Research progress on cancer immunotherapy targeting TIGIT，CD226 and CD112R / 中国肿瘤生物治疗杂志

@#[摘 要] 目前，针对抑制性受体或配体、PD-1/PD-L1及CTLA-4的靶向免疫治疗已经取得了显著的临床成果，然而仍有许多患者未从免疫治疗中获益。因此，有必要寻找新的靶点及治疗方法，以提高免疫治疗的应答率。淋巴细胞上的T细胞免疫球蛋白及其ITIM结构域（T-cell immunoreceptor with immunoglobulin and ITIM domains，TIGIT）、CD226和CD112R等均属于免疫球蛋白超家族受体，与不同配体结合后传递抑制或激活信号，他们复杂的相互作用形成的整合信号能够调节免疫细胞的功能。对靶向TIGIT、CD226和CD112R的免疫治疗研究进展进行综述，包括TIGIT、CD226、CD112R的生物学特性，抑制肿瘤或促进肿瘤的机制，靶向治疗的研究进展。

Preliminary study of cytomegalovirus infection and its correlation with NK cell subsets after renal transplantation / 器官移植

Objective To explore the variation trend of natural killer (NK) cell subsets in the recipients infected with cytomegalovirus (CMV) after renal transplantation. Methods Clinical data of 92 renal transplant recipients were retrospectively analyzed. All recipients were divided into the CMV infection group (n=43), CMV infection recovery group (n=13), stable renal function group (n=15), rejection group (n=11) and other infection group (n=10). In addition, healthy adult volunteers were enrolled in the healthy control group (n=15). The proportion of NK cells in peripheral blood, the expression proportion and the mean fluorescence intensity (MFI) of CD226 and CD16 in NK cells were observed and statistically compared among different groups. Results The proportion of NK cells was 4.9% (2.2%, 11.5%) in the CMV infection group and 3.7% (2.3%, 6.5%) in the CMV infection recovery group, which were significantly lower than those in the other groups (all P < 0.05). The expression proportion of CD226 and CD16 in NK cells in the CMV infection group was significantly lower compared with those in the healthy control group and stable renal function group(all P < 0.05). The expression proportion of CD226 and CD16 in NK cells in the CMV infection recovery group was remarkably higher than those in the CMV infection group (both P < 0.05). The MFI of CD226 and CD16 in the CMV infection group was significantly lower than those in the healthy control group (both P < 0.05). The MFI of CD226 and CD16 in the CMV infection recovery group was significantly higher than those in the CMV infection group (both P < 0.05). Conclusions The expression proportion and MFI of CD226 and CD16 in NK cells are down-regulated in CMV infection period, whereas up-regulated during the CMV infection recovery period, prompting that CD226 and CD16 expressed by NK cells are intimately correlated with the course of CMV infection.

Mechanism of CD226-dependant NK cell regulation in mouse obesity / 中华微生物学和免疫学杂志

Objective To investigate the effects of CD226 knockout ( KO) on obese mice fed with high fat diet and to analyze the composition of immune cells in CD226KO obese mice for further elucidating the immunological mechanism of CD226 involved in high fat diet-induced obesity. Methods Both wild-type ( WT) and CD226KO mice were randomly divided into two groups, high-fat and normal diet groups, and fed for 14 weeks to establish the type 2 diabetes model. Immune cells in mouse spleen and peripheral blood were analyzed by flow cytometry. In in vitro experiments, NK92-MI cells were infected with pshRNA-CD226 lenti-virus to silence CD226 expression, and then qPCR was performed to detect the expression of Foxp3, TNF-αand IFN-γ at mRNA level. Results In the high-fat diet groups, CD226KO mice had lower blood glucose, serum insulin and HOMA-IR than WT mice, but higher HOMA-IS and HOMA-β. CD226KO could reduce compensatory hyperplasia of islet tissue, and significantly down-regulate the proportion of spleen NK cells in mice. The proportion of CD3-CD49b+CD25+Foxp3+regulatory NK cells (NKreg) increased significantly in CD226KO mice. CD226KO could significantly increase Foxp3 expression in NK92-MI cells and decrease the expression of TNF-α and IFN-γ. Conclusions CD226KO can alleviate insulin resistance, increase the number of islet β-cell and improve islet β-cell function in obese mice. The mechanism might be related to the up-regulation of Foxp3+ NKreg ratio.

CD226 involved in regulation of CD4+T cell / 中国免疫学杂志

CD226 is a typeⅠtransmembrane glycoprotein expressed on various immune cell membranes,such as NK cells,T cells,monocytes and other cells.After binding to ligand CD112 or CD155, CD226 mediates the differentiation, proliferation and functional regulation of various immune cells to participate many physiological and pathological activities.This paper mainly focuses on two aspects,the first is CD226 on CD4+T cell immune regulation, the seconds is the CD226 involved in the progress of many dis-ease.Detailed explanation of CD226 involved in naive CD4+T cell proliferation and differentiation, Th1/Th2/Th17 cells polarization and regulatory function of Treg cells.

Relationship between viral load and expression of CD226 on the surface of T follicular helper cells in patients with chronic hepatitis C / 中华传染病杂志

Objective To explore the relationship between viral load and expression of CD 226 on the surface of peripheral blood T follicular helper cells (Tfh) in patients with chronic hepatitis C (CHC) . Methods One hundred and thirty-five CHC patients hospitalized at Wuxi Fifth People′s Hospital from March 2015 to April 2017 were collected ,and another 30 healthy blood donors were set as healthy control group .CHC patients were divided into two groups based on hepatitis C virus (HCV) RNA level ,with 49 cases (36 .3％ ) in low viral load group and 86 cases (63 .7％ ) in high viral load group .Expression of CD226 on the surface of peripheral blood Tfh cells , Tfh cells ,interleukin (IL )-21 and HCV specific cytotoxic lymphocyte (CTL) levels of two patient groups were compared .Categorical data were compared with chi-square test and normally distribute continuous data were compared with t test .Correlations between different factors were analyzed by Pearson correlation analysis .Results Expression of CD226 on the surface of peripheral blood Tfh cells in 135 cases of CHC patients was (77 .69 ± 5 .42)％ ,which was lower than that of healthy control ([90 .06 ± 5 .83]％ ) ,and the difference between the two groups was significant (t= 7 .541 , P < 0 .01) .The CD226 expression on the peripheral blood Tfh cells in low viral load group was (88 .75 ± 6 .68)％ ,which was higher than that of high viral load group ([69 .23 ± 5 .86]％ ) ,and the difference between the two groups was significant (t = 19 .232 , P< 0 .01) .The viral load was negatively correlated with Tfh cell surface CD 226 expression (r = - 0 .705 , P < 0 .01) .The peripheral blood Tfh cell level in 135 CHC patients was higher than that of healthy control ,and the difference between the two groups was significant (t= 13 .878 , P< 0 .01) .The peripheral blood Tfh cell level in low viral load group was higher than that in high viral load group ,and the difference between the two groups was significant (t= 26 .993 , P< 0 .01) .The IL-21 level of 135 CHC patients was lower than that of healthy control ([70 .35 ± 1 .6]％ ) ,and the difference between the two groups was significant (t=18 .322 , P< 0 .01) .The IL-21 level in peripheral blood of low viral load group was higher than that of high viral load group ,and the difference between the two groups was significant (t= 84 .54 , P< 0 .01) . HCV specific CTL level in peripheral blood of low viral load group was higher than that of high viral load group ,and the difference between the two groups was significant (t = 29 .869 , P< 0 .01) .The viral load was negatively correlated with levels of HCV specific CTL (r= -0 .734 ,P< 0 .01) .Conclusions In patients with chronic hepatitis C ,different levels of viral load can result in different levels of CD 226 expression on the peripheral blood Tfh cells .Patients with low viral load has high CD226 expression on Tfh cell surface , resulting in rise of Tfh cell level ,IL-21 level and HCV specific CTL level .

Preparation of an Ag85A-CD226 DNA vaccine and evaluation of its immunogenicity in a mouse mod-el / 中华微生物学和免疫学杂志

Objective To construct a eukaryotic expression plasmid of pcDNA 3.1-Ag85A-CD226, and to use it as DNA vaccine then further evaluate its immunogenicity through oral administration in a mouse model.Methods The CD226-PCR2.1-ToPo plasmid was used as the template to clone CD 226 gene by PCR.The CD226 gene was then inserted into pcDNA 3.1-Ag85A plasmid to construct the recombinant plas-mid of pcDNA3.1-Ag85A-CD226.After identified by restriction enzyme analysis and sequencing , the re-combinant plasmid was transfected into HEK 293 cells by using lipofection .The expression of Ag85A-CD226 gene in HEK293 cells was detected by RT-PCR, Western blot and indirect immunofluorescence assay .The purified recombinant plasmid was used to prepare the Ag 85A-CD226 DNA vaccine by liposomal encapsula-tion.The vaccine was administered intragastrically to mice .The activities of NK cells , the cytokine levels in the supernatants of spleen cell cultures and the mRNA level of cytokines in the intestines were evaluated to analyze the immunogenicity of Ag85A-CD226 DNA vaccine.Results The Ag85A-CD226 DNA vaccine was prepared successfully .The expression of Ag85A-CD226 fusion protein was detected in HEK293 cells.The activities of NK cells from mice vaccinated with Ag 85A-CD226 DNA vaccine were higher than those from other control groups (P0.05).Conclusion The Ag85A-CD226 DNA vaccine could significantly enhance Th1 type immune responses systemically and in the intestine as in comparison with those vaccinated with single dose of Ag 85A DNA vaccine or CD226 DNA vaccine.

Variantes do gene CD226 associadas com a susceptibilidade ao diabetes mellitus tipo 1 autoimune / CD226 gene variants associated with susceptibility to type 1, immune mediated, diabetes

Recentemente, estudos de Genome Wide Association (GWA) identificaram uma nova região cromossômica, 18q22, como de susceptibilidade ao Diabetes tipo 1 autoimune (DM1A). Nesta região localiza-se o gene CD226, responsável por codificar uma molécula de adesão leucocitária (CD226) envolvida no processo de adesão celular, diferenciação de células T CD4+ virgens, citotoxicidade induzida por células natural killer (NK) e produção de citocinas. Até o momento, apenas o polimorfismo rs763361 A/G foi relacionado ao diabetes autoimune e pouco é conhecido quanto ao envolvimento de outras variantes do CD226, associadas a outras doenças autoimunes, na patogênese do DM1A. Com o objetivo de definir as variantes polimórficas relacionadas à susceptibilidade ao DM1A, às suas características fenotípicas e outras manifestações de autoimunidade, 532 pacientes diabéticos tipo 1A e 594 controles normais foram envolvidos neste estudo. Inicialmente, em um subgrupo de 106 diabéticos e 102 controles, as regiões codificadoras e flanqueadoras do gene CD226, obtidas do DNA genômico de leucócitos do sangue periférico, foram amplificadas pela técnica de Reação em Cadeia da Polimerase e submetidas à sequenciamento direto. Em uma segunda etapa, os polimorfismos rs763361, rs1788101 e rs727088 foram genotipados pelo ensaio TaqMan nos demais pacientes e controles. Resultados: foram identificadas 12 variantes no gene CD226, sete com frequência acima de 5%. Nenhuma variante nova foi encontrada. A variante rs727088 não estava em equilíbrio de Hardy Weinberg no grupo controle. Os genótipos AA da variante rs763361 e CC do rs727088 foram associados ao risco de DM1A e estavam em desequilíbrio de ligação. O genótipo do haplótipo ACAC, formado pelas variantes de risco, predominou nos pacientes diabéticos. Tanto o genótipo AA do rs763361 como o CC do rs727088 e o genótipo do haplótipo ACAC foram associados com menores valores de peptídeo C em pacientes com até dois anos de duração da doença. Nenhum...

Recently, Genome Wide Association (GWA) studies identified a new locus, 18q22, as a canditate to Type 1 A, or immune mediated diabetes (T1AD) susceptibility. This locus harbors the CD226 gene, responsible for encoding the leukocyte adhesion molecule (CD226) involved in cell adhesion, differentiation of naïve CD4+T cells, cytotoxicity induced by natural killer (NK) cells and cytokine production. Although just one single nucleotide polymorphism (SNP) rs763361 A/G had been related to T1AD, little is known about the involvement of new variants of CD226, implicated in other autoimmune disorders, in the pathogenesis of T1AD. In order to identify polymorphic variants related to T1AD susceptibility and their influences in phenotypic characteristics and other manifestations of autoimmunity, 532 type 1A diabetic patients and 594 health controls were enrolled in this study. Initially, in a subset of 106 diabetics and 102 controls, coding and flanking regions of CD226 gene obtained from genomic DNA extraction were amplified by polymerase chain reaction technique and subjected to direct sequencing. In a second step, the polymorphisms rs763361, rs727088 and rs1788101 were genotyped by TaqMan assay in the remaining patients and controls. Results: 12 variants in CD226 gene, seven of them with frequency above 5 % where identified. We did not found new variants. The variant rs727088 was not in Hardy Weinberg equilibrium in the control group. The genotypes AA (OR=1.45; p=0.005) and CC (OR=1.41; p=0.01) related to rs763361 and rs727088 variants respectively, were associated with risk of T1AD. Both predominated in female (p<0.01). Further, these variants were in linkage disequilibrium. The genotype haplotype ACAC formed by the risk variants was more frequent in patients with diabetes (30.5% x 25.6%; OR=1.42; p=0.014). The AA genotype of rs763361, the CC genotype and ACAC genotype haplotype were associated with lower levels of C-peptide in patients with no more than two...

Expression of CD226 mRNA in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the expression level of CD226 mRNA in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) and explore the relation between the gene expression and disease activity, and the relation between the gene expression and Gly307Ser polymorphism of CD226 was also examined. Methods CD226 gene was measured with real-time polymerase chain reaction (qRT- PCR) in PBMCs. The expression levels of CD226 gene in PBMCs were compared between 90 SLE patients and 30 healthy individuals. One-way ANOVA and pearson correlation were used for statistical analysis. Results The expression level of CD226 in the PBMCs of SLE patients (6.8±1.1) was significantly decreased compared to healthy individuals (26.5±6.7) (P＜0.01), while there was no association between mRNA level and genotype (P＞0.05). No correlation between ESR, CRP, ANA, SLEDAI scores, C3 and the expression level of CD226 gene was discovered. Conclusion In Hubei Chinese Han population, CD226-Gly307Ser locus is associated with the development of SLE, while T allele does not impact the expression of CD226 gene, thus the role of CD226 gene in autoimmune diseases should be explored in the future.

Eukaryotic expression and identification of human pSecTag2B-CD226 fusion protein / 医学研究生学报

Objective: To construct and express the eukaryotic expression vector of human pSecTag2B-CD226(PTA1).Methods: The gene fragment encoding extracellular region of human CD226 was cloned into the eukaryotic expression vector pSecTag2B.After sequencing,the vector was transfected into COS-7 cells,and the expressed molecule was purified by affinity chromatography.Finally,the product was characterized by ELISA.Results: hCD226-6His was successfully expressed.After purification,the concentration of hCD226-6His was 50?g/ml.Conclusion: Human CD226-6His fusion protein involving the extracellular region of CD226cDNA has been successfully expressed and purified,which helps prepare the ground for further functional studies of this molecule.

Identification of 5-flank upstream regulation region of CD226 / 中国免疫学杂志

Objective:To identify the function of the 5′-flank upstream regulation region of human CD226 gene.Methods:The upstream regulation region of CD226 was cloned by PCR and ligated into pGL3 vector. Then the vector was transfected into Jurkat cell and luciferase activity was detected after 48 h culture.Results:CD226 gene may have two promoters, P1 and P2,which were located at the region of -843--319 bp and +1-+181 bp respectively, and PMA can up-regulate P1 while down-regulate P2. Both P1 and P2 can be up-regulated by A23187, especially P2.Conclusion:CD226 gene may have two promoters, and PMA and A23187 can regulate CD226 promoter activity in the similar pattern of protein level.

Expression and function of CD226 with CD16 on NK subsets / 中国免疫学杂志

Objective:To investigate the expression and function of CD226 on NK s ubsets and its coexpression with other activation receptor and inhibition recept or on NK cells. Methods:The expression of CD226 on CD56 bright and CD56 dim NK subsets and coe xpress ion with CD16 and NKG2A in PBMC and MLC in the presence or absence of IL-2 or IL-15 were detected by double fluorescent staining and flow cytometry analysis. The level of IFN-? in the supernatants of PBMC culture and MLC treated with o r without IL-2 or IL-15 were evaluated by ELISA. 51Cr release assay w as employed to measure the specific lysis of NK cells killing target K562 cells. Results:CD226 was mainly expressed on CD56 dim NK subsets in PBMC. When s imulated by IL-2, CD226 expression was shifted to CD56 bright NK subsets, while IL-15 increased the subpopulation of NKG2A+CD226+double positive cell s. In MLC-generated NK cells, CD226 was mainly expressed on CD56 dim NK su bsets, and also shifted to CD56 bright NK subsets in the addition of IL-15 . Furt hermore, the percentage of CD16+CD226+和NKG2A+CD226+ subsets were increa sed when stimulated by IL-2 or IL-15. There was great increase in IFN-? lev el in the supernatants of PBMC culture in the presence of IL-2 or IL-15, but no difference in the supernatants of MLC treated with or without two cytokine s. Moreover, the cytotoxicity of NK cells in PBMC and MLC were greatly enhanced by IL-2 or IL-15. Conclusion:CD226 is mainly expressed on CD56 bright NK subsets in IL-2 or IL-1 5 activated NK cells, and is coexpressed with CD16 and NKG2A preferentiatly, whi ch maybe involve in the modulation of cytotoxicity of NK cells based on the balance of coexpressed activation and inhibition receptors. [

Detection of Serum Soluble CD226/PTA1Level in Patients with Psoriasis Vulgaris / 中华皮肤科杂志

Objective To determine the serum soluble CD226/PTA1(sCD226/PTA1)level in pa-tients with psoriasis vulgaris.Methods Serum sCD226/PTA1level was measured by sandwich ELISA in30patients with psoriasis vulgaris before treatment(17patients in active stage and13in inactive stage),10patients after treatment,and15healthy individuals as a control.Results Serum sCD226/PTA1level was significantly increased in patients with psoriasis compared with that in healthy controls(P

Molecular cloning of mouse CD226(PTA1) and its isoforms / 中国免疫学杂志

Objective:To clone mouse CD226(platelet and T cell activation antigen1,PTA1).Methods:Specific primers were designed and sythesized according to the EST sequence from GenBank,which had 51% homology to human CD226 on the amino acid level.And then,the cDNA of mouse CD226 was cloned from the thymus of 4 week old BALB/c mouse by using RACE technique.Results:The length of mouse CD226 cDNA is 2 223 bp,with the open reading frame(ORF) of 1 002 bp,encoding 333 amino acids,which is 3 amino acids shorter than its counterpart in human.The mouse CD226 belongs to IgSF,and shares 53% homology with human PTA1 on amino acid level.Besides,three isoforms of mouse PTA1 were also cloned at the mean time.Conclusion:The molecular cloning of mouse PTA1 lays the foundation for the in vivo studies on the biological function of this molecule,as well as the studies of this molecule in gene knockout mouse and transgenic mouse.

CD226(PTA1) monoclonal antibody induced the cytotoxicity of NK cell clone in redirected cytotoxicity assay / 中国免疫学杂志

Objective:To investigate the expression and function of CD226 on NK cell clone Methods:NK cell clone was established by limited dilution, and identified by FCM The function of CD226 on the cytotoxicity of NK cell clone was detected by RCA and the NK cell clone secreted cytokines in the supernatants during the killing phase were measured by ELISA Results:One NK cell clone was obtained by limited dilution The cytotoxicity of this clone was upregulated markedly by CD226 mAb, and the secretion levels of IFN ? and GM CSF by NK cell clone increased obviously in RCA Conclusion:CD226 is a novel NK cell activation receptor, and the elevated IFN ? and GM CSF levels may be related to CD226 mAb enhanced NK function