Constitutive expression of interleukin-7 mRNA and production of IL-7 by a cloned murine thymic stromal cell line

Mouse interleukin-7 (IL-7) cDNA was cloned from mouse thymic stromal cell clone MRL 104.8a using a polymerase chain reaction (PCR) technique and expressed in COS-7 cells. The resulting recombinant interleukin-7 (rIL-7) supported the proliferation of mouse antigen-specific helper T cell (Th) clone 9-16 in the absence of IL-2 and antigen as well as mouse pre-B cell line DW34. It was also found that high levels of the mRNA for IL-7 were constitutively expressed in the MRL104.8a cells, and a potent amount of IL-7 was produced in its culture supernatant. These results provide the evidence for constitutive expression of IL-7 mRNA and for production of IL-7 by thymic stromal cells that have a critical role in intrathymic T cell development. The results are discussed in the context of the functional and molecular relationship between IL-7 and the previously described cytokines produced by thymic stromal cells.     

The influence of S17 stromal cells and interleukin 7 on B cell development

A clonal assay was used to study different stimuli involved in the progression of fetal liver B cell precursors to mature B lymphocytes. In this report we replaced fetal liver heterogenous feeder cells by a recombinant growth factor, interleukin 7 (IL 7), and a clonal stromal cell line, S17. Under those conditions we could clone 1 in 10 B220+ B cell precursors from fetal liver and the cells could differentiate to a mitogen-responsive, immunoglobulin-secreting stage. We found that IL 7 stimulates proliferation of B220+ precursors but is not sufficient to support maturation of those precursors to a stage of mitogen responsiveness. We show further that the cell line S17 does not produce IL 7 at functionally detectable level but provides support for B cell maturation. We conclude that this cell line supplies an exogenous stimulus required by B cell precursors to become mature lymphocytes. We describe therefore two stages in pre-B cell development: (a) IL 7-dependent proliferation and (b) S17-dependent maturation to mitogen reactivity. Further studies demonstrate that S17 has a profound effect on B cells by increasing the clonal efficiency of lipopolysaccharide-responsive cells to nearly 1:1 B cell in the spleen of adult C57BL/6 mice.     

B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation

B lymphopoiesis supporting activities of two stromal cell clones, MC3T3-G2/PA6 (PA6) and ST2, were compared. When normal bone marrow cells were cultured in these clones under Whitlock-Witte-type condition, mature B cells were generated only in the culture with the ST2 layer. The cells maintained on the PA6 layer, however, contained the precursor cells giving rise to mature B cells when transferred to the ST2 layer. Thus, PA6 is a stromal cell clone capable of supporting the early B progenitors but cannot support a further maturation step into pre-B cells. The immunoglobulin heavy chain gene configuration of B progenitors maintained on the PA6 layer diversified after their transfer onto ST2 layer. This suggests that they are actually the earliest progenitors. This marked difference in the stromal cell activities between PA6 and ST2 could also be distinguished by stromal cell-dependent pre-B cell lines. Among four ST2-dependent pre-B cell lines tested, two grew only on the ST2 layer, which is capable of supporting B lymphopoiesis, while the others grew both on the ST2 and PA6 layers. These results strongly suggest that the process of intra-marrow B cell development is controlled by more than one signal acting on different stages of B cell differentiation.     

Proliferation and differentiation of immature thymocytes induced by a thymic stromal cell clone

The monolayer of our established thymic stromal cell clone (MRL104.8a) exhibited the capacity to maintain immature double-negative thymocytes. Such capacity was also expressed by a factor produced by the MRL104.8a monolayer. This factor designated as thymic stroma-derived T cell growth factor (TSTGF) was found to be distinct from IL-2 or IL-4 but similar to IL-7 of the previously described cytokines from the functional and molecular aspects. The MRL104.8a monolayer also exerted its differentiation-promoting effect on double-negative thymocytes. Culture for one day of purified double-negative thymocytes on the monolayer resulted in the induction of an appreciable per cent of CD3-4-8+ cells. This differentiation could also be induced by a semipurified TSTGF sample but not by recombinant IL-7, suggesting that the MRL104.8a cells elaborate a factor(s) responsible for initiating the differentiation of double-negative cells in addition to the growth-promotion factor identical or closely related to IL-7. When the culture period of double-negative thymocytes was extended to 2 or 3 days, an appreciable number of double-positive (CD4+8+) and single-positive (CD4+8-) cells were generated on the MRL104.8a monolayer. Thus, these observations provide strong support for the proposition that a specialized thymic stromal component plays an essential role in the intrathymic T cell development in the context of T cell growth and differentiation.     

Interferon-γ arrests proliferation and causes apoptosis in stromal cell/interleukin-7-dependent normal murine pre-B cell lines and clones in vitro,but does not induce differentiation to surface immunoglobulin-positive B cells

Normal pre-B cells from fetal liver or bone marrow of the mouse proliferate for long periods of time in tissue culture on stromal cells in the presence of interleukin-7 (IL-7). Their IgH loci are partly in germ-line, partly in D_HJ_H-rearranged configuration, while their light chain loci are in germ-line configuration. They express the pre-B cell-specific genes V_preB and λ₅. Proliferation of these pre-B cells is inhibited by interferon (IFN)-γ, with half-maximal inhibition at concentrations between 0.1 and 1 unit/ml. Normal pre-B cells exposed to IFN-γ die by apoptosis, as is evidenced by the disintegration of pre-B cell DNA into oligonucleosomal multimers of 180-200 bp. While the proliferation of pre-B cells from Eμ-bcl-2 transgenic (tg) mice is inhibited by IFN-γ, these cells do not die by apoptosis. IFN-γ does not induce differentiation to more mature B lineage cells. In the absence of IL-7 normal pre-B cells differentiate to V_HD_HJ_H/V_LJ_L-rearranged, surface immunoglobulin-positive B cells expressing the a chain of the IL-2 receptor. They also down-regulate the expression of V_preB and X.5, and lose the capacity to proliferate on stromal cells in the presence of IL-7. In contrast, both normal and Eμ-bcl-2 tg pre-B cells exposed to IFN-γ in the presence of stromal cells and IL-7 fail to differentiate, i.e. do not express surface immunoglobulin, retain expression of V_preB and λ₅, do not express the α chain of the IL-2 receptor, and retain the capacity to proliferate on stromal cells in the presence of IL-7, once IFN-γ is removed. The potential usefulness of a treatment of acute lymphocytic leukemia of the B cell lineage (pre B-ALL) with IFN-γ is discussed.     

几种细胞因子对成年小鼠CD4^—CD8^—胸腺细胞的增殖作用

本实验观察了多种细胞因子对成年小鼠CD4~-CD8~-(Double negative,DN)胸腺细胞增殖和分化的影响。结果表明,IL-7能诱导DN细胞增殖;IL-2只在高浓度下轻度支持其增殖。IL-1α、IL-4、IL-6、SCF(干细胞因子)、TGF(转化生长因子)-β_1和TGF-β_2单独作用不能支持DN细胞生长。IL_2、IL—6和SCF与IL—7联合应用不同程度地协同促进DN细胞增殖。TGF—β_1和TGF—β_2则抑制IL—7诱导的DN细胞增殖。PMA活化的DN细胞能对IL—2、IL—4或IL—7应答进行增殖。     

Culturing of HIV-1-specific cytotoxic T lymphocytes with interleukin-7 and interleukin-15

The ability to study HIV-1-specific cytotoxic T cell (CTL) clones in models in vitro or to expand them for immunotherapeutic use is limited by the technical difficulty of propagating these cells. The factors that determine the survival and proliferation of the cells are incompletely understood and could include cytokines provided from feeder cells or serum. We therefore investigated the effects of adding two cytokines reported to have effects on T cell proliferation and function, interleukin (IL)-7 and IL-15. Four HIV-1-specific clones derived from infected persons were cultured under standard conditions with IL-2 compared to IL-7 or IL-15 alone or in combination with IL-2. Proliferation and survival, as reflected by cell numbers after stimulation, were poorly supported by IL-7 or IL-15 alone, and these cytokines appeared to provide no additional benefit when added to IL-2. Similarly, these cytokines alone did not support the functional status of these cells as measured by chromium release assays with peptide-pulsed target cells. Addition of IL-7 or IL-15 to IL-2 did not augment function of the cells. These data suggest that supplementing CTL cultures with these cytokines does not provide improvement of cell growth or function.     

Regulation of B cell precursor proliferation by aminopeptidase A

The BP-1/6C3 molecule expressed by early B lineage cells andsome stromal cells has been identified as aminopeptidase A (APA).We have previously demonstrated that IL-7 selectively inducesBP-1/APA expression by pre-B cells coincident with their growth.Here we directly demonstrate that BP-1 is preferentially expressedby the proliferating subpopulation of normal B cell progenitors.Furthermore, when non-adherent BALB/c bone marrow cells wereincubated with IL-7 in the presence of purified BP-1 antibody,B cell precursor proliferation was markedly inhibited. Modulationof the BP-1/6C3 antigen did not occur in the presence of theBP-1 antibody, but APA enzymatic activity was significantlyinhibited. The 6C3 antibody, which recognizes a different epitopeon the APA molecule, had no effect on either B cell precursorproliferation or APA enzyme activity. We hypothesized that neutralizationof APA by the BP-1 antibody results in inhibition of IL-7 drivenB cell precursor proliferation. However, when isolated 14.8⁺bone marrow cells were cultured with IL-7 in the presence ofthe BP-1 antibody, no inhibition of proliferation occurred.This data suggested that the effect of the BP-1 antibody mightbe related to the action of APA on peptides in the marrow microenvironmentwhich are not present in cultures of isolated B cell precursors.The addition of irradiated non-adherent bone marrow cells tothe 14.8⁺ cell cultures restored the inhibitory effect of theBP-1 antibody. Based on these observations, we propose thatAPA cleaves a small peptide which serves as a natural inhibitorof B cell precursor proliferation.     

Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7

B cell development is influenced by interactions between B cell progenitors and stromal cells. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. Flt3 ligand (FL) is a growth factor which stimulates the proliferation of stem cells and early progenitors. Mice deficient for the FLT3 receptor exhibit severe reductions in early B lymphoid progenitors. We have previously described a clonal assay in vitro which allows us to follow the entire B cell differentiation pathway from uncommitted progenitors to mature, immunoglobulin-secreting plasma cells. The growth factor combination of interleukin (IL)-11, mast cell growth factor (MGF) and IL-7 was shown to maintain the differentiation of these hematopoietic precursors into B cell progenitors capable of giving rise to functionally mature B cells in secondary cultures. Here, we show that FL in combination with IL-11 and IL-7 is sufficient to support the differentiation of uncommitted progenitors from day 10 yolk sac (AA4.1⁺) or day 12 fetal liver (AA4.1⁺ B220^? Mac-1^? Sca-1⁺) into the B lineage. The frequency of B cell progenitors obtained in these conditions was similar, if not better, than the frequency of B cell precursors that arose when cultured in IL-11+MGF+IL-7. Furthermore, the growth factor combination of IL-11+FL+IL-7 was able to maintain the potential of bipotent precursors giving rise to both the B and myeloid lineages in secondary cultures. We also show that FL synergizes with IL-7 in the proliferation of committed B220⁺ pro-B cells and may contribute to the maintenance of an earlier pro-B cell population. Together, these results show that FL is important in supporting the differentiation and proliferation of early B cell progenitors in vitro.     

Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo

B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB-1 and SAB-2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic mu chain-positive pre-B cells or mature B cells to the BM stromal cell line. Injection of both SAB-1 and SAB-2 antibodies into pregnant mice reduced by 90% the number of B220+n B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU-preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB-1 is present on CS 1.3 stroma cells and on a pre-B cell line while SAB-2 is present on pro-B and pre-B cell lines, but not on CS 1.3 stromal cells. SAB-1 and SAB-2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB-1 and SAB-2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB-1 and SAB-2 probably recognize novel cell-cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.     

Pre-pro-B cell growth-stimulating factor (PPBSF) upregulates IL-7Ralpha chain expression and enables pro-B cells to respond to monomeric IL-7.

Although pro-B cells are well represented in IL-7 knockout (KO) mice, they express abnormally low concentrations of the interleukin-7 receptor alpha-chain (IL-7Ralpha) and do not generate pre-B cells. Here, we demonstrate that pro-B cells from IL-7 KO mice can be induced to generate pre-B cells and immature B cells by exposure to recombinant IL-7 (rIL-7) in vivo but not in vitro. Experiments in recombinant activation gene-1 (RAG-1) KO mice indicate that the in vitro unresponsiveness of IL-7(-/-) pro-B cells to rIL-7 is unrelated to the absence of a functional pre-B cell receptor (pre-BCR). Rather, it appears to be due to the suboptimal expression of the IL-7Ralpha chain. Thus, IL-7(-/-) pro-B cells readily respond to rIL-7 in vitro if IL-7Ralpha chain expression is first upregulated by exposure to IL-7 in vivo or to IL-7(+/+) bone marrow (BM) stromal cells or conditioned medium (CM) therefrom in vitro. Similar results were obtained when pro-B cells from IL-7 KO mice were cultured on IL-7(-/-) BM stromal cells in the presence of rIL-7. This suggested that the recently described pre-pro-B cell growth-stimulating factor (PPBSF), a self-assembling hybrid cytokine comprising IL-7 and the stromal cell-derived hepatocyte growth factor beta-chain (HGFbeta), is required to stimulate pro-B cells from IL-7 KO mice. This inference was verified by demonstrating that purified PPBSF upregulates IL-7Ralpha chain expression on IL-7(-/-) pro-B cells in vitro and enables them to respond to rIL-7 in a stepwise manner. We, therefore, postulate that PPBSF is the operative form of IL-7 that normally induces IL-7Ralpha(lo) pre-pro-B cells to proliferate and differentiate into IL-7Ralpha(hi) pro-B cells, which then proliferate and differentiate into pre-B cells on stimulation with monomeric IL-7.     

Expression of interleukin (IL)-11 receptor by the human endometrium in vivo and effects of IL-11, IL-6 and LIF on the production of MMP and cytokines by human endometrial cells in vitro

Interleukin (IL)-6, leukaemia inhibitory factor (LIF) and IL-11 belong to the same family of cytokines whose receptors utilize gp130 as the signalling molecule. We have investigated the expression of the IL-11 receptor, IL-11Ralpha, protein in the human endometrium in vivo and the effects of IL-6, LIF and IL-11 on the production of metalloproteinases (MMPs) and cytokines by cultured endometrial epithelial and stromal cells. Immunostaining showed that IL-11Ralpha was expressed in both epithelial and stromal cells, with epithelial staining being more intense than stromal staining and little variation in staining in either compartment throughout the cycle. Incubation of both stromal and epithelial cells with IL-6, LIF and IL-11 had no effect on MMP-2, -7, -9, transforming growth factor (TGF)beta or IL-1beta production or cell growth. IL-6 and LIF also had no effect on tumour necrosis factor (TNF)alpha production, but IL-11 caused a dose-dependent decrease in TNFalpha production by epithelial cells. IL-6 receptor, LIF receptor and gp130 were all expressed by cultured stromal and epithelial cells, showing that the lack of effect is not due to lack of expression of the receptor components. The results show that although IL-6, LIF and IL-11 signal through the same molecule, they may have different effects in endometrial cells, suggesting the activation of different signalling pathways, which may ultimately be important in the control of endometrial function.     

IFN-gamma abrogates IL-7-dependent proliferation in pre-B cells, coinciding with onset of apoptosis.

These studies investigated the mechanism by which interferon-gamma (IFN-gamma) inhibits the interleukin-7 (IL-7)-dependent proliferation of BALB/c bone marrow B-cell precursors in vitro. Low concentrations (1 U/ml) of recombinant murine IFN-gamma (rmIFN-gamma) caused a approximately 80% suppression of IL-7 colony-forming units (CFU) formation in semi-solid media, in part through a direct affect on isolated B220+ pre-B cells. IFN-gamma did not induce apoptosis in small resting pre-B cells in BALB/c bone marrow. There was no difference in the proportion of apoptotic B220+ pre-B cells in IFN-gamma-treated cultures compared to cultures treated with IL-7 alone. However, IL-7-responsive pre-B cells generated from bone marrow had a 30-50% loss in cells in S+G2/M phases of the cell cycle and an increase of up to twice as many in apoptotic cells within 48 hr of exposure to IFN-gamma. Notably, expression of the tyrosine phosphatase B220 was increased in the IFN-gamma-treated pre-B cells. Interestingly, although there was no substantial change in IL-7 receptor mRNA expression upon IFN-gamma treatment, a small decrease in binding of biotinylated IL-7 to IFN-gamma-treated pre-B cells was observed. These results suggest that IFN-gamma inhibits IL-7 responsiveness in pre-B cells, resulting in a subtle down-regulation of IL-7 binding, inhibition of proliferation and, ultimately, apoptosis.     

New human embryo liver cell lines obtained by stabilization and immortalization enhance in vitro clonal growth of cordonal blood cells

We developed two new cell lines derived from embryo liver and tested their inductive capacity on in vitro clonal growth of cordonal blood (CB) hematopoietic cells. One line was stabilized and named BAEP2-WILD (W), and the other one was immortalized by retroviral transduction with SV40 Large T antigen and called BAEP2-SV40. Southern blot analysis demonstrated the integration of the Large T antigen gene in the BAEP2-SV40 cell genome, but this line did not display the expected growth arrest at the non-permissive temperature of 39 degrees C. Immunocytochemistry showed that BAEP2-SV40 cell line was positive for several cytokeratins and stromal markers (vimentin, desmin and laminin), as well as for epidermal growth factor (EGF), fibroblast growth factor (FGF) and their receptors (Rs). In contrast, BAEP2-W evidenced positivity only for cytokeratin-7 and laminin, and low positivity to EGF, EGF-R, FGF and FGF-R. BAEP2-SV40 cell line, but not BAEP2-W, expressed interleukin (IL)-1, IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor, stem cell factor and vascular-cell adhesion molecule-1 mRNAs, and secreted IL-6 and GM-CSF. Taken together, these findings could suggest that BAEP2-W cell line possesses the phenotype of fetal hepatocytes, while BAEP2-SV40 cell line has that of stromal cells. The supernatants conditioned by both cell lines stimulated the clonal growth of CB hematopoietic cells cultured on semisolid media deprived of growth factors and cytokines, the inductive capacity of the BAEP2-SV40 cell line being markedly higher than that of its wild counterpart, conceivably due to its ability to produce cytokines. Our study indicates that these two new cell lines, and especially BAEP2-SV40 one could be used in co-culture systems as feeder-layers for hematopoietic CB SC expansion in vitro.     

The murine T cell line CT6 provides a novel bioassay for interleukin-7

Like interleukin (IL)?2 and IL-4, IL-7 can act as a growth factor for activated T lymphocytes. Upon screening a panel of growth factor-dependent T cell lines, we found that only the cell line CT6 responded to IL-7, indeed as vigorously as to IL-2. Obviously, these findings challenge the validity of previous results on IL-2 production obtained using the CT6 cell line. However, they also demonstrate a novel and sensitive system for the bioassay of IL-7. The ability of the surveyed T cell lines to proliferate to IL-7 corresponded with the expression of IL-7 receptors (IL-7R) on the cell surface. The murine IL-7R on CT6 was shown to bind IL-7 with dual affinity and was visualized as an affinity cross-linked complex of 93 kDa. This IL-7R appears similar to that seen on murine splenic T cells and on 70Z/3, the pre-B cell line from which the murine IL-7R was cloned.     

Gamma 2b provides only some of the signals normally given via mu in B cell development

B cell development is a complex process involving interactions between B cell precursors, stroma, and known and unknown ligands and cytokines. In order to more fully understand the requirements for Ig in that development we have created transgenic mice that carry a gamma 2b transgene and express it early in B cell development. Previously it was believed that these B cells arrested in their development prior to the pro- to pre-B cell transition. We show here that in conventional gamma 2b mice, B cell development actually arrests later, at the pre-B cell stage. This shows for the first time that a constant region different from mu can allow signaling through the pre-B cell receptor, but cannot promote complete development. The pro- and pre-B cells in the conventional gamma 2b transgenics are not fully functional since they cannot grow in IL-7 without stromal cells. This is a novel phenotype, separating development from stroma independence. The few, mature B cells that do develop in these mice express both mu and gamma 2b simultaneously, and are CD5+. Expression of a Bcl-2 transgene allows survival of gamma 2b transgenic immature B cells, but does not promote full maturation, indicating that normally mu provides both an anti- apoptotic signal and a differentiation signal. One line of gamma 2b mice, the C line, does not have this phenotype. B cell development is accelerated in this unconventional line, and the developing B cells have a very different phenotype from both normal mice and conventional gamma 2b mouse lines, but are very similar to mu transgenics. Mature B cells are largely CD5-, gamma 2b-only expressing. This unique phenotype is apparently due to the activation in B cell precursors of a gene at the insertion site of the transgene, circumventing the need for mu. Comparison of conventional gamma 2b transgenics with the C line and mu transgenics reveals the multiple signals required throughout B cell development.      

A bone marrow-derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4−CD8−double negative to the CD4+CD8+ double positive differentiation stage in vitro

T-cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T-cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T-cell maturation, we examined the effect of a bone marrow-derived stroma cell line, ST2, on T-cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14.5 of gestation from a CD4- CD8- double negative (DN) to a CD4+ CD8+ double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti-interleukin-7 receptor (IL-7R) and anti-c-kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow-derived stroma cell, ST2, which supports B-cell differentiation, is also able to support T-cell development and may constitute one of the microenvironmental components for extrathymic T-cell development.     

Expression of cytokines and their receptors by human thymocytes and thymic stromal cells.

The repertoire of cytokine and cytokine receptor mRNA expressed by unstimulated human thymocytes and thymic stromal cells was explored by a quantitative polymerase chain reaction (PCR) using sequence specific internal standards. Of the 18 cytokines tested we found a considerable overlap in the expression of cytokines by human thymocytes and by thymic stromal cells; both cell types express the mRNA for interleukin-1 beta(IL-1, IL-6, IL-7 and tumour necrosis factor-alpha (TNF-alpha). However, there are substantial differences in the levels of cytokine mRNA expressed in these two types of cells as revealed by the quantitative PCR assay. Stromal cells express considerably higher levels of IL-1 beta and IL-6 than thymocytes (14- and 27-fold respectively). In addition, a number of cytokines such as lymphotoxin and interferon-gamma (IFN-gamma), are expressed exclusively in thymocytes whereas others such as stem cell factor (SCF), IL-1 receptor antagonist-2 (IRAP-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are produced only in stromal cells. There is a complete overlap in the expression of a group of cytokine receptors tested in thymocytes and thymic stromal cells; these include IL-1R, IL-2R, IL-6R, IL-7R, TNFR and stem cell growth factor receptor (c-KIT). The expression of specific cytokines by thymic stromal cells and the parallel expression of their receptors on thymocytes under physiological conditions, support the hypothesis that these cytokines participate in paracrine interactions between these two cell populations during thymocyte differentiation.