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.     

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V{alpha}1 gene

Previous studies of the TCR chain gene have located promoterelements 5' to the start of the various V genes. The only fullycharacterized enhancer for the entire chain gene (V, J andC genes) has been located {small tilde}3 kb from the 3' endof C. We now report the existence of additional regulatory elementslocated in the introns of several murine V genes (V1, V3 andVB6.2.16). In the case of V1, this element appears to be a promoterwith bidirectional activity that is not T cell specific. Interestingly,upstream of the promoter in the antisense strand, an open readingframe has been found that codes for a small molecular weightprotein ({small tilde}60 amino acids) that contains a prollne-richregion and a tyrosine-isoleucine motif that has homology toIgß (the B29 gene product). A rabbit antiserum madeagainst this sequence has confirmed its existence by Westernblot and immunoprecipitation. Thus this V1 intronic promoterhas the potential not only to induce the formation of a truncatedV1 gene product, but also regulates the expression of a smallmolecular weight protein that may be involved in lymphocyteantigen receptor signaling. The activity of this promoter isregulated by changes in intracellular calcium. In the presenceof ionomycin the promoter is down-regulated in the sense directionand its activity is enhanced in the antisense direction. Thisresult suggests that this promoter can act differentially toproduce two very different gene products. The bidirectionalV1 promoter appears to be the first in the Ig superfamily toinduce potentially functional proteins in both directions.     

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.     

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.     

Thymic stroma is required for the development of human T cell lineages in vitro

Development of the T cell lineage is characterized by the homingof hematopoietic precursors to thymus, followed by their acquisitionof receptors for antigen. T cell receptors are ß or heterodimers associated with CD3 (TCR-CD3). Very early T cellprecursors in humans have been characterized as CD7+45+ cellswhich lack the T cell differentiation antigens CD1, CD2, CD3,CD4, and CD8. A phenotypically equivalent early thymocyte populationalso occurs in postnatal life, and we have previously shownthat interleukin 2 (IL2) promotes the development in vitro ofboth the ß and the T cells from these early thymocytes.Here we have analyzed the requirements of the induction of theIL2 pathway in early thymocytes, and their developmental potential.We show that: (I) thymic stromal cells, which are present inthymocyte suspensions, are necessary to induce the IL2 pathwayand the development of ß or T cell lineages fromearly thymocytes in vitro; and (II) when removed from the invivo environment, early thymocytes can develop in vitro intoTCR-CD3^– cells of the natural killer (NK) lineage. Weconclude that CD7+45+, CD1–2–3–4–8–early thymocytes are multipotential progenitors that, at least,have the capacity to develop into ß or T cell andNK lineages. The analysis of the mechanisms of generation andselection of human T and NK cell diversity, not feasible inbone marrow cultures, is now possible.     

Phenotypic and functional properties of murine {gamma}{delta} T cell clones derived from malaria immunized, {alpha}{beta} T cell-deficient mice

Six murine T cell clones expressing TCR were generated frommalaria immunized, ß T celldeficient mice. Phenotypiccharacterization of these clones has revealed that, in contrastto conventional ß T cells, there is a considerabledegree of heterogeneity among these clones with regard to theirsurface markers and their lymphokine profile. One clone wasfound to display significant anti-parasite activity in vivoupon adoptive transfer. We attempted to determine whether theprotective clone differs in one or more key characteristicsfrom the non-protective clones. Although no obvious patternpeculiar to the protective clone was observed, it appears thatmore than one parameter may, in combination, define a distinctprotective phenotype, and thus explain the functional differencebetween the protective and non-protective clones.     

The development of dermatitis infiltrated by {gamma}{delta} T cells in IL-7 transgenic mice

Transgenic mice constitutively expressing IL-7 developed severedermatitis with erythroderma and alopecia. The skin lesionswere characterized by massive infiltration of mononuclear cells.Immunofluorescence staining showed that most of the infiltratingcells were T cells with the majority bearing the TCR otherthan the V5 moiety. Furthermore, the number of T cells hadincreased in the lymphold organs of the dermatitis animals.These findings idicate the strong relationship between the expressionof IL-7 and the development of T cells in vivo and the pathologicalinvolvement of proliferated and/or activated T cells in skindisease.     

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.     

Involvement of {alpha}1 and {alpha}4 integrins in gut mucosal injury of graft-versus-host disease

We have investigated the involvement of adhesion molecules inthe lymphocyte infiltration associated with acute intestinalgraft-versus-host disease (GVHD) induced by injection of C3Hlymph node cells into irradiated (C3H x DBA/2)F₁ mice. Firstwe analyzed the expression profile of adhesion molecules including1, 2, 4, 5, 6, L and ß7 integrins, CD44 and L-selectinof lymphocytes from lymph nodes and gut mucosa in normal mice.In normal mice, intraepithelial lymphocytes (IEL) and laminapropria lymphocytes (LPL) uniquely showed increased expressionof 1, 2 and ß7 integrins, and decreased expressionof L-selectin compared with that of lymphocytes of the lymphnodes and Peyer's patches. In mice with GVHD, IEL and LPL ofdonor lymph node cells origin underwent phenotyplc changes characterizedby the increased expression of 1, L and ß7 integrins,and the loss of L-selectin. The expression profile of adhesionmolecules on IEL and LPL of GVHD mice resembled that of normalmice except for the lack of 2 integrin. Treatment of GVHD micewith anti-1,-4 or-ß7 integrin antibody alone partiallyprevented the mucosal pathology of intestinal GVHD, whereasonly mice treated with anti-1 showed reduced donor lymphocyticinfiltration into the intestinal mucosa. In contrast, treatmentwith anti-L or anti-CD44 antibody did not affect the intestinalGVHD. Furthermore, dual blockade of both 1 and 4 integrins completelyinhibited the mucosal pathology and donor lymphocyte infiltrationof intestinal GVHD. These results indicate that 1 and 4 integrinsplay an important role in the pathology of intestinal GVHD.     

Intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 are involved in protection mediated by CD3+TCR{alpha}{beta} T cells at the early stage after infection with Listeria monocytogenes in rats

To Investigate the significance of Intercellular adhesion molecule-1(ICAM-1) and leukocyte function-associated antlgen-1 (LFA-1)In host defense against infection with Intracellular parasites,we examined the effects of In vivo pretreatment with mAbs toICAM-1 (1A29) and LFA-1 (WT-1) on the protection against Infectionwith Listeria monocytogenesIn Fisher F344/N rats. Expressionof ICAM-1 and LFA-1 molecules on T cells In spleen, liver andperitoneal cavity of rats was down-regulated after i.p. administrationwith daily doses of 300 µg of either 1A29 or WT-1 for10 days. The survival rate of rats inoculated with viable Listeriawas significantly reduced byIn vivo pretreatment with 1A29 togetherwith WT-1 for 10 days but not by In vivo pretreatment with controlmAb. The numbers of bacteria In the spleen In rats pretreatedwith both 1A29 and WT-1 were significantly increased on day3 and day 6 after Infection with 1 x 10⁷ of viable Listeriacorresponding to 1/30 of LD₅₀ to normal rats. Thus, the resistanceagainst llsterial Infection was severely Impaired by combinationalpretreatment with mAbs In ICAM-1 and LFA-1. As shown In ourprevious report, the early appearance of CD3⁺TCRß^–T cells, presumably TCR cells, was evident In the peritonealcavity and liver of control rats at the early stage after llsterialInfection, while this was suppressed at this stage in rats pretreatedwith both 1A29 and WT1. These results suggest that the ICAM-1and LFA-1 adhesion pathway may be critically involved in protectlveroles of CD3⁺TCR_ß– T cells at the early stageof rat listeriosis.     

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc{gamma}R) encoded by an alternatively spliced transcript of the Fc{gamma}RII gene

Low affinity FcR are a heterogeneous group of glycoproteinswhich exist in transmembrane (TM) as well as in soluble forms.Two membrane isoforms of the murine type II FcR, FcRilb1 andFc;Rilb2, have been described. They result from the translationof alternatively spliced premRNA, FcRilb2 lacking sequencesof the first intracytoplasmic domain (IC1). Soluble forms ofFcR (sFcR) have previously been shown to result from proteolysisof membrane receptors. We report here the identification, inmacrophages, of a mRNA derived from the FCRll gene by splicingexons encoding the TM and IC1 domains, i.e. corresponding toa TM-deleted FcRllb2 mRNA. A soluble protein possibly encodedby this mRNA was identified in macrophage supernatants. In accordancewith FcR nomenclature, we propose to name this new FcRll IsoformFcRllb3. It is the most abundant 8FcR present in serum, as comparedwith 8FcR resulting from cleavage of membrane FcR.     

Homing and in situ differentiation of resident pulmonary lymphocytes

At birth, T lymphocytes which colonize the lung are mainly ofthe subset, while ß T cells predominate in the spleen.Thus, the lung is a preferred site for the homing of T cellsin the perinatal period. However, after birth, the pattern ofV gene usage among resident pulmonary lymphocytes (RPL) changeswith age, from a predominance of V6 at birth to a predominanceof V4 in older mice. The generation of the V6 fraction appearsto be thymus dependent, since in athymic nude mice, the V6 populationpresent at birth is replaced by V4 T cells. In the postnatalperiod, both RAG-1 and RAG-2 genes are expressed at high levelsin the RPL population. TCR bearing cells are among those thatexpress RAG genes, indicating that maturation of T cells takesplace in this organ. In addition, transfer experiments revealthat lymphoid precursors are present in the lung. The stageof differentiation of these precursors will be characterizedin future studies. The data presented here indicate that pulmonaryT lymphocytes are derived from both migrants of thymic originand from precursors which have undergone differentiation andselection in the lung. The population that is generated in situand that has not been selected in the thymus may include cellsthat are typical for the pulmonary environment.     

Ligand-induced autoregulation of IL-2 receptor {alpha} chain expression in murine T cell lines

The IL-2 receptor (IL-2R) is composed of three chains a, ßand . In mice, contrary to the human system, we have previouslydemonstrated that the IL-2Rß complex does not bindIL-2. Therefore, mouse IL-2 response is completely dependenton the expression of the IL-2R gene product. T cell clones expressingmouse IL-2Rß and the human IL-2R transgene have beenstudied. When cells are grown in IL-4, mouse IL-2R is not expressed.However, exposure to IL-2 leads to the expression of the endogenousmurine IL-2R subunit. The T cell line expressing mouse IL-2Rand human IL-2Rß can grow in IL-2 but does not expressendogenous murine IL-2 R. Transfection of these cells with thehuman IL-2R gene restores the capacity to induce murine IL-2R.This result demonstrates that IL-2-IL-2R interactions are requiredfor induction of IL-2R. The kinetics of induction and deinductionof murine IL-2R have been studied using clone 18.III. From negativecells, expression of murine IL-2R is a very slow phenomenon.From cells fully expressing IL-2R, deinduction is a two-stepprocess: after a rapid decrease of IL-2R the cells continueto express, for a long period of time, basal levels of murineIL-2R. When cells expressing basal levels of IL-2R are exposedto IL-2, induction of IL-2R is a very rapid phenomenon. Theautoregulatory loop formed by IL-2-IL-2R therefore displaysdifferent levels of functioning.     

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.     

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.