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Sjögren’s syndrome
Deciphering the role of cDC2s in Sjögren’s syndrome: transcriptomic profile links altered antigen processes with IFN signature and autoimmunity
  1. Ana P Lopes1,2,
  2. Maarten R Hillen1,2,
  3. Anneline C Hinrichs1,2,
  4. Sofie LM Blokland1,2,
  5. Cornelis PJ Bekker1,2,
  6. Aridaman Pandit1,2,
  7. Aike A Kruize1,
  8. Timothy RDJ Radstake1,2,
  9. Joel A van Roon1,2
  1. 1Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
  2. 2Center for Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
  1. Correspondence to Dr Joel A van Roon, Rheumatology & Clinical Immunology/Lab Translational Immunology, UMC Utrecht, Utrecht, 3584 CX, The Netherlands; j.vanroon{at}umcutrecht.nl

Abstract

Objective Type 2 conventional dendritic cells (cDC2s) are key orchestrators of inflammatory responses, linking innate and adaptative immunity. Here we explored the regulation of immunological pathways in cDC2s from patients with primary Sjögren’s syndrome (pSS).

Methods RNA sequencing of circulating cDC2s from patients with pSS, patients with non-Sjögren’s sicca and healthy controls (HCs) was exploited to establish transcriptional signatures. Phenotypical and functional validation was performed in independent cohorts.

Results Transcriptome of cDC2s from patients with pSS revealed alterations in type I interferon (IFN), toll-like receptor (TLR), antigen processing and presentation pathways. Phenotypical validation showed increased CX3CR1 expression and decreased integrin beta-2 and plexin-B2 on pSS cDC2s. Functional validation confirmed impaired capacity of pSS cDC2s to degrade antigens and increased antigen uptake, including self-antigens derived from salivary gland epithelial cells. These changes in antigen uptake and degradation were linked to anti-SSA/Ro (SSA) autoantibodies and the presence of type I IFNs. In line with this, in vitro IFN-α priming enhanced the uptake of antigens by HC cDC2s, reflecting the pSS cDC2 profile. Finally, pSS cDC2s compared with HC cDC2s increased the proliferation and the expression of CXCR3 and CXCR5 on proliferating CD4+ T cells.

Conclusions pSS cDC2s are transcriptionally altered, and the aberrant antigen uptake and processing, including (auto-)antigens, together with increased proliferation of tissue-homing CD4+ T cells, suggest altered antigen presentation by pSS cDC2s. These functional alterations were strongly linked to anti-SSA positivity and the presence of type I IFNs. Thus, we demonstrate novel molecular and functional pieces of evidence for the role of cDC2s in orchestrating immune response in pSS, which may yield novel avenues for treatment.

  • Sjogren's Syndrome
  • Autoimmunity
  • Inflammation
  • Autoantibodies

Data availability statement

Data are available in a public, open access repository. The RNA sequencing datasets generated for this study can be found in NCBI’s Gene Expression Omnibus under the following accession number GSE200020.

https://creativecommons.org/licenses/by/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.

https://doi.org/10.1136/ard-2022-222728

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • Type 2 conventional dendritic cells (cDC2s) are central in the initiation and control of immune responses, but their functional role in primary Sjögren’s syndrome (pSS) is poorly understood.

    WHAT THIS STUDY ADDS

    • Transcriptomic profile of cDC2s reveals changes in important pathways in patients with pSS consistent with cell activation, presence of type I interferon (IFN) and altered antigen response.

    • Phenotypical validation shows increased fractalkine receptor (CX3CR1) expression on cDC2s from patients with pSS.

    • cDC2s from patients with pSS with anti-Ro/SSA autoantibodies demonstrate altered antigen uptake and processing, modulated by type I IFN.

    • cDC2s from patients with pSS activate and increase CXCR3 and CXCR5 expression on CD4+ T cells, facilitating migration to the inflammatory sites.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • Considering their key role in orchestrating inflammatory responses understanding the underlying molecular mechanisms that drive cDC2 function and activation may disclose novel targets to halt immunopathology in pSS.

    Introduction

    Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterised by prominent T-lymphocyte and B-lymphocyte infiltrations of the exocrine glands, which is associated with glandular destruction and dysfunction.1 2 The immunological hallmarks of pSS include B-cell hyperactivity, the presence of anti-SSA/Ro (SSA) and anti-SSB/La antibodies3 and a type I interferon (IFN) signature. Furthermore, the presence of a type I IFN signature in patients with pSS is associated with higher disease activity and higher levels of autoantibodies4 and reinforces the involvement of the innate immune system.5

    Type 2 conventional dendritic cells (cDC2s) are professional antigen-presenting cells with a unique ability to induce potent T-cell and B-cell responses.6 On stimulation, cDC2s take up antigens and migrate into the T-cell area of the draining lymph node to initiate immune responses.7 The internalisation, processing and presentation of antigens are a critical step for T-cell priming. cDC2s are potent activators of CD4+ T cells and induce T helper (Th) cell polarisation, thus directing the immune system in distinct directions.8 Moreover, cDC2s can affect B-cell differentiation and survival, mainly through the production of B-cell activating factor (BAFF) and a proliferation-inducing ligand.9 Activation and maturation of cDC2s to initiate adaptive immune responses can be potently amplified by inflammatory mediators, particularly type I IFNs.10 11

    Despite the important role of cDC2s to activate T and B cells, their contribution to pSS immunopathology has been poorly studied. Recently, transcriptomic analysis of minor salivary glands from patients with pSS confirmed the presence of a cDC2 gene signature in the inflamed salivary glands, which was strongly associated with CD4+ T cells.12 13 In patients with pSS, cDC2s are epigenetically altered with decreased miR-130a expression and increased expression of its target mitogen- and stress-activated protein kinase-1, important for proinflammatory cytokine production. Furthermore, on stimulation, cDC2s from patients with pSS produce more interleukin (IL)-12 and tumour necrosis factor alpha (TNF-α).14 In addition, in non-obese diabetic (NOD) and IQI/Jic, both spontaneous mouse models of Sjögren’s syndrome, dendritic cell (DC) influx precedes the presence of focal lymphocytic infiltrates into the submandibular glands.15 16 Furthermore, in aged mice lacking Dcir expression, a crucial negative regulator of DC function, mice spontaneously develop sialadenitis with elevated serum autoantibodies levels including anti-SSA, anti-SSB/La and antinuclear antibodies.17 Thus, cDC2s seem to play an important role in pSS pathogenesis as well as in T-cell and B-cell activation and in driving salivary gland inflammation.

    Here, we set out to investigate the role of cDC2s in pSS by exploiting RNA sequencing (RNAseq) to identify the transcriptional profile of circulating cDC2s from patients with pSS and non-Sjögren’s sicca (nSS) as compared with healthy controls (HCs). In addition, we performed phenotypical and functional validation to confirm identified altered pathways and mechanisms through which cDC2s could drive pSS.

    Materials and methods

    Patients and controls

    Patients and controls were age-matched and gender-matched and randomly allocated across the different experiments. All patients with pSS fulfilled the American-European Consensus Group (AECG) classification criteria for pSS.18 Patients who did not fulfil the pSS classification criteria but presented with dryness complaints without a known cause in the absence of any generalised autoimmune disease were classified as patients with nSS and subjected to minor salivary gland biopsy. Two independent cohorts were selected to establish the transcriptional profile of cDC2s: a discovery cohort (14 with pSS, 9 with nSS and 8 HCs) and a replication cohort (9 with pSS, 6 with nSS and 10 HCs) (table 1). For the validation experiments, additional independent cohorts of HCs and patients with pSS were recruited (online supplemental table 1).

    Supplemental material

    View this table:
    Table 1

    Characteristics of the patients and controls enrolled in the RNAseq cohort

    Patients and public involvement

    Patients and HCs recruited for this study were not involved in the design, conduct, reporting or dissemination plans of our research.

    Detailed information for all the methods, including phenotypical and functional validation, can be found in the online supplemental material and methods.

    Results

    Transcriptome of cDC2s from patients with pSS is distinct from patients with nSS and HCs

    In the discovery cohort, the majority of differentially expressed genes (DEGs) were found between pSS and HC cDC2s (figure 1A). Similarly, in the replication cohort, the larger number of DEG was also observed between pSS and HC cDC2s (figure 1B). In addition, cDC2s from patients with nSS partly exhibited changes in gene expression similar to pSS cDC2s in both cohorts. However, the magnitude of differences was generally larger in cDC2s from patients with pSS compared with patients with nSS. Overall, the cDC2 transcriptomic profile of patients with pSS overlaps to some extent to patients with nSS, but both are distinct from HCs (figure 1A,B).

    Figure 1
    Figure 1

    Transcriptomic characterisation of circulating cDC2s from patients with pSS and nSS. RNA sequencing of circulating cDC2s was performed independently for both discovery and replication cohorts. Venn diagrams show the overlap of the DEGs with a nominal p value of <0.05 between any of the three groups for the discovery cohort (A) and the replication cohort (B). Scatter plots show the FC (log2) of the DEGs between the two cohorts for the different comparisons, pSS versus HC (C), nSS versus HC (D) and pSS versus nSS (E). Volcano plots display the relationship between the FC (log2, x-axis) and the nominal p value (−log10, y-axis) of the DEGs consistently downregulated or upregulated in both cohorts for each comparison (F–H). cDC2, type 2 conventional dendritic cell; DEG, differentially expressed gene; FC, fold change; HC, healthy control; nSS, non-Sjögren’s sicca; pSS, primary Sjögren’s syndrome.

    To identify the most robust and consistently altered genes, those genes differentially expressed with a nominal p value of <0.05, with an average base mean expression (defined as the mean of normalised counts of all samples normalising for sequencing depth) higher than 100 in both cohorts, were selected (figure 1C–E). Of the DEGs identified in both cohorts, a large fraction (87% in pSS vs HC, 66% in nSS vs HC and 87% in nSS vs pSS) exhibited the same directionality (figure 1F–H) and therefore was considered to be replicated. The majority of the replicated DEGs were found between pSS and HC, and out of these, 30% (356 genes) were differentially expressed between nSS and HC (online supplemental figure 2). For the DEGs between patients with nSS and HC cDC2s, functional annotation did not reveal enriched pathways (online supplemental figure 3A). As for the DEGs between pSS versus nSS (figure 1H), functional annotation indicated that these genes were associated with viral and IFN-related pathways, nonsense-mediated decay and translation processes (online supplemental figure 3B-C).

    Transcriptomic analysis of pSS cDC2s reveals impaired expression of genes involved in cell trafficking and activation

    To gain further insight into the pSS cDC2s transcriptional profile, we identified the top 100 DEGs in both cohorts, based on fold-change differences. The majority of upregulated genes identified in pSS cDC2s included IFN-inducible genes (eg, MX1, IFITM1 and DDX58) and molecules involved in cell migration (eg, CCR2, CX3CR1 and CCR5) and activation (TLR7). Likewise, the top downregulated genes in pSS cDC2s comprise important regulators of cell activation like NFKBIA, a member of the nuclear factor kappa B (NF-κB) inhibitor family; PLXNB2 and PLXND1, both negative regulators of IL-12p40 production19; ITGB2, a negative regulator of toll-like receptor (TLR) activation20; and PELI1, which negatively regulates non-canonical NF-κB signalling21 (figure 2A).

    Figure 2
    Figure 2

    Transcriptomic profile and protein validation of cDC2s from patients with pSS display altered expression of key molecules involved in cell trafficking, activation and interferon signalling. Heatmap visualisation of the top 100 DEGs (50 upregulated and 50 downregulated genes, rows) across the two cohorts (discovery and replication) and the studied groups (HC, nSS and pSS; columns) clustered by Euclidean distance and Ward’s method (A). Dot plots depict the expression of selected DEGs in discovery and replication cohorts in both HCs and patients with pSS (B). Protein expression of the selected DEGs was assessed on cDC2s by flow cytometry in HCs (n=22) and patients with pSS (n=22) (C). *, ** and *** represent nominal p values of <0.05, <0.01 and <0.001, respectively. cDC2, type 2 conventional dendritic cell; DEG, differentially expressed gene; HC, healthy control; nSS, non-Sjögren’s sicca; pSS, primary Sjögren’s syndrome.

    As DC migration, cell–cell interaction and activation are crucial steps in the initiation and regulation of the immune response, we sought to further investigate the expression of potential mediators of these processes at protein level (figure 2B and online supplemental figure 4A). To this end, the protein expression of CX3CR1, a key chemokine receptor involved in trafficking of cDC2s,22 TNFSF13B (BAFF), integrin beta-2 (ITGB2) and plexin-B2 (PLXNB2), both regulators of cell activation, was assessed by flow cytometry. In line with the transcriptomic data, the surface expression of CX3CR1 was significantly higher and the expression of ITGB2 and PLXNB2 was significantly lower on cDC2s from patients with pSS when compared with HCs. BAFF surface expression was not significantly different (figure 2C). Together, these results corroborate transcriptional changes in key mediators of migration and activation in pSS cDC2 and identify a possible mediator of cDC2 recruitment to the inflamed salivary glands.

    To further understand the functional pathways altered in pSS cDC2s, we performed annotation of the consistent DEGs (figure 1F). Pathways involved in inflammation, including IFN signalling, class I major histocompatibility complex (MHC)-mediated antigen processing and presentation, TLR cascade and mitochondrial translation were enriched in pSS cDC2s compared with HC cDC2s (figure 3A). As TLRs are crucial receptors for cDC2 activation, we sought to investigate the phosphorylation profile of downstream TLR signalling mediators. However, we did not observe changes in the phosphorylation profile of p38, ERK1/2, JNK, ATF2 and NF-kB p65 between pSS cDC2s and HC cDC2s, both ex vivo and after TLR4 activation (online supplemental figure 5).

    Figure 3
    Figure 3

    cDC2s from patients with pSS are functionally different in interferon-associated pathways and in antigen processing. Reactome pathway enrichment analysis was used for functional annotation of the DEGs between pSS versus HC (selected in figure 1F). The top significantly enriched reactome pathways are depicted. The x-axis shows the number of DEGs found within the pathway over the total number of pathway components (ratio); dot size depicts the number of genes used for enrichment and colour indicates the statistical significance (A). Isolated PBMCs were incubated with DQ–BSA for 10 min and antigen processing was followed for the indicated time points (B). Representative histograms (C) and quantification (D) of processed DQ-BSA, represented as MFI normalised to T=0, in HC (n=11) and non-treated patients with pSS (n=6) at different time points determined by flow cytometry. Quantification of DQ-BSA processing in patients with pSS with (pSS-SSA+, n=4) or without (pSS-SSA−, n=2) anti-SSA antibodies (E). Violin plots depict TAP1, LNPEP and PSMA3 gene expressions in HC and patients with pSS from discovery and replication cohorts combined (F). cDC2, type 2 conventional dendritic cell; DEG, differentially expressed gene; DQ-BSA, fluorescent BODIPY dye labelled bovine serum albumin HC, healthy control; MFI, median fluorescence intensity; PBMC, peripheral blood mononuclear cell; pSS, primary Sjögren’s syndrome.

    cDC2s from patients with pSS display a less effective antigen processing capacity

    Antigen uptake and processing are crucial pathways in cDC2s that affect their antigen presentation to CD4+ and CD8+ T cells. As functional annotation of pSS cDC2 DEGs indicated altered antigen processing in pSS cDC2s, we performed in vitro validation experiments to assess the capacity of pSS cDC2s to degrade phagocytosed protein. To this end, we used bovine serum albumin (BSA) as an antigen model labelled with a fluorescent BODIPY dye (DQ-BSA). DQ-BSA is not fluorescent due to self-quenching, but on endocytosis and degradation, the self-quenching is abolished and a fluorescent signal can be detected using flow cytometry (figure 3B). As immunosuppressive treatment affects antigen processing, patients who were on treatment at the time of sample collection were excluded from this analysis (online supplemental figure 6A). We observed significantly reduced antigen processing in cDC2s from patients with pSS as compared with HCs, particularly at t=60 min (figure 3C–D). Next, we investigated the association between the presence of anti-SSA autoantibodies and the antigen processing capacity of cDC2s. Interestingly, cDC2s from patients with pSS with anti-SSA autoantibodies (pSS-SSA+) displayed a significantly lower processing capacity compared with HC, while patients without anti-SSAautoantibodies (pSS-SSA−) demonstrated antigen processing similar to that of HC (figure 3E). No differences were found in the frequency of circulating cDC2s between patients with pSS and HCs (online supplemental figure 4B).

    As decreased capacity to process antigen has been associated with prolonged antigen survival, facilitating MHC-I supply,23 and enhanced cross-presentation to CD8+ T cells, we further investigated the expression of genes involved in class I-mediated processing. The expression of TAP1, a key transporter associated with MHC-I loading,24 the peptidase LNPEP, implicated in endosomal trimming of cross-presented peptides and interaction with MHC-I molecules,25 as well as the expression of proteasome subunits like PSMA3, involved in protein degradation in a ubiquitin-independent manner, were upregulated in cDC2s from patients with pSS compared with HCs (figure 3F). Thus, our results suggest that pSS cDC2s are possibly more efficient at storing (auto-)antigens, enhancing cross-presentation to CD8+ T cells.

    Increased antigen uptake of pSS cDC2s is related with anti-SSA autoantibodies and type I IFNs

    As cDC2 antigen processing and presentation are importantly impacted by antigen uptake, we next investigated the capacity of cDC2s from patients with pSS and HCs to uptake BSA (figure 4A–B). Time-course analyses of BSA uptake demonstrated that pSS cDC2s have an increased antigen uptake compared with HC cDC2s, particularly at later time points (t=60 and t=120 min) (figure 4C). No differences were observed in cDC2 antigen uptake between patients who were treated with immune-suppressive treatment and those who were not (online supplemental figure 6B). Interestingly, cDC2s from pSS-SSA+ uptake more antigen compared with pSS-SSA− and HCs. A significantly higher uptake capacity was observed in pSS-SSA+ compared with HCs at t=60 min, and this further increased at t=120 min (figure 4D). In addition, at t=120 min, pSS-SSA+ cDC2 s uptake more antigen compared with pSS-SSA−, who show similar BSA uptake to HC (figure 4D).

    Figure 4
    Figure 4

    Enhanced uptake of antigen and apoptotic cells by pSS cDC2s is associated with autoimmunity and type I IFN. PBMCs from HC and patients with pSS were incubated with AF647–BSA for the indicated time points and the uptake by cDC2s, represented as MFI, was assessed by flow cytometry (A). Representative histograms (B) and quantification of BSA uptake by cDC2s of HCs (n=11) and patients with pSS (n=14) (C) and patients with pSS with (pSS-SSA+, n=10) or without (pSS-SSA−, n=4) anti-SSA antibodies (D). IFN signature calculated as the mean Z-score of five IFN-induced genes was determined by qPCR in HC (n=13), pSS-SSA− (n=5) and pSS-SSA+ (n=10) (E). HC-PBMCs were primed for 3 hours without (medium) or with IFN-α, exposed to AF647–BSA and chased for the indicated times by flow cytometry (F). The effect of IFN-α priming on BSA uptake was analysed in HC cDC2s (n=5) by flow cytometry (G). Quantification of BSA uptake by cDC2s of patients with pSS with (pSS-IFN+, n=9) or without (pSS-IFN−, n=3) IFN signature (H). Apoptotic CFSE labelled HSG-epithelial cells were added to PBMCs from HCs (n=9) and patients with pSS (n=13) at a 1:1 ratio for 120 min. Representative histogram (I) and quantification of apoptotic cell uptake of cDC2s from HCs and patients with pSS (J) and pSS-SSA− (n=4) and pSS-SSA+ (n=9) (K) measured by flow cytometry. HC-PBMCs were primed for 3 hours with IFN-α or without (medium) and exposed to apoptotic CFSE labelled HSG-epithelial cells for 2 hours. The effect of IFN-α priming on apoptotic cell uptake was analysed in HC cDC2s (n=4) by flow cytometry (L). Results are represented as mean±SEM. *, ** and *** represent p values of <0.05, <0.01 and <0.001, respectively. cDC2, type 2 conventional dendritic cell; HC, healthy control; HSG, human salivary gland; IFN, interferon; MFI, median fluorescence intensity; PBMC, peripheral blood mononuclear cell; pSS, primary Sjögren’s syndrome; qPCR, quantitative PCR.

    Given the strong association between the presence of anti-SSAautoantibodies and the IFN signature,26 we confirmed that the majority of the pSS-SSA+ patients (8 out of 10) exhibited an IFN signature (pSS-IFN+), reflected by a higher IFN score (figure 4E). In addition, as functional annotation of DEGs in pSS cDC2s indicated altered IFN signalling, we investigated whether the presence of type I IFN affects cDC2 antigen uptake and processing. For this, HC cDC2s were left untreated or primed with IFN-α for 3 hours to mimic the IFN-signature and next challenged with labelled BSA (figure 4F). IFN-α priming of HC cDC2s increased antigen uptake by HC cDC2s to similar levels as those seen for pSS-SSA+ cDC2s (figure 4G) but did not alter antigen processing of cDC2s (data not shown). In addition, an increased antigen uptake was also observed in cDC2s from pSS-IFN+ patients compared with pSS-IFN− patients, particularly at t=120 min (figure 4H). Together, these results show that cDC2s from anti-SSA+ patients with pSS have an increased antigen uptake capacity related to the IFN signature, as exposure of HC cDC2s to type I IFN induces an increase uptake profile similar to pSS-SSA+ cDC2s.

    Next, we tested whether pSS-SSA+ cDC2s also uptake increased amounts of autoantigens derived from apoptotic human salivary gland (HSG)-epithelial cells, which is a relevant mechanism to drive pSS. To this end, PBMCs from patients with pSS and HC were incubated with apoptotic carboxyfluorescein succinimidyl ester (CFSE)-labelled HSG-epithelial cells for 2 hours, and the phagocytic capacity of cDC2s was assessed by flow cytometry (online supplemental figure 7). Similar to BSA uptake, pSS cDC2s displayed a significantly enhanced ability to uptake apoptotic HSG-epithelial cells (figure 4I–J) compared with HC cDC2s. Furthermore, the increased uptake of apoptotic HSG-epithelial cells was only observed in cDC2s of pSS-SSA+ patients (figure 4K). Moreover, IFN-α priming of HC cDC2s increased uptake of apoptotic HSG-epithelial cells by HC cDC2s, as observed in BSA uptake, although without statistical significance (figure 4L). Thus, cDC2s from patients with pSS with anti-SSA antibodies also uptake autoantigens more efficiently, possibly associated with the presence of type I IFN.

    pSS cDC2s increase proliferation of CD4+ T cells with a tissue homing signature

    In the context of the enhanced antigen uptake observed in pSS cDC2s, we next evaluated whether in vivo priming by these cells differently affects CD4+ T-cell activation in vitro. CD4+ T cells from HCs were allogenic cocultured with cDC2s from either patients with pSS or HCs for 3 days (figure 5A). No significant differences were observed among the CD4+ T-cell subsets between the different donors at the start of the experiment (figure 5B and online supplemental figure 8A). Coculture of CD4+ T cells with pSS cDC2s significantly increased the frequency of proliferating CD4+ T cells compared with coculture with HC cDC2s (figure 5 C-D, and online supplemental figure 8B). In addition, we observed an increased percentage of proliferating CD4+ T cells expressing CXCR3 and CXCR5 in the presence of pSS cDC2s when compared with HC cDC2s. No differences were observed on CCR4 expression (figure 5E–F). Furthermore, CD4+ T cells cocultured with pSS cDC2s produced substantially increased levels of TNF-α but not IFN-γ (figure 5G). Thus, the altered transcriptional profile and observed differences in antigen uptake and processing in pSS cDC2s are associated with an increased capacity to activate CD4+ T cells to express markers that allow them to migrate to the salivary glands and produce proinflammatory cytokines to drive local autoimmune response.

    Figure 5
    Figure 5

    cDC2s from patients with pSS efficiently induce CD4+ T-cell proliferation with a tissue homing signature. Total CD4+ T cells from HCs were cultured alone (n=3) or cocultured either with cDC2s from HCs (n=3) or patients with pSS (n=3) at a 5:1 ratio (T cells:cDC2s) for 3 days (A). The frequency of CD4+ T-cell subsets (naïve; CM, EM and EF) was assessed by flow cytometry directly after cell isolation (B). Quantification (C) and representative flow cytometry dot plot (D) of proliferating CD4+ T cells measured by flow cytometry at day 3. Representative histograms of CXCR3, CXCR5 and CCR4 expression on proliferating CD4+ T cells (E) and quantification of the percentage of chemokine receptor expressing CD4+ T cells measured by flow cytometry (F). TNF-α and IFN-γ production during T cells: cDC2s coculture was measured by ELISA (G). cDC2, type 2 conventional dendritic cell; CM, central memory; EF, effector; EM, effector memory; HC, healthy control; IFN-γ, interferon gamma; pSS, primary Sjögren’s syndrome; TNF-α, tumour necrosis factor alpha.

    Discussion

    In-depth transcriptional profiling was performed in two independent cohorts and identified consistent transcriptional alterations in cell trafficking, activation, IFN signalling and class I-mediated antigen processing and presentation in cDC2s from patients with pSS compared with patients with nSS and HCs. Using in vitro cultures with primary isolated cDC2s from patients with pSS, we confirmed that pSS cDC2s display altered uptake and processing of (auto-)antigens. We observed that these differences were most pronounced in patients with pSS with anti-SSA antibodies, linked with type I IFN and possibly associated with enhanced antigen presentation to CD4+ and CD8+ T cells. Indeed, pSS cDC2s mediated increased proliferation of CD4+ T cells, associated with increased expression of chemokine receptors and enhanced production of TNF-α.

    Our transcriptomic analysis revealed that the altered molecular signature found in cDC2s from patients with pSS is, at least in part, shared by cDC2s from patients with nSS. Although patients with nSS do not display signs of systemic autoimmune disease, this heterogeneous group of patients shares severe objective dryness and occasionally presents single systemic features similar to patients with pSS. The overlap of clinical features and DEGs between pSS and nSS suggests that patients with nSS might have a low-grade inflammatory environment similar to patients with pSS and thus might share immune mediated pathological processes driving symptom burden. In accordance, we previously showed that the transcriptomic profile of plasmacytoid dendritic cells and monocytes of patients with nSS display an intermediate phenotype that largely overlaps with that of patients with pSS.27 28 In addition, patients with nSS also share locally and systemically signs of immune activation with patients with pSS such as presence of effector T cells (Th1, Th17 and Tfh cells) in the salivary glands and similar proteomic profile and increased circulating U6-sncRNA in circulation.29–31 However, differences in IFN signalling and nonsense-mediated decay pathways in cDC2s of patients with nSS could indicate that these patients still maintain some regulatory mechanisms to ensure cellular homeostasis.32 Altogether, the transcriptional profile of patients with nSS reveals an intermediate phenotype between HCs and patients with pSS. As such, longitudinal studies and further investigation in the regulation of the immune profile of patients with nSS are essential to gain more insight into the mechanisms driving immune activation and development of pSS immunopathology.

    The presence of cDC2s in the salivary glands of patients with pSS is clear12 13; however, the mechanisms that regulate the migration of cDC2s to the inflamed glands are unknown. Given the increased CX3CR1 expression in pSS cDC2s and since fractalkine levels (CX3CL1, the CX3CR1 ligand) are increased in the salivary gland of patients with pSS,33 the CX3CL1–CX3CR1 axis could mediate the presence of cDC2s in the inflamed glands of patients with pSS. In fact, recruitment and adhesion of CX3CR1-expressing cDC2s mediated by fractalkine lead to their accumulation in inflamed kidneys.22 Furthermore, reduction of PLXNB2, a negative regulator of IL-12/IL-23p40 response,19 is in line with increased IL-12 levels found in patients with pSS.34 Hence, a reduction of PLXNB2 expression may contribute to increase IL-12 production in patients with pSS. Moreover, the decreased ITGB2 expression in pSS cDC2s is in accordance with an activated state of these cells, in view of its role as negative regulator of TLR activation20 and mediator of T-cell priming by DC.35 As such, the enhanced CX3CR1 expression combined with changes in cell-activating pathways indicates that cDC2s could significantly contribute to the maintenance and possibly the initiation of inflammatory responses in pSS.

    The less efficient antigen processing observed in cDC2s from patients with pSS can possibly lead to long-term antigen storage, shown to occur in mature DCs.36 This antigen retention capacity by cDC2s, together with their migratory profile, suggests that pSS cDC2s favour the coordination of an efficient antigen presentation/cross-presentation to T cells at specific inflammatory sites. Long-term antigen storage can promote more effective cross-presentation via MHC-I, which is in line with the increased TAP1 and LNPEP expression observed in pSS cDC2s. These molecules can potentiate cross-presentation by enhancing the transport of antigen-derived peptides for binding to MHC-I37 and by trimming peptides in optimal size for loading on MHC-I,38 providing potent CD8+ T-cell activation that in turn can contribute to exacerbate the immune inflammation in the salivary glands. Hence, the activated CD8+ T cells present in the salivary glands of patients with pSS, which are associated with increased lymphocytic focus score, disease severity and autoimmunity,39 can be, in part, a consequence of an impaired regulation of cross-presentation by cDC2s.

    Type I IFNs are pleiotropic cytokines that can affect not only the maturation and activation of cDC2s11 but also antigen-related processes. We showed that IFN-α priming increased the uptake capacity of HC cDC2s to levels observed in pSS-SSA+ patients and that impaired antigen processing was also related with type I IFNs. Although antigen processing by cDC2s remained unaltered after short IFN-α priming (3 hours), stimulation for 24 hours was effective to upregulate genes involved in class I antigen processing and cross-presentation in a subset of cDC2s,11 suggesting that the modulation by IFN might be a long-lasting process. In line with this, IFN-α-driven monocyte-derived DCs were characterised by upregulation of genes linked to antigen processing and degradation, resembling cDC2 from patients with pSS with anti-SSA antibodies.23 40 Thus, the autosustaining feedback loop that, in part, maintains IFN production and anti-SSA antibodies alters cDC2 functions and possibly cDC2s-mediated T-cell activation.

    Apoptosis represents an important source of self-antigens for DCs, and it has recently emerged as a possible mechanism to expose autoantigens in pSS.41 42 In fact, this process can trigger immune cell activation to self-proteins43 as challenging of DCs with apoptotic cells induces their maturation, IL-12 production and Th1/Th17 responses.44 45 Moreover, apoptosis of epithelial cells leads to cleavage and translocation of Sjögren’s autoantigens, including α-fodrin and SSA, into apoptotic particles.46 The increased uptake of apoptotic cells by cDC2s of anti-SSA+ patients with pSS could represent a mechanism through which cDC2s activate CD4+ T cells and B cells to maintain the autoantibody production observed in pSS. Although the mechanisms underlying the antigen uptake and processing by pSS cDC2s need to be further clarified, our results suggest that these processes may be driven by type I IFN and could significantly contribute to the chronic inflammatory response observed in pSS.

    Interestingly, the increased CD4+ T-cell proliferation induced by pSS cDC2s with a tissue homing signature corroborates that cDC2s are also important in reshaping the CD4+ T-migratory profile in pSS. Blocking of CXCR3 function in a NOD mouse model of pSS reduces effector CD8+ T-cell infiltration and TNF-α expression in the salivary glands.47 Moreover, the increased CXCR5 expression induced by pSS cDC2s is in line with an increase frequency and recruitment to the salivary glands of patients with pSS, despite the differences observed in the peripheral blood.29 48–50 Recruitment of CXCR5 expressing CD4+ T cells is mediated by CXCL13 to ensure preferential B-cell interaction and activation.51 In the salivary glands of patients with pSS, CXCR5-expressing Tfh cells strongly correlated with CXCL13 expression and were associated with B-cell frequency and lymphocytic infiltration.29 Thus, these findings suggest that pSS cDC2s, through the modulation of CD4+ T-cell chemokine receptors, favour their migration to the salivary glands contributing to B-cell hyperactivity. However, whether CD4+ T cells undergo expansion in the gland, or whether it occurs elsewhere, for example, in the lymphoid organs, with subsequent migration still remains unclear.

    In conclusion, cDC2s from patients with pSS are transcriptionally altered, display an aberrant antigen uptake and processing, including self-antigens derived from salivary gland epithelial cells, and induce increased proliferation of tissue-homing CD4+ T cells. Although our study has limitations related with sample size and heterogeneity of the RNAseq analysis cohorts, we were able to successfully corroborate these results using an independent set of patients with pSS and HC to experimentally validate our hypothesis. Future studies in larger cohorts of patients with integrated analysis of different multiomics data from different tissues would be valuable to support our study. These data represent the first evidence of molecular and functional alteration of cDC2s in pSS, highlighting a novel pathway via which cDC2s contribute to the pSS pathogenesis.

    Data availability statement

    Data are available in a public, open access repository. The RNA sequencing datasets generated for this study can be found in NCBI’s Gene Expression Omnibus under the following accession number GSE200020.

    Ethics statements

    Patient consent for publication

    Ethics approval

    This study involves human participants and was approved by the University Medical Center Utrecht (METC no. 13-697). The participants gave informed consent to participate in the study before taking part in accordance with the declaration of Helsinki.

    Acknowledgments

    We thank Dr Lynne Bingle for kindly providing the human submandibular salivary gland epithelial cell line (HTB-41). Schematic representations were created with BioRender (biorender.com).

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    Footnotes

    • Handling editor Josef S Smolen

    • Correction notice This article has been corrected since it was first published. The open access licence has been updated to CC BY.

    • Contributors APL, MRH, TRDJR and JAvR were involved in the conception and design of the study. APL, MRH, ACH, SLMB, CPJB and AAK were involved in data acquisition. APL, MRH, CPJB, AP, AAK, TRDJR and JAvR were involved in data analysis and interpretation. APL drafted the manuscript. All authors contributed to the article and approved the submitted version. APL and JAvR are responsible for the overall content as the guarantors.

    • Funding APL was supported by a PhD grant from the Portuguese national funding agency for science, research and technology: Fundação para a Ciência e a Tecnologia (SFRH/BD/116082/2016). TRDJR received research funding from GlaxoSmithKline (GSK) for his work on Sjögren’s syndrome as part of the GSK Immune Catalyst Program. The funding sources had no role in study design; data collection, analysis, and interpretation; writing; or in the decision to submit the manuscript for publication.

    • Competing interests TRDJR was the principal investigator in the immune catalyst programme of GlaxoSmithKline, which was an independent research programme. He did not receive any financial support other than the research funding for the current project. Currently, He is an employee of Abbvie, where he holds stock. He had no part in the design and interpretation of the study results after he started at Abbvie.The remaining 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.

    • Patient and public involvement Patients and/or the public were not involved in the design, conduct, reporting or dissemination plans of this research.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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