Agonistic anti-DCIR antibody inhibits ITAM-mediated inflammatory signaling and promotes immune resolution

DC inhibitory receptor (DCIR) is a C-type lectin receptor selectively expressed on myeloid cells, including monocytes, macrophages, DCs, and neutrophils. Its role in immune regulation has been implicated in murine models and human genome-wide association studies, suggesting defective DCIR function associates with increased susceptibility to autoimmune diseases such as rheumatoid arthritis, lupus, and Sjögren’s syndrome. However, little is known about the mechanisms underlying DCIR activation to dampen inflammation. Here, we developed anti-DCIR agonistic antibodies that promote phosphorylation on DCIR’s immunoreceptor tyrosine-based inhibitory motifs and recruitment of SH2 containing protein tyrosine phosphatase-2 for reducing inflammation. We also explored the inflammation resolution by depleting DCIR+ cells with antibodies. Utilizing a human DCIR–knock-in mouse model, we validated the antiinflammatory properties of the agonistic anti-DCIR antibody in experimental peritonitis and colitis. These findings provide critical evidence for targeting DCIR to develop transformative therapies for inflammatory diseases.


Introduction
Immune regulator receptors play a crucial role in immune homeostasis by balancing activating vs inhibitory immune responses.The inhibitory receptors usually transduce the inhibitory signal through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the intracellular domains to counteract the functions of immunoreceptor tyrosine-based activation motifs (ITAMs)-bearing receptors in a proximity.This fine-tuned regulation from two opposed signaling modules can be initiated by receptors interacting with antibodies, immune complexes, or opsonized particles in a multivalency manner to control the ultimate cellular responses.The insufficient inhibitory signals by the dysfunctional ITIM-bearing receptors can affect the immune responses and eventually lead to autoimmune disorders (1,2).Dendritic cell inhibitory receptor (DCIR) belongs to the C-type lectin receptor (CLR) family and is predominantly expressed on myeloid-derived cells including monocytes, dendritic cells (DCs), macrophages and neutrophils.
Studies have demonstrated that DCIR receptor in dendritic cells and macrophages inhibits TLR-dependent inflammatory cytokine production, such as IL-1β, IL-6, TNFα, IL-12, and IFNα (10)(11)(12).Ligation of DCIR by sialylated IgG generates tolerogenic DCs and Tregs (4).A recent study demonstrated that an asialo-biantennary N-glycan(s) (NA2) binding to DCIR on DCs ameliorate CIA and EAE symptoms (13).These studies indicate that engagement of DCIR with ligands mediates the resolution of inflammation.However, the mechanisms that govern this inhibitory receptor biology by agonism are not well understood.In addition, an optimized agonistic antibody needs to be developed to induce potent and selective myeloid cell inhibition via targeting DCIR.
Here, we generated a panel of anti-human DCIR (huDCIR) monoclonal Abs (mAbs) and identified two strongly agonistic mAbs (clone 3A4 and 9D9).We found that the agonistic anti-DCIR mAbs induced ITIM phosphorylation and SHP2 recruitment to DCIR in human monocytes and DCs, diminished the Syk-SHP2-ITAM association triggered by the ITAM-bearing receptors activation, and reduced the proinflammatory cytokine production in both human monocytes and peripheral blood mononuclear cells (PBMCs).Additionally, in huDCIR knock-in (huDCIR-KI) mice peritonitis and colitis models, there was more DCIR+ cell accumulation in the peritoneal fluid and colonic crypts respectively compared to naïve mice.Importantly, treatment of agonistic but not non-agonistic mAbs significantly reduced the DCIR+ cell accumulation in the peritonitis and colitis models.Moreover, one weakagonistic clone, 5E11 with an engineered mouse IgG2b Fc to enhance the antibody-dependent cell depletion also suppressed leukocyte/neutrophil accumulation and the proinflammatory cytokine production in the peritonitis model.Taken together, these findings reveal the immune inhibitory mechanisms through agonistic and cell depletion effects provided by anti-DCIR mAbs and lay a solid foundation for targeting DCIR in the pursuit of transformative therapies for inflammatory diseases.

DCIR expression is induced in the disease-associated myeloid antigen-presenting cells and neutrophils
To confirm DCIR expression, we first compared DCIR mRNA expression in unperturbed blood immune cells based on the BLUEPRINT data set (Figure 1A).DCIR expression is enriched in not only immature and mature conventional DCs (cDCs) but also on neutrophils, classical monocytes, and macrophages.However, DCIR expression is low on B cells and T cells.Like other immune check point receptors, elevated DCIR after stimulation can be a feedback mechanism to prevent excessive inflammation in activated immune cells (14).Indeed, a RNAseq meta-study shows elevated levels of DCIRin autoimmune patients, including Crohn's disease (CD), ulcerative colitis (UC), and hidradenitis suppurativa (HS) compared to normal samples (Supplementary Figure 1A).Consistent with the mRNA profiling results, we confirmed DCIR protein expressed on the surface of neutrophils and monocytes from human whole blood by FACS (Supplementary Figure1B), which was further induced in the inflammatory condition due to LPS stimulation (Figure 1B).While little DCIR was identified on the B and T cells even when stimulated with LPS (Figure 1B).
To scrutinize the cells contributing to the increased DCIR in the disease-associated tissues, we analyzed public single-cell RNA-Seq (scRNAseq) datasets derived from the diseased skin tissues of HS patients (GSE155850) (Figure 1C and 1D) and mucosal tissues of CD patients (GSE134809) (Supplementary Figure 1C and 1D).
The scRNAseq analysis revealed DCIR is enriched in the tissue infiltrating neutrophils, monocytes, macrophage, and tissue resident myeloid cells including cDC1/2 and Langerhans cells in the lesional HS samples (Figure 1D).Beyond HS, mucosal tissue scRNAseq data (GSE134809) from CD patients with resistance to anti-TNFα therapy revealed that DCIR is selectively expressed on classical, intermediate monocytes, and DCs in the lesions (Supplementary Figure 1C-E).Mucosal tissue bulk RNAseq data further supported that enhanced DCIR expression is associated with anti-inflammatory treatment resistance (Supplementary Figure 1F), as patients who responded to anti-TNFα therapy displayed significantly lower DCIR levels before treatment compared to the treatment non-responders.In agreement with RNAseq results, immunohistology staining also confirmed increased DCIR+ cell accumulation in both epidermis and dermis of the HS skin biopsy, CD mucosal tissue, and a cutaneous lupus skin lesion (Figure 1E and 1F).The highly increased DCIR expression in disease lesions provides the rationale for developing DCIR-based therapy to restrain the inflammatory myeloid cells migration and activation.

Characterization of humanized anti-DCIR mAbs
To investigate the potential of DCIR as a therapeutic target for treating inflammatory diseases, we initiated an antibody generation campaign to produce anti-DCIR mAbs (Supplementary Figure 2A).Briefly, we immunized rats with a huDCIR cDNA vector and generated hybridoma cells by fusing the resulting spleen B cells with myeloma cells.We screened the culture supernatants of isolated hybridoma clones for secreted antibodies that bound to huDCIR-overexpressing HEK293 cells and monocyte-derived dendritic cells (MoDCs) (Supplementary Figure 2B).The heavy and light chain variable regions (VH and VL) of the hybridomas that secreted strong DCIR binders were cloned into a human IgG1 backbone and expressed as chimeric mAbs.Their cross-reactivity to cynomolgus (cyno) DCIR and huDCIR, but not murine DCIR1 (muDCIR1) were also confirmed (Supplementary Figure 2C).Finally, fully humanized antibodies were produced after removing rat-derived sequences and codon-optimization suggested by the high-throughput antibody humanization design software developed at AbbVie.The resultant humanized antibodies passed quality controls for aggregation and thermal stability (data not shown) were confirmed to be specific to human and cyno DCIR, but not other selected human C-type lectin receptors, pattern recognition receptors, and muDCIR1 expressed on immortal murine DC cell line JAWS II (Supplementary Figure 2D).

Agonistic anti-DCIR mAb induces tyrosine phosphorylation and SHP2 recruitment to DCIR ITIM motif
Although the suppressive role of DCIR has been reported in multiple inflammatory disease models (6,7,15), its intracellular signaling remains unclear.Previous studies have shown that the ITIM motif of checkpoint receptors PD1, SIGLEC9, and SIRPα can be phosphorylated at tyrosine residues upon agonistic ligand binding, which then recruits SHP2 (16)(17)(18)(19).DCIR association with SHP2 was also observed in the DCIR-overexpressed bone marrow-derived dendritic cells (BMDCs) (20).Based on these findings, we hypothesize that agonistic anti-DCIR mAbs could trigger tyrosine phosphorylation and promote SHP2 recruitment to DCIR (Figure 2A).To evaluate this hypothesis, we screened anti-DCIR mAbs in huDCIR-overexpressing HEK293 cells based on tyrosine phosphorylation and SHP2 interaction of DCIR identified by immunoprecipitation.We identified three mAbs, clones 9D9, 3B4, and 3A4, induced strong agonistic effects (Figure 2B and 2C).
To enable high-throughput comparison of agonistic signaling triggered by in-house generated humanized anti-DCIR mAbs, we created a DCIR agonistic signaling reporter cell line by co-transfecting HEK293 cells with an NF-κB luciferase reporter vector and a chimera DCIR vector carrying the intracellular ITAM domain from Dectin-1 to replace the DCIR ITIM motif (Figure 2D).We quantified antibody-mediated agonistic effects by measuring luciferase activity through ITAM-NF-κB signaling activation.Using this system, we selected two strong agonistic anti-DCIR mAbs, 9D9 and 3A4, as well as a non-and a weak-agonistic anti-DCIR mAbs, 3F7 and 5E11, for scale-up production and downstream mechanistic studies (Figure 2E and 2F).Mannose and NA2-glycan have been reported as DCIR's ligand for inducing DC tolerance (13,21).We confirmed mannose and NA2-glycan conjugated with BSA and immobilized on plates can induce agonistic effect, which was saturated at 50 µg/ml using the DCIR agonistic signaling reporter system (Figure 2G and 2H).However, pre-treatment of mannose-BSA or NA2-glygan-BSA (50 µg/ml), followed by the addition of agonistic anti-DCIR antibody (9D9), did not compromise the signaling induced by 9D9 (Figure 2I), indicating that our agonistic anti-DCIR antibody further amplify immune tolerance signaling from DCIR even when occupied by natural ligands.

Agonistic anti-DCIR mAb provides immunosuppressive function by sequestering SHP2 away from ITAM receptors
After selecting the lead humanized agonistic anti-DCIR mAbs, we investigated how DCIR agonistic signaling influences human primary cell activation.To ensure that the agonistic effect was not due to antibodies' binding discrepancy, we confirmed their comparable binding to human monocytes (Figure 3A).SHP2 has been reported to regulate inflammatory signaling in a dual manner.On one hand, SHP2 functions as a phosphatase and negatively modulates inflammatory signaling (22)(23)(24).On the other hand, SHP2 can act as an adaptor protein and recruit SYK to ITAM-containing receptors, such as Dectin-1 and Fc receptor γ chain, promoting cell activation (25).Therefore, we hypothesize that DCIR's ITIM can compete with ITAM for SHP2, and SHP2 binding to DCIR prevents SYK activation and interaction with ITAM-bearing receptor (Figure 3B).
Consistent with the antibody screening results using DCIR-overexpressing HEK293 cells, agonistic anti-DCIR mAbs 9D9 and 3A4 triggered DCIR and SHP2 interaction in human monocytes (Figure 3C).Stimulating the monocytes with anti-human serum albumin immune complex (IC: HSA) in the presence of Fc-matched isotype control or non-agonistic anti-DCIR mAb 3F7 increased the interaction between SYK and SHP2 (Figure 3D).
However, treating the immune complex-stimulated monocytes with agonistic anti-DCIR mAbs 9D9 and 3A4 significantly reduced the SHP2-SYK interaction to a similar level observed in cells with no stimulation, while promoting the SHP2 and DCIR interaction (Figure 3D).These data suggest that DCIR-ITIM signaling might overcome ITAM activation and compete for SHP2 recruitment.Indeed, agonistic anti-DCIR clones 9D9 and 3A4 also compromised the interaction between SYK and Fc receptor γ chain (Figure 3E).Another ITAM receptor, DECTIN1, also requires the formation of an ITAM-SHP2-SYK signaling complex (25).We found that agonistic mAbs 9D9 and 3A4 suppressed DECTIN1 activation in response to zymosan-D (ZymD) stimulation, as indicated by the reduced TNFα and IL6 secretion from monocytes (Figure 3F and 3G) and PBMC (Supplementary Figure 3A-D), whereas the non-agonistic 3F7 showed no effect.Beyond pattern-recognition receptors, SHP2 is also required for the function of granulocyte-macrophage colony-stimulating factor (GM-CSF) (26), regulating the differentiation and activation of neutrophils and macrophages.Given the rapid neutrophils' activation and ROS production kinetics, we analyzed anti-DCIR antibodies' impact on the oxidative burst of neutrophils stimulated with GM-CSF/phorbol myristate acetate (PMA) in real-time by measuring oxygen consumption rate (OCR), which reflects the oxygen consumption by NOX2 to generate ROS (27).Both agonistic antibodies (9D9 and 3A4) reduced OCR compared to the isotype and non/weak-agonistic antibodies (3F7 and 5E11) (Figure 3H).

Infiltrated DCIR-positive cells are evident in huDCIR-KI mice during acute peritonitis and colitis
We then generated a huDCIR-KI mouse strain aiming to evaluate the potential of targeting huDCIR as a treatment for inflammatory diseases.To do so, we inserted huDCIR cDNA adjacent to an internal ribosome entry site (IRES)-tdTomato cassette into the muDcir1 (Clec4a2) exon4 (Figure 4A).We confirmed successful cloning of huDCIR in the heterozygous (HET) huDCIR-KI mice by Southern blot analysis (Supplementary Figure 4A and 4B) of genomic DNA isolated from mice tail tips.We observed similar huDCIR expression compared to the endogenous WT muDCIR1 in CD115-Ly6G+ bone marrow neutrophils isolated from HET mice (Supplementary Figure 4C), indicating the promoter activity and translational efficiency between the huDCIR and endogenous muDCIR1 are at the comparable level.Subsequently, gene expression of huDCIR, muDcir1, and tdTomato reporter in blood monocytes from HET huDCIR-KI mice were also validated by qPCR analysis compared to the wildtype (WT) mice and human monocytes (Supplementary Figure 4D).Before inbreeding the HET huDCIR-KI mice to obtain homozygous (HO) huDCIR-KI mice, huDCIR-KI mice were backcrossed to the WT C57BL/6 mice for 7 generations to minimize the chance of non-target genetic modification.The resultant HO huDCIR-KI mice show stable endogenous huDCIR expression on CD11b+ cells purified from bone marrow, spleen, and blood compared to the WT mice (Figure 4B).Bone marrow CD11b+Ly6G-monocytes and CD11b+Ly6G+ neutrophils from huDCIR-KI mice displayed stable expression of huDCIR and tdTomato compared to WT mice (Figure 4C and 4D).
In addition, both huDCIR+ cells frequency and tdTomato expression were higher from HO compared to HET huDCIR-KI mice (Supplementary Figure 4E).TdTomato+ cells from both HO and HET huDCIR-KI mice express huDCIR (Supplementary Figure 4F) and tdTomato is solely expressed by huDCIR+ cells but not huDCIR-cells in HO huDCIR-KI mice (Figure 4E).Furthermore, we confirmed the intracellular signaling of the transgenic huDCIR is intact when binding to an agonistic anti-huDCIR antibody, clone 3A4, indicated by an increased interaction between huDCIR and mouse endogenous SHP2 in the BMDCs from HO huDCIR-KI mice (Supplementary Figure 4G).These results confirm the success of huDCIR-KI mice generation.
Based on our previous findings that huDCIR expression in neutrophils and monocytes can be significantly induced during inflammatory perturbations, we evaluated huDCIR expression in a murine ZymD-induced peritonitis model and a dextran sodium sulfate (DSS)-induced colitis model, where neutrophil and monocyte activation and infiltration are evident during the disease phase (28,29).Consistent with our previous findings, we observed a robust induction of huDCIR+ cells in the peritoneal fluid during peritonitis (Figure 4F and 4G) and in the colon crypts during DSS-induced colitis of huDCIR-KI mice (Figure 4H and 4I; Movie S1 and S2).In agreement with our hematoxylin and eosin (H&E) staining results on human tissues, few DCIR+ cells were detected in the disease-free state.These results demonstrate the translational potential of our huDCIR-KI mouse for developing huDCIR-based therapies.

Agonistic anti-DCIR mAb ameliorates experimental acute peritonitis
To investigate the anti-inflammatory function of DCIR, we assessed the efficacy of agonistic and non-agonistic mAbs in a ZymD-induced peritonitis model (Figure 5A).Prophylactic treatment with the agonistic clones 3A4 and 9D9 significantly reduced the severity of peritonitis in huDCIR-KI mice, as indicated by the reduced accumulation of leukocytes and neutrophils in the peritoneal lavage fluid, compared to the Fc-matched isotype control treatment group.Noteworthily, we did not observe significant effects for the agonistic clone 3A4 treatment in WT mice (Figure 5B and 5C).Although the non-agonistic clone 3F7 showed some inhibitory effect in reducing peritoneal leukocyte and neutrophil accumulation, only the agonistic clones significantly suppressed cytokine production in the peritoneal fluid (Figure 5D-G) and serum (Supplementary Figure 5A-C).As human IgG1 binds to mouse Fc receptors with lower affinity, our anti-DCIR mAbs might still modulate cell activation through binding to the ITIM-and ITAM-containing Fc receptors.To eliminate the influence of the antibody's Fc portion in evaluating the agonistic effects, we compared WT 3A4 to a LALA (L234A and L235A) mutated 3A4 variant with compromised FcR-binding ability (30).The results indicated that the protective effect from strong agonism on DCIR is more dominant compared to the FcR binding, as there were no significant differences in peritoneal cell accumulation (Figure 5H and 5I) or cytokine secretion in the peritoneal fluid and serum (Figure 5J-M; Supplementary Figure 5D-G) between the wild-type and LALA-mutated 3A4 treatments.However, we cannot totally rule out the importance of the Fc portion without thoroughly evaluating the antibody-mediated cell clearance effect.

Anti-DCIR antibody promotes neutrophils clearance via antibody dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC)
The engagement of agonistic anti-DCIR mAb initiates immune inhibitory signaling that effectively suppresses the inflammatory response.However, it remains unclear whether the Fc-mediated ADCP and ADCC also contribute to resolving inflammation.Given that DCIR is highly expressed on activated neutrophils and other myeloid cells, ADCP and ADCC could facilitate the clearance of inflammatory cells and prevent chronic inflammation.
Therefore, we sought to investigate whether the Fc fragment of anti-DCIR mAb promotes the resolution of inflammation.
To investigate whether anti-DCIR mAbs induce ADCP, we labeled LPS-primed neutrophils with CellTracker Green dye and co-cultured them with human monocyte-derived macrophages in the presence of a panel of anti-DCIR mAbs or Fc-matched isotype control (Figure 6A).We found that both the agonistic clones 3A4 and 9D9, as well as the weak-agonistic clone 5E11 and to a lesser extent non-agonistic 3F7, displayed an enhanced ADCP effect, as indicated by the increased proportion of CellTracker Green-positive macrophages (Figure 6B; Supplementary Figure 6).Moreover, using a commercial ADCC reporter system (Figure 6C), we found that 3A4, 9D9, and 5E11 can promote neutrophil clearance via Fc-mediated ADCC as well.However, the 3A4 LALA mutant failed to trigger the ADCC-associated signaling (Figure 6D).
Internalization of the cell surface target can impair the clinical benefit of ADCC-dependent therapeutic antibodies (31).Lectin receptors, such as CD209 and CD206, have been reported to promote antigen uptake through endocytosis (32,33).Similarly, DCIR, as a C-type lectin, can also be endocytosed, resulting in reduced ADCCmediated cell clearance.To assess the potential internalization effects of our humanized anti-DCIR mAbs, we compared the ADCC-inducing clones (3A4, 9D9, and 5E11) to an anti-TNFα mAb after their binding to the overnight LPS-primed human MoDCs for up to 1 hour.It is consistent with our previous finding (34), anti-TNFα mAb binding to membrane TNFα can trigger rapid internalization, indicated by the increased overlap between the AF647 labeled anti-TNFα mAb with AF488-stained LAMP1, an endosome marker.However, we did not observe obvious endocytosis of anti-DCIR mAb, as most AF647 labeled anti-DCIR mAbs still localized on the plasma membrane (Figure 6E).This data suggests that our anti-DCIR mAb can provide an approach to overcome the endocytosis of DCIR and promote the antibody-mediated clearance of inflammatory DCIR+ cells.
To assess the in vivo antibody-mediated clearance of neutrophils, we compared the effects of WT and LALA mutant anti-DCIR mAbs in a ZymD-induced peritonitis mouse model (Figure 5A).To diminish the confounding effects of inhibitory signaling via the DCIR receptor, we intraperitoneally injected the mice with a weak-agonistic clone 5E11, which significantly promotes both ADCP and ADCC effects.We observed a significant reduction in leukocyte and neutrophil accumulation in the peritoneal fluid of mice treated with WT clone 5E11, compared to the Fc-matched isotype control group.However, the introduction of the LALA mutation into this clone (5E11-LALA) compromised its ability to suppress peritoneal cell accumulation (Figure 6F and G).
To fully exploit the ADCP and ADCC effects mediated by mouse Fc receptors CD16 and CD32, which are only partially activated by the human IgG1 Fc portion of our anti-DCIR mAbs due to suboptimal binding affinity (35), we generated an anti-DCIR mAb, clone 5E11, with mouse IgG2b (5E11-mIgG2b) and evaluated its efficacy in the ZymD-induced peritonitis model.Compared to the Fc-matched isotype control, 5E11-mIgG2b significantly reduced peritoneal leukocyte and neutrophil accumulation to a level comparable to that achieved by anti-Gr1 mAb treatment, a standard approach for depleting mouse neutrophils (Figure 6H and 6I).Consistent with the reduction in neutrophils, 5E11-mIgG2b treatment also suppressed proinflammatory cytokines, IL6 and TNFα, production in the peritoneal lavage (Figure 6J and 6K).Unfortunately, non-agonistic mAbs like 1G3 and 3F7 fail to induce robust ADCC effects, hindering our ability to dissect the distinct contributions of the Fab versus Fc portion separately, and 5E11 still exhibits a weak agonistic effect as depicted in (Figure 2C).In summary, by employing a potent agonistic mAb with abrogated Fc (3A4-LALA) and a weaker agonistic mAb (5E11) with potent ADCP/ADCC effects, we deduce that both the Fab and Fc portion contribute to the protective effects in our peritonitis model.

Agonistic anti-DCIR mAb attenuates the experimental colitis
Our results suggest that engagement of agonistic anti-DCIR mAb triggers immune inhibitory signaling, providing a suppressive effect; meanwhile the Fc fragment of anti-DCIR mAb facilitates clearance of inflammatory cells, promoting resolution of inflammation.These findings support the potential use of anti-DCIR mAbs as therapeutic agents for chronic inflammatory diseases such as IBD.To evaluate this potential, we treated mice with agonistic anti-DCIR mAb (9D9), non-agonistic anti-DCIR mAb (3F7), or isotype control prior to (on Day 0) and during (on Day 3) DSS exposure (Figure 7A).Administration of agonistic anti-DCIR mAb significantly ameliorated DSSinduced colitis, indicated by less body weight loss (Figure 7B).We have found that the extent of neutrophil activation and crypt abscesses correlates with the disease severity in the DSS-induced colitis model by confocal laser endoscopy (CLE) (Figure 7C).Since huDCIR highly expresses on neutrophils accumulated in the colon crypts of huDCIR-KI mice during DSS-induced colitis (Figure 4I), we analyzed the infiltrated neutrophil activation and the integrity of crypts by CLE and found administration of agonistic anti-DCIR mAb (9D9) decreased neutrophil elastase staining (NE680) and increased integrity of crypts (Figure 7D; Movie S3-S6).In the histology of colon, agonistic antibody (9D9) treatment reduced erosion and inflammatory cell infiltration with less hyperplastic and expanded mucosal glands and submucosa compared to non-agonistic anti-DCIR mAb (3F7) and isotype control treatments (Figure 7E).In addition, agonistic antibody (9D9) significantly reduced the MIP-2 secretion (Figure 7F) compared to non-agonistic anti-DCIR mAb (3F7) and isotype control treatments, indicating a reduced chemotaxis to the source of inflammation and activation of neutrophils.Overall, administration of agonistic anti-DCIR mAb significantly ameliorated DSS-induced colitis.

Discussion
Immune inhibitory receptors play a crucial role in maintaining immune homeostasis by regulating excessive immune responses and preventing the development of inflammatory diseases.As a result, targeting these receptors with antagonist antibodies has emerged as a widely adopted approach for cancer treatment.However, utilization of agonistic antibodies to restore immune tolerance in inflammatory diseases remains poorly understood.In this study, we investigated the effects of DCIR agonistic antibodies on immune signaling and discovered their ability to trigger immunosuppressive pathways.Specifically, we found that DCIR agonistic antibodies recruit SHP2 to the intracellular ITIM domain, thereby inhibiting the activation of the immunestimulating SHP2-SYK pathway that is typically initiated by other proinflammatory pattern recognition receptors.
SHP2 can function as an immunosuppressor by dephosphorylating and inactivating downstream molecules involved in the proinflammatory signaling pathways.For instance, SHP2 can dephosphorylate TCRζ chains, ZAP70, and the costimulatory receptor CD28, thereby preventing T cell activation and reducing the production of proinflammatory cytokines (22)(23)(24).Interestingly, other studies suggested that the phosphatase activity of SHP2 is not essential for immune checkpoint receptors' function (16), indicating the involvement of additional regulatory mechanisms beyond its enzymatic activity.Additionally, SHP2 has been reported to act as an adaptor protein that mediates protein-protein interactions, such as ITAM-SHP2-Syk interaction, thereby promoting the activation of immune pathways (25).Hence, even elevated SHP2 expression has been observed in various autoimmune diseases (36)(37)(38).SHP2 can be hijacked to facilitate proinflammatory signaling when strong agonists of inhibitory receptors are absent.Our study provides proof of concept that agonistic signaling through ITIM-bearing DCIR can compete with ITAM-containing pattern recognition receptors for SHP2 binding and restrain proinflammatory signaling.
Given its lectin-like properties, DCIR can recognize various glycoproteins, suggesting a potential overlap in antigens recognized by other lectin receptors (21).It is plausible that DCIR might form co-inhibitory complexes through cross-linking with these shared antigens.Notably, previous studies have shown that cross-linking multiple immune inhibitory receptors using multivalent antibodies can lead to a more robust suppressive effect compared to targeting individual receptors (39)(40)(41).Although we confirmed that our agonistic anti-DCIR antibody can further amplify DCIR signaling even when it is occupied by its natural ligands, such as mannose and NA2glycan, our study did not directly investigate the impact of our agonistic anti-DCIR antibody on the binding of DCIR to other unknown ligands or DCIR's potential cross-linking with other immune receptors.We acknowledge the possibility that our agonistic antibody may disrupt the formation of co-inhibitory complexes in the presence of DCIR's natural ligands.To address this limitation, future studies can explore the application of a bispecific agonistic antibody design to cluster DCIR with another immune inhibitory receptor, such as PD-1, or utilize engineered Fc regions to enhance cross-linking with FcγIIB.These strategies have potential to significantly improve the immune regulatory function by strengthening the co-inhibitory complex.
In addition to its agonistic effect, our anti-DCIR antibody may promote inflammation resolution through Fcmediated neutrophil clearance.While neutrophils play a critical role in antimicrobial defense, they can also contribute to tissue damage by releasing proinflammatory factors such as ROS, granule proteases, and cytokines.Efficient clearance of tissue-infiltrated neutrophils by phagocytes is essential for the prevention of chronic inflammation.Multiple mechanisms have been reported for inducing immunotolerant phagocytes.One such mechanism involves lipid metabolism initiated by engulfed apoptotic cells, which activates β-oxidation and the translocation of PPARs and LXR/RXR.Activation of these pathways can upregulate pro-resolving factors like IL-10 and TGF-β (42,43).Accelerating neutrophil clearance not only prevents the systemic release of proinflammatory ligands and the formation of neutrophil extracellular traps (NETs), but also supports the development of tolerogenic macrophages.Therefore, agonistic anti-DCIR antibodies could provide therapeutic benefits for chronic diseases driven by neutrophil-mediated pathology, including chronic obstructive pulmonary disease (COPD), neutrophilic asthma, rheumatoid arthritis, and gouty arthritis.
Inconsistencies in DCIR-/-mouse phenotypes during DSS-induced colitis have been noted, with Sun et al. (44) reporting protection and Hütter et al. (45) indicating increased susceptibility.This variability suggests potential influences of environmental factors, such as commensal microbes and diet, on muDCIR's natural ligands, altering gut glycan structure and affecting native agonistic effects (46)(47)(48).Sun's study attributes reduced MIP-2 to increased GM-CSF from ILC3 in response to excessive IL-1b secreted by hyperactivated DCIR-/macrophages, suggesting an indirect effect.Our investigation, using huDCIR-KI mice and potent agonistic mAbs, consistently reveals reduced MIP-2 and neutrophil accumulation in peritonitis and DSS-colitis models.This supports our hypothesis and agrees with our findings that potent antibody agonism may overcome the weak effects seen with natural ligands.Notably, differences between mouse and human DCIR could contribute to this variation, underscoring the need to explore huDCIR biology in humanized models.While our huDCIR-KI mice and anti-huDCIR mAbs offer valuable tools for translational animal studies supporting drug development.
Nevertheless, we acknowledge that our prophylactic drug dosing strategy may be less applicable to clinical practices.The acute nature of the peritonitis and colitis models we utilized, with rapid kinetics for neutrophil and monocyte/macrophage activation, limits the testing window for our anti-DCIR mAbs once the disease has developed.Employing appropriate animal models that represent chronic diseases driven by neutrophils and monocytes/macrophages-mediated pathology, such as COPD and neutrophilic asthma, would yield more informative results for examining the therapeutic effects of targeting DCIR.

Sex as a biological variant
For studies involving mice models, male and female mice were studied with no clear differences discerned by sex.Results from the male and female were pooled.DSS in drinking water for 7 days.For the vessel staining, mice were i.v.administrated with 100 μl of 1 mg/ml 10kDa AF680 dextran 2 days before feeding DSS water.For the DCIR+ cells detection, AF488 anti-DCIR mAb were i.v.injected into the mice 3 days before and 6 days after DSS feeding.CLE analyses were performed on day 0 (naïve) and day 7 (colitis) during the DSS-feeding phase.(I) Representative CLE of naïve mice and mice with colitis as described in (F).DCIR-positive cells in the colonic crypts were stained with AF488 anti-DCIR mAb (green) and blood vessels run along the crypt wall were stained with AF680 dextran (red).Scale bars represent 20 μm.

Figure 2 :
Figure 2: Agonistic anti-DCIR mAb induces tyrosine phosphorylation and SHP2 recruitment to DCIR ITIM motif.(A)Graphic of the agonistic effect induced by anti-DCIR mAbs.(B, C) WT and huDCIR transfected HEK293 cells were treated with anti-DCIR mAbs (10μg/ml) for 30 min, followed by immunoprecipitation assay (IP) with antiphosphorylated tyrosine (B) or anti-SHP2 (C) antibodies.DCIR levels were analyzed by WB and quantitated by densitometry.Representative data from 2 independent studies are shown.(D) Illustrate of the huDCIR agonistic effect reporter cell generation.HEK293 cell was co-transfected with a huDCIR vector containing an ITAM motif cloned from Dectin-1, and a luciferase reporter vector with NF-кB response element.(E) Reporter cells as described in (D) were treated with 10μg/ml anti-DCIR mAbs or isotype control for 30 min, followed by luciferase assay analysis.Agonistic effects induced by the antibodies were quantitated by the luminescent signal.(F) Reporter cells as described in (D) were treated with a serial dilution of agonistic or non-agonistic anti-DCIR antibodies, or isotype control for 30 min, followed by luciferase assay analysis.Dose-dependent induction of agonistic signaling was quantitated by the luminescent signal.(G-H) DCIR ligands, mannose and asialo-

Figure 3 :
Figure 3: Agonistic anti-DCIR mAb provides immunosuppressive function by increasing SHP2 binding to the ITIM.(A)Human monocytes were treated with 5 µg/ml antibodies for 30 min.Antibodies' binding were quantitated by the 10g/ml PE anti-human IgG secondary antibody.Data are normalized to the isotype group.Representative data from 2 independent studies are shown.(B) Scheme of the immunosuppressive effect induced by the agonistic anti-DCIR mAb.(C) Human monocytes were treated with 5 µg/ml anti-DCIR mAbs or isotype for 30 min, followed by immunoprecipitation (IP) using anti-SHP2 antibody.SHP2-DCIR interaction was evaluated by the DCIR level analyzed by WB.SHP2 from IP lysate and GAPDH from whole cell lysate were probed as loading controls.(D, E) Human monocytes were pretreated with 5 µg/ml antibodies for 30 min, followed by 50g/ml antihuman IC: HSA stimulation for 30 min.Syk's interactions with SHP2 and FcRγ chain were evaluated by