The objective was to evaluate whether ILIT can be an alternative to SCIT and SLIT. We also aimed to determine its safety, efficacy and immune modulatory effects, including changes in spontaneous and allergen-induced cytokine and chemokine production, and proportions of circulating T helper cell subsets.
A 3-year double-blind randomized clinical trial in 72 patients with rhinoconjunctivitis due to sensitization with birch and grass pollen allergens. The patients were given active treatment with birch or grass in one inguinal lymph node and active treatment with the other allergen or placebo in an inguinal lymph node on the other side. The study was without a clean placebo group as the pollen seasons are clearly separated in Sweden.
Study population eligibility criteria
In all, 126 patients were assessed for eligibility. Forty-four did not meet the inclusion criteria, 7 withdrew consent before treatment and 1 was excluded for unknown reasons (Fig. 1). Fifty-seven patients with allergic rhinoconjunctivitis due to birch and timothy pollen allergens were randomized in 2014 and 17 patients 2015. The 74 patients, including 35 females, were 19–53 years old and had seasonal allergic symptoms to birch and grass (Table 1), whereof 28 were randomized and followed in the Department of Medicine, County Hospital Ryhov, Jönköping. All participants were given ILIT at Allergy Center, University Hospital, Linköping, Sweden. Their skin prick test was >3 mm and displayed IgE to birch and timothy >0.35 kU/L. Exclusion criteria were pulmonary disease, < 80 % of predicted forced expiratory volume at the end of the first second (FEV1), use of more than 800 µg inhaled budesonide (or equivalent) per day, pregnancy, severe arterial hypertension, autoimmunity, cardiovascular, hepatic, renal, upper airway or metabolic disease, mental incapacity, alcohol abuse, medication interfering with immune response or beta-blockers. From earlier studies we expected 8 out of 9 patients would improve at least 40%. With 40 active treated and 20 in the placebo group, with an alpha of 0.05 the power was calculated to 92%.
The patients were randomized into three groups receiving three doses at four-week intervals of 0.1 ml of birch pollen allergen on aluminum hydroxide (10,000 SQ-U/ml; ALK-Abelló, Hørsholm, Denmark) and/or 0.1 ml of 5-grass pollen allergen on aluminum hydroxide (10,000 SQ-U/ml; ALK-Abelló, Hørsholm, Denmark. 5-grass is a mix of equal amounts of SQ-U of Alopecurus pratensis (meadow foxtale), Dactylis glomerata (cocks’s foot), Festuca pratensis (meadow fescue), Lolium perenne (English ryegrass), and Phleum pratense (timothy). Each allergen dose was 1.000 SQ-U. A diluent from ALK was used as placebo. Thus the paricipants received two injections, one in each groin on three occasions. Patients were randomized in blocks of six, facilitated by Forum Östergötland. An unblinded nurse prepared and marked each syringe with a label providing randomization number, injection number and injection site. ILIT was administered by three clinicians (LA, PR, and UN). Ultrasound-led technique was used whereby a lymph node was punctured with a 27G (0.4 x 40 mm) needle. Histamine-1 blocker desloratadine tablet 5 mg was given 15 minutes prior to the injections.
Primary outcome measures
Symptoms and drug consumption were primary outcome measures. Symptoms were validated based on the rhinoconjunctivitis total symptom score (RTSS) questionnaire (21). Drug consumption was measured using an MS questionnaire (see Additional file 1). The RTSS and MS were recorded by the patients at the end of the birch pollen season (approximately June 1st) and at the end of the grass pollen season (approximately September 1st) before treatment and for the following three and four year’s altogether. The birch- and grass pollen seasons are quite separate in Sweden. Also, when we planned the study, no daily symptom score was recommended.
Safety was assessed as the recording of adverse events from the time of the first injection to three years after the last injection. A research nurse called the patients to assess adverse events during the first 5 days after each injection. Safety laboratory parameters were assessed at screening, after the third ILIT injections and after the first pollen season following treatment (Table 2).
Secondary outcome measures
Effects on quality of life were measured using the rhinoconjunctivitis quality of life questionnaire (RQLQ) (22) recorded as RTSS and MS, see above. Skin prick test reactions (Soluprick SQ Birch and Timothy, ALK-Abelló), allergen-specific IgE and allergen-specific IgG4 levels were analyzed (ImmunoCAP ThermoFisher, Uppsala, Sweden) before ILIT and in the fall the following three years. Conjunctival allergen provocation tests (CAPT) (23) were performed with timothy (Aquagen SQ Timothy, ALK-Abelló) before treatment and after the first pollen season after treatment. Due to lack of extract from the company planned CAPT were not performed after the third pollen season (Table 2).
Immune laboratory methods
Flow cytometry was used to analyze the CD4+ Th cell population in whole blood from the patients at randomization, and one and three years after completed ILIT. Peripheral blood mononuclear cells obtained from the patients at randomization and one year after ILIT were stimulated in vitro with birch and timothy allergen. Levels of IL-5, IL-10, IL-13, IFN-g, CCL17 and CXCL10 were quantified using Luminex. For logistic reasons immune tests were only analyzed from the 45 participants from the Allergy Center, University Hospital, Linköping, Sweden. For detailed methods, experimental protocols, and statistical analyses, see the Methods section in this article’s supplementary information.
Descriptive statistics for RQLQ, RTSS and MS are presented in medians and percentiles (p25 and p75), and in the graphs with medians and 95% confidence interval. Paired comparisons over time were calculated with Friedman’s test and adjusted with the Bonferroni correction for multiple comparisons. Descriptive statistics for IgE, IgG4, SPT and CAPT are presented in mean values and standard deviation (SD). Paired comparisons over time were calculated with repeated measures ANOVA with Bonferroni confidence interval adjustment. The answers to the 28 questions in RQLQ were explored with an item analysis, rendering a Cronbach’s Alpha at 0.933; thus the changes within the different domains of RQLQ were consistent. All analyses above were performed in SPSS version 25 (IBM Corp. Armonk, NY, USA).
All flow cytometry, cytokine and chemokine data were analyzed using GraphPad Prism, version 8.3.1 (GraphPad software, Inc., La Jolla, CA, USA), and non-parametric tests were used. Comparisons at the different time points within the treatment groups were calculated using paired Wilcoxon signed ranks test. Unpaired Mann Whitney U test was used to compare differences between the treatment groups at the different time points. The significance level was set at p < 0.05.