Thymic stroma-derived T cell growth factor (TSTGF). I. Functional distinction of TSTGF from interleukins 2 and 4 and its preferential growth-promoting effect on helper T cell clones

A recently established thymic stroma-derived cell line (TSCL) supported the growth of the interleukin (IL) 2-dependent, antigen-specific helper T cell (Th) clone, 9-16, without requirement for IL-2 and antigen, and such growth was substituted by a factor produced into cultures by this established TSCL. This substance, thymic stroma-derived T cell-growth factor (TSTGF), was capable of inducing the proliferation of various Th clones including 9-16 Th clone, but not of cytotoxic T cell clones. TSTGF-induced growth promotion was obtained in a dose-dependent fashion and in maintaining antigen specificity of Th clones. The culture supernatant from the TSCL did not contain detectable level of IL-1, IL-2, IL-3, IL-4, or interferon activity. The proliferation of 9-16 Th clone was stimulated by recombinant IL-2 and IL-4 as well as TSTGF, but not by IL-1, IL-3, or interferons. However, the proliferation of this Th clone by IL-2 or IL-4 was almost completely inhibited by anti-IL-2 receptor or anti-IL-4 monoclonal antibody, respectively, whereas TSTGF-induced growth of 9-16 Th clones was not affected by either type of antibody, demonstrating that TSTGF is functionally distinct from IL-2 and IL-4. In addition, TSTGF activity was also obtained from the culture supernatant of the primary thymic explant, which was freshly prepared. These results indicate that the primary thymic explant as well as an established TSCL produce factors capable of promoting the growth of helper but not cytotoxic type of T cells in the absence of T cell growth factors thus far defined.     

Responsiveness of fetal and adult CD4-, CD8- thymocytes to T cell activation

Day-14 fetal CD4-, CD8- thymocytes showed a greater proliferative response to PMA + IL-4 than did adult double-negative thymocytes. In contrast, adult double-negative thymocytes were more responsive to PMA + IL-1 + IL-2 or to IL-1 + IL-2 alone. The adult double-negative thymocytes showed significantly greater proliferation than fetal thymocytes after stimulation via anti-CD3 or anti-Thy-1 in the presence or absence of interleukins (IL-1 + IL-2 or IL-4). Adult CD4-, CD8- thymocytes also exhibited greater calcium mobilization following anti-CD3 stimulation IL-2-dependent activation with anti-Thy-1 or IL-1 + IL-2 in the absence of PMA resulted in marked expansion of CD 3+, F23.1+, CD4-, CD8- thymocytes, a population absent in fetal thymocytes but constituting 4% of pre-cultured CD4-, CD8- adult thymocytes. IL-4 + PMA failed to expand this CD 3+ population. It is hypothesized that before expression of functional TCR, T cell development may be more dependent on activation pathways not using IL-2; after TCR expression, IL-2-dependent pathways, including Thy-1-mediated stimulation, become functional.     

Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.     

Expression and function of a 90-kilodalton homodimeric molecule (10D1 antigen) on human thymocytes

The 10D1 Ag is a 90-kDa homodimeric molecule specifically expressed on a subpopulation of human T cells, and is involved in an alternative pathway of T cell activation. In the present study, we have examined the expression and function of the 10D1 Ag on human thymocytes. Three-color FMF analysis showed that the 10D1 Ag was highly expressed on minor but distinct subpopulations of double-negative and CD4 single-positive thymocytes, and weakly on a part of double-positive thymocytes, but not on CD8 single-positive thymocytes. In double-negative thymocytes, the vast majority of 10D1+ cells were immature thymocytes of CD7+2+3- phenotype. Interestingly, 10D1 mAb could induce the proliferation of CD4 single-positive thymocytes in the presence of goat anti-mouse Ig to cross-link the 10D1 Ag. The treatment of thymocytes with OKT4 mAb plus C but not with OKT8 mAb plus C totally abrogated the proliferative response induced by 10D1 mAb, indicating that the 10D1-responsible thymocytes were of CD4+8- phenotype. This 10D1 mAb-induced thymocyte proliferation was perfectly dependent on the endogenous IL-2/IL-2R system since a complete inhibition was observed with anti-IL-2 and anti-IL-2R mAb. The proliferating CD4 single positive thymocytes predominantly expressed the IL-2R alpha (p55) but not a detectable level of the IL-2R beta (p75). These results indicate that, although the 10D1 Ag can be detected on the CD7+2+3-4-8- thymocytes, its functional expression is restricted to a minor more mature CD4+ thymocyte population as well as in peripheral blood T cells, and the implications of these findings are discussed.     

IL-7 maintains the T cell precursor potential of CD3-CD4-CD8- thymocytes.

We and other investigators have reported that IL-4 (in the presence of PMA) or IL-7 (used alone) induce proliferation of both adult and fetal (gestation day 15) CD4-CD8- thymocytes. These results suggested that these cytokines may be growth factors for pre-T cells. However, we recently observed that among adult CD4-CD8- thymocytes, only the CD3+ subset proliferates in response to IL-7, whereas IL-4 + PMA induces proliferative responses in both CD3- and CD3+ subsets. Thus, we concluded that IL-7 used alone is not a potent growth stimulus for adult thymic CD3-CD4-CD8- triple negative (TN) T cell precursors. Interestingly, the viability of adult TN thymocytes in culture was improved by IL-7 for up to 1 wk, in spite of the inability of IL-7 to induce significant [3H]TdR incorporation in these cells. After culture in IL-7 for 4 days, the viable cells remained CD4-CD8-, but 25 to 35% expressed CD3 whereas the rest remained CD3-. In contrast, most of the cells cultured with IL-4 + PMA for 4 days remained TN. To investigate whether adult TN thymocytes that survive in vitro in the presence of IL-4 + PMA or IL-7 retain T cell progenitor potential, we tested whether they could reconstitute lymphoid cell-depleted (2-deoxyguanosine-treated) fetal thymus organ cultures. Our results demonstrate that TN cells cultured in IL-7 retain T cell progenitor potential.     

Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2

Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

IL-4/B cell stimulatory factor 1 stimulates T cell growth by an IL-2-independent mechanism

Resting T cells are stimulated to synthesize DNA by IL-4 and phorbol myristate acetate (PMA). This response of T cells to IL-4 plus PMA is independent of the action of IL-2 as judged by 1) the lack of IL-2 in supernatants of stimulated cells, 2) the failure to detect IL-2 mRNA in stimulated cells by in situ hybridization, 3) the inability of anti-IL-2R antibody and of anti-IL-2 antibody to block responses to IL-4 plus PMA, and 4) the failure of cyclosporin A to block responses. T cells also respond to anti-CD3 antibodies and IL-4 in the presence of anti-IL-2R antibodies. IL-4 stimulation of growth of the long term T cell line HT-2 also appears to be independent of the action of IL-2. No IL-2 mRNA is found in IL-4-stimulated HT-2 cells by Northern blotting; the response of HT-2 cells to IL-4 is not blocked by anti-IL-2R antibodies; the response of HT-2 cells to IL-4 is not inhibited by cyclosporin A. Although IL-4 stimulation of T cells is independent of IL-2, IL-4 plus PMA treatment of resting T cells does cause enhanced expression of IL-2R and prepares cells to proliferate to IL-2 alone. In both these properties IL-4 resembles IL-2. These experiments lead us to conclude that IL-4 can act as an alternative to IL-2 as authentic T cell growth factor.     

Co-stimulation of T cell proliferation by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) exhibits diverse regulatory roles in the immune system and has been described as a potent inhibitor of lymphocyte and hemopoietic progenitor cell growth. The present studies investigated the effects of TGF-beta 1 on murine T cell proliferation triggered through the T cell receptor/CD3 complex. In contrast to previously reported T cell growth inhibition, TGF-beta 1 costimulated splenic T cell proliferation in the presence of immobilized anti-CD3 antibody 2C11, with maximal effect at anti-CD3 concentration of 50 micrograms/ml. Although TGF-beta 1 induced a modest increase in IL-2R display, TGF-beta 1 co-stimulated proliferation was largely independent of IL-2 and/or IL-4. Anti-IL-2 and/or anti-IL-4 antibody did not significantly block the TGF-beta 1 co-stimulated T cell growth, and no IL-2 or IL-4 bioactivity was detected in TGF-beta 1 co-stimulated cultures. TGF-beta 1 did not enhance IL-2 mRNA expression beyond control levels. However, TGF-beta 1 co-stimulation caused an accelerated evolution of a memory or mature T cell population phenotype. Both CD4+ and CD8+ T cell subsets were significantly enriched for cells of the mature CD45RBloPgp-1hi phenotype, in comparison with T cells stimulated by anti-CD3 alone or with PMA, and CD8+ T cells predominated. These results thus provide initial evidence that TGF-beta 1 is capable of bifunctional T cell growth regulation, which occurs largely via an IL-2- and IL-4-independent pathway.     

Growth-promoting activity of IL-1 alpha, IL-6, and tumor necrosis factor-alpha in combination with IL-2, IL-4, or IL-7 on murine thymocytes. Differential effects on CD4/CD8 subsets and on CD3+/CD3- double-negative thymocytes

Many cytokines (including IL-1, IL-2, IL-4, IL-6, and TNF-alpha) have been shown to induce thymocyte proliferation in the presence of PHA. In this report, we demonstrate that certain cytokine combinations induce thymocyte proliferation in the absence of artificial comitogens. IL-1 alpha, IL-6, and TNF-alpha enhanced the proliferation of whole unseparated thymocytes in the presence of IL-2, whereas none of them induced thymocyte proliferation alone. In contrast, of these three enhancing cytokines, only IL-6 enhanced IL-4-induced proliferation. We also separated thymocytes into four groups based on their expression of CD4 and CD8, and investigated their responses to various cytokines. The results indicate that each cytokine combination affects different thymocyte subsets; thus, IL-1 alpha enhanced the proliferation of CD4-CD8- double negative (DN) thymocytes more efficiently than IL-6 in the presence of IL-2, whereas IL-6 enhanced the responses of CD4+CD8- and CD4-CD8+ single positive (SP) thymocytes to IL-2 or IL-4 better than IL-1 alpha. TNF-alpha enhanced the proliferation of both DN and both SP subsets in the presence of IL-2 and/or IL-7. None of these combinations induced the proliferation of CD4+CD8+ double positive thymocytes. Finally, DN were separated into CD3+ and CD3- populations and their responsiveness was investigated, because recent reports strongly suggest that CD3+ DN thymocytes are a mature subset of different lineage rather than precursors of SP thymocytes. CD3+ DN proliferated in response to IL-7, TNF-alpha + IL-2, and IL-1 + IL-2. CD3- DN did not respond to IL-7 or to IL-1 + IL-2, but did respond to TNF-alpha + IL-2. Finally, we detected TNF-alpha production by a cloned line of thymic macrophages, as well as by DN adult thymocytes. These results suggest that cytokines alone are capable of potent growth stimuli for thymocytes, and indicate that different combinations of these molecules act selectively on thymocytes at different developmental stages.     

IL-4 (B cell stimulatory factor 1) exhibits thymocyte growth factor activity in the presence of IL-2

The proliferative activity of thymocytes cultured with IL-2 and submitogenic concentrations of PHA is increased by 3- to 10-fold in the presence of IL-4. In contrast, IL-4 alone is unable to induce proliferative activity in thymocyte cultures and its synergistic activity is only apparent to concentrations of IL-2 above 1 U/ml. The costimulatory activity of IL-4 is abrogated by the monoclonal anti-IL-4 antibody 11B11. Furthermore, potentiation of the IL-2-mediated thymocyte proliferation is not seen with IL-1, IL-3, IFN-gamma, and granulocyte-macrophage CSF. Thymocytes are at least as responsive to IL-4 as B cells and the IL-4 costimulatory activity in fractionated thymocytes appears to be restricted mainly to the Lyt-2+/L3T4- population. In contrast, purified resting mature T cells do not respond to IL-4 plus IL-2, although they did proliferate in response to IL-4 in combination with PMA. These findings indicate that thymocytes and mature T cells are responsive to the costimulatory activity of IL-4 under quite different conditions, and that IL-4 may play an important role in thymocyte maturation in the thymus.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

IL-2 and IL-4 stimulate different subpopulations of double-negative thymocytes

Double-negative (CD4-/CD8-) thymocytes from young adult mice can be separated into two distinct subpopulations on the basis of the binding of mAb 7D4 directed against the receptor for IL-2. The 7D4+ cells have predominantly nonrearranged TCR beta-chain genes and express incomplete 1.0-kb beta-messages, whereas the 7D4- cells have rearranged beta-genes and express complete 1.3-kb as well as incomplete 1.0-kb beta-messages. These two populations of double-negative thymocytes also differ in their responses to IL-2 and IL-4. The 7D4+ cells are nonresponsive to IL-2 alone or IL-2 plus PMA but they are stimulated to proliferate by the combination of IL-4 and PMA. In contrast, the 7D4- cells vigorously proliferate in response to IL-2 alone or IL-2 plus PMA but they respond poorly to IL-4 alone or IL-4 plus PMA. These results suggest that IL-2 and IL-4 may be involved in the stimulation of immature thymocytes at distinct steps of their differentiation. IL-4 together with PMA stimulate immature thymocytes which seem to express the IL-2R but do not respond to IL-2.     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

Mitogen-induced IL-2 production and proliferation at defined stages of T helper cell development.

Th cell development inside the thymus can be defined on the basis of qualitative and quantitative CD4 and CD8 marker expression and follows the pathway of CD4-8- cells----CD4+8+ cells----CD4+8low cells----CD4+8- cells, which presumably emigrate to seed the periphery and serve as functionally mature Th cells. The various cell subpopulations at defined developmental stages were isolated by electronic cell sorting and examined for mitogen induced IL-2 production and cell proliferation responses. For TCR-alpha beta-bearing CD4+8+ and CD4+8low thymocytes that are actively engaged in positive and negative selection processes, negligible to low levels of IL-2 production and cell proliferation were observed in response to TCR:CD3 triggering or to the combined activation of protein kinase C and calcium mobilization mediated by PMA and ionomycin, respectively. For CD4-8- TCR-alpha beta early thymocytes that have not yet entered the selection process, PMA + ionomycin induced significant cell proliferation but little IL-2 production, in the absence of added IL-1. However, addition of IL-1 caused a powerful induction of IL-2 production that was accompanied by increased cell proliferation. Triggering of the TCR:CD3 complex had no effect on CD4-8-TCR(-)-alpha beta thymocytes as they do not express detectable levels of TCR-alpha beta. For thymus CD4+8- Th cells, the first cells that have completed TCR repertoire selection, vigorous proliferation was observed in response to TCR:CD3 triggering in the presence of added IL-2. However, the development of IL-2 responsiveness was not accompanied by high level IL-2 inducibility as TCR:CD3 triggering caused only marginal IL-2 production. In contrast, spleen CD4+8- T cells, the most "mature" representatives of Th cells, expressed high levels of IL-2 production as well as IL-2 responsiveness in response to TCR:CD3-mediated stimulation. The lack of anti-TCR-induced IL-2 production by thymus CD4+8- T cells was not due to an intrinsic defect as high levels of IL-2 production was induced by PMA + ionomycin. Possible reasons for the temporal acquisition and differential control of IL-2 inducibility and IL-2 responsiveness are discussed in the context of established Th cell development pathway.     

Expression of ets genes in mouse thymocyte subsets and T cells

The cellular ets genes (ets-1, ets-2, and erg) have been identified by their sequence similarity with the v-ets oncogene of the avian erythroblastosis virus, E26. Products of the ets-2 gene have been detected in a wide range of normal mouse tissues and their expression appears to be associated with cell proliferation in regenerating liver. In contrast, the ets-1 gene was previously shown to be more highly expressed in the mouse thymus than in other tissues. Because the thymic tissue contains various subsets of cells in different stages of proliferation and maturation, we have examined ets gene expression in fetal thymocytes from different stages of development, in isolated subsets of adult thymocytes, and in peripheral T lymphocytes. Expression of the ets-1 gene was first detected at day 18 in fetal thymocytes, corresponding to the first appearance of CD4+ (CD4+, CD8-) thymocytes, and reaches maximal/plateau levels of expression in the thymus at 1 to 2 days after birth. The ets-2 gene expression is detected at least 1 day earlier, coinciding with the presence of both double-positive (CD4+, CD8+) and double-negative (CD4-, CD8-) blast thymocytes and reaches maximal/plateau levels 1 day before birth. In the adult thymus, ets-1 and ets-2 mRNA expression is 10- to 8-fold higher respectively in the CD4+ subset than in the other subsets examined. Higher levels of p55 ets-1 protein were also shown to exist in the CD4+ subset. Because the CD4+ thymic subset is the pool from which the CD4+ peripheral, helper/inducer T cells are derived, the ets gene expression was examined in lymph node T cells. Both the CD4+ and the CD8+ T cells subsets had lower ets RNA levels than the CD4+ thymocytes. These results suggest that ets-2 and more particularly ets-1 gene products play an important role in T cell development and differentiation and are not simply associated with proliferating cells, which are observed at a higher frequency in fetal thymocytes, or dull Ly-1 (low CD5+), and double-negative (CD4-, CD8-) adult thymocytes. Selectively enhanced expression of ets-1 gene may be observed in thymic CD4+ thymocytes because these cells have uniquely encountered MHC class II or other Ag in the thymic environment. These cells may have been subsequently stimulated to activate the ets genes in conjunction with their differentiation of helper/inducer function(s) and expression of mature TCR.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proliferation in vitro and interleukin production by 14 day fetal and adult Lyt-2-/L3T4- mouse thymocytes

When 14 day fetal mouse thymocytes, which are phenotypically Lyt-2-/L3T4-(2-4-), were stimulated in vitro with a combination of phorbol myristate acetate (PMA) and the calcium ionophore ionomycin, they proliferated without addition of exogenous interleukins and/or growth factors. Addition of exogenous IL 2 resulted in a slight enhancement of fetal thymocyte proliferation. By using factor-dependent indicator cell lines, this proliferation was shown to be accompanied by the production of IL 2 and IL 3. However, phenotypic analysis by using flow microfluorometry and monoclonal antibodies to Lyt-2 and L3T4 showed little differentiation among proliferating 2-4-fetal thymocytes. Interestingly, the in vitro growth of PMA + ionomycin-stimulated fetal thymocytes appeared to be IL 2 dependent in that it was inhibited by a monoclonal antibody to the IL 2 receptor. The results obtained with fetal thymocytes were compared with those obtained when using 2-4- thymocytes from adult mice.     

A novel T cell-activating molecule (THAM) highly expressed on CD4-CD8- murine thymocytes

Recent studies have focused on the potential role of accessory molecules such as CD2, CD28, Thy-1, or TAP in the delivery of activating signals to thymocytes through antigen-independent pathways. To better understand the molecular interactions involved in the expansion of early thymic immigrants, rat mAb were raised against murine thymocyte-surface molecules and screened for their capacity to trigger thymocyte proliferation. One of these mAb (H194-112, IgG2a) was found to recognize a novel heterodimeric thymocyte-activating molecule (THAM) of Mr = 110,000 to 128,000. Flow cytometric analyses and staining patterns on frozen thymus sections subdivided adult thymocytes in three subsets expressing THAM at either low (10%), moderate (80%), or high (5 to 8%) cell-surface density; these cell groups were found to correspond, respectively, to the medullary, the cortical, and the immature CD4-CD8-, J11d+ thymocytes, in which the T cell precursor pool is included. Moreover, most (90%) day 16 fetal thymocytes were also found to upregulate THAM cell-surface expression. The THAMhigh cells were localized in the subcapsular area of the neonatal thymus and scattered throughout the adult organ. Cross-linked mAb H194-112 induced the proliferation of both immature and mature thymocytes in the presence of either PMA or IL-1 and IL-2. The observation that early thymocytes up-regulate THAM along with the IL-2R suggests that this molecule might be involved in an important activation pathway during thymocyte differentiation.     

Mechanism of T cell proliferation in vivo: analysis of IL-2 receptor expression and activation of c-myc and c-myb oncogenes during lymphatic regeneration

The mechanism of T cell proliferation was studied using in vivo lymphatic regeneration as the model. Lymphatic regeneration was induced by injecting a sublethal dose (300 mg/kg) of cyclophosphamide (Cy) into mice. Majority of the regenerating splenic T cells were found to be in the cell cycle, nearly 30% being found in S/G2+M phases resembling the ratio obtained for mitogen activated T cells in vitro. Expression of interleukin-2 receptor (IL-2R) was defined by the monoclonal anti-IL-2R antibody, AMT-13. Only 1-3% of regenerating T cells were IL-2R positive (while about 30% of the in vitro activated T cells were IL-2R positive). Accordingly, these cells did not respond to IL-2 in vitro. However, when the freshly isolated regenerating T cells were cultured in the presence of Con A or PMA + ionophore A 23187, IL-2R was readily induced. The regenerating T cells were further analyzed for the expression of the cellular oncogenes c-myc and c-myb. These cells expressed about three times more c-myb mRNA than Con A-stimulated T cells and the levels were comparable to those seen in thymocytes. By contrast, the amount of c-myc mRNA was similar in the regenerating T cells and in Con A-activated T cells, but weak or barely detectable in splenocytes and thymocytes. Taken together, our results imply that the vigorous T cell proliferation during cyclophosphamide-induced lymphatic regeneration is independent of the IL-2/IL-2R hormone system, like T-cell precursor proliferation in the thymus, and is characterized by both high c-myb expression typical for thymocytes and high c-myc expression typical for in vitro proliferation-activated T cells.     

Regulatory T cell properties of chicken CD4+CD25+ cells

Chicken CD4(+)CD25(+) cells were characterized for mammalian regulatory T cells' suppressive and cytokine production properties. Anti-chicken CD25 mAb was produced in mice and conjugated with a fluorescent tag. The specificity of the Ab against chicken CD25 was confirmed by evaluating Con A-induced CD25 upregulation in thymocytes and by quantifying the CD25 mRNA content of positive and negative cells identified by anti-chicken CD25 Ab. The percentage of CD4(+)CD25(+) cells, expressed as a percentage of CD4(+) cells, in thymus and blood was ～3-7%, in spleen was 10%, and in cecal tonsil, lung, and bone marrow was ～15%. Bursa had no detectable CD4(+)CD25(+) cells. CD25(+) cells were mostly CD4(+) in the thymus, whereas in every other organ studied, CD25(+) cells were distributed between CD4(+) and CD4(-) cells. Chicken thymic CD4(+)CD25(+) cells did not proliferate in vitro in the absence of recombinant chicken IL-2 (rCIL-2). In the presence of rCIL-2, PMA plus ionomycin or Con A stimulated CD4(+)CD25(+) cell proliferation, whereas anti-CD3 plus CD28 did not stimulate CD4(+)CD25(+) cell proliferation. Naive CD4(+)CD25(+) cells had 29-fold more IL-10 mRNA and 15-fold more TGF-β mRNA than the naive CD4(+)CD25(-) cells. Naive CD4(+)CD25(+) had no detectable IL-2 mRNA. Both naive and PMA plus ionomycin-stimulated thymic CD4(+)CD25(+) cells suppressed naive T cell proliferation. The suppressive properties were partially contact dependent. Supplementing CD4(+)CD25(+) cell coculture with rCIL-2 reversed the suppressive properties of CD4(+)CD25(+) cells. Chicken CD4(+)CD25(+) cells have suppressive properties similar to that of mammalian regulatory T cells.