Broad-Based CD4+ T Cell Responses to Influenza A Virus in a Healthy Individual Who Lacks Typical Immunodominance Hierarchy

Influenza A virus (IAV) infection is a significant cause of morbidity and mortality worldwide. CD4+ T cell responses have been shown to be important for influenza protection in mouse models and in human volunteers. IAV antigen-specific CD4+ T cell responses were found to focus on matrix 1 (M1) and nucleoprotein (NP) at the protein antigen level. At the epitope level, only several epitopes within M1 and NP were recognized by CD4+ T cells. And the epitope-specific CD4+ T cell responses showed a typical immunodominance hierarchy in most of the healthy individuals studied. In this study, we reported one case of atypical immunodominance hierarchy of CD4+ T cell responses to IAV. M1 and NP were still the immunodominant targets of CD4+ T cell responses. However, CD4+ T cell responses specific to 11 epitopes derived from M1 and NP were detected and showed no significant immunodominance hierarchy. Such an atypical pattern is likely determined by the individual’s HLA alleles. These findings will help us better understand the anti-IAV immunity as a whole and improve future vaccines against IAV.


Influenza virus [influenza A virus (IAV)] infection is a global threat to human health.
Each year, about half a billion human beings have symptomatic influenza illness (1), and three to five million subjects suffer from severe influenza, causing approximately half a million deaths annually worldwide (2). Frequent mutation in hemagglutinin and neuraminidase of the circulating viruses and the mismatch between the circulating and vaccine viruses significantly affected the effectiveness of antibody-based vaccine strategy (3). Novel vaccines that are more effective and covering a broader spectrum of influenza viruses are urgently needed. T cell immunity has an important protective role against IAV, and T cell-based vaccines represent an important new development, worldwide, in efforts to combat influenza (4).
Study of IAV-specific T cell immunity has focused more on CD8 + T cells (5,6) partly due to the lack of accurate prediction algorithms for CD4 + T cell epitopes that often show promiscuous length requirement (7). Otherwise, specific CD4 + T cell responses were proven to be indispensable for the clearance of IAV in both animal models (8,9) and in human volunteers (10). CD4 + T cells can exert their protective effect directly through cytotoxic activity (11) or indirectly through providing "help" to both CD8 + T cells and B-cells to eliminate virus and virus-infected cells via cytotoxicity and antibody neutralization, respectively (12,13). Furthermore, the generation of strong memory CD4 + and CD8 + T cell responses are also CD4 + helper T cell dependent (14,15). Thus, stimulating robust CD4 + T cell response is critical for both developing effective T cell-based and antibody-based IAV vaccine (16). To realize that and to be able to properly appreciate the future IAV vaccine efficiency, antigen specificity of IAV-specific CD4 + T cell responses need to be properly understood and finely characterized.
Immunodominance refers to the phenomenon that the cellular immunity tends to focus on a very limited number of antigenic epitopes even during immune responses to complex antigens or pathogens in infected individuals. Immunodominance in CD4 + T cell responses have been widely observed in many viral systems, including HIV, EBV, HTLV1, and others (17)(18)(19) and such immunodominance hierarchies are often long lasting (20). Using in vitro expanded multi-specificity IAV-specific T cell lines and individual IAV protein antigens produced by recombinant vaccinia viruses (rVVs), we have demonstrated that matrix 1 (M1) and nucleoprotein (NP) are the immunodominant antigens targeted by IAV-specific CD4 + T cells in healthy individuals (21). We further finely characterized 10 immunodominant epitopes derived from these antigens using synthetic overlapping peptides (21). Although some of these have been previously reported, their immunodominance status was confirmed for the first time. The epitope-specific CD4 + T cell responses showed a typical immunodominance hierarchy in most of the healthy individuals we studied. In some individuals, the CD4 + T cell responses even focused on a single epitope (21).
In the present study, using the same approach as mentioned above, we found atypical CD4 + T cell responses to IAV in a healthy individual. Although M1 and NP were still the immunodominant targets of these CD4 + T cell responses and up to 11 epitopes derived from nine antigenic regions were recognized, the magnitude of these epitope-specific CD4 + T cell responses were relatively equal, and no significant immunodominance was observed. From this, one highly conserved epitope, M1240-252 restricted to DPB1*0501, was identified. The potential implication of these findings to T cell-based vaccine development is further discussed.

PBMc samples
Buffy coats were obtained with informed written consent from the Australian Red Cross donors under the agreement of 12-07VIC-17 Material Supply Agreement V15.1. PBMC were isolated by Ficoll-Hypaque gradient and stored in liquid nitrogen until use. HLA typing was performed by the Victorian Transplantation and Immunogenetics Service (VTIS, Melbourne, VIC, Australia).

synthetic Peptides
All peptides were synthesized by Mimotopes (Melbourne, VIC, Australia); IAV-M1 and NP overlapping 18mers with 6-aa shifts, and 13mers with either 1-or 2-aa shifts were synthesized as cleaved peptide libraries. All peptides were dissolved in DMSO.

Viruses
The Mount Sinai strain of PR8 (A/Puerto Rico/8/1934 H1N1) IAV was prepared as previously described (21). Virus aliquots were stored at −80°C until use. rVV for the generation of individual IAV proteins were gifts from Drs. Jonathan Yewdell and Jack Bennink (National Institutes of Health, Bethesda, MD, USA). The viruses were propagated using a TK − cell line and were stored at −80°C until use. These proteins are all derived from the PR8 sequences.

cell culture
Donor EBV BLCLs (Epstein-Barr virus-transformed B lymphoblast cell lines) were established using standard EBV transformation. The other human BLCL lines were made available from the International HLA Workshop and the Victorian Transplantation and Immunogenetics Service (Melbourne, VIC, Australia). P815 cells were kind gifts from Drs. Jonathan Yewdell and Jack Bennink (National Institutes of Health, Bethesda, MD, USA). All cells were cultured in RF-10 consisting of RPMI-1640 supplemented with 10% FCS, 2-ME (5 × 10 −5 M), and antibiotics (penicillin 100 U/ mL, streptomycin 100 µg/mL).

Preparation of iaV-and rVV-infected P815 cell lysates
Influenza A virus and rVV infection of P815 cells were conducted as previously described (21). Infected cells were pelleted and lysed by 8 M urea. The lysates were aliquoted and preserved at −20°C until use.
generating single Peptide-specific cD4 + T cell lines FigUre 1 | generation of cD4 + T cell lines for the identification of antigen specificity of influenza a virus (iaV)-specific cD4 + T cell responses. (a) To identify the dominant IAV antigen, multi-specificity IAV-specific CD4 + T cell lines were generated by pulsing PBMCs with IAV-infected P815 cell lysates and then cultured in the presence of rIL-2 for 12-15 days. The immunodominant IAV antigens were identified using an IFN-γ intracellular cytokine staining (ICS) assay in response to autologous BLCLs pulsed with recombinant vaccinia viruses (rVV)-infected P815 cell lysates, which were engineered to express a single IAV protein.
Following the identification of immunodominant antigens, antigenic regions were determined by 18mer overlapping peptides covering the corresponding antigens. (B) To identify the epitopes buried in the antigenic regions, single 18mer peptide-specific CD4 + T cell lines were generated and screened for 13mer overlapping peptides using ICS assay. The HLA restriction was identified by partially HLA-matched BLCLs pulsed with target peptide in an ICS assay. RP-5 containing 10 U/mL rIL-2 on day 5 and then 50% replaced by RP-5 containing 20 U/mL rIL-2 when required.

identification of antigenic regions and epitopes
IFN-γ intracellular cytokine staining (ICS) was performed to identify antigenic regions and epitopes as previously described (21). In brief, autologous BLCLs were pulsed with IAV-or rVVinfected P815 cell lysates overnight and then cocultured with IAV-specific T cell lines for 5 h in the presence of 10 µg/mL Brefeldin A (BFA) or single peptide-specific T cell cultures were incubated with peptide at 10 µg/mL at 37°C for 5 h in the presence of BFA. First, the cells were harvested and stained with anti-CD3 (FITC) and anti-CD4 (APC) and then washed, fixed, and stained with anti-IFN-γ (PE-Cy7) as described previously (23). The flow cytometry mAbs were purchased from eBioscience. Samples were acquired on a FACS Canto II flow cytometer (Becton Dickinson), and FACS data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

hla restriction assay
For antibody-blocking assay, T cells were incubated with 10 µL of anti-HLA class II antibody for 30 min before addition of peptide and BFA. Pan anti-DR (L243), anti-DP (B7/21), and anti-DQ (SPV-L3) antibodies were used as culture supernatants (22). For identifying restriction HLA, BLCLs were pulsed with the peptide of interest at 10 µg/mL for 1 h, washed extensively, and then cocultured with peptide-specific T cells for 5 h in the presence of BFA. Then, IFN-γ ICS was performed as described above.

Bioinformatics analysis
Protein sequences were aligned and amino acid differences were scored to determine the sequence conservation between IAV strains for the newly identified peptides. The National Center for Biotechnology Information (NCBI) Influenza virus database 1 was used (accessed on November 7, 2016) with the search criteria set as Australia, M1/NP, H1N1/H3N2 [or Any (Country/region), M1/NP, H5N1] identical sequences were represented by the oldest sequence in the group and full length only, which identified H1N1 (n = 19 for M1, N = 43 for NP), H3N2 (n = 24 for M1 and n = 74 for NP), and H5N1 (n = 36 for M1 and n = 96 for NP) sequences. Protein sequences were aligned using the NCBI database, peptide regions were mapped, and frequency of mutation was determined across the various sequence groups.  resUlTs approach for systematical identification of iaV-specific cD4 + T cell responses A two-step approach (21) is established for systematic identification of IAV-specific CD4 + T cell responses.
Step one is the identification of the dominant IAV antigen that stimulated CD4 + T cell responses ( Figure 1A), and then, step two is the determination of potential minimal sequence of epitopes together with their HLA restriction ( Figure 1B). To identify the dominant virus protein, multi-specificity, IAV-specific CD4 + T cell lines were generated by stimulating PBMCs in the presence of IL-2 with a soluble IAV antigen source generated by lysing IAV-infected P815 cells in 8 M urea. The cell lines were then screened with a panel of lysates generated by P815 cells infected with rVV, which were engineered to express individual IAV proteins (21,23,24). To further identify the immunodominant epitope regions within the dominant proteins, 18mer overlapping peptides covering the full protein sequence were screened by using an ICS assay measuring interferon-γ production ( Figure 1A). Next, the same PBMCs were stimulated with 18mer peptides covering the immunogenic regions to establish single epitope-specific T cell lines. The potential minimal epitope sequences were determined using ICS assays in response to overlapping 13mer peptides. The HLA restrictions were identified by HLA class II-blocking antibodies and further confirmed by partially HLA-matched antigen-presenting cell (APC) lines ( Figure 1B).

M1 and nP are Dominant antigens recognized by iaV-specific cD4 + T cells
To identify dominant antigens recognized by IAV-specific CD4 + T cells, multi-specificity IAV-specific CD4 + T cell lines were generated by stimulating PBMCs with a urea dissolved soluble IAV antigen (Figure 2A). In response to 12 rVV (11 rVVs encoding 11 individual IAV proteins including PB1-F2 and one wild type rVV)-infected P815 lysates, only M1 and NP stimulated specific IFN-γ producing CD4 + T cells over background ( Figure 2B). Therefore, M1 and NP were dominant targets recognized by IAV-specific CD4 + T cells in this donor.
atypical immunodominance hierarchy of iaV-specific cD4 + T cell responses M1 and NP have been demonstrated to be the most dominant targets of IAV-specific CD4 + T cell responses in healthy individuals by others (25) and by us (21), and it seemed no exception in this donor. To further define IAV-specific CD4 + T cell responses in this donor, M1 and NP 18mer overlapping peptides were screened using the multi-specificity T cell line. As shown in Figure 3, unlike most of the IAV-specific CD4 + T cell responses generally focusing on one or two antigenic regions and displaying a typical immunodominance hierarchy (21), no significant immunodominant region was observed in this donor although up to nine antigenic regions in M1 and NP were recognized. The magnitudes of the CD4 + T cell responses revealed by the 18mer peptides were relatively equal, including M1(37-60), M1(97-120), M1(229-252), NP19-42, NP97-120, NP223-246, NP403-426, NP457-480, and NP469-492. Thus, there was no typical immunodominance hierarchy of IAV-specific CD4 + T cell responses observed in this subject.

Fine characterization of epitopes Derived from M1 and nP
As MHC is one of the important determining factors of the immunodominance, to further explore such an atypical immunodominance hierarchy of IAV-specific CD4 + T cell responses, potential minimal sequences of epitopes were determined by overlapping 13mer peptides within the antigenic 18mer regions.
To further increase the accuracy of epitope identification, the antigenic neighboring 13mers with single amino acid difference in sequence were quantitatively assessed by the T cell lines in a peptide titration assay. HLA restriction was further identified. Three antigenic regions were identified from M1 protein ( Figure 3A). The CD4 + T cells responding to the M1(37-54) and M1(43-60) 18mer peptides ( Figure 3A) recognized seven 13mer peptides [ Figure 4A Figure 4C) and M1240-252 ( Figure 4D). Although they were both restricted to HLA-DPB1*05:01 (Figures 4C,D), no cross-reactivity was observed [ Figure 4C (i); Figure 4D (i)]. Six antigenic regions were identified in NP protein ( Figure 3B). Among them, four epitopes within three antigen regions were finely characterized. The epitope in antigenic region NP97-120 was shown to be NP102-114 [ Figure 5A (i, ii)]. It was shown to be restricted to HLA-DP [ Figure 5A (iii)]; however, we were not able to further resolve whether that was HLA-DPB04:01-or 05:01-restricted. Epitope NP409-421 restricted to HLA-DRB1*12:02 was identified within the antigenic region NP403-426 ( Figure 5B). Two independent epitopes, NP460-472 and NP463-475, were identified in the antigenic region NP457-480, and interestingly, both restricted to HLA-DRB1*09:01 ( Figure 5C). The epitopes in the remaining three antigenic regions, NP19-42, NP223-246, and NP469-492, were not finely characterized due to limited PBMC availability. Several potential key 13mers and corresponding 18mers were titrated in FCS-containing condition to compare their T cell stimulating capacity, which led to the identification of the core peptide M139-51 (ii). HLA restriction of M139-51 was then determined by HLA-class II antibody blocking assay (iii) and partial HLA matching BLCLs (iv, v). (B) The 13mer peptides within 18mer M1(97-114) and M1(103-120) were screened by ICS (i). M1105-117 was titrated to be the core peptide (ii). HLA restriction of M1105-117 was determined (iii-v). (c) The 13mer peptides within 18mer M1(229-246) were screened (i). The core 13mer peptide M1232-244 was identified by titration (ii), and HLA restriction of M1232-244 was analyzed (iii-v). (D) The 13mer peptides within 18mer M1(235-252) were screened as in panel (a) (i). The core 13mer peptide M1240-252 was identified by titration (ii), and its HLA restriction was determined (iii-v). Some of the assays were performed after the T cell lines were restimulated in vitro for two to three times.
It is possible that the infection history or even previous vaccination may influence the T cell response, and therefore the immunodominance hierarchy in the studied samples. However, without knowing the exact IAV exposure history of the individual, it would be difficult to approach this concern using just a few IAV strains as stimulating antigens. To our knowledge, there is no report showing a systematical evaluation of one individual's IAV T cell response using various IAV strains.
We believe that the HLA combination in an individual is very important in determining the outcome of CD4 + T cell immunodominance hierarchy. For example, when DPB1*05:01 was co-expressed with DRB1*01:01, the dominant response to IAV changed to M1129-141/DRB1*01:01 [donor 4 in Ref. (21)]. It seems that DRB1*01:01 has the priority to present dominant epitopes when DRB1*01:01 was co-expressed with other HLA-class II alleles. This was partially supported by the data in the Immune Epitope Database (IEDB). 2 So far, 88 IAV epitopes restricted to DRB1*01:01 were indexed, while only six epitopes were reported to be restricted to DRB1*09:01, and no epitope was found to be DPB1*05:01 restricted, indicating that DRB1*01:01 might play a bigger role in presenting IAV epitopes than many other HLA alleles.
Many IAV-derived CD4 + T cell epitopes have been identified and indexed in the IEDB. 3 As shown in Table 1, some epitopes identified in the present study were reported previously. However, we found a portion of the epitopes restricted to more than one HLA allele. For example, M1232-244 and M1240-252, although reported to be restricted to DRB5 and DQ1, respectively (29), were found in our study to be presented by DPB1*0501 (Figures 4C,D); NP409-421 restricted to DRB1*12:02 ( Figure 5B) was once reported to be restricted to DRB1*15:01 (IEDB) and DRB1*08:01 (26), etc. These results further confirmed that many CD4 + T cell epitopes may be presented by multiple HLA molecules (Table 1). However, since most of the previously identified epitopes were not defined to their minimal core sequences, the reported sequences might contain two or more different epitopes restricted to various HLA molecules. All of the epitopes, including the novel epitope NP460-472, identified in the present study were defined to their most potent core sequences by 13mer overlapping peptides and peptide titration. Interestingly, we also identified an epitope M1240-252/DPB1*05:01, previously reported to be restricted to DQ1, located in the C-terminal of M1. It is highly conserved in H1N1, H3N2, and even H5N1 strains ( Table 2), indicating that these 13 amino acids are likely critical for M1 binding to ribonucleocapsids. DPB1*05:01 was highly expressed in the population of Australia Cape York Peninsula Aborigine (45.3%) and Kimberly Aborigine (68.4%) based on the data of HLA Allele Frequencies Database. 4 The Aboriginal population has been shown to be more susceptible to influenza (32). Thus, this might be a good candidate for IAV vaccine development in the Australian Aborigine population.
In conclusion, using a systematic screening approach, we confirmed that IAV-specific CD4 + T cell responses in the studied individual focus on M1 and NP as we previously reported (21). Eight epitopes were finely characterized for their core sequences, HLA restriction, and sequence conservation. Interestingly, the broad T cell responses were largely equal, and we failed to observe the typical immunodominance hierarchy. We believe HLA allele expression might be the major mechanism that leading to such broad-based and less-focused CD4 + T cells response as in principle immunodominance hierarchy (and potentially the lack of it) should be HLA-dependent. To the best of our knowledge, no such atypical pattern of immunodominance hierarchy was reported in CD4 + T cell responses to IAV before. 4 http://www.allelefrequencies.net/hla6006a.asp.
The identification of such immune response pattern may help us further understand cellular immunity against IAV and development of T cell-based vaccines.

aUThOr nOTe
In the text, all published and defined minimal epitopes are shown as subscribed amino-acid positions, such as NP463-475 or M139-51; other peptide sequences are shown as normal text, such as M1(37-60) or NP19-42.