α2-3 Sialic Acids-Decorated Allergens Exert a Tolerogenic Effect on CD4+ T Cells from Der p 2-Allergic Patients

Allergen-specific immunotherapy (AIT) has been used for over a century for the effective management of allergy [1]. AIT reduces allergic symptoms in patients and prevents the progression from mild-to-severe manifestations of allergic asthma [1, 2]. However, the build-up to a therapeutic dose of allergen during AIT still presents a risk of serious adverse events, including anaphylaxis [3, 4]. Moreover, there is low patient adherence to the full AIT course due to the long duration of treatment (3–5 years) necessary for significant effects [5, 6]. Furthermore, due to the safety concerns and difficulties in universal standardization [7] associated with allergen extracts currently used in AIT, more and more research is being carried out assessing the potential use of single allergens as alternatives. Consequently, novel approaches to further prevent undesired allergic reactions and to shorten the duration of AIT by accelerating the development of anti-inflammatory processes are needed. Several approaches have been explored so far including but not limited to the use of alternative routes of administration, development of hypo-allergens, the attachment of adjuvants to allergens, and the chemical modification of allergens [1, 8‒10]. Approaches that include the modification of allergens are intended to modulate the response of dendritic cells (DCs) and subsequently direct T-cell responses toward a regulatory state.

To successfully direct DCs toward immunosuppression, novel strategies that modify allergens with molecules that target tolerogenic receptors on DCs are being explored. These inhibitory receptors often contain immunoreceptor tyrosine-based inhibitory motifs [11]. The family of glycan-binding receptors that belong to the sialic-acid-binding immunoglobulin-like lectins (Siglec) has these inhibitory properties [12]. Siglecs promote cell-cell interactions upon binding sialic acids and regulate the functions of immune cells [12, 13]. Siglecs with tyrosine-based inhibitory motifs, namely, Siglec-2, Siglec-7, Siglec-8, Siglec-9, and Siglec-10, the so-called inhibitory Siglecs, have gained a lot of attention in recent years as functional parallels with the T-cell checkpoint receptors CTLA-4 and PD-1. As such, they suppress the activity of immune cells, leading to an anti-inflammatory effect [12, 14]. Consequently, a few studies have explored the targeting of inhibitory Siglecs as a viable and promising therapeutic option in immune-mediated pathologies. In this regard, we have previously reported that modifying ovalbumin with α2-3 sialic acids to target Siglec-E (Siglec-9 homolog) on mouse DCs led to the induction of a regulatory program in DCs that inhibited T-cell proliferation and induced Tregs in an antigen-specific manner [15]. Moreover, we recently demonstrated that α2-3 sialic acids downregulated the production of pro-inflammatory cytokine secretion human monocyte-derived dendritic cells (moDCs) [16] and T helper (Th) 2 cytokines (interleukin [IL]-5 and IL-13) in human PBMCs [17]. Finally, we have shown that treating grass pollen-sensitized mice with Phl p 5a peptides modified with sialic acids resulted in the suppression of allergic airway inflammation, suppression of Th2 cell activity and expansion of Foxp3+ Tregs [18].

Most studies investigating the therapeutic potential of sialic acids in allergy have focused on in vivo mouse models which often poorly reflect the actual physiological conditions in human settings. The aim of the present study was to extend the above findings to a preclinical human setting. We make use of a major house dust mite (HDM) allergen (Der p 2), as model antigen, which is of public health importance, and to which approximately 30% of the world population is sensitized [19]. Moreover, we aimed to assess whether modifying Der p 2 with sialic acids alters the nature of cellular immune cell responses in HDM-allergic individuals. The HDM-induced allergic response is orchestrated by DCs that take up allergens and activate T cells toward producing Th2 cytokines such as IL-4, IL-5, IL-9, and IL-13 [20]. Together, these cytokines stimulate the production of HDM allergen-specific IgE and the recruitment of inflammatory cells, leading to structural changes in the lung, nose, and skin [20]. We therefore modified a recombinant version of Der p 2 with α2-3 sialic acids and explored whether this altered moDCs and CD4+ T-cell function of Der p 2 allergic volunteers compared to non-atopic volunteers. We observed that α2-3 sialic acids-conjugated Der p 2 suppressed moDC activation, suppressed CD4+ T-cell activation and proliferation, and suppressed IL-13 and IFNγ secretion from CD4+ T cells in mite-allergic patients.

α2,3 sialic acids were conjugated to Der p 2 as described previously [17] with maleimide-activated 3′-sialyl-N-acetyllactosamine (LSTd; Neu5Acα2,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc; Elicityl Oligotech, Rhone-Alpes, France) and thio-activated Der p 2 (Amsterdam University Medical Centers, Amsterdam, The Netherlands) through a thiol-maleimide reaction. The protein concentration of Der p 2 was quantified before and after sialylation using the Pierce BCA Protein Assay Kit (Thermo Fisher, MA, USA) following the manufacturer’s instructions.

Monocytes from buffy coats of HDM-allergic patients, and of non-atopic controls, were isolated first by Ficoll gradient centrifugation and then by CD14-positive MACS bead (Miltenyi Biotec, CA, USA) and subsequently cultured for 5 days at 37°C, 5% CO₂ in RPMI 1640 (Thermo Fisher) supplemented with 10% fetal calf serum (FCS) (Biowhittaker, Switzerland), 100 U/mL penicillin (Thermo Fisher), 100 U/mL streptomycin (Lonza, Switzerland), 2 mm glutamine (Lonza), 500 U/mL IL-4 (ImmunoTools, Germany), and 500 U/mL granulocyte-macrophage colony-stimulating factor (ImmunoTools) to generate moDCs, as previously described [21]. Blood donors gave informed consent, in accordance with the Amsterdam University Medical Centers Medical Ethics Committee guidelines (NL71330.018.19).

Day 5 immature moDCs were seeded (2 × 10⁵ cells/well) in sterile 48-well plate in RPMI-1640, supplemented with 10% FCS, l-glutamine (2 mm), and penicillin/streptomycin (100 U/mL) and incubated overnight with 10 μg/mL each of Der p 2 or sia-Der p 2 at 37°C, 5% CO₂. Lipopolysaccharide (LPS) (LPS from E. coli 0111:B4; Sigma-Aldrich) and Pam3CysSerLys4 (Pam3) (Invitrogen, CA, USA) were added at 10 ng/mL or 10 µg/mL, respectively, and when indicated, 5 μg/mL of anti-Siglec-9 blocking antibody (clone 191240; R&D Systems) was used, and the plate incubated overnight. Supernatant was harvested for cytokine determination by ELISA. Expression of surface molecules was determined using the following antibodies: anti-CD80-FITC (clone 2D10; BioLegend), anti-CD83-PE-Cy7 (clone HB15e; BD Biosciences), anti-CXCR5-BV711 (clone J252D4; BD Biosciences, San Jose, CA), anti-ILT3-BV510 (clone ZM4.1; BioLegend), anti-ICOSL-PE-CF594 (clone C398.4A; BD Biosciences), and anti-CCR7-PE (clone 3D12; MBL, Nagoya, Japan). The moDCs were harvested, washed, and reseeded in a sterile 96-well flat bottom plate at a concentration of 1 × 10⁴ cells/well. Total CD4+ T cells were isolated from autologous donors with a CD4+ T-Cell Isolation Kit (Miltenyi Biotec) and frozen. Autologous CD4+ T cells were thawed and left to rest overnight at 37°C, 5% CO₂ before use. The CD4+ T cells were added to moDCs in a 1:10 ratio and incubated for 7 days at 37°C, 5% CO₂. These cocultures were performed in IMDM (Thermo Fisher) with 10% FCS, l-glutamine (2 mm), and penicillin/streptomycin (100 U/mL). At day 7, supernatant was harvested for cytokine determination by ELISA and T cells were restimulated with phorbol 12-myristate 13-acetate (0.5 μg/mL)/ionomycin (1 μg/mL; Sigma-Aldrich) in the presence of brefeldin A (BD Biosciences) and monensin (BD Biosciences) for 4 h at 37°C, 5% CO₂. Effects on T-cell function were evaluated by intracellular cytokine staining using anti-IFNγ FITC (clone 25723.11; BD Biosciences), anti-IL-13 BV421 (clone JES10-5A2; BioLegend), and anti-Ki67 APC (clone Ki-67; BioLegend), and surface staining of anti-CD25 BUV737 (clone 2A3; BD Biosciences), anti-CD69 PE-Cy7 (clone FN50; BioLegend), anti-HLA-DR BV510 (clone L243; BioLegend). Anti-CD3 BV605 (clone OKT3; BioLegend), anti-CD4 PerCP (clone OKT4; BioLegend), and anti-CD8 Pacific Orange (clone 3B5; Thermo Fisher) were used to delineate CD4+ T cells. Transcription factor staining buffer set (Thermo Fisher) was used to permeabilize cells for intracellular staining. Stained cells were acquired on the Aurora 5 Laser Flow Cytometer (Cytek Biosciences). FCS files were analyzed with FlowJo v10 (BD Biosciences, OR, USA).

IL-6, IL-10, IL-12p70 (eBioscience), and IL-8 (Invitrogen) secretion was measured in moDC supernatant, and IL-5 (BioLegend), IL-13 and IL-17 (Invitrogen), and IFNγ (eBioscience) secretion was measured in moDC-CD4+ T-cell coculture supernatant using a standard ELISA. Briefly, monoclonal antibodies to the respective cytokines were coated on a 96-well NUNC maxisorp plate and incubated overnight at 4°C. After washing plates with 0.05% PBS/Tween 20 and blocking with 1% bovine serum albumin (Roche, Basel, Switzerland)/PBS, with 0.05% PBS/Tween 20, supernatant was added together with biotin-conjugated antibodies to respective cytokines and incubated at room temperature for 2 h. Quantification of cytokines following addition of 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, MO, USA) and sulfuric acid to stop the reaction was done using human recombinant (rh) cytokines as standards. RhIL-6 (ImmunoTools), rhIL-8 (eBioscience), rhIL-10 (BD Biosciences), rhIL-12 (eBioscience), rhIL-17 (eBioscience), rhIL-13 (Invitrogen), rhIL-5 (BioLegend), rhIFNγ (Invitrogen) were used as standards for quantification of cytokine levels. Optical density was measured at 450 nm on an iMark™ Microplate Absorbance Reader (Bio-Rad, CA, USA).

All data were tested for normality with the Shapiro-Wilk normality test. In the case of normal distributed data, groups were compared with a paired Student’s t test. Groups with non-normal distributed data were compared with the Wilcoxon matched-pairs signed rank test. Because we did planned comparisons (medium vs. Der p 2 and Der p 2 vs. sia-Der p 2), p values or confidence intervals were not corrected for multiple comparisons. All tests were performed using GraphPad Prism version 10 (GraphPad, CA, USA). p values <0.05 were considered significant.

To evaluate the effects of sia-Der p 2 on moDC maturation and expression of inhibitory receptors (ICOSL and ILT3), known to expand T cells with regulatory capacities [22, 23], moDCs were matured with LPS or Pam3 in the presence of sia-Der p 2, Der p 2. In Pam3-matured moDCs, treatment with sia-Der p 2 (sia-Der p 2-moDC) resulted in a lower proportion of CD83+ (shown in Fig. 1a) and CXCR5+ (shown in Fig. 1b) but not CD80+ moDCs (shown in Fig. 1c) compared to moDCs treated with Der p 2 (Der p 2-moDCs) in the mite-allergic group. LPS-matured moDCs were not modulated by sia-Der p 2 (shown in Fig. 1a, f; online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000543157). The changes in moDC maturation were however not accompanied by significant changes in the production of cytokines (shown in online suppl. Fig. 2). For both ICOSL and ILT3, the presence of sialic acids on Der p 2 did not modulate the proportion of cells expressing them (shown in online suppl. Fig. 3). However, there was a trend toward higher frequencies in LPS-treated sia-Der p 2-moDCs in the mite-allergic group (shown in online suppl. Fig. 3). Surprisingly, Der p 2-moDCs are present with a higher frequency of ICOSL+ moDCs in the non-atopic group of the Pam3-matured moDCs (shown in online suppl. Fig. 3). These results indicate that compared to Der p 2, sia-Der p 2 prevented the upregulation of CD83 and CXCR5 induced by Pam3 in mite-allergic volunteers.

Given the phenotypic changes exerted by sia-Der p 2 on moDCs, we next determined how moDCs exposed to sia-Der p 2 affected T-cell function. The activation and proliferative status of T cells at day 7 of coculture was analyzed by measuring expression of classical activation markers CD25, CD69, and HLA-DR and proliferation marker, Ki67. While the early T-cell activation marker CD69 was not affected by sia-Der p 2-moDCs (shown in online suppl. Fig. 4), the proportion of CD25+ T cells (shown in Fig. 2a, b) and to a lesser extend HLA-DR+ T cells (shown in online suppl. Fig. 4) was lower in T cells of the mite-allergic group. This observation was true in both cocultures of Pam3 and LPS-matured moDCs with T cells (shown in Fig. 2a, b). The levels of expression of CD25 were also reduced in these cells (shown in online suppl. Fig. 4). Moreover, compared to Der p 2-moDCs, sia-Der p 2-moDCs limited the proliferation of T cells of mite-allergic patients (shown in Fig. 2c, d), as indicated by lower percentages of Ki67+ T cells. No alterations were observed in the non-atopic group (shown in Fig. 2a, d). Since α2-3 sialic acids are known ligands for Siglec-9, we next tested the involvement of Siglec-9 in the observed inhibitory effects. We have previously shown that sia-Der p 2 binds to Siglec-9 and to DCs [17]. We thus treated moDCs with an anti-Siglec-9 antibody prior to treatment with the allergen as previously tested in cellular inhibition studies [24]. We could then no longer detect the expression of Siglec-9 (shown in online suppl. Fig. 5). However, the inhibitory effects of sia-Der p 2 on T-cell activation and proliferation were not reverted by blocking Siglec-9 (shown in online suppl. Fig. 5). Taken together, these data demonstrate that sia-Der p 2 unlike Der p 2 reduced CD4+ T-cell activation and proliferation in mite-allergic patients but non-atopic individuals.

Besides proliferation, cytokine production is an important feature of T-cell function. Since cytokines like IL-13 and IL-5 are major players in the development of allergies [25], we evaluated how sia-Der p 2-moDCs affect IL-13-expressing T cells and their production of IL-13. Compared to Der p 2-moDCs, sia-Der p 2-moDCs matured with Pam3 significantly limited the frequency of IL-13+ T cells in the mite-allergic group (shown in Fig. 3a, b). These changes were not observed in the non-atopic group (shown in Fig. 3a), suggesting an antigen-specific response. In LPS-matured DCs, sia-Der p 2-moDCs did not significantly alter the frequency of IL-13+ T cells in both volunteer groups (shown in Fig. 3a, e). We did not observe significant differences in the levels of IL-13 (shown in Fig. 3c, f), or IL-5 secretion (data not shown), between Der p 2 and sia-Der p 2 conditions in both volunteer groups in either Pam3 (shown in Fig. 3c) or LPS (shown in Fig. 3f)-matured moDCs. Thus, these data show that Pam3-matured moDCs treated with sia-Der p 2 but not with Der p 2 limit the expansion of IL-13+ T cells in an antigen-specific fashion.

Th1 responses are often characterized by the secretion of IFNγ in the immune response against allergens [26]. We therefore examined the frequency of IFNγ+ T cells in our study. Coculturing T cells from allergic volunteers with sia-Der p 2-moDCs matured with either LPS or Pam3 resulted in a lower frequency of IFNγ+ T cells when compared to Der p 2-moDCs (shown in Fig. 4a, c). This effect was not observed in T cells from non-atopic volunteers. Concomitantly, we observed a decrease in the levels of IFNγ secretion from T cells of the allergic group when cocultured with sia-Der p 2-moDCs matured with LPS (shown in Fig. 4f). We next assessed whether Siglec-9 was involved in this response by treating moDCs with a blocking antibody to Siglec-9 prior to adding the allergens. There were no significant changes in the frequency of IFNγ+ T cell or IFNγ secretion upon blocking Siglec-9 on moDCs (shown in online suppl. Fig. 5). It must be pointed out that in 3 out of the 5 mite-allergic volunteers tested, the proportion of IFNγ+ T cells were slightly increased in the sia-Der p 2 group in the presence of the Siglec-9 blocking antibody (shown in online suppl. Fig. 5). We can conclude that sia-Der p 2 prevents the expansion of IFNγ+ T cells and IFNγ secretion from T cells when moDCs are matured with LPS.

The role of IL-10 in the success of AIT cannot be overstated. In fact, the regulatory response observed in HDM sublingual immunotherapy is attributed to IL-10 and TGF-β [27]. We therefore assessed the immunomodulatory effects of sia-Der p 2 on IL-10 secretion CD4+ T cells cocultured with (sia)-Der p 2-moDCs. In the mite-allergic group, LPS-matured moDCs treated with sia-Der p 2 significantly increased IL-10 secretion from CD4+ T cells compared Der p 2-moDCs (shown in Fig. 5a). Of note, Der p 2-moDCs significantly decreased IL-10 production from T cells from mite-allergic donors (shown in Fig. 5a). No effects on IL-10 production were observed when moDCs were matured with Pam3 in both volunteer groups (shown in Fig. 5b). Using an anti-Siglec-9 blocking antibody to block Siglec-9 on moDCs did not alter the production level of IL-10 from T cells cocultured with sia-Der p 2-moDCs (shown in online suppl. Fig. 5). In summary, sia-Der p 2 induced higher levels of IL-10 secretion in moDC T-cell cultures than Der p 2 in LPS maturation conditions.

In this study, we investigated the effect that a sialic acid-conjugated version of Der p 2 would have on immune cells from both mite-allergic and non-atopic volunteers. We focused on the activation of DCs and, ultimately, on the inhibition of T-cell proliferation. We further investigated sia-Der p 2’s potential to modulate Th1 and Th2 cytokines and Treg responses (assessed by the production of IL-10). Also, this study aimed to extend previous findings from us and others showing that using sialic acids to target DCs can dampen inflammation [15, 18]. We demonstrated that treating moDCs with α2-3 sialic acid-conjugated Der p 2 resulted in the down modulation of CD4+ T-cell activation and proliferation, and limited the expansion of IL-13+ and IFNγ+ CD4+ T cells. Notably, the immune modulating effects produced by sia-Der p 2 were restricted to mite-allergic donors, suggesting an antigen-specific response.

Phenotypic changes in DCs are suggested to drive the corresponding changes in T cells associated with successful AIT [4, 28, 29]. We therefore investigated the effects of sia-Der p 2 on moDCs. In Pam3-matured sia-Der p 2-moDCs, we observed a suppression of moDC maturation compared to Der p 2-moDCs, while in LPS-matured moDCs, no changes in maturation were observed. Very little is known about the involvement of well-known DC markers like CD83 in AIT and even less of CXCR5. Nevertheless, it is acknowledged that CD83 on DCs potentiates their capacity to induce allogeneic T-cell responses [30] and DCs treated with tolerizing pharmacological agents decrease the expression of CD83. This is commonly associated with T-cell unresponsiveness [31]. On the other hand, CXCR5 on DCs accelerates the development of both T follicular helper cells and Th2 effector cells [32]. We have demonstrated here that sia-Der p 2 does not increase the expression of these markers on moDCs, unlike Der p 2 and allergen extracts [33]. Consequently, it may allow for the rapid development of tolerance. No alteration was observed in the expression of CD80, which is in line with another study showing that the generation of tolerogenic DCs using IL-10 did not lead to alterations in CD80 expression levels [34].

The upregulation of inhibitory receptors like ICOSL and ILT3 on moDCs can render them tolerogenic and reduce their capacity to activate effector CD4+ T cells [35] or promote their capacity to induce CD4+ T-cell anergy [36]. Although we did not observe significant differences in the frequency of sia-Der p 2-moDCs or Der p 2-moDCs expressing ICOSL and ILT3, other tolerogenic markers, which were not measured in this study, may play a role in the resulting changes in T cells observed, specifically because the effects here reported cannot be attributed entirely to a reduction of activation markers which were unaltered in LPS-matured DCs. The absence of significant differences in ICOSL and ILT3 expression could either be due to the heterogeneity between donors or the small number of samples tested or that sia-Der p 2 did not have the optimal amount of sialic acids necessary to significantly induce the upregulation of these markers. Nonetheless, the fact that we also observed a significant decrease in ILT3+ moDCs in Pam3-matured moDCs treated with Der p 2 stresses out that using an allergen-like Der p 2 without any form of modification may not be ideal to accelerate the development of tolerance in AIT.

Functional and phenotypic changes on DCs are important for the successful polarization of T cells toward appropriate effectors required to either mount a restorative immune response or regulate ongoing immune responses. In this study, the effects of moDCs on autologous CD4+ T-cell responses were measured by assessing their activation (CD25, CD69, HLA-DR), proliferative status (Ki67), and functional (IL-13, IL-10, and IFNγ) activity. With the exception of CD69 and IL-10, the expression of all the above-listed T-cell functional markers was limited upon interaction with sia-Der p 2-moDCs, but the reverse was true upon exposure to Der p 2-moDCs. Sustained or increased expression of molecules like CD25 and HLA-DR on T cells has been linked to enhanced T-cell proliferation [37] and bias toward a Th2 phenotype [38]. Since CD25 is also a marker for Tregs, it is necessary to further investigate how sia-Der p 2 affects its expression in this context. CD69 has previously been implicated in the exacerbation of airway inflammation [39], but sia-Der p 2-moDCs failed to modulate its expression. Similar to reports on HDM allergen extracts [40‒44], Der p 2 increased CD4+ T-cell proliferation and production of IL-13 in allergic individuals. This effect was lost when Der p 2 was decorated with α2-3 sialic acids. Consequently, CD4+ T cells treated with sia-Der p 2-moDCs proliferated less and produced less IL-13. It is known that inhibition of CD4+ T-cell proliferation and IL-13+ T cells are features of successful AIT [1, 29, 45, 46]. Therefore, it is possible that lower activation and proliferation of T cells by sia-Der p 2 may allow for accelerated development of tolerance.

The effects of sia-Der p 2 on IFNγ+ T cells and IFNγ secretion in the present study were contrary to what would be ideal for more effective AIT. Based on previous reports, successful AIT is often associated with an increase in IFNγ+ T cells [1, 46‒49]. Nonetheless, other studies [50, 51] like that of O’Brien and colleagues demonstrated that subcutaneous AIT resulted in a reduction in expression of IFNγ in circulating lymphocytes [51]. Furthermore, even though IFNγ+ T cells are assumed to be protective against the development of allergy, an excessive induction of Th1-dominated responses in allergy could result in significant tissue injury [52]. This signifies that IFNγ+ T cells may play a double-sided role in the success of AIT depending on the route of administration and the extent of induction. Several reports have demonstrated that allergen extracts significantly decreased the levels of IL-10 in immune cells from allergic patients [53‒56], contributing to the pathogenesis of allergy. We observed a similar decrease in IL-10 levels of mite-allergic volunteers when their cells were treated with Der p 2 but not with sia-Der p 2, suggesting that sia-Der p 2 unlike Der p 2 and allergen extracts does not affect the IL-10 regulatory arm of the immune response, which may thus allow for rapid development of tolerance during AIT.

We used a blocking antibody to Siglec-9 to investigate whether the observed differences between Der p 2 and sia-Der p 2 were mediated by sialic acid-Siglec-9 interactions but our data are not conclusive to demonstrate the involvement of Siglec-9. We did however observe a possible involvement of Siglec-9 in 2 out of 6 allergic donors tested, where blocking Siglec-9 on moDCs reversed the suppressive effect of sia-Der p 2 on CD25+ and Ki67+ T cells. One explanation could be that the activity of the anti-Siglec-9 antibody used in this study was not strong enough to block the signaling of Siglec-9. Moreover, the present sample size was quite small and might lack sufficient power necessary to make definite conclusions.

In conclusion, we demonstrated that unlike Der p 2, sia-Der p 2 did not enhance the activation and proliferation of CD4+ T cells in mite-allergic patients but not in non-atopic volunteers. Also, sia-Der p 2 did not alter the IL-10 response as did Der p 2. These findings showed that if whole allergens are to be used in AIT, modifying them with immunosuppressive agents like sialic acids may make them more efficacious. There is however a need for further investigations to [1] make sia-Der p 2 more immunosuppressive (by increasing the multivalency of sialic acids) [2], decipher the mechanism involved, and [3] ensure its safety.

The authors would like to thank Yasmin te Winkel, Inge Bruins, Katarina Olesek, Fabio Balzarini, Nadia van der Meijs, Babet Springer, Celine Sewnath, Stefanie Busold, Charlotte Castenmiller, Noemi Nagy, Sabine Haggenburg, Quincy Hofsink, and Toni van Capel for helping out with recruiting and processing samples from donors. We say a big thank you to all the blood donors for providing blood. We are grateful to the members of the Microscopy & Cytometry Core Facility, Department of Molecular Cell Biology and Immunology, Amsterdam University Medical Center, for assisting in all flow cytometry work.

This study protocol was reviewed and approved by the Amsterdam University Medical Centers Medical Ethics Committee guidelines, Approval No. NL71330.018.19. Written informed consent was obtained from all participants to participate in the study.

Y.K. and E.E.P. are involved in DC4U technologies, which develops glycan-based technologies that enable steering the human immune response. R.R. receives consultancy fees from HAL Allergy BV, Citeq BV, Angany Inc., Reacta Healthcare Ltd, AB Enzymes, Mission MightyMe, and The Protein Brewery, receives speaker’s fees from HAL Allergy BV, ALK, and Thermo Fisher Scientific, and possesses stock options at Angany Inc. All other authors declare no conflict of interest.

This study was supported by grants from HEALTH HOLLAND (HH LSHM19073), DC4U Technologies, and Cytek Biologics.

B.-C.K.D., R.R., E.E.C.J., and Y.K. conceived the study; R.R. provided crucial reagents; B.-C.K.D. designed the experiments, analyzed the results, and wrote the manuscript; B.-C.K.D., E.N., E.E.P., H.K., and S.A.V. performed experiments; R.R., E.C.J., and Y.K. critically edited and revised the manuscript, and supervised the work.

Edited by: H.-U. Simon, Bern.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author (Y.K.) upon reasonable request.