Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo.

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4+CD8lo intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class I.

The MHC class II-restricted T cell response of C57BL/6 mice to human C-reactive protein: homology to self and the selection of T cell epitopes and T cell receptors.

The T cell response of C57BL/6 mice to human C-reactive protein (hCRP), an inducible acute phase protein, was analysed. Two I-A(b)-restricted epitopes at positions 79 95 (epitope A) and 87-102 (epitope B) were identified using a panel of CD4+ T cell clones. Human C-reactive protein shares considerable homology with mouse C-reactive protein and mouse serum amyloid P component. Interestingly, the two epitopes map to the region of lowest homology between human CRP and its mouse homologues. Human CRP-specific T cell clones express a restricted T cell receptor (TCR) repertoire, both with regard to usage of TCR germline gene segments (V alpha, J alpha, V beta, J beta) and certain TCR alpha beta combinations. Therefore, epitope-A specific clones preferentially use TCR V beta8.3 and V alpha3 J alpha15 V beta8.3-J beta2.3 and epitope-B specific clones use V beta2 and V alpha1-J alpha24/30-V beta2. This bias is even more pronounced when TCR usage is correlated with epitope fine specificity. A role for homology of hCRP to self components in selecting these particular T cell epitopes and TCR is discussed.

Insertional mutagenesis affecting programmed cell death leads to thymic hyperplasia and altered thymopoiesis.

The mouse mutant Ft displays thymic hyperplasia and fused toes (Ft) of the forelimbs. Both phenotypic abnormalities are caused by transgene insertion in the D region of chromosome 8. While the forelimb defect is probably caused by developmentally dysregulated programmed cell death, the mechanism underlying thymic hyperplasia has not been characterized. In this work, we show that expansion of the thymocyte compartment progresses with time, is polyclonal, and affects all major thymocyte subsets, including the earliest CD4-8- subset, i.e., CD44+ CD25- cells; hyperplasia is not an autonomous property of mutant T cells, but is caused indirectly by a primary defect in thymic stromal. The rate of cell division and the cell turnover of immature CD4-8- and CD4+8+ thymocytes under steady state conditions are not altered in hyperplastic Ft thymi. Immature CD4+8+ thymocytes of mutant mice, however, are less susceptible to induction in vitro of programmed cell death by different modes (TCR cross-linking, cortisone, or radiation). Increased production of thymocytes results in increased export of T cells, yet the size and composition of the peripheral T cell pool are normal. Overproduction of immature CD4+8+ thymocytes is offset partly by a reduced conversion rate of CD4+8+ double positive to single positive thymocyte growth control by epithelial cells, and may serve as a model to study the regulation of early thymopoiesis.

Intrathymic T cell receptor (TcR) targeting in mice lacking CD4 or major histocompatibility complex (MHC) class II: rescue of CD4 T cell lineage without co-engagement of TcR/CD4 by MHC class II.

A critical step during intrathymic T cell development, termed positive selection, is associated with rescue of short-lived, immature thymocytes from programmed cell death, T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TcR)-major histocompatibility complex (MHC) interactions during positive selection can be closely mimicked by targeting TcR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. Here, we show that antibody-mediated TcR signaling in mice deficient for CD4 or MHC class II expression induces polyclonal differentiation of the CD4 T cell lineage. Following a single TcR signal pulse in situ, a temporal sequence of phenotype changes can be discerned: CD69 up-regulation (< 1 day), CD8 down-regulation, TcR up-regulation (1-1.5 days) and down-regulation of the heat-stable antigen (1.5-2 days). Differentiation of phenotypically and functionally mature CD4 T cells in situ is attained within 3 days. Rescue of CD4 lineage T cells in the absence of TcR/CD4 co-engagement by MHC class II in this experimental system supports the stochastic/selective model of T cell lineage commitment.

Half-life of antigen/major histocompatibility complex class II complexes in vivo: intra- and interorgan variations.

We have determined the half-life in vivo of antigen/MHC class II complexes in different organ microenvironments. Mice were "pulsed" with myoglobin intravenously and MHC class II-positive antigen-presenting cell (APC) populations from different organs were isolated after various time intervals. Specific antigen/MHC complexes were quantitated by co-cultivation of the APC subsets with myoglobin-specific T-T hybridoma cells in vitro. Half-lives of antigen/MHC complexes differed both between organs and between compartments of the same organ. Half-lives in peripheral organs (spleen and bone marrow) ranged between 3 and 8 h, whereas in the thymus half-lives between 13 h (cortical epithelial cells) and 22 h (medullary dendritic cells) were observed. Half lives in vivo were independent of antigen processing, since intact protein or antigenic peptides yielded similar values. The considerably longer half-life of peptide/MHC complexes in the thymus as compared to peripheral organs may reflect the distinct role which antigen presentation plays in both organs, i.e. induction of tolerance versus induction of immunity.

T cell receptor targeting to thymic cortical epithelial cells in vivo induces survival, activation and differentiation of immature thymocytes.

We report that targeting of T cell receptors (TcR) to non-major histocompatibility complex (MHC) molecules on thymic cortical epithelial cells by hybrid antibodies in vivo and in fetal thymic organ cultures results in phenotypic and functional differentiation of thymocytes. A single pulse with hybrid antibodies rescues immature, CD4/8 double-positive thymocytes from their programmed death in vivo, induces expression of the early activation antigen CD69 followed by TcR up-regulation, concomitant down-regulation of CD8 or CD4 and their conversion to functional mature T cells by day 3. This temporal sequence of maturation only affects small thymocytes without co-induction of blastogenesis. TcR targeting to MHC class II-positive epithelial cells predominantly induces CD4-positive T cells. This generation of CD4 single-positive T cells occurs also in MHC class II-deficient mice and thus is independent of CD4-MHC class II interactions. Moreover, in the presence of a specific deleting antigen (Mls 1a), TcR targeting results in transient activation of immature thymocytes, however, not in subsequent TcR (V beta 6) up-regulation and development of single-positive T cells. Our findings imply that TcR cross-linking to cortical epithelial cells is sufficient to confer a differentiation signal to immature thymocytes. Furthermore, this approach distinguishes two independent TcR-mediated intrathymic events: activation and subsequent deletion of the same thymocyte subset.

Thymocytes can tolerize thymocytes by clonal deletion in vitro.

Clonal deletion of thymocytes bearing TCR for self antigens is one major mechanism of T cell tolerance induction. Peptide antigen-induced deletion of thymocytes from alpha beta TCR transgenic mice has been studied using single cell suspension cultures. The results show that antigen-presenting immature CD4+CD8+ thymocytes can tolerize antigen-reactive immature thymocytes in vitro by programmed cell death (apoptosis) 6-8 h after antigen exposure. Antigen-induced apoptosis of immature thymocytes was inhibited by antibodies specific for the alpha beta TCR, CD3, CD8, and LFA-1 molecules. This implies that clonal elimination of self-reactive CD4+CD8+ thymocytes does not depend on specialized deleting cell types in the thymus and occurs whenever the TCR of immature thymocytes bind antigen fragments presented by MHC molecules.

Anti-CD2 antibodies induce T cell unresponsiveness in vivo.

The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo.

Anti-CD2 monoclonal antibodies alter cell-mediated immunity in vivo.

The antimurine CD2 mAb 12-15 was administered intravenously to investigate the role of CD2 in cell-mediated immunity in vivo. The anti-CD2 mAb was able to diminish the contact sensitivity response to the hapten trinitrophenyl and was most effective when administered during the efferent or elicitative phase of immunity. Antibody treatment was also able to inhibit in vivo priming for subsequent generation of secondary, TNP-specific CTL in vitro. This inhibitory effect was most effective in the afferent or early phase of immunity. Indeed antibody could be injected at least 3 weeks prior to in vivo antigen priming, and the subsequent CTL response was still suppressed. Additional experiments showed a well-defined dose-response relationship between the amount of anti-CD2 administered and subsequent immunosuppression. Control experiments showed that other isotype-matched antibodies were not suppressive and that the anti-CD2 was not merely shifting the kinetics of the CTL response. Further experiments revealed that in vivo mAb treatment could also inhibit the subsequent development of primary, alloantigen-specific CTL in vitro while the mixed lymphocyte reaction (MLR) remained unchanged. FACS analysis revealed a marked downmodulation of CD2 in vivo, a small and variable decrease in CD8, and essentially no change in CD3 or CD4 after treatment with anti-CD2. The F(ab')2 fragment was not able to downmodulate CD2 or to suppress CTL activity at the doses tested. These results support a major role for CD2 in diverse aspects of cell-mediated immunity affecting both CD4+ and CD8+ effector T cells. The anti-CD2 mAb functions not by deleting or depleting relevant cell populations but rather by altering the array of cell surface receptors and subsequent responses to antigenic challenge.

Antibodies against the T cell receptor/CD3 complex interfere with distinct intra-thymic cell-cell interactions in vivo: correlation with arrest of T cell differentiation.

Postnatal treatment of mice with antibodies against the T cell receptor complex (TcR) prevents the differentiation of mature T cells in the thymic medulla without affecting the generation of most immature cortical thymocytes, thus interfering with a discrete stage of intra-thymic T cell differentiation at the cortex/medulla transition. This result has been interpreted as indicating a direct role of the TcR in the differentiation of immature to mature T cells, possibly via TcR-ligand interactions during direct cell-cell contact. Here we analyze the effect of anti-TcR (V beta 8 family) and anti-CD3 (epsilon chain) antibodies on distinct intra-thymic cell-cell interactions in vivo. We find that the maturation arrest of thymocytes correlates with a nearly complete abrogation of interactions of corresponding immature thymocyte with I-A/E+ cortical epithelial cells and I-A/E+ medullary dendritic cells, while preserving interactions with adherent I-A/E- macrophages. It is proposed that the blockade of thymocyte-epithelial cell recognition in the cortex by anti-TcR antibodies prevents the translocation of thymocytes into the medulla and their subsequent differentiation and selection, including interactions with dendritic cells. Interestingly, the anti-CD3 mAb treatment seems to spare the intra-thymic development of the CD3+, CD4-/CD8- T cell lineage.

The effects of anti-CD2 antibodies on the differentiation of mouse thymocytes.

Using a rat monoclonal antibody against mouse CD2, we determined the expression of this marker on thymocytes during ontogeny. CD2 expression becomes detectable at day 15 and reaches adult levels (approximately 95% positivity) by day 19. Furthermore, the effect of anti-CD2 antibodies on T cell differentiation was analyzed by addition of antibodies to thymus organ cultures or repeated injection into newborn mice. Anti-CD2 antibodies inhibit CD2 expression in organ cultures and drastically reduce its expression on thymocytes and peripheral lymphocytes in vivo. In either situation, suppression of CD2 expression does not significantly alter the generation of T cells expressing CD3, CD4, CD8 and T cell receptor V beta 8. These results do not support a role for CD2 in early steps of thymocyte differentiation.

Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells.

We describe monoclonal antibodies (MAB) reactive with subsets of mouse and human thymic epithelial cells. Rat MAb CDR1 reacts with mouse but not human cortical epithelial cells. Immunologic staining of thymic nurse cells in suspension indicates the CDR1 antigen is located on the cell surface. Mouse MAb CDR2 reacts with human but not mouse cortical thymic epithelial cells. Rat MAb MD1 and MD2 detect different determinants expressed by most medullary epithelial cells in mouse thymus but fewer such cells in human thymus. In addition, MD1 detects flattened subcapsular cells rarely in mouse thymus but frequently in human thymus. Two-color stains using an anti-keratin antiserum demonstrate the epithelial nature of the cells reactive with these antibodies. The antigens detected by CDR1 and MD1 first appear during the neonatal period, achieving adult distribution by postnatal days 14 and 4, respectively. The extra-thymic staining of these MAb is described. On the basis of their intra- and extra-thymic reactivities, these MAb differ from those previously reported and may permit dissection of the thymic microenvironment.

Seeding of thymic microenvironments defined by distinct thymocyte-stromal cell interactions is developmentally controlled.

Seeding of distinct intrathymic microenvironments defined by direct thymocyte-stromal cell interactions was correlated with T cell development in situ using radiation and nonradiation chimeras of Thy-1.1/1.2 congenic mice. The results identify associations of thymocytes with I-A- macrophages in the cortex as the earliest discernible cell-cell interactions during thymopoiesis. After a significant delay, this recognition stage is followed by concomitant interactions of T cells with I-A+ epithelial cells in the cortex and bone marrow-derived I-A+ dendritic cells in the medulla. All three types of T cell-stromal cell interactions occur after seeding of the intrathymic precursor cell subset and before development of mature medullary-type T cells. The seeding kinetics imply that recognition of cortical epithelial cells by thymocytes in situ represents a relatively late stage of cortical T cell development, whereas thymocyte-dendritic cell interactions denote a very early stage of T cell development in the medulla. The relative positioning of these cell-cell recognition stages during the course of T cell maturation pertains to a putative role of these microenvironments in selection and tolerization of the T cell repertoire.

Phenotype of stromal cell-associated thymocytes in situ is compatible with selection of the T cell repertoire at an "immature" stage of thymic T cell differentiation.

Murine thymocytes interacting with cortical macrophages, cortical epithelial cells and medullary dendritic cells in situ express T cell receptors at low to intermediate density. They co-express the lineage markers Lyt-2 and L3T4 and are not enriched for cells expressing high-density interleukin 2 and lymph node homing receptors. Thus, recognition of stromal cells in situ in distinct thymic microenvironments occurs at a common stage of T cell maturation which is phenotypically intermediate between intrathymic precursor cells and mature medullary-type thymocytes. The surface phenotype of dendritic cell-associated thymocytes indicates the presence of thymocytes with a "cortical" phenotype within the antigen-exposed ("nonsterile") phase of T cell maturation in the medulla.

Intrathymic presentation of circulating non-MHC antigens by medullary dendritic cells. An antigen-dependent microenvironment for T cell differentiation.

We present evidence for intrathymic presentation of soluble circulating antigens in vivo. Our results show that proteins of different molecular weight enter the mouse thymus rapidly after i.v. injection. The intrathymic presence of antigen was assayed by proliferation of cloned antigen-specific T helper cells, which were cocultured with purified thymic stromal cells; stromal cells were isolated and purified as lymphostromal cell complexes, which preexist in vivo. Antigen presentation copurified with non-adherent medullary dendritic cells (DC) (interdigitating cells). I-A- cortical macrophages forming thymocyte rosettes in vivo and I-A+ cortical epithelial cells forming thymic nurse cells (TNC) in vivo did not act as antigen presenting cells (APC) after antigen pulsing in vivo or in vitro. Thymic APC turn over physiologically and are rapidly replaced (within 2-5 wk) after lethal irradiation by donor bone marrow-derived cells. The frequency of thymocyte-DC interactions in vivo strictly correlates with thymic T cell differentiation, and is independent of the immune status of the animal. Fetal thymic APC seem to be secluded from antigen in the maternal circulation. Thymic DC-ROS probably represent the microenvironment where maturing T cells first encounter non-MHC antigens in the context of self-MHC antigens.

Thymic nurse cells: possible sites of T-cell selection.

The precise role of the thymic microenvironment in induction and selection of the T-cell repertoire, a matter of considerable interest to immunologists, has been difficult to define. Direct cell-cell interactions between thymocytes and distinct stromal cells are an important component of this thymic microenvironment. Thymic epithelial cells, in particular, have been suspected to select immature T cells for self MHC-specificity via direct cell-cell contact. Here, Bruno Kyewski discusses the type of epithelial cells known as thymic nurse cells and their remarkable association with the thymocytes which are completely enclosed within them.