Loss of core fucosylation enhances the anticancer activity of cytotoxic T lymphocytes by increasing PD-1 degradation

As an immune checkpoint, programmed cell death 1 (PD-1) and its ligand (PD-L1) pathway plays a crucial role in CD8⁺ cytotoxic T lymphocytes (CTL) activation and provides antitumor responses. The N-glycans of PD-1 and PD-L1 are highly core fucosylated, which are solely catalyzed by the core fucosyltransferase (Fut8). However, the precise biological mechanisms underlying effects of core fucosylation of PD-1 and PD-L1 on CTL activation have not been fully understood. In this study, we found that core fucosylation was significantly upregulated in lung adenocarcinoma. Compared to those of Fut8^+/+OT-I mice, the lung adenocarcinoma formation induced by urethane was markedly reduced in Fut8^−/−OT-I mice. De-core fucosylation of PD-1 compromised its expression on Fut8^−/− CTL, resulted in enhanced Fut8^−/− CTL activation and cytotoxicity, leading to more efficient tumor eradication. Indeed, loss of core fucosylation significantly enhanced the PD-1 ubiquitination and in turn led to the degradation of PD-1 in the proteasome. Our current work indicates that inhibition of core fucosylation is a unique strategy to reduce PD-1 expression for the antilung adenocarcinoma immune therapy in the future.     

Enhancement of humoral and protective immune response induced by live-attenuated Salmonella typhi by ampicillin

Efficacy of live-attenuated Salmonella vaccines delivered by the mucosal route is limited by the dose and interference from mucosal flora of the alimentary tract. In a mouse model, the total antibody response towards lipopolysaccharide of S. typhi was significantly enhanced at day 21 post-immunization with live-attenuated S. typhi (Ty21a) when ampicillin was concomitantly administered (p<0.005), and the lethal dose 50 of mice in the ampicillin and control groups immunized with Ty21a after wild-type S. typhi challenge on day 24 was 4x10(7) and 1x10(7), respectively. The faecal bacterial counts of the ampicillin group at days 1 and 3 were significantly lower than those of the control group (p<0.01 and <0.05). On day 1, the number of mice with > or =10 Ty21a colonies isolated from the spleen was significantly higher in the ampicillin group than the control group (p<0.05). Furthermore, on the same day, Ty21a was isolated from the faeces of three mice from the ampicillin group, but only one from the control group. We conclude that ampicillin may have enhanced the humoral and protective immune responses by giving the Ty21a a selective advantage over the normal bacterial flora. This concept of antibiotic enhancement of immunization could have important implications for other live-attenuated vaccines, as well as the delivery of microbial antigens and DNA vaccines by live-attenuated Salmonella carriers.     

阻断核心岩藻糖基化修饰抑制肾小管上皮细胞的间充质转化

目的: 探讨阻抑核心岩藻糖基化修饰对肾小管上皮细胞间充质转化(EMT)过程的影响。方法: 利用转化生长因子β₁(TGF-β₁)建立肾小管上皮HK-2细胞EMT的模型,应用RNAi技术沉默HK-2细胞的α-1,6-岩藻糖基转移酶(FUT8)基因表达,光镜下观察FUT8 基因沉默后细胞形态变化,免疫印迹及免疫细胞化学方法测定细胞表型标记物蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、α-平滑肌肌动蛋白(α-SMA)和成纤维细胞特异性蛋白-1(FSP-1)的表达变化,流式细胞仪测定细胞凋亡。 结果: TGF-β₁孵育48 h后,HK-2细胞失去原有的上皮细胞形态,呈现纤维细胞形态,纤维细胞表型标记蛋白α-SMA、FSP-1及N-cadherin表达明显升高,而上皮细胞表型标记蛋白E-cadherin表达明显下降,同时伴有 FUT8 基因表达上调,细胞凋亡增加,而提前转染FUT8 siRNA能明显减弱上述这些反应。结论: FUT8催化的核心岩藻糖基化修饰参与HK-2细胞的EMT过程;阻断核心岩藻糖基化修饰,能有效阻断肾小管上皮细胞的EMT过程。     

Dysregulation of the humoral immune response in old mice

The increase in autoantibodies with age of both experimentalanimals and humans has been thought to reflect a shift in theantibody repertoire from foreign to self antigens. In mice,before immunization, the age-associated increase in antibodiesreactive with a prototypic autoantigen, bromelain-treated autologouserythrocytes (BrMRBC), reflected a 3-fold increase in serumIgM and the number of IgM-secreting spleen cells in old comparedwith young mice. However, the percentage of the IgM-secretingspleen cell repertoire reactive with BrMRBC in old mice wasactually {small tilde}50% that in young mice. In contrast, afterimmunization with sheep erythrocytes (SRBC), old mice showeda 5-fold increase in the percentage of IgM-secreting cells reactivewith BrMRBC while young mice showed no significant increase.The converse is true for the percentage of IgM-secreting spleencells in old mice specific for SBRC, which is 10% the numbergenerated by young mice. The increased autoantibody responseof old mice is not, however, linked to their poor response tothe nominal antigen. Thus, immunization with phosphorylcholine(PC) conjugated Keyhole limpet hemocyanin, an antigen that inducesa comparable anti-PC response in old and young mice, also inducedmore autoantibody forming cells in old than young mice. Theincreased autoantibody response of old mice after immunizationcan be accounted for by both an increased number of Ig-secretingspleen cells as well as an increased percentage of the expressedrepertoire of IgM-secreting spleen cells that react with autoantigens.In contrast, prior to immunization the age-associated increasein serum autoantibodies and autoantibody-secreting spleen cellscan be accounted for by the increased concentration of serumIg and the polyclonal activation of splenic B cells.     

Humoral immune response to 2,4-dinitrophenyl -- keyhole limpet hemocyanin in antigen-free,germ-free and conventional BALB/c mice

The B cell immune response to 2,4-dinitrophenyl (DNP) keyhole limpet hemocyanin was compared in antigen-free, germ-free and conventional BALB/c mice. The numbers of total and of DNP-specific IgM-, IgG- and IgA-secreting cells in the spleen were determined by enzyme-linked immunosorbent plaque assays after primary, secondary and hyperimmunization. Three days after primary immunization a peak of DNP-specific IgM-secreting cells was seen in conventional mice only. However, this specific response was accompanied by a rise in the total number of IgM-secreting cells. At day 6 after primary immunization the total number and the frequency of DNP-specific IgG-secreting cells were higher in antigen-free mice, compared to germ-free and conventional mice. After secondary immunization in conventional mice only, a considerable bystander IgG response was seen together with the DNP-specific IgG response. At the end of the secondary response 90% of all IgG-secreting cells were DNP specific in antigen-free mice, while the corresponding figure in germ-free and conventional mice was 63% and 14%, respectively. After hyperimmunization, the absolute number of DNP-specific IgG-secreting cells in the spleen was 5-fold and 11-fold higher in antigen-free mice then in germ-free and conventional mice, respectively. Approximately 48% of all IgG-secreting cells were DNP specific in antigen-free mice, while the corresponding figure in germ-free and conventional mice was 17% and 12%, respectively. The results show that the exogenous antigenic load of animals influences the immune response to newly introduced antigens. The higher absolute and relative numbers of antigen-specific IgG-secreting cells after hyperimmunization in antigen-free mice compared to germ-free and conventional mice may provide a better source for antigen-specific B cells that eventually can be used for hybridoma production.     

pCD-Em10对泡球蚴感染小鼠免疫应答的影响

目的：探讨pCD-Em10免疫对泡球蚴感染小鼠免疫应答的影响。方法：将pCD-Em10肌肉注射免疫BALB/c鼠,免疫后8周用Em原头节进行攻击感染,感染后18周剖杀小鼠,计算减蚴率;取脾并分离脾细胞,流式细胞仪检测脾CD4^＋和CD8^＋T淋巴细胞亚群的百分比;用EmAg或ConA刺激培养脾细胞,收集脾细胞培养上清液,用试剂盒检测脾细胞培养上清液的IL-2、IFN-γ、TNF-α和IL-4水平;流式细胞仪检测脾细胞的凋亡发生率,同时设有BCG和PBS对照。结果：pCD-Em10免疫组的减蚴率为71.17%,脾CD4^＋T细胞亚群显著增加,IFN-γ和TNF-α水平升高,IL-4水平降低,脾细胞凋亡发生率明显低于PBS对照组。结论：pCD-Em10可诱导泡球蚴感染小鼠产生一个保护性的免疫反应。     

Pregnancy and humoral immune response in mice chronically infected by Trypanosoma cruzi.

The effect of pregnancy on the humoral immune response induced by Trypanosoma cruzi was studied in groups of chronically infected and pregnant mice (IP) or chronically infected and nonpregnant mice (INP) of strain BALB/c. Groups of noninfected and nonpregnant mice (NINP) or noninfected and pregnant mice (NIP) served as controls. The pregnant mice were killed on day 17 of pregnancy. Anti-T. cruzi immunoglobulin G (IgG) and IgM antibodies, detected by immunofluorescence or enzyme-linked immunosorbent assay or both, underwent a pregnancy-associated decrease of 20 to 40%, whereas complement-mediated lytic antibodies were unaffected by pregnancy. Immunoblotting analysis indicated identical specificities of the anti-T. cruzi antibodies in IP and INP groups. The levels of all the immunoglobulin isotypes (particularly IgG2a and IgG3), circulating immune complexes, rheumatoid-like factor, and anti-DNA antibodies were considerably increased during chronic infection (NINP versus INP), which could be related to the high degree of polyclonal B-cell activation occurring in T. cruzi infection. However, pregnancy significantly decreased (by 20 to 60%) such parameters. IgG levels were particularly affected (by 40 to 60%), and the decreases could be ordered as follows: IgG3 greater than IgG2a greater than IgG1 greater than IgG2b for IP versus INP. Comparisons between the noninfected groups indicated differences only in IgG levels. These results indicate the following. (i) The specific humoral anti-T. cruzi immune response is weakly affected by pregnancy, which is not sufficient to modify the course of the mother's infection. (ii) Pregnancy does not modify the expression of the anti-T. cruzi antibody repertory. (iii) Pregnancy reduces the polyclonal B-cell activation, particularly the levels of the IgG isotypes undergoing the greatest activation.     

驱虫斑鸠菊注射液对小鼠免疫功能的影响

目的:探讨驱虫斑鸠菊对小鼠免疫功能的影响。方法:采用[3H]-TdR掺入法和脾细胞介导羊红细胞定量溶血分光光度法以及迟发型超敏反应试验等观察了驱虫斑鸠菊对小鼠兔疫功能的作用。结果:驱虫斑鸠菊在体内外均可以明显抑制ConA刺激的小鼠T淋巴细胞的增殖反应和LPS刺激的小鼠B淋巴细胞的增殖反应（P<0.01）;对小鼠胸腺指数和脾脏指数、绵羊红细胞（SRBC）诱导的正常小鼠脾抗体细胞生成反应以及小鼠皮肤迟发型超敏反应都显示出明显的抑制作用,而且上述抑制作用与药物浓度有一定的剂量效应关系。结论:驱虫斑鸠菊对机体体液免疫、细胞免疫都有明显的抑制作用。     

Loss of HIV-specific memory B-cells as a potential mechanism for the dysfunction of the humoral immune response against HIV

A central, yet unresolved issue in the pathogenesis of HIV disease is the mechanism of antibody perturbation. In this study, HIV-specific memory B-cells were quantified in groups of infected subjects and compared with memory responses to other antigens and antibody titers. HIV-specific memory B-cell responses were vigorous in individuals with CD4⁺ T-cell counts > 350/μl and weak or undetectable in subjects with CD4⁺ T-cell numbers < 200/μl. Memory B-cell loss was permanent, because antiretroviral therapy failed to restore HIV-specific memory responses while influenza- and tetanus toxoid-specific memory B-cells remained unaffected or recovered. Antibody titers to Gag strongly correlated with memory B-cell frequencies. In contrast, Env-specific antibodies were maintained in advanced disease despite low or undetectable levels of memory B-cells. These results provide a potential mechanism by which destruction of HIV-specific CD4⁺ T-cells affects the humoral immune response against HIV and compromises the ability to maintain an effective antibody response.     

Dissecting the humoral immune response to simian immunodeficiency virus

The ultimate goal of an AIDS vaccine is to elicit potent cellular and humoral immune responses that will result in broadly enduring protective immunity. During the past several years, we have focused on characterizing the quantitative and qualitative properties of the antibody response, principally working to define the mechanism(s) of antibody-mediated neutralization in vitro. We have utilized a panel of monoclonal antibodies generated from monkeys infected with attenuated SIV for more than 8 mo to dissect the early events of virus infection involved in antibody-mediated neutralization. Presented herein are highlights from our studies that have identified potential mechanisms by which antibodies neutralize SIV in vitro.     

Respective roles of immune and nutritional factors in the priming of the immune response in the elderly

Ninety-six institutionalized elderly (greater than 70 years old) (mean age: 82 +/- 7 years) subjects, negative for tetanus toxoid antibodies were primed with tetanus toxoid vaccination and dinitrochlorobenzene (DNCB). Correlations were studied between some immunological parameters, nutritional parameters prior to immunization and the immune response intensity after it. Levels of tetanus toxoid specific IgG (ELISA assay) were positively correlated with monocyte phagocytosis, DNCB response and prealbumin levels, and negatively correlated with total IgG, monocyte immune degradation and tetanus toxoid lymphocyte stimulation. No correlation was observed with IgA, IgM, PHA stimulation. Tetanus toxoid lymphocyte stimulation correlated positively with response to DNCB, and negatively with tetanus toxoid IgG as well as total IgG. DNCB response correlated with prealbumin, tetanus toxoid IgG and tetanus toxoid lymphocyte stimulation. Therefore, it appears that malnutrition, as measured by prealbumin level, is one of the main factors contributing to the inconstant senile immunodeficiency. Monocyte antigenic degradation function unaltered with age can impair immune response while conserved or increased phagocytosis enhances immune response in the elderly. High total IgG levels were linked with low specific responses to priming antigens. High specific antibody levels also correlated negatively with cellular specific response. It is assumed that regulatory IgG antibody accumulation, likely anti-idiotypic antibodies, play an important role in senile immunological depletion.     

T细胞免疫BALB/c小鼠诱导调节性体液免疫应答的研究

目的 :研究T细胞免疫后正常小鼠的调节性体液免疫应答。方法 :用经γ射线照射的体外扩增的活化OVA特异T细胞克隆免疫BALB c小鼠 ,FACS法分析血清中抗T细胞抗体水平 ,免疫共沉淀法分析靶抗原。结果 :T细胞免疫能诱导BALB c小鼠产生抑制T细胞增殖的抗T细胞抗体 ,这种抗体不是多克隆活化的结果 ,其靶抗原可能是T细胞上 93、87、5 5和 4 5kD的蛋白。结论 :T细胞免疫能诱导正常小鼠产生调节性体液免疫应答     

Analysis of immunization route-related variation in the immune response to heat-killed Salmonella typhimurium in mice.

In examinations of the factors regulating the quality and quantity of the immune response to Salmonella typhimurium, we have shown previously that live and heat-killed preparations of S. typhimurium can induce gamma interferon-dominant and interleukin-4-dominant immune responses, respectively, upon intraperitoneal (i.p.) immunization of BALB/c mice. Using this system to investigate the role of the route of immunization in the immune response, we show in the present study that i.p. immunization with heat-killed S. typhimurium generates a quantitatively better immune response than does intradermal (i.d.) immunization. The quantitative differences observed between the i.p. and i.d. routes are apparent in the amount of S. typhimurium-specific antibodies produced, the extent of responses in T-cell proliferation assays, and the quantities of lymphokines generated. However, the ratios of immunoglobulin (Ig) isotypes [IgG1/IgG2a] are comparable and the relative dominance of interleukin-4 over gamma interferon is seen in both i.p.- and i.d.-immunized mice, suggesting that the predominant T-cell effector pathways triggered are not qualitatively dependent on the route of immunization. An examination of the antigenic profile recognised by the B-cell and T-cell responses in i.p.- versus i.d.-immunized mice shows that while the Western immunoblot patterns recognized by serum antibodies from the two groups of mice were not significantly different, T cells from i.p.-immunized mice recognized a broader spectrum of antigens in an immunoblot assay than did those from i.d.-immunized mice. These data suggest that there may be a significant difference in the antigen-processing ability of peritoneal and dermal antigen-presenting cells for complex antigenic formulations such as bacterial vaccines.     

Modulation of orientation and exploratory behavior in mice during development of humoral immune response

We demonstrated opposite changes in the orientation and exploratory behavior of (CBA× C57Bl/6)F1 mice in the open field test during the formation of primary humoral immune response. These changes depended on initial behavioral activity: the exploratory behavior was suppressed in animals with initially high activity, stimulated in animals with medium reaction, and remained unchanged in mice with initially low activity. The detected changes in the exploratory behavior during the formation of immune response were leveled in the total population (not divided by initial behavioral status).     

Phases of development of the humoral immune response

Research Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR. All-Union Research Center for Maternal and Child Health Care, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 12, pp. 635–637, December, 1990.     

含HIV-1核心蛋白基因的重组质粒的构建及其免疫应答的研究

目的构建含有HIV-1核心蛋白gag基因的真核表达质粒,并研究其免疫应答反应。方法采用PCR方法从1例HIV感染者中扩增出gag基因,构建重组质粒pcDNA-GAG。接种小鼠后,检测其抗体及抗体亚类,并对小鼠的淋巴细胞亚群进行分析,采用3H-TdR法、ELISPOT方法和51Cr释放法分别检测T淋巴细胞的增殖反应性、特异性分泌IFN-γ的CD8+T淋巴细胞以及特异性CTL杀伤活性。结果重组质粒体外表达目的蛋白的相对分子质量(Mr)约为55×103。免疫小鼠后可诱导产生特异性抗体,其中IgG2a与IgG的比例显著高于IgG1与IgG的比例;T淋巴细胞体外经ConA刺激后SI达到160.67,显著高于对照组(14.04,P<0.05);产生IFN-γ的CD8+T淋巴细胞数目以及特异杀伤率均显著高于对照组小鼠(P<0.05)。结论重组质粒pcDNA-GAG可诱导小鼠产生特异性体液和细胞免疫应答反应,而且ELISPOT方法检测特异性细胞免疫应答反应的结果与51Cr释放法一致,提示可用此法对DNA疫苗诱导的细胞免疫进行评价。     

Follicular dendritic cells in aging, a "bottle-neck" in the humoral immune response

Senescence leads to the appearance of atrophic follicular dendritic cells (FDCs) that trap and retain little immune complexes (IC), generate few memory B cells, and induce a reduced number of germinal centers (GC). Deficiencies in antibody responses to T cell dependent exogenous antigens such as pneumonia and influenza vaccines may reflect intrinsic FDC defects or altered FDC-B cell interactions. We recently studied antigen handling capacity and co-stimulatory activity of old FDCs and determined age-related changes in the expression or function of FcgammaRII or CR1 and 2 on FDCs. Here, we present an overview of FDC function in recall responses with known deficiencies in FDCs and GC development. Then, we review our recent work on aged FDCs and discuss age-related changes in molecular interactions between FDCs and B cells. We also discuss the causes underlying the impaired humoral immune response with respect to age-related molecular changes in FDC and B cell interactions. In vitro evidence suggests that FcgammaRII on aged FDCs is regulated abnormally and this in turn might cause the development of a defective FDC-network (reticulum) that retains few ICs, promotes ITIM signaling, prevents B cell proliferation and GC formation, and antibody production.     

Erythropoietin enhances immune responses in mice

Erythropoietin (Epo) is the main erythropoietic hormone. Recombinant human Epo (rHuEpo) is thus used in clinical practice for the treatment of anemia. Accumulating data reveals that Epo exerts pleiotropic activities. We have previously shown an anti-neoplastic activity of Epo in murine multiple myeloma (MM) models, and in MM patients. Our findings that this anti-neoplastic effect operates via CD8+ T lymphocytes led us to hypothesize that Epo possesses a wider range of immunomodulatory functions. Here we demonstrate the effect of Epo on B lymphocyte responses, focusing on three experimental models: (i) tumor-bearing mice, (5T2 MM mouse); (ii) antigen-injected healthy mice; and (iii) antigen-injected transgenic mice (tg6), overexpressing human Epo. In the MM model, despite bone marrow dysfunction, Epo-treated mice retained higher levels of endogenous polyclonal immunoglobulins, compared to their untreated controls. In both Epo-treated wild type and tg6 mice, Epo effect was manifested in the higher levels of splenocyte proliferative response induced in vitro by lipopolysaccharide. Furthermore, these mice had increased in vivo production of anti-dinitrophenyl (DNP) antibodies following immunization with DNP-keyhole limpet hemocyanin. Epo-treated mice showed an enhanced immune response also to the clinically relevant hepatitis B surface antigen. These findings suggest a potential novel use of rHuEpo as an immunomodulator.     

The early immune response in the liver of BALB/c mice infected with S. typhimurium

Gram-negative bacteria acquired through gastrointestinal infection can be a serious cause for the development of septic shock especially in immunosuppressed patients. Thus, the aim of this study was to examine the early events of the immune reaction against S. typhimurium. Bacteria were injected into mice at different concentrations. Four animals from each group were killed at five different points of time. Liver cytokine mRNA expression was determined by semiquantitative rt-PCR and liver histology was examined. Serum cytokine levels of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-4 and IL-10 were determined. intravenous (i.v.) infection with 109 bacteria led to lethal septic shock within 24 h. A delayed production of IFN-gamma, TNF-alpha, IL-18 and IL-10 and milder histological alterations in the liver were observed in these animals. The highest expression of cytokines in the liver and the strongest histological alterations were seen after infection with 107 bacteria. Here, an increased mRNA expression of all proinflammatory cytokines began 1 h after infection. Animals infected with 1 x 102 bacteria had the highest detectable serum levels of IL-6 and IL-10. These data indicate that the immediate events in the immune reaction within the liver after infection with S. typhimurium are associated with the outcome of the subsequent sepsis.