Enhanced benefit from house dust mite immunotherapy in subjects with low serum levels of LILRA5 or CCL25

Allergen immunotherapy (AIT) is recognized as an etiologic treatment of allergy with long-lasting effects.1 Although AIT has been associated with changes in cellular and humoral parameters (e.g. ILC2, Th2A, Treg, IgE, IgG4 and IgA), no predictive or follow-up marker of AIT efficacy has been validated as of today.2-4 The aim of the present study was to identify, using OLINK® proximity extension assay, markers predictive of AIT efficacy in the serum of house dust mite allergic subjects having completed a phase 3 clinical trial evaluating AIT with sublingual tablets.5 Briefly, a sub-cohort of 176 participants having received 300 IR tablet daily, mono or polysensitised, with or without concomitant asthma and having exhibited either high or low clinical improvement was selected. Serum levels at baseline of 543 proteins were analysed using OLINK® Target 96 panels (Supplementary Material and Methods, Table S1 and Figure S1). When considering the whole dataset and correcting for multiple comparisons, Leukocyte Immunoglobulin-Like Receptor A5 (LILRA5) and C-C motif chemokine ligand 25 (CCL25) were found to display statistically significant different levels (adjusted p < .05) in high compared to low responders. Focusing on markers individually, both were confirmed to have highly significantly (p < .001) lower levels in high compared to low responders despite an overlap between the two populations (Figures 1A and 2A). In order to confirm results obtained with OLINK®, we enquire about alternative assays allowing absolute quantification. No suitable method was found for LILRA5. However, we were able to perform confirmatory analyses for CCL25 with a specific Luminex® assay and confirmed mean serum amounts of CCL25 to be lower in high responders (p < .05) with a good correlation between the two datasets (Spearman's ρ = 0.64, p < .001) (Figure S2). We further analysed expression of LILRA5 and CCL25 taking into consideration sensitisation status (mono- or polysensitisation), asthma status and combination thereof and performed complementary receiver operating characteristic (ROC) analyses (Figures 1 and 2). We found that statistically significant differential levels in polysensitised participants with concomitant asthma was associated in ROC analyses with a large increase in area under curve from 0.66 to 0.85 for LILRA5 (Figure 1E–H) and a more limited one for CCL25 from 0.65 to 0.70 (Figure 2E–H), thus reflecting better discriminating performances of high and low responders when taking other co-morbidities into account associated with a larger effect size (Figure S3). Both LILRA5 and CCL25 have been reported to play a role in inflammation-related diseases,6-8 including allergy and asthma. Specifically, LILRA5 is expressed on various immune cells, including monocytes, macrophages, neutrophils and B-lymphocytes.6 Cross-linking of LILRA5 on peripheral blood monocytes (PBMCs) induces the production of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6, suggesting that LILRA5 plays a role in inflammation. Two soluble forms of LILRA5 have also been described.6 However, it is unclear whether they exhibit similar functions or could act as regulators of effector responses such as the inhibition of LILR/HLA-class I complexes.9 On the other hand, both in vivo murine and in vitro human studies have highlighted that CCL25 and its receptor, CCR9, are key drivers of airway inflammation. Particularly, CCR9+ cells and CCR9+CD4+ T cells were more frequent in PBMCs from patients with perennial allergic conjunctivitis than in healthy controls.10 In the context of allergic rhinitis, polysensitisation and concomitant asthma are likely to increase the inflammatory status of the patients. The trend for AIT-responsive patients to have lower levels of both LILRA5 and CCL25 markers in serum may thus reflect a less inflammatory status more favorable to an immune system reorientation. To our knowledge, this is the first report of potential predictive serum marker of AIT efficacy using large-scale multiplex analyses. Some limitations related to the number of patients analysed, notably in subgoups, should be however noted. Confirmatory prospective studies on larger cohorts should be conducted to validate such markers and confirm clinical utility for clinical practice. In the future, clusterisation of patients using combination of markers will help unravel patients' response profiles and address patients' needs with a personalized treatment regimen. V.B.-L.F. and L.M. designed the research. L.M., V.B.-L.F. and C.A. supervised the research. A.H. performed the OLINK® experiments. S.L. performed Luminex® experiments. D.Y. and V.B.-L.F. analysed the data. V.B.-L.F. and L.M. wrote the paper. C.A. reviewed the paper. This work has been financed by Stallergenes Greer. Co-authors (V.B.-L.F., L.M., S.L.) are employees at Stallergenes Greer. C.A. reports grants or contracts from Swiss National Science Foundation, EU CURE EU SynAir-G, Novartis Research Institutes, Stanford University and consulting fees from Sanofi/Regeneron, Stanford University Sean Parker Asthma Allergy Center, Novartis, GlaxoSmithKline, Bristol-Myers Squibb and SciBase. The data that support the findings of this study are available from the corresponding author upon reasonable request. 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