Composition of TCR-CD3 complex in human intestinal intraepithelial lymphocytes: lack of Fc{varepsilon}Rl{gamma} chain

Human intestinal intraepithelial lymphocytes (DEL) are a uniquepopulation of predominantly CD8ß⁺ TCRß⁺lymphocytes and, to a lesser extent, TCR⁺ lymphocytes that proliferatepoorly to anti-CD3 mitogenic signals but display significantcytolytic activity. Studies in mouse model systems have shownthat the chain of the high-CD3 affinity receptor for IgE (FcRl)may substitute for the chain in the TCR-CD3 complex of iIEL.This has suggested that the functional properties of these cellsmay be associated with an altered composition of the TCR-CD3complex. We therefore analyzed the TCR-CD3 complex of normalhuman iIEL. One-and two-dimensional non-reducing/reducing SDS-PAGEanalysis of CD3, CD3, CD3, and FcRr chain immunopreclpitatesof cell surface radiolabeled proteins with subunit-specificantibodies revealed a TCR-CD3 complex without associated FcRrchains. Thus, normal human NEL contain a TCR-CD3 complex thatconsists predominantly of , homodimers in association with theß TCR and CD3, and , similar to the majority of peripherallymphocytes. This indicates that the distinct properties ofhuman DEL are not associated with substitutions of the FcRlchain in the TCR-CD3 complex.     

TNF-{alpha} regulates IL-4-induced Fc{varepsilon}RII/CD23 gene expression and soluble Fc{varepsilon}RII release by human monocytes

We examined the regulatory effects of TNF- on IL-4-induced geneexpression of the low-affinity receptor for IgE (FcRII/CD23)in human monocytes and IL-4-induced soluble FcRII (sFcRII) releasefrom monocytes. IL-4-induced FcRII expression on the surfaceof monocytes was reduced by TNF- as early as 1 day after cultureand the effect of TNF- increased with prolonged culture. Thepresent analysis was designed to examine whether or not TNF-could suppress IL-4-induced FcRII mRNA expression and enhancedIL-4-induced sFcRII release. The addition of TNF- to monocytecultures with IL-4 significantly reduced FcRII expression onthe surface of monocytes and significantly increased sFcRIIrelease from monocytes. Over time, there was an inverse relationshipbetween the disappearance of cell surface FcRII and the appearanceof sFcRII in culture supernatants. FcRII mRNA expression inmonocytes cultured with IL-4 was not affected by TNF- when examinedat 6 h after cultivation. When the cells were cultured withTNF- for more than 24 h, however, TNF- down-regulated IL-4-inducedFcRII mRNA levels. This correlated with the kinetics of down-regulationof IL-4-induced FCRII expression on the surface of monocytesby TNF-. These results suggest that TNF-dependent reductionof IL-4-;induced FcRII expression on the surface of monocytesresulted, at least in part, from the suppression of FcRII mRNAexpression and the enhancement of sFcRII release.     

Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells

The T-cell antigen receptor (TCR) consists of a glycoproteinheterodimer (/ß or /) which is non-covalently associatedwith at least four or five invariant polypeptides (CD3 ,,, ;and ). In T-cell variants lacking TCR ,ß or , it hasbeen shown that incomplete TCR/CD3 complexes are retained withinthe cell. To examine requirements for cell surface expressionof TCR/CD3, we transfected COS monkey kidney cells with cDNAsencoding TCR ,ß and CD3 , , and . We report thatcell surface appearance of TCR/CD3 on COS cells requires coordinateexpression of all six proteins. In the absence of the chain,subcomplexes comprising from two to five chains were readilydemonstrable In COS cells, but they failed to reach the cellsurface or to acquire N-llnked oligosaccharide side chains indicatingfailure to reach the medial Golgl. Pulse-chase, metabolic labellingof transfected COS cells showed that three chains (CD3 , CD3, and ) were stable while three (TCR , TCR ß and CD3) were rapidly degraded. In two- and three-chain co-transfectionsspecific intracellular subcomplexes were formed between TCR and CD3 , TCR and CD3 , or TCR ß and CD3 . Binarysubcomplexes having at least one stable chain (CD3 –TCRß) were stable while one formed by two unstable chains(TCR –CD3 ) was still degraded. Assembly of the TCR/CD3complex in COS cells thus appears centered around the metabolicallystable CD3 and CD3 proteins. Site-specific mutations of thenegatively-charged transmembrane amino acid of residues of theCD3 chains to alanines served to either abolish (for TCR –CD3 and TCR ß–CD3 ) or diminish (for TCR –CD3) these TCR-CD3 interactions. These mutations had no effect,however, on CD3–CD3 Interactions or upon synthesis, metabolism,or intracellular distributions of the CD3 proteins. The transmembranedomains of CD3 , , and thus appear to play a major role inassociations of CD3 with TCR chains.     

Soluble human high-affinity receptor for IgE abrogates the IgE-mediated allergic reaction

The high-affinity receptor for IgE (FcRI) has a tetrameric structurecomposed of one, one ß, and two disulfide-linked subunits, of which the subunit binds IgE with high affinity.A recombinant soluble form of the ectodomain of the human FcRIsubunit (rsFcRI) was recently generated by gene engineeringand was verified to bind IgE with an affinity as high as thatof native FcRI on the cell surface. rsFcRI was prepared on alarge scale in order to analyze its biological function. rsFcRIcompletely inhibited IgE binding to the cell surface, resultingin abrogation of the chemical mediator release from RBL-2H3cells. Furthermore it completely abolished the passive cutaneousanaphylaxis (PCA) response by trapping IgE specifically whenitwas administered into rats prior to IgE sensltizatlon. Evenafter IgE sensitizatlon, treatment of rsFcRI substantially reducedthe PCA response. It was finally shown that rsFcRI inhibitedIgE binding to human peripheral blood basophils and the histaminerelease from them. In this paper we address the ability of rsFcRIto specifically prevent the IgE-mediated allergic reaction.     

Different cytoplasmic structure of the CD3{zeta} family dimer modulates the activation signal and function of T cells

The TCR complex transduces the antigen recognition signal throughcommon activation motifs present in both CD3 chains and , dimerswithin the complex. We have investigated functional roles ofthe cytoplasmic domain in and CD3 for T cell activation inearly and late responses by comparing the signaling capabilityof the TCR complexes containing mutant lacking some or allmotifs, or chain, another family molecule. The results withthe mutant , lacking all motifs indicated that CD3 can transducesignals to cause earty activation events and production of IL-2upon antigen stimulation in the absence of , motifs. However,any one of the ; motifs was required to respond to Thy-1 stimulationand this requirement cannot be replaced by other CD3 chains.Such , motif-dependent responses were also observed in tyrosinephosphoryiation of a 90 kDa protein upon TCR stimulation. Furthermore,we found that the C-terminal unique region of the chain exhibitsinhibitory function in phosphoryiation and Ca2⁺ response uponTCR stimulation as well as IL-2 production upon Thy-1 stimulation.Collectively, the present analyses suggest that two types ofsignals are induced through the TCR-CD3 complex: (I) the commonmotif-dependent signals which are mediated equally through ,dimers and CD3, and (II) , specific motif-dependent signals.Differences in the cytoplasmic domain of , family moleculesmay modulate the cooperation of these two signals, resultingin alteration of T cell functions.     

Cloning of cDNA coding for low-affinity Fc receptors for IgE on human T lymphocytes

Using a cDNA probe corresponding to the membrane-bound formof the B cell receptor for IgE, we have isolated, sequenced,and expressed a cDNA clone which codes for a human T lymphocyteFcR from HUT-78 cells. This T cell FcR cDNA codes for 320 aminoacid residues, and shows high homology to the B cell FcR sequence.The major differences between this T cell and the B cell FcRcDNA sequences are (i) a limited stretch of nucleotides at the5' segment of the coding region which encodes a putative cytoplasmicregion of the FcR molecule and the untranslated 5' end; and(ii) an additional 64 bp segment in the untranslated 3' endcontaining two repeats in tandem with three existing repeatsin the same region. The expression of FcR on T lymphocytes mayreflect involvement of the FcR in regulation of IgE-mediatedresponses. The cytoplasmic difference implies functional activityof the FcR in T lymphocytes that is mechanistically differentfrom the FcR of B lymphocytes.     

Expression of genes encoding the pre-TCR and CD3 complex during thymus development

The mature TCR is composed of a clonotypic heterodimer (ßor) associated with the invariant CD3 components (, , and ).There is now considerable evidence that more immature formsof the TCR-CD3 complex (consisting of either CD3 alone or CD3associated with a heterodimer of TCR ß and pre-T)can be expressed at the cell surface on early thymocytes. Thesepre-TCR complexes are believed to be necessary for the orderedprogression of early T cell development. We have analyzed indetail the expression of both the pre-TCR and CD3 complex atvarious stages of adult thymus development. Our data indicatethat all CD3 components are already expressed at the mRNA levelby the earliest identifiable (CD4¹⁰) thymic precursor. In contrast,genes encoding the pre-TCR complex (pre-T and fully rearrangedTCR ß) are first expressed at the CD44¹⁰CD25⁺CD4^–CD8^–stage. Detectable surface expression of both CD3 and TCR ßare delayed relative to expression of the corresponding genes,suggesting the existence of other (as yet unidentified) componentsof the pre-TCR complex.     

CD3{varepsilon}-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2 / mice in the absence of TCR {beta} chain expression

Recent studies have shown that TCR ß chain expressioncan effect the differentiation of CD4^–CD8^– double-negative(DN) thymocytes to CD4⁺CD8⁺ double-positive (DP) thymocytes.The TCR ß chain is expressed on the surface of DPthymocytes in association with CD3, and chains, suggestinga potential role for CD3 components in this signaling process.We now report detection of a very tow level of surface expressionof CD3 on adult DN RAG-2^–/–; thymocytes. This surfaceCD3 was associated with CD3 and chains, as detected by anti-CD3immunoprecipitation analyses. Significantly, injection of anti-CD3mAb into RAG-2^–/– mice led to the accumulation ofan IL-2R^– CD2⁺ DP cell population and a nearly 100-foldincrease in thymic cellularity to essentially normal levels.Together, these data strongly indicate that TCR ßchain-mediated developmental signals are transduced by CD3 componentsand provide potential insights into mechanisms by which TCRß chain expression may effect this process.     

Over-expression of CD3{varepsilon} transgenes blocks T lymphocyte development

We have reported previously that mice carrying >30 copiesof the human CD3 transgene completely lose their T lymphocytesand NK cells (36). Here we demonstrate by immunohistology thatin the most severely immunodeficient mouse, tg26, the thymusis very small, has sizeable vacuoles and does not contain recognizableT lymphocytes except for a small percentage of Thy- 1⁺ cellsand B cells. Cell surface phenotyping and TCR and -ßrearrangement studies confirm that the arrest in T lymphocytedevelopment precedes the arrest in rag-1^null, rag-2^null andTCRß^null mice. Since the T cell progenitors in whichthe arrest occurred were absent in the transgenic mice, indirectapproaches were taken to examine the causes of the block inT cell development. Analyses of 12 independently establishedmutant mouse lines, generated with five different transgenicconstructs, revealed that the severity of the abrogation inT cell development was dependent on the number of copies oftransgenes. Since the number of transgene copies generally correlatedwith the levels of expression of the transgenic CD3 proteins,we concluded that over-expression of the CD3 protein was thelikely cause of the block in T lymphocyte development. The Tcell immunodeficiency was caused by either the human or themurine CD3 protein. Since transgene coded mRNAs were found insignificantly higher quantities than endogenous CD3 mRNAs infetal thymi on days 13 and 14 of gestation, over-expressiontook place very early in development, probably prematurely.Over-expression of the CD3 transgene in thymocyte precursorsmay therefore affect T lymphocyte development in the absenceof TCR and possibly in the absence of the other CD3 proteins.More importantly, over-expression of the CD3 protein in thymocytesof mice with a low copy number of transgenes had a significanteffect on late thymic development Over-expression of the CD3protein in immature thymocytes mimicked the effects caused byexposure of CD4^–; CD8^– thymocytes to anti-CD3 treatment:apoptosis and lack of TCRß expression. We thereforespeculate that in the homozygous tg26 animals the arrest inT cell development was caused by excessive signal transductionevents rather than by a toxic effect of the transgenic protein.     

TCR isoform containing the Fc receptor {gamma} chain exhibits structural and functional differences from isoform containing CD3{xi}

The structure and function of the TCR-CD3 complex containinga homodimer of the gamma chain of the high affinity receptorfor IgE (FcR) (FcR⁺ TCR) was investigated by transfecting theFcR gene into a CD3^–, CD3^–, FcR^– T cell line.Introduction of FcR, as well as CD3, induced a high expressionof the TCR-CD3 complex on the cell surface. Transfected FCRformed a homodimer and associated firmly with the TCRßdimer but only weakly with the CD3. Stimulation of both FcRand CD3 transfectants by antibodies against TCR or CD3 inducedaccumulation of inositol phosphates, the Ca²⁺ response, IL-2production, and growth inhibition. On the other hand, antigenstimulation of transfectants expressing FcR as well as CD3 inducedIL-2 production, but only the latter exhibited the antigen-inducedgrowth inhibition. In vitro kinase assay suggested that theCD3 dimer but not the FcR dimer associates with the Fyn kinase.These results indicate that the FcR homodlmer Is able to forma functional TCR complex but that the mode of assembly and thesignaling function of FcR⁺ TCR, including its association withtyrosine klnase(s), may differ from the TCR-CD3 complex containingCD3 homodimers (⁺ TCR). This provides an example which illustratesthat different TCR isoforms mediate distinct signals and functions.     

Molecular cloning and characterization of guinea pig Fc{gamma}RIII: expression but not function is independent of the {gamma} chain of Fc{gamma}FceRI

We have isolated two cDNA clones encoding the guinea pig receptorfor the Fc portion of lgG2 (Fc2R) from a guinea pig peritonealmacrophage cDNA library. Analysis of the predicted amino acidsequence of the one cDNA clone indicated that the guinea pigFc2R Is a type I transmembrane protein and has 72% DNA sequencehomology and 57% protein sequence homology with the human FcRIII.Therefore, we propose that the guinea pig Fc2R Is referred toas guinea pig FcRIII. The most important finding In this reportis that the obtained cDNA directed the cell surface expressionof the Fc2R on COS-7 cells without association with the chainof the high-affinity IgE receptor (FcRly) which is requiredfor human and mouse FcRIII to be expressed on the cell surface.Furthermore, we demonstrated that the endocytosis activity ofFcRIII is dependent upon the association with FcRl, suggestingthat FcRl is Involved in the functions of guinea pig FcRIII.The other clone was found to lack the sequence encoding transmembraneand cytoplasmic domains, suggesting the presence of a solubleform of guinea pig FcRIII. Northern blot analysis and RT-PCRshowed that a transmembrane form of guinea pig FcRIII was expressedin peritoneal macrophages, but not in neutrophils In spite ofthe fact that they express Fc2R, indicating that the Fc2R onneutrophils is a product of a distinct gene.     

{gamma}{delta} T cells promote CD4 and CD8 expression by SCID thymocytes

Molecular studies of the TCR, which is expressed by a minorsubpopulatlon of T lymphocytes in all vertebrate species, havedefined a subset which expresses a receptor with extreme junctionaldiversity and a second subset, most commonly found in eplthella,which expresses a receptor of very limited diversity. In thedeveloping murine thymus, T cells appear in an ordered sequenceof specific v rearrangements, V3V, 1 on day 14, V2V1 on day17, and subsequently V4V5, V6, or V7. We demonstrate that thetransfer of expanded populations of cells from newborn thymusand cell lines expressing the invariant V3V1 receptor into SCIDmice, which lack T and B cells, results in the appearance ofCD3^–CD4⁺CD8⁺ thymocytes. Thus, one role of the early appearingV3V1 T cells in thymlc development in vivo is to promote CD4and CO8 surface expression on precursor cells.     

Granulocyte macrophage colony stimulating factor induces Fc{varepsilon}RII/CD23 expression on normal human polymorphonuclear neutrophils

Three major molecules have been recognized as IgE-binding structureson hematopoletic cells: the heterotrimeric high-affinity receptorfor IgE (FcRI), the low-affinity receptor for IgE (FcRII/CD23)and the Mac-2/IgE-bindlng protein (BP). The latter has beenshown to be expressed on polymorphonuclear neutrophils (PMN),where it regulates IgE-dependent activation. Experiments wereundertaken to determine whether the IgE-binding capacity ofPMN is mediated exclusively by this molecule. No detectablebinding of human myeloma IgE to unstimulated PMN from normalvolunteers could be evidenced. In contrast, PMN stimulated withgranulocyte macrophage colony stimulating factor (GM-CSF) (500U/ml) for 24 h displayed positive IgE binding. This bindingwas significantly inhibited in the presence of mAb directedagainst Mac-2/BP and also in the presence of anti-CD23 mAb,but not of anti-FcRI mAb or isotype-matched controls. By flowcytometry, CD23 expression was detected on GM-CSF-primed PMNby several anti-CD23 mAb, including EBVCS-5, BB10 or Mab135,which recognize different epitopes. CD23 was also evidencedby immuno cytochemistry in GM-CSF-primed PMN. By in situ hybridization,GM-CSF-treated PMN exhibited a hybridization signal for CD23mRNA and the presence of the CD23b isoform-specific mRNA wasdetected by RT-PCR. These findings Indicate that PMN can synthesizeCD23 molecules under GM-CSF induction. This strong CD23 expressionmight be of physiopathological relevance in IgE-dependent activationduring allergic processes.     

Expression and function of fibronectin binding integrins on rat mast cells

Adhesion molecules of the integrin family are implicated notonly in leukocyte migration but also in leukocyte activation.Here we characterize the expression and function of fibronectinreceptor integrins on rat mast cells. A rat basophilic leukemiacell line (RBL-2H3) and phorbol esterstimulated rat peritonealmast cells adhered to fibronectin (FN), vitronectin and fibrinogen.These mast cells expressed fibronectin receptor integrins, Includingvery late antigen (VLA)-4, VLA-5 and vitronectin receptor (VNR),as estimated by immunofluorescent staining and inhibition ofFN adherence by newly established mAbs reactive with the rat4 (MR4-1), 5 (HM5-1) or ß3 (HMß3-1) chainsof the integrin molecules. The ß-hexosaminidase release,a marker for mast cell degranulation, triggered by high affinityIgE receptor (FcRI)-medlated stimulation, was enhanced by adhesionof RBL-2H3 cells to either immobilized FN, MR4-1, HM5-1 or HMß3-1.This FN enhancement of ß-hexosaminidase release wasinhibited by soluble MR4-1, HM5-1 and HMß3-1 as wellas by GRGDSP and DELPQLVTLPHPNHLGPEILDVPST peptides which abrogateVLA-5/VNR and VLA-4 binding to FN respectively. In vivo, passivecutaneous anaphylaxis induced by IgE anti-DNP and DNP-BSA wasinhibited by concurrent s.c. injection of MR4-1, HM5-1 and HMß3-1.These results demonstrate that FN receptor integrins expressedon rat mast cells play an important role in regulating mastcell activation both in vitro and in vivo.     

{alpha}{beta} and {gamma}{delta} T cells in the immune response to the erythrocytic stages of malaria in mice

Mice lacking T cells with ß TCR (TCR ß–/–)or TCR (TCR –/–) were infected with the erythrocyticstages of the malaria parasite, Plasmodium chabaudi chabaudi(AS). Mice without T cells could control and reduce a primaryinfection of P. chabaudi with a slight delay in the time ofclearance of the acute phase of infection and significantlyhigher recrudescent parasitaemias compared with control intactmice. TCR –/– mice had higher levels of both serumIg and malaria-specific antibodies of the isotypes IgG3 andIgG1 compared with control mice. TCRß–/–mice, despite a striking increase in NK1.1⁺ cells and the presenceof T cells, were unable to clear their infection. Althoughthe plasma of TCR ß–/– mice containedall Ig isotypes before and during a primary infection, theywere unable to produce significant levels of malaria-specificIgG antibodies, suggesting that in the absence of ßT cells T cells are not able to provide efficient help forantibody production.     

Differential regulation of antigen-specific IgG4 and IgE antibodies in response to recombinant filarial proteins

Having identified two recombinant filarial proteins (Ov27 andOvD5B) that induced patient peripheral blood mononuclear cellsto produce antigen-specific lgG4/lgE antibodies in vitro, weassessed the role these filarial antigens play in inducing antigen-specificisotype switching (4 and iv) in the absence of T cells. PurifiedCD19⁺ s^–/s^– B cells were cultured with either ofthese antigens in the presence of anti-CD40 mAb and human IL-4.Both antigen and polyclonal signals delivered by IL-4 (or IL-13)were necessary for the induction of specific lgG4/lgE antibodies.To assess the role played by cytokines produced by B lymphocytesin antigen-driven selection of the 4 or isotype, neutralizinganti-cytokine antibodies were used in vitro. While anti-IL-12antibodies did not alter the antigen-specific lgG4/lgE production,anti-IL-6, anti-IL-13 and anti-tumor necrosis factor- antibodiessignificantly inhibited the production of lgG4/lgE. Anti-IL-2and anti-IL-10 antibodies appeared to down-regulate antigen-specificlgG4 antibodies without affecting antigen-specific IgE antibodies.Although anti-CD21 antibodies had no effect on specific IgEantibodies, they up-regulated specific lgG4 antibodies, a findingparalleled by anti-CD23 antibodies. These data suggest thatcertain filarial antigen-specific lgG4/lgE responses can bedifferentially regulated and that certain endogenously producedmolecules from B cells—such as IL-2, IL-10, CD23 and CD21–playa significant role in the induction of specific isotypes ofantigen-specific antibodies.