Unique influenza A cross-reactive memory CD8 T-cell receptor repertoire has a potential to protect against EBV seroconversion
2017; Elsevier BV; Volume: 140; Issue: 4 Linguagem: Inglês
10.1016/j.jaci.2017.05.037
ISSN1097-6825
AutoresLevi B. Watkin, Rabinarayan Mishra, Anna Gil, Nuray Aslan, Dario Ghersi, Katherine Luzuriaga, Liisa K. Selin,
Tópico(s)Viral Infections and Outbreaks Research
ResumoThere is extensive direct evidence in murine viral challenge studies that heterologous immunity facilitated by cross-reactive CD8 T-cell responses can mediate either beneficial or detrimental effects.1Welsh R.M. Che J.W. Brehm M.A. Selin L.K. Heterologous immunity between viruses.Immunol Rev. 2010; 235: 244-266Crossref PubMed Scopus (208) Google Scholar Studies defining the role of heterologous immunity during human viral infection are more challenging. A classic example of protective heterologous immunity in humans is smallpox vaccination. Immunological memory to vaccinia virus (cowpox) protects against human smallpox (variola) infection. More recent studies have shown that children vaccinated with live measles virus or Bacille-Calmette-Guerin have unexpectedly lower susceptibility and decreased mortality to other pathogens than do nonvaccinated children and decreased atopic disease.2Benn C.S. Netea M.G. Selin L.K. Aaby P. A small jab—a big effect: nonspecific immunomodulation by vaccines.Trends Immunol. 2013; 34: 431-439Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar Heterologous immunity and cross-reactive CD8 T cells in humans are associated with enhanced pathology such as dengue shock syndrome during dengue virus (DENV) infection, necrotizing fulminant hepatitis during hepatitis C virus (HCV) infection, and acute infectious mononucleosis (AIM) during EBV infection.1Welsh R.M. Che J.W. Brehm M.A. Selin L.K. Heterologous immunity between viruses.Immunol Rev. 2010; 235: 244-266Crossref PubMed Scopus (208) Google Scholar, 2Benn C.S. Netea M.G. Selin L.K. Aaby P. A small jab—a big effect: nonspecific immunomodulation by vaccines.Trends Immunol. 2013; 34: 431-439Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 3Cornberg M. Wedemeyer H. Hepatitis C virus infection from the perspective of heterologous immunity.Curr Opin Virol. 2016; 16: 41-48Crossref PubMed Scopus (17) Google Scholar Although there are multiple mechanisms that can be involved in heterologous immunity, previous research in mice has shown that CD8 T-cell cross-reactivity can mediate both beneficial and detrimental effects.1Welsh R.M. Che J.W. Brehm M.A. Selin L.K. Heterologous immunity between viruses.Immunol Rev. 2010; 235: 244-266Crossref PubMed Scopus (208) Google Scholar, 4Cornberg M. Clute S.C. Watkin L.B. Saccoccio F.M. Kim S.-K. Naumov Y.N. et al.CD8 T cell cross-reactivity networks mediate heterologous immunity in human EBV and murine vaccinia virus infections.J Immunol. 2010; 184: 2825-2838Crossref PubMed Scopus (62) Google Scholar Primary EBV infection is suited for translational studies investigating potential effects of T-cell heterologous immunity because approximately 95% of individuals globally are infected by their fourth decade and infection is life-long5Luzuriaga K. Sullivan J.L. Infectious mononucleosis.N Engl J Med. 2010; 362: 1993-2000Crossref PubMed Scopus (382) Google Scholar; CD8 T-cell responses are extensively defined and established to be important in controlling the virus5Luzuriaga K. Sullivan J.L. Infectious mononucleosis.N Engl J Med. 2010; 362: 1993-2000Crossref PubMed Scopus (382) Google Scholar; and there is large variability in clinical presentation of primary infection, ranging from asymptomatic to severe AIM.5Luzuriaga K. Sullivan J.L. Infectious mononucleosis.N Engl J Med. 2010; 362: 1993-2000Crossref PubMed Scopus (382) Google Scholar There are strong causal relationships between EBV infection and certain malignancies (nasopharyngeal carcinoma and Burkitt's lymphoma) or autoimmune disorders.5Luzuriaga K. Sullivan J.L. Infectious mononucleosis.N Engl J Med. 2010; 362: 1993-2000Crossref PubMed Scopus (382) Google Scholar We have previously identified a population of cross-reactive memory T cells that recognizes highly conserved influenza A virus (IAV)-encoded influenza-derived matrix protein 1 (M1)58-66 epitope and immunodominant EBV-lytic BMLF1 ([EBV-derived early lytic protein] BamHI M fragment leftward open reading frame 1)280-288 epitope in HLA-A2.01+ patients with AIM.6Clute S.C. Watkin L.B. Cornberg M. Naumov Y.N. Sullivan J.L. Luzuriaga K. et al.Cross-reactive influenza virus-specific CD8+ T cells contribute to lymphoproliferation in Epstein-Barr virus-associated infectious mononucleosis.J Clin Investig. 2005; 115: 3602-3612Crossref PubMed Scopus (132) Google Scholar Here, we identified a rare group of middle-aged adults who remain EBV-seronegative (MA-EBV-SN) without detectable EBV genome despite likely constant exposure, because EBV infects most people and is actively shed at high titers during life-long chronic infection.5Luzuriaga K. Sullivan J.L. Infectious mononucleosis.N Engl J Med. 2010; 362: 1993-2000Crossref PubMed Scopus (382) Google Scholar This HLA-A2.01+ MA-EBV-SN cohort gives us a unique opportunity to determine whether there is any evidence that these individuals have cross-reactive memory responses that could recognize EBV antigens. Because mouse models of heterologous immunity show that the same epitope cross-reactive responses can be either protective or detrimental depending on the history of infection and T-cell receptor (TCR) repertoire,1Welsh R.M. Che J.W. Brehm M.A. Selin L.K. Heterologous immunity between viruses.Immunol Rev. 2010; 235: 244-266Crossref PubMed Scopus (208) Google Scholar, 4Cornberg M. Clute S.C. Watkin L.B. Saccoccio F.M. Kim S.-K. Naumov Y.N. et al.CD8 T cell cross-reactivity networks mediate heterologous immunity in human EBV and murine vaccinia virus infections.J Immunol. 2010; 184: 2825-2838Crossref PubMed Scopus (62) Google Scholar we sought to determine whether MA-EBV-SN HLA-A2.01+ adults had any evidence of potentially protective IAV-M1+EBV-BMLF1+ (M1+BMLF1+) cross-reactive T-cell responses. To address whether T-cell cross-reactivity was associated with seronegative status, we identified both MA-EBV-SN and young adult EBV-seronegative (YA-EBV-SN) donors and determined whether cross-reactive CD8 T-cell responses could be detected. Lack of EBV-specific serum antibodies and genomic EBV-DNA was confirmed in 5 HLA-A2.01+ healthy MA-EBV-SN adults and YA-EBV-SN college students (see Table E1 in this article's Online Repository at www.jacionline.org). EBV-SN donors lacked or exhibited minimal dim-staining, with EBV-BMLF1 (BMLF1) or EBV-BRLF1 ([EBV-derived immediate-early lytic protein] BamHI R fragment leftward open reading frame 1109-117) (BRLF1)-loaded tetramers as assayed directly ex vivo on sorted CD8 cells from freshly isolated PBMCs (see Fig E1, A, i-iii, in this article's Online Repository at www.jacionline.org; Table E1). These MA-EBV-SN donors had 5.4-fold higher levels of IAV-M1-tetramer+ cells directly ex vivo versus YA-EBV-SN donors (Fig E1, A, iii), who were similar to EBV-seropositive (EBV-SP) donors.7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar CD8 T cells sorted from fresh PBMCs of MA-EBV-SN donors were cultured in vitro for 3 weeks with IAV-M1, BMLF1, BRLF1, or control peptides. Using this short-term culture system, we have shown nearly identical TCR repertoires at clonal levels in culture versus tetramer+ CD8 T cells sorted directly from fresh PBMCs ex vivo.7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar, 8Clute S.C. Naumov Y.N. Watkin L.B. Aslan N. Sullivan J.L. Thorley-Lawson D.A. et al.Broad cross-reactive TCR repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes.J Immunol. 2010; 185: 6753-6764Crossref PubMed Scopus (44) Google Scholar IAV-M1-tetramer+ cells from MA-EBV-SN donors cultured with EBV-lytic antigens expanded as well as those cultured with IAV-M1 (Fig E1, B); IAV-M1-tetramer+ T cells from YA-EBV-SN donors did expand to IAV-M1, but not to EBV peptides (Fig E1, B). Antigen-presenting cells alone or pulsed with control tyrosinase (Fig E1, B) or cytomegalovirus (CMV)-pp65 peptides (see Fig E2 in this article's Online Repository at www.jacionline.org) did not induce expansion of IAV-M1-tetramer+ cells either in CMV seropositive or in seronegative MA-EBV-SN donors. Low frequencies of functionally cross-reactive IAV-M1-tetramer+ cells are frequently observed in BMLF or BRLF1 peptide–stimulated cultures of EBV-SP donors,4Cornberg M. Clute S.C. Watkin L.B. Saccoccio F.M. Kim S.-K. Naumov Y.N. et al.CD8 T cell cross-reactivity networks mediate heterologous immunity in human EBV and murine vaccinia virus infections.J Immunol. 2010; 184: 2825-2838Crossref PubMed Scopus (62) Google Scholar, 6Clute S.C. Watkin L.B. Cornberg M. Naumov Y.N. Sullivan J.L. Luzuriaga K. et al.Cross-reactive influenza virus-specific CD8+ T cells contribute to lymphoproliferation in Epstein-Barr virus-associated infectious mononucleosis.J Clin Investig. 2005; 115: 3602-3612Crossref PubMed Scopus (132) Google Scholar, 8Clute S.C. Naumov Y.N. Watkin L.B. Aslan N. Sullivan J.L. Thorley-Lawson D.A. et al.Broad cross-reactive TCR repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes.J Immunol. 2010; 185: 6753-6764Crossref PubMed Scopus (44) Google Scholar while almost none were observed in YA-EBV-SN donors (Fig E1, B, ii-iii). IAV-M1–specific cells were at 122- and 145-fold greater frequency in BMLF cultures or BRLF1 cultures, respectively, in MA-EBV-SN versus YA-EBV-SN donors (Fig E1, B, iii). Antigen-experienced memory IAV-M1–specific T cells were required for BMLF1- or BRLF1-induced expansion, because this expansion was not detected in immunologically naive (never exposed to IAV or EBV) HLA-A2.01+ cord-blood PBMCs (Fig E1, C). Most of IAV-M1-tetramer+ T cells in IAV-M1-, BMLF1-, and BRLF1-stimulated short-term cultures from MA-EBV-SN donors produced IFN-γ (Fig 1, A and B) in response to IAV-M1 peptide-pulse. In donor 1, 37% (21/(21 + 36)), 88%, and 20% of IAV-M1-tetramer+ cells from IAV-M1-, BMLF1-, and BRLF1-stimulated cultures, respectively, produced IFN-γ in response to BMLF1 peptide-pulse (Fig 1, A). This cross-reactivity was specific and unique to these 3 epitopes, as peptide stimulation with 4 other viral- and 1 self-epitope (tyrosinase) did not induce cytokine production (Fig 1, A and B). Similar experiments in YA-EBV-SN donors showed no IFN-γ production to BMLF1 peptide from IAV-M1-tetramer+ cells in BMLF1- or IAV-M1–stimulated cultures (Fig 1, B). B-cell transformation from MA-EBV-SN individuals confirms that B cells from these individuals can be infected with EBV. Control autologous B-cell lymphoblastoid cell lines (BLCLs) were created by infecting donor B cells with BZLF1-knock out (KO) EBV. BZLF1 is required for reactivation from latent to lytic cycle and leads to expression of lytic proteins BMLF1 and BRLF1, which encode BMLF1 and BRLF1 epitopes, respectively. CD8 T-cell cultures grown with IAV-M1, BMLF1, or BRLF1 peptides lysed wild type (WT) autologous BLCL targets, but not BZLF1-KO autologous BLCL targets (Fig 1, C, i; see Fig E3 in this article's Online Repository at www.jacionline.org). These short-term cultures also lysed IAV-M1, BMLF1, or BRLF1 peptide–loaded HLA-A2.01+ targets, but not control targets (Fig 1, C, ii; Fig E3). The ability of MA-EBV-SN CTL to kill EBV-infected and EBV-peptide–loaded targets suggests that these IAV-M1 cross-reactive cells may function to protect against EBV infection. Costaining studies showed a 6-fold higher frequency of CD103-expressing (an integrin molecule associated with migration into mucosal sites and resident memory T cells [TRM]) IAV-M1-tetramer+ cells in MA-EBV-SN versus YA-EBV-SN donors (Fig 1, D) (see this article's Methods section in the Online Repository at www.jacionline.org). Thus, when EBV initially infects tonsillar epithelium, cross-reactive TRM in MA-EBV-SN donors could eradicate EBV before it establishes chronic B-cell infection and seroconversion. Do MA-EBV-SN IAV-M1–specific TCR repertoires have unique features that potentially confer protective immunity? YA-EBV-SN donors such as EBV-SP6Clute S.C. Watkin L.B. Cornberg M. Naumov Y.N. Sullivan J.L. Luzuriaga K. et al.Cross-reactive influenza virus-specific CD8+ T cells contribute to lymphoproliferation in Epstein-Barr virus-associated infectious mononucleosis.J Clin Investig. 2005; 115: 3602-3612Crossref PubMed Scopus (132) Google Scholar, 8Clute S.C. Naumov Y.N. Watkin L.B. Aslan N. Sullivan J.L. Thorley-Lawson D.A. et al.Broad cross-reactive TCR repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes.J Immunol. 2010; 185: 6753-6764Crossref PubMed Scopus (44) Google Scholar had highly diverse7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar IAV-M1–specific responses restricted to Vβ19, which maintained public xRSx complementary determining region 3 (CDR3) β motif without any dominant clones. In contrast, IAV-M1 responses from all 3 cultures in 3 representative MA-EBV-SN donors showed highly private oligoclonal variable region of TCR β chain (Vβ) 19 usage (see Fig E4, B, i-iii, and Table E2 this article's Methods section in the Online Repository at www.jacionline.org). The single dominant clonotype in donor 1 contained a rare noncanonical IVGG motif with uncommon junctional region of TCR β chain 2.1 (Jβ2.1). YA-EBV-SN donors had a typical polyclonal variable region of TCR α chain (Vα) repertoire predominantly using Vα27 and Vα38 often combined with junctional region of TCR α chain 42 (see Table E3 in this article's Methods section in the Online Repository at www.jacionline.org) like EBV-SP.7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar However, in 3 representative MA-EBV-SN donors, the Vα repertoire was dominated by 1 or 2 clonotypes (see Fig E4, C, i-iii, and Table E2). MA-EBV-SN donor IAV-M1–specific Vα and Vβ TCR repertoires were significantly less diverse and more oligoclonal versus YA-EBV-SN donors (Fig E4, D). Circos plot analysis (pairs V and J regions) of sorted IAV-M1-tetramer+ clonotypes of MA-EBV-SN donors show near-identical highly restricted distributions of Vα and Vβ repertoires strongly dominated by Vα12 and Vβ19 (Fig 2, A). This contrasted with typical M1-specific repertoires of YA-EBV-SN (Fig 2, B) and EBV-SP donors,7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar which are highly polyclonal, including using multiple different Vα families that differ between donors. MA-EBV-SN had 6-fold greater usage of Vα12 and almost no usage of Vα38 versus YA-EBV-SN donors (Fig 2, C). This uncommon Vα12 family is used by EBV-BMLF1 responses8Clute S.C. Naumov Y.N. Watkin L.B. Aslan N. Sullivan J.L. Thorley-Lawson D.A. et al.Broad cross-reactive TCR repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes.J Immunol. 2010; 185: 6753-6764Crossref PubMed Scopus (44) Google Scholar and in narrowed IAV-M1 repertoires of elderly adults, who perhaps maintain cross-reactive responses with EBV.7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar CDR3 motif sequence analysis of clonotypes in both groups showed diversity in amino acid content in CDR3α/β regions (Fig 2, D and E). Both donor groups had unique features in their CDR3α motif, which suggests that they may bind M1/MHC and cross-reactive ligands such as BMLF1/MHC complexes differently.7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar In both groups although CDR3β motifs differed in length and amino acid content, arginine was dominant at P6, but MA-EBV-SNs uniquely also had a second dominant arginine in P8, perhaps enhancing the plasticity of binding. These results suggest that this near-identical Vα usage in MA-EBV-SN IAV-M1 TCR repertoire may be a driving factor in this strong functional cross-reactivity with BMLF1. Thus, highly functionally cross-reactive responses against EBV-lytic antigens were detected in IAV-M1–specific CD8 T-cell memory of HLA-A2.01+ MA-EBV-SN, but not YA-EBV-SN donors. Functional cross-reactivity was demonstrated by proliferation and cytokine production to EBV-lytic antigens. Most importantly, IAV-M1–specific T cells from MA-EBV-SN donors killed autologous EBV-infected targets expressing BMLF1 and BRLF1 epitopes. YA-EBV-SN donors, who are susceptible to EBV infection, did not demonstrate any cross-reactive responses. IAV-M1 responses in MA-EBV-SN donors were so consistent between donors and dramatically different from YA-EBV-SN donors that 5 donors were sufficient for highly significant differences in this rare population. These 2 groups dramatically differed in the structure of their IAV-M1–specific TCR repertoires. MA-EBV-SN donors had highly unusual oligoclonal TCR repertoires that were nearly identical between donors particularly in the Vα compartment, which in YA-EBV-SN donors mirrored published EBV-SP donors7Song I. Gil A. Mishra R. Ghersi D. Selin L.K. Stern L.J. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope.Nat Struct Mol Biol. 2017; 160: 2842Google Scholar in being highly polyclonal and variable between donors. A completely different cross-reactive IAV-M1 memory TCR repertoire correlates with the severity of EBV-induced AIM (Aslan N et al, 2017 unpublished data). This difference in the TCR repertoire would be part of the explanation for why cross-reactivity between the same 2 epitopes can be either protective or detrimental.4Cornberg M. Clute S.C. Watkin L.B. Saccoccio F.M. Kim S.-K. Naumov Y.N. et al.CD8 T cell cross-reactivity networks mediate heterologous immunity in human EBV and murine vaccinia virus infections.J Immunol. 2010; 184: 2825-2838Crossref PubMed Scopus (62) Google Scholar How common is protective heterologous immunity during human infection? Su et al9Su L.F. Kidd B.A. Han A. Kotzin J.J. Davis M.M. Virus-specific CD4+ memory-phenotype T cells are abundant in unexposed adults.Immunity. 2013; 38: 373-383Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar found HIV-, CMV-, and herpes simplex virus–specific CD4 tetramer+ memory cells in uninfected adults. A beneficial cross-reactive response in human subjects may go undetected. Cross-reactive responses are more likely to be detected where they contribute to illness and come to medical attention as in AIM. Investigators have reported apparent resistance to HIV or HCV infection in certain high-risk groups and detected circulating antigen-specific CD8 T cells in these exposed, uninfected individuals3Cornberg M. Wedemeyer H. Hepatitis C virus infection from the perspective of heterologous immunity.Curr Opin Virol. 2016; 16: 41-48Crossref PubMed Scopus (17) Google Scholar, 9Su L.F. Kidd B.A. Han A. Kotzin J.J. Davis M.M. Virus-specific CD4+ memory-phenotype T cells are abundant in unexposed adults.Immunity. 2013; 38: 373-383Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar (see the Methods section). These HIV and HCV responses may be cross-reactive memory responses.1Welsh R.M. Che J.W. Brehm M.A. Selin L.K. Heterologous immunity between viruses.Immunol Rev. 2010; 235: 244-266Crossref PubMed Scopus (208) Google Scholar Perhaps continuous reexposure to antigen maintains cross-reactive T cells at higher frequencies and as activated memory effectors or TRM in tissues. Thus, protective heterologous immunity may play a role in resistance to infection in these high-risk individuals. We thank L. Lambrecht, R. Brody, and M. McManus for assistance and Drs R. Welsh, L. Stern, S. Waggoner, E. Szomolanyi-Tsuda, M. Cornberg, and A. Prince for manuscript review. The study was supported by the National Institutes of Health (grant nos. AI-49320 to L.K.S. and K.L., AI-046629 to L.K.S., and AI-109858 to L.K.S.), the Center for Diabetes Research Core (grant no. DR32520), the National Institute of Allergy and Infectious Diseases (grant no. T32-AI-007349-17 to L.B.W.), and the UMass Center for Clinical and Translational Science (grant no. UL1-TR001453). Contents of this publication are solely the responsibility of authors and do not represent the official view of the National Institutes of Health. 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