Effect of murine thymic epithelial cell line (MTEC1) on the functional expression of CD4+CD8 thymocyte subgroups
Qing Ge and
Wei-Feng Chen
Department of Immunology, Peking University Health Science Center, Beijing 100083, PRC
Correspondence to:
W.-F. Chen
 |
Abstract
|
---|
To determine the effect of thymic stromal cells on the functional maturation of CD4 single-positive (SP) thymocytes, the functional status of isolated CD4 SP thymocyte subgroups was investigated by means of cell proliferation and cytokine production in response to concanavalin A (Con A) prior and after co-culturing with a murine thymic epithelial cell line (MTEC1). Mouse medullary CD4 SP thymocytes were phenotypically divided into seven discrete subgroups predicted to reflect the maturation pathway from newly emerging CD4 SP thymocytes to terminally differentiated cells. For functional analysis, six major subgroups (6C10+CD69+, 6C10CD69+, 6C10CD693G11+Qa-2, 6C10CD693G11+Qa-2+, 6C10CD693G11Qa-2 and 6C10CD693G11Qa-2+) cells were isolated and their functional status in response to Con A stimulation assessed. A functional hierarchy is revealed among these subgroups, consistent with their phenotypic maturation status, which may imply that these cells undergo a functional maturation process within thymic medulla. The function of cytokine production by CD4 SP thymocytes is acquired in a stepwise manner from a low to high level and characterized by Th0-type cytokines in the main stream of differentiation pathway. However, a minor subgroup that appeared at the late stage as 3G116C10 cells was biased to produce Th2-type cytokines. Nevertheless, the functional capacity of the final two Qa-2+ subgroups of CD4 SP thymocytes was still significantly lower than that of spleen CD4+ T cells. After co-cultivation with MTEC1 cells, four subgroups of TCR
ß+CD4+CD8 thymocytes exhibited significantly higher levels of proliferation capability and modulation in cytokine production capability. However, co-culturing with MTEC1 cells did not change the pattern of Th0- or Th2-like cytokine production by respectively medullary CD4 SP thymocyte subgroups nor could MTEC1 induce CD4 SP thymocytes to secrete Th1-type cytokines. The results suggest that MTEC1 can regulate the functional status of these thymocyte subgroups.
Keywords: CD4+CD8 thymocyte subgroup, cytokine, proliferation, thymic stromal cells
 |
Introduction
|
---|
The thymus is the primary site dedicated to the development and maturation of T lymphocytes. Within the thymus, thymocyte progenitors undergo a program of cell proliferation, TCR gene rearrangement, TCR repertoire selection and functional maturation, ready for emigration to the periphery (14). At which stage thymocytes acquire functional capacity and where they are equipped with functional machinery is a matter of controversy. In RelB/ mice with deficiency in the thymic medullary stroma, functional CD4+CD8 and CD4CD8+ single-positive (SP) thymocytes can be generated (5). This seems to exclude the importance of the thymic medulla for SP thymocyte functional maturation. However, the thymic medulla is commonly regarded as a site for negative selection (3,6,7). Apart from the existence of residual stromal cells in the thymic medulla in RelB/ mice (5,8), which may contribute to the generation of SP thymocytes, in the case of normal mice, all the SP thymocytes are localized in the thymic medulla and hence functional maturation program should be accomplished in the thymic medulla. It is known that newly emerging CD4+CD8 and CD4CD8+ SP thymocytes, surviving after positive selection, reside in the thymic medulla for 14 days (9). During this time, not only are the SP thymocytes imposed for further negative selection, they also undergo functional maturation to acquire immunocompetence. The evidence is that the newly emerged SP thymocytes are non-functional or low-functional, whereas the thymic emigrants are virtually fully functional, and there are many SP thymocyte subgroups that possess distinct functions at levels between these two extremes (10,11). To understand the true function of the thymic medulla, it is necessary to estimate if there is a functional maturation pathway of SP thymocytes. The changes in functional status are often accompanied with phenotypic alterations, such as the loss of cell surface molecules of 6C10, CD69 and heat-stable antigen (HSA), and expression of Qa-2 (1218). It is therefore possible to separate SP thymocytes into subgroups according to their phenotypes for both functional analysis and the study of the factors that contribute to the functional maturation of SP thymocytes.
Although the thymus provides a unique environment for thymocyte development, the role of the thymic stroma in regulating the functional maturation of SP thymocytes, is not well understood. Dyall and Nikolic-Zugic found that immature CD3highCD4+CD8low cells expressing CD69 and HSA were not able to survive in the periphery (19). They still require the thymic environment for them to reach the end stage of functional maturation. Res et al. also reported that mouse thymic microenvironments could support differentiation of human progenitors into CD3+CD4+CD1a+ SP cells, which could not be expanded in vitro with phytohemagglutinin and IL-2 (20). Upon co-cultivation with human thymic stromal cells, however, these CD1a+CD4 SP could differentiate into functional CD1a thymocytes. These data suggest that the thymic microenvironment (including stromal cells and cytokines) plays an important role in the functional maturation of SP thymocytes.
We have previously reported that mouse medullary CD4 SP thymocytes could be phenotypically divided into seven discrete subgroups predicting a maturation pathway from newly emerging CD4 SP thymocytes to terminally differentiated cells (21). Functional analysis including cell proliferation and cytokine production showed a functional hierarchy among these subgroups, consistent with their phenotypic maturation status (manuscript in preparation). We asked if the cells of these subgroups could be induced by thymic medullary epithelial cells for further functional maturation. In the previous report we showed that isolated mouse Qa-2CD4 SP thymocytes could give rise to Qa-2+CD4 SP thymocytes and acquire full functional competence under the foster of MTEC1, a murine thymic medullary-type epithelial cell line, in the in vitro assay system (17). We therefore adopted this in vitro system to study the role of MTEC1 in the induction of functional maturation of CD4 SP thymocytes of different subgroups. In this report we demonstrate that CD4 SP thymocytes within various subgroups could be induced by MTEC1 cells to elevate the capacity of proliferation and cytokine production in the presence of concanavalin A (Con A). More interestingly, co-cultivation with MTEC1 could not change the Th0 pattern of cytokine secretion of the CD4 SP thymocytes along with the mainstream of functional differentiation pathway. However, CD4 SP thymocytes expressing the TCR
ß+3G116C10 CD69+/HSAmed/lo phenotype could be induced by MTEC1 to regulate their cytokine production levels by up-regulating IL-6 production, but down-regulating their IL-4 and IL-10 production.
 |
Methods
|
---|
Mice
Specific pathogen-free BALB/c mice (68 weeks old) were obtained from the Laboratory Animal Center, Institute of Genetics, Chinese Academy of Sciences, Beijing, China.
mAb and reagents
The following mAb were used. Anti-mCD8 (3.155), anti-mCD4 (GK1.5), anti-m3G11 (SM3G11), anti-m6C10 (SM6C10), anti-CD69 (H1.2F3) and anti-mIL-4 (11B11) mAb were produced in our laboratory as ascites or tissue culture supernatants. Biotinanti-IL-4 (Bud24G2-3Biotin) and biotinanti-IL-10 (2A5 and Sxc1Biotin) were kindly provided by Dr Ying Liu. Anti-mCD8FITC, anti-mCD4phycoerythrin (PE), anti-CD69-biotin, anti-m3G11-biotin, anti-mQa-2-biotin and sheep anti-rat IgGFITC were purchased from PharMingen (San Diego, CA) and Sigma (St Louis, MO) respectively. Con A (Pharmacia Piscataway, NJ), mitomycin C (MMC; Kyowa, Tokyo, Japan), MTT (Sigma) and [3H]thymidine (Shanghai Atomic Energy Research Institute, sp. act. 22 mCi/mmol/l) were purchased from the respective manufacturers.
Thymic stromal cell lines
All mouse thymic stromal cell lines were established in our laboratory and cultured in 10% FCS-DMEM. For the primary cultures of whole thymic stroma cells, mouse thymus was finely minced and cultured in 10% FCS-DMEM. The cultures were replenished with fresh medium once a week. After one month, all the thymocytes died off and left the healthy whole thymic stromal cells ready for use.
Cell preparation and separation
Fresh adult thymi were homogenized to a single-cell suspension in cold 2% NCS/RPMI 1640 medium using a steel mesh. The cells were subjected to two cycles of cytotoxic killing by anti-CD8 mAb and complement to obtain CD4CD8 and CD4+CD8 thymocytes. The CD8-depleted cells were panned at 4°C for 15 min on plates precoated with anti-CD4 mAb (GK1.5) at 1:100 dilution. The adherent cells were collected as CD4 SP thymocytes. For the isolation of 6C10+ cells, the CD4 SP cells were first incubated with mouse anti-6C10 mAb (1 µg/106 cells) at 4°C for 30 min. After washing twice, the cells were incubated with biotin-labeled anti-mouse Ig (1 µg/106 cells). The cells bound with biotin-labeled Ig were further panned at 4°C for 30 min on plates precoated with anti-biotin antibody at the concentration of 10 µg/ml. The non-adherent cells were collected as 6C10 cells, while the adherent cells were collected as 6C10+ cells. For the isolation of 6C10CD69+ cells, 6C10CD4+CD8 cells were incubated with biotin-labeled anti-CD69 mAb (1 µg/106 cells) and then panned at 4°C for 30 min on plates precoated with anti-biotin antibody. The non-adherent cells were collected as CD69 cells, while the adherent cells were collected as 6C10CD69+. For the isolation of CD69Qa-2 and CD69Qa-2+ cells, the CD69 cells were incubated with biotin-labeled anti-Qa-2 mAb (1 µg/106 cells) and then panned at 4°C for 30 min on plates precoated with anti-biotin antibody. The non-adherent cells were collected as CD69Qa-2 cells, while the adherent cells were collected as Qa-2+ cells. For the isolation of 3G116C10CD4 SP thymocytes, total thymocytes were subjected to two cycles of cytotoxic killing by mixed mAb for anti-CD8, anti-3G11, anti-6C10 and complement. The viable cells were panned at 4°C for 30 min on the anti-CD4 mAb precoated plates. The adhered cells were collected as 3G116C10CD4 SP. Then, these cells were incubated with biotin-labeled anti-Qa-2 mAb (1 µg/106 cells) and panned at 4°C for 30 min on the plates precoated with anti-biotin antibody. The nonadherent cells were collected as Qa-2 cells, while the adherent cells were collected as Qa-2+ cells.
Flow cytometry analysis
For FACS analysis, the separated CD4 SP thymocytes were stained with PE-labeled anti-CD4 and FITC-labeled anti-CD8 mAb. The isolated thymocytes of the 6C10+CD69+, 6C10CD69+, CD69Qa-2 and CD69Qa-2+ subgroups were stained with anti-6C10-biotin, anti-CD69-biotin and anti-Qa-2-biotin, followed by staining either with FITCsheep anti-rat IgG or with FITCavidin at 4°C for 30 min. 3G116C10 cells were stained with anti-3G11 and anti-6C10 antibody at 4°C for 30 min. After washing twice, the cells were stained with FITC-labeled sheep anti-rat IgG. For negative controls the separated total CD4 SP thymocytes were stained with FITCsheep anti-rat IgG. The stained cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA) with an argon-ion laser tuned at 488 nm. The parameters of forward light scattering, orthogonal scattering and fluorescence signals were determined for 10,000 cells, and stored in list mode data files. Data acquisition and analysis were performed with Lysys 2.0 software (Becton Dickinson).
MMC treatment of MTEC1 cells
MTEC1 cells growing to confluence were treated with MMC (50 µg/ml) at 37°C in a 5% CO2 incubator for 1 h, washed twice with 5% NCS/BSS and treated with TE (0.05% trypsin/0.02% EDTA) at room temperature to detach the adhering cells. The MMC-treated cells were marked as mMTEC1.
Proliferation assay
A total of 1x105 purified cells was incubated in flat-bottomed wells of tissue culture plates in the presence of Con A (2.5 µg/ml). The cultures were incubated for 3 days at 37°C in a humidified atmosphere of 5% CO2. During the last 12 h, 0.5 µCi of [3H]thymidine was added. At the end of incubation, the cultures were harvested onto fiberglass filters and [3H]thymidine incorporation was determined by liquid scintillation counting. All values represent the mean of triplicate wells.
Lymphokine production and activity assays
A total of 12x105 cells was stimulated with 2.5 µg/ml Con A. After 48 h of incubation at 37°C in a humidified atmosphere of 5% CO2, supernatants were harvested from each well and kept frozen until use. IL-2 and IL-6 were assayed by bioassays supporting HT-2 and 7TD1 cell proliferation respectively. IL-4 and IL-10 were assayed by sandwich ELISA. IFN-
activity was determined in an inhibition assay of cytopathic effect using cells grown in 96-well plates and infected with vesicular stomatitits virus (22). IFN titers are expressed in actual laboratory units.
 |
Results
|
---|
Effect of MTEC1 on the proliferation capacity of six subgroups of CD4+CD8 medullary thymocytes
Using the procedures described in Methods, we isolated six subgroups, i.e. 6C10+CD69+, 6C10CD69+, 3G11+6C10CD69Qa-2, 3G11+6C10CD69Qa-2+, 3G116C10CD69Qa-2+ and 3G116C10CD69Qa-2+, of purity 95, 96, 97, 97, 98 and 98% respectively. The CD4 SP thymocytes of isolated subgroups were cultured with or without Con A stimulation. In the absence of Con A stimulation, cells of all the subgroups cultured alone were virtually unable to proliferate, while cells co-cultured with mMTEC1 showed different levels of weak proliferation (Table 1
), in which the two subgroups of CD69+ cells including 6C10+ and 6C10 cells showed the highest level of growth. Under Con A stimulation, the isolated cells exhibited a high proliferation response. As shown in Table 1
, the phenotypically most immature subgroup of 6C10+CD69+ had the least proliferation capacity. Compared with 6C10+CD69+, cells in the 6C10CD69+ subgroup exhibited sharply increased proliferation capacity, whereas in the remaining four CD69 subgroups, cells responded vigorously by proliferation in a stepwise increasing manner with Con A stimulation. The Con A-activated proliferation capability was progressively increased in the cells with consecutive down-regulation of their cell surface markers 6C10, CD69 and HSA, and up-regulation of Qa-2. This proliferation response was further increased by 1.4- to 1.9-fold in co-cultures with MTEC1 and activated by Con A. In view of the phenotypic differentiation pathway of CD4 SP thymocytes, a significant feature is that MTEC1 cells could only induce the cells of each individual subgroups to increase their functional capacity to the level possessed by the naive cells positioned at the next differentiation stage. After induction by MTEC1, the 3G11+6C10Qa-2+ cells reached the same proliferation capacity as that of spleen T cells. The 3G116C10CD69Qa-2 thymocytes belong to a branched subgroup derived from 3G11+6C10CD69CD4 SP cells. As shown in Table 1
, the proliferation capacity of 3G116C10Qa-2+ cells was similar to that of the 3G11+6C10Qa-2+ subgroup, but much higher than its Qa-2 counterpart. Co-culturing with mMTEC1 also increased the proliferation capability of 3G116C10Qa-2+ cells and made them comparable to mature spleen T cells. These data demonstrated that mMTEC1 cells enhanced the proliferation capability of different subgroups of CD4+CD8 cells remarkably.
Effect of MTEC1 on cytokine production of six subgroups of CD4+CD8 thymocytes
We have previously investigated the spectrum and the level of cytokines produced by CD+ SP thymocytes of four major subgroups in response to Con A stimulation. 6C10+CD69+CD4 SP could produce IL-2 only and its level was marginal. After co-cultivation with the epithelial cell line mMTEC1, as shown in Table 2
, the cells of this subgroup produced not only IL-2 at a little higher level, but also IL-4 at a low level. 6C10CD69+ cells produced low levels of both IL-2 and IL-4. Cultivation with MTEC1 increased the capability of 6C10CD69+ cells to produce IL-2, IL-4, IL-6 and IFN-
. When cells developed into the 3G11+6C10CD69Qa-2 stage, they started to secrete intermediate levels of multiple types of cytokines including IL-2, IFN-
, IL-4, IL-10 and IL-6. The level of cytokine production was further increased when cells developed to the Qa-2 stage, which showed the highest cytokine-producing activity among all the CD4 SP thymocyte subgroups assessed, consistent with its highest proliferation capacity. Whereas in the co-cultures with mMTEC1 cells for 48 h, the level of IL-2, IFN-
, IL-4, IL-10 and IL-6 produced by the thymocytes of these two subgroups of 3G11+6C10CD69Qa-2 and 3G11+6C10CD69Qa-2+ was further elevated 1.5- to 3.3-fold in comparison with the these cytokines produced by the respective cells cultured alone. In terms of cytokine production, it is once again shown that, significantly, MTEC1 cells could only induce the cells of each individual subgroups to increase their functional capacity to the level possessed by the naive cells positioned at the next differentiation stage along with the phenotypic differentiation pathway of CD4 SP thymocytes.
The pattern of cytokines produced by 6C103G11+ CD4 SP thymocytes was mainly Th0 type. The presence of MTEC1 could not change the Th0 pattern of cytokine secretion by these CD4 SP thymocytes composing the mainstream of the maturation pathway within thymic medulla.
Different effects of MTEC1 on Th2-like cytokine production by 3G116C10CD4+CD8 thymocytes
The 3G116C10CD4+CD8 thymocytes including Qa-2 and Qa-2+ belonging to the branched subgroup derived from 3G11+6C10CD69CD4 SP cells exhibited a prominent feature of producing Th2-like cytokines, such as IL-6, IL-4 and IL-10, in response to Con A stimulation. Table 3
shows that after co-cultivation with mMTEC1 cells for 48 h, the levels of IL-6 production by thymocytes of these two subgroups were elevated 5.7-fold, whereas the level of IL-4 and IL-10 production was reduced 0.3- and 0.7-fold, compared with the levels produced by these CD4 SP thymocytes cultured alone. Therefore, mMTEC-1 cells have different effects on the production of individual Th2-type cytokines by 3G116C10Qa-2CD4 SP and 3G116C10Qa-2+CD4 SP thymocytes.
View this table:
[in this window]
[in a new window]
|
Table 3. Th2-type cytokine production by 3G116C10CD4+CD8 thymocytes to Con A stimulation with or without co-cultivation with MTEC1 cellsa
|
|
Effect of MTEC1 on Th1-type cytokine production by 3G116C10CD4+CD8 thymocytes
Upon Con A stimulation, both Qa-2 and Qa-2+ of the 3G116C10CD4+CD8 thymocytes could produce a small amount of IFN-
, but no detectable IL-2, whereas upon co-cultivation with mMTEC1, the level of IFN-
secretion was not changed and there was no detectable IL-2 (Table 4
).
The effect of different types of mouse thymic stromal cells on cytokine production by 3G116C10CD4 SP thymocytes
MTEC1 is a murine thymic medullary-type epithelial cell line cultured in vitro for a long time. The above data indicates that MTEC1 has regulatory effects on the cytokine production of 3G116C10CD4 SP thymocytes, i.e. up-regulated IL-6, down-regulated IL-4 and IL-10, and no effect on Th1-type cytokine production. To see if it is a unique function of the MTEC1 cell line, several mouse thymic stromal cell lines and fresh mouse total thymic stromal cells were added to the in vitro co-culture system. As shown in Table 5
, all the thymic stromal cells could up-regulate IL-6 secretion but could not increase the pattern of Th1 cytokine production. The effect on IL-4 and IL-10 production was different when co-cultured with different types of thymic stromal cells. Both MTEC1 and MTSC4 down-regulated IL-4 and IL-10 production, whilst MTEC5 had a slight enhancing effect on the production of IL-4 and IL-10. Only fresh MTSC with all types of thymic stromal cells could support 3G116C10CD4 SP thymocytes to produce significantly higher levels of IL-4 and IL-10. These results indicate that different types of thymic stromal cells may play different roles in the regulation of cytokine production by developing 3G116C10CD4 SP thymocytes during functional maturation.
 |
Discussion
|
---|
We have previously separated murine CD4 SP medullary thymocytes into seven subgroups based on their differential expression of CD69, 3G11, 6C10, HSA and Qa-2 (21), and assessed their functional status by the parameters of cell proliferation and cytokine production capacities. A functional hierarchy among these subgroups of CD4 SP thymocytes was observed (manuscript in preparation), which implies that the virgin CD4 SP cells are predisposed to a functional maturation process programmed in the thymic medulla. To study the role of the thymic microenvironment in this thymocyte functional maturation process, an in vitro system has been established by co-culturing the isolated subgroups of cells with MTEC1 cells, a murine thymic medullary epithelial cell line, and the outcomes evaluated. The main results in our experiments are: MTEC1 cells induce the main subgroups of 3G11+6C10 of CD4 SP to become functionally mature by up-regulation of proliferation and Th0-type cytokine production; MTEC1 cells regulate the functional expression of the minor subgroup of 3G116C10 CD4 SP thymocytes by upregulation of cell proliferation and IL-6 production, but down-regulation of the IL-4 and IL-10 production; MTEC1 cells could not induce the subset of CD4 SP thymocytes to develop into Th1-type cytokine-producing cells.
A significant feature is that MTEC1 cells could only induce the cells of each individual subgroup to increase their functional capacity to the level possessed by the naive cells positioned at the next differentiation stage, as shown by the assessment of both cell proliferation capacity and the profiles and levels of cytokines produced. It implies that the cells at each individual stages have the inherent potential to develop into the next individual stages along with the differentiation pathway. Apparently, the functional maturation of CD4 SP thymocytes is a programmed process executed in a stepwise maturation manner.
The mechanisms for the effects of MTEC1 in the functional up-regulation of CD4 SP thymocytes may lie on the impact of cytokines and/or cell surface co-stimulatory molecules. We have reported before that MTEC1 cells produce a panel of cytokines (22), including IL-1, IL-6, IL-7, granulocyte macrophage colony stimulating factor, IFN and chemokines, of which IL-1, IL-6 and IL-7 are growth factors for CD4 SP thymocytes. In the proliferation assay, MTEC1-SN (data not shown) enhances the proliferation capability of CD4 SP thymocytes in response to Con A stimulation, although the enhancing activity is not as high as that of the direct effect of MTEC1 cells through cellcell interaction. By contrast, in the cytokine production assay, only through direct contact with mMTEC1 cells could CD4+CD8 thymocytes produce higher level of cytokines, whereas MTEC1-SN does not have such a regulatory effect. As we have reported previously that MMC-treated MTEC1 cells could barely produce cytokines, it is possible that signals produced by cellcell interaction between CD4 SP thymocytes and MTEC1 cells generated such positive effects on the functional expression of the CD4 SP thymocytes. The nature of such a signal given by the stromal cell is not known at present. MHC class II signal is probably insufficient by itself, since class II molecules cannot induce TCRhi double-positive thymocytes from bcl-2 transgenic mice to mature into CD8CD4+ cells (23). Turda et al. (24) reported that CD3hi SP thymocytes express a high level of CD28, which could act as the second signal to increase the proliferation response upon TCRCD3 stimulation. Other cell surface molecules may also be involved in such an effect. The exact cell surface molecules mediating such an effect awaits further investigation. However, our data demonstrate that most of the CD4 SP thymocytes still need thymic stromal cells to provide intrathymic signals for final maturation.
Our finding that the subgroup of 3G116C10CD4+CD8 thymocytes produce Th2-like cytokines has raised a theoretical issue important in understanding CD4+ Th cell development and functional expression. The naive CD4+ T cells in the periphery are known to produce Th0-type cytokines. The development to Th1- and Th2-type CD4+ T cells from Th0 cells is regarded as a process that occurs only when they are activated in the periphery (2528). Our results argue that the differentiation from Th0 to Th2 CD4+ T cells may have already started in the thymic medulla. At present, we have not found that Th0 CD4 SP thymocytes can give rise to Th1-type cells within the thymic medulla with the markers we have used to define the subgroups of these CD4 SP thymocytes.
At present, we cannot determine the nature of MTEC1 cell-induced functional modulation in the cells of CD4 SP thymocyte subgroups, i.e. if it is a transient effect or a true induction of functional maturation, because of the difficulty of long-term maintenance of such thymocytes in in vitro cultures. At least we show here that one type of medullary thymic epithelial cell line (MTEC1) could induce one step forward in the functional maturation of 6C103G11+CD4 SP thymocytes and regulate the levels of individual cytokines produced by 6C103G11CD4 SP thymocytes. As MTEC1 cells are only one cell line, this may have limitations in the induction capability of CD4 SP thymocyte functional maturation; therefore, we have assessed the inducing capacity of different types of thymic stromal cells on cytokine production by 6C103G11CD4 SP thymocytes. Although different types of thymic stromal cells have different capacities for the induction of 6C103G11CD4 SP thymocytes to produce cytokines, the pattern of Th2-like cytokines does not change. However, it is not certain if multiple types of thymic stromal cells are required for the induction of CD4 SP medullary thymocyte maturation or only a longer time for cells at earlier stages to develop through a stepwise process into late-stage cells with full functions. More experiments need to be performed to estimate the capability of multiple defined types of thymic stromal cells in the induction of the functional maturation process of CD4 SP thymocytes
 |
Acknowledgments
|
---|
This work was supported by the Natural Science Foundation of China (grant no. 39730414).
 |
Abbreviations
|
---|
Con A concanavalin C |
HSA heat shock antigen |
MMC mitomycin C |
PE phycoerythrin |
SP single positive |
 |
Notes
|
---|
Transmitting editor: J. Banchereau
Received 3 August 1999,
accepted 12 April 2000.
 |
References
|
---|
-
Fischer, A. and Malissen, B. 1998. Natural and engineered disorders of lymphocyte development. Science 20:237.
-
Muller, K. and Kyewski, B. A. 1995. Intrathymic T cell receptor (TCR) targeting in mice lacking CD4 or major histocompatibility complex (MHC) class II rescue of CD4 T cell lineage without co-engagement of TCR/CD4 by MHC class II. Eur. J. Immunol. 25:896.[ISI][Medline]
-
Dautigny, N., Campion, A. L. and Lucas, B. 1999. Timing and casting for actors of thymic negative selection. J. Immunol. 162:1294.[Abstract/Free Full Text]
-
Derek, B., Angelo, S., Lucas, B., Waterbury, P. G., Cohen, B., Brabb, T., Goverman, J., Germain, R. N. and Janeway, C. A. 1998. A molecular map of T cell development. Immunity 9:179.[ISI][Medline]
-
DeKoning, J., DiMolfetto, L., Reilly, C., Wei, Q., Havran, W. L. and Lo, D. 1997. Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice. J. Immunol. 158:2558.[Abstract]
-
Kishimoto, H. and Sprent, J. 1997. Negative selection in the thymus includes semimature T cells. J. Exp. Med. 185:263.[Abstract/Free Full Text]
-
Van Meerwijk, J. P. M., Marguerat, S., Lees, R. K., Germain, N., Fowlkes, B. J, and MacDonald, H. R. 1997. Quantitative impact of clonal deletion on the T cell repertoire. J. Exp. Med. 185:377.[Abstract/Free Full Text]
-
Naquet, P., Naspetti, M. and Boyd, R. 1999. Development, organization and function of the thymic medulla in normal, immunodeficient or autoimmune mice. Seminars in Immunol. 11:47.[ISI][Medline]
-
Scollay, R. and Godfrey, D. I. 1995. Thymic emigration: conveyor belts or lucky dips? Immunol. Today 16:268.[ISI][Medline]
-
Ramsdell, F., Jenkins, M., Dinh, Q. and Fowlkes, B. J. 1991. The majority of CD4+CD8 thymocytes are functionally immature. J. Immunol. 147:1779.[Abstract/Free Full Text]
-
Vicari, A., Abehsira-Aamar, O., Papiernik, M., Boyd, R. L. and Tucek, C. L. 1994. MTS-32 monoclonal antibody defines CD4+8 thymocyte subsets that differ in their maturation level, lyymphokine secretion, and selection patterns. J. Immunol. 152:2207.[Abstract/Free Full Text]
-
Bendelac, A., Matzinger, P., Seder, R. A., Paul, W. E. and Schwartz, R. H. 1992. Activation events during thymic selection. J. Exp. Med. 175:731.[Abstract]
-
Bendelac, A. and Schwartz, R. H. 1991. CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation. Nature 353:68.[ISI][Medline]
-
Wilson, A., Day, L. M., Scollay, R. and Shortman, K. . 1988. Subpopulations of mature murine thymocytes: properties of CD4CD8+ and CD4+CD8 thymocytes lacking the heat-stable antigen. Cell Immunol. 117:312.[ISI][Medline]
-
Hayakawa K. and Hardy, R. R. 1988. Murine CD4+ T cell subsets defined. J. Exp. Med. 168:1825.[Abstract]
-
Hayakawa K. and Hardy, R. R. 1991. Murine CD4+ T-cell subsets. Immunol. Rev. 123:145.[ISI][Medline]
-
Lu, L. S. and Chen, W. F. 1996. Functional maturation of mouse CD4+CD8 thymocytes induced by medullary-type thymus epithelial cells. Science in China (C) 39:427.
-
Vanheck, D., Verhasselt, B., Debacker, V., Leclercq, G., Plum, J. and Vandekerckhove, B. 1995. Differentiation to T helper cells in the thymus. Gradual acquisition of T helper cell function by CD3+CD4+ cells. J. Immunol. 155:4711.[Abstract]
-
Dyall, R. and Nikolic-Zugic, J. 1995. The majority of postselection CD4+ single-positive thymocytes requires the thymus to produce long-lived, functional T cells. J. Exp. Med. 11:235.
-
Res, P., Blom, B., Hori, T., Weijer, K. and Spits, H. 1997. Down regulation of CD1 marks acquisition of functional maturation of human thymocytes and defines a control point in late stages of human T cell development. J. Exp. Med. 185:141.[Abstract/Free Full Text]
-
Ge, Q. and Chen, W. F. 1999. Phenotypic identification of the subgroups of murine TCR
ß+CD4+CD8 thymocytes and its implication in the late stage of thymocyte development. Immunology. 97:665.[ISI][Medline]
-
Chen, W. F., Fan, W., Cao, L. X., Pang, X. W., Zhang, P. X. and Yu, Q. 1992. Multiple types of cytokines constitutively produced by an established murine thymic epithelial cell line. Eur. Cytokine Netw. 3:43.[Medline]
-
Petrie, H. T., Strasser, A., Harris, A. W., Hugo, P. and Shortman, K. 1993. CD4+8 and CD4-8+ mature thymocytes require different post-selection processing for final development. J. Immunol. 151:1273.[Abstract/Free Full Text]
-
Turda, L. A., Ledbetter, J. A. and Lee, K. 1990. CD2 is an inducible T cell surface antigen that transduces proliferative signal in CD3+ mature thymocytes. J. Immunol. 144:1646.[Abstract/Free Full Text]
-
Mosmann, R. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348.[Abstract/Free Full Text]
-
Cherwinski, H. M., Schumacher, J. H., Brown, K. D. and Mosmann, T. R., 1987. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J. Exp. Med. 166:1229.[Abstract]
-
Mosmann, T. R. and Coffman, R. L. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7:145.[ISI][Medline]
-
Del Prete, G. F., De Carli, M., Mastromauro, C., Biagiotti, R., Macchia, D., Falagiani, P., Ricci, M. and Romagnani, S. 1991. Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen (s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J. Clin. Invest. 88:346.[ISI][Medline]