Dendritic cells and macrophages are required for Th1 development of CD4+ T cells from {alpha}{beta} TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-{gamma} production is IFN-{gamma}-dependent

We have examined the antigen presenting cell (APC) requirementsfor primary T cell activation and T helper (Th) cell phenotypedifferentiation using naive CD4⁺ T cells from ß TCRtransgenic mice. Purified dendritic cells were the principalcell required for induction of primary ovalbumln peptide specificT cell activation and clonal expansion. However, dendritic cellsdid not induce differentiation of T cells toward Th1 or Th2phenotype. Addition of IL-4 during primary dendritic cell stimulationsof T cells resulted in the development of a Th2 phenotype whichproduced high levels of IL-4 during secondary and tertiary stimulation.In contrast, development of Th1 cells producing high levelsof IFN- could not be induced with dendritic cells alone butrequired the addition of appropriately activated macrophages.Addition of splenic or peritoneal B cells did not induce Th1development. Activated splenic macrophages induced Th1 developmentvia a non-MHC restricted mechanism. Thus, requirements for inductionof proliferation of naive CD4⁺ T cells are distinct from thosedirecting Th1 phenotype development. IL-12 could replace therequirement for macrophages to induce Th1 development when Tcells were activated with dendritic cells. Furthermore, thisIL-12 mediated development of Th1 cells producing high levelsof IFN- was dependent on IFN-.     

Transforming growth factor-{beta} inhibits IL-4 and IFN-{gamma} production by stimulated human T cells

The present study investigates the effect of transforming growthfactor (TGF-ß on the production of IL-4 and IFN- bythe leukemia T_h0 type cell line HUT78, by freshly Isolated humanT cells, and by antigen specific human T cell clones. We foundthat IL-4 and IFN-ß, but not IL-2, production by stimulatedHUT78 cells was inhibited by TGF-ß1. TGF-ß1also reduced the accumulation of IL-4 and IFN- specific mRNAin stimulated HUT78 cells. However, IL-2 and IL-7 co-stimulatedIL-4 and IFN- production, whereas IL-1, IL-3, IL-5, IL-6, IL-8,tumor necrosis factor- or granulocyte macrophage colony stimulatingfactor had no effect. Because IL-2 is an Important helper cytokinefor the production of IL-4 and IFN-, we investigated whethersignal transductlon through the IL-2 receptor is Impaired byTGF-ß1. We found that tyrosine phosphorylatlon inresponse to IL-2 In HUT78 cells was strongly inhibited by ashort prelncubatlon with TGF-ß1. Evidence for an antagonisticrole for TGF-ß1 and IL-2 comes from the finding thathigh doses of IL-2 could partially overcome TGF-ß1mediated inhibition of IL-4 and IFN- production. Similar toIts effect on HUT78 cells, TGF-ß1 also inhibited IL-4and IFN- production by freshly Isolated T cells as well as byhuman T cell clones. Taken together, our experiments show thatthe IL-2 dependent cytokines IL-4 and IFN- are both negativelycontrolled by TGF-ß under conditions where IL-2 productionIs unaffected by a mechanism which partially involves an inhibitionof IL-2/1L-2R signal transductlon. These data Identify TGF-ßand IL-2 as mutual antagonists in the regulation of IL-4 andIFN- production.     

CD40配基化的肿瘤特异性树突状细胞介导Th1细胞分化的研究

目的：探讨CD40配基化的肿瘤特异性DCs在介导Th1细胞分化中的作用。方法：采用GM-CSF和IL-4联合方案体外诱导小鼠髓系DCs，并利用mCD40L-CHO和TNF-α分别刺激凋亡肿瘤细胞负载的DCs制备DCs瘤莆；^3H-TdR掺入试验检测DCs对T淋巴细胞的促增殖效应；ELISA测定细胞培养上清中IL-10、IFN-1、IL-12的含量；胞内染色和流式细胞术检测经成熟DCs活化的T细胞中CD4^＋IFN-γ^＋T和CD4^＋IL-4^＋T的比例。结果：体外刺激T细胞增殖能力在CD40配基化DCs组最高（P〈0．05），CD40配基化DCs能更有效地促进活化T细胞分泌IFN-γ和介导CD4^＋IFN-γ^＋T细胞的分化（P〈0．05）。同时，CD40配基化DCs分泌IL-12的量也明显高于TNF-α组（P〈0．05）。结论：CD40配基化的肿瘤特异性DCs体外能有效介导Th1细胞的分化。     

Th1- and Th2-type lymphokine-assisted induction and release of chemokine receptors from primary human trophoblast cells

Chemokine receptors (CCRs) have been demonstrated to facilitate the entry of HIV in different cell types of infected individuals, including CD4(+) T cells and dendritic cells. The natural or inducible expression of CCRs on trophoblast cells could provide a valid mechanism for the in utero transmission of HIV from mother to fetus. Because of the rapid turnover of these receptors, we attempted to define the natural and inducible expression of surface CCR3 and CXCR4 on primary human trophoblasts during short periods of cell culture. In the absence of any external stimulus the expression of CCR3 and CXCR4 varied from 1% to 24%. Kinetic experiments show that the levels of both CCR3 and CXCR4 reach a peak of expression after 6 h of culture, whereas by 24 h they have almost disappeared. In the presence of IFN-gamma, CCR3 is showing an increasing pattern of expression after 4 h of incubation, reaching highest levels after 24 h of culture, whereas CXCR4 is kept at lower levels as compared with nontreated cells. Furthermore, in the presence of IL-4, CCR3 expression declines from 2 to 8 h of culture to increase again at 24 h, where 50% of the cell population is expressing the receptors. Under the IL-4 stimulus, CXCR4 shows a peak of expression at 8 h of culture. An interesting feature of this study is that we were able to detect soluble CCR activity in the culture supernatants of trophoblast cells, which followed an inverse pattern of this of surface expression. Thus, the inability of many laboratories to detect high levels of CCRs in placentae of HIV infected mothers may be due on these fast turnover of these receptors, which by the assaying time have either been released in the culture medium or been internalized to the cell.     

Allergen-driven limiting dilution analysis of human IL-4 and IFN-{gamma} production in allergic rhinitis and clinically tolerant individualsz

Difficulties in detecting human IL-4 synthesis in antigen-drivenprimary culture have led to widespread reliance on less physlologicappraoches to T cell activation. Although there is general agreementof a T 2-like blas,l the precise defects in ctokine responsivenessremain controverslal. Analysis of cytokine protein productionby fresh, unselected cell populations in response to gognate,antigen-driven stimulation remains an important goal. Here,limitting dilution analysis (LDA) was sued to evaluate antigen-stimulatedcytokine gene expression by fresh peripheral blood mononuclearcells(PBMC)(.PBMC from 19 grass pollen sensitive, allergicrhinitis subjects and normal, non-atopic contrls were evaluated1 mont after natural relmmunization (the peak of te local grasspollen season). Surprisingly, highly atopic subjects and cllnicallynon-allergic individuals exhiboited virtually equlvalent antigen-specific,CD4-dependent cytokine production in response to short-termculture with these common environmental antigens. As anticipated,pronounced increases in T_h2-like activity were evidnet in tecirculating immune repertolre of grass of 117:1 among normalsubjects versus 4:1 among those with allergic rhinitis (Mann-WhitneyU-test, P=0.0067). This T_h2-like blas reflected both a lowerfrequencey of IFN-prducing cells and a markedly increased frequencyof IL-4-producing cells in the circulatin grass-pollen specificrepertoire of atopic donors. The observation that every atopicand normal subject produced IFN- (±IL-4) following antigenre-stimulation arguses that the decision as to whether allergyor (clinical) tolerance results, hinges not on a geneticallydetermined capacity of whether allergen-reactive T cells canbe stimulated in any given individual by cronic exposure toubiqultous enveronmental antigens, but on the nature of thecokine response that comes to dominate that individual's response     

Quantitation of human influenza virus-specific cytotoxic T lymphocytes: correlation of cytotoxicity and increased numbers of IFN-{gamma} producing CD8+ T cells

To study virus-specific cytotoxic T lymphocyte (CTL) activityat the single cell level, an IFN-     

IFN-{gamma}is a potent inducer of Ig secretion by sort-purified murine B cella activated through the mlg, but not the CD40,signaling pathway

To study the influence of the quaternary structure of the outermembrane protein PhoE of Escherlchla coli on the resentationof an inserted T cell epttope, an epttope comprising amino acidresidues 72–85 of myeiin basic protein (MBP) was insertedat dmerent shes in PhoE. This quence is the crtticai T cellepitope in experimental autoimmune encephalomyelttis (EAE) inLewis rats. The antigenicity and immunogenicity of two dtfferentconformational forms of the chimeric PhoE constructs, i.e. thedenatured monomeric form and the native Mmeric form, were studied.tt appeared that the monomeric form, but not the natlve trimericform of such PhoE constructs induced proitferation of the MBP7245spectficT cell line Zla. This conformational discrepancy was independentof the site in PhoE in which the epitope was inserted. lmmunlzationwith the monomeric form of PhoE constructs resutted in the primingof MBW2-85-spectfic T cells. in contrast, the Mmeric form ofthese constructs was much less efficient in priming such cells.differences between the monomeric and trimeric forms were mostapparent when induction of EAE was studied. The monomeric formwas encephalitogenic whlle the trimeric form was not Furthermore,the antigen fine spectficity, Vg usage .and encephalttogenicityof T cells triggered by immunization with a monomeric PhoE constructappead to be the same as those of T cell line Zla, which wasobtained after immunization with MBP, indicating that similarcells are triggered by immunization with the epitope ether insertedin PhoE or in its native context.     

Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells

In this study we have investigated the capacity of human fetalthymocytes to differentiate in vitro into subsets of T cellswith polarized T_h1 or T_h2 cytokine profiles. Stimulation offreshly isolated human fetal thymocytes with anti-CD3 mAb, cross-linkedonto CD32,CD58,CD80-expressing mouse fibroblasts and subsequentculture in the presence of exogenous rIL-2 for 6 days, inducedthe production of both IL-4 and IFN-, which was mainly producedby CD4⁺ single-positive (SP) and CD8⁺ SP cells respectively.Addition of rIL-4 during priming augmented IL-4 production incultures of human fetal thymocytes, which was mainly due toan increased production of IL-4 by CD8SP cells. In contrast,addition of IL-4 to the cultures only slightly enhanced IL-4production and had little effect on frequencies of IL-4-producingCD4SP cells. Both CD4SP and CD8SP cells produced IL-5, IL-10and IL-13 at comparable levels, following priming in the presenceof rIL-4. Priming in the presence of rIL-12 strongly enhancedthe production of IFN- in both CD4SP and CD8SP cells. No correlationbetween expression of CD27, CD30 and CD60, and a particularcytokine profile of differentiated thymocytes could be demonstrated.Together, these results demonstrate the full capacity of fetalhuman thymocytes to differentiate into cytokine-producing Tcells in a priming milieu with appropriate stimulatory moleculesand exogenous cytokines. In addition, CD4SP thymocytes rapidlydifferentiate into polarized T_h2 cells following stimulationin vitro in the absence of exogenous rIL-4.     

天花粉蛋白联合CD40信号激发的人MoDC介导Th2细胞分化的研究

探讨天花粉蛋白(Tk)联合CD40信号诱导人单核细胞来源DC(MoDC)的成熟活化及其介导Th2细胞分化的作用和机制。采用GM-CSF和IL-4联合方案体外诱导人MoDC,并利用鼠抗人CD40激发型单抗(5C11)刺激负载了Tk的DC制备成熟DC。采用免疫荧光标记和流式细胞术分析DC表型(CD80、CD83、CD86、CD14、PDL1)及其摄取FITC-Dextran的能力;ELISA法测定DC上清中IL-12p70的含量;胞内染色和流式细胞术检测经成熟DC活化的T细胞中CD4~+IFN-γ~+T和CD4~+IL-4~+T的比例。实验结果显示单独Tk不能刺激DC完全成熟,用Tk负载DC后必须联合外源性成熟刺激信号(如5C11),才能有效促进DC成熟,表现在CD80、CD83、CD86的上调表达,CD14下调表达,DC摄取FITC-Dextran的能力降低。与CD40信号单独诱导组相比,Tk联合CD40信号诱导的成熟DC表面PDL1分子呈下调表达,分泌IL-12的能力显著降低,明显提高T细胞中CD4~+IL-4~+T的比例。由此表明负载了Tk的成熟MoDC体外能有效介导Th2细胞分化。     

Kinetics and expression patterns of chemokine receptors in human CD4+ T lymphocytes primed by myeloid or plasmacytoid dendritic cells

We investigated the kinetics of expression of 12 chemoattractant receptors as a function of cell division following priming of human naive CD4+ T cells by different populations of dendritic cells (DC) and under conditions favoring Th1 or Th2 differentiation. Two chemokine receptors, CXCR3 and CXCR5, were rapidly up-regulated following T cell activation by either monocyte-derived DC, myeloid DC (mDC) or plasmacytoid DC (pDC). While CXCR5 expression was transient, expression of CXCR3 at advanced cell divisions was dependent on differentiation, being expressed at high levels on Th1 cells. Several other receptors (CCR2, CCR3, CCR4, CCR5, CXCR6 and CRTh2) were acquired progressively as a function of cell division and in a fashion that was influenced by polarizing cytokines. The Th2-associated chemoattractant receptors CRTh2 and CCR3 were up-regulated with slower kinetics compared to the Th1-associated receptors CXCR3 and CXCR6, consistent with a different kinetics and efficiency of polarization. Moreover, CCR4 and CXCR6 were preferentially induced in T cells activated by mDC and pDC, respectively. Finally, CXCR5 and CCR7 were also rapidly and transiently up-regulated in memory T cells following TCR stimulation. These results indicate a complex chemokine receptor regulation dependent on both T cell activation and differentiation state. In addition, they reveal the existence of DC-specific cues for the regulation of T cell migratory capacity.     

Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-{gamma} in vivo

2 Is a heterodlmerlc cytoklne that has been shown to enhancenatural killer (NK) and cytotoxic T lymphocyte (CTL) responses,and to induce IFN- production in vitro. In this study, we haveexamined the effects in vivo of administering purified murinerlL-12 to normal mice. Dally injections of riL-12 I.p. (1 ngto 10 µg/day) caused dose-dependent enhancement of NKcell lytic activity in the spleens and livers of treated mice.Histologlc examination of the livers of IL-12-treated mice revealedfocal mononuclear cell infiltrates, and flow cytometry studiesindicated that the livers of IL-12-treated mice contained increasednumbers of NK cells, CD8⁺ T cells, and monocytes. Liver andsplenic lymphold cells from IL-12-treated mice, unlike liverand splenic lymphoid cells from control mice, spontaneouslysecreted IFN- in vitro, suggesting that they had been inducedby IL-12 to produce IFN- in vivo. Consistent with this, IFN-could be detected in the serum of IL-12-treated mice. In micewhich had been immunized by footpad injection of allogenelcsplenocytes, dally administration of rlL-12 I.p. was shown toenhance the specific CTL response in the draining lymph nodes.Thus, these studies demonstrate that IL-12 can enhance NK andCTL activity and induce IFN- production in vivo, as well asin vitro, and suggest possible mechanisms by which IL-12 mayexert therapeutic effects in the treatment of some tu more andinfectious diseases.     

Differential production of IL-12 in BALB/c and DBA/2 mice controls IL-4 versus IFN-{gamma} synthesis in primed CD4 lymphocytes

The profile of cytokines produced by CD4 T cells is profoundlyinfluenced by the presence of IL-12. Here we demonstrate thatduring re-stimulation of antigen-specific immune responses invitro, antigen-primed lymph node cells from DBA/2 mice produced3- to 30-fold more IL-12 than did cells from BALB/c mice, whichare identical at the major histocompatibility locus. The straindifferences in IL-12 production were observed only in antigen-drivenresponses (and not in responses induced by bacterial products),and were dependent upon an interaction between CD4 T cells andlymph node adherent cells. In addition, differences in the quantityof IL-12 produced by DBA/2 and BALB/c antigen-presenting cells(APC) was not dependent on differential production of IFN- byT cells, since APC from DBA/2 mice still produced much greaterquantities of IL-12 than did BALB/c APC when each was culturedwith the same H-2^d-restricted T_h2 clones, in the complete absenceof IFN, or when each was cultured with primed (BALB/c x DBA/2)F₁T cells. The level of IL-12 produced in the cultures criticallyaffected cytokine production in CD4 T cells, since neutralizationof endogenous IL-12 in DBA/2 cultures, which are predisposedtowards developing T_h1 responses, reduced IFN- production andenhanced IL-4 synthesis to levels normally seen in BALB/c cultures,which are predisposed toward developing Th2 responses. We proposetherefore that differential production of antigen-driven IL-12is a mechanism by which the genetic background in DBA/2 andBALB/c mice can affect the pattern of cytokine synthesis byT cells during the development of adaptive immune responses.     

Ocular immune privilege is circumvented by CD4+ T cells, leading to the rejection of intraocular tumors in an IFN-{gamma}-dependent manner

Although intraocular tumors reside in an immune-privileged site, they can circumvent immune privilege and undergo rejection, which typically follows one of two pathways. One pathway involves CD4(+) T cells, delayed-type hypersensitivity (DTH), and the culmination in ischemic necrosis of the tumor and phthisis (atrophy) of the eye. The second pathway is DTH-independent and does not inflict collateral injury to ocular tissues, and the eye is preserved. In this study, we used a well-characterized tumor, Ad5E1, to analyze the role of IFN-gamma in the nonphthisical form of intraocular tumor rejection. The results showed that IFN-gamma induced tumor cell apoptosis, inhibited tumor cell proliferation, and promoted rejection by inhibiting angiogenesis. Microarray analysis revealed that IFN-gamma induced up-regulation of five antiangiogenic genes and down-regulation of four proangiogenic genes in Ad5E1 tumor cells. Although IFN-gamma knockout (KO) mice have progressively growing intraocular tumors, IFN-gamma was not needed for the elimination of extraocular tumors, as all IFN-gamma KO mice rejected s.c. tumor inocula. This represents a heretofore unrecognized role for IFN-gamma in circumventing ocular immune privilege and eliminating intraocular tumors. The findings also reveal that some IFN-gamma-independent tumor rejection processes are excluded from the eye and may represent a new facet of ocular immune privilege.     

Distinct effect of CD40 and TNF-signaling on the chemokine/chemokine receptor expression and function of the human monocyte-derived dendritic cells

A key and limiting step in the process of human monocyte-derived dendritic cells （mDCs） for clinical use is their in vitro maturation and in vivo migration. We previously observed that CD40 signal facilitated human mDC growth and maturation. To further explore this process, mDCs generated with GM-CSF and IL-4 were co-cultured with apoptotic tumor cells for 24 hours, followed by incubating with anti-CD40 monoclonal antibody or TNF-a for 48 hours to generate mature DCs. The chemokine/chemokine receptor expression and functions of mature DCs upon various stimuli were determined. The expression of costimulatory molecules on apoptotic tumor cell-loaded mature DCs co-cultured with either anti-CD40 antibody （anti-CD40-DCs） or TNF-a （TNF-DCs） were up-regulated compared to immature DCs, consistent with the abilities of these cytokine to drive DC maturation in vitro. The mRNA levels of chemokines such as stromal cell-derived factor-1a （SDF-1a）, EBV-induced molecule 1 ligand chemokine （ELC）, and IFN inducible protein-10 （IP-10） in anti-CD40 activated DCs were increased and the dendritic cell-specific chemokine 1 （DC-CK1） was moderately up-regulated as compared with other mature DCs. The corresponding chemokine receptors CXCR4 and CCR7 of anti-CD40-DCs were significantly expressed. The CXCR3 expression on activated T cells stimulated by anti-CD40-DCs was also increased. Moreover, the anti-CD40-DCs had a stronger ability to stimulate T cell proliferation than any other DCs. The NF-xB activity was much higher in anti-CD40-DCs than that of TNF-DCs. These results offer further evidence of the importance of the CD40 signal in developing efficient human DC vaccines for cancer immune therapy. Cellular ＆ Molecular Immunology.     

Herpesvirus saimiri immortalization of {alpha}{beta} and {gamma}{delta} human T-lineage cells derived from CD34+ intrathymic precursors in vitro

Herpesvirus saimiri (HVS), an agent that can infect many humancell types, has been shown to immortalize selectively TCR ß⁺CD3⁺T lymphocytes. Human T cell precursors defined as CD34⁺CD3^–CD4^–CD8^–were isolated from thymic samples and exposed to HVS in thepresence of either IL-2 or IL-7. Cultures lacking the viruswere non-viable by day 15. Test cultures, in contrast, showeda sustained proliferative activity lasting >5 months, allowingthe phenotypical and molecular analysis of the cellular progeny.In the presence of IL-7, TCR ß⁺ cells with three differentphenotypes (mainly CD4⁺CD8^–, but also CD4⁺CD8⁺ and CD4^–CD8⁺)were immortalized, whereas no TCR ⁺ cells were recovered. Kineticstudies showed that the expansion of immortalized TCR ß⁺cells was preceded by a gradual loss of CD34⁺ cells followedby a transient accumulation of two distinct cell subsets: firstCD1⁺CD4⁺CD3^– cells and then CD4⁺CD8⁺ thymocytes. Thisresembles early phenotypic changes occurring during normal intrathymicT cell development. In the presence of IL-2, in contrast, onlyTCR ⁺ cells were immortalized (mainly CD4^–CD8⁺, but alsoCD4^–CD8^–). The results show that HVS can be usedto read the CD3⁺ cellular outcome of T cell differentiationassays, including ⁺ CD4^–CD8⁺, ⁺CD4^–CD8^–, ß⁺CD4⁺CD8^–,ß⁺CD4^–CD8⁺ and ß⁺CD4⁺CD8⁺ T cells.A clear role for different cytokines (IL-2 for ⁺ cells, IL-7for ß⁺ cells) in early T cell commitment was alsoapparent.     

PGE_2促进人脐血CD34~+造血祖细胞向淋巴样树突状细胞分化

为了探讨前列腺素E2(protaglandin E2,PGE2)对人外周血CD14+单核细胞和脐血CD34+造血祖细胞(hematopoieticprogenitor cells,HPC)体外诱导pDC分化的影响。分别采用IL-4+GM-CSF+TNF-α和SCF+Flt-3L+GM-CSF+TNF-α诱导人外周血CD14+单核细胞和CD34+HPC向pDC分化,采用流式细胞仪分析细胞表型,并观察PGE2加入培养体系后对pDC分化的影响。结果:以CD34+HPC为来源,比CD14+单核细胞能诱导出更多数量且具有功能的pDC。培养体系中加入PGE2显著增强了CD34+HPC向pDC的分化,而对CD14+单核细胞的分化却无明显促进作用。PGE2可有效促进脐血CD34+HPC在多种细胞因子的作用下向pDC的成熟分化。