Macrophage-derived macrophage migration inhibitory factor mediates renal injury in anti-glomerular basement membrane glomerulonephritis.

Macrophages are a rich source of macrophage migration inhibitory factor (MIF). It is well established that macrophages and MIF play a pathogenic role in anti-glomerular basement membrane crescentic glomerulonephritis (anti-GBM CGN). However, whether macrophages mediate anti-GBM CGN via MIF-dependent mechanism remains unexplored, which was investigated in this study by specifically deleting MIF from macrophages in MIFf/f-lysM-cre mice. We found that compared to anti-GBM CGN induced in MIFf/f control mice, conditional ablation of MIF in macrophages significantly suppressed anti-GBM CGN by inhibiting glomerular crescent formation and reducing serum creatinine and proteinuria while improving creatine clearance. Mechanistically, selective MIF depletion in macrophages largely inhibited renal macrophage and T cell recruitment, promoted the polarization of macrophage from M1 towards M2 via the CD74/NF-κB/p38MAPK-dependent mechanism. Unexpectedly, selective depletion of macrophage MIF also significantly promoted Treg while inhibiting Th1 and Th17 immune responses. In summary, MIF produced by macrophages plays a pathogenic role in anti-GBM CGN. Targeting macrophage-derived MIF may represent a novel and promising therapeutic approach for the treatment of immune-mediated kidney diseases.

CD74 supports accumulation and function of regulatory T cells in tumors.

Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observe that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs reveals that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intratumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they are associated with accelerated tumor rejection in preclinical models in female mice. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a target to interfere with Treg anti-tumor activity.

Comprehensive analysis of macrophage-related genes in prostate cancer by integrated analysis of single-cell and bulk RNA sequencing.

Macrophages, as essential components of the tumor immune microenvironment (TIME), could promote growth and invasion in many cancers. However, the role of macrophages in tumor microenvironment (TME) and immunotherapy in PCa is largely unexplored at present. Here, we investigated the roles of macrophage-related genes in molecular stratification, prognosis, TME, and immunotherapeutic response in PCa. Public databases provided single-cell RNA sequencing (scRNA-seq) and bulk RNAseq data. Using the Seurat R package, scRNA-seq data was processed and macrophage clusters were identified automatically and manually. Using the CellChat R package, intercellular communication analysis revealed that tumor-associated macrophages (TAMs) interact with other cells in the PCa TME primarily through MIF - (CD74+CXCR4) and MIF - (CD74+CD44) ligand-receptor pairs. We constructed coexpression networks of macrophages using the WGCNA to identify macrophage-related genes. Using the R package ConsensusClusterPlus, unsupervised hierarchical clustering analysis identified two distinct macrophage-associated subtypes, which have significantly different pathway activation status, TIME, and immunotherapeutic efficacy. Next, an 8-gene macrophage-related risk signature (MRS) was established through the LASSO Cox regression analysis with 10-fold cross-validation, and the performance of the MRS was validated in eight external PCa cohorts. The high-risk group had more active immune-related functions, more infiltrating immune cells, higher HLA and immune checkpoint gene expression, higher immune scores, and lower TIDE scores. Finally, the NCF4 gene has been identified as the hub gene in MRS using the "mgeneSim" function.

Immunohistochemical study of CD74 biomarker in normal and malignant breast tissues.

OBJECTIVE: Aim: To detect the role of CD74 expression in breast carcinoma as a predictive marker for identifying the biological behavior of malignancy in Iraqi women.. PATIENTS AND METHODS: Materials and Methods: The study used technique of immunohistochemistry for detection CD74 protein role in breast cancer, and its expression in breast cancer tissue samples. Samples were collected in Al-Najaf city in Iraq, from Al-Forat Al-Awsat Oncology Center. The study was achieved at the Laboratories of the Faculty of Science in the University of Kufa. Fifty samples of breast cancer tissue, and twenty controls benign tissue were included in the study. The study has investigated relationship between expression of biomarker with grade, age of patient and tumor size. RESULTS: Results: The study showed that the cytoplasmic expression of CD74 with more clear and intensive staining in the cytoplasm, and reported that CD74 positivity rate was 52%. A significant association between CD74 expression and grade and size of tumor, so CD74 can be considered as a biomarker for prediction of breast cancer in women. No association was found between CD74 expression and each of patients' age and node metastasis. CONCLUSION: Conclusions: The study represents an important step in our region because there are a few studies about this topic; more efforts are required to approve the function of this biomarker.

MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study.

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 µg); or MIF mAb (15 µg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

Blocking the MIF-CD74 axis augments radiotherapy efficacy for brain metastasis in NSCLC via synergistically promoting microglia M1 polarization.

BACKGROUND: Brain metastasis is one of the main causes of recurrence and death in non-small cell lung cancer (NSCLC). Although radiotherapy is the main local therapy for brain metastasis, it is inevitable that some cancer cells become resistant to radiation. Microglia, as macrophages colonized in the brain, play an important role in the tumor microenvironment. Radiotherapy could activate microglia to polarize into both the M1 and M2 phenotypes. Therefore, searching for crosstalk molecules within the microenvironment that can specifically regulate the polarization of microglia is a potential strategy for improving radiation resistance. METHODS: We used databases to detect the expression of MIF in NSCLC and its relationship with prognosis. We analyzed the effects of targeted blockade of the MIF/CD74 axis on the polarization and function of microglia during radiotherapy using flow cytometry. The mouse model of brain metastasis was used to assess the effect of targeted blockade of MIF/CD74 axis on the growth of brain metastasis. RESULT: Our findings reveals that the macrophage migration inhibitory factor (MIF) was highly expressed in NSCLC and is associated with the prognosis of NSCLC. Mechanistically, we demonstrated CD74 inhibition reversed radiation-induced AKT phosphorylation in microglia and promoted the M1 polarization in combination of radiation. Additionally, blocking the MIF-CD74 interaction between NSCLC and microglia promoted microglia M1 polarization. Furthermore, radiation improved tumor hypoxia to decrease HIF-1α dependent MIF secretion by NSCLC. MIF inhibition enhanced radiosensitivity for brain metastasis via synergistically promoting microglia M1 polarization in vivo. CONCLUSIONS: Our study revealed that targeting the MIF-CD74 axis promoted microglia M1 polarization and synergized with radiotherapy for brain metastasis in NSCLC.

CXCR4 and CD74 together enhance cell survival in response to macrophage migration-inhibitory factor in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small, mature CD5+ B lymphocytes in the blood, marrow, and lymphoid organs. Cell survival depends on interaction with the leukemic microenvironment. However, the mechanisms controlling CLL cell survival are still incompletely understood. Macrophage migration-inhibitory factor (MIF), a pro-inflammatory and immunoregulatory chemokine-like cytokine, interacts with CXCR4, a major chemokine receptor, as well as with CD74/invariant chain, a single-pass type II receptor. In this study, we analyzed the roles of CXCR4, CD74, and MIF in CLL. Mononuclear cells from patients with hematological malignancies were analyzed for coexpression of CXCR4 and CD74 by flow cytometry. Strong co- and overexpression of CXCR4 and CD74 were observed on B cells of CLL patients (n = 10). Survival and chemotaxis assays indicated that CXCR4 and CD74 work together to enhance the survival and migration of malignant cells in CLL. Blockade of the receptors, either individually or in combination, promoted cell death and led to an abrogation of MIF-driven migration responses in murine and human CLL cells, suggesting that joint activation of both receptors is crucial for CLL cell survival and mobility. These findings indicate that the MIF/CXCR4/CD74 axis represents a novel therapeutic target in CLL.

Identification of invariant chain CD74 as a functional receptor of tissue inhibitor of metalloproteinases-1 (TIMP-1).

Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chain-associated protein kinase-70 (ZAP-70) signaling-associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1-CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1-CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide-abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1-CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis.

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

Nanoparticles Displaying Allergen and Siglec-8 Ligands Suppress IgE-FcεRI-Mediated Anaphylaxis and Desensitize Mast Cells to Subsequent Antigen Challenge.

Siglec-8 is an inhibitory receptor expressed on eosinophils and mast cells. In this study, we took advantage of a novel Siglec-8 transgenic mouse model to assess the impact of modulating IgE-dependent mast cell degranulation and anaphylaxis using a liposomal platform to display an allergen with or without a synthetic glycan ligand for Siglec-8 (Sig8L). The hypothesis is that recruitment of Siglec-8 to the IgE-FcεRI receptor complex will inhibit allergen-induced mast cell degranulation. Codisplay of both allergen and Sig8L on liposomes profoundly suppresses IgE-mediated degranulation of mouse bone marrow-derived mast cells or rat basophilic leukemia cells expressing Siglec-8. In contrast, liposomes displaying only Sig8L have no significant suppression of antigenic liposome-induced degranulation, demonstrating that the inhibitory activity by Siglec-8 occurs only when Ag and Sig8L are on the same particle. In mouse models of anaphylaxis, display of Sig8L on antigenic liposomes completely suppresses IgE-mediated anaphylaxis in transgenic mice with mast cells expressing Siglec-8 but has no protection in mice that do not express Siglec-8. Furthermore, mice protected from anaphylaxis remain desensitized to subsequent allergen challenge because of loss of Ag-specific IgE from the cell surface and accelerated clearance of IgE from the blood. Thus, although expression of human Siglec-8 on murine mast cells does not by itself modulate IgE-FcεRI-mediated cell activation, the enforced recruitment of Siglec-8 to the FcεRI receptor by Sig8L-decorated antigenic liposomes results in inhibition of degranulation and desensitization to subsequent Ag exposure.

Induction of the endogenous sialoglycan ligand for eosinophil death receptor Siglec-8 in chronic rhinosinusitis with hyperplastic nasal polyposis.

Siglec-8, an immune-inhibitory sialoglycan binding lectin (S8), is expressed on the surface of eosinophils and mast cells, which are potent mediators of allergic inflammation. When S8 engages endogenous sialoglycan ligands, eosinophils undergo apoptosis and mast cell mediator release is inhibited. In the human airway, Siglec-8 ligands (S8L) are sialylated keratan sulfate chains carried on isoforms of the protein Deleted in Malignant Brain Tumors-1 (DMBT1), an immunoregulatory protein that we recently identified as the endogenous ligand for S8, DMBT1S8. We herein report that S8L is overexpressed in chronic rhinosinusitis with nasal polyposis (CRSwNP), a prevalent eosinophilic laden airway disease. Quantification and comparison of the degree to which DMBT1 carries the S8L by immunoblot analysis and lectin blot overlay, respectively, from nasal lavage showed that the S8L/DMBT1 ratio was significantly increased in CRSwNP vs. control or CRS patients. We identified the histological sites of S8L and DMBT1 expression in fresh surgically resected human nasal polyps. Histochemistry of diseased polyps and adjacent nondiseased middle turbinate (MT) tissue from CRSwNP demonstrated colocalization of S8L and DMBT1 with highest staining in submucosal glands >> epithelium > stoma. S8L expression was specifically elevated in the submucosal glands and epithelium of polyp tissue compared to MT. We hypothesize that expression of the isoform of DMBT1 carrying the Siglec-8 binding sialoglycan, DMBT1S8, is induced in polyps of CRSwNP specifically at the site of disease, is produced in the submucosal glands of polyps and secreted into the lumen of the sinonasal cavity as a host response to mitigate eosinophil-mediated inflammation.

Interplay Between Sialic Acids, Siglec-E, and Neu1 Regulates MyD88- and TRIF-Dependent Pathways for TLR4-Activation During Leishmania donovani Infection.

TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How Leishmania donovani suppresses these pathways is not well studied. We earlier reported, TLR4 is hypersialylated due to reduced membrane-bound neuraminidase (Neu1) on infected-macrophages. We hypothesized that such enhanced sialoglycoconjugates on host cells may modulate the interactions with siglecs- which are the inhibitory receptors. Here, we examined the impact of such sialylation on overall TLR4 activation both in murine cell line J774A.1 and primary bone marrow derived macrophages (BMDM). Supporting this hypothesis, we demonstrated siglec-E engages hypersialylated TLR4 during infection. Such sialic acids-siglec-E interaction enhanced siglec-E phosphorylation that mediated its strong association with SHP1/SHP2 and also upregulated their phosphorylation in both types of macrophages. Pre-treatment of parasites and host cells with neuraminidase reduced SHP1/SHP2 phosphorylation and triggered TLR4 activation respectively through enhanced nuclear translocation of p-65. Moreover, a reciprocal interplay between Neu1 and siglec-E differentially regulates MyD88- and TRIF-pathways through sialic acids on TLR4 as their common substrate during infection. Correspondingly, Neu1 overexpression enhanced MyD88-signaling while still suppressing TRIF-activation. However, silencing siglec-E specifically activated TRIF-signaling. Pro-inflammatory cytokines corresponding to MyD88 and TRIF pathways were also upregulated respectively. Additionally, Neu1 overexpression or siglec-E silencing prevented TLR4 ubiquitination and subsequent degradation by Triad3A. Neu1-overexpression and siglec-E-silencing together followed by infection activated both MyD88 and TRIF-signaling through their enhanced TLR4-association. This elevated the MyD88-specific cytokines and TRIF-mediated IRF3 and IFN-ß genes, thus upregulating the pro-inflammatory cytokines and nitric oxide levels and reduced anti-inflammatory cytokines. All these significantly inhibited parasite survival in macrophages thus demonstrating a previously unidentified dualistic regulation of TLR4signaling pathways activation through sialic acids by interplay of Neu1 and siglec-E during Leishmania infection.

CD74 is a regulator of hematopoietic stem cell maintenance.

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.

Hsp90-stabilized MIF supports tumor progression via macrophage recruitment and angiogenesis in colorectal cancer.

Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity, but its expression is increased in some cancers via stabilization with HSP90-associated chaperones. Here, we show that MIF stabilization is tumor-specific in an acute colitis-associated colorectal cancer (CRC) mouse model, leading to tumor-specific functions and selective therapeutic vulnerabilities. Therefore, we demonstrate that a Mif deletion reduced CRC tumor growth. Further, we define a dual role for MIF in CRC tumor progression. Mif deletion protects mice from inflammation-associated tumor initiation, confirming the action of MIF on host inflammatory pathways; however, macrophage recruitment, neoangiogenesis, and proliferative responses are reduced in Mif-deficient tumors once the tumors are established. Thus, during neoplastic transformation, the function of MIF switches from a proinflammatory cytokine to an angiogenesis promoting factor within our experimental model. Mechanistically, Mif-containing tumor cells regulate angiogenic gene expression via a MIF/CD74/MAPK axis in vitro. Clinical correlation studies of CRC patients show the shortest overall survival for patients with high MIF levels in combination with CD74 expression. Pharmacological inhibition of HSP90 to reduce MIF levels decreased tumor growth in vivo, and selectively reduced the growth of organoids derived from murine and human tumors without affecting organoids derived from healthy epithelial cells. Therefore, novel, clinically relevant Hsp90 inhibitors provide therapeutic selectivity by interfering with tumorigenic MIF in tumor epithelial cells but not in normal cells. Furthermore, Mif-depleted colonic tumor organoids showed growth defects compared to wild-type organoids and were less susceptible toward HSP90 inhibitor treatment. Our data support that tumor-specific stabilization of MIF promotes CRC progression and allows MIF to become a potential and selective therapeutic target in CRC.

The MHC-II antigen presentation machinery and B7 checkpoint ligands display distinctive patterns correlated with acute myeloid leukaemias blast cells HLA-DR expression.

Acute Myeloid Leukaemia (AML) is a neoplasia characterised by rapid proliferation and an increased rate of relapses. The AML blasts display features of antigen-presenting cells (APC), and thus can directly modulate the anti-tumour T cell responses. The bone marrow of a group consisting of 30 newly diagnosed patients and four healthy donors (HD) was investigated for the expression of HLA-DR, several molecules involved in MHC-II antigen-presentation and MHC-II groove editing, like HLA-DM, CD74 and CLIP, as well as a set of immune checkpoint ligands, like ICOS-L, B7.2, PD-L2 and B7-H3. The patients were further characterised for their genetic anomalies and distributed to favourable, intermediate and adverse ELN risk categories. We were able to show that while 23% of our patients displayed a low level of HLA-DR surface expression, all patients displayed higher HLA-DM and CD74 expression compared to HD. However, a higher CLIP expression was noticed only in the HLA-DR low patients. The co-inhibitory PD-L2 and B7-H3 molecules were increased in the cases with normal HLA-DR expression; oppositely, the co-stimulatory ICOS-L and the dual function B7.2 were significantly increased in the cases with HLA-DR low expression. Furthermore, no favourable ELN risk cases were found within the HLA-DR low group. All in all, these data show that the AML with low versus normal HLA-DR expression display different profiles of MHC class II machinery molecules and B7 ligands, which are correlated with distinct ELN stratification. Furthermore, as our study included healthy individuals, it offers valuable information about the expression levels that should be considered as normal for these markers known to cause differences in peptide repertoires, reflected further in distinct T-cells polarisation pathways.

Permissive HLA-DPB1 mismatches in HCT depend on immunopeptidome divergence and editing by HLA-DM.

In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-ß (TCRß) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCRß clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.

A monoclonal antibody to Siglec-8 suppresses non-allergic airway inflammation and inhibits IgE-independent mast cell activation.

In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.

[Expression of cluster of differentiation 74 in prostate cancer and its clinical significance].

OBJECTIVE: To study the expression of cluster of differentiation 74 (CD74) in PCa and its clinical significance. METHODS: Using immunohistochemical staining, we detected the expression of CD74 in 56 samples of PCa tissue and 55 samples of the adjacent BPH tissue. RESULTS: The rates of CD74 expression in the epithelial and stromal cells were 48.2% and 82.3% in the PCa tissue compared with 62.7% and 83.6% in the adjacent BPH tissue, with statistically significant difference between the two types of cells (P < 0.05 or P < 0.01), and those in the stromal cells of the PCa tissue with Gleason score ≤7 or >7 were 44.0% versus 80.8% (P < 0.05), while those in the epithelial cells of the PCa tissue with Gleason score ≤7 or >7 were 36.7% versus 61.5% (P > 0.01). CD74 was overexpressed in the poorly differentiated PCa cells and the surrounding stromal cells. CONCLUSIONS: CD74 may play an important role in the development and invasiveness of PCa, and is expected to be a prognostic indicator of the malignancy.

Bacterial lipopolysaccharide induces the intracellular expression of trophoblastic specific CD74 isoform in human first trimester trophoblast cells: Correlation with unsuccessful early pregnancy.

OBJECTIVE: During first trimester of human pregnancy, the maternal system develops immunity against infection and to provide protection of allogeneic foetus from abortion. This study was undertaken to determine the role of trophoblast specific CD74 isoforms in first trimester trophoblast derived cells under normal and lipopolysaccharide (LPS) stimulated conditions. METHODS: Gene and protein of CD74 were determined in first trimester trophoblast derived cells, JEG-3 and ACH-3 P and also in human placenta by PCR, western blotting and immunoprecipitation. Effect of LPS mediated infection on the regulation of CD74 isoforms was studied intracellularly and also on the cells surface by flow cytometry. RESULTS: Data demonstrated that JEG-3 and ACH-3 P cells under normal conditions have not expressed CD74 isoforms neither intracellularly or nor on the surface. These results were further validated directly in human placenta. However, treatment of these trophoblast cells with a bacterial LPS, significantly upregulated CD74 mRNA expression (p < 0.05). Furthermore, expression of CD74 on the surface was not detected even after stimulation with LPS. Interestingly, CD74 isoform at 35 kDa was significantly detected intracellularly upon stimulation with LPS (p < 0.05). These results were further confirmed by western blotting followed by immunoprecipitation. CONCLUSIONS: To the best of our knowledge, this is the first study concluded that the bacterial LPS induce infection in the first trimester trophoblasts via intracellular upregulation of CD74. Data indicated that the lack of cell surface expression of trophoblastic specific isoforms of CD74 may provide protection for human pregnancy in the first trimester.

The antifibrotic adipose-derived stromal cell: Grafted fat enriched with CD74+ adipose-derived stromal cells reduces chronic radiation-induced skin fibrosis.

Fat grafting can reduce radiation-induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose-derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74- and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor ß3 (TGF-ß3) and decreased levels of TGF-ß1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74- or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation-induced fibrosis.