Expression Levels Of CD86 Research Articles

Phase 2 Study of E7777, a Diphtheria Toxin Fragment-Interleukin-2 Fusion Protein, in Japanese Patients with Relapsed or Refractory Peripheral and Cutaneous T-Cell Lymphoma

Phase 2 Study of E7777, a Diphtheria Toxin Fragment-Interleukin-2 Fusion Protein, in Japanese Patients with Relapsed or Refractory Peripheral and Cutaneous T-Cell Lymphoma

Serum Soluble CD86, Still a Prognostic Factor in the Novel Agent Era in Multiple Myeloma Patients, Is Produced By Myeloma Cells with High CD86 Variant 3 Expression

Introduction: We previously reported that CD86 expressed on tumor cells from multiple myeloma (MM) patients is associated with a proliferative advantage of tumor cells and suppresses the antitumor immune response by inducing the immunosuppressive cytokine IL-10 from CD4+ T cells via CD86-CD28 interaction [Yamashita T, Clin Cancer Res 2009]. Furthermore, CD28 expressed on MM cells can mediate pro-survival signaling through CD86-CD28 interaction, resulting in chemotherapeutic resistance [Murray ME, Blood 2014]. Hock et al. reported that serum soluble CD86 levels (sCD86) were a significant independent prognostic marker in MM patients treated with conventional chemotherapy [Br J Haematol 2006]. However, it is unknown whether serum soluble CD86 is still a prognostic marker in the novel agent era and how sCD86 is produced in serum. In this study, we investigated the association of clinical characteristics and prognosis with serum sCD86 and elucidated the mechanism by which sCD86 is produced in MM patients. Materials and Methods: 1) Peripheral blood and bone marrow (BM) samples were obtained from 294 newly diagnosed (42 asymptomatic and 252 symptomatic) MM patients, 16 patients with monoclonal gammopathy of undetermined significance (MGUS), and 16 healthy controls. 2) sCD86 levels were measured using ELISA. 3) The expression of cell-surface antigen on plasma cells identified as CD138-positive and CD38-strong positive cells were analyzed using flow cytometry. Expression of the CD86 full-length (variant 1) and alternatively spliced variant deleting exon 6 (variant 3), which encodes for the transmembrane domain, in MM cell lines and CD138+ plasma cells isolated from BM samples from MM patients were analyzed using real-time PCR. Results: 1) sCD86 levels were significantly increased in MM patients compared with MGUS patients and healthy controls. Among MM patients, the levels were significantly higher in symptomatic than in asymptomatic patients and markedly increased in advanced stages (International Staging System [ISS] stage II/III and revised ISS II/III). We next investigated the differences in clinical characteristics between two groups according to serum sCD86 levels: high (≥2.16 ng/mL); and low (<2.16 ng/mL). In the high group, plasma cell percentages and corrected calcium, creatinine, and β2-microglobulin levels were markedly higher, and hemoglobin, hematocrit, and estimated glomerular filtration rate significantly lower than those in the low group. Furthermore, MM patients in the high group had significantly shorter overall survival (OS) times than those in the low group (hazard ratio, 0.385; 95% confidence interval, 0.1572-0.9425; P=0.032). 2) CD86 variant 1 and variant 3 were found to be expressed in plasma cells from MM patients. The mRNA levels of CD86 variant 3 in the sCD86-high group were significantly increased in comparison with the low group. These levels were significantly correlated with the concentration of sCD86 (r=0.635, P=0.0003). High levels of CD86 variant 3 mRNA were detected in primary MM cells, but not in hematopoietic BM cells, although the expression of CD86 variant 1 was the same in both MM and hematopoietic cells. On the other hand, sCD86 in cell cultures and mRNA of CD86 variant 3 were not detected in CD86-expressing MM cell lines, suggesting that these phenomena may be seen only in primary MM cells from patients. 3) There was a weak positive correlation between serum sCD86 concentration and CD86 expression levels on MM cells. However, progression-free survival (PFS) and OS were not significantly different between MM patients with high and low expression of CD86. Further, we confirmed that recombinant human CD86 could bind to MM cell lines through CD86-CD28 interaction, which might induce aggressive disease with chemotherapeutic resistance in MM cells. Consistent with this hypothesis, patients with low expression of sCD86/its receptor CD28 had longer OS times than others. In addition, receptor CD28 expression in patients with high CD86 expression was significantly higher than in those with low expression levels. Conclusions: We demonstrated that sCD86 is produced via expression of CD86 variant 3 in primary MM cells and that sCD86 is a prognostic marker in the novel agent era. These results suggest that serum sCD86 levels may reflect disease progression through CD28 expressed on myeloma cells and be a useful prognostic factor. Disclosures Inokuchi: Novartis: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria. Handa:Ono: Research Funding. Komatsu:Fuso Pharmaceutical Industries, Ltd.: Research Funding; Novartis K.K: Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau; Wako Pure Chemical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau. Imai:Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Ito:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Ono: Honoraria. Tamura:Celegene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical: Honoraria; Takeda Pharmaceutical: Honoraria.

INTERDEPENDENCE BETWEEN THE PHENOTYPE OF DENDRITIC CELLS AND AMOUNTS OF BLOOD PROINFLAMMATORY MONOCYTES IN PATIENTS WITH KIDNEY CANCER

The aim of the study was to investigate an interdependence between the phenotype of dendritic cells (DC) differentiated from monocytes and the number of pro-inflammatory monocytes in peripheral blood of patients with kidney cancer (KC). The study involved 28 patients at the age of 40-55 years suffering with KC (Т3N0М0, clear cell type) before surgical treatment. The diagnosis was verified histologically. 31 healthy agematched persons were examined as a control group. Mononuclear cells were isolated from heparinized venous blood by centrifugation in a Histopaque®-1077 density gradient followed by plastic adsorption in RPMI 1640 medium supplied with 10% autologous serum. Immature DCs (iDCs) were generated from blood monocytes by culturing for 5 days with GM-CSF and IFNα. Activation of DCs (mDCs) was induced by incubation with the tumor cell lysate and TNFα, followed by incubation for 48 hours. A tumor fragment was used to prepare the lysate of autologous tumor cells. Phenotyping of blood monocytes and DC at various maturation stages was performed by flow cytometry. The numbers of CD14+CD16+ monocytes in peripheral blood of KC patients were decreased (up to 42% of the total monocyte level) against the control ranges. In this regard, the analysis of the dependence between the phenotype of DCs differentiated from monocytes and the number of pro-inflammatory blood monocytes was carried out by comparing the groups with a high content of pro-inflammatory monocytes in the blood in KC patients (> 42%, near-control range) and low content (resp., < 42%). We have found that the contents of tolerogenic iDC in cell culture are increased in KC patients with low amounts of pro-inflammatory monocytes in blood (< 42%). A relatively increased expression of antigen-presenting and co-stimulatory molecules proved to be the specific feature of iDC phenotype in patients with high contents (> 42%) of proinflammatory monocytes in blood. The phenotype of dendritic cells in KC patients with different content of proinflammatory monocytes during maturation/activation showed more differences. In the patients with low levels of pro-inflammatory monocytes, the cell pool of in vitro maturing DCs was characterized by low level of CD86 and HLA-DR receptor expression, thus reflecting a weak co-stimulating and antigen-presenting activity. In the patients with high levels of pro-inflammatory monocytes in blood, the in vitro activated DCs showed higher level of functional activity using the above markers. The revealed differences in the DC phenotype and interrelations with amounts of blood monocyte subpopulations in KC patients may presume the programmed cell differentiation mechanisms depending on the microenvironment, under pathogenic conditions (i.e., in presence of malignant tumor growth).

Cervical cancer patients that respond to chemoradiation therapy display an intense tumor infiltrating immune profile before treatment

Cervical cancer (CC) is a major cause of death and suffering to women globally with 570,000 new cases in 2017. It disproportionately affects those living in resource-constrained countries such as Brazil, with 90% of the deaths from CC happening in low and middle-income countries. Early detection is still the best strategy for improving response to therapy and survival and cases detected in advanced stages show variable response rates to the standard chemoradiation therapy protocols. Both the genetic landscape and the immune status of patients can dramatically affect cancer progression and response to therapy, as well as disease recurrence. Here we performed a comprehensive sequencing analysis using the cancer gene panel - Ion AmpliSeq™ Cancer hotspot Panel V2 CHPv2, as well as determined the immune infiltrate composition of a group of locally advanced CC patients with the goal of identifying genetic and immune characteristics associated with a clinical response to therapy. The expression levels of CD68+ tumor-associated macrophages (TAMs) and CD8+ tumor-infiltrating lymphocytes (TILs), as well as the immune checkpoint molecules PD-1, PD-L1 and PD-L2 in stroma and in tumor regions were analyzed by immunohistochemistry (IHC). The HPV infection status with high-risk strains was also determined. Twenty-one samples from patients with squamous cell carcinoma segregated into responder (11) and non-responder (10) groups according to standard chemoradiation therapy response were studied. Our findings indicate that responder patients showed an increase of an inflammatory tumor microenvironment as indicated by higher numbers of CD8+ and PD-L2+ TILs, as well as higher expression of PD-L1 immunoreactive area, as compared to the non-responder group. Additionally, our results demonstrate a correlation between the number of gene mutations and PD-L2+ TILs in the responder group. The genes PIK3CA and KDR/VEGFR were the most mutated genes, corroborating past findings. Together, these findings indicate an inflammatory tumor microenvironment present in patients that will respond to future chemoradiation treatment as compared to those that will not. This points to possible future predictors of response to therapy in CC patients.

Short Call Abstracts

Short Call Abstracts

6-Sulfo LacNAc (Slan) as a Marker for Non-classical Monocytes.

Monocytes are subdivided into three subsets, which have different phenotypic and functional characteristics and different roles in inflammation and malignancy. When in man CD14 and CD16 monoclonal antibodies are used to define these subsets, then the distinction of non-classical CD14low and intermediate CD14high monocytes requires setting a gate in what is a gradually changing level of CD14 expression. In the search for an additional marker to better dissect the two subsets we have explored the marker 6-sulfo LacNAc (slan). Slan is a carbohydrate residue originally described to be expressed on the cell surface of a type of dendritic cell in human blood. We elaborate herein that the features of slan+ cells are congruent with the features of CD16+ non-classical monocytes and that slan is a candidate marker for definition of non-classical monocytes. The use of this marker may help in studying the role of non-classical monocytes in health and in diagnosis and monitoring of disease.

Activation of mucosal-associated invariant T cells in the lungs of sarcoidosis patients

Although the pathogenesis of sarcoidosis is not fully understood, immunological characterization has elucidated highly polarized expression of the type 1 T helper cell response. Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize bacterial riboflavin and rapidly produce cytokines such as interferon γ and tumor necrosis factor α. We prospectively evaluated the proportion of MAIT cells and the expression levels of cell surface markers in peripheral blood from 40 sarcoidosis patients and 28 healthy controls. MAIT cells in bronchoalveolar lavage fluid (BALF) were also examined in 12 sarcoidosis patients. In peripheral blood, the proportion of MAIT cells was lower (P = 0.0002), but the expression levels of CD69 and programmed death 1 on MAIT cells were higher in sarcoidosis patients than in healthy controls. Moreover, CD69 expression levels were significantly correlated with clinical biomarkers. Sarcoidosis patients with parenchymal infiltration in the lungs showed a significantly higher proportion and number of MAIT cells in BALF compared to patients without parenchymal infiltration. CD69 expression levels on MAIT cells in BALF were higher than levels in peripheral blood. The activation status of MAIT cells might reflect the disease activity of sarcoidosis. Therefore, it is a potential target for sarcoidosis treatment.

Silencing of CD86 in dendritic cells by small interfering RNA regulates cytokine production in T cells from patients with allergic rhinitis invitro.

The aim of the present study was to investigate the expression and role of the co‑stimulatory molecule T‑lymphocyte activation antigen CD86 (CD86) in dendritic cells (DCs) from the peripheral blood of patients with allergic rhinitis (AR) compared with those from healthy individuals. It was observed that mature DCs from the peripheral blood of patients with AR expressed high levels of the co‑stimulatory molecule CD86, but not CD80, compared with healthy control subjects. CD86 expression levels in DCs decreased significantly following transfection with siRNA in a lentiviral vector. Furthermore, the level of transforming growth factor‑β1 produced by T cells co‑cultured with DCs was significantly increased in the siRNA group, while interleukin (IL)‑4 and IL‑5 production was significantly decreased. The findings of the present study indicated that CD86 may play a pivotal role in the regulatory T cell/type2 helper T cell imbalance in allergic inflammation.

LOX-1+ PMN-MDSC enhances immune suppression which promotes glioblastoma multiforme progression.

Background/aimsPatients with glioblastoma multiforme (GBM) that is the most common brain cancer in adults have a rather poor prognosis. The accumulation of immune suppressive myeloid-derived suppressor cell (MDSC) is negatively associated with clinical outcomes in various cancers. A recent study identified that lectin-type oxidized LDL receptor 1 (LOX-1) may serve as a specific marker of human polymorphonuclear neutrophil (PMN)-MDSC. Thus, herein we focused on exploring the role of LOX-1+ PMN-MDSC in GBM progression.MethodsLOX-1, IFN-γ, dichlorodihydrofluorescein diacetate (DCFDA), CD15, CD4 and CD8 expression levels were examined by flow cytometry. ARG1 and iNOS expression levels in PMN were examined by quantitative real-time PCR. LOX-1 and CD15 expression levels in tumor tissue were determined by immunofluorescent microscopy. T cell proliferation was determined by 3H-thymidine incorporation.ResultsWe identified a protumorigenic subset of PMN, which constitutively expressed LOX-1 and accumulated in the peripheral blood of GBM patients. Compared to LOX-1− PMN, the LOX-1+ PMN exhibited a PMN MDSC profile, with a significant increase in the expression of DCFDA, ARG1 and iNOS, and the capacity of inhibiting the CD3+ T cell proliferation in a dependent-ARG1/iNOS way. Additionally, we found that LOX-1+ PMN negatively correlated with effector immune cells in GBM patients, accumulated in GBM tissues, and was related to early recurrence and disease progression tightly.ConclusionOur study revealed that LOX-1+ PMN-MDSC inhibited the T cell proliferation to enhance immune suppression, which may play a key role in driving the GBM progression.

Gastrodin, a traditional Chinese medicine monomer compound, can be used as adjuvant to enhance the immunogenicity of melanoma vaccines

Gastrodin (GAS) is a Chinese medicine with wide application for the treatment of nervous system disease. Previous studies reported that GAS exhibited non-specific immunomodulatory activities. To explore the effects of GAS as a vaccine adjuvant, the expression levels of CD80, CD86, MHCI and MHCII activated markers were detected after GAS treatment in vitro and in vivo, and the expression levels of IL-2 and TNF-α in splenocytes were detected after GAS treatment in vivo. Besides, the expression levels of IL-2 and IFN-γ in CD4+T cells and perforin, TNF-α and IFN-γ in CD8+T cells were detected. The effects of GAS on the survival rate and tumor size of tumor-challenged mice and the effect of cytotoxicity on CD8+T cells were also investigated. Our data showed that GAS ameliorated CD8+T cell mediated immune response and significantly improved protection of tumor-challenged animals. The results demonstrated that GAS is a potential adjuvant contributing to anticancer immunomodulation.

The Stat3 inhibitor, S3I-201, downregulates lymphocyte activation markers, chemokine receptors, and inflammatory cytokines in the BTBR T+ Itpr3tf/J mouse model of autism

Autism is a complex neurodevelopmental disorder with a high incidence rate. It is characterized by deficits in communication, a lack of social skills, cognitive inflexibility, and stereotypical behaviors. Autism has been gradually increasing in children over the past several years, without the existence of an effective treatment. BTBR T+ Itpr3tf/J (BTBR) mice serve as an accepted model to evaluate autistic-like behaviors as they display core behavioral symptoms displayed in autism. Previous findings showed that S3I-201, a selective Stat3 inhibitor, can be used to treat neuroinflammation disorders. Previously, we showed that S3I-201 treatment has therapeutic effects on autism-like behaviors, and Th1/Th17 and regulatory T cells in BTBR mice. The objective of the present study was to further explore the role of S3I-201 in BTBR mice, and this was performed by investigating the effects of S3I-201 treatment on lymphocyte activation markers (CD4+CD25+ and CD4+CD69+), chemokine receptors (CD4+CCR6+, CD4+CCR7+, CD4+CXCR4+, and CD4+CXCR5+), and proinflammatory cytokines (CD4+IL-6+ and CD4+TNF-α+) in the spleen cells of BTBR and C57BL/6 (C57) mice. The mRNA and protein expression levels of CD69, CCR6, CCR7, CXCR4, CXCR5, IL-1β, IL-6, and TNF-α were examined in the brain tissues, and in BTBR mice, a significant decrease in CD25, CD69, CCR6, CCR7, CXCR4, CXCR5, IL-6, and TNF-α producing CD4+ T cells was observed. The present findings suggest that treatment with S3I-201 may be a therapeutic approach to improve immune abnormalities in a subgroup of autistic subjects.

High Expression of Angiopoietin-like Protein 4 in Advanced Colorectal Cancer and its Association with Regulatory T Cells and M2 Macrophages.

Colorectal cancer (CRC) is one of the most aggressive tumours in the human digestive system. Most CRC patients have poor prognosis due to metastasis and recurrence. Angiopoietin-like 4 (ANGPTL4) is involved in tumour development. Regulatory T (Treg) cells and M2 macrophages promote tumour growth and metastasis. Herein, we explored the changes of ANGPTL4 expression in CRC patients at different stages and observed whether in situ tumour-Treg and -M2 macrophages are correlated with ANGPTL4 expression. Serum ANGPTL4 (sANGPTL4) levels of 70 CRC patients and 10 healthy controls were detected by ELISA. ANGPTL4, Foxp3 and CD163 expression levels in CRC tissues were measured by immunohistochemistry. Recombinant ANGPTL4 (rANGPTL4) proteins were further added into cell-culture systems for induction of Treg cells and M2 macrophages. The results showed both sANGPTL4 and in situ tumour-ANGPTL4 expression levels increased in Dukes C-D stage CRC patients. Foxp3+ and CD163+ cells in tumour tissue sections were also more intensive in Dukes C-D stage patients than in Dukes A-B stage patients. Foxp3+ and CD163+ cells in tumour tissues were positively correlated with both tissue and sANGPTL4 expression (P < 0.01). Recombinant ANGPTL4 promoted the induction of murine Treg cells and M2 macrophages ex vivo. Therefore, elevated ANGPTL4 expression could be a marker for advanced CRC. Treg cell and M2 macrophage induction could be one of the mechanisms of tumour promotion mediated by ANGPTL4.

Abstract 3091: Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions

Abstract Background: Exosomes are a subset of small (30-150nm) extracellular vesicles (EVs) of the endocytic origin that mediate intercellular communication in health and disease. Exosomes are actively produced by tumor cells, circulate freely in body fluids and carry messages that alter functions of normal cells in the tumor microenvironment (TME), including immune cell functions. The ability of melanoma exosomes to reprogram immune cell functions might contribute to disease progression. Methods: To evaluate effects of melanoma cell-derived exosomes (MTEX) on human immune cell subsets, we isolated exosomes from plasma of 12 patients with melanoma and separated MTEX from non-MTEX by immune capture using anti-CSPG4 mAb on beads. These exosome fractions, and exosomes from plasma of 6 normal donors used as controls (NC), were studied by on-bead flow cytometry to quantify their surface protein profiles and in co-incubation assays with immune cell subsets for functional attributes. Phenotypic and functional profiles of MTEX and non-MTEX were compared. Results &amp; Conclusion: MTEX were enriched (p&amp;lt;0.005)( in FasL and TRAIL, induced apoptosis of CD8+T cells, down-regulated CD69 expression levels on T cells and inhibited their proliferation (all p&amp;lt;0.0005). MTEX also interfered with NK cell functions (p&amp;lt;0.001). Non-MTEX were enriched (P&amp;lt;0.005) in co-stimulatory proteins, CD40L, OX40L, OX40 and, similar to exosomes of NC, were not immunosuppressive. Thus, MTEX, present in excess in plasma of patients with metastatic melanoma, mediate immune suppression and contribute to immune escape of the tumor. Citation Format: Priyanka Sharma, Brenda Diergaarde, Soldano Ferrone, John M. Kirkwood, Theresa L. Whiteside. Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3091.

Synthesis and biochemical studies of novel organic selenides with increased selectivity for hepatocellular carcinoma and breast adenocarcinoma

Nineteen organoselenides were synthesized and tested for their intrinsic cytotoxicity in hepatocellular carcinoma (HepG2) and breast adenocarcinoma (MCF-7) cell lines and their corresponding selective cytotoxicity (SI) was estimated using normal lung fibroblast (WI-38) cells. Most of the organic selenides exhibited good anticancer activity, and this was more pronounced in HepG2 cells. Interestingly, the naphthoquinone- (5), thiazol- (12), and the azo-based (13) organic selenides demonstrated promising SI (up to 76). Furthermore, the amine 4c, naphthoquinone 5, and azo-based 13 and 15 organic selenides were able to down-regulate the expression of Bcl-2 and up-regulate the expression levels of IL-2, IL-6 and CD40 in HepG2 cells compared to untreated cells. Moreover, most of the synthesized candidates manifested good free radical-scavenging and GPx-like activities comparable to vitamin C and ebselen. The obtained results suggested that some of the presented organoselenium candidates have promising anti-HepG2 and antioxidant activities.

Effect of electroacupuncture combined with Schwann cell transplantation on limb locomotor ability, regional remyelination and expression of spinal CD4 and CD8 proteins in compressive spinal injury rats

To observe the effect of electroacupuncture (EA) combined with transplantation of Schwann cells (SCs) on limb locomotor, myelin sheath repair and expression of CD4 and CD8 in compressed spinal cord injury (CSCI) rats, so as to explore its mechanisms underlying improvement of CSCI. A total of 45 female SD rats were randomly divided into normal control, model, EA, Schwann cell (SC) transplantation, and EA＋SC transplantation groups (n＝9 rats in each group). The CSCI model was established by laminectomy at T12-L2 and clip compression. Rats of the SC transplantation group accepted injection of the cultured SC suspension (2×106/6 µL) into the central, upper and lower sites of the injured spinal cord (5 mm in depth) 7-8 days after CSCI modeling. EA (2 Hz) was applied to bilateral "Zusanli" (ST36) and "Sanyinjiao" (SP6) for 10 min, once daily and 6 days a week for 3 weeks. The Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) was used to evaluate the function state of CSCI. Morphological changes of the regional injured tissue were observed under light microscope after H．E. staining. The myelin sheath repair state and survival of SCs were detected by Luxol fast blue (LFB) staining and immunofluorescence histochemistry, and the expression of CD4, CD8 and P0 of the injured spinal cord was detected by Western blot. Compared with the normal control group, the BBB scores at the time-points of 0 d, and 1, 2, and 3 weeks were significantly decreased in the model group (P<0.001), and those of the EA＋SC transplantation group at the 2nd and 3rd week were significantly higher than those of the model group (P<0.05). No significant changes of BBB scores were found after EA and SC transplantation relevant to the model group (P>0.05). LFB staining showed a disordered arrangement of the nerve fibers in the white matter, myelinociasis and obvious decrease of the medullated fibers in the model group, and these situations were relatively milder in both EA and SC transplantation groups and obviously milder in the EA＋SC transplantation group. H．E. staining displayed that the structure of the injured region of the spinal cord was incomplete, accompanied with a large number of defect cavities and neuronal karyopyknosis in the model group, while the structure was relatively clear, with an increase of the normal neurons and fewer neuronal karyopyknosis in the EA＋SC transplantation group. Compared with the normal control group, MBP in the model group was significantly decreased (P<0.001)，and P0 was significantly increased (P<0.001). Compared with the model group, the expressions of MBP and P0 were significantly increased in the EA, SC transplantation, and EA＋SC transplantation groups (P<0.01, P<0.001), and was significantly higher in the EA＋SC transplantation group than in both EA and SC transplantation groups (P<0.001). The average immunofluorescence intensity of Hoechst33342-labeled SCs was significantly higher in the EA＋SC transplantation group than in the SC transplantation group (P<0.05). After CSCI, the expression levels of spinal CD4, CD8 and P0 proteins had no significant changes in comparison with the normal control group (P>0.05), while after the intervention and in comparison with the model group, the expression levels of P0 protein were significantly increased in the EA, SC transplantation and EA＋SC transplantation groups (P<0.05), and was significantly higher in the EA＋SC transplantation group than in both EA and SC transplantation groups (P<0.05). The expression levels of CD4 and CD8 proteins were significantly lower in the EA＋SC transplantation group than in the SC transplantation group (P<0.05)．. EA＋SCs transplantation can improve the locomotor function in CSCI rats, which may be related to its effects in increasing the survival of transplanted SCs to promote the remyelination and in reducing the immune rejecting reaction.

Human Tissue-Resident Mucosal-Associated Invariant T (MAIT) Cells in Renal Fibrosis and CKD.

Mucosal-associated invariant T (MAIT) cells represent a specialized lymphocyte population associated with chronic inflammatory disorders. Little is known, however, about MAIT cells in diseases of the kidney, including CKD. To evaluate MAIT cells in human native kidneys with tubulointerstitial fibrosis, the hallmark of CKD, we used multicolor flow cytometry to identify, enumerate, and phenotype such cells from human kidney tissue biopsy samples, and immunofluorescence microscopy to localize these cells. We cocultured MAIT cells and human primary proximal tubular epithelial cells (PTECs) under hypoxic (1% oxygen) conditions to enable examination of mechanistic tubulointerstitial interactions. We identified MAIT cells (CD3+ TCR Vα7.2+ CD161hi) in healthy and diseased kidney tissues, detecting expression of tissue-resident markers (CD103/CD69) on MAIT cells in both states. Tissue samples from kidneys with tubulointerstitial fibrosis had significantly elevated numbers of MAIT cells compared with either nonfibrotic samples from diseased kidneys or tissue samples from healthy kidneys. Furthermore, CD69 expression levels, also an established marker of lymphocyte activation, were significantly increased on MAIT cells from fibrotic tissue samples. Immunofluorescent analyses of fibrotic kidney tissue identified MAIT cells accumulating adjacent to PTECs. Notably, MAIT cells activated in the presence of human PTECs under hypoxic conditions (modeling the fibrotic microenvironment) displayed significantly upregulated expression of CD69 and cytotoxic molecules perforin and granzyme B; we also observed a corresponding significant increase in PTEC necrosis in these cocultures. Our findings indicate that human tissue-resident MAIT cells in the kidney may contribute to the fibrotic process of CKD via complex interactions with PTECs.

Atractylodin Suppresses Dendritic Cell Maturation and Ameliorates Collagen-Induced Arthritis in a Mouse Model.

The aim of this study was to evaluate the immunomodulatory effects of atractylodin, a polyethylene alkyne, on the maturation of bone marrow-derived dendritic cells (BM-DC) as well as its antirheumatic effect on collagen-induced arthritis (CIA) in DBA/1 mice. Our results indicate that atractylodin effectively suppressed the secretion of pro-inflammatory cytokines, expression of costimulatory molecules, and p38 MAPK, ERK, and NF-κBp65 signaling pathways in LPS-incubated dendritic cells (DCs). Additionally, the proliferation and cytokine secretion (IFN-γ and IL-17A) of CD8+ and CD4+ T cells were reduced. In a murine CIA model, intraperitoneal injection of atractylodin significantly alleviated the severity of the disease progression, as indicated by reduced paw swelling, clinical arthritis scores, and pathological changes of joint tissues. In addition, the overall proliferation of T cells stimulated by type II collagen and the abundance of Th1 and Th17 in the spleens were also significantly decreased with atractylodin treatments. Furthermore, atractylodin significantly downregulated the expression levels of CD40, CD80, and CD86 of DCs in the spleens. In conclusion, this study shows for the first time that atractylodin has potential to manipulate the maturation of BM-DCs and should be further explored as a therapeutic agent in the treatment of rheumatoid arthritis (RA).

Expression of Toll-like receptors and costimulatory molecules in splenic B cells in a normal and abortion-prone murine pregnancy model.

The regulatory role of B lymphocytes in the pregnancy-induced maternal immune response is not well recognized. B lymphocytes function as antigen-presenting cells (APCs) and regulate Toll-like receptors and costimulatory molecule expression in response to intrinsic and extrinsic signals. Therefore, the aim of this study was to determine the expression of TLR2, TLR4, TLR9, and MHC class II and the costimulatory molecules CD80, CD86, and CD40 in splenic B cells in a normal and abortion-prone murine pregnancy model. The expression level of these molecules on female splenic B cells was investigated using real-time PCR and flow cytometry. The analysis was performed on the 3rd and 14th day of normal (CBA/JxBALB/c) and abortion-prone (CBA/JxDBA/2J) murine pregnancy. The expression of Tlr9, Cd86, and H2-Ab1 in splenic B cells on the 3rd day after mating was upregulated, whereas Tlr2 was downregulated in abortion-prone females. On day 14, we observed lower expression levels of Tlr4 and Cd80 and higher expression levels of Cd86 in CBA/J females mated with DBA/2J males. At the protein level, the differences were observed only on day 3 of pregnancy. TLR4 and CD40 molecules were upregulated in splenic B cells, while TLR9 and CD86 were downregulated in abortion-prone mice. Differential expression of TLRs and costimulatory molecules in splenic B cells in abortion-prone and normal pregnancies suggests the involvement of these cells in the regulation of the immune response at the periphery in pregnant females.

Hyperactivated peripheral invariant natural killer T cells correlate with the progression of HBV-relative liver cirrhosis.

Invariant NKT (iNKT) cells express markers of both T and NK cells and may produce various cytokines to regulate liver immunity. However, the role of iNKT cells in the progression of HBV-relative liver cirrhosis (HBV-LC) is incompletely understood. Here, we investigated the impact of peripheral iNKT cells on a cohort of patients with HBV-LC. The frequency, number, activation status, apoptosis and proliferation ability of peripheral iNKT cells were detected with flow cytometry. The impact of peripheral iNKT cells on the proliferation of hepatocyte cell line (MIHA) and activation of hepatic stellate cell line (LX-2) was detected with flow cytometry and PCR. In HBV-LC patients, the frequency and absolute number of peripheral iNKT cells significantly reduced, but the expression levels of CD25, interleukin (IL)-4, IL-13 and interferon (IFN)-γ increased. No difference was observed in the proliferation and apoptosis of circulating iNKT cells between patients and healthy controls (HCs). CXCR6 (CD186), known to be closely associated with iNKT cells migration from the periphery to the liver, was highly expressed on peripheral iNKT cells in HBV-LC patients. Furthermore, peripheral iNKT cells had a profound impact on MIHA cell proliferation and LX-2 cell activation through IL-4 or IL-13. Our data suggest that in HBV-LC patients, highly activated peripheral iNKT cells may migrate to the liver and affect hepatocyte cell line (MIHA) proliferation and hepatic stellate cell line (LX-2) activation through the expression of type 2 cytokines, which may result in excessive healing and contributing to the progression of fibrosis toward cirrhosis in liver.

ESTIMATION OF LEVEL OF SUBPOPULATION CD14&lt;sup&gt;+&lt;/sup&gt;/CD16&lt;sup&gt;+&lt;/sup&gt; MONOCYTES IN PATIENTS WITH JUVENILE DEPRESSIONS

At present, at least two phenotypically different subpopulations of blood monocytes have been identified: "classical" monocytes with the phenotype CD14 ++ /CD16 - and "nonclassical" (pro-inflammatory) with the phenotype CD14 + /CD16 + . It has been established that monocytes CD14 + /CD16 + participate in latent or so-called systemic non-infectious inflammation in the human body along with blood neutrophils and microglial cells of the brain, which are the analogue of monocyte/macrophage system in the brain. Monocyte function disorders are considered as one of the links in the neurodegenerative process underlying the neuroinflammatory hypothesis of the development of schizophrenia. In this connection, the aim of this research was to study in patients with juvenile depression the amount of pro-inflammatory monocytes in terms of the expression level of CD14 + /CD16 + receptors as well as the activity of leukocyte elastase and a 1-proteinase of elastase inhibitor. It was also interesting to determine the mechanisms of their interaction in the pathogenesis of immunosuppression, in particular, to identify a possible connection of pro-inflammatory monocytes with the activity of leukocyte elastase and a 1-proteinase of elastase inhibitor in patients with juvenile depression at the early stage of disease. 27 male patients aged 17-23 years were examined. The disease diagnostics was carried out in accordance with the criteria of ICD-10 according to which all patients have depression with the syndrome of attenuated psychotic symptoms ( Attenuated Psychotic Syndrome) in the structure of psychopathological disorders. 12 mentally healthy age- and gender-matched persons were examined as controls. Monocytes were obtained by the conventional method of adhesion to the plastic surface from mononuclear cells isolated from peripheral venous blood of patients and healthy controls by centrifugation in a ficoll-urographin density gradient (ρ = 1.077). In the blood of patients and healthy controls, the level of monocytes with the phenotype CD14 + /CD16 + , the enzymatic activity of leukocyte elastase and the functional activity of α1-proteinase inhibitor were studied. The clinical and laboratory study showed an increase of more than twofold in the level of the pro-inflammatory subpopulation of CD14 + /CD16 + monocytes in patients compared with its level in healthy individuals. The increase was accompanied by an elevation of leukocyte elastase and a 1-proteinase inhibitor activity. A positive correlation was found between the increase of surface receptors CD14 + /CD16 + expression on monocytes and elevation of the leukocyte elastase activity. These results confirm participation of CD14 + /CD16 + monocytes in the development of latent or non-infectious immune inflammation, and determine the mechanisms of possible interaction between monocytes and neutrophils in the development of immune inflammation in patients with juvenile depressions with the Attenuated Psychotic Syndrome.