T‐cell responses against rhinovirus species A and C in asthmatic and healthy children

Abstract Background Infections by rhinovirus (RV) species A and C are the most common causes of exacerbations of asthma and a major cause of exacerbations of other acute and chronic respiratory diseases. Infections by both species are prevalent in pre‐school and school‐aged children and, particularly for RV‐C, can cause severe symptoms and a need for hospitalization. While associations between RV infection and asthma are well established, the adaptive immune‐mechanisms by which RV infections influence asthma exacerbations are yet to be defined. Objective The aim of this study was to characterize and compare T‐cell responses between RV‐A and RV‐C and to test the hypothesis that T‐cell responses would differ between asthmatic children and healthy controls. Methods A multi‐parameter flow cytometry assay was used to characterize the in vitro recall T‐cell response against RV‐A and RV‐C in PBMCs from children with acute asthma (n = 22) and controls (n = 26). The responses were induced by pools of peptides containing species‐specific VP1 epitopes of RV‐A and RV‐C. Results Regardless of children's clinical status, all children that responded to the in vitro stimulation (>90%) had a similar magnitude of CD4+ T‐cell responses to RV‐A and RV‐C. However, asthmatic children had a significantly lower number of circulating regulatory T cells (Tregs), and healthy controls had significantly more Tregs induced by RV‐A than RV‐C. Conclusions and Clinical Relevance The comparable recall memory T‐cell responses in asthmatic and control children to both RV‐A and RV‐C show that differences in the antibody and inflammatory responses previously described are likely to be due to regulation, with a demonstrated candidate being reduced regulatory T‐cells. The reduced Treg numbers demonstrated here could explain the asthmatic's inability to appropriately control immunopathological responses to RV infections.


Introduction
Rhinovirus (RV) infections are the major cause of acute asthma exacerbations [1,2] and are also important causes of acute lower respiratory infections in cystic fibrosis [3] and chronic obstructive pulmonary disease [4]. School-aged children are likely to have recurrent RV infections [5,6], thus are at particular risk for RV-induced asthma exacerbations. Host genetics in combination with environmental factors (i.e., atopy) and characteristics of the virus are known to influence the severity of RV-induced asthma [7,8]. From the three RV species currently identified, RV-A and RV-C are the most prevalent amongst children [9][10][11] and RV-C has been associated with more severe symptoms of infection and a higher frequency of asthma exacerbations [12,13]. While the association between RV infections and asthma exacerbations are now well established, the mechanisms by which RV infections induce asthma exacerbations are yet to be defined.
A recent study utilizing peptide/MHC class II tetramerguided epitope mapping has identified circulating epitopespecific memory CD4þ cells to a genotype of RV-A, indicating that recall memory CD4þ T cell responses drive the immune response against RV infections. Furthermore, intra-species cross-reactivity was evidenced by in vitro proliferation of RV-A16-induced-memory T cells to peptides from another RV-A genotype, RV-A39 [14], indicating that the memory response to RV is less type-specific than what was previously expected. This study was conducted in healthy adult donors, therefore the adaptive response to RV in asthmatic children remains unknown. The only study on adaptive responses of asthmatic children to RV-C focused on antibody responses and reported markedly lower and often undetectable species specific IgG1 antibody titres to their VP1 proteins, contrasting to the high specific titres to the VP1 proteins of RV-A [15]. Furthermore, the anti-RV-A antibody responses but not those specific to RV-C were higher in asthmatics compared with controls. These unexpected and intriguing differences might be due to overall poor immune response to RV-C that could be evident in the T-cell compartment which is examined here.
The current study describes T-cell responses to RV-A and RV-C in a pediatric clinical cohort of asthma-diagnosed children. The responses measured are induced by pools of peptides containing epitopes of the RV-A and RV-C VP1 proteins that are both species-specific and representative of memory T-cell responses made to each species [16]. We aimed to characterize and compare T-cell responses between RV-A and RV-C and to test the hypothesis that T-cell responses would differ between asthmatic children and healthy controls.

Study participants
Biobanked peripheral blood mononuclear cells (PBMCs) and plasma of 48 children (aged 5 months to 14 years) were collected between December 2009 and March 2015 as part of the ''Mechanisms of Viral Infection in Children'' (MAVRIC) cohort, from Princess Margaret Hospital for Children (Perth, Western Australia). The MAVRIC study is a continuation of the Perth childhood acute asthma study began by Professor Peter Le Souëf where the role of virus infection in childhood respiratory illness is being investigated in a range of genetic and inflammation related studies. Children presenting with an acute wheezing episode to the emergency department (ED) were recruited into the study and examined by an independent physician for asthma and wheeze assessments and nasal samples were taken and submitted to routine virus diagnosis and testing for RV species. Blood sample have been taken at admission and during the follow-up which is part of the study. The blood samples (5-10 mL), used here were collected after the children had recovered from their acute episode of asthma, were diluted with an equal volume of RPMI-1640 medium (Sigma-Aldrich, St Louis, USA) and 10 IU/mL preservativefree heparin (Pfizer, New York, USA). The PBMCs were isolated by density gradient Lymphoprep TM (Nycomed, Oslo, Norway) and washed three times in RPMI media prior to cryopreservation. From the cohort, 22 children (asthmatics, mean age in years 7.7 AE 3.7) had been diagnosed with asthma, requiring hospitalization (Table 1). Children with no medical history of doctor-diagnosed asthma or other respiratory illness were included as controls (mean age in years 5.9 AE 3.7, n ¼ 26). All 48 children (asthmatics and controls) had nasal secretion specimens tested for RV using a molecular detection and typing assay [12] to determine the RV genotypes. As shown in Table 1 and reported previously [1,15,17], most asthmatic children presenting to the ED following an epidose of asthma exacerbation had detectable RV (70%) and the majority of the infections were caused by RV-C. 27% of the nasal swabs, taken at the time of blood collection, from the healthy controls were PCRþ for RV in keeping with other studies of disease free children.
PBMCs from asthmatics were collected at their follow-up visit, 2-9 months (mean 5 months) after an acute episode of asthma resulting in an ED presentation. The choice of using samples collected at the follow-up visit, when the children were clinically well, was to avoid external confounders (i.e., corticosteroid administration) that could potentially impact the in vitro adaptive immune responses. The research has been approved by the Princess Margaret Hospital Human Ethics Committee (approval number 1761/EP) and written informed consent was obtained from each parental/guardian of all participants.

Rhinovirus peptides
Immunodominant peptides of the VP1 capsid protein of rhinoviruses A and C, recently identified as specific for and representative of each RV-A and RV-C species by an epitopemapping study [16], were combined into pools representing each species (Table 2). From a measure that considered the size of the stimulations of individual subjects and the frequency of stimulation in the sample (reactivity score) these peptides accounted for almost 50% of the reactivity score achieved with all the peptides for both RV-A and RV-C. The peptides representing each species were in different positions of the VP1 sequence and in regions where there was little response to peptides of the other species and where the sequences between species were 40-85% disparate.

T-cell response against RV-A and RV-C
Each step of the T-cell activation process was followed by evaluating three parameters of the CD4þ and CD8þ activation in the in vitro RV response: (i) the co-expression of the T-cell activation markers CD25 hi HLA-DR hi ; (ii) the expression of the co-stimulatory molecule ICOS-I hi ; and (iii) effective proliferation, measured by the dilution of the cellproliferation dye CellTrace TM Violet into daughter cells.

CellTrace TM labeling
Thawed PBMCs were labeled with CellTrace TM Violet proliferation kit (Invitrogen, Mulgrave/USA) as follows: PBMCs were re-suspended at 6 Â 10 6 cells in 1 mL of PBS in a 15 mL conical tube. The tube was laid horizontally and 110 mL of PBS was added to the non-wetted portion of the plastic, at the top of the tube. To this, 1.1 mL of CellTrace TM stock solution (5 mM) was re-suspended to obtain a 5 mM final working concentration. The cell suspension was quickly vortexed to ensure uniform labeling prior to incubation for 5 min at RT, protected from light. Unbound dye was quenched by three consecutive washes with RPMI supplemented with 10% heat inactivated fetal calf serum (HI-FCS; SAFC, Brooklyn, Australia). At the final washing step, CellTrace TM labeled cells were re-suspended in warm AIM-V serum free culture media (Life Technologies, Mulgrave/ Australia) supplemented with 50 mM 2-mercaptoethanol (Invitrogen, Mulgrave/Australia) and seeded at 3 Â 10 5 cells per well in a round bottom 96-well plate.
In vitro stimulation of RV-A and RV-C-specific T cells PBMCs' responses measured after a 6-day stimulation period in the absence of added co-stimuli (i.e., exogenous cytokines) or enrichment of dendritic cells necessary for in vitro priming of na€ ıve CD4þ cells [18,19] are memory Tcell responses. This is also confirmed by the expression of HLA-DR (see results) known to be expressed by memory and not na€ ıve T cells [20]. Seeded wells were cultured in sextuplicate without stimulus (negative control) or stimulated with either RV pools ( Table 2)   were washed out and cells were fixed using Stabilizing Fixative (Becton Dickinson Pharmingen, NJ, USA). Flow cytometry data was acquired using FACS Diva Software on a LSR Fortessa flow cytometer (all from BD Biosciences). At least 250,000 total events were acquired per each stimulation condition. Data was acquired using FlowJo X version 10.0.7r2 (Tree Star). ICOS-I readings are not available for two cases and two controls due to a shortage of antibody markers from supplier delays.

Data analysis
Following cell population gating (Fig. 1) on lymphocytes, singlets, live cells, CD3 þ , CD4 þ /CD8 þ , circulating Tregs (CD4 þ CD25 hi CD127 low ) from total CD4þ, and stimulated Tregs (sTregs: CD4 þ CellTrace dim CD25 hi CD127 low ) from the proliferative CD4þ subset, the percentage of activated (CD25 hi HLA-DR hi and ICOS-I hi ) and proliferating (Cell-Trace dim ) CD4þ and CD8þ cells was recorded for each condition. Activated CD4þ or CD8þ cells were defined as [D % of CD25 hi HLA-DR hi ] ¼ (% of CD25 hi HLA-DR hi cells among a pool of six antigen-stimulated wells)À(% of CD25 hi HLA-DR hi cells among a pool of six unstimulated control wells) and the same analysis was repeated for ICOS-I hi . The proliferative response was given by the % of CD4þ and CD8þ cell proliferation above background, which was calculated as [D % of proliferating cells] ¼ (% of CellTrace dim cells among a pool of six antigen-stimulated wells)À(% of CellTrace dim cells among a pool of six unstimulated control wells). The threshold for proliferation and activation of 0.01% was used as detection limit of the assay for all , and the proliferative response (CellTrace dim ). Tregs (CD4 þ CD25 hi CD127 low ) and sTregs (CD4 þ CellTrace dim CD25 hi CD127 low ) were measured in unstimulated and RVstimulated cultures, respectively.
antigens for both CD4þ and CD8þ cell subsets. Although there are several cell markers recognized for characterizing different Treg subsets, here we adopted the combination of the cell surface markers CD4 þ CD25 hi CD127 low to identify frequency of circulating Tregs in peripheral blood, as described previosuly [21][22][23].

Statistical methods
Non-parametric RV-specific CD4þ and CD8þ T cell data was statistically analyzed using independent samples Mann-Whitney U-tests when comparing between asthmatics and controls, while all paired samples (comparison between RV-A and RV-C responses within asthmatics or controls) were compared using related samples Wilcoxon signed rank tests. Chi^2 was used for two sample prevalences comparisons. Differences and associations were considered significant ( Ã ) if the p value was less than 0.05, while non-significant results are represented by ''ns.'' Statistical analyses were performed using the statistical packages Prism (GraphPad Software Inc., La Jolla, USA).

Prevalence of the recall T-cell responses to RV-A and RV-Cs in children
The prevalence of CD4þ T-cell responses to RV-A and RV-C was evaluated by investigating the prevalence of antigen specific CD4þ cells expressing the activation and costimulatory markers CD25 hi HLA-DR hi and/or ICOS-I hi and the prevalence of cells progressing to proliferation (CellTrace dim ). Approximately 70% of the asthmatic children had RV-A and RV-C specific CD4þ activation and proliferation, which was similar to the prevalence of RV-A and RV-C specific CD4þ activation and proliferation found in control children (Table 3). Proliferation and activation for the CD8þ cells was also found but it was numerically less prevalent than the CD4þ cells, being about 75% for activation and about 50% for the proliferation to both the RV-A and RV-C peptides, although not statistically significantly different from the corresponding CD4þ responses (chi2 > p ¼ 0.1) ( Table 3).

Magnitude of the CD4þ and CD8þ T-cell response to RV
CD4þ T-cells were the main subset to proliferate (Cell-Trace dim ) in response to the RV stimulus in both asthmatic and control groups ( Fig. 2A). The magnitude of the memory T-cell response of asthmatics and controls were compared by analyzing the extent of the expression of activation markers (CD25 hi HLA-DR hi and ICOS-I hi ) and proliferation (Cell-Trace dim ) of CD4þ and CD8þ cells in the response to the RV-A and RV-C. There were no significant differences between the magnitude of in vitro CD4þ (Fig. 2B) and CD8þ (not shown) response to rhinoviruses, at both activation and proliferation levels, although the average proliferation of controls was 70% and 55% of that found for the averages of the asthmatic RV-A and RV-C responses (Fig. 2B). The characteristic in vitro CD4þ and CD8þ T-cell recall response to RV-A and RV-C for two asthmatic and two control children is shown in Figures 3 and 4, respectively. The proliferation and activation markers for the controls with (27%) and without positive PCR tests to RV at bleeding did not differ significantly, with the values from PCR positive subjects being found across the whole range of results. These results show that children, independent of their clinical status, have a competent recall of CD4þ memory response to both RV-A and RV-C. Tables S1 and S2 summarise the three parameters of CD4þ and CD8þ T cell responses, respectively, against RV-A and RV-C in the childhood cohort.
Although of a significantly lower magnitude than the CD4þ T-cell population, as shown by the activation markers and the frequency of T-cell proliferation ( Fig. 2A), especially in response to RV-C peptides, the magnitude of the CD8þ proliferative response was significantly higher when compared to unstimulated cultures and was highly correlated with the magnitude of the CD4þ proliferation for both cases (RV-A, r ¼ 0.74, p < 0.0001 and RV-C, r ¼ 0.61, p < 0.005) and controls (RV-A, r ¼ 0.43, p < 0.05 and RV-C, r ¼ 0.55, p < 0.005).

T regulatory cells in the recall T-cell response to rhinoviruses
Total Tregs (CD4 þ CD25 hi CD127 low ) were calculated as a percentage of the total CD4þ population and were measured Table 3. Prevalence of in vitro T-cell response to RV-A and RV-C, given by the prevalence of RV-activated and proliferating T cells in asthmatic and control children. in unstimulated PBMCs to compare the frequency of circulating Tregs in asthmatic and control children. The sTregs (CD4 þ CellTrace dim CD25 hi CD127 low ) were measured in the CD4þ proliferating fraction of RV-stimulated cultures to evaluate the ability of asthmatic and control children to develop Tregs under the stimulus of RV.

RV-
In agreement with the literature for the frequency of total circulating Tregs in peripheral blood of control children [24], Tregs were found at a frequency of 5.8% AE 1.9 in the control group; the frequency of Tregs in cases was significantly lower (4.0% AE 1.3, p 0.001) (Fig. 5A). Asthmatic children had a higher stimulated Treg response to RV-A above background (p < 0.05), that was not found for the control children, possibly as they already had higher numbers of background sTreg cells compared to asthmatics (p < 0.05) (Fig. 3B). The number of sTregs responding to RV-C was significantly lower than the response to RV-A for the control group (p < 0.02) (Fig. 3B).

Discussion
This is the first study to compare the T-cell responses to the different RV species and the peripheral blood responses of healthy and doctor-diagnosed asthmatic children. A previous study has shown that anti-RV induced IFN-g responses could be detected in cells from broncho-alveolar lavage of healthy, but not asthmatic subjects upon experimental RV infection, although the data shows that the reduced detection was due to the production of high levels of IFNg without stimulation by the lavage cells from asthmatics but not healthy subjects [25]. The study did not find differences in other RV induced cytokines, although the small number of samples analyzed might have precluded a significant estimation of these cytokines. We found indistinguishable recall responses of T-cells from asthmatics and healthy controls, and for responses to RV-A and RV-C peptides. Differences in the magnitude of the RV-specific T cell memory responses and the activation marker expression are therefore not responsible for the increased anti-RV-A IgG in asthmatic children and the anomalously low IgG titres specific to RV-C found in all subjects [15]. The latter indicates that RV-C does not evade adaptive immune responses per se, but rather that responses may be subject to regulatory events that affect either the specificity of the antibodies or the ability of the response to induce them. The lack of differences in the viral load of healthy and asthmatic adults found after experimental RV-A infection [25] would be in agreement with the similar T-cell responses here and suggest that the higher anti-RV-A antibody responses might be due to the higher inflammation described by Message et al. [25] or due to more frequent infections. Interestingly, the asthmatics are known to have reduced IL-15 production [26], a cytokine known to increase antibody responses and induce B-cell differentiation [27].
CD4þ T-cell responses are believed to be critical in determining the outcome of many viral infections, including RV [28][29][30][31]. In this study, that used peptides selected for the stimulation of CD4þ responses, greater than 70% of all children showing some level of CD4 proliferation. Furthermore, asthmatics' PBMCs had similar magnitudes of CD4 proliferation as those of control children. In order to evaluate the degree of functionality of this response, three markers of mid to late T-cell activation were analyzed: ICOS-I, CD25, and HLA-DR. ICOS-I plays a fundamental role in all stages of T-cell antigen-depend responses, proving costimulatory signaling at T cell growth, differentiation and effector functions [32][33][34]. ICOS deficiency results in a reduced memory T cell compartment and defective recall of

T-cell responses to rhinoviruses in children
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memory T-cell response [34]. Similarly, HLA-DR and CD25 are highly expressed on activated memory T cells and participate in effector turnover and persistence of memory T cells [20]. Similar levels of CD25, HLA-DR, and ICOS-I expression in response to the two RV species were found in both cases and controls and together with the outcomes of CD4þ proliferation, these results suggest that children, independent of their clinical status, have a competent recall of CD4þ memory response to both RV-A and RV-C. Additional studies measuring cytokine production by RVmemory T cells and studies in transcriptome analysis exploring cellular gene-pathways associated with recall antigenic responses to RVs would complement our findings on functional memory T-cell response to both RV species. The levels of pro-inflammatory T-cell cytokines IFN-g, IL-5, and IL-13 found in the lung fluids of asthmatics after RV infection [25] were all associated with decreased lung function in acccord with the known effects of these cytokines on lung tissues and in the recruitment of inflammatory cells.
The peptides stimulated much smaller responses from the CD8þ cells. This justified the precaution of measuring CD4þ and CD8þ cells separately but the significance should be viewed recognizing that the assays were optimized for CD4þ responses.
Comprising only 5-10% of the total CD4þ population, Tregs are critically important in maintaining immune homeostasis and preventing exacerbated immune responses. Tregs are fundamental in regulating induction of de novo and memory immune responses to allergens and pathogens, all of which in excess can result in chronic inflammation and damage to epithelial tissue in the airways [35]. This would be especially important in the upper and lower respiratory tracts, where a large surface area is constantly exposed to potential antigens. A recent study utilizing class II tetramers to track primary and recall of influenza-specific memory Tcell response, showed rapid accumulation of antigen-specific Tregs in the lungs and associated lymph nodes when compared to the primary infection [36]. Furthermore, high CD8þ response and pulmonary inflammation was observed after influenza-specific memory Treg depletion prior to reinfection [36] demonstrating the importance of Tregs in controlling immune-inflammation in response to respiratory viral infections. For rhinoviruses in asthma the Tregs could reduce the Th2 responses associated with adverse lung function and eosinophilic inflammation [25] and reduce inflammatory cytokine production such as IL-13 [25] and produce anti-inflammatory effects via mediators such as adenosine and TGF-b that act on inflammatory and endothelial cells. In asthma, a decline in the number of circulating Tregs is associated with increased symptoms of asthma [37], whereas the use of corticosteroids increases the frequency of circulating Tregs in asthmatic children and control symptoms of exacerbation [38]. The finding that the RV-C peptides induced a considerably lower Treg response than the RV-A peptides not only show a difference in the responses between the two RV species, but is consistent with reports of increased immunopathology produced by RV-C in lower respiratory tract infections [1,2]. The difference between the Treg responses to RV-A and RV-C was however apparently masked in the asthmatics who tended to have much lower overall Treg cell stimulation to RVs.
Our findings indicate that all children, independent of their asthma status, have a competent CD4þ T-cell recall response to RV-A and RV-C. However, we identified significantly lower numbers of circulating Tregs in asthmatic children in comparison to their healthy counterparts and differences of Tregs induced by the antigens of different species. These, and possibly other regulatory immune mechanisms could explain the reduced ability of asthmatics to suppress viral-induced inflammatory responses in asthma exacerbations. and Drs Steve Oo and Franciska Prastanti for recruitment of subjects. The study was funded by the Telethon-Perth Children's Hospital Research Fund/Telethon Kids Institute grant and NHMRC grant APP1087700.

SUPPORTING INFORMATION
Additional supporting information may be found in the online version of this article at the publisher's web-site. Table S1. Measurement of CD25 hi HLA-DRhi (intermediate activation), ICOS-Ihi (late activation), and CellTrace dim (proliferation) above background (4 value) in CD4þ of asthmatic and control children in the in vitro recall response to RV-A and RV-C epitopes. Table S2. Measurement of CD25 hi HLA-DRhi (intermediate activation), ICOS-I hi (late activation), and CellTrace dim (proliferation) above background (4 value) in CD8þ of asthmatic and control children in the in vitro recall response to RV-A and RV-C epitopes.