New insights into the immunomodulatory potential of sialic acid on monocyte-derived dendritic cells

doi:10.21203/rs.3.rs-3981826/v1

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New insights into the immunomodulatory potential of sialic acid on monocyte-derived dendritic cells

https://doi.org/10.21203/rs.3.rs-3981826/v1

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published 02 Nov, 2024

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Sialic acids at the cell surface of dendritic cells (DCs) play an important immunomodulatory role, and their manipulation enhances DC maturation, leading to heightened T cell activation. Particularly, at the molecular level, the increased stability of surface MHC-I molecules in monocyte-derived DCs (MoDC) underpins an improved DC: T cell interaction. In this study, we focused on the impact of sialic acid remodeling by treatment with C. perfringens sialidase on MoDCs' phenotypic and functional characteristics. Our investigation juxtaposes this novel approach with the conventional cytokine-based maturation regimen commonly employed in clinical settings.

Notably, C. perfringens sialidase remarkably increased MHC-I levels compared to other sialidases having different specificities, supporting the idea that higher MHC-I is due to the cleavage of specific sialoglycans on cell surface proteins. Sialidase treatment induced rapid elevated surface expression of MHC-I, MHC-II, and CD40 within an hour, a response not fully replicated by 48h cytokine cocktail treatment. These increases were also observable 48h post sialidase treatment. While CD86 and PD-L1 showed significant increases after 48h of cytokine maturation, 48h post sialidase treatment showed a higher increase of CD86 and shorter increase of PD-L1. CCR-7 expression was significantly increased 48h after sialidase treatment but not significantly affected by cytokine maturation. Both treatments promoted higher secretion of the IL-12 cytokine. However, the cytokine cocktail induced a more pronounced IL-12 production. SNA lectin staining analysis demonstrated that the sialic acid profile is significantly altered by sialidase treatment, but not by the cytokine cocktail, which causes only slight sialic acid upregulation. Notably, the lipid-presenting molecules CD1a, CD1b, and CD1c remained unaffected by sialidase treatment in MoDCs, a finding also further supported by experiments performed on C1R cells. Inhibition of endogenous sialidases Neu1 and Neu3 during MoDC differentiation did not affect surface MHC-I expression and cytokine secretion. Yet, sialidase activity in MoDCs was minimal, suggesting that sialidase inhibition does not significantly alter MHC-I related functions. Our study highlights the unique maturation profile induced by sialic acid manipulation in MoDCs. These findings provide insights into the potential of sialic acid manipulation as a rapid immunomodulatory strategy, offering promising avenues for targeted interventions in inflammatory contexts.

Immunomodulation

Dendritic cells

Sialic acid

Antigen-presentation

MHC-I

Dendritic cells (DCs) play a pivotal role in orchestrating immune responses, as professional antigen-presenting cells, crucial to activate T cells and generate immunological memory. Due to this role in stimulating the immune response, DCs emerged as a promising resource for immunotherapy, capable of initiating immune response against infected and cancer cells or dampening the immune response in autoimmune diseases and transplantation^1,2.

The sialic acid content on the cell surface emerges as a finely tuned regulatory mechanism of immune cells, exhibiting variations across cell types, differentiation states, and environmental stimuli³. In monocyte-derived dendritic cells (MoDCs), the activity of sialyltransferases during differentiation and maturation dynamically modulates the content of cell surface sialylated structures (sialoglycans)^4,5. Although much of sialylation regulation has been attributed to sialyltransferases, sialidases also play a role, modulating sialoglycan content. During their differentiation from monocytes, MoDCs overexpress Neu1 and Neu3⁶, which respectively act on glycoproteins and gangliosides. Previous studies show that cell surface sialic acids in DCs contribute to dampen maturation and down-regulate the ability to activate T cells^7–11. This immunomodulation may be operated through the interactions between sialoglycans and sialic acid binding immunoglobulin type lectins (Siglecs), occuring in cis (within the same cell) or in trans (with another cell), triggering inhibitory signaling pathways¹². Consequently, sialic acid-Siglec engagements are now regarded as novel immune checkpoints, with ongoing development of strategies targeting these axes¹³. These strategies entail using sialidases to remove sialic acids from cancer cells^14,15, or the inhibition of sialyltransferase activity ¹⁶.

Maturation is essential for DCs to effectively respond to pathogens, allowing them to recognize and fight off infection. Microbial sialidases have been found to exert an immunomodulatory effect, particularly in infections, by cleaving sialic acid from the cell surface and unleashing protective barriers on host cells^17,18.

Either in infection or as experimental set up, sialic acid removal from the cell surface of human DCs by sialidases was shown to induce maturation^4,19, increasing antigen-presenting and co-stimulatory abilities, resulting in higher polarization of T cells towards a T helper type (Th) 1 phenotype. This improves the ability of autologous T cells to mediate tumour cell killing¹⁰. Additionally, we have demonstrated that the human major histocompatibility complex class I (MHC-I) is one of the sialylated proteins and its function is modulated by the removal of sialic acids, improved cell surface stability in DCs due to reduced turnover rates. This desialylation of DCs resulted in more and earlier immunological synapses with autologous CD8 + T cells, significantly increasing activation of T cells⁹. Additionally, increased general binding avidity between DCs and T cells was observed when a sialic acid-blocking mimetic was used to reduce the sialic acid content of DCs⁷, confirming that sialic acid content affects DC: T cell synapse.

DC-based vaccines typically imply isolating DCs’ precursors from the patient’s blood, followed by differentiation, maturation and antigen loading before re-introduction into the patient's body. These vaccines can be tailored to different production methods and therapeutic purposes, namely, to promote immunity against cancer or infectious cells or inducing tolerance to specific antigens²⁰.

In this work, we aimed to further elucidate the effect of sialidase treatment on the maturation features of MoDCs. Our findings reveal that sialic acid manipulation with a sialidase from C. perfringens generates MoDCs with a unique maturation profile, not achieved by other sialidases. This profile is characterized by a significant increase in the expression of MHC-I, MHC-II and CD40 immediately after 1h of sialidase treatment, which extends for 48h. These effects could not be replicated with a 48h of conventional cytokine-based maturation cocktail, typically used in clinical settings. A significant increase was also observed for PD-L1, CD86 and CCR-7, 48h after sialidase treatment. On the other hand, cytokine maturation only affected PD-L1 and CD86 expression, while not being able to significantly increase the expression of CCR-7. IL-12 secretion was significantly increased by both treatments, although maturation with the cytokine cocktail achieved much higher levels. Interestingly, the sialidase treatment improved stabilization of the MHC-I, and MHC-II molecules for up to 48h, without affecting the levels of other antigen-presentation molecules.

2.1. Cells

Human primary DC were differentiated from monocytes (MoDCs) as previously described¹⁰.. In brief, monocytes were isolated by immunomagnetic separation and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (ThermoFisher Scientific, cat. no. 1870025), supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific, cat. no. 10500064), 2 mM L-glutamine, 100 g/mL penicillin/streptomycin (ThermoFisher Scientific cat. no. 15140122), 1 mM sodium pyruvate (ThermoFisher Scientific, cat. no. 11360070) and 1% non-essential amino acids (ThermoFisher Scientific, cat. no. 11140050), in the presence of 750U/ml of human recombinant interleukin-4 (IL-4) (R&D Systems, cat. no. BT-004) and 1000U/ml of human recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) (Miltenyi Biotec, cat. no. 130-095-372), for 5 to 6 days. At the third day of differentiation, the culture medium was refreshed and supplemented with 750U/ml of IL-4 and 1000U/ml of GM-CSF. The C1R cell lines (human B-cell lymphoblastoid line) transfected with the expression vector containing cDNAs encoding either CD1a, CD1b, or CD1c. Cells were cultured in RPMI-1640 medium, supplemented as described above.

2.3 Cell treatments

2.3.1 Sialidase

Enzymatic removal of sialic acid from the cell surface was performed using sialidases from Clostridium perfringens (Merck/Roche, cat. no. 11585886001), from Vibrio Cholera (Merck/Roche, cat. no.7885), Macrobdella decora (Sigma-Aldrich Merck, cat. no. 480706) and O-sialoglycoprotrein endopeptidase (Accurate, cat. no. ACLE100) as described²¹. Briefly, cells (5 × 10⁶ / mL) were resuspended in RPMI medium and 100 mU of sialidase for each 10⁶ cells and incubated for 1h at 37ºC. After incubation, 1 ml of RPMI medium containing 10% FBS was added to stop the enzymatic reaction, and the cells were centrifuged, discarding the supernatant. Mock-treated control was performed in parallel using the same incubation conditions but with boiled sialidase.

2.3.2 Cytokine Cocktail

For the cells treated with the cytokine cocktail, after the differentiation step, the medium was refreshed and a cytokine cocktail comprising Interleukin (IL)-1β (Sigma-Aldrich, cat. no. IL038) (10 ng/ml), IL-6 (Merck, cat. no. I1395) (1000 U/ml), Prostaglandin E2 (PGE2), (Merck, cat. no. P6532), (1 µg/ml) and TNF-α, (Sigma-Aldrich, cat. no. T6674) (10 ng/ml) was added for 48 hours.

2.3.3 Sialidase Inhibitors

To inhibit endogenous sialidase activity, 1 mM of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), (Merck, cat. no. D9050) or 1 mM of 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (Zanamivir), (Merck, cat. no. SML0492) were added to the culture medium during the differentiation period.

2.4 Flow cytometry analysis

For staining with antibodies and lectins, cells were resuspended (1x10⁶ cells/mL) in RPMI supplemented with 10% FBS and stained with the following antibodies or lectins: fluorescein isothiocyanate (FITC)- conjugated anti-MHC-I (W6/32) (Immunotools, cat. no. 21159033); allophycocyanin (APC) conjugated anti-MHC-II (GRB-1) (Immunostep, cat. no. PHLADRF-100T); anti-CD1a (OKT6) (Immunotools, cat. no. 21159033); APC conjugated anti-CD1b (SN13) (Biolegend, cat. no. 32919110); phycoerythrin (PE) conjugated anti-CD1c (AD5-8E7) (Miltenyi Biotec, cat. no. 1200-000-889); (FITC)- conjugated anti-CD86 (BU63) (Immunotools, cat. no. 21480863); APC conjugated anti-CD40 (HI40a) (Immunotools, cat. no. 21270406); FITC conjugated anti-CCR-7 (G043H7) (Biolegend, cat. no. 353216), PE-conjugated anti- PD-L1 (MIH1) (BD Biosciences, cat. no. 557924), and biotin conjugated Sambucus nigra lectin (SNA) (Vector labs, cat. no. B-1305-2). Staining was performed at 4ºC for 30 minutes in the dark and cells were washed after. For the SNA staining streptavidin-PE was added and incubated at 4ºC for 30 minutes in the dark. Quality control of the flow cytometer’s performance and coefficient of variation (CV) values were monitored on a day-to-day basis using performance tracking beads (Thermofisher, cat. no. 4449754).

2.5 Gene expression assay

The RNA was extracted from 1×10⁶ MoDCs using the GenElute Mammalian Total RNA Miniprep Kit (Sigma, cat. no. RTN70-1KT). RNA concentration of each sample was determined spectrophotometrically. The RNA was reverse transcribed with random primers using the HighCapacity cDNA Reverse Transcription Kit (Thermoscientific/Applied Biosystems, cat. no. 4368814).

Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) was performed in Rotor-Gene 6000 Series (Corbett Research Ldt., UK) using TaqMan Universal PCR Master Mix II, TaqMan probes and primers from TaqMan Gene Expression Assays (Thermofisher Scientific). The assay ID provided by the manufacturer are the following: IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1); IL-10 (Hs00174086_m1); IL-12a (Hs00168405_m1); tumor necrosis factor (TNF-a; Hs00174128_m1); glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 4333764F); β-actin (4352935E). Each reaction was performed in duplicate. Thermal cycling conditions were 95ºC for 20 seconds followed by 40–50 cycles of 95ºC for 3 seconds, and 60ºC for 30 seconds.

Messenger RNA (mRNA) expression was normalized using the geometric mean of the expression of the GAPDH and β-actin genes as a reference. The relative expression of each gene was calculated according to the 2^−ΔCT* 1000 method²². The efficiency of the amplification reaction for each primer/probe is above 95% (as determined by the manufacturer).

2.6 Cytokine production evaluation

The production of IL-12 was assessed in culture supernatants by an enzyme-linked immunosorbent assay (ELISA) technique using Human Interleukin-12p40-total development kit (Immunotools, cat. no. 31679129), following the manufacturer’s instructions. Cytokine concentration was calculated using the specific standard curves.

2.7 Measurement of endogenous sialidase activity.

Sialidase activity was measured in lysates from MoDCs (0.5x10⁶ cells) that were not stimulated or stimulated with 5 or 50 µg/mL of lipopolysaccharide (LPS) for 60 minutes. The assay mixture consisted of 200 mM citrate-phosphate buffer (pH 4.5) containing 0.1% BSA (w/v), 0.5 mM4MU-Neu5Ac (TCI Chemicals), and the sample fractions (10 µL) in a final volume of 100 µL. After incubation for 30 min at room temperature, the reaction was terminated by the addition of 100 µL of 1M Na₂CO₃ (pH 10.4), and the amount of 4-MU released was determined fluorometrically with a FLUOstar Optima microplate reader (BMG Labtech) using an excitation wavelength of 360 nm and emission wavelength of 450 nm. One unit of sialidase activity was defined as the amount of enzyme that released 1 µmol sialic acid/min at 37ºC.

2.8 Confocal laser scanning microscopy

To perform confocal laser scanning microscopy, cells were plated on 12-mm diameter polylysine-coated glass coverslips and incubated for 5 min at room temperature. Coverslips were then centrifuged at 100 × g for 1 min to promote cell adhesion, fixed for 30 min with 4% paraformaldehyde (PFA) and washed using 1% bovine serum albumin (BSA) in PBS. Mouse anti-human HLA-ABC, clone 246-B8.E7 (Invitrogen. cat. no. 11504371) was used for staining human MHC-I, followed by a fluorescently conjugated secondary antibody. Images were acquired on a Zeiss LSM710 confocal microscope (Zeiss). Illustrative confocal cross-section pictures were selected after Z-stacking processing. Staining intensity was analytically quantified using the corrected total cell fluorescence (CTCF) = Integrated Density – (Area of selected cell × Mean fluorescence of background readings).

2.9 Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, USA). Unless otherwise stated, Statistical significance (p-value) was calculated using the ratio paired t-test. Statistical significance was defined as p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

2.10 Data availability

Data was generated by authors and is available on request.

3.1 The modulation of MHC-I expression by sialic acid removal in MoDCs is dependent on the nature of the sialidase

In our previous study, we demonstrated that treatment of immature MoDCs with a sialidase from C. perfringens led to an increase in the cell surface expression of MHC-I, attributed to enhanced stability of MHC-I molecules at the cell surface⁹. To assess which sialoglycans influenced MHC-I stability, we used sialidases from various sources as described in the Material and Methods section. These sialidases modulate the cell surface sialome with different specificity: sialidase from C. perfringens cleaves α2,3, α2,6 and α2,8-linked sialic acids; sialidase from V. cholerae removes α2,3-linked sialic acids decorating gangliosides (sialic acid-containing lipids); sialidase from M. decora acts on sialic acid α2,3-linked to galactose; and O-sialoglycoprotein endopeptidase specifically cleaves O-sialoglycoproteins. The activities of these enzymes towards sialoglycans in MoDCs surface was confirmed by staining with PNA lectin and evaluation by Flow cytometry (Supplementary Figure S1).

Treatment with the sialidase from C. perfringens resulted in a remarkable 5-fold increase in MHC-I levels compared to untreated cells, whereas other sialidases showed no significant effect (Figure 1A). This increase in the expression of MHC-I at the cell surface was confirmed by confocal microscopy images showing higher fluorescence intensity for MHC-I staining in C. perfringens sialidase-treated MoDCs, compared to untreated cells (Figures 1B-C). Notably, C. perfringens sialidase treatment also augmented MHC-I expression in THP-1 cells, a monocyte cell model (Supplementary Figure S2), suggesting that the observed mechanism is shared by different antigen-presenting cells. These findings suggest that the modulation of MHC-I expression in MoDCs is intricately linked to the trimming of α2,3, α2,6-linked sialic acids, mostly from proteins. C. perfringens sialidase was hereby used to manipulate the sialic acid content of MoDCs in the subsequent assays.

3.2 Sialidase-treated MoDCs show differential phenotypic and functional characteristics when compared to a cytokine maturation cocktail used in clinical trials

We have previously shown that the removal of sialic acids by sialidase treatment induces DC maturation within an hour, as evidenced by increased expression of antigen-presenting and co-stimulatory molecules, suggesting a potential new technology to mature DC to be used in clinical settings⁹. To better understand the clinical potential of the sialidase effect, we compared its effect with the maturation protocol used in clinics, namely the “gold standard” maturation cocktail composed of IL-1β, IL-6, PGE2 and TNF-α for 48h²³. As shown in Figure 2, both treatments increased the surface expression of MHC molecules and maturation markers. Immediately after sialidase treatment, there was a significant 5-fold, 3-fold, and 5-fold increase of MHC-I, MHC-II and CD40, respectively, while cytokine cocktail led to a non-significant 2-fold increase for the same molecules. On the other hand, with cytokine cocktail, there was a significant 37-fold and 11-fold increase of CD86 and PD-L1, respectively, whereas, immediately after sialidase treatment, there was only 15-fold and 2-fold marginal increase of CD86 and PD-L1, respectively. For CCR-7, there was a negligible 1.6-fold increase with the cytokine cocktail and 1.5-fold increase immediately after sialidase treatment (Figure 2A). To assess the effect of sialidase treatment over the same time as the cytokine cocktail, we analyse its impact after 48h. 48h post sialidase treatment, CD86, PD-L1 and CCR-7 levels significantly increased by 167-fold, 4-fold and 2.6-fold, respectively (Table 1).

The Th1-inducing cytokine, IL-12 was measured in the culture supernatants after 48h of cell culture post-with either sialidase or with the cytokine cocktail. The levels of IL-12 increased approximately 26 times with the sialidase treatment, and 223 times with the cytokine cocktail (Figure 2B). To evaluate sialic acid content after sialidase and cytokine cocktail treatments, MoDCs were stained with the SNA lectin and evaluated by flow cytometry. While, as expected, sialidase treatment decreased the staining with SNA resulting from the removal of α-2,6 sialic acids, cytokine maturation caused a non- significant increase in SNA staining (Figure 2C).

Table 1. Cell surface expression of maturation markers in MoDCs after sialidase treatment (0h and 48h) or cytokine maturation (48h).

	Control	Sialidase treatment				Cytokine maturation
	0h^#	0h^#	48h^#			48h^#
	MFI (mean±SEM)	MFI (mean±SEM)	P	MFI (mean ±SEM)	P	MFI (mean±SEM	P
CD86	71±26	1094±191	ns	11879±750	*	2632±368	*
PD-L1	2718±1153	3954±737	ns	12080±3892	***	28870±4828	*
CCR-7	1757±441	2570±217	ns	4588±110	*	2774±59	ns
MHC-I	7312±4370	37141±5172	*	51769±8430	*	13961±1700	ns
MHC-II	22043±5377	59476±8158	*	450290±79416	*	37681±20079	ns
CD40	7574±3064	33978±4643	*	420429±	*	17611±5741	ns

ns- not significant, # time post-treatment. Cytokine maturation represents MoDCs differentiated from monocytes and then maturated for 48h with a cytokine cocktail composed of IL-1β (10ng/mL), IL-6 (1000 U/mL), TNF-α (10 ng/mL), and PGE2 (1µg/mL). Statistic significance was performed against the control, using ratio paired t-test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

To understand whether sialidase treatment induces significant transcriptional changes of cytokines, we evaluated the expression of genes (IL-12, TNF-α, IL-1β, IL-10 and IL-6) by RT-PCR. At 3h, the sialidase-treated MoDCs have a more pronounced transcription of IL-12 than MoDCs treated with a cytokine cocktail (Figure S3). After 6h, the transcriptional levels were lowered for sialidase-treated cells and were highly increased for MoDCs treated with the maturation cocktail. For TNF-α, the mRNA levels are higher at 3h and 6h for DCs treated with the cytokines, while for IL-1β, the mRNA levels are higher for sialidase treated MoDCs than for the cytokine maturated MoDCs. For IL-10 and IL-6, the mRNA levels are very low for all the tested conditions (Supplementary Figure S3). These cytokine expression profiles highlight critical differences between sialidase and cytokine stimulation, with potential clinical interest. Altogether, this data reinforces the potential clinical benefit of the sialidase effect, as a rapid strategy to improve peptide presentation and CD40 co-stimulation and a better ability to control PD-L1 expression in MoDC maturation.

3.3 Sialidase treatment differently affects the expression of CD1 lipid- presenting molecules

We then investigated the impact of sialidase treatment on the expression of CD1 lipid-presenting molecules, to determine whether it parallels the observed enhancement in MHC class I molecules. CD1a, CD1b and CD1cshare sequence and structural homology with MHC class I, and are also decorated with sialic acid-containing glycans⁹. To assess the impact of sialic acid removal on the levels of CD1 molecules over time, the DCs were evaluated immediately after sialidase treatment and at subsequent time points during culture (Figures 3A, B). As expected, the augmented MHC-I and MHC-II expression levels of sialidase-treated cells was kept over time (Figure 3B). However, for the lipid-presenting molecules CD1a, CD1b, and CD1c there were no significant differences between the non-treated and treated cells at any of the points tested (Figures 3A, B). Additionally, we tested the effect of sialidase treatment on the human B-cell lymphoblastoid line C1R, transfected with CD1a, CD1b or CD1c. While the levels of CD1a, CD1c were not affected by sialidase treatment, a negligible impact was observed on CD1b levels (Figures 3C, D). From these observations, we can conclude that sialic acid removal in MoDCs improves the expression of MHC but not CD1 molecules, suggesting that it affects only peptide but not lipid antigen presentation.

3.4 Inhibition of endogenous sialidases during monocyte differentiation does not affect MoDCs

MoDCs express the endogenous sialidases Neu 1 and Neu 3, which act preferentially on glycoproteins and gangliosides, respectively⁶. To assess the potential impact on MoDCs’ phenotype of inhibiting Neu 1 and Neu 3 activity during their differentiation from monocytes, we used two inhibitors: DANA, a potent Neu1 and Neu3 inhibitor, or Zanamivir, a strong Neu3 inhibitor. Monocytes were differentiated in the presence of these inhibitors and the expression of cell surface markers (MHC-I, MHC-II, CD40, CD86, PD-L1) was assessed by flow cytometry. Interestingly, the inhibition of endogenous sialidases by DANA or Zanamivir did not alter the expression of the DC surface markers (Figures 4A). Accordingly, the levels of the cytokine IL-12, measured in the culture supernatants, showed no difference for the DCs differentiated in the presence of the inhibitors when compared to DCs cultured with the control differentiation medium (Figures 4B). To evaluate MoDCs endogenous sialidase activity 4MU-Neu5Ac was used as the substrate. The activity detected for immature MoDCs (control) was 0.020 ±0.002 mU and for MoDCs stimulated for 60 minutes with LPS 5 and 50 µg/mL was 0.049 ±0.018 mU and 0.044±0.013 mU, respectively (Figures 4C). Interestingly, when sialidase inhibitors were added to the differentiation medium, the staining of MoDCs with SNA showed no significant differences. However, in comparison to the control, there was a slight increase in α-2,6-linked sialic acids, especially in the presence of DANA, the inhibitor of Neu1 and Neu3 (Figures 4D). While the staining results hint at subtle alterations in α-2,6-linked sialic acids, the overall impact of sialidase inhibitors appears negligible. In conclusion, in these experimental conditions, MoDCs showed very low sialidase activity, supporting the observation that the use of sialidase inhibitors does not significantly affect their phenotype.

The field of DC-based immunotherapy has witnessed a very promising but also challenging development with clinical trials evolving slowly due to concerns about effectiveness. Innovative approaches which improve DC-based vaccines are needed to increase their efficacy²⁴. Consequently, there is a pressing need to identify and understand mechanisms and complementary forms of maturing DCs.

While different methods exist for DC production, particular emphasis is placed on refining the methodology for generating DC derived from monocytes (MoDCs) due to their clinical potential. This method benefits from the existence of reliable techniques for large-scale production and widespread application in clinical settings^25,26.

Since DC's ability to present antigens is dependent on the MHC-I, understanding the dynamic of MHC-I, including its recycling and stabilization mechanisms in DCs is critical to improve effective clinical applications.

DCs exhibit a cell surface adorned with sialic acid, tightly regulated, and dynamically altered during differentiation and stimulation. We have previously shown that enforced desialylation improves expression of MHC-I at DC’s surface. Interestingly, here, through our experiments with different sialidases, we have shown that the modulation of MHC-I expression in MoDCs is linked to the trimming of α2,6-linked sialic acid by Clostridium perfringens sialidase, which also trims α2,3 and α2,8-linked sialic acids from cell glycoproteins. Our results corroborate previous studies in leukaemia cells comparing sialidase effects, including Clostridium perfringens, and demonstrated enhanced stimulatory capacity linked to the removal of specific membrane structures, likely α2-6 linked to N-linked glycosyl moieties²⁷. These findings add further insights into the already-known role of N-glycans in the dynamics of MHC-I²⁸, its conformation and strength of the TCR- MHC-I complex^29,30, highlighting the role of α2-6 sialylated N-glycans. These results extend to the recently revealed sialic acid impact on neoglycosphingolipids, indirectly influencing the function of MHC-I, by shielding and hindering its recognition³¹.

Our findings uniquely position the role of sialic acids as immune regulators, besides engaging with Siglecs on immune cells, and serving as immune checkpoints, akin to PD-1 and CTLA-4 receptors³². Furthermore, our findings underscore the relevance of sialidase technology as a tool for manipulating sialic acid content, thereby influencing the immune response.

Structurally similar to Siglecs, the T cells co-stimulation receptor CD28 also engages with sialic acids, either in cis or trans. This interaction with sialic acids competes with binding to the activator ligand CD80 on antigen-presenting cells, leading to attenuation of T cell responses¹¹. Although there are reports on the immunoregulatory role of sialic acids in DCs³³, we were the first to report their influence in MHC-I, leading to reduced internalization and higher stability accounting for the increased expression on the cell surface.

Interestingly, others demonstrated that depleting sialic acid in cancer cells disrupts intracellular trafficking, resulting in diminished recycling of internalized receptors and enhanced endosomal delivery³⁴. Therefore, it is likely that besides MHC-I other cell surface molecules also experience delayed trafficking in desialylated cells.

Surprisingly, the sialidase treatment did not affect the levels of the MHC-I-like glycoproteins- CD1 molecules, pointing to distinctive features of these molecules or neighboring molecules at the cell surface that make them unaffected by the action of sialidase. CD1 molecules present lipid antigens and, in DCs, do not undergo the same changes in intracellular trafficking as MHC-I during DC maturation ^35,36.

The role of glycosylation of MHC-I in assembling, epitope selection and antigen presentation, which occurs through chaperones and modifying enzymes, is well documented³⁷. The assembly of MHC-I is dependent on calreticulin binding to the monoglycosylated branch of the N-glycan, and its interaction with tapasin is also glycan dependent involving ERp57 and calreticulin³⁷. Importantly, the sialic acid removal using sialidase, described in this work, is unlikely to affect these processes because it is restricted to the cell surface when the MHC-I complex is already formed, being advantageous over other more drastic sialic acid removal strategies. Sialic acid is a charged monosaccharide capping glycan chains, whose role in protein turnover has been debated since desialylation does not affect the internalization of several cell surface glycoproteins³⁸. Yet, these studies have not enclosed MHC-I in human DCs, which, collectively with our observation that other molecules are not affected by sialidase, suggest that sialic acid affects the turnover of only specific proteins, potentially present in specific cell contexts.

Comparative analyses between sialidase treatment and cytokine maturation underscored distinct phenotypic and functional characteristics in MoDCs. DC-vaccines ex-vivo production aims for the expression of co-stimulatory molecules, low expression of immune inhibitory molecules, capacity for T cell polarization towards Th1 characterized by their ability to produce IL-12 upon contact with responding T cells, expression of CCR-7 to improve migration to lymph nodes, and superior capacity to activate cytotoxic T cell responses³⁹. By comparing the effects of sialidase treatment with cytokine maturation we essentially observe differences on the levels and the rate of expression of surface molecules. Sialidase treatment produced a very rapid (immediately after 1h) and steady increase of MHC-I, and other molecules for at least 48h, without significantly increasing PD-L1. Strategies to upregulate MHC-I without increasing the PD-L1 are very important. Therefore, sialidase treatment may be a very efficient strategy to rapidly improve antigen presentation through MHC-I as well as other critical molecules without increasing inhibitory effects. Another very important molecule that was significantly upregulated only by sialidase treatment and not by cytokine cocktail was CCR-7, which enhances DC migration to lymph nodes to encounter T cells.

Furthermore, the production of cytokines by DCs during the maturation process determines the immune responses generated by CD4⁺ T cells⁴⁰. Both sialidase treatment and maturation with cytokines upregulated the secretion of the Th1-inducing cytokine IL-12 at transcriptional and protein level. IL-12 is critical for the proliferation and lytic activity on T cells, stimulating the secretion of IFN, and anti-tumor function^39,41. Besides IL-12, the expression of cytokines such as TNF-α, IL1β, IL-6 are also critical for the activation of anti-tumor immune responses⁴². Importantly, the treatment with sialidase significantly increased the transcription of IL-1β and IL-6 genes which are inhibited by the cytokine cocktail. The signaling induced by these cytokines activates immune cells and drives polarization of CD4+ T cells towards Th1 and Th17 cells with a largely beneficial role in initiating adaptive anti-tumor responses⁴³. It is likely that the cytokine cocktail, containing IL-1β and IL-6, inhibits the expression of those cytokines by DCs by negative feedback, in agreement with the known IL-1β and IL-6 interplay⁴⁴. In contrast, sialidase treatment overcomes negative feedback, and the NF-κB pathway required for IL-6 and IL-1β expression is activated upon sialidase treatment of MoDCs¹⁹. Thus, sialidase treatment emerges as a reliable alternative strategy to induce DC maturation and sustain appropriate cytokine intracellular signaling.

In MoDC, endogenous sialidase activity has been associated with LPS induced cytokine production⁶. In our study, the expression of MoDCs surface markers was unchanged after treatment with inhibitors DANA and Zanamivir. Regarding the effects of these inhibitors, although it was reported that the LPS-induced cytokine production by MoDCs was affected by them, the expression of surface markers was unchanged,⁶which is in agreement with our results. Moreover, in our experimental conditions, we observed very little sialidase activity in control conditions and that activity was only slightly increased by LPS, which may explain the null effect of sialidase inhibitors on the phenotype of MoDCs. From these observations, we conclude that endogenous sialidase inhibition cannot be considered as a strategy to increase sialic acid content in MoDCs. However, it is possible to modulate the sialic acid content in MoDCs ex-vivo either by removing sialic acids using sialidase as shown in this work or by increasing its content by employing sialyltransferases externally, producing a destabilizing effect on MHC-I, opposite to stabilizing effect produced by sialic acid removal⁹.

Finding the optimized maturation protocol has been a challenge not yet surpassed due to the difficulty to replicate the natural body environment ex-vivo, which endows DCs with ideal antigen-presentation, co-stimulatory abilities and also migratory capacity towards the lymph nodes⁴⁵. In this work, we present new insights on the potential of the manipulation of sialic acid content as a means to tailor the production of DCs for clinical applications.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement

The buffy coats of human peripheral blood from healthy volunteers were provided by the Portuguese Blood and Transplantation Institute, after approval by institutional ethical committee. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Author contributions

Conceptualization and data curation: ZS, PV. Formal Analysis: ZS, JR, SS, PV. Funding acquisition: PV. Investigation: ZS, JR, PV. Methodology: ZS, JR, VL, ACO, RAL, PB. Project administration: ZS, PV. Resources: PV, MS, SS, PB. Supervision: ZS, PV. Validation: PV. Visualization: ZS, RAL, PV. Writing – original draft: ZS, PV. Writing – review & editing: all authors. All authors contributed to the article and approved the submitted version.

Funding

This work received financial support from Fundação para a Ciência e a Tecnologia (FCT) Portugal, under grants UIDP/04378/2020 and UIDB/04378/2020 (provided to the Applied Molecular Biosciences Unit – UCIBIO), LA/P/0140/2020 (provided to the Associate Laboratory Institute for Health and Bioeconomy – i4HB), from the European Commission through the GLYCOTwinning project (Grant Agreement: 101079417), and from the European Union’s Horizon 2020 research and innovation programme under the EJPRD COFUND-EJP N 825575 (EJPRD/0001/2020). VL for her fellowship through the DCMatters project (Portugal2020 (SI I&DT)- copromoção) and RAL (SFRH/BD/148480/2019) and ACO (UI/BD/153068/2022) thank FCT for their grant.

Acknowledgments

We thank all healthy volunteers for their participation in the study. We are grateful to the staff at IPST for assistance with processing of blood products. We thank Danielle Almeida for technical assistance and Tiago Carvalho for helping in materials and methods writing and for proofreading the manuscript.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Mariolina Salio is currently employed by Immunocore LTD.

Gardner A, de Mingo Pulido Á, Ruffell B (2020) Dendritic Cells and Their Role in Immunotherapy. Front Immunol 11:924. https://doi.org/10.3389/fimmu.2020.00924
Moreau A, Alliot-Licht B, Cuturi MC, Blancho G (2017) Tolerogenic dendritic cell therapy in organ transplantation. Transpl Int Off J Eur Soc Organ Transpl 30(8):754–764. https://doi.org/10.1111/tri.12889
Lewis AL, Chen X, Schnaar RL, Varki A et al (2022) Sialic Acids and Other Nonulosonic Acids. In: Varki A, Cummings RD, Esko JD, eds. Essentials of Glycobiology. 4th ed. Cold Spring Harbor Laboratory Press; Accessed February 15, 2024. https://doi.org/10.1101/glycobiology.4e.15
Videira PA, Amado IF, Crespo HJ et al (2008) Surface alpha 2-3- and alpha 2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis. Glycoconj J 25(3):259–268. https://doi.org/10.1007/s10719-007-9092-6
Cabral MG, Piteira AR, Silva Z, Ligeiro D, Brossmer R, Videira PA (2010) Human dendritic cells contain cell surface sialyltransferase activity. Immunol Lett 131(1):89–96. https://doi.org/10.1016/j.imlet.2010.02.009
Stamatos NM, Carubelli I, van de Vlekkert D et al (2010) LPS-induced cytokine production in human dendritic cells is regulated by sialidase activity. J Leukoc Biol 88(6):1227–1239. https://doi.org/10.1189/jlb.1209776
Balneger N, Cornelissen L, a. M, Wassink M et al (2022) Sialic acid blockade in dendritic cells enhances CD8 + T cell responses by facilitating high-avidity interactions. Cell Mol Life Sci CMLS 79(2):98. https://doi.org/10.1007/s00018-021-04027-x
Büll C, Collado-Camps E, Kers-Rebel ED et al (2017) Metabolic sialic acid blockade lowers the activation threshold of moDCs for TLR stimulation. Immunol Cell Biol 95(4):408–415. https://doi.org/10.1038/icb.2016.105
Silva Z, Ferro T, Almeida D et al (2020) MHC Class I Stability is Modulated by Cell Surface Sialylation in Human Dendritic Cells. Pharmaceutics 12(3):249. https://doi.org/10.3390/pharmaceutics12030249
Silva M, Silva Z, Marques G et al (2016) Sialic acid removal from dendritic cells improves antigen cross-presentation and boosts anti-tumor immune responses. Oncotarget 7(27):41053–41066. https://doi.org/10.18632/oncotarget.9419
Edgar LJ, Thompson AJ, Vartabedian VF et al (2021) Sialic Acid Ligands of CD28 Suppress Costimulation of T Cells. ACS Cent Sci 7(9):1508–1515. https://doi.org/10.1021/acscentsci.1c00525
Lübbers J, Rodríguez E, van Kooyk Y (2018) Modulation of Immune Tolerance via Siglec-Sialic Acid Interactions. Front Immunol 9. https://doi.org/10.3389/fimmu.2018.02807
Murugesan G, Weigle B, Crocker PR (2021) Siglec and anti-Siglec therapies. Curr Opin Chem Biol 62:34–42. https://doi.org/10.1016/j.cbpa.2021.01.001
Palleon Pharmaceuticals, Inc O, Identifier NCT05259696. (November 2022 -). https://clinicaltrials.gov/study/NCT05259696
Gray MA, Stanczak MA, Mantuano NR et al (2020) Targeted glycan degradation potentiates the anticancer immune response in vivo. Nat Chem Biol 16(12):1376–1384. https://doi.org/10.1038/s41589-020-0622-x
Büll C, Boltje TJ, van Dinther EAW et al (2015) Targeted Delivery of a Sialic Acid-Blocking Glycomimetic to Cancer Cells Inhibits Metastatic Spread. ACS Nano 9(1):733–745. https://doi.org/10.1021/nn5061964
Wang Yhua (2020) Sialidases From Clostridium perfringens and Their Inhibitors. Front Cell Infect Microbiol 9. https://doi.org/10.3389/fcimb.2019.00462
Jennings MP, Day CJ, Atack JM (2022) How bacteria utilize sialic acid during interactions with the host: snip, snatch, dispatch, match and attach. Microbiology 168(3):001157. https://doi.org/10.1099/mic.0.001157
Cabral MG, Silva Z, Ligeiro D et al (2013) The phagocytic capacity and immunological potency of human dendritic cells is improved by α2,6-sialic acid deficiency. Immunology 138(3):235–245. https://doi.org/10.1111/imm.12025
Sabado RL, Meseck M, Bhardwaj N (2016) Dendritic Cell Vaccines. Methods Mol Biol Clifton NJ 1403:763–777. https://doi.org/10.1007/978-1-4939-3387-7_44
Luz VCC, Silva Z, Sobral P, Tanwar A, Paterson RL, Videira PA (2023) Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes. J Vis Exp JoVE 200https://doi.org/10.3791/65525
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods San Diego Calif 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Cunningham S, Hackstein H (2020) Recent Advances in Good Manufacturing Practice-Grade Generation of Dendritic Cells. Transfus Med Hemotherapy 47(6):454–463. https://doi.org/10.1159/000512451
Patente TA, Pinho MP, Oliveira AA, Evangelista GCM, Bergami-Santos PC, Barbuto JAM (2018) Human Dendritic Cells: Their Heterogeneity and Clinical Application Potential in Cancer Immunotherapy. Front Immunol 9:3176. https://doi.org/10.3389/fimmu.2018.03176
Wimmers F, Schreibelt G, Sköld AE, Figdor CG, De Vries IJM (2014) Paradigm Shift in Dendritic Cell-Based Immunotherapy: From in vitro Generated Monocyte-Derived DCs to Naturally Circulating DC Subsets. Front Immunol 5. https://doi.org/10.3389/fimmu.2014.00165
Berger TG, Feuerstein B, Strasser E et al (2002) Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories^™. J Immunol Methods 268(2):131–140. https://doi.org/10.1016/S0022-1759(02)00189-8
Powell LD, Whiteheart SW, Hart GW (1987) Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction. J Immunol 139(1):262–270. https://doi.org/10.4049/jimmunol.139.1.262
Rollins Z, Harris B, George S, Faller R (2023) A molecular dynamics investigation of N-glycosylation effects on T-cell receptor kinetics. J Biomol Struct Dyn 41(12):5614–5623. https://doi.org/10.1080/07391102.2022.2091660
Barb AW, Prestegard JH (2011) NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic. Nat Chem Biol 7(3):147–153. https://doi.org/10.1038/nchembio.511
Harbison AM, Brosnan LP, Fenlon K, Fadda E (2018) Sequence-to-structure dependence of isolated IgG Fc complex biantennary N-glycans: A molecular dynamics study. Published online August 14:392001. https://doi.org/10.1101/392001
Jongsma MLM, de Waard AA, Raaben M et al (2021) The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses. Immunity 54(1):132–150e9. https://doi.org/10.1016/j.immuni.2020.11.003
Ibarlucea-Benitez I, Weitzenfeld P, Smith P, Ravetch JV (2021) Siglecs-7/9 function as inhibitory immune checkpoints in vivo and can be targeted to enhance therapeutic antitumor immunity. Proc Natl Acad Sci U S A 118(26):e2107424118. https://doi.org/10.1073/pnas.2107424118
Duan S, Paulson JC (2020) Siglecs as Immune Cell Checkpoints in Disease. Annu Rev Immunol 38:365–395. https://doi.org/10.1146/annurev-immunol-102419-035900
Tsui CK, Barfield RM, Fischer CR et al (2019) CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity. Nat Chem Biol 15(10):949–958. https://doi.org/10.1038/s41589-019-0342-2
van der Wel NN, Sugita M, Fluitsma DM et al (2003) CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation. Mol Biol Cell 14(8):3378–3388. https://doi.org/10.1091/mbc.e02-11-0744
Hunger RE, Sieling PA, Ochoa MT et al (2004) Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells. J Clin Invest 113(5):701–708. https://doi.org/10.1172/JCI200419655
Domnick A, Winter C, Sušac L et al (2022) Molecular basis of MHC I quality control in the peptide loading complex. Nat Commun 13(1):4701. https://doi.org/10.1038/s41467-022-32384-z
Reichner JS, Whiteheart SW, Hart GW (1988) Intracellular trafficking of cell surface sialoglycoconjugates. J Biol Chem 263(31):16316–16326. https://doi.org/10.1016/S0021-9258(18)37595-1
Saxena M, Balan S, Roudko V, Bhardwaj N (2018) Towards superior dendritic-cell vaccines for cancer therapy. Nat Biomed Eng 2(6):341–346. https://doi.org/10.1038/s41551-018-0250-x
Sabado RL, Balan S, Bhardwaj N (2017) Dendritic cell-based immunotherapy. Cell Res 27(1):74–95. https://doi.org/10.1038/cr.2016.157
Palucka K, Banchereau J (2013) Dendritic-cell-based therapeutic cancer vaccines. Immunity 39(1):38–48. https://doi.org/10.1016/j.immuni.2013.07.004
Lee S, Margolin K (2011) Cytokines in Cancer Immunotherapy. Cancers 3(4):3856–3893. https://doi.org/10.3390/cancers3043856
Bent R, Moll L, Grabbe S, Bros M (2018) Interleukin-1 Beta—A Friend or Foe in Malignancies? Int J Mol Sci 19(8):2155. https://doi.org/10.3390/ijms19082155
Zhang Z, Fuller GM (2000) Interleukin 1beta inhibits interleukin 6-mediated rat gamma fibrinogen gene expression. Blood 96(10):3466–3472. https://doi.org/10.1182/blood.V96.10.3466
Castiello L, Sabatino M, Jin P et al (2011) Monocyte-derived DC maturation strategies and related pathways: a transcriptional view. Cancer Immunol Immunother CII 60(4):457–466. https://doi.org/10.1007/s00262-010-0954-6

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New insights into the immunomodulatory potential of sialic acid on monocyte-derived dendritic cells

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Abstract

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1 Introduction

2 Materials and methods

2.1. Cells

2.3 Cell treatments

2.3.1 Sialidase

2.3.2 Cytokine Cocktail

2.3.3 Sialidase Inhibitors

2.4 Flow cytometry analysis

2.5 Gene expression assay

2.6 Cytokine production evaluation

2.7 Measurement of endogenous sialidase activity.

2.8 Confocal laser scanning microscopy

2.9 Statistical analysis

2.10 Data availability

3 Results

Discussion

Declarations

Data availability statement

References

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