RSV Downregulates IL-21/IL-21R on TFH cells via PD-L1 induction in APCS impairing protective humoral responses

Respiratory syncytial virus (RSV) is the major cause of hospitalization for children under two years of age. RSV vaccines are currently unavailable, and children suffering from multiple reinfections by the same viral strain, fail to develop protective memory responses. Follicular helper T (TFH) cells specialize in providing B cell help to antibody production and affinity maturation, mainly via IL-21 secretion. Although RSV-specific antibodies can be detected upon infection, how they are generated and their relevance against disease protection has not been fully examined. Here, we observed that RSV expands a functionally impaired murine TFH cell population in vitro and vivo, with downregulated IL-21R expression and IL-21 production. IL-21 treatment of RSV-infected mice, however, increased TFH cells frequency, enhanced the germinal center reaction and improved protective humoral immune responses by increasing viral protein F specific antibody avidity and neutralization capacity. In vivo, it protected from RSV infection, decreasing lung inflammation. Passive immunization with purified IgG from IL-21 treated RSV-infected mice protected against RSV infection. Both viable and UV-inactivated RSV induced PD-L1 expression on B cells and DCs, however, only in DCs a direct effect of RSV was detected. Blocking PD-L1 during infection recovered IL-21R expression in TFH and B cells and increased secretion of IL-21 by TFH cells in a DC-dependent manner. Our results unveil a novel pathway by which RSV affects TFH cells activity, reducing levels of IL-21 and its receptor, by increasing PD-L1 expression on APCs. These results highlight the PD-L1/IL-21 axis importance for the generation of protective responses to RSV infection. GRAPHICAL ABSTRACT RSV infection impairing IL-21 secretion by TFH cells via PD-L1induction in dendritic cells and B cells. Low levels of IL-21 lead to poor RSV-specific humoral immune responses, low antibody titer, avidity and neutralization capacity. PD-L1 blockade can upregulate IL-21 secretion, and IL-21 treatment restores the entire immune humoral responses, resulting in protection against RSV infection.


INTRODUCTION
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants, responsible for about three million hospitalizations and 66,000 deaths every year in children under two years of age (1). RSV-infected infants develop upper respiratory tract symptoms, often progressing to bronchiolitis and/or pneumonia, and even respiratory failure, which frequently needs mechanical ventilation and intensive care unit therapy (2). There is no effective vaccine available against RSV, and passive immunization with monoclonal antibodies is used only in high risk infants, in which RSV infection can be deadly. However, it is not only in premature children that RSV infection may be problematic or deadly; infants, elderly and pregnant women are also target populations (3), reinforcing the need for development of an effective vaccine .
In humans, neutralizing RSV-specifics antibodies are formed in upper respiratory tract, however, re-infection with the same RSV strain is frequent in healthy, immune competent individuals (4,5). The protective relevance of serum antibody levels remains unclear (6,7). Serum antibody response was loosely correlated with protection; however, there was a correlation of nasal preexisting RSV-specific humoral response with resistance to re-infection (8). Other studies have correlated the presence of serum high avidity RSV-specific IgG with protection (9); although these antibodies have a short life in children (10) and adults (11). Nevertheless, RSV morbidity and mortality are mainly associated with 2-4 months of age infants when titers of maternal antibodies are decreasing and have not yet been replaced by an endogenous antibody responses (12). In mice, the RSV antibody response induced by formalin-inactivated RSV (FI-RSV) is non-protective (13). Formalin inactivation results in conformational changes of the RSV fusion protein (14), which is required for the generation of effective neutralizing antibody responses (15). Moreover, antibodies induced by FI-RSV are low affinity due to poor toll-like receptor (TLR) stimulation (16).
Effective B cells responses require help from follicular helper T (TFH) cells. TFH cells are predominantly found in germinal centers (GC) of secondary lymphoid organs (17)(18)(19) and produce high levels of IL-21. This cytokine is known to increase the affinity of antibodies and induce immunoglobulin class switching (20,21). IL-21 acts on naive B cells in conjunction with co-stimulatory signals which drive differentiation of either GC B cells or plasma cells (22). The specific provision of TFH cell-derived helper signals to GC B cells is proposed to be the major driver of antibody affinity maturation (23,24). Therefore, we hypothesized that RSV modulates TFH cells, preventing B cell help, targeting humoral immune response efficiency.
By employing a murine model of RSV infection, our results unravel an immunological mechanism by which RSV modulates TFH cells and GC reactions. We found that RSV can induce PD-L1 (Programed Death-ligand 1) in dendritic cells (DCs) and B cells, decreasing B and TFH cells functions. This correlated with a decreased ability of TFH cells to produce IL-21 and downregulation of IL-21R, leading to low avidity RSVspecific humoral immune response. Treatment with recombinant IL-21 reduced RSVmediated impairment of GC and TFH cell functions as well as improved animal survival.
Our results underline the importance of the PD-1/PD-L1 pathway and IL-21 adjuvant activity in the generation of effective anti-RSV protective antibody responses.
Lytic plaques assay was used to calculate PFU of HSV-1. Inactivation of the viruses was performed at UV light for 30 min, at room temperature.

Animals
Female BALB/c mice ranging from 6 to 8 weeks old were purchased from the Biological

Infection and treatment
For in vitro infection, splenocytes were isolated after lysis of red blood cells. Cells were seeded at 5x10 5 cells per well in a 96 well-plate and were stimulated either with 10 2 PFU/ml of HSV-1 or RSV or the same virus inactivated by UV for 30 min. As negative controls, uninfected Vero cells were used and processed similarly to RSV infected cells. Alternatively, cells were treated with 0.5 µg of anti-PD-L1 antibody (clone MHI5, eBioscience) or control IgG (BioXcell) 1 hour before infection. The supernatant was collected at 12, 24, 48, 72 and 96 hours post-infection for analysis. After four days, cells were labeled with specific antibodies and analyzed by flow cytometry.
Rabbit anti-mouse antibody HRP-conjugated (Invitrogen) and TMB (Life Technologies) was used to development. Plates were read at 450nm in EZ Read 400 Microplate reader (Biochrom).
ELISA to measure the avidity of anti-RSV antibody was conducted in the same manner, however the plate was washed with a 6M Urea solution. The results were expressed as an Avidity Index (AI), which was calculated as previously described by Freitas et al (9). The avidity of RSV-specific total IgG was classified according to values that had been predetermined arbitrarily defined as low (<50%), intermediate (50-70%), or high (>70%).

Neutralization assay
Antibody neutralization capacity assay was performed as previously described by Zielinska et al (26), with modifications. Four-fold serial dilutions from 1:10 to 1:10240 were prepared in virus diluent (DMEM 0% FCS and 1% gentamicin (0.08 mg/ml, NOVAFARMA). Serially diluted serum was challenged with an equal volume of the RSV-A2 strain, previously tittered to give ~150 PFU per 50 µl of inoculum. The serum/virus mixtures were incubated at 37°C, 5% CO2 for 1 h. 96-well plates plated with Vero cell monolayers were infected with 50 µl/well (in triplicates) of the serum/virus mixture. Plates were blotted with 0.5% methyl cellulose, prepared in DMEM with 2% FBS and incubated at 37°C, 5% CO2 for 3 days to allow plaque formation. To detect the syncytium formation, wells were incubated with primary anti-RSV antibodies (Millipore) and secondary antibody HRP-Rabbit anti-mouse IgG (Millipore) for 1 hour at 37ºC. Blocking occurred with blotto (5% milk, 0.05% tween in PBS 1X buffer). For revelation, we used 4-chloro-1-napthol with 0.01% H2O2 for 20 min in dark. Syncytium was counted in optical microscope and PFUs calculated by ratio of the number of syncytium by multiplying the dilution with virus medium volume in the plate.

Histology and Immunohistochemistry
Lungs and spleens were fixed with 10% formalin, embedded in paraffin and cut into 5 μm-thick sections. Hematoxylin and eosin staining was performed in slide sections to evaluated inflammation scores. For immunohistochemistry, slides sections were deparaffinized with xylol and endogenous peroxidase activity was blocked by incubation with 3% H2O2 in methanol. Antigen unmask was performed by incubating the slides in 0.1 mol/L citrate buffer, pH 6, for 30 min at 95°C, followed by cooling at room temperature for 1h. Sections were blocked in PBS with 4% bovine serum albumin (BSA), 5% mouse serum, and incubated with primary antibody anti-PD-L1 (clone MIH5, eBioscience) at 1:1000 dilution in PBS, 1% BSA, and 1.25% mouse serum.

Passive immunization
Mice were separated into 3 groups, the first group received naive IgG serum, the second group received IgG serum from RSV-infected mouse and the latter group received IgG serum from RSV-infected and IL-21 treated mouse. For IgG purification was used Protein A-Sepharose column (Sigma) following manufacturer's instructions.
Each mouse received intraperitoneal 300µg of purified IgG. After two days, animals were infected with 10 7 PFU of RSV. Mice were euthanized at day 5 post-infection and lungs were harvesedt for further analysis.

Statistical analysis
The significance for differences between the groups was analyzed with one-way ANOVA test followed by a Bonferroni post-test or t-test were applied to parametric data

RSV induces TFH proliferation in vitro.
Given the importance of TFH cells in helping B cells to produce antibodies, we analyzed whether RSV could modulate TFH cells proliferation in vitro. Mice splenocytes were infected with 10 2 PFU/ml of HSV-1 or RSV for 4 days. HSV-1 was used as a positive control since it induces high levels of high affinity HSV-specific antibodies (27)(28)(29). We found that both RSV and HSV-1 increase the frequency of TFH (CD3 + CD4 + CXCR5 + PD-1 + ) cells compared to uninfected controls (Fig. 1, A and B).

RSV decreases IL-21 secretion and IL-21R expression by TFH and B cells
We next investigated whether RSV could affect the function of TFH cells. Production of IL-21 is an important indicator of TFH activity (30). IL-21 is necessary for antibody affinity maturation and B cell differentiation, and is known to upregulate the expression of its receptor on CD4 + T cells. No IL-21 production was detected when RSV was added to splenocyte cultures, in contrast to what was observed for HSV-1 (Fig. 2 A).
In vivo, IL-21 serum levels were undetectable even 4 days post-infection (Fig. 2 B), again contrasting to the abundant IL-21 induction observed during HSV-1 infection. In vitro, RSV also reduced IL-21R expression on TFH cells (Fig. 2, C and D) and B cells (CD3 -CD19 + ) (Fig. 2, E and F), while HSV-1 increased IL-21R expression on these cell populations (Fig. 2 C-F), suggesting the regulation of the IL-21/IL-21R axis was specific to RSV. These data indicate that RSV could negatively modulate the function of TFH cells by downregulation of IL-21R expression, as well as of IL-21 production.

IL-21 treatment increases B cell follicle size, IgG production, antibody avidity and neutralization capacity
We hypothesized that the low levels of IL-21 observed upon RSV infection (Fig. 2 B) would well as increased frequencies of B and TFH cells (Fig 3B and C). Finally, treatment with IL-21 also improved titers (Fig. 3 D), avidity ( Fig. 3 E), and neutralization capacity ( Fig. 3 F) of anti-F protein specific antibodies.

IL-21 treatment protects RSV infected animals and decreases lung inflammation by specific humoral immune response
To evaluate the protective roles of IL-21 in vivo, similar experimental design was performed (Supplementary figure 2). While pulmonary injury was observed in lungs from untreated RSV-infected mice, (Fig. 4 A, arrows show cell infiltration and reduction of bronchi lumen space), treatment with IL-21 reduced inflammation as well as improved survival compared to untreated infected mice, reducing weight loss in infected animals (Fig. 4 B and C). IL-21 treatment also decreases numbers of RSV copies in lungs in RSV-infected mice (Fig. 4 D). These results suggest that IL-21 is essential for protection from RSV infection by activating TFH and B cells function.
To further investigate the immunologic mechanisms of protection, we pretreated (two days before RSV infection) mice i.p. with 300µg of purified IgG from naïve serum and RSV-infected mice serum, either treated or not with IL-21. The results, depicted in Fig.   4 E, indicate that protection can be mediated by IgG alone elicited by IL-21 treatment in infected mice. Thus, low production of IL-21 during RSV infection affect GC reactions, dramatically impairing the generation of a protective, anti-RSV humoral response.

IL-21 secretion by TFH cells
We  (44). Studies in humans suggest that RSV can modulate IL-21 secretion. In a cohort study that characterized the primary and secondary cytokine response to RSV infection, IL-21 was not detected in swab nasal secretion samples from infants recruited during two consecutive winters (45). Our data indicated that treatment with IL-21 successfully enhanced humoral responses to RSV, leading to increases in B cell follicles, anti-F (fusion RSV protein) IgG titers, antibody avidity and neutralization capacity. High-avidity anti-RSV IgG antibodies have been shown to confer protection in infants under 3 months old (9). We observed that the antibodies generated upon IL-21 treatment in infected mice were highly protective upon transfer to naïve animals that were challenged in vivo by RSV. Our data agrees with reports that IL-21 is most important at the beginning of a humoral response, activating AID (Activation-induced cytidine deaminase), promoting immunoglobulin switch class and somatic hypermutation.
In addition, IL-21 increases germinal center reactions and induces IL-21R expression on activated B cells and TFH cells (46)(47)(48). IL-21R is required to generate TFH cells, GC reaction, B cells, plasma cells and plasmablasts in mice (49). We observed that IL-