Mass cytometry profiling the response of basophils and the complete peripheral blood compartment to peanut

To the editor

Anaphylaxis is a serious, life-threatening immediate hypersensitivity reaction that is caused by cross-linking of IgE bound to FcεRI on mast cells and basophils. However, it has been described that anaphylaxis can also be induced by alternative pathways, mediated by IgG-antigen immune complexes, FcγRIII and basophils, neutrophils and/or macrophages ^1-3. The availability of human data regarding the acute response to allergens by different leukocyte populations in allergic patients is scarce. Mass cytometry uses heavy metal-labeled antibodies and time of flight mass spectrometry for detection, allowing for simultaneous detection of more than 40 markers. This multiplexing capacity allows for simultaneous profiling of all hematopoetic cells, and provides a unique platform with which to comprehensively study the human immune response to allergens.

Whole blood from peanut-allergic patients (n=6) or healthy controls (n=3) was stimulated with peanut extract (PN), anti-IgE, or media control for 15 or 30 minutes prior to fixation and red blood cell lysis. For detailed methods see the online repository. Samples were barcoded (Fluidigm, San Francisco, CA), pooled, blocked with heparin, stained with cell surface and intracellular phospho-protein antibodies listed in the online repository (Table E1) and acquired on a CyTOF2 mass cytometer (Fluidigm). Following data normalization and debarcoding, CD45+ cell events were manually gated and analyzed using SPADE (spanning-tree progression analysis of density-normalized events), an algorithm that organizes multidimensional single-cell data to clusters displayed as a 2D tree plot^{4, 5}. Differences between groups were analyzed by Mann-Whitney U test. Data analysis was done by using Prism software (GraphPad, San Diego, CA). Results are expressed as mean ± SEM and a value of p<0.05 was considered significant.

Identification of cell subsets on the SPADE tree was performed manually by iteratively evaluating the expression of specific cell markers (Fig E1, Table E2). This approach unambiguously identified basophils, eosinophils, neutrophils, monocyte subsets, plasmacytoid DCs (pDCs), conventional DCs (cDCs), NK subsets, NK T cells, CD4 and CD8 T cells, and B cells. We compared the frequencies of populations with conventional flow cytometry in a separate group of subjects, and could identify the same subsets by both techniques (data not shown). Eosinophils and B cells were increased in frequency in peanut-allergic subjects versus healthy controls, while neutrophils were reduced (Fig E2).

Basophils were clearly the most responsive cells in blood to peanut stimulation in peanut-allergic patients (Fig 1A and E3). In addition to upregulation of CD63 (Fig 1A), a conventional marker of basophil degranulation, basophils from peanut-allergic subjects responded to peanut with regulation of IgE (FcεRI, CD23) and IgG (CD16, CD32) receptors, which could modulate the ability of basophils to respond to a second challenge. CD25 and CD38 were downregulated upon peanut exposure in basophils (Fig 1A). Basophils from healthy controls had minimal response to peanut stimulation. However, they responded similarly to anti-IgE stimulation (Fig E4), indicating that they are able to respond in the same extent as those from allergic subjects. The response of peanut-allergic subjects to peanut stimulation was identical in pattern to the anti-IgE response.

Figure 1. Basophil activation and association with platelets after peanut stimulation.

(A) Expression of each marker by basophils stimulated with peanut (Δ median intensity (MI) vs. media control). Expression at 15min is shown for all markers except FcεRI, CD32 and CD38 where 30min is shown. (B) Expression of CD63 and CD61 (MI) by basophils after 15min stimulation. *p < 0.05 vs media. (C) Representative plot of CD61 expression (%) by basophils from peanut-allergic subjects. (D, E) Expression of CD63 and pp38 (%) by free basophils (CD61-) or basophils+platelets (CD61+) from peanut-allergic patients. *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. free basophils. (F) Imaging flow cytometry of basophils-platelet complexes from a healthy control stimulated with anti-IgE for 30min. Two representative images of activated basophils are shown. BF, bright field. (G) Expression of pERK (15min), pp38 (15min) and pS6 (30min) by basophils. *p < 0.05, **p < 0.01.

Basophils from peanut-allergic patients but not healthy controls became strongly positive for CD61, a platelet-specific marker, after peanut or anti-IgE stimulation (Fig. 1A and 1B). In addition there was an upregulation of CD141 (Fig 1A), correspondent to thrombomodulin, a cofactor of thrombin, that is expressed in platelets among other cell types. These results suggest that after activation, there is a physical interaction between basophils and platelets. The same result was observed by conventional flow cytometry using another platelet-specific marker, CD42b (Fig E5). Furthermore, we confirmed the physical interaction between basophils and platelets using imaging flow cytometry (Fig 1F). Platelet-basophil complexes had substantially greater CD63 expression and phospho-p38 than free basophils (Fig 1C,D,E). This suggests that either basophils must be activated to form complexes with platelets, or that the formation of complexes contributes to basophil activation. Although activated platelet adhesion to basophils has been described as a potential pitfall or artifact of basophil activation assays⁶ we observed that platelet adhesion to basophils was only observed on basophils activated by allergen or anti-IgE. Furthermore, there was no evidence of CD63 expression by platelets not adherent to basophils. By imaging flow cytometry, we observed that CD63 signal was increased in the area of platelet adherence suggesting that platelets contribute to CD63 expression (Fig 1F). On the contrary, phospho-p38 signal was observed only within the basophils (Fig 1F and E6). Platelet-neutrophil complexes have been shown to contribute to aspirin-exacerbated respiratory disease through cooperative production of leukotriene C4⁷. We speculate that similar cooperative function between platelets and basophils in vivo may contribute to anaphylaxis, and that this platelet basophil interaction may be biologically meaningful. It has been described that the levels of the platelet activating factor (PAF) correlates with severity of anaphylaxis and that deficiency of the enzyme that hydrolizes PAF, the PAF acetylhydrolase, has been linked to death from anaphylaxis⁸. The fact that platelets are the major target of PAF suggests the importance of platelets in anaphylactic responses.

After stimulation with peanut, basophils from peanut-allergic patients showed uniquely increased phosphorylation of ERK and p38 (Fig. 1G), which was not observed in any other cell population (Fig E3). There was also increased phosphorylation of S6 (Fig 1G) that was also observed in monocytes, cDCs and pDCs (Fig 2A and 2B) of peanut-allergic subjects but not healthy controls in response to peanut, and in all subjects in response to anti-IgE. Although cDCs and pDCs express FcεRI (Fig E1), phospho-S6 in monocytes did not correlate with FcεRI or FcεRII expression, suggesting that this may be a secondary response perhaps downstream of basophil activation. Human neutrophils have been shown to be activated by allergen-IgG complexes⁹. We observed a very modest but significant downregulation of CD66a in neutrophils in response to peanut in peanut-allergic subjects, but also in response to anti-IgE (Fig 2C and 2D). As neutrophils do not express FcεRI, this may therefore be a secondary effect of basophil activation.

Figure 2. Myeloid cells are activated by peanut in peanut-allergic patients.

(A) Representative SPADE trees of pS6 expression by hematopoietic cells in a peanut-allergic subject and a healthy control after peanut stimulation. Colors represent fold change vs. media control. (B) Expression of pS6 (MI) by monocytes, pDCs, cDCs and basophils from peanut-allergic patients after 30min stimulation. (C) Representative SPADE trees of CD66a expression by the different cell populations in a peanut-allergic and a healthy control after peanut stimulation. Colors represent fold change vs. media control. (D) Expression of CD66a (MI) by neutrophils after 15min stimulation. Monocytes: a, CD11c+CD16+CD14+; b, CD11c+CD16+CD14−; c, CD11c+CD16−CD14+. NK cells: a, CD56bright; b: CD56dim. DP, double positive. *p < 0.05, **p < 0.01 with respect to the media.

The major novel finding of this study is that basophils and platelets physically interact after peanut allergen exposure. The functional consequence of this interaction in anaphylaxis will be addressed in future studies. It is possible that by modulating the formation of these complexes anaphylaxis responses could be reduced in severity. Secondly, we observe that myeloid cells are activated upon allergen exposure in allergic individuals. Additional studies are needed to determine if these cells directly respond to antigen, and the consequence of this activation in anaphylaxis and antigen presentation.

Methods

Subjects and samples

Peanut allergic subjects were recruited through the Jaffe Food Allergy Institute, and as controls we recruited healthy adult volunteers. The study and consent forms were approved by the Institutional Review Board at the Icahn School of Medicine at Mount Sinai. Blood was drawn in heparinized vacutainer tubes and used for experiments within 3 hours of blood draw.

Whole blood stimulation and antibody staining

All the antibodies used in this study were either purchased pre-conjugated from Fluidigm (San Francisco, CA) or were conjugated using X8 MaxPar conjugation kits (Fluidigm) according to the manufacturer’s protocol. All antibodies were added after fixation except CD63 and CRTH2, which were added during stimulation.

Blood (1mL/condition) was added to 1mL of RPMI and stimulated with 1ug/mL of peanut extract, 1ug/mL of anti-IgE (Bethyl Laboratories, Montgomery, TX) or media control for 15 or 30min at 37°C in the presence of 2ng/mL IL-3 (R&D systems, Minneapolis, MN). Then, the samples were fixed and lysed using BD Phosflow Lyse/Fix Buffer (BD Biosciences, San Diego, CA) and barcoded with the Cell-ID 20-Plex Pd Barcoding Kit (Fludigm) following manufacturer's instructions.

Samples were pooled and blocked with 100U/mL of heparin to inhibit non-specific binding to eosinophils¹, and stained with a cocktail of metal-conjugated antibodies for surface staining for 20min at RT. Then samples were washed and permeabilized with ice-cold methanol for 30min, washed and stained with intracellular phospho-protein antibodies for 30min on ice, with prior heparin blocking.

After washing, the samples were then incubated with 0.125nM Ir nucleic acid intercalator (Fluidigm) to enable cell identification based on DNA-content, and stored in PBS with freshly diluted 2% formaldehyde (Electron Microscopy Sciences) until acquisition.

Data acquisition and analysis

Immediately prior to acquisition, the samples were washed once with PBS, once with deionized water and resuspended at a concentration of 600,000 cells/ml in water containing a 1/20 dilution of EQ 4 element beads (Fluidigm). Following routine autotuning according to the manufacturer’s recommendations, the samples were acquired on a CyTOF2 mass cytometer (Fluidigm) using a SuperSampler (VictorianAirships) fluidics system at a flow rate of 0.045ml/min. For quality control, the acquisition event rate was maintained under 400 events/s, and the EQ beads were confirmed to have a median Eu151 intensity of over 1000 to ensure appropriate mass sensitivity. The resulting FCS files were concatenated, normalized and debarcoded using the bead-based normalization and debarcoding tools in the CyTOF2 software and uploaded to Cytobank for analysis. Cells events were identified as Ir191/193 DNA+ Ce140-events, and doublets were excluded on the basis of higher DNA content and longer event length.

Imaging flow cytometry

Whole blood from healthy controls was stimulated with anti-IgE for 30min and processed as described above. All antibodies were from BioLegend (San Diego, CA) unless otherwise stated. After lysis/fixation, cells were stained with anti-CD123 (6H6), anti-Bdca2 (201A), anti-CD42b (HIP1) and anti-CD63 (H5C6). Cells were permeabilized with methanol and stained with phospho-p38 (4NIT4KK; eBioscience, San Diego, CA). Before acquisition nuclei were stained with Hoechst (BD Pharmingen, San Jose, CA). Imaging flow cytometry was performed by acquiring cells in the Image Stream 100 (Amnis, Seattle, WA) imaging flow cytometry. Complexes between basophils and platelets as wells as the expression of the different markers were visualized using the IDEAS software (Amnis).