Intralymphatic immunotherapy with one or two allergens renders similar clinical response in patients with allergic rhinitis due to birch and grass pollen

Abstract Introduction There is a need for a fast, efficient and safe way to induce tolerance in patients with severe allergic rhinitis. Intralymphatic immune therapy has been shown to be effective. Methods Patients with severe birch and timothy allergy were randomized and received three doses of 0.1 ml of birch and 5‐grass allergen extracts (10,000 SQ units/ml, ALK‐Abelló), or birch and placebo or 5‐grass and placebo by ultrasound‐guided injections into inguinal lymph nodes at monthly intervals. Rhinoconjunctivitis total symptom score, medication score and rhinoconjunctivitis quality of life questionnaire were evaluated before treatment and after each birch and grass pollen season during three subsequent years. Circulating proportions of T helper subsets and allergen‐induced cytokine and chemokine production were analysed by flow cytometry and Luminex. Results The three groups reported fewer symptoms, lower use of medication and improved quality of life during the birch and grass pollen seasons each year after treatment at an almost similar rate independently of treatment with one or two allergens. Mild local pain was the most common adverse event. IgE levels to birch decreased, whereas birch‐induced IL‐10 secretion increased in all three groups. IgG4 levels to birch and timothy and skin prick test reactivity remained mainly unchanged. Conjunctival challenge tests with timothy extract showed a higher threshold for allergen. In all three groups, regulatory T cell frequencies were increased 3 years after treatment. Conclusions Intralymphatic immunotherapy with one or two allergens in patients with grass and birch pollen allergy was safe, effective and may be associated with bystander immune modulatory responses. Clinical Trial Registration: EudraCT (2013‐004726‐28).


| INTRODUC TI ON
Nearly 30% of the adult population of Sweden report allergic rhinitis. 1 In addition, the prevalence of allergic sensitization, as determined as the presence of circulating IgE antibodies to birch, timothy, mugwort, cat, dog, horse, Dermatophagoides pteronyssinus, Dermatophagoides farinae and Alternaria, is up to 45% in Sweden and in many other European countries. 2,3 The total cost of allergic rhinitis in Sweden, with a population of 9.5 million (in 2014), has been estimated at €1.3 billion annually. 4 Treating these patients with allergen immunotherapy (AIT) is cost-effective. 5 To date, AIT is the only treatment that affects the long-term development of allergic rhinoconjunctivitis. It induces clinical tolerance primarily by stimulating regulatory T (Treg) cells, attenuating T helper 2 (Th2) responses and inducing blocking antibodies. 6 Conventional AIT with subcutaneous injections is effective but consumes time and resources as it lasts more than 3 years and requires about 40 injections to complete. 7,8 Another method is sublingual immunotherapy (SLIT), where tablets containing allergens are given daily for 3 years 9 Allergen immunotherapy for allergic rhinoconjunctivitis improves symptom, medication and combined symptom and medication scores (MS) in patients with allergic rhinoconjunctivitis. 10 A randomized placebo-controlled trial with subcutaneous immunotherapy (SCIT) with birch pollen allergen reduced the symptom score by 40% over placebo. 11 In a review article of SCIT and SLIT trials, SCIT was reported to reduce nasal and ocular symptoms by 32-36% compared with placebo, whereas SLIT produced a reduction in 26-36% compared with placebo. 12 Furthermore, both clinical and immunological bystander effects of specific AIT on other allergens have been reported in animal studies and one case report. [13][14][15] In an open intralymphatic immunotherapy (ILIT) study, 16  relief of symptoms were observed after ILIT than after SCIT. The study was followed by a few smaller studies of which five [17][18][19][20][21] pointed in the same direction as the original trial, whereas one showed no benefit of ILIT. 22 These studies were performed with timothy or birch pollen or both. Small ILIT studies with cat, house dust mite and dog have also shown positive clinical results. 23

| Aim
The objective was to evaluate safety and efficacy after ILIT with one or two allergens: birch-or grass pollen or both. We also aimed to determine its immune modulatory effects including changes in spontaneous and allergen-induced cytokine and chemokine production, and proportions of circulating T helper cell subsets.

| Study population eligibility criteria
In all, 126 patients were assessed for eligibility. All patients signed written informed consent to participate in the study. Forty-four did not meet the inclusion criteria, 7 withdrew consent before treatment and 1 was excluded for unknown reasons ( Figure 1). Informed consent x  The diary had space for description of the adverse events (AEs) since the last visit. f Two to five days after visit 3-5, telephone contact was made considering symptoms after the allergen injections. 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%.

| Intralymphatic immunotherapy
The patients were randomized into three groups receiving three

| Primary outcome measures
Symptoms and drug consumption were primary outcome measures. Symptoms were validated based on the rhinoconjunctivitis total symptom score (RTSS) questionnaire. 26 Drug consumption was measured using an MS questionnaire (see File S1). Medication was not provided by our study, but participants bought it themselves over the counter or prescribed as in normal health care. 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 3 and 4 year's altogether, no daily symptom score was recorded. The birch-and grass peak pollen seasons are separate in Sweden.

| Safety assessment
Safety was assessed as the recording of adverse events from the time of the first injection to 3 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) 27 was recorded by the patients at the end of the birch pollen seasons (approximately June 1st) and at the end of the grass pollen seasons (approximately September 1st) thus mirroring the last season rather than the last week. Skin prick test reactions (Soluprick SQ Birch and Timothy, ALK-Abelló), allergen-specific IgE and allergen-specific IgG4 levels were analysed (ImmunoCAP ThermoFisher) before ILIT and in the fall the following 3 years. Conjunctival allergen provocation tests (CAPT) 28 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).

| Symptoms and medication
The symptoms measured by the RTSS and MS were significantly

| Adverse events
A total of 438 injections were given, of which 285 had an active substance containing allergen extract. Mild local pain at the injection site was the most common adverse event (AE) ( Table 3). However, on three occasions, patients recorded severe pain from ILIT. One patient had moderate breathing problems without any fall in peak flow 30 min after the second ILIT (birch and 5-grass) and received the third treatment without any AEs. One patient experienced breathing problem 2 h after physical activity, 4 days after the first injections and was relieved with salbutamol inhalations, antihistamine and oral corticosteroids. The remaining injections followed without breathing problems. During the follow-up, 3 years after ILIT, nine patients reported severe AEs, diverticulitis, miscarriage, burn injury, disc hernia, abdominal pain, hysterectomy, concussion, chest pain and heart failure, but none was judged to be related to ILIT. One patient showed hypothyroidism with elevated levels of antithyroid peroxidase, and thyroid-stimulating hormone receptor antibodies diagnosed in 2018, possibly related to the therapy given in 2014. No anaphylactic reaction was observed or reported.

| Health-related quality of life
The impact on health-related quality of life as measured by RQLQ was significantly reduced during the birch and grass pollen seasons, regardless of active ILIT with birch, 5-grass or both allergens. When combining all three groups, the RQLQ score was significantly reduced from 3.15 to 1.50 (−52%) and from 2.82 to 1.25 (−56%) 3 years after treatment during the birch and grass pollen seasons, respectively (p < .01, Figure 2, and File S4). similar, but non-significant differences were determined after ILIT with 5-grass and placebo. IgE levels to both birch and timothy decreased slightly (p < .01 and <.05) after ILIT with both birch and 5grass. There was no significant decrease in the levels of total IgE (see

| Circulating T helper cell subsets
Flow cytometry data revealed that the proportion of Th1 cells, defined as CD3 + CD4 + CD45RA − Tbet + cells, decreased between baseline and 3 years after treatment in the groups receiving ILIT with birch and placebo and 5-grass and placebo (p < .05, Figure 3A,B). A significant increase was observed after 1 year in the group receiving birch and 5-grass treatment and it seemed to decrease, | 753 AHLBECK Et AL.
but not significantly so, to 3 years (p < .05, Figure 3C). The proportion of Th2 cells, defined as CD3 + CD4 + CD45RA − GATA3 + cells, increased 3 years after treatment in the group receiving birch and placebo ILIT ( Figure 3D, p < .01) and in the group receiving both treatments ( Figure 3F, p < .05). This change was not significant in the group that received 5-grass and placebo ILIT ( Figure 3E

| Allergen-induced cytokine and chemokine production
At baseline and 1 year post ILIT, peripheral blood mononuclear cells were harvested and stimulated with birch and grass allergen in vitro to measure cytokine and chemokine secretion (see Files S6 and S7).
Both birch and grass allergens induced an increase in IL-5 production after birch and placebo ILIT (p < .05, Figure 5A,B), while IL-5 secretion did not change in the other treatment groups. Increased birch allergen-induced IL-10 secretion was also observed after birch and placebo ILIT and 5-grass and placebo ILIT, whereas no significant change occurred in the birch and 5-grass group ( Figure 5C). ILIT did not affect grass-allergen IL-10 production ( Figure 5D). The spontaneous production of the CCL17 chemokine decreased after 5-grass and placebo ILIT and birch and 5-grass ILIT but not after birch and placebo ILIT (p < .05 Figure 5E).

| DISCUSS ION
The current study is to our knowledge the largest double-blind randomized clinical ILIT trial to date. Patients with hayfever due to sensitization to both grass and birch pollen allergens bystander immunological effects of SCIT in animal studies and one case report [13][14][15] As clinical improvement was sustained throughout our study, we suggest the results are not only due to the placebo used in the single treatment groups, although placebo effects may be strong in AIT-clinical trials. 30 The positive clinical effect did not seem to be mediated by allergen-specific IgG4, as these antibody levels were not clearly elevated between pre-ILIT baseline and any of the timepoints thereafter. AIT efficacy has, however, often been reported to correlate with increased IgG4 in SCIT and SLIT. 6 Moreover, in previous grass-or birch-pollen ILIT studies, only moderate or no changes in IgG4 were determined, [18][19][20][21][22] whereas a significant increase in allergen-specific IgG4 was determined after ILIT with cat dander allergen. 31 However, different results regarding IgG4 levels in SCIT, SLIT and ILIT may depend on the route of administration of the allergen.  AEs were very common (>1/10) but most were judged as mild or moderate, generally at the allergen injection site ( Table 3). These are also normal reactions after SCIT. 32  The proportion of Treg cells, defined as CD4 dim CD25 hi FoxP3 + cells, increased over time in all treatment groups (A-C). The same trend was observed in the activated Treg cell population, defined as CD3 + CD4 + CD45RA − FoxP3 ++ (D-F). *p < .05; **p < .01; ***p <.001 from Wilcoxon signed rank tests. The lines indicate median and interquartile range (IQR, 25th and 75th percentile values). Only patients randomized in Linköping included F I G U R E 5 Spontaneous and allergeninduced cytokine and chemokine production after intralymphatic immunotherapy with birch and/or 5-grass. (A) birch-induced IL-5 production (B) grass induced IL-5 production, (C) birch-induced IL-10 production, (D) grass induced IL-10 production and (E) spontaneous CCL17 production. *p < .05 from Wilcoxon signed rank tests. The lines indicate median and interquartile range (IQR, 25th and 75th percentile values). Only patients randomized in Linköping included AIT renders significant improvements in rhinoconjunctivitis and conjunctival sensitivity that persist at least 7 years after termination of treatment. 7 In addition, AIT can prevent development of asthma 37,38 and new sensitizations in mono-sensitized patients. 12,39 After an updosing phase of 7-15 weeks in SCIT, a maintenance phase of 3 years with injections every six to eight weeks is typically required to render long-term tolerance. For SLIT, 55-82% of patients were reported to abandon the treatment before completing the recommended course of therapy. 40 With only three injections over eight weeks, ILIT may overcome the disadvantages of long duration of treastment and poor compliance.

TA B L E 3 Cumulative data for adverse events
The results of this study add to the hitherto positive studies and suggest that ILIT may be effective and safe as treatment for pollen allergy. We will follow the patients in this study for symptoms, health-related quality of life and adverse events in an open study until 2024 (EPN 2017/302-31). ILIT may be an opportunity to make AIT more easily accessible to patients at a lower cost and less risk.
However, there is a need for further studies to establish the optimal dose for efficacy and side effects. 25

| CON CLUS IONS
Intralymphatic immunotherapy against grass and birch pollen allergy was safe and seems effective. The unspecific effect of one allergen ILIT may be associated with bystander immune modulatory responses.

ACK N OWLED G EM ENTS
We would like to thank Dr Camilla Janefjord for outstanding sup-

E TH I C S S TATEM ENT
The study was approved by the Regional Ethics Committee in Linköping (EPN Dnr 2013/487-31, amendments 2014/55-32 and 2015/296-31). Informed signed consent was obtained from the participants before inclusion.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.