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Randomized Controlled Trial
. 2016 Apr 19;113(16):4440-5.
doi: 10.1073/pnas.1603106113. Epub 2016 Mar 31.

Molecular basis for universal HLA-A*0201-restricted CD8+ T-cell immunity against influenza viruses

Sophie A Valkenburg  1 , Tracy M Josephs  2 , E Bridie Clemens  1 , Emma J Grant  1 , Thi H O Nguyen  1 , George C Wang  3 , David A Price  4 , Adrian Miller  5 , Steven Y C Tong  6 , Paul G Thomas  7 , Peter C Doherty  8 , Jamie Rossjohn  9 , Stephanie Gras  10 , Katherine Kedzierska  11
Affiliations
Randomized Controlled Trial

Molecular basis for universal HLA-A*0201-restricted CD8+ T-cell immunity against influenza viruses

Sophie A Valkenburg et al. Proc Natl Acad Sci U S A. .

Abstract

Memory CD8(+)T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158and the hypervariable HLA-B*3501-NP418antigens. The TCRαβs for HLA-B*3501-NP418 (+)CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19(+)TCRαβ was selected in HLA-A*0201(+)donors responding to M158 This public TCR cross-recognized naturally occurring M158variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19(+)TCR specificity for the WT and mutant M158peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201(+)individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158 Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs.

Keywords: T-cell receptor; human CD8+ T cells; influenza infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
M158–66 mutants emerge during influenza virus infection in HLA-A2.1 HHD mice. (A) CD8+ T-cell responses to M158, PA46, and NS122 at the acute influenza phase (day 10 after HK-H3N2 infection) were assessed by TNFα/IFNγ ICS in spleen and BAL cells from HHD-A2.1 mice. Representative dot plots are shown. (B) The numbers of spleen A2-M158+CD8+, A2-PA46+CD8+, and A2-NS122+ CD8+ T cells at days 10, 30, and 60 following HK infection in HHD-A2.1 mice. (C) HHD-A2.1 mice were infected intranasally with HK-H3N2 virus, and lungs were harvested 15 d after infection. RNA was extracted from infected lungs, and viral cDNA was synthesized and amplified by PCR, cloned, subjected to a second round of PCR, and sequenced; (D and E) Data represent the mutation frequency (%) of total sequences. As a control, the input HK viral stock M158 peptide region was sequenced with no additional mutations isolated. The MHC-I anchor position is shown in grey. (F) A panel of PR8 viruses with specific mutations within M158–66 generated by reverse genetics. (G) The replicative fitness of the mutated viruses was compared with that of the WT-PR8 virus in MDCK cells. MDCK monolayers were infected with WT PR8 (rescued by reverse genetics) or mutant PR8-M1-I2M, M1-F5L, M1-V6I, and M1-F7Y viruses at a multiplicity of infection of 0.01. Culture supernatants were harvested at various time points ranging from 5 min to 72 h. Data represent the mean ± SD.
Fig. 1.
Fig. 1.
Ex vivo immunodominance of A2-M158+ over B35-NP418+ CTLs. (A) Costaining of A2-M158+ and B35-NP418+ CTLs directly ex vivo by tetramer enrichment showing (i) single tetramer+CD8+CD4−CD14−CD19− cells and (ii) fold-increase of the A2-M158 above the B35-NP418 CTL response. (B and C) Recognition of naturally occurring M158 (B) and NP418 (C) variants by human PBMCs from A2+B35+ donors was assessed 10 d after restimulation using intracellular staining for IFN-γ production in response to the indicated peptides. Data show individual subjects (S).
Fig. S2.
Fig. S2.
Differential immunodominance hierarchies of B35-NP418+ CTLs in healthy A2−B35+ donors. Costaining of B35-NP418+ CTLs with A3-NP265+ (subjects 40 and 61) or A1-NP44 pool+ CTLs (subjects 43 and 44) directly ex vivo by tetramer enrichment are shown. (A) Tetramer+ cells were gated on viable CD4−CD14−CD19−CD3+CD8+ cells following a sequential gating strategy. (B–D) Representative FACS plots of post-enriched (B), pre-enriched (C), and flowthrough (D) fractions are gated on CD8+ T cells to show tetramer-costaining profiles. The A3-NP265 epitope encodes for the ILRGSVAHK peptide sequence, and the A1-NP44 pool consists of the WT CTELKLSDY and the S7N-variant.
Fig. 2.
Fig. 2.
The A2-M158+ TCRαβ repertoire is dominated by a public clonotype. A2-M158+ CTLs were isolated directly ex vivo from non-Indigenous healthy donors (n = 5) by magnetic enrichment and flow cytometric sorting of single tetramer+ cells. Populations were gated on viable Dump−tetramer+CD3+CD8+ events. (A) Representative flow cytometry profiles showing tetramer+CD8+ T cells after ex vivo enrichment. (B) TRBV and TRAV use. (C) Frequency of CDR3αβ clonotypes. Corresponding data for healthy Indigenous Australian donors (n = 3) are shown in Fig. S3. P, public.
Fig. S3.
Fig. S3.
The A*0201-M158 TCRαβ repertoire is dominated by a shared public TCRαβ clonotype in Indigenous Australians. A2-M158+CD8+ T cells were isolated from healthy donors by magnetic enrichment of A2-M158 tetramer-binding cells and single-cell sorting. Populations were gated as viable Dump−CD3+CD8+A2-M158tetramer+ cells. Paired amino acid CDR3αβ diversity analysis was performed for HLA-A*0201-M158+CD8+ T-cell response directly ex vivo from Indigenous Australians (n = 3) (A–D) and nonindigenous donors (n = 5) (E). (A) FACS profiles of A2+M158+CD8+ T cells after ex vivo tetramer enrichment. (B) TRBV and TRAV gene use. (C) The frequency of CDR3α-CDR3β clonotypes. (D and E) The abundance of particular CDR3β/CDR3α clonotypes in Indigenous Australian (D) and nonindigenous (E) donors. The prominent BV19+ population is highlighted in blue. TCRαβ repertoires of sorted cells were analyzed using a TCRαβ multiplex protocol.
Fig. S4.
Fig. S4.
The total-NP418 TCRαβ repertoire is private and lacks dominant clonotypes. PBMCs from B*3501+ healthy donors were subjected to magnetic enrichment with a pool of NP418 tetramers [1918 (LPFERATIM), 1934 (LPFDRTTIM), 1947 (LPFDKTTIM), 1980 (LPFEKSTVM), and 2002 (LPFEKSTIM)] and then were stained with anti–CD8-APC. Single B35+NP418+CD8+ T cells were sorted and analyzed by single-cell multiplex TCRαβ RT-PCR. Paired amino acid CDR3αβ diversity profiles for HLA-B*3501-NP418+CD8+ T-cell response directly ex vivo from three healthy donors are shown. (A) The frequency of TRAV and TRBV use for the total NP418 TCR repertoire. (B and C) CDR3αβ sequences repeated across donors are depicted in bold, and sequences repeated within donors are depicted in italics. “x” indicates the sequence could not be determined.
Fig. 3.
Fig. 3.
The A2-M158+ TCRαβ repertoire cross-recognizes M158 peptide variants. (A) A2+ PBMCs were restimulated for 10 d in vitro with the M158 variant peptides M1-G4E, M1-F5L, or M1-L3W and then stained with the M158 WT tetramer. Thus, TCRs were selected to recognize the mutant by the 10-d restimulation and to recognize the cross-reactive WT epitope by tetramer sort. Representative flow cytometry plots are shown gated on CD8+ T cells after the exclusion of CD4+CD14+CD19+ events. (B) Summary of public TCRαβ use.
Fig. 4.
Fig. 4.
Structural analysis of M158 variants in complex with HLA-A*0201 and the JM22 TCR. (A–C) HLA-A*0201 is represented as a white cartoon with the peptide in stick form (M158 in white, M1-G4E in orange, M1-L3W in green, M1-F5L in pink). The glycine Cα is represented as a sphere. (D–F) The JM22 TCR footprint on the surface of HLA-A*0201 (white) in complex with M158 (D), M1-F5L (E), and M1-G4E (F) peptide (gray). The HLA and peptide atoms are colored teal, green, and purple when contacted by CDR1α, CDR2α, and CDR3α, respectively, and red, orange, and yellow when contacted by CDR1β, CDR2β, and CDR3β, respectively. The black spheres represent the JM22 TCR center of mass for the Vα and Vβ domains. (G) Superimposition of HLA-A*0201-M1-F5L free (pink) and bound to the JM22 TCR (blue). (H) Superimposition of HLA-A*0201-M1-G4E free (orange) and bound to the JM22 TCR (red). (I) Superimposition of HLA-A*0201-M158 (gray), HLA-A*0201-M1-F5L (blue), and HLA-A*0201-M1-G4E (red) bound to the JM22 TCR. (J and K) Superimposition of JM22 TCR-HLA-A*0201-M158 (dark gray) with the JM22 TCR-HLA-A*0201-M1-F5L (blue) and JM22 TCR-HLA-A*0201-M1-G4E (red) complexes.
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