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Animal and human studies have demonstrated the importance of influenza A virus (IAV)-specific CD8+ cytotoxic T lymphocytes (CTLs) in heterosubtypic cross-protective immunity. Using peripheral blood mononuclear cells obtained intermittently from healthy HLA-typed blood donors between 1999 and 2012, we were able to demonstrate that IAV-specific CTLs are long-lived. Intercurrent IAV infections transiently increase the frequency of functionally distinct subsets of IAV-specific CTLs, in particular effector and effector memory T cells.
Annually, influenza A virus (IAV) infections cause excess mortality and morbidity in the human population. Neutralizing antibodies (nAbs) induced by IAV infection or vaccination are mainly directed to the globular head of the hemagglutinin (HA) molecule . Lifelong protection by these nAbs is hampered by continuous antigenic drift in this region of the HA molecule [1, 2] and the introduction of antigenically distinct (pandemic) IAVs into a serologically naive population . However, IAV-specific T cells induced by infection with seasonal IAV contribute to protective immunity against these novel viruses. IAV-specific CD8+ cytotoxic T lymphocytes (CTLs) are predominantly directed against more-conserved internal proteins and are therefore cross-reactive and provide protection against antigenically distinct IAVs [3–5]. However, the longevity of the IAV-specific human CTL response is largely unknown. Studies in mice showed that CTLs induced by IAV infection are relatively long-lived [6, 7]. Here, we investigate the longevity of the human IAV-specific CD8+ T-cell response, using uniquely biobanked samples obtained from HLA-typed healthy study subjects.
Peripheral blood mononuclear cells (PBMCs) were obtained intermittently from 9 HLA-typed healthy blood donors (age, 18–64 years) between 1999 and 2012 (Sanquin Bloodbank, Rotterdam, the Netherlands) and cryopreserved (Supplementary Table 1) . For most time points, blood plasma specimens were obtained and stored at −20°C. Plasma specimens were analyzed by a virus neutralization (VN) assay to assess whether reinfections had likely occurred between 1998 and 2012 , using 17 representative H1N1 and H3N2 IAV strains that circulated in the Netherlands in these years. PBMCs and plasma specimens obtained from blood donors after they provided informed consent were approved by the Sanquin Bloodbank for use in scientific research.
PBMCs were stimulated with IAV H3N2 strain Resvir-9 (a reassortant strain containing the HA, nucleoprotein, and neuraminidase of IAV A/Nanchang/933/95 and all other genes of IAV A/Puerto Rico/8/34) to assess the frequency of IAV-specific CD8+ T cells by CD69 and intracellular interferon γ (IFN)-γ staining (ICS), as described previously and shown in Figure Figure11A . Staphylococcus enterotoxin B (Sigma-Aldrich, Zwijndrecht, the Netherlands) was used as positive control to confirm the functional integrity of the cells after thawing; a strong response was detected with PBMCs from all donors (data not shown). The frequency of IAV-specific CD8+ T cells was also determined by staining with HLA-peptide oligomers (Dextramers; Dms), using the following cocktail of R-phycoerythrin–labeled Dms for highly conserved IAV CTL epitopes corresponding to the HLA haplotypes of the blood donors (Supplementary Table 1): HLA-A*0101-PB1591–599(VSDGGPNLY), HLA-A*0201-M158–66(GILGFVFTL), HLA-A*0301-NP265–273(ILRGSVAHK), HLA-B*0801-NP380–388(ELRSRYWAI), and HLA-B*2705-NP174–184(RRSGAAGAAVK) (Immudex, Copenhagen, Denmark). Briefly, 2 × 106 cells were washed extensively with phosphate-buffered saline containing 5% fetal bovine serum (Sigma-Aldrich) and incubated for 10 minutes at room temperature with the Dm mixture. CD8+Dm+ cells were further functionally phenotyped as naive cells (CD45RA+CD28+CCR7+CD27+), effector cells (CD45RA−CD28−CCR7−CD27−), effector memory T cells (TEM; CD45RA−CD28+CCR7−), effector memory RA T cells (TEMRA; CD45RA+CD28−CCR7−), and central memory T cells (TCM; CD45RA−CD28+CCR7+), using fluorochrome-labeled antibodies directed to the respective CD antigens (BD Biosciences, Breda, the Netherlands, and eBiosciences, Vienna, Austria; Figure Figure11B). Remaining cells were defined as “other” and consisted of cell subsets that have not been defined previously (eg, CD45RA−CD28−CCR7−CD27+ or CD45RA−CD28−CCR7+CD27+ T cells).
Based on Dm staining, donors 4564, 7482, 5878, 6358, and 5891 displayed an increase in the number of IAV-specific CD8+ T cells at one time point (Figure (Figure22A–C, ,22E, and and22I). Expansion of effector T-cell, TEM, and TEMRA populations mainly accounted for this increase, which is typically observed after a recent infection . For 3 of these donors (4564, 5878, and 6358), the increase in IAV-specific CD8+ T-cell numbers coincided with an antibody response directed against contemporary IAV strains (H3N2 and/or H1N1), suggesting that IAV infection was responsible for the increase in IAV-specific CTLs (Figure (Figure22A, ,22C, and and22E). However, donor 5891 did not seroconvert, despite an increase in IAV-specific CTL numbers (Figure (Figure22I). Of note, since the PBMCs and corresponding plasma sample were obtained early in 2010, a possible seroconversion later that year could not be excluded (Supplementary Table 1). Unfortunately, for donor 7482, no plasma sample was available for the year 2008, which precluded correlating T-cell and antibody responses (Figure (Figure22B).
A subsequent decrease in the frequency of IAV-specific CD8+ T cells was found in donors 4564, 7482, 5878, and 6358 (Figure (Figure22A–C and and22E), which was accompanied with a contraction of the effector T cell, TEM, and TEMRA subsets. In years following the contraction phase, small numbers of TEM, TEMRA, and TCM persisted in these study subjects.
As shown by Dm-staining and ICS, the frequency of IAV-specific CD8+ T cells remained relatively stable over the years in the other 4 donors (8801, 6888, 8904, and 6877). Although VN antibody testing indicated a possible IAV infection for donor 6888 during 1998–1999, this did not correspond with a conclusive increase in IAV-specific CD8+ T cells (Figure (Figure22D, ,22F, ,22G, and and22H)
In most cases (4564, 7482, 8801, 6358, 6888, 8904, and 6877), the results obtained with Dm-staining and ICS correlated well. For donors 5878 and 5891, the correlation was less obvious. Since we used a cocktail of selected Dms to stain influenza virus–specific CD8+ T cells, it is likely that CD8+ T cells with specificity for other (unknown) epitopes were not detected, so the use of a Dm cocktail may have underestimated the number of IAV-specific CD8+ T cells, for donors 8801 and 6877.
Although PBMCs and/or plasma samples were not available for each donor and every year, these data are the first to indicate that the IAV-specific CD8+ T-cell immunity persists for a prolonged period. Although indication of recent infections was not found for all donors tested (7482, 8801, 8904, 6877, and 5891), it is likely that all subjects had experienced multiple infections with IAV H1N1 and H3N2 since childhood . The various CD8+ T-cell subsets were relatively stable over the years. However, IAV infection may induce a transient increase in the frequency of IAV-specific CD8+ T cells, which can mainly be attributed to an increase of effector T cell, TEM, and TEMRA subsets. Of interest, the proportion of these subsets decreased in the contraction phase. The proportion of IAV-specific CD8+ T cells, as detected by Dm-staining and ICS, was small but comparable to that of memory T cells against other viruses causing acute infections [12, 13]. Only during chronic virus infections can larger virus-specific T-cell populations be observed . Of note, none of our study subjects experienced an acute IAV infection at the sampling time points, as illness in the 2 weeks before blood donation was an exclusion criteria. The contraction of the IAV-specific CD8+ T-cell response occurs rapidly within 1–2 weeks after clinical onset, as was demonstrated in patients acutely infected with 2009 pandemic IAV H1N1 , and similar contraction of CD8+ T cells was demonstrated after vaccination with live attenuated yellow fever (YFV-17D) and smallpox (Dryvax) vaccines . The real number of persisting TCM may be higher than that shown in Figure Figure2,2, since these cells preferentially reside in the lymph nodes rather than in peripheral blood .
Collectively, we demonstrated that adult subjects possess IAV-specific CD8+ T cells and that the presence of this cell-mediated immunity in the blood is long-lived. Since the majority of these T cells are highly cross-reactive, they will respond to infection with antigenically related and unrelated IAVs. The presence of these cells correlated with protection against severe disease caused by IAV, as was shown recently . Thus, repeated boosting of IAV-specific cross-reactive CD8+ T-cell responses, such as by the use of live attenuated vaccines or alternative T-cell antigen delivery systems, may be a means by which to induce broadly protective immunity against future pandemic influenza viruses.
Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Acknowledgment.We thank R. D. de Vries for excellent technical advice and assistance.
Financial support.This work was supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health (contract HHSN272201400008C to R. A. M. F.); the European Union (project 602604 to G. F. R. on behalf of FLUNIVAC); and the European Commission (project 101920 to A. D. M. E. O. on behalf of ERC project FLUPLAN).
Potential conflicts of interest.A. D. M. E. O. and G. F. R. are employed partially by Viroclinics Biosciences. All other authors report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.