Targeting the ICOS/ICOS‐L pathway in a mouse model of established allergic asthma disrupts T follicular helper cell responses and ameliorates disease

Abstract Background Allergic asthma is characterized by chronic inflammation and remodelling of the airways, associated with dysregulated type 2 immune responses and allergen‐specific IgE. T follicular helper cells (TFH) are crucial in T‐dependent B‐cell responses and have been implicated in allergic airway disease (AAD). TFH, unlike other CD4+ T cells, are uniquely reliant on continuous ICOS signalling to maintain their phenotype after T‐cell priming; therefore, disrupting this signal can impair TFH responses. However, the contribution of TFH to disease during chronic aero‐allergen exposure and the therapeutic potential of targeting these cells have not been evaluated. Methods To establish AAD, female BALB/c mice were repeatedly exposed to house dust mite or Alternaria alternata three times a week for up to 5 weeks. To examine the impact of TFH on AAD, mice were allergen exposed for 5 weeks and co‐administered anti‐ICOS Ligand‐targeted antibodies, three times a week for the last 2 weeks. Results TFH were first observed in the lung‐draining lymph nodes and with further exposure were also found locally within the lungs. TFH accumulated with sustained allergen exposure, alongside germinal centre (GC) B cells. Blockade of ICOS signalling after AAD establishment successfully depleted TFH but did not affect the differentiation of other CD4+ T‐cell subsets. This reduced GC responses, allergen‐specific IgE, inflammation, pulmonary IL‐13 and airway hyper‐responsiveness. Conclusions TFH are crucial in the regulation of AAD and the ICOS/ICOS‐L pathway could represent a novel therapeutic target in allergic asthma.

hyper-responsiveness (AHR), 1 . T helper 2 cells (Th2), and the type 2 cytokines they produce, have traditionally been thought of as the central drivers of the disease. It is now clear, however, that many other immune cells can produce these cytokines and play vital roles in the regulation of distinct asthma phenotypes. 1 T FH are a distinct CD4 + T-cell subset specialized to provide help to B cells, resulting in the production of high affinity, isotypeswitched antibodies and the differentiation of B cells into memory B cells and plasma cells. 2 They can be identified by their expression of CXCR5, PD1, Bcl-6 and ICOS and are predominantly located within the B-cell follicles of secondary lymphoid organs (SLOs), such as lymph nodes and the spleen. 2 T FH differentiation is a multi-step process. First dendritic cells (DCs) present antigen and co-stimulatory signals to naive CD4 + T cells within the T-cell zone of SLOs. 3,4 Next, pre-T FH migrate towards the T-B border 5,6 where antigen presentation and inducible T co-stimulator ligand (ICOS-L) co-stimulation is provided by activated B cells. 7,8 Fully differentiated T FH migrate into the B-cell follicle, further migrating into newly formed anatomical structures called germinal centres (GC). 6 Here, T FH form tight, cognate interactions with GC B cells, providing survival and differentiation signals to the B cells in return for T-cell receptor (TCR) signalling and co-stimulation. 2,5,7 The co-stimulatory molecule ICOS is required for T-cell activation and is upregulated on CD4 + T cells following TCR engagement. 9 Importantly, however, T FH uniquely require sustained ICOS/ICOS-L signalling throughout an immune response and after DC priming to maintain their phenotype, unlike other CD4 + T-cell subsets. 10 The role of T FH in allergic asthma appears to be complex. T FH are found in the SLOs of mice that have undergone sensitization and challenge with HDM [11][12][13] and are required for allergen-specific IgE production. 14,15 T FH isolated from the lung draining lymph nodes can migrate to the lungs and become Th2 cells, enhancing allergic airway disease (AAD), when injected intravenously. 13 In contrast, IL-21 + T FH failed to generate Th2 cells when adoptively transferred into naïve mice subsequently challenged with HDM, but do cause airway eosinophillia. 11 After helminth infection Th2 cells can become T FH, 16 retaining a Th2-like phenotype, and T FH themselves can obtain effector functions related to other CD4 + T-cell

G R A P H I C A L A B S T R A C T
Chronic allergen exposure drives T follicular helper cell (TFH) formation in the lung draining lymph node after 1 week and the lung tissue after 3 weeks TFH at both sites interact with germinal centre B cells and are critical for the production of allergen specific IgE Allergic disease is also hallmarked by cellular inflammation and airway hyperresponsiveness driven by IL-13 Blocking ICOS and ICOS-L interactions between TFH and B cells reduces allergen specific IgE production and also dampens other hallmark symptoms of allergic airway disease UWADIAE ET AL.

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lineages. 17 Conversely, CD4 + T cells lacking the T FH master transcriptional regulator Bcl6, which cannot become T FH , preferentially differentiate into lung resident Th2 cells and promote AAD. 12 Thus, T FH appear to be important in AAD but their exact contribution and the therapeutic potential of targeting them once disease is established are uncertain.
Here, using chronic allergen exposure models of AAD, to mimic the frequent, low dose, allergen exposures allergic asthmatics experience, Mice were culled at the end of week 5. All animals were harvested 18 hours after the final allergen dose.

| Flow cytometry assessment
Cell suspensions were acquired as previously described 18

| Assessment of lung function
Airway hyper-responsiveness was measured in anesthetized and tracheotomized mice in response to increasing doses of methacholine (3-100 mg/mL; Sigma-Aldrich, MO, USA) using the flexiVent system (Scireq, Montreal, Canada) as previously described. 19

| Antibody assessment
Allergen-specific IgE and IgG1 levels were measured by coating plates with 50 μg/mL HDM then adding serially diluted serum and biotinylated IgG1 or IgE antibodies (BD Pharmingen ™ , Oxford, UK). Endpoint titre was calculated using baseline+2xSD based on naïve animals.

| Repeated aero-allergen exposure generates lung-and lymphoid-resident T FH
To replicate the repeated low dose aeroallergen exposure experienced by allergic asthmatics, mice were exposed to two common aeroallergens; HDM or ALT three times a week for up to 5 weeks ( Figure 1A).
T FH , defined as CXCR5 + PD1 + Foxp3 − CD4 + ( Figure S1) were observed in the lung-draining mediastinal lymph nodes (mLN) of allergen-exposed animals after 1 week but were not found in the mLNs of PBS-treated controls ( Figure 1B). Continued allergen exposure further increased T FH proportions in the mLN after 3 and 5 weeks ( Figure 1B). T FH were also observed in the spleen but not in the circulation of allergen-treated animals ( Figure S2).
Interestingly, T FH were identified in the lung tissue itself after 3 weeks of HDM inhalation and remained elevated at 5 weeks of exposure ( Figure 1C). Lung T FH frequencies were also significantly increased following 3 weeks of ALT treatment and further increased after 5 weeks ( Figure 1C). Therefore, prolonged allergen exposure induced both local and systemic T FH responses that increased in frequency over time.
F I G U R E 1 T follicular helper cells (T FH ) accumulate over time in the mediastinal lymph nodes and lung tissue. Adult female BALB/c mice were exposed to either 25 μg house dust mite (HDM), 10 μg Alternaria alternata (ALT) or 25 μL phosphate-buffered saline (PBS), three times a week for up to 5 weeks. Flow cytometry was used to determine the frequency of T FH within cellular compartments. Representative flow plots of T FH in PBS, ALT or HDM-treated animals are displayed, pregated on CD4 + CD3 + Foxp3 − CD44 hi CD62L − lymphocytes. Data are quantified. T FH were defined as CXCR5 + PD1 + Foxp3 − CD4 + lymphocytes. A, Experimental set-up. B, mediastinal lymph nodes (mLN), C, lung tissue. Statistical significance was determined using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, n = 5 per time-point. Representative data from two independent experiments UWADIAE ET AL.

| T FH precede the development of humoral immunity during AAD
T FH direct B-cell responses, driving GC formation, isotype switching, affinity maturation and B-cell differentiation. 2

GC B cells defined as
CD38 − GL7 + FAS + IgD − IgM − B220 + CD19 + lymphocytes ( Figure S3) were absent and comparable to PBS controls in the mLN and lungs after 1 week of aero-allergen inhalation (Figure 2A and B). However, after 3 weeks of HDM exposure, GC B-cell frequencies were significantly elevated in the mLN, lungs and spleen, remaining consistently raised between weeks 3 and 5. (Figure 2A, Figure S4). Similar results were observed following ALT exposure ( Figure S4).
Allergen-specific antibodies, especially IgE, are a key feature of AAD. Allergen-specific IgE and IgG1 were detectable in allergenexposed mice from 3 weeks onward ( Figure 2C and D). Sustained exposure to HDM did not further alter the levels of allergen-specific IgE ( Figure 2C), but did increase IgG1 ( Figure 2D). These data show chronic allergen exposure to generate local and systemic GC B-cell responses, alongside allergen-specific antibody which increase over time and are preceded by T FH responses.

| α-ICOS-L treatment dampens lung inflammation and reduces AHR
Airway hyper-responsiveness and inflammation are key indicators of AAD progression. α-ICOS-L administration successfully reduced aeroallergen induced cellular infiltration into the lungs compared to IgGtreated controls ( Figure 4A). The total number of lung eosinophils was reduced with allergen and α-ICOS-L co-administration ( Figure 4B); however, the proportion of lung eosinophils were unchanged ( Figure 4C). A similar trend was observed during ALT-driven AAD ( Figure S8).
Allergen-treated mice showed increased AHR, with raised airway resistance and elastance, and reduced compliance compared to PBS controls in response to increasing doses of methacholine ( Figure 4D). Treatment of HDM-exposed mice with α-ICOS-L resulted in reduced airway resistance and elastance and increased compliance, indicative of improved lung function compared to HDM-treated control mice ( Figure 4D). This was also observed for ALT-induced AAD ( Figure S6). Despite this α-ICOS-L administration had no impact on HDM-induced goblet cell hyperplasia, collagen deposition or airway smooth muscle hyperplasia and hypertrophy ( Figure S9A-I). Taken together the data shows that therapeutic administration of α-ICOS-L after disease establishment improved airway inflammation and lung function.

| α-ICOS-L reduces overall inflammation without specifically targeting Th2 cells or ILC2s
To determine the mechanism by which α-ICOS-L treatment could be impacting pulmonary inflammation and AHR, the release of concentrations compared to IgG aeroallergen exposed mice (Figure 5A and B). IL-5 was reduced but did not reach significance, while no change was observed for Eotaxin-2 ( Figure 5D and E).
As IL-13 release was reduced by ICOS-L blockade and IL-13 is central to AAD pathogenesis the cellular sources of IL-

ICOS-L intervention (
Although ILC2s are the major ILC subset induced during HDMdriven chronic AAD, 22 Figure 6D). Lung Tregs, however, were reduced both in proportion and total number ( Figure 6D). Similar results were observed in ALT-driven AAD ( Figure S12). This suggests α-ICOS-L may limit lung Foxp3 + CD4 + T-cell responses but despite this, the treatment still dampens hallmark features of chronic AAD.
T FH have been shown to accumulate and become dysregulated during sustained antigen exposure, 24 and to be capable of differentiating into Th2 cells after adoptive transfer. 13 Here, T FH were reduced together with both secreted IL-13 and IL-13 + CD4 + T cells, therefore the capacity of T FH to produce IL-13 was examined. Using IL-13 GFP reporter mice, CXCR5 expression was found to be separated from IL-13 GFP expression in CD4 + T cells within the mLN and lungs after HDM treatment ( Figure 6E and F). IL-13 GFP+ cells were, however, readily identified within the non-T FH T effector (T EFF ) population ( Figure 6E and F). This suggests that T FH are not directly responsible for the increased IL-13 observed during aeroallergen-driven AAD or the reduction in IL-13 observed after α-ICOS-L intervention. Overall α-ICOS-L treatment was beneficial as a therapeutic during established AAD, targeting both pathogenic humoral immunity intervention and other key features of AAD.

| DISCUSSION
Allergic airway disease is characterized by Th2-biased lung inflammation and dysregulated humoral immunity in response to recurrent exposure to environmental aero-allergens. 1 Here, we examine the IgG remains stable. 34 Consequently, a more prolonged α−ICOS-L protocol may be required to sufficiently alter IgG1. 21 Nonetheless serum MCPT1, an important indicator of mast cell activation, 35 also decreases suggesting the observed IgE reduction to adequately F I G U R E 6 IL-13 + CD4 + T cells and ILCs are not directly targeted by ICOS-L blockade. A-B, Adult female BALB/c mice were exposed to either 25 μg house dust mite (HDM) or 25 μL phosphate-buffered saline (PBS), three times a week for up to 5 weeks. From the start of week 4, mice were also administered 150 μg anti-ICOS-L (α-ICOS-L) or isotype control (IgG) antibody (i.p) three times a week. Mice were culled at the end of week 5. Flow cytometry was used to determine the frequency of lung cellular populations. A, Representative gating of IL-13 + CD4 + T cells and IL-13 + ILC2s following HDM and IgG or α-ICOS-L treatment. Data are quantified for all groups. B, Proportions of lung IL-13 + T cells and ILCs. C, Numbers of lung IL-13 + T cells and ILCs. Data are pooled from two independent experiments, n = 8 for PBS-treated groups, n = 12 for HDM-treated groups. D, Representative flow cytometry of Foxp3 + CD4 + cells in allergen-treated mice given IgG or α-ICOS-L. Pregated on CD4 + CD3 + lymphocytes. The number of Foxp3 + CD4 + T cells is quantified for all groups, E-F, Adult female IL-13 GFP reporter mice were exposed (i.n) to 25 μg HDM or 25 μL PBS, three times a week for 3 weeks. Flow cytometry was used to determine the frequency of IL-13 GFP cells. E, Representative flow plots of CXCR5 + T cells and IL-13-GFP + cells in PBS and HDM-treated animals, pre-gated on CD4 + CD3 + CD44 hi CD62L − lymphocytes. F, Quantification of mLN and lung IL-13 GFP+ T FH (CXCR5 + PD1 + CD4 + CD44 hi CD62L − ) and T EFF cells (CXCR5 − CD4 + CD44 hi CD62L − ). Statistical significance was determined using a Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001. Data are pooled from two independent experiments, n = 6 for PBS-treated groups, n = 12 for HDM-treated groups affect the inflammatory response. As T FH are required for antibody generation including allergen-specific IgE, 14,15 and we observe reduced allergen-specific IgE even after a short intervention, this highlights the potential of transiently targeting T FH to abrogate IgE-mediated clinical features not only in AAD but also in other IgE-mediated diseases.
Along with its' effects on antibody-mediated immunity target- Previous work blocking ICOS or ICOS-L has been done prophylactically, during disease inception [40][41][42] or prior to an exacerbation 40 using less clinically relevant ovalbumin-induced AAD models, [40][41][42] which are difficult to deliver clinically. Here, using a more realistic ongoing exposure model and clinically relevant aeroallergens, we show α-ICOS-L to reduce disease at the height of pathology. Additionally, we show for the first time in AAD that the prevailing outcome of therapeutic ICOS blockade is not only depletion of T FH and their associated GCs, but also improvements in multiple disease facets. This approach could, therefore, be complimentary or advantageous to currently approved biological therapies, such as omalizumab (anti-IgE mAb), reslizumab (anti-IL-5 mAb) and mepolizumab (IL-5 antagonist), which generally favour one arm of the allergic response and thus are only effective in specific asthma endotypes. 43

CONFLI CTS OF INTEREST
The authors declare that they have no conflicts of interest.