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.     

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.     

Emigration of selected subsets of {gamma}{delta}+ T cells from the adult murine thymus

Cells bearing the form of the TCR make up only 1–3% ofT cells in the adult murine thymus and peripheral lymphold organs.Evidence from studies of nude mice suggests that the developmentof at least some T cells is thymus dependent; however, untilnow it has not been directly demonstrated that cells are exportedfrom the thymus. In this paper we have used the technique oflabelling thymocytes in vivo with FITC, followed by flow cytometrlcanalysis to trace cells emigrating from the thymus to the spleen.Using this approach we have been able to demonstrate for thefirst time that T cells are exported from the adult murinethymus to the spleen. We also demonstrate that the cells emigratingto the spleen are a selected subset of thymocytes being heatstable antigen positive, Thy-1⁺, and expressing low levels ofCD44 (Pgp-1). In addition, investigation of TCR V; gene usageamong adult ⁺ thymocytes, recent emigrants, and spleen cells,indicated a selective emigration of cells expressing certainVgenes.     

{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.     

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.     

Bovine {gamma}{delta} T cells express high levels of functional peripheral lymph node homing receptor (L-selectin)

Lymphocyte migration from the blood into specific tissues Isdirected by their expression of adhesion molecules referredto as homing receptors. The homing receptor L-selectln, forexample, directs the migration of lymphocytes into peripherallymph nodes (PLN). Since bovine T cells, a major lymphocytesubset in peripheral blood (25–50%), represent only aminor subset in PLN, we examined whether these cells lack expressionor function of L-selectin. We found that bovine T cells expressedL-selectln at levels higher (2- to 5-fold) than ßT cells and B cells. Furthermore, T cells accumulated alongthe vascular wall of venules that support lymphocyte extravasationinto PLN (MECA-79⁺ venules) in vivo and bound mouse PLN highendothelial cell venules in an In vivo binding assay. In contrastto this primary adhesive event, we directly demonstrate that T cells in vivo do not appreciably extravasate from the bloodinto the parenchyma of lymph nodes. Since the lack of functionalL-selectln expression could not account for the inability of T cells to enter PLN, we tested for other differences between T cells and PLN homing lymphocytes related to the processesfollowing primary adhesion; for instance, the down-regulationof L-selectin expression following short-term activation andthe expression of accessory adhesion molecules necessary fortransendothellal migration. We found that and ß Tcells demonstrate differential down-regulation of L-selectinafter PMA activation. Kinetic analysis revealed that, at alltime points after PMA treatment, L-selectin expression remainedsignificantly higher on T cells and was down-regulated at aslower rate compared with ß T cells. However, theexpression levels of CD44 and CD18 on and ß T cellswere found to be equivalent. This study Is the first to demonstratefor lymphocytes that the expression of L-selectln alone doesnot predict a PLN homing capacity. Our results suggest thatthe T cells' reduced ability to enter PLN may be due to inefficientdown-regulation of L-selectln compared with non- lymphocytes,thus potentially disrupting the dynamics of the extravasationevent.     

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.     

{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.     

Cytokine dependence of V{gamma}3 thymocytes: mature but not immature V{gamma}3 cells require endogenous IL-2 and IL-7 to survive evidence for cytokine redundancy

It has previously been described that V3 cells can proliferateextensively in vitro in the presence of different cytokines.Here, the role of cytokines in the maintenance of V3 cells inthe thymus has been determined. Culture of fetal thymocytesin cell suspension for 24 h showed that, whereas immature TCR^lowHSA^highV3cells remained viable, all mature TCR^highHSA_lowV cells died.These cells died by apoptosis since protein synthesis was requiredand flow cytometric analysis as well as DNA gel electrophoresisshowed that the DNA was degraded to oligonucleosomal bands.Addition of IL-2, IL-4 or IL-7 to suspension cultures of fetalthymocytes rescued V3 cells from dying. Addition of IL-1, IL-3,IL-5, IL-6, IL-9, TNE- or IFN- was without effect. Phenotypicanalysis showed that the -chain of the IL-2 receptor (IL-2R)was expressed by part of the immature V3 thymocytes, all matureV3 cells expressed the p-chain of the IL-2 receptor (IL-2RP).Addition of anti-IL-2R mAb to fetal thymic organ culture (FTOC)resulted in a moderate reduction of the cell number of matureV3 thymocytes. Addition of anti-IL-2Ra, anti-IL-4 or anti-IL-7mAb had no effect. The cell number of mature V3 cells was highlyreduced when both anti-IL-2Rp and anti-IL-7 mAb were added toFTOC. These results show that IL-2 and IL-7 are actively involvedin the maintenance of mature V3 cells in the thymus. This cytokinedependence of mature Vthymocytes may explain their selectivelocalization in skin epithelium.     

Phenotypic and functional assessment of intraepithelial lymphocytes bearing a 'forbidden' {alpha}{beta} TCR

Differences in the surface antigen phenotype, such as the expressionCD8 as an homodimer or the lack of Thy-1, on Intestinal Intraepitheliallymphocytes (IEL) are related, In part, to alternative differentiationpathways. The relationship of IEL lacking the pan-T cell markerCD5 to these IEL, their TCR repertoire and function has notbeen examined directly. We explored the TCR repertoire and functionof the CD5^– IEL subset In relation to the expression ofthe ‘autospecific’ V_ß6 TCR in Mls-1^a miceand to TCR. The results indicate that CD5 expression was absenton the majority of TCR IEL (96.9%) and on a significant proportionof TCR ß IEL (25.0%). Virtually all IEL In DBA/2 (Mls-1^a)mice that expressed the ‘autospecific’ V_ß6TCR were CD5^–, and this correlated with the expressionof CD8 . To assess the functional capacity of this subset ofIEL, we examined proliferation and IL-2 production in responseto TCR activation. Although CD5^– IEL proliferated in responseto anti-CD3, IEL bearing TCR V_ß6, In Mls-1^a mice,were not responsive to TCR-mediated activation. Similarly, TCR IEL were not responsive to stimulation by anti-TCR antibodies.The addition of exogenous IL-2, however, reconstituted the prollferativeresponse of both TCR IEL and the TCR V_ß6 expressingIEL. We conclude that the lack of CD5 defines a unique subsetof intraepithelial T cells expressing either TCR or ßthat Include potentially autoreactive cells that remain anergicin the absence of IL-2.     

An IFN-{gamma}-dependent pathway plays a critical role in the pathogenesis of murine immunodeficiency syndrome induced by LP-BM5 murine leukemia virus

The murine acquired immunodeficiency syndrome (MAIDS) causedby a defective murine leukemia virus produces severe immunodeficiencywith abnormal lymphoproliferation and hypergammaglobulinemia.The presence of both CD4⁺ T cells and B cells is critical forthe development of this disease. Remarkably elevated mRNA expressionfor IFN- and IL-10 was observed in spleen cells of C67BU6 micestarting from the early phase of viral infection. IFN- productionwas induced by spleen cells from virus-infected mice upon stimulationwith concanavalln A or lipopolysaccharide in both the earlyand late phases of MAIDS progression. When mice that had beenpassively administered anti-IFN- mAb were infected with thevirus, the development and progression of lymphadenopathy, immunodeficiencyand elevated levels of serum lgG2a associated with MAIDS weredelayed. Treatment with anti-IL-4 or anti-IL-10 mAb in placeof anti-IFN- mAb did not induce the delayed progression of MAIDS.These data support the concept that IFN--dependent pathway maybe involved in the development of MAIDS.     

TCR-mediated hyper-responsiveness of autoimmune Galphai2(-/-) mice is an intrinsic naïve CD4(+) T cell disorder selective for the Galphai2 subunit

Heterotrimeric Gi signaling regulates immune homeostasis, sinceautoimmunity occurs upon disruption of this pathway. However,the role of the lymphocyte-expressed Gi subunits (Gi2 and 3)on T cell activation and cytokine production is poorly understood.To examine this role, we studied T lymphocytes from mice deficientin the Gi2 or Gi3 subunits. Gi2^-/- but not Gi3^-/- splenocyteswere hyper-responsive for IFN- and IL-4 production followingactivation through the TCR. Gi2^-/- T cells had a relaxed costimulatoryrequirement for IL-2 secretion and proliferation compared towild-type cells. Purified naïve Gi2^-/- T cells producedmore IL-2 than naïve wild-type T cells following TCR activation,indicating that the hyper-responsive cytokine profile was notdue to the expanded Gi2^-/- memory T cells, but involved an intrinsicT cell alteration. Cytokine hyper-responsiveness was not seenwhen purified Gi2^-/- T cells were stimulated with phorbol myristicacetate/ionomycin, localizing the alteration to a proximal TCR-specificsignaling pathway. Gi2^-/- CD4⁺ T cells were distinguished fromwild-type or Gi3^-/- T cells by a globally augmented TCR-inducedcalcium response. These findings indicate that Gi2^-/- mice havean intrinsic CD4⁺ T cell abnormality in TCR signaling whichmay be one cause of augmented T cell effector function and Gi2^-/-autoimmune susceptibility.     

Re-evaluation of the probabilities for productive rearrangements on the {kappa} and{lambda}loci

V-J rearrangements at Ig light chain (IgL) genes occur in restingsmall pre-B cells. In the absence of cell division, the probabilityof productive and rearrangements is proportional to the outputof ⁺ B and ⁺ B cells in bone marrow. The kinetics and probabilityof productive or rearrangements was assessed in three groupsof mice carrying two (wild-type), one or no intact Ig gene,and the following conclusion are drawn, and rearrangementsoccur independently at different kinetics, and rearrangementsare initiated at a time when rearrangements are stopping. Theprobability of productive and rearrangements per chromosomeis calculated to be –60 and –20% respectively. Thus,a gene can attempt rearrangements up to three times per chromosomeduring B cell development. These findings explain that the observedratio of ⁺ B/⁺ B cell production in wild-type mice is 95/5.     

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.     

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.     

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.     

CD2 expression on murine intestinal intraepithelial lymphocytes is bimodal and defines proliferative capacity

The CD2 molecule is normally expressed on nearly all murinelymphocytes, and is co-stimulatory in T cell activation viathe antigen receptor (TCR). A naturally occurring T lymphocytepopulation that is bimodal for CD2 expression was found in theintestinal intraepithelial lymphocytes (IEL). TCRß⁺IEL contain CD2^– and CD2⁺ cells of approximately equalproportion, while TCR⁺ IEL are predominantly CD2^–. Theproliferative response of IEL to stimulation with an anti-CD3mAb or with PMA plus ionomycin co-segregated with CD2 expression;the CD2⁺ subset proliferated vigorously under these conditionswhile the CD2^– subset was much less responsive. The respondingCD2⁺ IEL contained both TCRß⁺ and TCR⁺ cells. However,activation of the CD2^– IEL with anti-CD3 mAb resultedin only the expansion of TCR⁺ IEL, while activation with PMAplus ionomycin did not promote expansion of either the TCRß⁺or the TCR⁺ IEL. These findings parallel observations in theautoimmune lpr mouse, where massive numbers of peripheral TCRß⁺CD4^–CD8^–T cells that lack CD2 expression are also hyporesponsive tomltogenic stimulation. The apparent energy of CD2^–TCRß⁺IEL, as well as CD2^– T cells from lpr mice, demonstratesthat the absence of CD2 on TCRß⁺ T lymphocytes co-segregateswith nonresponsiveness.