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.     

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 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.     

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.     

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.     

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.     

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.     

TCR repertoire in early fetal mouse thymus

We investigated the rearrangement and expression of TCR genesin mouse fetal thymus organ culture, a system that avoids subsequententry of hematopoietic precursor cells. The first observablerearranged TCR gene was homogeneous V2-J2, detectable as earlyas fetal day 11 (d11) in the thymic primordla. The productiveTCR was homogeneous V5-J1, first detectable in d13 thymocytes,followed by adult-type TCR (V4 and V7). Sequence analysis ofTCR revealed five types of V-J junctional sequences. In thevery early stage, a homogeneous V-J junction is generated viaa short homology sequence in the coding region (Type I), whilea short homology sequence in the P-nucleotlde rather than thecoding region is used in the following stage (Type II). In thelater embryonic stages, diverse V-J junctions are generatedby well-known mechanisms, such as P-nucleotide (Type III), N-regioninsertion (Type IV) or trimming of the coding ends (Type V).These findings suggest that the generation of homogeneous TCR (V2 and V5) in the early fetal stages is due to the intrinsicrearrangement mechanisms and is in stage specific manner.     

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.     

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.     

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.     

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.     

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.