | N3A | NP | N3A | NP | N3A | NP | N3A | NP | N3A |
SGGANTGQL | 2S2 | 9 | 67 | 70 | 53 | 3 | 63 | 22 | 10 | 15 | 36 | 48 | 24 | 22 |
SGGSNTGQL | 2S2 | 9 | 22 | 16 | 2 | 17 | 27 | 50 | 13 | 2 | 50 | 16 | ||
SGGGNTGQL | 2S2 | 9 | 11 | 7 | 5 | |||||||||
SGGGSTGQL | 2S2 | 9 | 17 | 7 | ||||||||||
RGGSNTGQL | 2S2 | 9 | 2 | 4 | ||||||||||
RGGANTGQL | 2S2 | 9 | 5 | 13 | 15 | 36 | 6 | 11 | ||||||
RGGGNTGQL | 2S2 | 9 | 4 | |||||||||||
RGGAPTGQL | 2S2 | 9 | 10 | |||||||||||
KGGSNTGQL | 2S2 | 9 | 63 | 3 | 38 | 2 | ||||||||
KGGGNTGQL | 2S2 | 9 | 25 | 60 | 4 | 4 | 3 | |||||||
SGGQGNSPL | 2S2 | 9 | 2 | |||||||||||
KAGGSTGQL | 2S2 | 9 | 48 | |||||||||||
KAGGGTGQL | 2S2 | 9 | 5 | |||||||||||
RALGRNTEV | 1S1 | 9 | 2 | 3 | ||||||||||
SDAGKTEV | 1S1 | 8 | 6 | 30 | ||||||||||
RDSANTEV | 1S1 | 8 | 2 | 15 | 8 | |||||||||
SDAGAEQ | 2S1 | 7 | 2 | 5 | ||||||||||
SDWGWQNTL | 2S4 | 9 | 40 | 27 | ||||||||||
M: individual mouse; NP: DbNP366 tetramer; N3A: DbNPN3A tetramer;
10 responses were generated by i.n. HK infection of mice; 20 responses were generated by priming mice with i.p. PR8 viruses then challenging i.n. with the HK virus; DbNP366: complex of H2Db and NP366–374 peptide; DbNPN3A: complex of H2Db and NPN3A366–374 peptide.
*Predominant: ≥15%, #Common: present in all mice sampled.
Following clonotype selection with the DbNP366 and DbNPN3A tetramers, sequencing of the secondary TCRβ repertoire (M11–M13) induced by challenge with the homologous (PR8, then HK) viruses showed less divergence within the wt DbNP366+CD8+ T cell specific population. Interestingly, clonotypes like KGGSNTGQL were enriched by the DbNPN3A tetramer in some but not other mice (
We further analyzed the DbNPN3A+Vβ9+CD8+ set that was prominent in 2 of the HK-NPN3A secondarily challenged mice. An average of 1.5±1 TCRβ clonotypes was found within this population (
Analysis of Vα chain usage for the mutant DbNPN3A+CD8+ and wt DbNP366+CD8+ T cells by PCR with a panel of Vα specific primers established that those two T cell responses indeed tend to utilise different Vα chains. The wt DbNP366+CD8+ T cell population
As there was substantial cross-reactivity
Naïve (A) B6 or (B) μMT mice were primed i.p with the mutant PR8-NPN3A and challenged i.n. 6 weeks later with 1×104 p.f.u. of either the wt HK or the mutant HK-NPN3A virus. Alternatively, naïve mice were primed i.p. with the wt PR8 virus and challenged i.n. 6 weeks later with 1×104 p.f.u. of the wt HK or the mutant HK-NPN3A virus. (A) The magnitude of the CD8+ T cell response measured 8d after 20 infection was determined by the ICS assay for IFN-γ. Data represent mean±SD (n = 5) (B) Viral clearance after the secondary homologous (PR8->X31 and PR8-NP-N3A>X31-NPN3A) or heterologous (PR8->X31-NPN3A and PR8-NP-N3A>X31) challenge. Lungs were sampled at days 5 and 6 after secondary infection and homogenized for virus titration by plaque assay on MDCK cell monolayers. Data represent the mean and n = 3–5 mice per group. * = p<0.01 # = p<0.05.
To determine whether such decreased CD8+ responses elicited after heterologous challenge would affect influenza virus clearance, we performed experiments to examine the protective efficacy of cross-reactive CD8+ T cell repertoires. We performed prime-and-challenge studies in μMT mice lacking B cells to ensure that antibody responses did not mask any possible inhibitory effects of “suboptimal” TCRs on viral clearance. As suggested by CD8+ T cell data, assessment of lung viral titres showed delayed viral clearance on d6 after the secondary infection in case of heterologous prime-and-boost (PR8->X31-NPN3A and PR8-NPN3A->X31) compared to homologous infections (PR8->X31 or PR8-NPN3A->X31-NPN3A) (
These patterns of complete, or partial, cross-stimulation following
(A–C) Responsiveness of CD8+ T cells to varying peptide concentrations was determined as a measure of functional TCR avidity for the cognate pMHCI complex. (A,B) DbNP366+ CD8+ and (A,C) DbNPN3A CD8+ T cells recovered from mice 2o-challenged with wt or NPN3A viruses were assessed for functional TCR avidity using the ICS assay. The enriched splenocytes analysed by IFN-γ ICS were stimulated
To determine the pMHC-I avidity of the responding DbNP366+CD8+ and NPN3A+CD8+ T cells, we additionally performed tetramer dilution (
pMHC-TCR avidity was assessed by three measures: (A–C) the overall pMHC avidity (tetramer “on” and “off” rates) by tetramer dilution, (D–F) tetramer “off”-rate by tetramer dissociation and (G–I) CD8β-dependence by anti-CD8β blocking combined with ICS. Splenocytes were obtained from mice 2o-challenged with wt or NPN3A viruses. (A–C) DbNP366+ CD8+ and DbNPN3A CD8+ T cells were stained with 2-fold dilutions of PE-conjugated tetramers, followed by anti-CD8 staining. (D–F) Cells were stained with either the DbNP336 or DbNPN3A tetramers at the saturating concentration, then incubated at 37oC with a mAb to H2Db to prevent rebinding of dissociated tetramer. The progressive diminution in tetramer staining was measured by flow cytometric analysis. (G–I) Lymphocytes were pre-cultured in the presence or absence of anti-CD8β antibody (53.5-8) (10 µg/ml). Cells were then stimulated for 5 h with peptide, IL-2 and GolgiStop also in the presence or absence of anti-CD8β antibody (5 µg/ml). Following stimulation, cells were analysed for CD8 and IFNγ expression. Shown is (G) the percentage of CD8+ T cells producing IFN-γ in the presence or absence of anti-CD8β blocking mAb, (H) mean fluorescence intensity (MFI) of IFN-γ staining, (I) the percentage of CD8+ cells dependent on anti-CD8β for IFN-γ production. Tetramer staining was performed in the presence of NaAz, then washed and incubated with anti-CD8 mAb. The progressive diminution in tetramer staining was measured. The Td50 value defining the time to 50% tetramer loss. Data represent mean±SD (n = 4–5 mice per group), *p<0.01; #p<0.05.
The P3N within the immunodominant influenza virus-specific DbNP366 epitope is a partially solvent exposed, and non-prominent for TCR binding, residue that is predominantly buried within the MHC cleft
Surprisingly, despite the loss of several contacts between NPN3A peptide and H-2Db, the stability of the peptide-MHC-I complex remains constant for both NP366 and NPN3A. This suggests that the Asn3 as a secondary anchor does not play an important role in stabilizing the peptide within the MHC-I. The structural basis for the diminished recruitment of DbNPN3A-specific CD8+ T cells is thus likely to rest in the way that the partially-solvent exposed residue contacts MHC-I and modifies TCR ligation.
The emerging DbNPN3A+CD8+ T cell population was characterised by different Vα and Vβ preference, distinct CDR3β sequences and a lower overall TCR diversity in comparison to wt DbNP366+CD8+ T cells. These findings suggest that a very limited spectrum of CD8+ T cells can recognise the DbNPN3A mutant structure. Interestingly, a similar P3 substitution in the influenza virus DbPA224 epitope had no affect on DbPA224+CD8+ T cell recognition and recruited a diverse array of TCR clones comparable to the spectrum found for the wt response
Interestingly, there were no differences in function or phenotype characteristics for the DbNP366+CD8+ and NPN3A+CD8+ T cells, although those two CTL sets had a high proportion of different TCR clones. This is in accordance with previous studies showing that the simultaneous production of antiviral cytokines
The TCR repertoires specific for DbNP366 and DbNPN3A appear to be quite distinct. The response overall for wt DbNP366+CD8+ T cells is characterised by conserved, “public” clonotypes that constitute the majority (83.5% in 10 response and 92.3% in 20 response) of the selected TCR repertoire
Overall, the results indicate that a loss of a number of contacts between the NPN3A peptide and the MHC-I molecule and lower functional and structural pMHC-I avidity (for wt DbNP366) DbNPN3A selects a narrowed TCR repertoire of “best fit” TCRs from a spectrum of naïve clonotypes that, once activated, clonally expanded and engaged in an immune response, have sufficient avidity to be recalled by exposure to the wt DbNP366 epitope. Conversely, the “better” fit DbNP366 finds a sufficient spectrum of high avidity TCRs within that available naive repertoire and does not (likely because of clonal competition) select most of the TCR αβ pairs that interact optimally with DbNPN3A. Priming with the wt virus thus establishes memory for only a very limited secondary response to the mutant. Similar to our results, subtle variations within the anchor residue of Hb peptide/I-Ek also decreased peptide-MHC class II affinity and the activation of responding T cells
Thinking about this in terms of possible vaccination strategies for use against rapidly changing viruses or tumor epitopes, it appears that priming with cross-reactive mutants that have characteristics comparable to NPN3A would be of no benefit (or even could be detrimental as evidenced by delayed viral clearance) as the same level of T cell immunity against such mutants can be elicited by exposure to the wt epitope. On the other hand, changes like the non-cross-reactive NP-M6A mutation
A further reason for defining the structural rules governing TCR cross-recognition is that similar effects have been found for different epitopes derived from unrelated viruses. Published studies provide evidence for cross-reactive CD8+ T cell responses between influenza A virus and Epstein-Barr virus (EBV)
All animal experimentation was conducted following the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes guidelines for housing and care of laboratory animals and performed in accordance with Institutional regulations after pertinent review and approval by the University of Melbourne Animal Ethics Experimentation Committee in Melbourne.
C57BL/6J (B6, H2b) and μMT mice were bred and housed under specific pathogen free conditions at the Department of Microbiology and Immunology, University of Melbourne. For the generation of acute influenza responses mice were lightly anaesthetised by inhalation of methoxyflurane and infected intranasally (i.n.) with 1×104 plaque forming units (p.f.u.) of HK-X31 (H3N2, X31) or modified HK-X31 (HK-NPN3A) influenza A viruses in 30µl of PBS. For recall responses mice were first primed intraperitoneally (i.p.) with 1.5×107 p.f.u. of the serologically distinct PR8 (H1N1) or modified PR8 (PR8-NPN3A) influenza A viruses, in 500µl of PBS. Viruses share the same internal components for NP and PA from which CD8 epitopes are derived
Recombinant influenza viruses with the single amino acid substitution (N3A) within the NP366 peptide, ASNENMETM, were generated using the eight-plasmid reverse genetics system
Lungs taken from mice after primary viral infection (
Spleen and bronchoalveolar lavage (BAL) samples were recovered from mice at acute phases of the primary and secondary infections (d10 and d8 respectively), and the BAL samples were incubated on plastic petri-dishes for 1hr at 370C to remove macrophages. The spleens were disrupted and enriched for CD8+ T cells using goat anti-mouse IgG and IgM antibodies (Jackson ImmunoResearch Labs, West Grove, PA, USA). For assessment of naïve precursor frequency of N3A366+CD8+ T cells, spleens and lymph nodes (inguinal, brachial, axillary, superficial cervical, and mesenteric) were collected from naïve mice and processed to single-cell suspensions.
Lymphocytes from the BAL and spleen were stained with tetramers conjugated to Strepavidin-APC or PE (Molecular Probes, Eugene, OR, USA) at optimal staining concentrations (10 µg/ml DbNP366, 40 µg/ml DbN3A366, and 10 µg/ml DbPA224 tetramers) for 1hr at room temperature. Cells were washed twice in FACS buffer, and stained with 1 µg/ml CD8-PerCP Cy5.5, 5 µg/ml CD62L-FITC and 5 µg/ml CD127-APC mAbs (BD Biosciences) for 30 mins on ice, washed twice and analysed by flow cytometry on the FACS Calibur (BD Immunocytometry) and analysed by CellQuest Pro software (BD Immunocytometry). We titrated all the batches of all the tetramers used in this study. We used tetramers at optimal concentrations (10–40µg/ml) based on both the percentage of epitope-specific CD8+ T cells and the mean fluorescence intensity (MFI) of tetramer staining. A Scatchard analysis
CD8+ T cells from spleen were stained with the DbNP366 and Db-NPN3A tetramers conjugated to Streptavidin-PE (Molecular Probes, Eugene, OR) for 60mins at room temperature. For a tetramer dilution assay, 2-fold dilutions of PE-conjugated tetramers were used at a range of concentrations (0.15–40µg/ml). For a tetramer dissociation assay, lymphocytes were stained at the optimal concentration of PE-conjugated tetramers as assessed by tetramer titration as determined by both the percentage of tetramer+CD8+ T cells and mean fluorescence intensity (MFI). Cells were washed twice in FACS buffer (10%BSA/0.02% NaAz in PBS), stained with a FITC-conjugated mAb to CD8α (BD Biosciences Pharmingen) for 30mins on ice, washed and analysed by flow cytometry. As a measure of pMHC avidity, splenic T cells were used in tetramer dissociation assay
Enriched T cell populations from spleen and BAL were stimulated with one of the NP366, N3A366, PA224 or PB1703 peptides (AusPep) for 5 hrs at 37°C, 5% CO2 in the presence of 1µg/ml Golgi-Plug (BD Biosciences Pharmingen) and 10U/ml recombinant human IL-2 (Roche, Germany) (BD Biosciences). Cells were washed twice with FACS buffer, stained with CD8-PerCP Cy5.5 for 30 mins on ice, fixed, permeabilised and stained with anti-IFN-γ-FITC (5µg/ml), TNF-α-APC (2µg/ml), and IL-2-PE (2µg/ml) mAbs (Biolegend). Samples were acquired using flow cytometry, and the total cytokine production calculated by subtracting background fluorescence using no peptide controls. In selected experiments, lymphocytes were stimulated with varying concentrations of peptides, three-fold dilutions ranging from 300nM to 0.0008nM to determine the sensitivity specific peptides, defined as ‘functional avidity’
Splenocytes were obtained from mice sampled on d6 after secondary infection. Lymphocytes were pre-cultured in the presence or absence of anti-CD8β antibody (53.5–8) (10 µg/ml). Cells were then stimulated for 5 h with peptide, IL-2 and GolgiStop also in the presence or absence of anti-CD8β antibody (5 µg/ml). Following stimulation, cells were analysed for CD8 and IFNγ expression. Shown is the percentage of CD8+ cells producing IFN-γ after stimulation in the presence of anti-CD8β blocking mAb.
Naïve N3A366-specific CD8+ T cells were identified as described
LacZ-inducible T cell hybridomas specific for NP366 peptide
Splenocytes were stained with 10 µg/ml DbNP366 or 40 µg/ml DbN3A366-PE tetramer in sort buffer (PBS with 0.1% BSA) for 60 mins at room temperature, washed and stained with 1 µg/ml anti-CD8-Allophycocyanin and 10 µg/ml of either anti-Vβ 8.3 or anti-Vβ9 for 30 mins on ice, washed twice with sort buffer. Single lymphocytes were isolated by sorting with a FACS Aria (BD Immunocytometry), into 80 wells of an empty 96 well twin-tec plate (Eppendorf). mRNA transcripts were reversed transcribed to cDNA, using a Sensiscript kit (Qiagen) according to manufacturers instructions, and the CDR3β region amplified by a nested hot start PCR using Vβ primers
H2-Db and β2-microglobulin molecules were expressed in
The crystals were flash frozen to a temperature of 100K before data collection in-house with a Rigaku RU-200 rotating-anode X-ray generator. The data were processed and scaled with the XDS
Circular Dichroism Spectra were measured on a Jasco 815 spectropolarimeter using a thermostatically controlled cuvette. A far-UV spectra was collected from 190nm to 250nm. The UV minimum was determined as 219 nm for H2Db-NP-N3A. The measurements for the thermal melting experiments was made at the minimum for H2Db-NP-N3A, at intervals of 0.1°C at a rate of 1°C/min from 20°C to 90°C. The Jasco Spectra Manager software was used to view and smooth the traces and then the GraphPad Prism software was used to plot Temperature versus % unfolded. The midpoint of thermal denaturation (Tm) for each protein was determined as the point at which 50% unfolding was achieved. The measurements were done in duplicate at two concentrations (4µM and 2.2µM) in a solution of 10mM Tris pH 8, 150mM NaCl.
The atomic coordinates have been deposited in the Protein Data Bank,
Phenotypic and functional characteristics of NPN3A+CD8+ T cells. (A–D) Tetramer+ CD8+ T cells were characterized for (A, B) CD62L and (C, D) IL-7Rα expression following primary (A, C; d10) or secondary (B, D; d8) challenge. (E–H) The ICS assay was used to measure cytokine production following in vitro stimulation with the cognate peptide following primary (E, G) or secondary (F, H) challenge for TNF-α+ (E, F) and IL-2+ within the IFN-γ+ set (G, H). Data represent mean±SD from 5 mice per group, * = p<0.01.
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TCR Vβ usage in primary and secondary NPN3A+ CD8+ and DbNP366+CD8+ T cell responses. Primary (A, C) or secondary (B, D) responses were generated by infection with either the (A, B) HK-NPN3A or (C, D) HK virus. Splenocytes were stained with the (A, B) DbNPN3A or (C, D) DbNP366 tetramer, anti-CD8 and anti-Vβ mAbs conjugated with FITC, then the tetramer+CD8+ cells were analysed for profiles of Vβ staining. Shown are results for (A–D) individual mice (n = 4). S: spleen.
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N3A substitution within NP366 does not affect antigen presentation or the rate of viral clearance. Effects of N3A substitution within NP366 on (A) viral clearance and (B, C) antigen presentation was assessed. (A) Naïve mice were infected with either the wt HK or mutant HK-NPN3A virus. Lungs were sampled at days 3, 6, or 8 after infection and homogenized for virus titration by plaque assay on MDCK cell monolayers. Data represent the mean and n = 5 mice per group. (B, C)
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Scatchard analysis of TCR avidity for pMHC by tetramer dilution assay at the acute secondary time point (d8). Scatchard analysis of tetramer dissociation and correlation coefficient (R2) based on tetramer binding MFI (Holmberg K et al, J Immunol 171:2427, 2003) are shown. Memory mice primed with either (A, D) PR8 or (B, C) PR8-NPN3A viruses were challenged with either (A, D) HK or (B, C) mutant HK-NPN3A virus. Data represent mean of MFI/tetramer concentration (µg/ml) versus MFI of tetramer staining, from 4 mice per group.
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Summary of TCRβ and TCRα repertoire for DbNP366 and DbNPN3A Vβ8.3+ T cells following 10 and 20 infection with the wt or mutant NPN3A influenza virus
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Frequency of TCRβs in DbNPN3A+ Vβ9+CD8+ T cells after 10 mutant HK-NPN3A infection detected with either the DbNP366+ or DbNPN3A+ tetramer
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We thank Dr John Stambas for review of the manuscript, Dr Richard Webby for help with NPN3A viruses, Ken Field and Jason Waithman for FACS sorting.