Lysis solution

Fresh peripheral blood (100 µl) was collected in EDTA-anticoagulation tubes and reacted at room temperature for 30 min in the dark with an anti-PRTN3 antibody (PR3G-2, Abcam; undiluted) and anti-CD66b antibody (G10F5, BioLegend, San Diego, CA; undiluted) according to the manufacturer’s protocol. Erythrocytes were lysed in a lysis solution (BD Biosciences, San Jose, CA, USA) at room temperature (24°C)for 10 min in the dark and centrifuged at 1500 rpm for 10 min. The cells were then washed with PBS. Finally, the cells were resuspended in 500 µl cell FIX (BD Biosciences; dilution, 1:10). Flow cytometric data were acquired using a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA) with the NovoExpress software (version 1.3.0, ACEA Biosciences) and analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).

The basophil activation test
was performed using 100 mL of heparinized whole blood, incubated with
20 μL of stimulation buffer (1 M HEPES buffer containing 0.78%
NaCl (w/v), 0.037% KCl (w/v);, 0.078% CaCl₂ (w/v), 0.033%
MgCl₂ (w/v), 0.1% HSA (w/v), 10 μL/mL of IL-3 (1
mg/mL), and 1 μL of monoclonal antibody CCR3-APC (1 mg/mL) (BioLegend
INC, San Diego, CA) for 10 min at 37 °C. Then, 100 μL of
each purified Sola l 7, Sola l 6, and their digests was added at different
concentrations following a 10-fold dilution pattern (0.1–0.00001
μg/mL) and incubated for 30 min at 37 °C. Anti-human IgE
(0.5 mg/mL) (BD Biosciences, Franklin Lakes, NY) or PBS were used
as a positive and negative controls, respectively. After 5 min on
ice to stop the degranulation process, samples were incubated with
1 mg/mL of monoclonal antibodies anti-CD 203c-PE and CD63 FITC (BioLegend)
for 15–20 min at 4 °C. Finally, lysis solution (BD Biosciences)
was used for red cells disruption. Cells were analyzed using a FACSCalibur
flow cytometer (BD Biosciences), acquiring at least 500 basophils
per sample. Results are presented as the percentage of activated basophils
(CD63⁺CD203c⁺CCR3⁺). Due to the low
availability of patients, only the sera of one tomato allergic patient
sensitized to Sola l 6 and Sola l 7 and displaying anaphylaxis (patient
numbers 13 and 7, respectively) was used for this experiment.

Peripheral blood samples were collected from the included patients around 45 days post-haploHCT. Briefly, 300 μl of fresh peripheral blood per sample was stained with the following fluorochrome-labelled antibodies: PE-Cy7-CD3, BV510-TCRαβ, BV605-CD4, APC-CD8, PE-CF594-CXCR4 (BioLegend, San Diego, CA). After incubation, red blood cells were lysed with a lysis solution (BD Biosciences, San Jose, CA) and then were washed twice with PBS. Polychromatic flow cytometric analyses were performed on a BD LSRFortessaTM Cell Analyser and further analyzed using BD FACSDivaTM software.

For immunophenotypic analysis, whole blood samples were stained by four-color technique using anti-CD45RA-FITC, anti-CD31-PE, anti-CD4-PerCP, and anti-CD3-APC monoclonal antibodies and appropriate isotope controls (Becton Dickinson, USA). Erythrocytes were lysed with lysis solution (Becton Dickinson, USA). Lymphocyte data were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, USA). After gating for CD3⁺ and CD4⁺ cells, the percentage of CD31⁺ cells among all CD45RA⁺CD4⁺ T cells was analyzed with CELLQuestTM software (Becton Dickinson, USA) as previously described (33 (link)). Whole blood lymphocyte counts were performed with an automated analyzer, and the absolute numbers of CD31⁺ RTEs were determined by multiplying the total lymphocyte count by the percentage of CD31⁺CD45RA⁺CD4⁺ T cells.

Immunofluorescence using monoclonal antibodies was used to determine the percentages of CD68+, CD4+, CD8+, CD4+/CD25+, CD8/CD25+, CD8+/CD69+, CD4+/CD69+ and granzyme⁺ cells in the peripheral blood samples, (Biolegend, USA). Briefly, 30 μl of blood were incubated for 30 min with 5 μl of the corresponding monoclonal antibody (1:100 dilution) afterwards, 200 μl of lysis solution were added (Becton Dickinson, California), incubated in darkness for 10 min and washed twice with 0.1 M PBS (pH 7.2), 0.1 % BSA and 0.1 % NaN3. The cells were then fixed in 1 % paraformaldehyde solution and stored at 4 °C until examination by flow cytometry (FACSCalibur, Becton Dickinson) using the Cell Quest software. 10,000 events in the region corresponding to lymphocytes were analyzed. From this region, the percentage of positive cells from each sample was determined. Results were expressed as means (±SD) for each experimental group.

Blood was collected into tubes with heparin and red blood cells (RBCs) were lysed with lysis solution (Becton Dickinson San Jose, CA, USA), prior to staining with standard procedures. Splenic, thymic and BM single-cell suspensions were immunophenotyped using standard methodology. Briefly, 10⁶ cells were stained for 20 min at 4 °C in PBS with 2% FBS with specific antibodies (Supplementary Data 14). When lineage-positive cells were excluded, biotin coupled anti-Gr-1, CD11b, CD19, Ter119, NK1.1, and CD11c were used and subsequently stained with BV711 streptavidin. Proliferation was analyzed by intracellular staining of Ki67 (APC-conjugated, Biolegend), using the Foxp3 staining kit from eBioscience and following the manufacturer’s instructions. Cell viability was determined using an annexin V-based apoptosis detection kit and following the manufacturer’s instructions (eBioscience). 10-color analyses were performed on LSR Fortessa II (Becton Dickinson San Jose, CA, USA) flow cytometers. Results were analyzed with FlowJo (Tree StarInc., Ashland, OR, USA) software. Gating strategies used are exemplified in Supplementary Figs. 26–30.