Role of IL-2 in rat fetal thymocyte development

Early during rat thymus ontogeny, an important proportion of thymocytes expresses IL-2R and contains IL-2 mRNA. To investigate the role of the IL-2-IL-2R complex in rat T cell maturation, we supplied either recombinant rat IL-2 or blocking anti-CD25 mAb to rat fetal thymus organ cultures (FTOC) under several experimental conditions. The IL-2 treatment initially stimulated the growth of thymocytes and, as a result, induced T cell differentiation, but the continuous addition of IL-2 to rat FTOC, as well as the anti-CD25 administration, resulted in cell number decrease and inhibition of thymocyte maturation. These results indicate that immature rat thymocytes bear functional high- affinity IL-2R and that IL-2 promotes T cell differentiation as a consequence of its capacity to stimulate cell proliferation. Modifications in TCR alpha beta repertoire and increased numbers of NKR- P1+ cells, largely NK cells, were also observed in IL-2-treated FTOC. Furthermore, IL-2-responsiveness of different thymocyte subsets changed throughout thymic ontogeny. Immature CD4-CD8-cells responded to IL-2 in two stages, early in thymus development and around birth, in correlation with the maturation of two distinct waves of thymic cell progenitors. Mature CD8+ thymocytes maximally responded to IL-2 around birth, supporting a role for IL-2 in the increased proliferation of mature thymocytes observed in vivo in the perinatal period. Taken together, these findings support a role for IL-2 in rat T cell development.      

Pre-TCR signaling regulates IL-7 receptor alpha expression promoting thymocyte survival at the transition from the double-negative to double-positive stage

The pre-T cell receptor (pre-TCR) and IL-7 receptor (IL-7R) are critical mediators of survival, proliferation and differentiation in immature thymocytes. Here we show that pre-TCR signaling directly maintains IL-7Ralpha expression as developing thymocytes undergo beta-selection. Inhibition of IL-7/IL-7R signaling in (CD44-CD25-) DN4 cells results in decreased generation of double-positive thymocytes due to increased death of rapidly proliferating beta-selected cells. Thus, we identify a mechanism by which pre-TCR signaling controls the selective survival of TCRbeta+ thymocytes, and define a further stage of T cell differentiation in which signaling from a TCR regulates the ability of that cell to respond to cytokine.     

bcl-2 transgene expression promotes survival and reduces proliferation of CD3-CD4-CD8- T cell progenitors

Proliferative expansion and apoptotic cell death play prominent roles in T cell development. The molecular control of cell cycle progression and apoptosis appear to be inter-connected since the Bcl-2 protein can inhibit apoptosis and slow cell cycle progression in cortical thymocytes and mature T cells, particularly during the transition from the quiescent state into the cell cycle. Here the impact of bcl-2 transgene expression on CD3-CD4-CD8- T cell progenitors was assessed. Bcl-2 enhanced the survival of these progenitors at all of the four major differentiation stages, CD25- CD44+ (pro-T1), CD25 + CD44+ (pro- T2), CD25 + CD44- (pro-T3) and CD25-CD44- (pro-T4). However, it reduced cell cycling and slowed turnover only in the pro-T4 subset. From an analysis of bcl-2 transgenic mice expressing a TCR transgene or bearing a mutation in the scid or rag-1 gene we conclude that Bcl-2 inhibits proliferation only of T cell progenitors that are activated via the pre- TCR, not those stimulated via c-Kit and the IL-7 receptor.      

Delineation of human thymocytes with or without functional potential by CD1-specific antibodies

Hematopoietic precursors lacking T cell antigen receptors (TCR-CD3-) and CD4 and CD8 surface markers (i.e. double-negative thymocytes) give rise to functionally mature T lymphocytes. Yet their major progeny are immunologically unresponsive thymocytes in spite of having acquired TCR-CD3 and CD4-CD8. Because only mature thymocytes migrate to peripheral lymphoid organs and most thymocytes die in situ, the knowledge of the events associated with functional maturation in the double-negative thymocyte progeny is a fundamental question in T cell development. We reasoned that a clue to trace the fate of early human thymocytes may perhaps come from the study of the developmental acquisition of CD1 antigen, currently used to define better the functionally inert CD4+8+ (double-positive) stage and absent in mature, medullary thymocytes and peripheral T cells. By using antibodies specific for CD1 (HTA 1/T6) we show here that a large fraction of double-negative thymocytes also express CD1. CD1+3-, CD1+3+, CD1-3+, and CD1-3- subsets all exist. The CD1+3- subset generates CD1+3-4-8+ precursors of CD1+ double-positive cells. A large portion of the CD1+3+ subset bears TCR gamma delta-CD3 complexes. The CD1- subsets are responsive in assays of function, in which they can be stimulated to use the interleukin 2 pathway of proliferation and to mediate cytotoxicity. In contrast, all CD1+ thymocytes behave as functionally inert cells. Thus, the CD1 surface marker delineates human thymocyte precursors and their products which lack, or possess, functional potential in vitro, on both alpha beta and gamma delta lineages.     

Jak3 activation in human lymphocyte precursor cells

Although expression of the Jak3 tyrosine kinase in T lymphocytes has been thought to be restricted to mature, activated cells, mutations of Jak3 can lead to the development of a human severe combined immunodeficiency (SCID) characterized by an absence of peripheral T lymphocytes. We therefore examined in detail the expression of Jak3 throughout human T cell differentiation and show that Jak3 is in fact present throughout the entire developmental process, with high levels expressed in thymocytes. Jak3 is highly expressed in double negative (CD4⁻CD8⁻) cells, one of the earliest stages of thymocyte differentiation, and can be activated via the IL-7 receptor. IL-7 is known to stimulate thymocyte proliferation and initiate re-arrangement of the T cell receptor (TCR) β gene, suggesting that the failure of mutated Jak3 proteins to transduce this signal may be responsible for failures in T cell development. While Jak3 SCID patients possess mature peripheral B cells, we demonstrate that the Jak3 tyrosine kinase is also expressed in human pre-B cells and can be activated by the pre-B cell growth factor IL-7.     

Triggering of thymocyte function by IL-2 as the only exogenous stimulus; analysis of two distinct modes of IL-2-induced thymocyte proliferation and IL-3 secretion in vitro.

Addition of recombinant interleukin-2 (rIL-2) to normal adult murine thymocytes in vitro as the only exogenous stimulus leads to a dose-dependent mitogenic response characterized by two distinct dosage kinetic components. The high-affinity IL-2 thymocyte response is mounted by in vivo-activated (IL-2 receptor light chain positive) thymocytes, while the low-affinity IL-2 response, of larger amplitude, is carried out by resting thymocytes. Addition of IL-2 to thymocytes also triggers intense IL-3 secretory responses with both high and low IL-2 affinity components. Addition of high IL-2 dosages to thymocyte bulk cultures results in a dramatic increase in IL-2 responsiveness for both proliferation and IL-3 secretion on a per viable cell basis and with tightly coupled temporal kinetics. The low-affinity component of IL-2-proliferative and IL-3-secreting responses is carried out by resting mature CD4+ thymocytes, as assessed by negative selection with monoclonal antibodies (mAb) plus complement. The mechanism of resting thymocyte activation by high doses of IL-2 is partially characterized. Depletion of endogenous thymus-adherent cells abolished both proliferation and IL-3 secretion, and addition of splenic accessory cells or peritoneal macrophages to depleted thymocytes restored IL-2 responsiveness. Mature CD4+ thymocytes spontaneously form rosettes with adherent accessory cells, while CD8+ thymocytes do so with much less efficiency. Rosette formation of CD4+, but not of CD8+ thymocytes, can be blocked by anti-CD4 mAb GK1.5. At the same dosage as it prevents rosette formation, mAb GK1.5 also blocks the low-affinity thymocyte response to IL-2. The high-affinity IL-2 response is completely resistant to the action of cyclosporin A (CsA), but the low-affinity IL-2 response, although of much larger amplitude, can be almost completely suppressed by CsA. Together, these results demonstrate that resting CD4+ thymocytes can be induced to proliferation and lymphokine secretion by IL-2 alone in a process that is dependent on interaction with accessory cells, involves CD4 adhesion molecules and triggers activation through a CsA-sensitive pathway. In addition, the results demonstrate that IL-2 alone is able to enhance thymocyte IL-2 responsiveness and IL-3 secretory responses in vitro. The ability of IL-2 to induce and maintain thymocyte function is discussed in the light of these results.     

Interleukin-7 treatment promotes the differentiation pathway of T-cell-receptor-alpha beta cells selectively to the CD8+ cell lineage.

In this report we have studied the influence of interleukin-7 (IL-7) on thymocyte differentiation by evaluating the effects of IL-7 on the generation of T-cell receptor-alpha beta (TCR-alpha beta) and TCR-gamma delta thymocyte subpopulations in rat fetal thymus organ culture. IL-7 enhanced the differentiation pathway of TCR alpha beta thymocytes, first increasing the numbers of immature CD8+ cells, and later those of both CD4+ CD8+ and mature thymocytes. The kinetics of thymocyte migration out of thymic lobes was also accelerated, and the average number of mature TCR-alpha beta phi emigrants per day was increased in the presence of IL-7. Moreover, mature CD4- CD8+ thymocytes were preferentially generated after IL-7 administration. This TCR-alpha beta hi cell population was not actively dividing, indicating that IL-7-promoted thymocyte differentiation was selective to the CD8 cell lineage. Distribution of some TCR-V alpha and TCR-V beta segments among mature thymocytes was also modified in IL-7-treated thymic lobes. On the contrary, the maturation of TCR-gamma delta was not affected by IL-7 addition during the first days of culture, but their numbers sharply increased by day 6 of culture. These results were confirmed with IL-7-treated cultures for 24 hr, showing that IL-7 responsiveness was acquired by TCR-gamma delta cells late in thymus ontogeny. The present results thus indicate a key role for IL-7 in the maturation of TCR-alpha beta thymocytes and the expansion of thymic TCR-gamma delta cells.     

The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiation

We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCRbeta and surface TCRalpha/beta and TCRgamma/delta expressing T cells. Selection for cytoplasmic TCRbeta-positive cells is dependent on preTalpha expression. Both gamma/delta and alpha/beta TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7.     

Endogenously secreted IL-4 is required for mouse thymocytes to become cytotoxic. Human, but not mouse, IL-2 induces a functionally immature thymic subset to secrete IL-4 and become CTL.

Experiments described here demonstrate that the differentiation of mouse thymocytes into cytotoxic T lymphocytes (CTL) requires interleukin-4 (IL-4). To reach this conclusion, we took advantage of our discovery that human and mouse IL-2 have very different effects on the development of CTL from a functionally immature subset of thymocytes. The lobster agglutinin 1 (LAg1)-negative subpopulation of thymocytes proliferated when cultured with concanavalin A (Con A)+ human or mouse IL-2, but these cells became CTL only when cultured with Con A+ human IL-2. Furthermore, Con A+ human IL-2, but not mouse IL-2, stimulated IL-4 production by cells within this population. Con A-induced cytotoxicity by mature LAg1-positive thymocytes and normal thymocytes was also accompanied by secretion of IL-4. The anti-IL-4 mAb 11B11 inhibited induction of cytotoxicity by all thymocyte populations tested. Taken together these experiments indicate that stimuli which induce cytotoxicity by mouse thymocytes also induce the secretion of IL-4, which is necessary for the differentiation of thymocyte CTL precursors into CTL.     

Similar co-stimulation requirements of CD4+ and CD8+ primary T helper cells: role of IL-1 and IL-6 in inducing IL-2 secretion and subsequent proliferation.

Primary murine CD4+ and CD8+ T helper (Th) cells provide help for various immune responses by secreting lymphokines which activate effector cells. The purpose of the present study was to investigate the co-stimulatory signals that, together with T cell receptor (TCR) cross-linking, induce phenotypically distinct primary Th cells to secrete IL-2 and proliferate. We isolated highly purified populations of primary CD4+ or CD8+ T cells and stimulated them in vitro with platebound anti-CD3 mAb. TCR cross-linking by anti-CD3 mAb induced both IL-2 receptor expression and responsiveness to exogenous IL-2, but was not sufficient to induce either IL-2 secretion or T cell proliferation. Rather, for both CD4+ and CD8+ primary Th cells, IL-2 secretion and proliferation required both TCR cross-linking and antigen presenting cell (APC)-derived co-stimulatory signals. Based on G-10 adherence and sensitivity to gamma-irradiation, the APC populations able to induce primary CD4+ Th cells and primary CD8+ Th cells to secrete IL-2 were indistinguishable. In addition, we found that either IL-1 or IL-6 could replace the requirement for APC-derived co-stimulatory signals for IL-2 secretion and proliferation by both primary CD4+ Th cells and primary CD8+ Th cells. Thus, the present study has examined and compared the co-stimulatory requirements of rigorously purified subsets of IL-2-secreting primary CD4+ and primary CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-6 enhances the generation of cytolytic T lymphocytes in the allogeneic mixed leucocyte reaction.

Cytolytic T lymphocytes (CTL) require soluble proteins termed lymphokines to develop lytic activity. In this report we have studied two of the lymphokines involved in the development of CTL during the allogeneic mixed leucocyte reaction (MLR). High doses of dendritic cells induced lytic activity from purified CD8+ cells in both the murine and human MLR. Under these conditions, IL-2 and IL-6 were endogenously produced and secreted. Antibodies to IL-2 or the IL-2 receptor blocked CTL formation; however, anti-IL-6 receptor antibodies only partially inhibited the response while anti-IL-6 antibodies were largely ineffective. When limiting numbers of antigen-presenting cells were used CTL failed to develop, and neither IL-2 nor IL-6 was secreted into the culture supernatant. Although the addition of IL-6 to such cultures was ineffective in generating CTL, the combination of IL-2 and IL-6 resulted in a 4-5-fold increase in lytic activity over that of IL-2 alone. We conclude that in the allogeneic MLR, IL-2 and IL-6 contribute to the generation of lytically active CD8+ cells, and the effect of IL-6 is evident when the dose of antigen-presenting cell is limited.     

Duration of TCR signaling controls CD4-CD8 lineage differentiation in vivo

The duration of T cell receptor (TCR) signaling is thought to be important for thymocyte differentiation into the CD4 or CD8 lineage. However, the in vivo relevance of this hypothesis is unclear. Here we divided T cell positive selection into genetically separable developmental steps by confining TCR signal transduction to discrete thymocyte developmental windows. TCR signals confined to the double-positive thymocyte stage promoted CD8, but not CD4, lineage differentiation. Major histocompatibility complex (MHC) class II-restricted thymocytes were, instead, redirected into the CD8 lineage. These findings support the hypothesis that distinct kinetics of MHC class I- and MHC class II-induced TCR signals direct intrathymic developmental decisions.     

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.     

Glycosyltransferase regulation mediated by pre-TCR signaling in early thymocyte development

CT1 is a carbohydrate moiety of CD45 that is expressed on fetal thymocytes in vivo. Examination of CT1 expression on thymocyte subsets revealed that primarily pro-T cells (CD44+ CD25+) and pre-T cells (CD44- CD25+) expressed CT1. Interestingly, non-T-lineage committed lymphoid progenitors (CD44+ CD25-) lacked CT1 indicating temporal regulation of expression of this determinant in early T-lineage committed development. In addition, CT1 was expressed by the majority of thymocytes in RAG-2(-/-) mice where thymocyte development is blocked at the CD44- CD25+ stage. Since late pre-T cells (CD44- CD25-) lacked the CT1 epitope we tested whether pre-TCR triggering regulated CT1 expression. Injection of CD3epsilon-specific mAb into RAG-2(-/-) mice induces differentiation of immature thymocytes to the double-positive stage of thymocyte development. Using this system, we demonstrated that expression of CT1 by RAG-2(-/-) thymocytes was rapidly lost from pre-T cells following anti-CD3 mAb treatment. Furthermore, the decline in CT1 expression induced by CD3 signaling paralleled a loss of mRNA for the glycosyltransferase responsible for the addition of CT1 to CD45. Flow cytometric analysis also revealed that the loss of the CT1 epitope was inversely correlated with an increase in peanut agglutinin ligand expression, demonstrating a complex regulation of cell surface glycosylation at a critical juncture in thymocyte development.      

Accelerated transition from the double-positive to single-positive thymocytes in G alpha i2-deficient mice

Deletion of alpha i2 subunit of heterotrimeric G proteins induces a 2-4-fold increase in the proportions of CD4 and CD8 single-positive (SP) thymocytes as compared with wild-type littermates, but how G alpha i2 is involved in thymocyte development is unknown. To determine a role for G alpha i2 in a specific developmental stage of thymocyte differentiation, we studied the ontogeny of thymocytes in G alpha i2-deficient mice. Our data show that an accelerated transition from the double-positive (DP) to SP thymocytes, rather than impairment in thymic emigration, accounts for a high proportion of the SP thymocytes in the absence of G alpha i2. Lack of G alpha i2 greatly augmented a response of thymocytes to TCR-mediated stimulation, as evidenced by enhanced proliferation of the DP thymocytes upon ligation of the TCRs. The augmented response may be the reason behind the expedited transition from the DP to SP thymocytes in the animal. In accordance with this, effects of G alpha i2 deficiency on CD8 or CD4 SP thymocyte differentiation required engagement of the TCRs with either MHC class I or MHC class II molecule. The abnormal thymocyte development resulted in an increase in positive selection, altered usage of TCR Vbeta gene, aberrant development of CD4+ CD25+ T regulatory cells and untimely thymic involution, the contribution of which to colitis development in the animal is discussed. These findings reveal a previously unappreciated role for G alpha i2 protein in clonal selection and functionality of thymocytes.     

The RhoA- and CDC42-specific exchange factor Dbs promotes expansion of immature thymocytes and deletion of double-positive and single-positive thymocytes

Specific members of the Rho family of GTPases exert unique influences on thymocyte proliferation, differentiation and deletion. Dbs is a guanine nucleotide exchange factor which is expressed throughout thymocyte development and is able to activate the Rho family GTPases CDC42, RhoA and RhoG. Transgenic mice expressing an activated form of Dbs had increased numbers of double-negative thymocytes. The Dbs transgene promoted expansion of double-negative thymocytes in the absence of pre-TCR, but had no effect on pre-TCR-dependent differentiation of double-negative thymocytes into double-positive thymocytes. Transgenic double-positive thymocytes were proliferative in vivo, but were also susceptible to apoptosis in vivo and in vitro. The transgenic single-positive thymocytes had attenuated proliferative responses following TCR ligation, and were depleted rather than expanded during culture in the presence of anti-CD3. When expressing a positively selectable TCR, transgenic double-positive thymocytes were increased in number and activated, but the output of single-positive thymocytes was reduced. Transgenic double-positive thymocytes were acutely sensitive to deletion by TCR ligation in vivo. These results indicate that activation of Dbs has the potential to promote proliferation throughout thymocyte development, but also sensitizes double-positive and single-positive thymocytes to deletion.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

Interleukin-4 induces expression of the CD45RA antigen on human thymocyte subpopulations

Interleukin-4 (IL-4) is a multifunctional lymphokine which promotes the growth and/or maturation of multiple cell types. We have examined the ability of IL-4 to promote the phenotypic maturation of subsets of human thymocytes. When cultured in serum-free medium supplemented with recombinant IL-4, a subset of immature CD3-CD45RA- human thymocytes ceased to express the CD1 common thymocyte antigen and acquired phenotypic features characteristic of relatively mature thymocytes, such as high-density expression of the CD3 antigen and de novo expression of the CD45RA isoform of the common leukocyte antigen family. These changes, which were not seen in cells cultured in medium alone, occurred over an 8-9 day period and were accompanied by a significant increase in cell size. The CD45RA+ cells that derived from these immature CD3-CD45RA- precursors were mainly CD4-CD8- or CD8+ cells, and a significant proportion of these cells expressed the T cell receptor delta chain. IL-4 also increased expression of the CD45RA antigen on the more mature CD3+ thymocyte population. However, the CD45RA+ cells derived from IL-4 stimulated CD3+ thymocyte precursors expressed either the CD4 or the CD8 antigen, and virtually all expressed alpha/beta TCR chains. Studies of cell viability and cell growth indicated that these findings were due to direct changes in the phenotype of responsive cells rather than the growth or selective survival of a small number of mature thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.