Expression of interleukin-1 receptors in the later period of foetal thymic organ culture and during suspension culture of thymocytes from aged mice

Interleukin-1 has been reported to be involved in thymocyte development by exerting a variety of effects on immature CD4-CD8- double-negative (DN) thymocytes. In contrast to the well-documented involvement of IL-1 in thymocyte development, expression of IL-1 receptors (IL-1R) on thymocytes has not been well demonstrated. In the present study, expression of IL-1R on the developing thymocytes was investigated. Although normal thymocytes barely express IL-1R, expression of IL-1R (type I) substantially increased at days 12-15 of foetal thymic organ culture (FTOC), with an increase of the DN subset. The CD4/CD8 profile of the IL-1R (type I)+ cells showed that these cells were mostly restricted to the DN and CD4+CD8+ subsets. Interestingly, in vitro culture of the thymocytes from an aged mouse, but not those from young adult or newborn mice, revealed similar results to those of FTOC. In addition, half of the IL-1R+ cells that increased in the later period of FTOC were gammadelta thymocytes. These results demonstrate IL-1R expression on thymocytes during ex vivo culture and suggest that IL-1R is expressed in a certain environment during normal thymocyte differentiation.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

Cytokine production by mature and immature CD4-CD8- T cells. Alpha beta-T cell receptor+ CD4-CD8- T cells produce IL-4.

This study follows our previous investigation describing the production of four cytokines (IL-2, IL-4, IFN-gamma, and TNF-alpha) by subsets of thymocytes defined by the expression of CD3, 4, 8, and 25. Here we investigate in greater detail subpopulations of CD4-CD8- double negative (DN) thymocytes. First we divided immature CD25-CD4-CD8-CD3- (CD25- triple negative) (TN) thymocytes into CD44+ and CD44- subsets. The CD44+ population includes very immature precursor T cells and produced high titers of IL-2, TNF-alpha, and IFN-gamma upon activation with calcium ionophore and phorbol ester. In contrast, the CD44- subset of CD25- TN thymocytes did not produce any of the cytokines studied under similar activation conditions. This observation indicates that the latter subset, which differentiates spontaneously in vitro into CD4+CD8+, already resembles CD4+CD8+ thymocytes (which do not produce any of the tested cytokines). We also subdivided the more mature CD3+ DN thymocytes into TCR-alpha beta- and TCR-gamma delta-bearing subsets. These cells produced cytokines upon activation with solid phase anti-CD3 mAb. gamma delta TCR+ DN thymocytes produced IL-2, IFN-gamma and TNF-alpha, whereas alpha beta TCR+ DN thymocytes produced IL-4, IFN-gamma, and TNF-alpha but not IL-2. We then studied alpha beta TCR+ DN T cells isolated from the spleen and found a similar cytokine production profile. Furthermore, splenic alpha beta TCR+ DN cells showed a TCR V beta gene expression profile reminiscent of alpha beta TCR+ DN thymocytes (predominant use of V beta 8.2). These observations suggest that at least some alpha beta TCR+ DN splenocytes are derived from alpha beta TCR+ DN thymocytes and also raises the possibility that these cells may play a role in the development of Th2 responses through their production of IL-4.     

Selective stimulation of thymocyte precursors mediated by specific cytokines. Different CD3+ subsets are generated by IL-1 versus IL-2.

The sequence of activation signals that stimulate proliferation, differentiation, and selection of mature T cell subsets from immature, dull-CD5+/CD4-, CD8- double negative (bCD5), (dCD5/DN) thymocytes are still unclear. However, it is likely that cytokines play integral roles in these events. Here we report that IL-1, in the presence of Con A, supports the proliferation and differentiation of highly purified dCD5/DN precursors into bright-CD5+ DN, CD2- lymphocytes with an apparently mature phenotype. These cells express CD3 and preferentially express the products of two TCR gene families, V beta 8 and V beta 6, whose expression is dependent on the allelic expression of the Mls-1 locus. Experiments, using DN thymocytes mixed with purified dCD5 subset of DN cells from a congenic strain of mice (i.e., expressing two different alleles of CD5) have shown that the cells that are stimulated by IL-1 and comitogen are derived from the immature dCD5 subset and not from the mature bCD5 cells contained within the DN subset. In contrast, IL-2 with the co-mitogen stimulates three- to fourfold higher levels of proliferation, from the same purified immature precursor population, and nearly a twofold increase in cell yield. However, the cells that were generated from precursor thymic cells stimulated with IL-2 represent a completely different T cell subset compared to IL-1-generated cells; these IL-2-stimulated cells express comparable levels of CD3, but also express substantial levels of CD2 and the TCR-gamma/delta, and a subset expresses CD8. These data suggest that these two TCR-alpha/beta and TCR-gamma/delta subsets of mature thymocytes use different cytokines and therefore possibly different stromal interactions to initiate differentiation.     

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.     

Contrasting effect of transforming growth factor type beta 1 (TGF-β1) on proliferation and interleukin-2 receptor expression in activated and rapidly cycling immature (CD3−CD4−CD8−) thymocytes

Transforming growth factor β (TGF-β) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4–6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin-1 (IL-1), we recently found that it up-regulates interleukin-2-receptor (IL-2R) expression. Since EL 4–6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF-β1 on the IL-2R 55kD α chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF-β1 was able to increase both the percentage of CD3^?DN cells expressing IL-2Rα chains and the expression of IL-2Rα chain in these cells. This stimulatory effect of TGF-β1 was distal from early transduction events. In addition, TGF-β1 was found to modulate CD3^?DN cell proliferation. During differentiation in the thymus, CD3^?DN cells transiently express the IL-2Rα chain of the IL-2R and these IL-2R⁺ CD3^?DN cells are preprogrammed to down-regulate the IL-2Rα chain and up-regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF-β1 on IL-2Rα chain expression in these in vitro differentiating CD3^?DN cells. We found that TGF-β1 neither significantly affected IL-2R expression nor changed CD4 or CD8 expression. Hence, in CD3^?DN cells, the effect of TGF-β1 on IL-2R expression seems to be restricted to proliferating cells. © 1993 Wiley-Liss, Inc.     

In vitro induction of CD8 expression on thymic pre-T cells. I. Transforming growth factor-beta and tumor necrosis factor-alpha induce CD8 expression on CD8- thymic subsets including the CD25+CD3-CD4-CD8- pre-T cell subset.

We previously reported that IL-7 maintains the viability and differentiation potential of CD25 (IL-2R p55) positive CD3-CD4-CD8- thymic pre-T cells in vitro. This culture system is suitable for studying signals that regulate differentiation of T cell precursors in the thymus. In this study, we screened cytokines for their capacity to induce CD4 or CD8 in murine thymic pre-T cells cultured with IL-7. Of 15 cytokines tested, only transforming growth factor (TGF-beta) and TNF-alpha induced CD8 (Lyt-2), while no cytokine was able to induce CD4 on CD25+CD3-CD4-CD8- thymocytes. The combination of TGF-beta and TNF-alpha was synergistic, and the majority of cells recovered after 2 to 3 days in culture expressed CD8 (but not CD3 or CD4). A similar effect of TGF-beta and TNF-alpha was observed using day-15 fetal thymocytes, CD3+CD4-CD8- or CD3+CD4+CD8- adult thymocytes, although the combination of these cytokines resulted in an additive rather than a synergistic effect in these subsets. In contrast, neither TGF-beta nor TNF-alpha induced CD8 expression on splenic CD4+CD8- T cells. These observations suggest a role for these cytokines in the induction of CD8 expression in CD8- thymocyte subsets including CD3-CD4-CD8- thymic pre-T cells.     

Human thymocytes do not respond to interleukin-2 after removal of mature "bright" CD5 positive cells

Interleukin-2 receptors (IL-2R) are expressed on minor populations of immature and mature human thymocytes. These studies were designed to determine if immature T cells could respond to the mitogen phytohemagglutinin (PHA-P) plus IL-2 in vitro by increasing the expression of IL-2R and by proliferation. Using monoclonal antibodies to CD5 and magnetic immunobeads we were able to remove all mature, "bright" CD5+ cells from nylon wool-purified thymocytes and to obtain less mature cells which consisted almost completely of cells with the CD4+CD8+ phenotype. These immature cells were mostly "dim" CD5+ and less than 5% CD5- and a small percentage expressed the IL-2R. After culture in serum-free medium with PHA-P, these cells showed only a slight increase in the percentage of IL-2R+ cells and the addition of IL-2 did not increase the percentage of IL-2R+ cells and no proliferation was observed. Unseparated, nylon wool-purified thymocytes contained 14% bright CD5+ cells. These bright CD5+ cells had a mature phenotype of CD4+CD8- (52%) and CD4-CD8+ (27%) cells. A small percentage of these cells were IL-2R+. These bright CD5+IL-2R+ cells were predominantly mature CD4+CD8- cells as measured by three-color flow cytometry. After culture with PHA-P and IL-2, the percentage of IL-2R+ cells increased and they were now found not only on CD4+CD8- but also on CD4-CD8+ and on CD4+CD8+ cells. IL-2 plus PHA-P increased proliferation of these cells as compared to those cultured in medium with PHA-P without IL-2. Thus, we show that human immature thymocytes in contrast to mature thymocytes are not responsive to IL-2 as measured by a lack of IL-2R expression and proliferation. These data indicate that mature thymocytes can express a functional high affinity receptor for IL-2 and suggest that immature thymocytes may not possess a (functional) p75 chain of the IL-2R.     

Immature CD4- CD8+ murine thymocytes

Mature thymocytes are usually defined and separated from other less mature thymocytes on the basis of their mutually exclusive expression of either CD4 or CD8. However, such murine "single positives" include a subpopulation of immature cells with properties resembling CD4- CD8- thymocytes or CD4+ CD8+ cortical blasts. Most of these immature single positives are CD4- CD8+, some expressing relatively low levels of CD8. They are large, dividing cortisone-sensitive cells found in the outer cortex. They express high levels of the heat-stable antigen (recognized by the monoclonals M1/69, B2A2, and J11d) but they are MEL-14-. The absence of detectable surface CD3, the absence of alpha-chain messenger RNA, and the predominance of the truncated form of the beta-chain messenger RNA all indicate that they do not express the T-cell antigen-receptor complex. Strategies for eliminating such immature cells from preparations of mature thymocytes are given, and their developmental significance is discussed.     

Intrathymic radioresistant stem cells follow an IL-2/IL-2R pathway during thymic regeneration after sublethal irradiation

Sublethally irradiated mice undergo thymic regeneration which follows a phenotypic pattern of events similar to that observed during normal fetal development. Thymic regeneration after irradiation is the product of a limited pool of intrathymic radioresistant stem cells undergoing simultaneous differentiation. We show that in this model of T cell development, thymic regeneration follows a pathway in which the IL-2R is transiently expressed on CD4-/CD8- cells. IL-2R expression occurred during the exponential growth period of thymic regeneration, and IL-2R blocking prevented this explosive growth. Flow cytometry analysis revealed that the IL-2R blockade affected primarily the development of the immature CD3-/CD4-/CD8- (triple negative) cells and their ability to generate CD3+/CD4+/CD8+ or CD3+/CD4+/CD8- and CD3+/CD4-/CD8+ thymocytes. Thus, our findings demonstrate that blocking of the IL-2R resulted in an arrest in proliferation and differentiation by intrathymic radioresistant stem cells, indicating that the IL-2/IL-2R pathway is necessary for the expansion of immature triple negative T cells.     

CD45R as a primary signal transducer stimulating IL-2 and IL-2R mRNA synthesis by CD3-4-8- thymocytes

CD45R is a high molecular weight (p205/220) form of a series of transmembrane glycoproteins, collectively known as CD45 and present in some form on all lymphoid cells. We have proposed that CD45R+ thymocytes, a minority (15 to 30%) of total thymocytes, represent the generative thymic lineage whereas CD45 p180+ thymocytes are destined for intrathymic death. To test this hypothesis, we prepared human thymus fractions enriched for the expression of CD45R by exhaustive depletion of CD45 p180+ cells, as well as progenitor CD3-4-8- "multinegative" thymocytes which are predominantly CD45R+. Northern analysis of RNA extracted from CD45 p180- and multinegative thymus fractions demonstrated that these populations are enriched for cells able to synthesize mRNA encoding IL-2 and IL-2R after mitogenic stimulation, as compared to unfractionated thymus, consistent with the properties expected for generative thymocytes. Postulating that the CD45R glycoprotein might represent an important signal delivery molecule, we analyzed the ability of mAb specific for CD45 epitopes to synergize with suboptimal amounts of PHA and PMA in the stimulation of IL-2 mRNA production by multinegative thymocytes. We found that CD45R-specific mAb synergizes strongly with PHA/PMA to stimulate IL-2 and IL-2R mRNA expression. In contrast, mAb to CD45 common determinants were unable to synergize. Multinegative thymocytes depleted of all CD45 p180+ cells were compared to total multinegative cells and found to synthesize fourfold greater levels of IL-2 mRNA after stimulation with anti-CD45R mAb. This CD45 p180- multinegative subset is enriched for cells expressing a high density of CD45R, and for CD45- thymus cells, suggesting a possible enrichment for nonlymphoid cells which may play a role in the stimulation process. Our results suggest that the extended amino acid insert of CD45R plays a fundamental role in transmembrane signalling, and that CD45R may be a primary signal transducer for developing thymic progenitor cells.     

Role of a thymic stromal cell clone in inducing the stage-specific differentiation of various subpopulations of double negative thymocytes

The monolayer of a thymic stromal cell clone termed MRL104.8a induced the differentiation of adult double negative (DN) thymocytes (CD3-4-8-) through a CD3-4-8+ intermediate into CD3- (or dull) 4+8+ stages. DN thymocytes were separated into three subpopulations depending on their cell-surface expression of Pgp-1 and IL-2R, namely, Pgp-1+IL-2R-, Pgp-1-IL-2R+, and Pgp-1-IL-2R-. The present study investigated the requirements of the MRL104.8a monolayer for inducing the differentiation of these DN thymocyte subpopulations. The following were revealed: i) the MRL104.8a monolayer failed to induce the differentiation of a Pgp-1+IL-2R- subpopulation; ii) whereas a Pgp-1-IL-2R+ subpopulation did not express either CD4 or CD8 Ag when cultured in medium, culturing this subpopulation on the thymic stromal cell monolayers resulted in the expression of CD8 but not CD4 Ag; and iii) a Pgp-1-IL-2R- DN subpopulation obtained through less extensive treatments with anti-CD4 and anti-CD8 antibodies in the presence of C before sorting procedures spontaneously differentiated into double positive cells in medium. In contrast, most of DN cells with the same phenotype obtained through extensive anti-CD4 and -CD8 treatments before sorting failed to express CD4 and/or CD8 Ag in medium but could differentiate through a CD3-4-8+ into more mature stages only when they were cultured on the thymic stromal monolayer. These results indicate differential requirements of thymic stromal cells for the differentiation of various DN subpopulations with qualitatively distinct phenotypes and different magnitudes (very low vs almost zero levels) of CD4/CD8 expression.     

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.     

Regulated costimulation in the thymus is critical for T cell development: dysregulated CD28 costimulation can bypass the pre-TCR checkpoint

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4- CD8- cells initiate expression of TCRbeta. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+ CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRbeta expression manifest in wild-type thymocytes, occurring even in Rag- or CD3epsilon- knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the "beta" checkpoint for pre-TCR/TCR-dependent thymic differentiation.     

Human T-cell leukemia virus type I-induced proliferation of human immature CD2+CD3- thymocytes.

The mitogenic activity of human T-cell leukemia virus type I (HTLV-I) is triggering the proliferation of human resting T lymphocytes through the induction of the interleukin-2 (IL-2)/IL-2 receptor autocrine loop. This HTLV-I-induced proliferation was found to be mainly mediated by the CD2 T-cell antigen, which is first expressed on double-negative lymphoid precursors after colonization of the thymus. Thus, immature thymocytes express the CD2 antigen before that of the CD3-TCR complex. We therefore investigated the responsiveness of these CD2+CD3- immature thymocytes and compared it with that of unseparated thymocytes, containing a majority of the CD2+CD3+ mature thymocytes, and that of the CD2-CD3- prothymocytes. Both immature and unseparated thymocytes were incorporating [3H]thymidine in response to the virus, provided that they were cultivated in the presence of submitogenic doses of phytohemagglutinin. In contrast, the prothymocytes did not proliferate. Downmodulation of the CD2 molecule by incubating unseparated and immature thymocytes with a single anti-CD2 monoclonal antibody inhibited the proliferative response to HTLV-I. These results clearly underline that the expression of the CD2 molecule is exclusively required in mediating the proliferative response to the synergistic effect of phytohemagglutinin and HTLV-I. Immature thymocytes treated with a pair of anti-CD2 monoclonal antibodies were shown to proliferate in response to HTLV-I, even in the absence of exogenous IL-2. We further verified that the proliferation of human thymocytes is consecutive to the expression of IL-2 receptors and the synthesis of IL-2. These observations provide evidence that the mitogenic stimulus delivered by HTLV-I is more efficient than that provided by other conventional mitogenic stimuli, which are unable to trigger the synthesis of endogenous IL-2. Collectively, these results show that the mitogenic activity of HTLV-I is able to trigger the proliferation of cells which are at an early stage of T-cell development. They might therefore represent target cells in which HTLV-I infection could favor the initiation of the multistep lymphoproliferative process leading to adult T-cell leukemia.     

The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets

Stem cell Ag 1 and 2 (Sca-1 and Sca-2), so named due to their expression by mouse bone marrow stem cells, were evaluated for expression by populations of cells within the thymus. Immunohistochemical analysis demonstrated that Sca-1 was expressed by cells in the thymic medulla and by some subcapsular blast cells, as well as by the thymic blood vessels and capsule. Sca-2 expression, which was limited to the thymic cortex, could be associated with large cycling thymic blast cells. Both Sca-1 and Sca-2 were expressed on a sub-population of CD4-CD8- thymocytes, and this subpopulation was entirely contained within the Ly-1lo progenitor fraction of cells. Sca-1 expression by a phenotypically mature subset of CD4+CD8- thymocytes was also noted. Conversely, Sca-2 expression was observed on a phenotypically immature or nonmature subpopulation of CD4-CD8- thymocytes. MEL-14, an antibody that defines functional expression of a lymphocyte homing molecule, identified a small population of thymocytes that contained all four major thymic subsets. Sca-2 split the MEL-14hi thymocyte subset into two Sca-2+ non-mature/immature phenotype fractions and two Sca-2- mature phenotype fractions. In peripheral lymphoid organs, Sca-1 identified a sub-population of mature T lymphocytes that is predominantly CD4+CD8-, in agreement with the thymic distribution of Sca-1. Peripheral T cells of the CD4-CD8+ phenotype were predominantly Sca-1-. In contrast, Sca-2 did not appear to stain peripheral T lymphocytes, but recognized only a subset of B lymphocytes which could be localized by immunohistochemistry to germinal centers. Thus, expression of Sca-1 is observed throughout T cell ontogeny, whereas Sca-2 is expressed by some subsets of thymocytes, including at least one half of thymic blasts, but not by mature peripheral T lymphocytes.     

Soluble factor-mediated differentiation of CD4+CD8+ thymocytes to single positives in vitro

Although the thymic microenvironment provides the necessary elements for T-cell differentiation, the precise role of individual components remains to be determined. In this paper, attempts were made to address the possibility that CD4 or CD8 single-positive (SP) thymocytes could be developed from immature CD4+CD8+ (double-positive; DP) thymocytes in a suspension culture in the presence of soluble factors. We observed that IL-4 and IFN-gamma weakly induced DP cells to differentiate to CD4 cells, but not to CD8. In contrast, IL-2 weakly induced differentiation to CD8. Interestingly, Con A sup strongly induced differentiation to CD8 SP from the purified DP thymocytes prepared from C57BL/6 or LCMV TCRtg mice. In particular, it was found that thymocyte culture with Con A sup generated CD69+DP cells, and the CD69+DP differentiated to CD8 SP under the suspension culture with soluble factors. Thus, Con A sup or combinations of IL-2, IL-4 and IL-7 strongly induced differentiation of CD69+DP to CD8 SP, whereas individual cytokines did not. These results suggest that soluble factors like cytokines play an important role in the generation of SP thymocytes in the absence of thymic stromal cells, at least from a distinctive subpopulation like CD69+DP thymocytes, and perhaps from those of broader range when in conjunction with TCR/MHC interaction.     

Developmentally regulated expression of the beta 4 integrin on immature mouse thymocytes.

Integrins are a superfamily of alpha beta heterodimers, most of which serve as cell surface receptors for extracellular matrix proteins. In this report, we demonstrate that the recently described alpha 6 beta 4 integrin, previously thought to be limited to epithelial cells and Schwann cells, is expressed on immature mouse thymocytes. The presence of alpha 6 beta 4 is controlled by regulation of beta 4 expression, because alpha 6 was expressed by virtually all cells examined, paired with the beta 1 integrin chain to form VLA-6. During fetal ontogeny, beta 4 was highly expressed by 35% of day-13 thymocytes, 75% of day-14 to -15 thymocytes, then rapidly declined to low levels by birth. In neonates and adults, beta 4 expression was highest on CD4- CD8- CD3- and TCR(+)-gamma delta subsets. Correlation of IL-2R, CD44 and beta 4 on CD4- CD8- thymocytes revealed maximal levels on the intermediate CD44- IL-2R+ subset. Most CD4- CD8+ TCR- thymocytes and a significant fraction of CD4+ CD8+ thymocytes were beta 4lo, whereas the most mature J11d- single positive thymocytes were beta-4. Overall, down-regulation of beta 4 was associated with up-regulation of CD4, CD8, and CD3 in the thymus. alpha 6 beta 4 was undetectable on fetal liver or bone marrow cells, lymphocytes from lymph node, spleen, or blood, and mitogen-activated splenic T cells cultured up to 10 wk with IL-2. The data suggest that alpha 6 beta 4 is up-regulated after pro-T cells enter the thymus and may have a thymus-specific function for T cells. The developmentally regulated pattern of expression and the prominence of alpha 6 beta 4 on day-13 to -16 fetal and adult CD4- CD8- CD3- thymocytes further suggest this unusual integrin may play a role in early T cell development, including stages before acquisition of the TCR.