Relationship between pollen levels and CSMS
The patients treated in Malmö in ILIT after SCIT- 10 000 (12 active and 7 placebo) were included in a subgroup analysis regarding responsiveness to allergen exposure measured by the local pollen counts. With a generalized additive model, we attempted to analyze if the relationship between the risk for worsening of symptoms and increased pollen concentration changed during the study. Before treatment, at an increase with 10 grain/m3 the RR for aggravated CSMS was 1.017 [SE 1.010-1.024] p= 0.029 in the active group. After treatment, the relationship between increased pollen concentration and worsening of the symptoms did not reach statistical significance. However, in the placebo group the pattern was the same. (See Table E4 and Figure E4.)

Discussion

These are the first studies that investigate dose-escalation ILIT with ALK Alutard®. Doses up to 10 000 SQ-U was safe after recent SCIT, but up-dosing to 5000 SQ-U caused serious adverse reactions in previously non-AIT treated patients. Grass-specific IgG4-levels were boosted in both studies but the CSMS was improved only in ILIT after SCIT- 10 000. ILIT de novo- 3000 showed increased activation of DCs in the lymph nodes but no induction of Treg cells in blood.
The dose-escalation 1000-3000-5000 SQ-U with one-month intervals in de novo-patients clearly seems hazardous and should be avoided. No obvious technical problems at the injections can explain the result. It is possible that the allergen bolus was drained fast to the hilus of the lymph node and further to the thoracic duct and systemically through the venous system. An even slower injection might prevent this, but that would confer more discomfort for the patient. Uncontrolled asthma and previous systemic reactions are known risk factors for adverse reactions in SCIT (23, 24 ). Both patients that had anaphylactic reactions in ILIT de novo- 3000 had seasonal asthma but normal lung function test and denied perennial symptoms. The patient with the serious anaphylaxis had reported heavy breathing after the previous ILIT-injection. This was at the time considered unspecific, but in retrospect it can be understood as a risk factor. Late systemic reactions after ILIT have been described preceding anaphylactic reactions at subsequent dose-escalated injections (19 ). However, the other patient in the present study with anaphylactic reaction had not reported any previous symptoms making up-dosing contraindicated.
At the planning stage of the trials, the only previous up-dosing ILIT study had used ALK Center-AL® Phleum pratense. Doses up to 250 PNU was given without any severe adverse events (18 ). We estimated that 5000-10 000 SQ-U ALK Alutard® could be in the same range, after comparing the conventional SCIT up-dosing protocols. However, it is hard to translate allergen doses between different extracts, which this study confirms. The intralymphatic dose escalation after SCIT could probably be carried out since the patients had a remaining tolerance to the allergen, even 20 months after the last SCIT-injection.
In ILIT after SCIT- 10 000, the median CSMS was 31% lower in the active group at the pollen season after treatment compared to the season before. The placebo group did not improve. In general, it is recommended to use between groups comparisons of active versus placebo in AIT-trials, and a difference of 20% or more is considered to be the minimal relevant level of improvement (25 ). When baseline registration of symptoms is available, as in our two studies, within group comparisons of before versus after treatment is also valid (25 ). Before-after comparisons cannot measure the relative treatment effect in relation to the placebo effect and different pollen counts during the two seasons of comparison is a confounding factor. However, the fact that the pollen levels were 17-21% lower the season after treatment compared to baseline did not improve the symptoms in the placebo group. This supports a true improvement in the active group. Our limited sample size, with unbalanced group at baseline, made it difficult to achieve a positive result in the comparison of the active versus placebo group after treatment. Among the secondary outcomes in ILIT after SCIT- 10 000, there was an absolute improvement of MS by 52% in the active group while the placebo group did not improve. This is above the reported relative improvement of MS in previous grass SLIT-studies, that have shown 27-38% reduction in medication (26 ).
In ILIT after SCIT- 10 000, the GAM regression analysis investigated the relationship between CSMS and increasing grass pollen concentration. The results before treatment were similar to what was found in a pediatric study in the Malmö area during the grass pollen season 2009 (27 ) and in a study of grass pollen sufferers in France and Switzerland (28 ). However, after treatment in our study, we couldn’t verify any relationship between pollen levels and symptoms. Few days with high pollen count and limited sample size may explain the result.
In ILIT de novo- 3000, the pollen counts were 25-46% lower the year after treatment compared to baseline. Low pollen counts might mask improvements after AIT (29 ). None of the parameters CSMS, MS, SS, QoL or NPT improved in the active group. This small study might have been underpowered for the secondary outcome measures. However, no trend for improvement could be seen in any of the parameters, which speak against a favorable effect of up-dosing ILIT among de-novo patients. If anything, at the peak pollen season, the placebo group but not the active group had improved CSMS, MS and QoL.
Immunologically, IgG4 increased after active ILIT in both studies, although accompanied with symptom improvement only in ILIT after SCIT- 10 000. The increase in IgG4 was larger in ILIT after SCIT, compared to in ILIT de novo- 3000. It seems that the SCIT treated patients were already primed towards inducible IgG4 production and responded after booster ILIT therapy with an expected recall response from the memory B cell population leading to the increased IgG4 levels. The raise in IgG4 in ILIT de novo- 3000 was modest and returned to baseline levels already after 8 months. It is possible that that the failure to improve the symptoms in ILIT de novo- 3000 is linked to the weak IgG4 induction. Another observation is that the IgE-levels increased in the active group in both studies, which reduced the IgG4/IgE ratio and theoretically could indicate an incomplete immunological skew (30, 31 ).
In the presented studies we used the same depot formulated Aluminum hydroxide adsorbed grass extract as in the first ILIT study from 2008 (5 ). The purpose of Aluminum adjuvant in AIT is to enhance the availability of the allergen for antigen presentation and to activate the immune system with its local pro-inflammatory properties (32 ). The advantage of intralymphatic administration is that the allergen is delivered directly to secondary lymphoid organs with high density of immunologically active cells. The need for Aluminum in ILIT has never been evaluated. There are ongoing investigations about the use of Alum in general, considering also the potential for an immune balance towards more activation of T helper (Th) type 2 cells (33 ). Future knowledge about the role of Alum in ILIT and evaluation of more specific Th1 skewing adjuvants would be of great interest.
In ILIT de novo, we could indeed see signs of Th2 type of activation. Lymph node derived DCs increased the expression of CD141, previously described on DC:s promoting differentiation of T-cells to Th2 (34 ). Our previous low dose ILIT study (16 ) showed activation of Th1 and T regulatory (Treg) cells in PBL. This could not be repeated in the present study. The negative clinical results in ILIT de novo-3000 might partly be explained by the limited sample size and low pollen levels. However, adding the immunological results, we must question if the dose-response relationship in ILIT with Alum adjuvant is linear. The positive effects of ILIT may get saturated at high doses and negative effects might stand out. Alternatively, there may be a maximum antigen dose per time interval that induces optimal immunological protective responses. This hypothesis is in theory supported by a previous ILIT trial with lack of symptom improvement when the time intervals between the injections were shortened, which increased the allergen dose per time unit (10, 35 ).
The mode of action of ILIT induced tolerance is only partly elucidated. It may include a combination of multiple mechanisms as in conventional AIT, such as Treg expansion, Th1 skewing and the induction of allergen specific IgG4 responses. Hypothetically, an additional mechanism for long term effect in ILIT can be a fine-tuned apoptosis of high affinity IgE memory B cells due to the abundance of allergen in in the lymph nodes upon ILIT-injections. Thus, it is not surprising that allergen dose, adjuvant composition and time intervals needs to be titrated to find the optimal tolerance inducing window.
This is not a consensus experience for other forms of AIT, but according to the literature, one birch SLIT study have shown that increasing the dose from 7 DU (developmental units) to 12 DU only increased the improvement in rhinoconjunctivitis total symptom score from 24 to 25% (36 ). Further, one earlier grass SLIT study have even shown a lower treatment effect in a high-dose group (500 IR[index reactivity]) compared to a moderate dose group (300 IR), resulting in further development of the moderate dose (37 ). In the present study of ILIT in de novo patients, the lack of clinical improvement and favorable immunological changes after 3000 SQ-U suggests that the optimal dose for ILIT with injections four weeks apart is 1000 SQ-U or below.
ILIT after SCIT- 10 000 shows that three intralymphatic injections up to 10 000 SQ-U might give additional symptom relief. Since SCIT rarely eliminates the symptoms completely, pre-seasonal ILIT after SCIT might play a role in the AIT toolbox in the future as a cost-effective supplementary treatment.
Future treatments of airborne allergies probably involve the expansion of sublingual at-home administration and the development of hypoallergenic allergoids or peptide vaccines (38, 39 ). However, these branches of AIT may not be suitable for all patients and health care systems due to the adherence problems, oral side effects and costs. If our knowledge about the mechanisms behind ILIT could be improved and doses, time interval and technique further optimized, ILIT might become a strong alternative AIT modality in the future.

Conclusion

De novo treatment with ILIT in patients with grass pollen induced allergic rhinitis should not exceed 1000 SQ-U, due to risk for severe side effects and limited improvement gain. An up-dosing schedule to 10 000 SQ-U after previous SCIT seems safe, appears to improve the seasonal symptoms and might be used to boost a previously given treatment.

Acknowledgement

We thank research nurses Maria Axelsson and Carina Israelsson for logistics and handling of patients and all other nurses at the ENT- and Allergology departments that helped to prepare the medical products. The studies were funded by grants from the Swedish Research Council, the Asthma and Allergy Association, Hesselman’s Research Foundation and ACTA Otolaryngologica.
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