Modulation of CRTh2 expression on allergen‐specific T cells following peptide immunotherapy

Abstract Background Allergen immunotherapy using synthetic peptide T‐cell epitopes (Cat‐PAD) from the major cat allergen Fel d 1 has been shown, in allergen exposure studies, to significantly reduce symptoms of allergic rhinoconjunctivitis in cat‐allergic subjects. However, the immunological mechanisms underlying clinical benefit remain only partially understood. Since previous studies of whole allergen immunotherapy demonstrated a reduction in the frequency of allergen‐specific (MHC II tetramer+) CD4+ T cells expressing the chemokine receptor CRTh2, we assessed the impact of Cat‐PAD on the frequency and functional phenotype of Fel d 1‐specific CD4+ T cells. Methods Using before and after treatment samples from subjects enrolled in a randomized, double‐blind, placebo‐controlled trial of Cat‐PAD, we employed Fel d 1 MHC II tetramers and flow cytometry to analyze the expression of chemokine receptors CCR3, CCR4, CCR5, CXCR3, and CRTh2, together with markers of memory phenotype (CD27 and CCR7) on Fel d 1‐specific CD4+ T cells. Results No statistically significant change in the frequency of Fel d 1‐specific CD4+ T cells, nor in their expression of chemokine receptors or memory phenotype, was observed. However, a significant reduction in cell surface expression of CRTh2 was observed between the placebo and active groups (P = 0.047). Conclusions Peptide immunotherapy with Cat‐PAD does not significantly alter the frequency or phenotype of Fel d 1‐CD4+ T cells, but may decrease their expression of CRTh2.

Allergen-specific immunotherapy (SIT) is the only therapy that corrects the dysregulated immune response that characterizes allergic inflammation. Traditionally, SIT involves administering the intact allergen for a period lasting from a few months to a few years. SIT has been shown to ameliorate allergic symptoms with relief lasting longer than the treatment period. 1,2 While the ability of SIT to ameliorate allergic symptoms is well documented, so too are the adverse events that can arise from administering intact proteinaceous allergens to allergic subjects. 3,4 The adverse events can be life-threatening and stem from the ability of allergens to cross-link allergen-specific IgE molecules on the surface of effector cells (such as basophils and mast cells). An alternative to SIT is the use of peptides containing CD4 + T-cell epitopes derived from clinically important allergens. 3 Peptide immunotherapy performed with short peptides is associated with fewer IgE-mediated adverse events than observed in conventional SIT, since short peptides have a markedly reduced ability to cross-link IgE on basophils (and by inference mast cells). 5 Peptide immunotherapy has been shown to effectively ameliorate symptoms of allergic disease in both mice and humans. 2,[5][6][7][8][9] Targeting allergen-specific Th2 CD4 + T cells is a rational immunotherapeutic strategy as allergen-specific Th2 cells play an important role in the development, maintenance, and exacerbation of allergic airway disease. Mouse models have demonstrated that Th2 cells are important for the class switching of allergenspecific B cells to IgE 10 and that Th2 cells may also facilitate the maintenance of plasma cells. 11 In addition, the Th2 cytokines IL-4, IL-5, and IL-13 have been implicated in goblet cell hyperplasia and mucus production, induction of airway hyperresponsiveness (AHR) and the recruitment of eosinophils to the airways. 10 Mouse models involving the adoptive transfer of Th2 cells, [12][13][14] or the acute depletion of CD4 + T cells prior to challenge, 15,16 have demonstrated that CD4 + T cells play a significant role in inducing allergic airway disease.
Sensitization to cats is one of the most common types of allergic sensitization and is strongly associated with the development of asthma. 17,18 In addition, children allergic to cats, as compared to other allergens, are more likely to develop severe asthma. 19 The principal cat allergen is Fel d 1 and peptides from Fel d 1 containing T-cell epitopes (Cat-PAD) capable of binding to commonly expressed class II HLA molecules have been identified and used to treat cat-allergic subjects. 5 The mechanisms responsible for improvements in clinical symptoms in cat-allergic subjects remain incompletely understood.
Previous studies of subcutaneous immunotherapy (SCIT), employing whole allergen extracts have described a shift from a Th2 signature to a Th1 signature following treatment, 20 while others have described the generation of putative regulatory T-cell populations. 21,22 Recently, deletion of allergen-specific T cells, and in particular terminally differentiated CD27 − Th2 cells, has been described. 7,23,24 Conceptually, altering the chemokine receptor profile of allergen-specific T cells could prevent the recruitment of these cells to sites of allergic inflammation. Chemokine receptors play a critical role in the trafficking of T cells and other leukocytes. 21

G R A P H I C A L A B S T R A C T
Peptide immunotherapy with Cat-PAD reduces the surface expression of Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) on Fel d 1-specific T cells. Neither the frequency, nor memory phenotype of Fel d 1-specific T cells in the blood were affected by peptide immunotherapy for cat allergy. Reduced expression of CRTh2 may modulate recruitment to sites of allergen exposure. and an intermediate population termed transitional memory T cells (T TM ). T CM cells express CCR7 while T TM and T EM cells do not. 22 It has also been reported that memory T cells can be divided by their expression of CD27. CD45RA − CD27 + cells behave as T CM (or T TM ) cells in that they do not readily proliferate or secrete cytokine upon stimulation. 25 In contrast, CD45RA − CD27 − cells do proliferate and secrete cytokines in response to antigen, which is characteristic of T EM cells. 25 The role of memory T cells in allergy has been the subject of a number of excellent reviews. 26,27 In the present mechanistic study, we sought to advance our understanding of the mechanism(s) governing the clinical impact of immunotherapy with Cat-PAD by measuring the frequency of Fel d 1-specific CD4 + T cells, their memory status, and their expression of Th1, and Th2-associated chemokine receptors. The hypothesis for this study was that peptide treatment would alter the chemokine receptor profile of Fel d 1-specific CD4 T cells. We isolated peripheral CD4 + T cells from cat-allergic, asthmatic subjects, before and after treatment with Cat-PAD and focused our analysis on CD4 + T cells using Fel d 1 MHC class II tetramers. Our analysis provides new insight into the mechanisms of action of peptide immunotherapy and highlights key differences with respect to whole allergen immunotherapy approaches.

| Ethics
The study was conducted in accordance with the Declaration of

| Subjects
The clinical trial providing samples for this study were registered at ClinicalTrials.gov (identifier: NCT00867906). The clinical trial received approval from Institutional Review Board Services (Aurora, Ontario, Canada). All subjects provided written, informed consent to participate in the clinical trial and to provide blood samples for mechanistic studies. The subjects were male or female, aged 18-65, with a history of controlled allergic asthma as defined in GINA (2007) on exposure to cats for at least 1 year. The subjects had a reliable history of rhinoconjunctivitis on exposure to cats for at least 1 year. In addition, the recruited subjects displayed a positive skin prick test to cat allergen with a wheal diameter at least 3 mm larger than the negative control, and a late-phase skin reaction of >25 mm in diameter to cat allergen, eight hours after intradermal injection.
The subjects were randomly assigned to the treatment and placebo groups. The primary objective of the clinical study from which the samples were obtained was to evaluate the safety and tolerability of multiple intradermal injections of Cat-PAD in cat-allergic subjects with controlled asthma. Secondary clinical surrogate outcomes included changes in the magnitude of the early-phase and late-phase skin response to intradermal allergen challenge, and change in tolerated allergen dose in a conjunctival provocation.
In total, 80 subjects were screened between March 2009 and October 2009; of these, 28 failed screening. The remaining 52 subjects were randomized with 45 subjects completing the clinical component of the study. Seven subjects withdrew from the study (4 withdrew consent; 2 withdrew for use of a prohibited medication, and 1 was lost to follow-up). For the mechanistic studies described herein, only 13 placebo-treated subjects and 12 Cat-PAD-treated subjects were analyzed as MHC class II tetramers were not available for the other trial participant samples.

| Tetramers
Recombinant HLA-DR proteins were generated as previously described. 28 Briefly, each HLA-DR was purified from the superna- The appropriate tetramer(s) were selected based on the subjects' expression of these HLA-DRB alleles.

| Isolation of Fel d 1-specific CD4 + T cells
Blood was taken from subjects at baseline (week 0) and 10-14 weeks after the final injection (week 24-28). PBMCs were isolated using a density gradient centrifugation and cryopreserved with RPMI media supplemented with 10% DMSO, 20% fetal bovine serum. Cryopreserved PBMCs were thawed (>90% viability) and CD4 + T cells were purified using magnetic selection with a negative selection cocktail according to the manufacturer's instructions (StemCell Technologies). Purified CD4 + T cells were incubated with 50 nmol/L dasatinib (to prevent T-cell receptor downregulation; Cedarlane) at 150 × 10 6 /mL in RPMI media supplemented with 10% fetal calf serum, 100 U/mL penicillin (Invitrogen), and 100 μg/mL streptomycin (Invitrogen) for 20 minutes at 37°C and 5% CO 2 . Fifteen µg of the appropriate phycoerythrin (PE)-labeled tetramer was added and incubated for another 2 hours with gentle mixing after 1 hour. The tetramer positive cells were then isolated using anti-PE magnetic beads according to manufacturer's instructions (StemCell Technologies). Calculation of the frequencies of tetramer + CD4 + T cells used the equation:

| Statistical analysis
Statistical analysis was performed using SPSS and GraphPad Prism.
A paired t test was used to compare pre-and posttreatment in the placebo and active groups. A repeated measures ANOVA was used to compare within-subject factors (ie, pretreatment vs posttreatment) and between-subject factors). All data sets were complete and did not contain missing values.

| Treatment with Cat-PAD does not affect the frequency of Fel d 1-specific CD4 + T cells
First, we assessed the frequency of all Fel d 1-specific CD4 + T-cells pre-and posttherapy, regardless of phenotype. Subsequently, we specifically examined changes in subsets of allergen-specific CD4+ T cells (CD27 − and CRTh2 + ) that have been shown to be absent following whole allergen immunotherapy. In contrast to previous studies of SIT, we did not observe a significant change in the frequency of tetramer + CD4 + T cells ( Figure 1A), percentage of tetramer + CD27 − (Figure 1B), or percentage of tetramer + CRTh2 + CD4 + T cells ( Figure 1C) following treatment, suggesting that the mechanisms of action of Cat-PAD do not involve the deletion of allergen-specific CD4 + T cells.

| Peptide immunotherapy does not alter the percentage of allergen-specific T cells expressing individual chemokines receptors
Next, we investigated the effect of treatment upon the percentage of tetramer + CD4 + T cells expressing individual Th1-and Th2-associated chemokine receptors: Th1 (CCR5, CXCR3), Th2 (CCR3, CCR4, CRTh2). We found no change in the percentage of tetramer + cells expressing any individual chemokine receptor (Figure 3). Similarly, no changes were observed following analysis of multiple chemokine receptors (data not shown). We conclude that peptide immunotherapy with Cat-PAD is not associated with a change in the frequency of allergen-specific T cells expressing any of the chemokine receptors analyzed in this study ( Figure 3).

| Treatment with Fel d 1 synthetic peptides does not alter the proportions of allergen-specific T Th1 and Th2 chemokine receptor phenotypes
Specific immunotherapy has been shown to shift the phenotype of the allergic response from Th2 to Th1. 20,24,32 It is generally accepted that Th1 and Th2 cells differ in their expression of chemokine receptors. Th2 cells predominantly express CCR3, CCR4, and CRTh2, while Th1 cells predominantly express CXCR3 and CCR5. We employed representatives of these surrogate markers to assess whether treatment with Cat-PAD affected the Th1/Th2 nature of the T-cell response Fel d 1 by comparing the ratio of tetramer + T cells expressing CXCR3 to those expressing CRTh2. As shown in Figure 4, peptide immunotherapy did not affect the ratio of tetramer + CXCR3 + to tetramer + CRTh2 + and therefore likely does not affect the overall Th1: Th2 phenotype of the response.

| Peptide immunotherapy modulates levels of surface expression of chemokine receptors on allergen-specific T cells
In addition to measuring the frequency of allergen-specific CD4 + T cells expressing individual chemokine receptors, we also quanti- which may indicate a suboptimal study design.
We focused on allergen-specific CD4 + T cells in the peripheral blood, and asked whether the frequency of these cells, their patterns of chemokine receptor expression, or memory T cell markers were changed. We isolated peripheral CD4 + T cells from cat-allergic subjects, before and after treatment with Cat-PAD. We were able to analyze Fel d 1-specific CD4 + T cells ex vivo with a panel of 14 MHC class II tetramers that target T cells restricted by 8 common F I G U R E 3 Chemokine receptor expression by allergen-specific CD4 + T cells. Panels show the % of CD4 + tetramer + T cells staining positive for chemokine receptors CCR3, CCR4, CCR5, CXCR3, and CRTh2, before and after treatment with Cat-PAD or placebo. Individual paired data from n = 12 (placebo) and n = 13 (active). Differences over time were analyzed using the paired t test. The difference between the placebo and active group was assessed by RM-ANOVA The effect of peptide immunotherapy on the ratio of CRTh2 + tetramer + T cells to CXCR3 + tetramer + T cells. Modulation, by peptide immunotherapy, of the ratio of allergen-specific (tetramer + ) Th2:Th1 T cells was modeled employing CRTh2 and CXCR3 as representative Th2 and Th1 markers, respectively. Individual paired data from n = 12 (placebo) and n = 13 (active). Differences over time were analyzed using the paired t test. The difference between the placebo and active group was assessed by RM-ANOVA HLA-DR molecules. 5,29 The ability to analyze T cells ex vivo is a sig- F I G U R E 5 Density of surface expression of chemokine receptor by allergen-specific T cells. Panels show the median fluorescence intensity (MFI) of tetramer + CD4 + T cells staining positive for chemokine receptors CCR3, CCR4, CCR5, CXCR3, and CRTh2, before and after treatment with Cat-PAD or placebo. Individual paired data from n = 12 (placebo) and n = 13 (active). Differences over time were analyzed using the paired t test. The difference between the placebo and active group was assessed by RM-ANOVA A potential weakness of this and other studies of antigen-specific CD4 + T-cell frequencies is that, as a result of the low frequency of tetramer+ T cells in peripheral blood and the wide variability in the range of baseline frequencies observed, few if any studies are adequately statistically powered to detect subtle changes that may, nonetheless, be biologically important. Flow cytometric analysis of tetramer+ T cells in this and other studies is based on capture of only a small number of events. As a result, we can only conclude with confidence that substantial changes in frequency did not occur following peptide immunotherapy. A further weakness of the study is the use of peripheral blood which may not be representative of treatment-induced changes in other compartments such as the lymphatics and the target organs.
Historically, whole allergen immunotherapy (eg, SCIT) has been associated with a shift in the response to allergen from Th2 to Th1, a phenomenon referred to as "immune deviation". 34 we did not see any evidence of a Th2 to Th1 shift in the allergen-specific CD4 + T-cell response in the peripheral blood. We conclude that immune deviation, at least at the level of peripheral blood responses to allergen, is unlikely to be a major mechanism of action of peptide immunotherapy.
There was a significant difference in the decrease in surface levels of CRTh2 on Fel d 1-specific CD4 + T cells when comparing subjects treated with peptide immunotherapy vs placebo. CRTh2 is the receptor for prostaglandin D 2 (PGD 2 ), which is produced by mast cells upon crosslinking of IgE on their surface, and is expressed primarily by Th2 cells. 40 cells. 23 Although we did not find a reduction in the percentage of Fel d 1-specific CD4 + T cells expressing CRTh2, the decrease in its level of expression is in line with the idea that SIT can affect the expression of CRTh2 by allergen-specific CD4 + T cells. However, further exploration with adequately powered studies will be required to elucidate whether PIT significantly alters the expression of CRTh2 on Fel d 1-specific CD4 + T cells and what the exact functional consequence this decrease might be.
In conclusion, peptide immunotherapy was not associated with substantial deletion of allergen-specific CD4 + T cells, including the CD27 subpopulation that has recently been implicated in the pathogenesis of allergic disease. A specific reduction in surface levels of the PGD 2 receptor CRTh2 was observed. We hypothesize that downregulation of CRTh2 might render allergen-specific CD4 + T cells relatively unresponsive to PGD 2 gradients emanating from mast cells activated at the site of allergen exposure. This could result in a failure to recruit and activate these cells, thereby reducing Th2 inflammatory responses in the airways.

ACK N OWLED G M ENTS
The authors are grateful to Lesley Wiltshire and Cheryl Kipling for expert technical assistance in the preparation and cryopreservation of blood cells. The authors acknowledge the generous support of Circassia Pharmaceuticals PLC in the provision of whole blood samples from clinical trial CP003.

CO N FLI C T S O F I NTE R E S T
ML is a co-Founder of Circassia Ltd, the company that sponsored the clinical trial that generated the blood samples employed in this study. ML is a consultant to Circassia Ltd and an associated