{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.     

Human {alpha}{beta} and {gamma}{delta} T cells from unexposed individuals respond to protein antigens of the yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, a dimorphic fungus, causes chronicgranuiomatous mycosis in susceptible individuals. Differentreports have shown that cell-mediated immunity is essentialfor protection against systemic mycosis, including paracoccldloidomycosis.We analyzed the reactivity of ß and T cells fromunexposed Caucasian donors to P. brasiliensis yeast form components.Our results indicate: (I) ß and T cells proliferateafter in vitro stimulation with lysates of P. brasiliensis;(II) similar numbers of ß T cells (f = 1/21,000) andof T cells (f = 1/8000) respond to P. braslllensls; (III) P.braslllensls-reactive T cells express the V9V2 TCR; (Iv) thestimulatory activity of P. brasilensis for both ßand T cells primarily resides in a high molecular weight (100kDa) and in a low molecular weight (<<1 kDa) fraction;(v) the ligands responsible for stimulation of both ßand T cells are sensitive to proteinase treatment We concludethat both ß and T cells from healthy individualsrespond to ubiquitous protein antigens of P. brasiliensis.     

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.     

{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.     

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.     

Peripheral lymphoid development and function in TCR mutant mice

We describe the development and function of the peripheral lymphoidsystem of mutant mice rendered deficient in either ßor T cells via targeting of TCR genes In embryonic stem cells.In the spleen of ß T cell-deficient mice, T cellsdo not compensate in numbers for the lack of ß Tcells, but B cells do. ß T cell-deficient mice areunable to mount an antibody response to ovalbumln and do notreject skin allografts. Natural killer cell function is notimpaired in any of the mutant mice. TCR mutant mice will proveuseful in dissecting differential functions of ßand T cells in vivo.     

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.     

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.     

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.     

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.     

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.     

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.     

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.