Positive selection as a developmental progression initiated by alpha beta TCR signals that fix TCR specificity prior to lineage commitment

During positive selection, immature thymocytes commit to either the CD4+ or CD8+ T cell lineage ("commitment") and convert from short-lived thymocytes into long-lived T cells ("rescue"). By formal precursor-progeny analysis, we now identify what is likely to be the initial positive selection step signaled by alpha beta TCR, which we have termed "induction". During induction, RAG mRNA expression is downregulated, but lineage commitment does not occur. Rather, lineage commitment (which depends upon the MHC class specificity of the alpha beta TCR) only occurs after downregulation of RAG expression and the consequent fixation of alpha beta TCR specificity. We propose that positive selection can be viewed as a sequence of increasingly selective developmental steps (induction-->commitment-->rescue) that are signaled by alpha beta TCR engagements of intrathymic ligands.     

Comparative analysis of CD8 expressed on mature CD4+ CD8+ T cell clones cultured with IL-4 and that on CD8+ T cell clones: implication for functional significance of CD8 beta

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.     

Analysis of CD3 and antigen receptor expression on T cell subpopulations of aged athymic mice

The expression of the T cell antigen receptor on subpopulations of extrathymically generated T cells from athymic mice was investigated and compared to antigen receptor expression in normal mice. To this end, spleen and lymph node cells from 18 individual athymic BALB/c nu/nu mice between 6 and 12 months of age and from normal controls were enriched for T cells by nylon wool filtration. Expression of the following cell surface markers was analyzed by two-color flow cytometry: Thy-1, CD4, CD8, V beta 8 and CD3. The distribution of subpopulations as defined by these markers varied much more among athymic than among normal mice. Some recurrent patterns did, however, occur that may be characteristic of the extrathymic pathway of T cell differentiation in nu/nu mice. Among Thy-1.2+ cells, CD8+ cells predominated over CD4+ cells. No CD4+8+ "double positive" cells were found, but CD4-8- "double negative" cell constituted 16% on average. All nu/nu nylon wool-nonadherent cells expressing Thy-1 at a normal level also expressed CD3, whereas Thy-1low and Thy-1- cells were CD3-. The fraction of V beta 8 T cells among the CD4+ and CD8+ subsets was near to normal in the majority of these animals. Most interestingly, the density of V beta 8 and CD3 expression was lower in athymic than in euthymic animals. This level of T cell antigen receptor expression was, however, higher than on immature CD4+8+ thymocytes. A fraction of the nude T cells presently characterized responded with proliferation to both anti-T cell receptor V beta 8 monoclonal antibody and to concanavalin A. Despite their apparently normal phenotype (with the exception of reduced T cell receptor expression), this response was, however, 4 to 10 times smaller than that of normal control cells. The presently described Thy-1+ T cell receptor intermediate cells may either be a(n) aberrant lineage(s) only generated extrathymically, or represent the accumulation of an immature intermediate stage of normal (i.e. thymic) T cell differentiation.     

Malignant histiocytosis. A phenotypic and genotypic investigation.

Ten cases of malignant histiocytosis (MH) were evaluated for clinical and histopathologic features, phenotype, and rearrangement of T cell receptor (TCR) beta, gamma, and alpha and immunoglobulin (Ig) genes (7/10). All cases were HLA-DR+ and CD30-positive. Four cases had molecular evidence of T cell lineage such as TCR beta, gamma, and alpha rearrangements, and one additional case synthesized the cytoplasmic TCR beta chain. The remaining five cases did not show unequivocal T, B, natural killer (NK) cell, or macrophagic origin, and three of them had germline TCR and Ig genes. Ultrastructural analysis was not helpful for the definition of the cell lineage. Most myelomonocytic markers (MAC387, CD13, CD14, CD64, CD68) were either negative on the MH cells or were expressed on cells with rearranged TCR gene. Precursor (CD34, CD7) and NK (CD16, CD56, and CD57) cell markers were not found. The lineage of a number of cases of MH remains unresolved.     

Gamma delta T cells expressing CD8 or CD4low appear early in murine foetal thymus development.

Three-colour flow cytometry was used to study the distribution of TCR gamma delta+ cells among CD4+CD8-, CD4-CD8+, CD4+CD8+, and CD4-CD8- cell populations during thymic development. Thymocytes were obtained either directly from embryos at different stages of gestation (ex vivo) or from organ cultures maintained in vitro. In both cases, TCR gamma delta+ cells were found predominantly among the double negative (CD4-CD8-) and CD8 single positive subsets. These cells were actively dividing as demonstrated by 7 amino actinomycin D (7AAD) labelling. A small population of TCR gamma delta+ cells expressing low levels of CD4 was identified early and transiently (days 15-18) during development, but this subset was rare in the adult thymus. In newborn mice, adult mice, and late during organ culture, TCR gamma delta+ cells were found mainly within the CD4-CD8- compartment of thymocytes, although a minor population of CD8+ cells (5-10%) bearing gamma delta receptor was routinely observed. In contrast, few gamma delta cells were contained among the CD4+CD8+ subset at any timepoint studied. These data highlight differences between the ontogeny of alpha beta and gamma delta cells in the thymus, and suggest that a CD4+CD8+ intermediate may not be a requisite for the intrathymic differentiation of murine gamma delta T cells.     

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2- type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross- linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.      

A critical role for the T cell receptor alpha-chain connecting peptide domain in positive selection of CD1-independent NKT cells.

Natural killer T (NKT) cells are a subset of mature alpha beta TCR(+) cells that co-express NK lineage markers. Whereas most NKT cells express a canonical Valpha14/Vbeta8.2 TCR and are selected by CD1d, a minority of NKT cells express a diverse TCR repertoire and develop independently of CD1d. Little is known about the selection requirements of CD1d-independent NKT cells. We show here that NKT cells develop in RAG-deficient mice expressing an MHC class II-restricted transgenic TCR (Valpha2/Vbeta8.1) but only under conditions that lead to negative selection of conventional T cells. Moreover development of NKT cells in these mice is absolutely dependent upon an intact TCR alpha-chain connecting peptide domain, which is required for positive selection of conventional T cells via recruitment of the ERK signaling pathway. Collectively our data demonstrate that NKT cells can develop as a result of high avidity TCR/MHC class II interactions and suggest that common signaling pathways are involved in the positive selection of CD1d-independent NKT cells and conventional T cells.     

Phenotypic changes accompanying positive selection of CD4+CD8+ thymocytes.

We know little about the way mature CD4 (helper) and CD8 (killer) T cells develop from thymic CD4+CD8+ precursors. Here we show that small but not large CD4+CD8+ cells with high levels of the alpha beta T cell receptor (TcRhigh) result from positive selection. Neither CD4+CD8+ cells with low TcR levels nor large CD4+CD8+ thymocytes with high TcR levels differentiate in vitro. However, small CD4+CD8+ cells with high TcR levels develop in vitro into mature cells by gradually decreasing the surface levels of one or the other co-receptor and acquiring the potential to respond with proliferation to ligation of the TcR. Small CD4+CD8+ cells with high levels of a major histocompatibility complex (MHC) class I-restricted transgenic TcR develop in vitro exclusively into CD4-CD8+ cells while small CD4+CD8+ TcRhigh cells with heterogeneous TcR from various mice yield both CD4 and CD8 T cells. While these experiments are consistent with an instructive model of CD4/CD8 lineage commitment they do not rule out other mechanisms which require multiple TcR-MHC ligand interactions in the generation of mature alpha beta T cells.     

Selection of phenotypically distinct NK1.1+ T cells upon antigen expression in the thymus or in the liver.

Unlike the main TCR alphabeta T cell lineage in which deletion occurs at the CD4+ CD8+ double-positive (DP) stage upon TCR engagement by antigen in the thymus, some T cells appear to require such engagement for their selection, either in the thymus or extrathymically. We used a transgenic TCR (tgTCR) model which, as we previously showed, led to selection upon expression of the corresponding antigen H-2Kb (Kb) in the thymus, of tgTCR/CD3(lo) CD4- CD8- double-negative (DN) thymocytes that expressed the NK1.1 marker (NK T cells) (Curnow, S. J., et al., Immunity 1995. 3: 427). We now report that antigen expression on medullary epithelial cells of the thymus failed to select the NK T cells, whereas its expression on thymocytes did, although tgTCR DP thymocyte development was affected under both conditions. Antigen expression on hepatocytes (Alb-Kb mice) did not perturb tgTCR DP thymocyte development. No enrichment in tgTCR NK T cells was detected in the periphery, except for the liver of the Alb-Kb/tgTCR mice. When reconstitution of thymectomized and irradiated H-2k hosts expressing or not Kb was performed with bone marrow from tgTCR H-2k mice, an enrichment in tgTCR+ NK T cells was found in the liver, but not in the spleen, of the hosts which expressed Kb, either selectively on hepatocytes or ubiquitously. Surprisingly, the majority of the hepatic tgTCR+ NK T cells also expressed the CD8 alpha/beta heterodimer. These results indicate that thymus-independent NK T cells with unique phenotypic characteristics can be selected upon antigen encounter in the liver.     

Co-ordinate expression of the pre-T-cell receptor complex and a novel immature thymocyte-specific antigen, IMT-1, during thymocyte development

Previously we described a monoclonal antibody (mAb) that reacted with a cell-surface antigen, immature thymocyte antigen-1 (IMT-1), which is expressed on thymocytes of late CD4- CD8- (double negative) to early CD4+ CD8+ (double positive) differentiation stages. In this study, we investigated the expression of IMT-1 on various cell lineages in thymus as well as in peripheral lymphoid organs. We found that IMT-1 is expressed on T-cell receptor (TCR)-betalo and TCR-deltalo thymocytes, but not on TCR-betahi, TCR-deltahi or natural killer (NK)1.1+ thymocytes, or on peripheral alpha beta or gamma delta T cells. We also investigated the kinetics of expression of IMT-1 during fetal thymocyte development and compared it with the expression of the pre-TCR complex, comprising CD3, pre-TCR-alpha (pTalpha) and TCR-beta. We found that expression of both was similar, starting at day 14.5 of gestation, peaking on day 16.5 and gradually decreasing thereafter. Furthermore, the expression of both IMT-1 and pTalpha was drastically reduced when DN thymocytes in recombination activating gene (RAG)-2-/- mice were challenged in vivo with anti-CD3 mAb. These results indicate that IMT-1 is expressed on not only immature thymocytes of alpha beta T-cell lineage but also on those of gamma delta T-cell lineage, and that the expression of IMT-1 and the pre-TCR complex is co-ordinately regulated during the alpha beta lineage thymocyte development.     

Proliferative properties of murine intestinal intraepithelial lymphocytes (IEL): IEL expressing TCR alpha beta or TCR tau delta are largely unresponsive to proliferative signals mediated via conventional stimulation of the CD3-TCR complex.

Murine intestinal intraepithelial lymphocytes (IEL) were studied for their capacity to proliferate in vitro following stimulation of the T cell receptor (TCR)-associated CD3 epsilon molecule, or upon direct stimulation of the TCR complex itself. Although IEL consisted primarily of CD3+ T cells which included activated cytotoxic T lymphocytes as demonstrated in CD3- and TCR-mediated redirected cytotoxic assays, IEL displayed minimal proliferative responses following stimulation with anti-CD3, anti-TCR alpha beta, or anti-TCR tau delta monoclonal antibodies under soluble conditions, or under conditions which effect membrane cross-linking, including the addition of accessory cells to IEL cultures. The lack of proliferation induction could not be overcome by stimulation of IEL in the presence of T cell-dependent cytokines, phorbol ester, or interleukin-4. Moreover, unlike splenic T cells, stimulation of IEL failed to result in expression of interleukin-2 receptor, further demonstrating an inability of IEL to respond to exogenous proliferative signals. This study is the first to examine the proliferative potential of murine IEL following direct CD3 or TCR stimulation. The findings described here: (i) identify an important functional distinction between intestinal IEL and other peripheral alpha beta or tau delta T cells which generally respond well to proliferative signals mediated through the CD3-TCR complex, and (ii) demonstrate that on murine IEL the CD3-TCR complex can discriminate signals of lytic activity from those of cell proliferation.     

Evidence against T-cell development in the adult human intestinal mucosa based upon lack of terminal deoxynucleotidyltransferase expression.

Several lines of evidence indicate that a subset of murine intestinal intraepithelial lymphocytes (iIEL), particularly those which express the CD8 alpha alpha homodimer, mature extrathymically. This study confirms that a small fraction of adult human iIEL also express the CD8 alpha alpha homodimer and demonstrates that most of these cells in the small intestine are T cells using the alpha beta T-cell receptor (TCR). Whether these cells or other subsets of adult human iIEL mature extrathymically in the intestine was assessed by measuring the expression of terminal deoxynucleotidyltransferase (TdT), an enzyme expressed exclusively by immature lymphocytes. Very low levels of TdT message could be detected by polymerase chain reaction (PCR) amplification in some iIEL samples. The level of TdT expression was assayed by competitive PCR amplification and compared with thymocytes and peripheral blood lymphocytes. These measurements indicated that the number of immature T cells expressing TdT in the intestinal epithelium was less than one cell per 10(7) lymphocytes. This demonstrates that there are few if any TdT expressing immature T cells in the adult human intestinal mucosa and indicates, therefore, that T-cell development in the intestinal mucosa does not contribute significantly to the T-cell repertoire of the adult human intestine.     

Development of CD8 alpha/beta + TCR alpha beta intestinal intraepithelial lymphocytes in athymic nu/nu mice and participation in regional immune responses.

On the basis of the CD8 coreceptor expression, T-cell receptor (TCR)alpha beta-bearing intestinal intraepithelial lymphocytes (i-IEL) segregate into two populations. The CD8 alpha alpha + TCR alpha beta i-IEL develop thymus independently, whereas the CD8 alpha beta + TCR alpha beta i-IEL are generally considered to be thymus dependent. Flow cytometry analysis revealed a distinct population of CD8 alpha beta + TCR alpha beta i-IEL in individual athymic nu/nu mice. The i-IEL encompassing CD8 alpha beta + TCR alpha beta cells expressed potent cytolytic and interferon-gamma-producing activities. These findings demonstrate that CD8 alpha beta + TCR alpha beta i-IEL can develop in nu/nu mice independently from a functional thymus and suggest that these cells, directly or indirectly, perform biological functions in the gut.     

Monoclonal antibody against murine T cell receptor V alpha 14 cross-reacts with human CD3 epsilon and detects disulfide-linked dimeric form.

A monoclonal antibody against V14+ alpha-chain of murine T cell receptor (TCR) was established by fusing spleen cells from a rat immunized with a soluble chimeric TCR/IgG3 protein containing murine TCR V alpha 14J alpha 281 in place of the VHDHJH of an IgG3. lambda 1, and subjected to screening on a human transfectant (Jurkat variant) expressing the murine V14J281 alpha-chain. The anti-mouse V alpha 14 antibody precipitated TCR alpha beta molecules from Triton X-100-solubilized extracts of 125I-labeled murine thymocytes and spleen cells. Unexpectedly, the antibody showed cross-reactivity to the human CD3 epsilon molecule and detected a disulfide-linked 20 kDa dimeric form of human CD3 epsilon, which is a novel family component of the CD3 complex and is associated closely with the CD3 zeta-zeta homodimer as well as TCR alpha beta or TCR gamma delta.     

Microbial colonization influences composition and T-cell receptor V beta repertoire of intraepithelial lymphocytes in rat intestine.

Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ-free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T-cell receptor (TCR) alpha/beta + IEL in that the proportions of CD4+ and CD8 alpha beta + TCR alpha/beta + cells were increased, while the double negative (CD4- CD8-) fraction was reduced. microbial colonization also influenced the TCR V beta repertoire of CD8+ IEL in that the proportions of V beta 8.2+ and V beta 10+ cells were increased, whereas V beta 8.5+ and V beta 16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same V beta subsets. Three-colour flow-cytometric analysis demonstrated that skewing of the V beta repertoire was most pronounced in the CD8 alpha alpha + subset, although the numerical increase of IEL mainly included the CD8 alpha beta + subset. In contrast to IEL, the TCR V beta repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCR alpha/beta + IEL subpopulations respond dynamically to the microbial gut flora and suggest that their V beta repertoire can be shaped by luminal microbial antigens.     

An appropriate in vitro culture condition for the induction of human TCR gamma delta + cells by heat-killed bacteria.

TCR gamma delta + cells proliferated when MNC were stimulated with various heat-killed bacteria. We investigated here the culture conditions for their maximum proliferation. MNC were cultured for 6 days with Streptococcus pyogenes, and for 3 days with T cell mitogens, PHA and anti-CD3 mAb, in medium supplemented with various concentrations (0.05-50%) of human sera. TCR gamma delta + and TCR gamma delta- CD2+3- double negative cells induced by Str. pyogenes required high concentrations of sera (greater than 6%) for their proliferation. Moreover, increased sera (up to 50%) greatly augmented their proliferation. On the other hand, TCR alpha beta + cell proliferation induced by T cell mitogens was supported by a small concentration (even 0.1%) of the sera, and the addition of high concentrations of sera (greater than 6%) somewhat suppressed responses. Similar serum requirement patterns were evident for the induction of cytotoxic cells. These results clearly demonstrated the existence of an appropriate culture condition for the proliferation of TCR gamma delta + cells induced in vitro.     

Unique CD4(+) T cells in TCR alpha chain-deficient class I MHC-restricted TCR transgenic mice: role in a superantigen-mediated disease process.

Mice carrying a transgenic TCR with targeted disruption of the TCR alpha chain (H-Y alpha(-/-)) possess CD4(+) T cells which express the transgenic TCR beta without the alpha chain. These mice developed the murine acquired immunodeficiency syndrome (MAIDS) after infection with LP-BM5 retroviruses, a process which requires CD4(+) T cells. These cells are negative for TCR delta chain and pre-TCR alpha chain expression, and thus express a unique surface receptor with the TCR beta chain as a component. The cells respond to MAIDS virus-associated superantigen and concanavalin A, but not to protein antigens such as ovalbumin. Thus, this novel surface receptor appears to play an important role in the pathogenesis of MAIDS.     

Analysis of clones derived from human CD7+CD4-CD8-CD3- thymocytes.

The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR gamma delta cells, 12% of clones were TCR alpha beta cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR alpha beta, TCR gamma delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR gamma, delta, and beta gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR alpha beta and TCR gamma delta lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR alpha beta and TCR gamma delta lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR gamma delta and NK cells.