Human CD4+ Memory T-cell Populations Secrete Th1 Cytokines in Response to Influenza Antigen Stimulation

Seasonal influenza vaccine efficacy is measured by the vaccine’s ability to elicit strain-specific antibody responses. Every year, resources are allocated into formulating new influenza vaccines. Candidate vaccines utilizing memory T-cells may afford long-term protection. Our study characterizes the human CD4+ memory T-cell response to influenza virus. Intracellular cytokine staining assay was used to assess T-cell production of several cytokines (IFN-γ, IL-2, TNF-α, IL-4, IL-5, and IL-17) found in human peripheral blood mononuclear cells after stimulation with influenza antigens. Production of Th1 cytokines (IFN-γ, TNF-α, and IL-2) was significant in activated CD4+ T-cells after stimulation, whereas Th2 cytokine secretion remained unchanged. In addition, a significant increase in multifunctional CD4+ T-cells that simultaneously secreted combinations of IL-2, IFN-γ, and TNF-α was observed. Our studies have revealed that CD4+ T-cell responses against influenza are Th1-biased, raising the possibility of identifying these populations as targets for successful influenza vaccination. Journal of Vaccines & Vaccination J o ur al of Va ccines & Vainati o n


Introduction
Influenza is an important health issue due to the emergence of several pandemic strains during the past century: 1918 H1N1, 1957 H2N2, 1968 H3N2 and 2009 H1N1 [1]. Current licensed vaccines protect health by eliciting antibody responses against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, influenza viruses evade host memory against HA and NA via antigenic drift and shift. Thus, vaccines are prepared annually containing three influenza strains predicted to be prevalent in the upcoming season [2]. Upon identification of a possible pandemic strain, it may take six months to develop a new vaccine [3]. The current licensed approach is insufficient.
New candidate vaccines attempt to address these issues by targeting conserved epitopes including influenza matrix protein 1 (M1), matrix protein 2 (M2), and nucleoprotein (NP) to elicit long-term protection. Humoral immunity prevents the spread of virus, while T-cell immunity mediates viral clearance. T-cell immunity against conserved proteins is heterosubtypic [4], reacting with multiple strains of influenza virus. M1, M2, and NP-specific T-cell epitopes have all been shown to be cross-strain reactive [5][6][7][8]. Furthermore, T-cell responses to M1 and NP are not only cross-reactive, but also immunodominant [7]. Therefore, influenza vaccines should be tested for their ability to elicit T-cell immunity.
CD4+ T-cells can be classified based on polarization. T helper 1 (Th1) cells will elicit a strong IFN-γ response; Th2 cells are characterized by IL-4 secretion; Th17 secrete IL-17, which enhances the inflammatory response; and regulatory T-cells (Treg) produce IL-10 and TGF-β to maintain peripheral tolerance and dampen immune responses [14].
Primary literature describing human memory CD4+ T-cell responses to influenza are relatively scarce. In our study, we utilized a complex multiparametric 14-color flow cytometry analysis to detect human influenza-specific memory T-cell responses. The scope of our study encompassed many aspects of CD4+ T-cell responses, testing for activated Th1, Th2, Th17, and T-regulatory cell subsets.

Peripheral blood mononuclear cell isolation and antigenic stimulation
Healthy human volunteers between the ages of 25 to 58 were enrolled for a single visit study after approval by the institutional ethics committee. Each volunteer provided written, informed consent in accordance with the principles of the Helsinki Declaration of 1975. Patient information on age, gender, and influenza vaccination status was collected (Table 1). 30 ml of blood was collected in 10 U/ ml heparin from each of seven donors. Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium (Invitrogen, Carlsbad, CA) as per protocol and cryopreserved in liquid nitrogen. Isolated cells were thawed and washed twice in RPMI medium (Invitrogen) supplemented with 8% fetal bovine serum (Invitrogen), 2 mM glutamine (Invitrogen), 1% penicillin/streptomycin (Invitrogen), and 10 µg/ml DNase (Invitrogen). Cells were then cultured in a 25 cm 2 flask at 2×10 6 cells/mL (same medium) at 37°C and 5% CO 2 overnight. Cell number and viability were assessed using Trypan Blue (Sigma, St. Louis, MO) exclusion. PBMCs were then transferred to a 96-well U-bottom culture plate, exposed to 0.1 µg of influenza antigens, and incubated at 37°C and 5% CO 2 for 2 hours. Cells were also stimulated in parallel and for the same duration with 1 µg/mL staphylococcal enterotoxin B (SEB) (Sigma) as a positive control and with medium alone as a negative control. Cells were then treated with 2 µM Monensine and 10 µg/ml Brefeldine A and incubated at 37°C and 5% CO 2 for 8 hours. The preparations were refrigerated at 4°C overnight.

Flow cytometry and analyses
14-color flow cytometric data were collected on a BD LSRII ® instrument (BD Biosciences) and analyzed using FlowJo software (TreeStar, Ashland, OR). Dead cells were excluded by forward and side scatter gating. A quadrant gating method was used (

Statistical analyses
Mann-Whitney tests were used to compare antigen-stimulated samples to medium-only stimulated (negative) samples using Prism 5.04 software (GraphPad, La Jolla, CA). P < 0.05 (*) was considered significant.

Th2, Th17, and T-regulatory CD4+ T-cell subsets did not respond to antigen stimulation
Stimulation with influenza antigens did not induce Th2 cytokines IL-4 and IL-5 (not shown, antigen stimulated cells did not exceed medium-only stimulated cells), and IL-17 production was not significantly increased after stimulation (not shown, p=1.0000). Also, stimulation did not increase the percentage of T-regulatory as defined by CD25+ /FoxP3+ expression (not shown, p=0.5224).

Discussion
In this study, we characterized the small subset of activated memory CD4+ T-cells upon stimulation with the pediatric trivalent FluZone vaccine. Previously, it has been difficult to study memory CD4+ T-cells due to low numbers that persist after influenza infection [13]. Our method is sensitive enough to reveal a robust induction of Th1 cytokines (IFN-γ, TNF-α, and IL-2) in memory CD4+ T-cells. IFN-γ promotes suppression of Th2 cell activity, Th1 differentiation via T-bet and increased expression of class I MHC molecules [17]. TNF-α is a pleiotropic cytokine with functions in the inflammatory response, apoptosis, and survival [18,19]. IL-2 is responsible for T-cell proliferation [20]. Thus, not only is it possible that memory CD4+ T-cells may play a role in inflammatory and antiviral responses to influenza, but they may also function in modulating the responses of other immune cells [13].
We did not detect influenza-specific Th2 (IL-4/IL-5) or Th17 (IL-17) cytokines in this study. While our stimulation time may be viewed as short, it has been shown that accessory-cell stimulation of IL-4 secretion by human T-cells in the presence of Brefeldin A peaks between 0-6 hours [24,25]. Moreover, in choosing an appropriate stimulation time, we took into account that multifunctional subsets are short-lived, with expression at a maximum between 0-6 hours, and disappear by 18-22 hours of stimulation [24]. We also did not detect influenza-specific T-regulatory cells (CD25+/FoxP3+ phenotype). However, as this initial study was modest (n = 7), we cannot definitively rule out the contribution to influenza memory immunity by these cell subsets.
In our donor pool, there were 4 donors who had received the seasonal influenza vaccine (2008-2009), and 3 donors that had not. We investigated if influenza-specific memory CD4+ responses differed. Statistically, there were no differences, which may be attributed to 1) sample size and 2) exposure to similar influenza strains, or 3) inability of the seasonal vaccine to significantly influence the cellular immune response. Recently, it has been shown that even live attenuated influenza vaccines fail to boost the cellular immune response in healthy adult subjects [26]. However, due to the small sample sizes, no meaningful conclusions can be derived.
Herein, we describe influenza specific CD4+ T-cells in the memory compartment. The response by these CD4+ T-cells derived from PBMCs was Th1-driven, producing IFN-γ, TNF-α, and IL-2 cytokines, but was, more importantly, multifunctional. Multifunctional, influenza-specific Th1 cells have previously been described in mice in the context of vaccine evaluations [27,28] and in resident memory T-cells from human lungs [29], and thus our study identifies additional subsets of multifunctional, human Th1 memory cells found in human peripheral blood that may function in host defense against influenza in healthy adults.