Basophil sensitivity through CD63 or CD203c is a functional measure for specific immunotherapy

The ethics committee of Aarhus County approved a project, in which patients aged 18 or older, registered to begin subcutaneous immunotherapy with Vespula Alutard (ALK-Abelló, Hørsholm, Denmark) were recruited. From January to April 2007, 20 patients were recruited and 18 patients completed the project (median age 54 years, interquartile range (IQR) 44-61 years, 8 women, 10 men). The clinical history of wasp allergy was confirmed by skin prick test (SPT) (ALK Abelló, Hørsholm, Denmark) and by measuring specific IgE with the CAP system (Phadia, Copenhagen, Denmark), with a threshold of 0,35 kU/L. A SPT of >3 mm was considered positive. The median concentration of specific IgE to vespula venom was 3,01 kU/L, with an IQR of 1,53 - 7 kU/L. Some of the patients (n = 5) were also sensitized to bee venom with a median and range of 0,95 kU/L, ranging from 0,88 - 1,23 kU/L. One patient was negative for both wasp and bee venom in the CAP test, but had a positive prick test for wasp. In SCIT increasing doses of an allergen, the patient is sensitized to, is injected subcutaneously once a week until a maintenance dose (prescheduled 100.000 SQU) is reached. Treatment continues for 3-5 years with injections every 6^th -8^th weeks. Allergen injections, in the up dosing phase, can be administered as clusters (11 injections at 7 visits) [15] or after the conventional regime with one injection at each visit (16 injections at 16 visits), if there is an increased risk of adverse effects to the treatment. It is possible to shift from the cluster up dosing regimen to the conventional. Six of the 16 patients that started on our cluster regimen completed it (Table 1). The remaining ten patients transferred to the conventional regimen, on which four patients had started. For generation of the ROC curve, additional patients (3 women, 3 men) with allergy to wasp venom (specific IgE > 0,35 kU/L), and healthy, stung controls (5 women, 4 men) that did not have specific IgE to wasp venom, were recruited. For determination of differences in LC50 (log10 of the allergen concentration resulting in 50% activation of basophils) as the protective component in patient plasma (see below) at maintenance dose, additional patients receiving maintenance dose of SCIT for wasp venom allergy (n = 3) or birch pollen allergy (n = 5) were recruited (3 women, 5 men).

For basophil activation, 4 ml venous blood was drawn into heparin tubes before allergen injection at visits 1, 2, 3, 4, 7 and at the first visit at which maintenance dose was injected. Basophil activation was done as previously described [6]; log dilutions of allergen (freeze-dried V. vespula allergen, a gift from ALK-Abello, Hørsholm, DK) ranging from 10 ug/ml final concentration to 10 pg/ml final concentration were compared with PBS only as control. Heparinised blood (100 ul) was added within 2 hours of venesection, and incubated with allergen at 37°C for 20 minutes. A dilution series of anti FceRI antibody CRA1 (Catalog Nr 334602, Biolegend, CA) was used as positive control (data not shown). Titrated amounts of CD63 FITC (5 ul, Invitrogen MHCD6301) and CD203c PE (5 ul, Beckmann-Coulter, IM3575) were added for the last 5 minutes. Activation was terminated by addition of 2 ml cold Saponin solution (0,15 mg/ml) in PBS for exactly 2 minutes, followed by 0,25 ml of 5% Methanol and 10% Paraformaledhyde in PBS and centrifugation at 400 g for 8 minutes at 4°C. Samples were washed once with PBS, suspended in PBS and 150 000 events were acquired on a FACS Calibur flow cytometer within 3 hours. On each day, compensation controls were acquired for CD63 FITC and CD203c PE with comp beads (Beckton-Dickinson, San Jose, CA). Data was compensated and in the analysis, the FS:SS region containing basophils was identified by back gating on CD203c⁺ cells (figure 1). A threshold set at 2% positive cells in the PBS tube was used to determine percent activated cells in the tubes incubated with allergen. The fraction of positive cells is plotted against allergen concentration.

The protective effect of plasma on basophil activation was calculated as difference in LC50 in full blood (autologous plasma) and LC50 of basophils in washed blood. Washed blood was prepared by diluting 4 ml blood with 46 ml PBS, centrifuging and diluting again with PBS up to 50 ml. The remaining cells were suspended in heterologous plasma (1 ml) from a nonatopic donor for 15 min. Washed blood was used in a BAT test as described above. In a control experiment, the LC50 of washed blood was reconstituted with autologous plasma was similar to that of whole blood (n = 5).

LC50 was calculated in R [16] by fitting a sigmoid curve to data from each visit, and determining the amount of allergen, that results in 50% of maximal activation.

The allergen concentration at which sensitivity and specificity were optimal for CD63 and for CD203c was determined with data from 24 clinically diagnosed wasp allergic patients and 9 stung controls with no specific IgE to vespula allergen, using plots of sensitivity and specificity [17], and confirmed with web based ROC software [18]. For SCIT patients, baseline visits were used for this analysis.

The difference in basophil activation as measured by up-regulation of CD63 and CD203c was investigated by a Bland Altman analysis [19].

Blood basophil concentration was determined by CVA [20]. Data is acquired for a constant, calibrated time, which appears to give a more precise measurement of volume than measurement with micro beads [20]. Briefly, 50 ul of blood was labelled with Lin1 FITC (BD Catalog Nr 340546), CD123 PE (BD Catalog Nr 340545), CD45 PE Cy7 (BD Catalog Nr 555484) and HLA DR APC (BD Catalog Nr 559866) for 15 min at room temp, and lysed with FACS Lyse (BD Catalog Nr 349202). Data was acquired for 4 minutes from all patients at all visits. Each day data was acquired, compensation controls were acquired, and data was compensated before analysis. Leukocytes were identified on a CD45 vs. SS plot. Basophils were identified as CD45^dim Lin1^neg CD123^bright HLA DR^neg cells, and the absolute concentration was calculated.

The effect of the up-dosing phase of subcutaneous immunotherapy on basophil up regulation of CD63 and CD203c (figure 1) was evaluated by fitting a sigmoid curve to data points obtained with a 7-step logarithmic dilution series of V. vespula allergen and blood of wasp allergic patients drawn at each of visits 1, 2, 3, 4, 7 and maintenance visit, if that did not coincide with visit 7. From this curve at each visit, the concentration of allergen resulting in half-maximal CD63 activation was determined (figure 1e). It is termed LC50. When normalised to the baseline visit, the LC50 was normally distributed. When plotted against visit, it increased significantly from the first to the third week of immunotherapy (visit 1 - 3; p = 0, 0098, visit 2 - 3 p = 0, 0259, visit 3 - 4 p = 0, 0274, visit 3-maint p = 0, 0095 for all in a mixed linear model, p = 0,029 for CD63, p = 0,015 for CD203c, n = 18), and tended toward baseline levels beyond that (figure 2a).

In a cross-sectional study to determine the contribution of humoral factors toward BAT performed in whole blood, blood from different patients initiating SCIT and patients on maintenance was washed with PBS and replaced by heterologous serum (termed HS in contrast to autologous plasma, AP) from a nonatopic donor as described in methods, and was used in a BAT. Replacing AP with HS dramatically increased basophil sensitivity. At baseline the sensitivity increased by 2,576 LC50 units (n = 8, p < 0,001), and at maintenance it increased by 3,595 LC50 units (n = 10, p < 0,001) (figure 2b). At the early stage of maintenance, the reduction in basophil sensitivity resulting from the autologous plasma had thus increased by 1,02 LC50 units (p = 0,04) (figure 2c).

The optimal allergen concentration used to detect allergy to V. vespula allergen was determined with sensitivity vs. specificity curves and is illustrated with a ROC curve (figure 3a) with 24 patients with documented allergy and 9 exposed controls. The optimal final V. vespula allergen concentration was 0,1 μg/ml. The sensitivity and specificity were 88% and 78% for CD63 at 3,28% basophil activation. For CD203c, the sensitivity and specificity were 88% and 89% at 3,85% basophil activation.

Agreement between activation measurements performed with CD63 as the predicate method and CD203c determined with a Bland Altman plot [19] (figure 3b). The median difference of CD63 - CD203c was -1,2%, IQR 23,5% to -28,6% (not normally distributed, n = 914).

Difference between CD63 and CD203c was determined with an approximate T test in a linear mixed model. For all concentrations there was no significant difference between CD63 and CD203c with all p-values greater than 0,11. When autologous plasma is replaced with heterologous serum, the value of CD63-CD203c increased from 4, 2% (IQR -20, 3 - 31, 7) to 41, 7% (IQR 8, 3 - 73, 5, n = 135. In the same linear mixed model that takes account for repeated measures the relative difference between activation through CD63 and CD203c was larger after plasma was replaced with serum (p = 0,0172). It increased significantly (n = 15, p < 0, 05) at four of the six highest allergen concentrations, suggesting that CD203c is up regulated less than CD63 when autologous plasma is replaced by heterologous serum. This was not due to an increase in baseline MFI for CD203c, as the baseline MFI for CD63 and CD203c increased 1,92 and 1,83 fold, respectively, after replacement of plasma with serum. This was not significant.

Blood basophil concentration measured using CVA was 37 (IQR 28 - 55, n = 18) cells/μl in allergic patients treated with SIT, and 35 (IQR 23 - 42, n = 6, p = 0, 06) cells/μl in controls. It increased by median 1 (IQR -2 - 6) cell/μl (5%) per 30 minutes for patients receiving allergen injections and by median 3 (IQR 2 - 5) cells/μl (12%) per 30 minutes in control persons after correction for time between sampling. The difference was significant in patients and controls when measured over 90 and 120 minutes (p < 0, 04), but not when measured over 30 minutes.

On the scale proposed by Brown [21], 14 patients reported moderate symptoms and 4 patients reported severe symptoms when stung. Seven patients had other allergies (2 latex, 3 bee, 2 cat, 3 house dust mites, 1 perfume, 1 kiwi, 1 nuts, 1 apples) and other allergic disease (3 asthma, 3 hay fever, 3 Urticaria).

For two patients the activity of the basophils for both CD203c and CD63 was only just above the threshold values (i.e. positive) at some of the visits. These two patients had almost no symptoms/adverse effects to treatment with SIT, but they had moderate symptoms as response to wasp sting, specific IgE at 3,01 kU/L and 6,0 kU/L respectively and positive skin prick tests (SPT) before the beginning of SIT. One patient had moderate symptoms to wasp sting and positive skin prick test but negative specific IgE against wasp before the beginning of SIT. The BAT of this participant was positive at all visits, and the patient had local reactions to the treatment and a systemic reaction at visit 2.