Background The same dosing schedule, 1000 SQ-U times three, with one-month intervals, have been evaluated in most trials of intralymphatic immunotherapy (ILIT) for the treatment of allergic rhinitis (AR). The present studies evaluated if a dose escalation in ILIT can enhance the clinical and immunological effects, without compromising safety. Methods Two randomized double-blind placebo-controlled trials of ILIT for grass pollen induced AR were performed. The first included 29 patients that had recently ended 3 years of SCIT and the second contained 39 not previously vaccinated patients. An up-dosage of 1000-3000-10 000 (5000 + 5000 with 30 minutes apart) SQ-U with one month in between was evaluated. Results Doses up to 10 000 SQ-U was safe after recent SCIT. The combined symptom-medication scores (CSMS) were reduced by 31% and the grass specific IgG4 levels in blood were doubled. In ILIT de novo, the two first patients that received active treatment developed serious adverse reactions at 5000 SQ-U. A modified up-dosing schedule; 1000-3000-3000 SQ-U appeared to be safe but failed to improve the CSMS. Flow cytometry analyses showed increased activation of lymph node derived dendritic but not T-cells. Quality of life and nasal provocation response did not improve in any study. Conclusion ILIT in high doses after SCIT appears to further reduce grass pollen induced seasonal symptoms and may be considered as an add-on treatment for patients that do not reach full symptom control after SCIT. Up-dosing schedules de novo with three monthly injections that exceeds 3 000 SQ-U should be avoided.

Dear Editor,CD4+ T helper 2 (Th2) cells play a key role in progression of allergic rhinitis (AR). Traditionally T-cells acquire the characteristics of Th2 cells in the presence of IL-4, a process that may be influenced by engagement of co-stimulatory factors expressed by antigen-presenting cells1. Notch is a co-stimulatory pathway for CD4+ T-cells, and the importance of Notch for generation of Th2 cells and the development of allergic rhinitis has been suggested in animal studies2,3. However, its potential role in the advancement of allergy in humans remains unknown. Our group has previously demonstrated that neutrophils and nasal epithelial cells promote allergic mucosal inflammation by interacting with T-cells. Based on these findings, we hypothesized that neutrophils and epithelial cells in allergic nasal mucosa induce Notch signaling in CD4+ T-cells, promoting allergic inflammation. The present study analyzed Notch ligand expression on primary nasal epithelial cells and neutrophils along with Notch receptor expression on CD4+ T-cells in patients with pollen-induced AR off season and compared with healthy control (HC). In the appendix of this article information regarding patient characteristics and methods is supplemented.Expression of Notch1-4 on T-cells was assessed by FACS. The fraction of CD4+ T-cells expressing Notch1,4 in mucosa was significantly higher in AR compared to HC (Fig 1B). No differences in Notch expression were detected in blood derived CD4+T-cells (Fig 1A). In animal models of allergic rhinitis, JAG1-Notch1,4 signaling has been demonstrated to polarize T-cell responses towards Th2, promoting an allergic inflammation3,4. Against the backdrop of our findings in CD4+ T-cells, it is tempting to suggest that the increase in Notch1,4 in allergic mucosa might be a sign of increased Th2 cell polarization and that Notch signaling maintain TH2 inflammation and consequently AR progression. In the present study no difference in the expression of Notch was detected on CD4+ T-cells in peripheral blood. However, this study was performed outside the pollen season, and it cannot be excluded that a similar signal can also be seen in the blood in patients with pollen induced symptoms. Further the expression of JAG1, JAG-2, DLL-1, and DLL-4 was also assessed with FACS. The fraction of epithelial cells expressing JAG-1 and DLL-1 was significantly elevated in AR compared to HC. (Fig 2A). The increased expression of JAG-1 and DLL-1 among patients with AR could be further corroborated by analyzing median fluorescence intensity (MFI) (Fig 2B,C). Although significant, the expression levels of DLL-1 on the surface of epithelial cells were notably lower than the corresponding expression levels of JAG-1. (Fig 2B,C). There are conflicting theories regarding in what way Notch signaling regulates T-cell functions. However, there are evidence for an instructive model where JAG-Notch1,2 interaction initiates Th2 cell differentiation, and DLL-Notch3 promotes T helper 1 (Th1) cell differentiation5. Our result illustrates a JAG-1>>DLL signaling gradient by epithelial cells supporting a Th2 outcome (Fig 2B,C). Further, the fraction of neutrophils expressing JAG-1 was significantly increased in AR compared to HC, in samples derived from mucosa and blood (Fig 2D,G). In addition, the expression levels of JAG-1 as measured by MFI, were significantly increased on neutrophils from AR compared to neutrophil from HC, in samples derived from mucosa and blood (Fig 2E,F and H,I).Together with our previous report of antigen-presenting epithelial cells, our data suggest that the increased expression of JAG-1 in epithelial cells in allergic mucosa induce a sustained Th2 polarization of Notch1 and Notch 4 expressing T-cells. In analogy, neutrophils have also been shown to present antigens to T-cells6. Hence, it might be that neutrophils in AR mucosa present antigen to T-cells promoting a Th2 polarization in CD4+ T-cells by increased expression of JAG-1.Wk 600Increased expression of jagged-1, Notch-1 and Notch-4 in nasal mucosa of patients with allergic rhinitisEric Hjalmarsson1, Marianne Petro1, Susanna Kumlien Georén1, Ola Winqvist2, Lars Olaf Cardell1,31. Division of ENT Diseases, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden2. ABClabs, Biomedicum, Stockholm Sweden3. Department of Otorhinolaryngology, Karolinska University Hospital, Stockholm, SwedenCorresponding author:Professor Lars Olaf CardellDivision of ENT Diseases, Department of Clinical Science Intervention and Technology, Karolinska Insitutet, Stockholm, SwedenPhone: +46 (0) 8 585 814 53E-mail: [email protected]

Background The same dosing schedule, 1000 SQ-U times three, with one-month intervals, have been evaluated in most trials of intralymphatic immunotherapy (ILIT) for the treatment of allergic rhinitis (AR). The present studies aimed to evaluate if a dose escalation in ILIT can enhance the clinical and immunological effects, without compromising safety. Methods Two randomized double-blind placebo-controlled trials of ILIT for grass pollen induced AR were performed. The first included 29 patients that had recently ended 3 years of SCIT and the second contained 39 not previously vaccinated patients. An up-dosage of 1000-3000-10 000 SQ-U with one month in between was evaluated. Results ILIT in doses up to 10 000 SQ-U was safe after recent SCIT. The combined symptom-medication scores (CSMS) were reduced by 31% and the grass specific IgG4 levels in blood were doubled. In ILIT de novo, the two first patients that received active treatment developed serious adverse reactions at 5000 SQ-U. A modified up-dosing schedule; 1000-3000-3000 SQ-U appeared to be safe but failed to improve the CSMS, quality of life and nasal provocation response. Flow cytometry analyses could not detect any T-cell changes, while lymph node derived dendritic cells showed increased activation. Conclusion ILIT in high doses after SCIT appears to further reduce grass pollen induced seasonal symptoms and may be considered as an add-on treatment for patients that do not reach full symptom control after SCIT. Up-dosing schedules de novo with three monthly injections that exceeds 3 000 SQ-U should be avoided.