Genes that code for T cell signaling proteins establish transcriptional regulatory networks during thymus ontogeny

Gene expression profiling by cDNA microarrays during murine thymus ontogeny has contributed to dissecting the large-scale molecular genetics of T cell maturation. Gene profiling, although useful for characterizing the thymus developmental phases and identifying the differentially expressed genes, does not permit the determination of possible interactions between genes. In order to reconstruct genetic interactions, on RNA level, within thymocyte differentiation, a pair of microarrays containing a total of 1,576 cDNA sequences derived from the IMAGE MTB library was applied on samples of developing thymuses (14-17 days of gestation). The data were analyzed using the GeneNetwork program. Genes that were previously identified as differentially expressed during thymus ontogeny showed their relationships with several other genes. The present method provided the detection of gene nodes coding for proteins implicated in the calcium signaling pathway, such as Prrg2 and Stxbp3, and in protein transport toward the cell membrane, such as Gosr2. The results demonstrate the feasibility of reconstructing networks based on cDNA microarray gene expression determinations, contributing to a clearer understanding of the complex interactions between genes involved in thymus/thymocyte development.     

Systematic development of distinct T cell receptor-gamma delta T cell subsets during fetal ontogeny

To elucidate the developmental pattern and diversity of murine cluster of differentiation (CD)3-associated TCR-gamma delta heterodimers, adult and fetal thymocytes were examined for cell-surface expression of various gamma- and delta-encoded TCR. Biochemical analysis, using antisera specific for distinct C gamma gene products, revealed the presence of T cells expressing C gamma 1 and/or C gamma 4 heterodimers in adult and fetal CD4- CD8- thymocyte populations. Although CD4-CD8- thymocyte populations express both C gamma 1 and C gamma 4 TCR-gamma delta heterodimers early in fetal thymus development, the relative level of C gamma 4-expressing T cells was significantly lower than previously observed in peripheral lymphoid organs. In addition, biochemical studies revealed the presence of TCR-gamma delta heterodimer(s) expressed during fetal ontogeny which were not detected in adult thymocyte or peripheral lymphoid populations. Studies of N-glycosylation patterns of one of these heterodimers suggested that it contained a rearranged V gamma 3/C gamma 1 gene product. To examine in detail individual TCR-gamma delta heterodimers, a panel of TCR-gamma delta expressing hybridomas was prepared. Biochemical analysis at the clonal level revealed that indeed three distinct TCR-gamma delta heterodimers were present at day 16 of fetal thymus development, with TCR-gamma-chains most likely encoded by V gamma 2/C gamma 1, V gamma 3/C gamma 1, and V gamma/C gamma 4. Together these findings suggest an ordered development of TCR-gamma delta T cells in the thymus and selective expression of distinct TCR-gamma delta subsets in peripheral lymphoid organs such as spleen and lymph nodes.     

The T cell receptor-associated protein is proteolytically cleaved in a pre-Golgi compartment

The TCR consists of at least seven transmembrane chains: the clonotypic disulfide-linked alpha- and beta-chains, the invariant gamma-, delta-, and epsilon-chains, termed the CD3 complex, and the zeta-zeta homodimer. We have recently described an additional 26-kDa protein, which is transiently associated with newly synthesized mouse CD3 chains in the endoplasmic reticulum. The exact function of this protein, which we called TRAP (for TCR-associated protein) is not yet known; studies suggest however, that it may play a role in the assembly of the TCR complex. Here we report the properties of another protein which has a Mr of 16,000 and, like TRAP, can be coimmunoprecipitated from metabolically labelled murine T cells with antibodies against the chains of the CD3 complex. Kinetic analysis suggests a precursor-product relationship between TRAP and the 16-kDa protein: the latter starts appearing once TRAP begin to disappear. Having reached a maximal level at approximately 1 h after biosynthesis, it is rapidly lost. Agents that slow or block the disappearance of TRAP, delay or prevent the appearance and eventual disappearance of the 16-kDa protein. Incubation of immunoprecipitates containing gamma, epsilon, and TRAP in vitro at 37 degrees C results in the appearance of the 16-kDa protein. Employing HPLC peptide mapping we demonstrate that this 16-kDa protein is structurally related to TRAP. These results suggest that the removal of TRAP from the newly synthesized CD3 chains is accompanied by its proteolytic cleavage in a pre-Golgi compartment.     

A T cell receptor-associated GTP-binding protein triggers T cell receptor-mediated granule exocytosis in cytotoxic T lymphocytes

To study the subcellular events occurring after T cell activation we used cloned human CTL permeabilized with alpha-toxin of Staphylococcus aureus. This method of permeabilization leads to stable transmembrane channels that permit the introduction of small molecules into the cell but preserves the cellular structures and macromolecular contents of the CTL. We used the exocytosis of CTL-specific serine esterases as a marker of T cell activation. The TCR-activated exocytosis is functioning in such permeabilized CTL. Introduction of the membrane impermeable guanosine nucleotide-binding protein (G-protein) activating GTP-analog GTP gamma S into CTL triggers exocytosis if Ca2+ is present. For optimal exocytosis ATP is required. The G-protein inactivating GDP-analog GDP beta S inhibited exocytosis triggered via the TCR-CD3 complex but not that triggered by activating the protein kinase C. If the protein kinase C was depleted in CTL by overnight incubation with phorbolester, the response to GTP-gamma S was reduced by more than 50%. These experiments demonstrate the presence of a G-protein involved in TCR-mediated CTL triggering. In the sequence of signaling steps this G-protein is localized after TCR-triggering but before the formation of the protein kinase C-activating phosphoinositol breakdown product diacylglycerol in the sequence of signaling steps.     

Development and ontogeny of hamster T cell subpopulations

The Syrian hamster is unique among laboratory animals because products of class I MHC genes are monomorphic. Thus, this species may be a model in which to test the relationship between MHC polymorphism and the T cell antigen receptor repertoire. Recently, cytotoxic and helper T cell subpopulations have been distinguished on the basis of cell surface phenotype detected with monoclonal antibodies (mAb). We used these reagents (mAb 110 detects all peripheral T cells and mAb 38 detects cytotoxic T cells) to dissect and categorize thymic populations according to relative maturational status. The two mAb divide thymocytes into four subpopulations in the young adult. Two (110+ 38+, 110+ 38-) were peripheral-like and were housed in the medulla, exclusively; another subset (110- 38+) consisted almost entirely of TdT+ cortical thymocytes. The fourth subset (110- 38-), bearing neither marker, was heterogeneous and consisted mostly of medium-large-size thymocytes, including cells with an early phenotype (nuclear TdT+). Cells with the cortical phenotype proved to be the most sensitive to cortisone treatment, whereas those which expressed the medullary marker, 110, were most resistant. To ascertain the relationship between 110- and 110+ T lineage cells, we followed the appearance of the four thymic subpopulations during ontogeny of the hamster thymus. Adult-like thymic architecture (delineation of cortex and medulla) as well as the two 110- subsets were established before expression of 110 antigen was apparent in the thymus. However, lymphocytes bearing the 110 antigen were found in lymph nodes prior to thymus during ontogeny, concomitant with developing T cell function in peripheral tissue. This finding implies that cells lacking 110 antigen were exported from the thymus and subsequently acquired expression of the molecule in the periphery, and we suggest that acquisition of 110 antigen may be a stage of postthymic maturation. Although 110+ cells appeared to be the most mature subset by several criteria, all functional thymocytes of adults or neonates were not 110+. Thus, we conclude that the 110 marker is acquired after T cells reach functional maturity. Moreover, the response profile of isolated 38+ thymocytes was analogous to peripheral 38+ T cells, suggesting that the dichotomy of function detected with our mAb also occurs before acquisition of 110 antigen. We have modeled what is known about hamster T cell development into a hypothetical scheme.     

Restoration of NK T cell development in fyn-mutant mice by a TCR reveals a requirement for Fyn during early NK T cell ontogeny

NK T cells are a unique lymphocyte population that have developmental requirements distinct from conventional T cells. Mice lacking the tyrosine kinase Fyn have 5- to 10-fold fewer mature NK T cells. This study shows that Fyn-deficient mice have decreased numbers of NK1.1(-) NK T cell progenitors as well. 5-Bromo-2'-deoxyuridine-labeling studies indicate that the NK T cells remaining in fyn(-/-) mice exhibit a similar turnover rate as wild-type cells. The fyn(-/-) NK T cells respond to alpha-galactosylceramide, a ligand recognized by NK T cells, and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell-specific TCR alpha-chain Valpha14Jalpha18 leads to a complete restoration of NK T cell numbers in fyn(-/-) mice. Together, these results suggest that Fyn may have a role before alpha-chain rearrangement rather than for positive selection or the peripheral upkeep of cell number. NK T cells can activate other lymphoid lineages via cytokine secretion. These secondary responses are impaired in Fyn-deficient mice, but occur normally in fyn mutants expressing the Valpha14Jalpha18 transgene. Because this transgene restores NK T cell numbers, the lack of secondary lymphocyte activation in the fyn-mutant mice is due to the decreased numbers of NK T cells present in the mutant, rather than an intrinsic defect in the ability of the other fyn(-/-) lymphoid populations to respond.     

First T cell receptor alpha gene rearrangements during T cell ontogeny skew to the 5' region of the J alpha locus

Fetal, neonatal, and early postnatal thymi were assessed for TCR J alpha gene rearrangements. Gene probes spanning the distance from 5' to 3' regions of the J alpha locus were used to determine the approximate location of gene rearrangements within hybridomas representing each of the early thymocyte populations. The predominant location of rearrangements was within the 5' region of the J alpha locus. Among the several cells in which rearrangements were found on only one chromosome, the one rearrangements was always in the 5' region. When two rearrangements were found, the rearrangements on homologous chromosomes were usually in the same region. The overall pattern among thymocytes was in great contrast to that previously observed among hybridomas derived from stimulated adult spleen cells within which rearrangements fell mostly to the 3' side of the alpha-locus. Results reveal the nonrandom nature of the TCR-alpha gene rearrangement event and may reflect an incidence of multiple V-J alpha joining events on each chromosome during T cell development in vivo. Due to the fact that most mature cells bear two J alpha joins, the allelic exclusion of alpha-chains cannot be explained by a mechanism whereby a functional rearrangement on one chromosome inhibits subsequent rearrangement on the second. Instead allelic exclusion may rely on a low frequency of productive vs nonproductive rearrangement events and an incompatibility between multiple alpha- and beta-protein pairs.     

Detection of expansin proteins and activity during tomato fruit ontogeny

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.     

The angiotensin Ⅱ type 1 receptor and receptor－associated proteins

The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.     

Distribution and ontogeny of CD2 expression by murine T cells

We have raised a polyclonal antiserum to murine CD2 by immunization of a rabbit with a synthetic peptide corresponding to a hydrophilic sequence in the extracellular domain of the murine CD2 gene. The antiserum immunoprecipitates a 55 kDa protein, consistent with the size predicted by the cDNA sequence. Flow microfluorometric analysis of a panel of T cell tumors and clones demonstrated concordance of reactivity of intact cells with the anti-CD2 serum and the presence of CD2 mRNA. Surprisingly, although splenic T cells were found to uniformly express high levels of CD2, several of a panel of functional T cell clones were found to lack CD2 expression. This suggests that the clones lost CD2 upon in vitro cultivation, and may not be required for activation or maintenance in culture. Adult thymocytes exhibited heterogeneous expression of CD2. The majority of CD4-8- thymocytes expressed low levels and CD4+8+ thymocytes intermediate levels, whereas all CD4+8- and the majority of CD4-8+ thymocytes expressed high levels of CD2. Multiparameter analysis of CD2 expression and that of CD3, CD5, JIId, and IL-2R p55 chain showed that expression of CD2 correlates with the maturational state of thymocytes. Finally, analysis of fetal thymuses from timed pregnancies revealed that expression of CD2 is preceded by that of IL-2R p55 chain.     

The ontogeny of territoriality during maturation

Territoriality drives the evolution of many mating systems, yet has remained an extremely difficult trait to measure in the wild. Classic studies rely on the theoretical framework of resource holding potential (RHP) as a predictor of success in territory acquisition. However, mounting evidence suggests that an individual's RHP may change over short time scales. Previous studies suggest that RHP is best understood by considering two categories of territoriality, resource defending and resource usurping potential (RDP and RUP, respectively). In a population of the side-blotched lizard, Uta stansburiana, blue-throated males defend territories near their natal site (RDP) while mature orange-throated males use their RUP to sequester high quality territories from defending territorial males. We tested differences in territoriality by releasing pairs of maturing male lizards onto experimentally altered territories that had improved thermal qualities owing to the addition of rock piles. Dyads of males competed for these thermal resources and the females that were released on rock piles. Early in the season, when throat colors were not yet fully expressed, large male body size predicted contest victories irrespective of throat color. This pattern changed however, with the onset of the breeding season and maturation of throat color. Orange males tended to usurp territories from blue males within 2 weeks of contest initiation. Large male body size still influenced these contests, but after one more week, throat color was the sole factor explaining variance in territory ownership. We demonstrate the ontogeny of territoriality relating to body size and throat color during maturation, and suggest a novel approach to assessing territoriality and aggression in the wild.     

Development of the dendritic cell system during mouse ontogeny

Based on the view that the efficacy of the immune system is associated with the maturation state of the immune cells, including dendritic cells (DC), we investigated the development and functional potential of conventional DC and plasmacytoid pre-DC (p-preDC) in spleen, thymus, and lymph nodes during mouse development. Both CD11c+ DC and CD45RA+ p-preDC were detected in small numbers in the thymus as early as embryonic day 17. The ratio of DC to thymocytes reached adult levels by 1 wk, although the normal CD8alpha+ phenotype was not acquired until later. Significant, but low, numbers of DC and p-preDC were present in the spleen of day 1 newborn mice. The full complement of DC and p-preDC was not acquired until 5 wk of age. The composition of DC populations in the spleen of young mice differed significantly from that found in adult mice, with a much higher percentage (50-60% compared with 20-25%) of the CD4-CD8alpha+ DC population and a much lower percentage (10-20% compared with 50-60%) of the CD4+CD8alpha- DC population. Although the p-preDC of young mice showed a capacity to produce IFN-alpha comparable with that of adult mice, the conventional DC of young mice were less efficient than those of their adult counterparts in IL-12p70 and IFN-gamma production and in Ag presentation. These results suggest that the neonatal DC system is not fully developed, and innate immunity is the dominant form of response. The complete DC system required for adaptive immunity in the mouse is not fully developed until 5 wk of age.     

The antibody response during amphibian ontogeny

The maturation of the anti DNP-response in adult and larval amphibians has been compared in two species of anurans,Rana catesbeiana andXenopus laevis. IgM responses were very similar in larvae and adults of both species. Specific IgG antibody synthesis, studied inRana catesbeiana only, was not obvious in larval primary response but was easily detectable in adults. Although metamorphosis results in profound changes in the lymphoid system associated with the apparent acquisition of self tolerance to new antigens, immunological memory of antigens injected during larval life ofRana andXenopus persists after the metamorphosis period.     

OX40-mediated memory T cell generation is TNF receptor-associated factor 2 dependent

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.     

Anatomy and topography of the tracheobronchial lymph nodes in man during the postnatal period of ontogeny

Tracheobronchial lymph nodes (TBLN) have been studied in 71 corpses of persons at different ages. The TBLN are revealed by means of polychromic injection of Gerota mass into the lung tissue, or directly into the lymph nodes revealed. The number of the inferior and superior (right and left) TBLN varies within a wide range. Longitudinal dimentions of the lymph nodes increase with age. A certain age dependence in topography of the inferior and superior TBLN is stated. In persons of mature and elderly age, dextrobronchial type in arrangement of the inferior TBLN is found more often, and at younger age periods--even type of their arrangement is specific. The right superior TBLN in newborns, children and adolescents possess an evenly concentrated type of localization, and at later age periods--dispersive and unevenly concentrated. The superior left TBLN in newborns and children are mainly situated in the TB angle area, in persons of mature and elderly age--on the lateral surface of the thoracic part of the trachea. The form of the TBLN depends on their localization. A certain relation is determined between the localization of the inferior TBLN and the number of the superior (right and left) TBLN.     

Role of phosphatidylinositol-anchored proteins in T cell activation

A novel class of cell surface proteins are attached to the plasma membrane via a phosphatidylinositol (PI)-glycan anchoring structure, and these proteins can be selectively removed from the cell surface by the enzyme PI-specific phospholipase C (PI-PLC). Enzyme treatment led to a prolonged reduction in cell surface expression of several PI-anchored proteins. Activation of T cells led to a marked decrease in the ability of PI-PLC to remove PI-anchored surface proteins from the activated T cells. This decrease in PI-PLC sensitivity may reflect an alteration in the PI-glycan anchoring structures, or in a general membrane property, which renders the PI-anchored proteins inaccessible to the enzyme. When murine T lymphocytes were treated with PI-PLC and then stimulated with either Con A, the calcium ionophore A23187 and PMA, or an anti-CD3 mAb, the response to Con A stimulation was inhibited by 90%, whereas the responses to ionophore and PMA or anti-CD3 were not affected. Removal of PI-anchored proteins inhibited an early event in the activation process in response to Con A because both IL-2 production and IL-2R expression were inhibited by the PI-PLC treatment. Inhibition of the Con A response was secondary to removal of a PI-linked protein from the responder T cell population because PI-PLC treatment of T-depleted spleen cells did not alter their ability to act as a source of accessory cells. It is unlikely that removal of the known PI-linked proteins on murine T cells, Thy-1 and Ly-6, can fully account for the inhibition of Con A response because the cell line M2B3, that lacks these surface proteins, responded normally to Con A stimulation. These studies demonstrate that one or more PI-anchored T cell proteins play an important role in an early step of Con A activation, perhaps involving T cell-accessory cell interactions. In contrast, the ability to stimulate T cells by direct cross-linking of TCR/CD3 complex is not dependent on the presence of these PI-anchored proteins.