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.     

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.     

Maturation of medullary thymic epithelium requires thymocytes expressing fully assembled CD3-TCR complexes

Unlike meduilary thymic epithelial cells (TEC) of normal mice,meduilary TEC of TCR^– SCID mice are immature and disorganized.In order to assess directly the role of TCR⁺ cells in the developmentof medullary TEC, we bred mice which co-expressed the SCID geneticdefect and transgenes encoding clonotypic TCR chains. Immunohistologicexamination revealed that meduilary thymic epithelial cellsfrom TCRß transgenic SCID mice, whose thymocytes onlyexpress TCRß chains that inefficiently associate withCD3 and , components, remained immature and disorganized. Incontrast, meduilary TEC from TCRß transgenic SCIDmice, whose thymocytes express fully assembled CD3--TCRßcomplexes were mature and organized. Interestingly, the abilityof TCRß⁺-⁺-CD3³ thymocytes to induce maturation ofmeduilary TEC appeared not to be related to the antigen specificityof the TCR as thyml from positively selecting, negatively selectingand non-selecting TCRß transgenic SCID mice all possessedinduced meduilary thymic epithelial cells. In addition, we foundthat induction of meduilary TEC cells was associated with thepresence of meduilary thymocytes, including those of the CD4-CD8-TCRß⁺phenotype. The present findings demonstrate that fully assembledCD3--TCR complexes are required to induce maturation of meduilarythymic epithelial cells and indicate that thymocyte inductionof meduilary thymic epithelial cells may result from signalingindependently of their clonotyplc chains.     

Predominant expression of invariant V{alpha}14+ TCR {alpha} chain in NK1.1+ T cell populations

A novel T cell subset characterized by cell surface NK1.1⁺ TCRß⁺expression was investigated for its TCR usage, particularlythat of invariant V14 TCR, which was found to be preferentiallyused in peripheral CD4^–CD8^–T cells developed atextrathymic sites. We found that NK₊ ß T cell subsetsaccount for 0.4% in thymocytes, 5% in the splenic T cells and40.5% in the bone marrow T cells. Among these NK⁺ ßT cells, two distinct subsets were detected; cell surface TCRV14⁺and V14^– subpopulations. Almost all of NK⁺ ßthymocytes express V14 mRNA; however, only<20% were positive,while >80% were negative or undetectable for V14 TCR expressionon the cell surface in the thymus. Similarly,50% of NK⁺ ßT cells in spleen and bone marrow are V14⁺; as revealed by FACS.TCR repertoire analysis by nucleotide sequences on inverse PCRproducts demonstrated that most NK⁺ ß T cells expressan invariant TCR encoded by the V14J281 gene with a 1 base N-regionin all tissues. Thus, invariant V14 TCR is uniquely expressedon NK T cells, and can be a marker to distinguish NK, NK T andT cells.     

IL-4 producing CD4+ TCR{alpha} {beta}int liver lymphocytes: influence of thymus, {beta}2-microglobulin and NK1.1 expression

The present report describes developmental, phenotypic and functionalfeatures of unconventional CD4⁺ TCRß lymphocytes.In C57BL/6 mice, the majority of liver lymphocytes expressingintermediate intensity of TCRß (TCRß^int)are CD4⁺NK1.1⁺ and express a highly restricted TCR V_ßrepertoire, dominated by V_ß8 with some contributionby V^ß7 and V^ß2. Although these cells expressthe CD4 co-receptor, they are present in H2-l Aß (Aß)^+/–gene disruption mutants but are markedly reduced in ß₂-microglobulin(ß₂m)^–/– mutant mice and hence are ß₂mdependent. Thymocytes expressing the CD4⁺NK1.1⁺ TCR ßphenotype are also (ß₂m) contingent, suggesting thatthese two T lymphocyte populations are related. The CD4⁺NK1.1⁺TCRßlymphocytes in liver and thymus share several markers such asLFA-1⁺, CD44⁺, CD5⁺, LECAM-1^– and IL-2Rßa. TheCD4⁺NK1.1 ⁺ TCRß^int liver lymphocytes were not detectedin athymic nuinu mice. We conclude that ß₂m expressionis crucial for development of the CD4⁺NK1.1⁺ TCRß^intliver lymphocytes and that thymus plays a major role. CD4⁺ TCRß^intliver lymphocytes were also identified in NK1.1⁺ mouse strains,there lacking the NK1.1 marker. We assume that the NK1.1 moleculeis a characteristic marker of the CD4⁺TCR"^int liver lymphocytesin NK1.1⁺ mouse strains, although its expression is not obligatoryfor their development. The liver lymphocytes from +₂m^+/–,but not from +₂m^–/–mice are potent IL-4 producersin response to CD3 or TCRß engagement and the IL-4production by liver lymphocytes was markedly reduced by treatmentwith anti-NK1.1 mAb. We conclude that the CD4+NK1.1⁺ TCRß^intliver lymphocytes are capable of producing IL-4 in responseto TCR stimulation.     

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.     

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.     

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.     

T cell receptor-triggered activation of intraepithelial lymphocytes in vitro

Intraepithelial lymphocytes (IEL) of the mouse small intestinewere examined for their potential to respond to TCR signallingin vitro. Purified IEL subsets were activated using mAbs specificfor CD3, TCRßor TCR&. Thy-1⁺IEL, regardless ofTCR type, proliferated equally well in response to anti-TCRmAb with or without exogenous IL-2. In contrast, Thy-1^–TCR, CD8^– IEL required exogenous IL-2 for proliferation.No such requirement was observed for Thy-1^– TCR& IELproliferation. IEL proliferation in the absence of added IL-2was due to an IL-2 secretion/IL-2 receptor (IL-2R) autocrinepathway, since mAbs specific for IL-2 and IL-2R inhibited IELproliferation. Thy-1⁺ CD8ß^– CD4⁺CD8⁺ IEL wereunresponsive to TCR-induced proliferation but exhibited highlevels of cytolytic activity upon TCR-triggerlng. Thy-1^–non-cytolytic IEL were induced to express Thy-1 and cytolytlcactivity following activation in vitro. In addition, the involvementof the co-stimulatory molecule CD28 in IEL activation was tested.CD28 was weakly expressed by fresh IEL and anti-CD28 mAb hadno effect on TCR-triggered proliferation. However, anti-TCRstimulation increased CD28 expression on a subset of TCRßIEL and the addition of anti-CD28 mAb resulted in increasedIL-2 production, but not in increased proliferation. Our resultsindicate that IEL, including the purported extrathymlc CD8ß^–subset, can respond to TCR-driven signals via proliferationand/or cytolytlc activity.     

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.     

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.     

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.     

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.     

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.     

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.     

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.