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.     

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.     

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.     

IFN-{gamma} expression in CD8+ T cells regulated by IL-6 signal is involved in superantigen-mediated CD4+ T cell death

Infection with pathogens containing superantigens (Sags) canresult in massive excessive CD4⁺ T cell activation and deathin such conditions as toxic shock, food poisoning and autoimmunediseases. We here showed how enhancement of IL-6 signaling suppressesSag-mediated activated CD4⁺ T cell death. Sag-induced CD4⁺ Tcell death increased in IL-6 knockout (KO) mice, whereas itdecreased in mice characterized by enhanced IL-6–gp130–STAT3signaling. The serum concentration of IFN- was inversely correlatedwith the magnitude of IL-6 signaling, and IFN- deficiency inhibitedSag-induced activated CD4⁺ T cell death, suggesting that IL-6suppresses CD4⁺ T cell death via IFN- expression. Interestingly,depletion of activated CD8⁺ T cells inhibited Sag-mediated increasesin IFN- expression in IL-6 KO mice as well as the augmentedCD4⁺ T cell death. The results demonstrate that IL-6–gp130–STAT3signaling in activated CD8⁺ T cells contributes to Sag-inducedCD4⁺ T cell death via IFN- expression, highlighting this signalingaxis in CD8⁺ T cells as a potential therapeutic target for Sag-relatedsyndromes.     

Implication of the common {gamma} chain of the IL-7 receptor in intrathymic development of pro-T cells

The effects of IL-7 on the growth and differentiation of thymocyteswere analyzed using murine fetal thymua organ cultures (FTOC)in the presence of mAbs specific for the conventional IL-7 receptor(1L-7R) and for the common (c) chain. In FTOC, the developmentof CD4^–CD8^– double-negative thymocytes to CD4+CD8+double-positive (DP) and CD4+ or CD8+ single-positive (SP) cellswas not completely blocked by adding these mAbs, although cellgrowth was reduced by the treatment. To define a developingstage sensitive to the mAbs, most immature thymocytes, Pgp-1⁺c-kit cells, were cultured in the 2-deoxyguartosine treatedfetal thymus. In the presence of both mAbs in the culture, neitherDP nor SP thymocytes developed whereas either of the mAbs partiallyblocked their development. These results indicate that the Cchain is involved in early T cell development as an indispensablesubunlt of the functional IL-7 receptor complex.     

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.     

CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of {alpha}{beta} TCR+CD4 CD8 thymocytes

We have examined CD38 expression on mouse lymphocytes usingthe rat mAb NIM-R5 and demonstrate that CD38 expression is restrictedto {small tilde}8% of thymocytes. Although CD38 is absent fromthe majority of CD4+ CD8^– and CD4^–CD8⁺ T cells,we detected a strong correlation between CD36 expression andß⁺CD4^–CD8^– T cells in the thymus, withnearly 80% of ß TCR⁺CD4^–CD8^– thymocytesbeing CD38⁺. Using heat stable antigen (HSA) and CD38, we dividedß⁺CD4⁺CD8^– thymocytes into four subsets: HSA⁺CD38^–,HSA^– CD38^hi, HSA^–CD3810^low and HSA^– CD38^–.Two established characteristics of ß TCR⁺CD4CD8^–cells, bias towards Vß 8.2 TCR expression and highlevels of IL-4 production, were used to establish a possiblerelationship between the above thymocyte subsets. Our presentdata show that the HSA⁺CD38^– subset is not biased towardsVß8.2 TCR expression whereas the HSA^– CD38^–subset does show this bias (–47%). Neither of these subsetsmake IL-4 upon CD3 mediated stimulation. In contrast, the CD38⁺subsets are heavily biased toward Vß8.2 expressionand produce large amounts of IL-4 upon stimulation, particularlythe CD38^low cells. Taken together, these data suggest that thesefour subsets represent various stages of a possible differentiationpathway for ß TCR⁺ CD4^–CD8^– cells, withthe HSA⁺CD38^– subset being the most Immature while theHSA^–CD38^low subset is the most functionally mature. Thesecharacteristics support the view that ap TCR⁺CD4^–CD8^–T cells represent an independent lineage with a distinct, butas yet obscure, role in immunity     

Developmentally regulated expression of the PD-1 protein on the surface of double-negative(CD4 CD8 ) thymocytes

PD-1, a member of the Ig superfamily, was previously isolatedfrom an apoptosis-induced T cell hybridoma 2B4.11 by subtractivehybridization. Expression of the PD-1 mRNA is restricted tothymus in adult mice. Using an anti-PD-1 mAb (J43), we examinedexpression of the PD-1 protein during differentiation of thymocytesin normal adult, fetal and RAG-2^-/- mice with or without anti-CD3mAb stimulation. While PD-1 was expressed only on 3–5%of total normal thymocytes, –34% of the CD4^-CD8^- double-negative(DN) fraction are PD-1⁺ cells with two distinct expression levels(low and high). PD-1^high thymocytes belonged to TCR lineagecells. In the DN compartment of the TCR ß lineage,PD-1 expression started at the low level from the CD44⁺CD25⁺stage and the majority of thymocytes expressed PD-1 at the CD44^-CD25^-stage in which thymocytes express TCR ß chains. Theanti-CD3 antibody administration augmented the PD-1 expressionas well as the differentiation of the CD44^-CD25⁺ DN cells intothe CD44^-CD25^- DN stage, not only in normal mice but also inRAG-2-deficient mice. The fraction of the PD-1^low cells in theCD4⁺CD8⁺ double-positive (DP) compartment was very small (>5%)but increased by stimulation with the anti-CD3 antibody, althoughthe total number of DP cells was drastically reduced. The resultsshow that PD-1 expression is specifically induced at the stagespreceding clonal selection.     

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.     

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.     

Preferential requirement of CD3{zeta}-mediated signals for development of immature rather than mature thymocytes

Antigen recognition signals by the TCR are transduced throughactivation motifs present in the cytoplasmic region of CD3 chains.In vitro analysis has suggested that the CD3 chain mediatesdifferent signals from other CD3 chains. To analyze the in vivofunction of CD3-mediated signals for T cell development, miceexpressing a mutant CD3 chain lacking all the activation motifswere generated by introducing the transgene into -knockout mice.Mature CD4⁺ single-positive (SP) thymocytes in these mice weregreater in number than in -deficient mice, and the promoteddifferentiation was indicated by the changes of CD69 and HSAphenotypes. We found that even in the absence of activationmotifs in CD3, these mature cells became functional, being ableto induce Ca²⁺ mobilization and proliferation upon stimulation.On the other hand, CD4^-CD8^- double-negative (DN) thymocytes,most of which were arrested at the CD44^-CD25⁺ stage similarlyto those in -deficient mice, could not be promoted for differentiationinto CD4⁺CD8⁺ double-positive thymocytes in these mice in spiteof the fact that the expression of the transgene in DN thymocyteswas higher than that of in wild-type mice. These results demonstratethe preferential dependence of the promotion of developmentand/or expansion of DN thymocytes rather than mature thymocytesupon the activation signals through the chain and suggest differentialrequirements of TCR signaling for mature SP and immature DNthymocyte developments in vivo.     

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.     

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.     

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.     

Cytokine production by CD16 CD56bright natural killer cells in the human early pregnancy decidua

Using a cell sorter, CD16^–CD56^bright natural killer (NK)cells were sorted from decidual mononuclear cells at an earlystage of pregnancy. These cells were examined by the reversetranscrlptase-polymerase chain reaction (RT-PCR) method fortheir expression of mRNA coding for the following 12 cytokines:IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), macrophage colonystimulating factor (M-CSF), tumornecrosis factor- (TNF-), interferond- (IFN-), and leukemia inhibitoryfactor (LIF). Although mRNA coding for every cytokine was detectedin decidual mononuclear cells, mRNAs coding for only G-CSF,GM-CSF, M-CSF, TNF-, IFN-, and LIF were detected In CD16^–CD56^brightNK cells. Also, the supernatant of CD16^–CD56^bright NKcell cultures was found to contain G-CSF, GM-CSF, M-CSF, TNF-,IFN-, and LIF. These findings indicate that CD16^–CD56^brightNK cells produce many different cytokines and that these cytokinesmay play an important role in a successful pregnancy.     

Entry of CD4 CD8 immature thymocytes into the CD4/CD8 developmental pathway is controlled by tyrosine kinase signals that can be provided through TCR components

Entry of thymus-migrated precursor cells into the CD4/CD8 developmentalpathway was analyzed by using the short-term organ culturesof day 14 fetal mouse thymus lobes. Organ cultures of CD4^–CD8^–day 14 fetal thymocytes for 1-2 days resulted in the generationof CD4^–CD8⁺ cells, which were mostly immediate precursorcells for CD4⁺CD8⁺ thymocytes. This differentiation of CD4^–CD8^–thymocytes into CD4^–CD8⁺ cells was strongly enhanced byanti-CD3 antibodies. The anti-CD3-induced generation of CD4^–CD8⁺cells was even found in the immunodeficient scid fetal thymuscultures, and the cell surface CD3 expression on the scid fetalthymocytes could be directly visualized, indicating that functionalCD3 could be expressed on CD4^–CD8^– immature thymocyteswithout being associated with rearranged TCR components. Theanti-CD3-lnduced generation of CD4^–CD8⁺ cells from scidand normal fetal thymus cultures was inhibited by tyrosine kinaseinhibitors Herblmycin A and Tyrphostin. The generation of CD4^–CD8⁺cells in unstimulated normal fetal thymus cultures was alsomarkedly inhibited by the tyrosine kinase inhibitors but notby Cyclosporin A, suggesting that tyrosine klnase-dependentbut calclneurin-lndependent signals were essential for the differentiationof CD4^–CD8^– thymocytes. Interestingly, the generationof CD4^–CD8⁺ cells from the normal fetal thymus cultureswas modestly but consistently enhanced by anti-TCRßantibody, suggesting that functional TCRß in additionto CD3 was expressed on normal CD4^–CDS⁺ immature thymocytes.On the other hand, anti-TCR antibody did not affect this differentiationin the normal fetal thymus cultures and the generation of CD4^–CD8⁺cells from the normal fetal thymus cultures of TCR-deficientmice was still enhanced by anti-TCRß or anti-CD3 antibodies,indicating that either TCR chains or TCR⁺ cells were not involvedin the control of the differentiation into CD4^–CD8⁺ cells.These results indicate that the entry of CD4^–CD8^–immature thymocytes into the CD4/CD8 developmental pathway iscontrolled by tyrosine kinase signals and that these signalscan be provided through the engagement of TCR-CD3 complexeswith or without TCRß chains expressed on the CD4^–CD8^–immature thymocytes.     

{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.     

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.