Reversible CD8 expression induced by common cytokine receptor
chain-dependent cytokines in a cloned CD4+ Th1 cell line
Cheung-Seog Park,
Yi-Fu Yang,
Xu-Yu Zhou,
Kazuhito Toyooka,
Yumi Yashiro-Ohtani,
Woong-Ryeon Park,
Michio Tomura,
Xu-Guang Tai,
Toshiyuki Hamaoka and
Hiromi Fujiwara
Department of Oncology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
Correspondence to:
H. Fujiwara; E-mail: hf{at}oncogene.med.osaka-u.ac.jp
 |
Abstract
|
---|
T cells that are intrathymically lineage committed are believed to maintain their CD4 or CD8 co-receptor expression. Here, we investigated whether intrathymic lineage commitment involves irreversible genetic modification or whether co-receptor expression can be reprogrammed depending on external stimuli. The CD4+ Th1 clone 2D6 established from splenic T cells as an IL-12-dependent line survived in culture with IL-2, IL-7 or IL-15 alone. Surprisingly, CD8 expression occurred in 2D6 cells upon replacement of IL-12 with any one of the three cytokines that stimulate the common cytokine receptor
chain, yielding CD4+CD8+ 2D6 cells. CD8 expression declined when IL-2 was replaced with IL-12 and CD8 induction was inhibited when IL-12 was included in IL-2 or IL-7 culture. Our observations show that even a lineage-committed mature T cell can be reprogrammed for co-receptor expression in response to particular external stimuli.
Keywords: CD4, CD8, cell surface molecules, cytokine, IL-2, IL-12
 |
Introduction
|
---|
Various mechanisms have been proposed to explain how signaled double-positive thymocytes ultimately differentiate into CD4+ or CD8+ T cells in the thymus (18). Notably, all models presume that intrathymic lineage commitment results in transcriptional termination of one or the other co-receptor molecule. After leaving the thymus, lineage-committed mature T cells are believed to maintain their CD4 or CD8 phenotype throughout their life. Here, we investigated whether intrathymic lineage commitment involves irreversible genetic modification or whether co-receptor reversion occurs even in lineage-determined T cells depending on the conditions. The results show that the CD4+ Th1 clone 2D6 (9) established as an IL-12-dependent cell line and maintained in culture containing IL-12 alone survived in cultures with IL-2, IL-7 or IL-15. Surprisingly, CD8 expression occurred in 2D6 cells when the cytokine IL-12 used for 2D6 maintenance was replaced with IL-2, IL-7 or IL-15 capable of stimulating the common cytokine receptor
chain (
c). This induction of CD8 was inhibited by addition of IL-12 to cultures of
c-dependent cytokines. Thus, co-receptor expression in lineage-committed T cells can be reprogrammed depending on the defined conditions.
 |
Methods
|
---|
Cell line and cell culture
The IL-12-responsive T cell clone, 2D6 (9), was previously established by maintaining cells of an allo-reactive Th1 clone (4-86; derived from C57BL/6 splenic T cells) (10) in the presence of rIL-12 alone without allogeneic antigen-presenting cells (APC). 2D6 cells have been maintained in culture medium (RPMI 1640 supplemented with 10% FCS and 2-mercaptoethanol) containing rIL-12 (250 pg/ml) that is replaced with fresh supplemented media every 3 days. In this study, 2D6 cells were also cultured in the presence of rIL-2 (200 U/ml), rIL-7 (200 U/ml) or rIL-15 (10 ng/ml) with a change of medium every 3 days unless otherwise indicated.
Reagents
Mouse rIL-12, rIL-2 and rIL-7 were kindly provided by Genetics Institute (Cambridge, MA), Shionogi Research Institute (Osaka, Japan) and Toray Institutes (Kamakura, Japan) respectively. Human rIL-15 was purchased from R & D Systems (Minneapolis, MN). FITC-conjugated anti-CD8 and phycoerythrin-conjugated anti-CD4 mAb were from PharMingen (San Diego, CA).
Flow cytometry
2D6 cells were stained doubly with FITCanti-CD8 and phycoerythrinanti-CD4 mAb, and stained cells were analyzed with a FACSCalibur (Becton Dickinson, Mountain View, CA).
Positive selection of CD8+ 2D6 cells
2D6 cells were labeled with superparamagnetic microbeads conjugated to rat anti-mouse CD8 mAb (Miltenyi Biotec, Sunnyvale, CA). Labeled cells were separated from unlabeled cells by magnetic cell sorting using a MiniMACS (Militenyi Biotec) according to a procedure described in detail elsewhere (11). The magnetic cells were retained in the MiniMACS column inserted into the MiniMACS magnet while the non-magnetic cells passed through. Labeled cells were eluted after the column was removed from the magnet.
RT-PCR
Total cellular RNA was prepared from C57BL/6 thymocytes or 2D6 cells by the acid guanidium thiocyanatephenolchloroform method. The synthesis of a single-stranded cDNA from the extracted RNA was performed in a final volume of 50 µl; 2 µl of the cDNA solution and 48 µl of PCR buffer containing 10 mM TrisHCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each deoxynucleoside 5'-triphosphate (dNTP), 100 pmol of each primer and 2.5 U of Taq DNA polymerase (Toyobo, Tokyo, Japan). The PCR for ß-actin evaluated the quality of the cDNA templates. The PCR was within the linear range. The PCR products were fractionated by 2% agarose gel electrophoresis in a volume of 10 µl, and visualized by ethidium bromide staining and UV illumination. The following sense and antisense primers were used: CD8
: sense, 5'-ATGGCCTCACCGTTGACCCG-3'; antisense, 5'-TTACACAATTTTCTCTGAAGGTCTGGGCTTG-3'; CD8ß: sense, 5'-CTCTCTGGAGCAGCTCTGCCC-3'; antisense, 5'-GGTTGGGGCAGTTGTAGGAAGG-3'; CD4: sense, 5'-GAGAGTCAGCGGAGTTCTC-3'; antisense, 5'-CTCACAGGTCAAAGTATTGTT G-3; ß-actin: sense, 5'-GTGGGCCGCTCTAGGCACCAA-3'; antisense, 5'-CTCTTTGATGTCACGCACGATTTC-3'.
 |
Results
|
---|
Induction of CD8 expression in a cloned CD4+CD8- Th 1 cell line
Cells of the IL-12-responsive CD4+ Th1 clone 2D6 (9) could be maintained with IL-12 alone in the absence of antigen/APC (9). Because 2D6 cells also responded to IL-2, they could be maintained with IL-2 instead of IL-12. Surprisingly, CD4+CD8+ cells emerged in culture containing IL-2 at a frequency that increased with the culture period (Fig. 1A
). The 2D6 clone was established from an allo-reactive CD4+CD8- Th1 clone (10). 2D6 cells, when established, were CD4+CD8- like a parental clone. However, they lost CD4 and became CD4-CD8- after >6-month continuous IL-12-culture, as shown in our previous report (9). Figure 2
shows that CD8 expression is induced in 2D6 cells irrespective of whether they express CD4. To exclude the possibility that a small number of CD4+CD8+ cells contaminated the original CD4+ 2D6 population and IL-2 expanded these cells more rapidly than CD4+ 2D6 cells, limiting dilution of the original population was performed in the presence of IL-12. Cells were generated in 33 of 480 wells (five 96-well microculture plates). Proliferating cells in each well were divided and maintained with either IL-12 or IL-2. CD4+CD8+ cells emerged in all 33 cultures that were maintained with IL-2 (profiles of cells from five representative sets of IL-2 or IL-12 cultures are shown in Fig. 1B
). This makes it extremely unlikely that CD4+CD8+ cells contaminated the original population. IL-2 induced comparable levels of cellular expansion in CD4+ and CD4+CD8+ cells (Fig. 1C
). Therefore, the possibility was also excluded that a few CD4+CD8+ cells generated by stimulation with IL-2 were preferentially expanded by IL-2. Instead, it is more likely that the generation of CD4+CD8+ cells occurred slowly but continuously during IL-2 culture.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 1. CD8 co-receptor induction on CD4+ T cells following continuous stimulation with IL-2. (A) 2D6 cells that had been maintained in culture with IL-12 (250 pg/ml) alone were cultured in the presence of IL-2 (200 U/ml) instead of IL-12 for 60 days. The culture medium was replaced every 3 days. The expression of CD4 and CD8 was determined at the time points indicated. (B) 2D6 cells in IL-12 maintenance culture were subjected to limiting dilution in the presence of IL-12. Cells were distributed to wells of five 96-well microculture plates at a cell frequency of 0.3/well. Thirty-three clones were obtained from 480 wells. Cells in each well were divided and cultured with either IL-12 or IL-2 for 30 days. The profiles of five representative clones are shown. (C) CD4+ and CD4+CD8+ 2D6 cells were cultured in the presence of IL-2. The numbers of proliferating cells are expressed as the mean ± SE of triplicate cultures.
|
|

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2. CD8 induction in CD4-CD8- 2D6 cells by stimulation with IL-2. CD4+CD8- and CD4-CD8- 2D6 cells were cultured in the presence of IL-2 (200 U/ml) for the indicated days.
|
|
To determine whether the above observation is common to most T cell clones or unique to 2D6, we cultured several T cell clones in the presence of IL-2 alone. This was done using T cell clones which well proliferated in response to IL-2 alone, including keyhole limpet hemocyanin-specific, ovalbumin-specific and allo-reactive Th clones. CD8 induction was not observed in these clones even after 2-month passages (data not shown).
Molecular basis for IL-2-stimulated CD8 induction
To understand the molecular basis for the modulation of co-receptor expression, we assessed co-receptor RNA transcripts in CD4+CD8- and CD4+CD8+ 2D6 cells obtained from cultures with IL-12 and IL-2 respectively by semiquantitative RT-PCR (Fig. 3
). Unlike the original CD4+ 2D6 cells expressing only CD4 RNA transcripts, CD4+CD8+ cells contained both CD4 and CD8 (
and ß chains) RNA transcripts. Thus, cultures with IL-2 induced CD8 RNA expression.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3. Molecular basis for CD8 induction in 2D6 cells. RT-PCR for CD8 , CD8ß, CD4 and ß-actin (control) mRNA transcripts was performed on the 2D6 cell populations together with unfractionated thymocytes as a positive control.
|
|
We also examined the time course of CD8 induction at early time points after the maintenance of 2D6 cells with IL-2. Figure 4(A)
shows that CD8 mRNA is detectable 7 days after the change of the cytokine from IL-12 to IL-2 and rapidly increases for an additional 4 days. Consistent with this, the frequency of surface CD8+ 2D6 cells increased at these time points (Fig. 4B
).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4. Time course of CD8 induction. CD4+CD8- 2D6 cells were cultured in the presence of IL-2 (200 U/ml) for indicated days. RNA was isolated from portions of the cells, and examined for CD8 and CD4 (control) mRNA expression (A). The rest of the cells were examined for surface CD8 expression (B). The numbers in upper right corners on each figure are the percentages of CD4+CD8+ cells.
|
|
The reversibility of CD8 expression
To determine whether IL-2-induced CD8 expression is reversible, cells expressing CD8 were positively selected from a CD4+CD8+ population obtained by continuous IL-2 culture. The population deleted of CD8- cells was returned to the culture with IL-12. The number of cells expressing CD8 decreased depending on the length of the culture period (Fig. 5A
). However, this might have been due to selective expansion of contaminating CD4+ cells by IL-12 stimulation. To rule out this possibility, cells expressing CD8 were positively selected from the above CD4+CD8+ population and subjected to limiting dilution in the presence of IL-2. Cells were generated in 39 of 288 wells (three 96-well microculture plates). Virtually all proliferating cells in these positive wells were confirmed to be CD8+ (CD4+CD8+) (data not shown). These cells were cultured with either IL-2 or IL-12. 2D6 cells in IL-2 culture continued to express CD8, but CD4+CD8- cells emerged in IL-12 culture from all of 39 wells (profiles of cells from three representative sets of cultures are shown in Fig. 5B
), indicating that CD8 expression is reversible depending on the cytokine used.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5. CD8 co-receptor induction is reversible depending on the cytokine stimulated. (A) 2D6 cells were cultured with IL-2. Twenty-four days later, the cells were divided and cultured with IL-2 or IL-12. At each time point, cells were examined for CD4 and CD8 expression, and the frequency of cells expressing CD8 co-receptor is shown. (B) Cells expressing CD8 (CD4+CD8+ or CD4-CD8+ if any) were positively selected from a CD4+CD8+ 2D6 population. A population deleted of CD8- cells were subjected to limiting dilution in the presence of IL-2. Thirty-nine clones were obtained from 288 wells of 96-well microculture plates. Cells in each well were divided and cultured with IL-2 or IL-12. The profiles for three representative clones are shown.
|
|
CD8 induction by T cell growth factors capable of stimulating
c
To determine whether the generation of CD4+CD8+ cells is inducible with growth factors other than IL-2, we cultured CD4+ 2D6 cells with IL-4, IL-7 or IL-15. Cells could be maintained with IL-7, but not with IL-4. The growth rate of 2D6 cells stimulated with IL-15 was very low during the initial 3 weeks, but after 1 month it became comparable to the growth rate of cells cultured with IL-2 or IL-7. Both IL-7 and IL-15 stimulation induced CD8 expression (Fig. 6A
). Because the receptors for IL-2, IL-7 and IL-15 all contain
c as a common component, CD8 induction may be related to the stimulation of and signaling through
c.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 6. CD8 co-receptor induction by stimulation with IL-2, IL-7 or IL-15. 2D6 cells were cultured in the presence of either IL-12, IL-2, IL-7 or IL-15 alone by standard passage (every 3 days) (A) or rapid passage (every other day) (B).
|
|
2D6 cells have thus far been maintained by passages of every 3 days (standard passage). When passages were performed more frequently (every other day), IL-2- or IL-7-stimulated CD8 induction was strikingly accelerated (Fig. 6B
). With either standard or more frequent passages, no substantial difference was observed in the rate and extent of CD8 induction between IL-2 and IL-7 cultures.
Prevention of IL-2- or IL-7-induced CD8 expression by IL-12 signals
To determine whether IL-12 signals influence IL-2- or IL-7-induced CD8 expression, CD4+ 2D6 cells were cultured with IL-12 plus IL-2 or IL-12 plus IL-7 (Fig. 7
). The coexistence of IL-12 in cultures with IL-2 or IL-7 almost completely inhibited CD8 induction. This is unlikely to be due to continuous enrichment of CD4+ 2D6 cells by stimulation with IL-12, because the 2D6 proliferation induced by IL-2 was more potent than that induced by IL-12 (9). These results suggest that CD8 expression in T cells is modulated by a cumulative effect of cytokines produced in the microenvironment.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 7. IL-12 inhibits IL-2- or IL-7-induced CD8 expression. CD4+ 2D6 cells were cultured in the presence of IL-12 (250 pg/ml) alone or together with IL-2 (200 U/ml) or IL-7 (200 U/ml).
|
|
 |
Discussion
|
---|
Contrary to the notion that intrathymically established co-receptor expression in T cells is maintained for the remainder of their life span, our results illustrate that CD8 expression is induced by particular conditions in mature T cells committed to the CD4 lineage. The modulation of co-receptor expression was previously reported in human peripheral blood lymphocyte T cells (12) and umbilical cord blood T cells (13). Stimulation of peripheral blood lymphocyte CD4+ T cells in the presence of IL-4 resulted in the generation of CD4+CD8+ T cells at a significant frequency, on the one hand (12). On the other, neonatal CD8+ T cells started to express CD4 within 24 h after anti-CD3/CD80 activation (13). Together, intrathymic lineage commitment in T cells does not involve irreversible genetic modification of co-receptor expression, but allows the potential for reprogramming.
Our present study showed that the modulation of co-receptor expression is induced based on the change in a single external stimulus. CD8 induction in the CD4+ 2D6 Th1 clone occurred when IL-12 was replaced with either of the
c-dependent cytokines such as IL-2. The change was not associated with TCR stimulation or the presence of feeder cells (APC). Virtually all CD4+ Th clones are maintained by stimulation with antigen plus APC and addition of exogenous IL-2. Despite stimulation with exogenous and/or endogenous IL-2, they continue to be CD4+ and CD8-. In addition to the presence of APC, moderate amounts of IL-12 are produced during interactions between Th1 clones and antigen/APC (10,14). Therefore, most Th1 clones might not have had the opportunity to be stimulated in long-term cultures with IL-2 in the absence of IL-12 and TCR signals. Because of this, we attempted to maintain several CD4+ T cell clones with IL-2 alone for a long period (~2 months) and to detect CD8 expression on these clones. However, we did not observe IL-2-induced CD8 expression on T cell clones other than the 2D6 clone (our unpublished observations). In this context, the 2D6 clone is unique, and this feature may be related to the fact that 2D6 cells have long been maintained with IL-12 alone in the absence of IL-2 and TCR signals. It will be necessary to determine whether CD8 expression is also induced in other cell lines exposed to IL-12 for a long period and then stimulated with IL-2.
While the induction of CD8 molecules in CD4+ 2D6 cells is unequivocal, this phenomenon should be considered in the context of the biological relevance, particularly of the presence and generation of TCR
ß+CD4+CD8+ T cells in the periphery. In contrast to conventional T cells present in lymph nodes and spleen, intraepithelial T lymphocytes (IEL) located in intestinal epithelial layers exhibit a markedly different pattern of CD4 and CD8 expression (reviewed in 15). IEL consist of TCR
and TCR
ß T cell populations, the latter of which is further divided into various subsets depending on the expression of CD4 and/or CD8 as well as CD8
or CD8
ß (15). Thus, TCR
ß+ IEL include CD4-CD8
, CD4-CD8
ß, CD4+CD8
and CD4+CD8
ß cells. The observation that both TCR
+ and TCR
ß+ IEL are generated in athymic radiation chimeras (16) has suggested the thymus-independent development of IEL and the role of the intestinal epithelium as a site of T lymphopoiesis (15). However, evidence that the generation of TCR
ß+ IEL is impaired in nude mice (17) and the report that CD8
ß+ IEL are thymus derived (18) have shown the role of the thymus in the generation of TCR
ß+CD8
ß+ IEL. Nevertheless, it is noteworthy that the number of TCR
ß+ IEL, particularly TCR
ß+CD4+CD8+ IEL, strikingly increases with age (7), and that this age-associated increase is strictly dependent on exposure of the animal to a conventional environment (diet/antigens) and is not induced when the animal is maintained within a germ-free environment (19). These observations may be consistent with the notion that IEL are indeed generated in situ in the intestinal epithelium while the presence of a thymus is required (15). Thus, it is obvious that TCR
ß+CD4+CD8
ß+ T cells with similar phenotypes to those of IL-2-stimulated 2D6 cells are generated in vivo depending on continuous exposure of individuals to conventional environment.
c-dependent cytokines have been shown to play a critical role in IEL development (reviewed in 20). For example, the requirement of IL-7 and IL-15 was demonstrated for the development of TCR
(2123), although a contribution of IL-2 to IEL development has not yet been excluded. Strikingly,
c-deficient mice lack all IEL subsets, including TCR
ß+CD8
ß+ (24,25). Thus, irrespective of whether TCR
ß+CD4-CD8
ß or TCR
ß+CD4+CD8
ß IEL are thymus derived, their expansion in the intestinal epithelium likely requires any of
c-dependent cytokines. While it is unknown where CD4/CD8 co-receptor expression in TCR
ß+ IEL occurs, recent studies (2629) suggest that CD8 induction is regulated differently in developing thymocytes and IEL. However, it also remains to be investigated which signals are responsible for CD8 induction in IEL.
In the present 2D6 cell model, CD8 induction in 2D6 cells is likely based on the stimulation of the
c chain considering CD8 expression induced by IL-7 or IL-15 besides IL-2. It should be noted that
c stimulation does not always lead to CD8 induction because simultaneous stimulation with IL-12 canceled the effect of
c-dependent cytokines. This suggests that CD8 induction occurs as a result of the cumulative effect of
c-dependent cytokines and non-
c-dependent cytokines such as IL-12. Even in the absence of IL-12 signals,
c stimulation in a 2D6 cell population gradually increases the frequency of CD8+ cells. This phenomenon observed in a long-term in vitro culture resembles an age-dependent (18) and
c-associated development of TCR
ß+CD8+ IEL in vivo (24,25). 2D6 cells are themselves intrathymically developed T cells. However, CD8 induction in 2D6 cells upon stimulation with
c-dependent cytokines may represent an aspect of the mechanism underlying CD8 expression in extrathymically generated/expanded IEL.
It is totally unknown how
c stimulation in 2D6 cells results in signaling for CD8 induction. Instead of STAT3/STAT4 activation by IL-12 (30,31),
c stimulation induces STAT5 phosphorylation (32,33). Therefore, it may be simply speculated that STAT5 activation is related to CD8 induction. However, this is not the case because a detectable level of STAT5 activation is observed during IL-12 stimulation in 2D6 cells (34) and high levels of IL-2-induced STAT5 activation are not down-regulated by simultaneous stimulation with IL-12 (our unpublished observations). The binding of IL-2 to the IL-2 receptor complex initiates the activation of various signaling molecules besides STAT5 (35,36). Further studies will be required to investigate what signal(s) from cytokine receptor complexes bearing the
c chain as a component is responsible for inducing CD8 in 2D6 cells.
In summary, the 2D6 cell system provides biologically important evidence that even a mature T cell committed to the CD4/CD8 lineage can be reprogrammed to change its co-receptor expression when the T cell is maintained in a given microenvironment and then exposed to particular external stimuli. This demonstrates the reversibility of intrathymic lineage commitment. The characterization of the promoter/enhancer elements that regulate expression of CD8 is at an early stage. The present cell system provides a quite useful model for investigating the molecular mechanisms underlying the induction of CD8 co-receptor expression that occurs independently of the thymus.
 |
Acknowledgments
|
---|
The authors are grateful to Drs Gene Shearer, Avinash Bhandoola and Qing Yu for their critical review of this manuscript, and to Mrs Mami Yasuda for her secretarial assistance. This work was supported by Grants-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
 |
Abbreviations
|
---|
APC antigen-presenting cell |
IEL intraepithelial T lymphocytes |
Received 6 August 2001,
accepted 26 November 2001.
 |
References
|
---|
-
Chan, S., Correia-Neves, M., Benoist, C. and Mathis, D. 1998. CD4/CD8 lineage commitment: matching fate with competence. Immunol. Rev. 165:195.[ISI][Medline]
-
Hedrick, S. M. and Sharp, L. L. 1998. T-cell fate. Immunol. Rev. 165:95.[ISI][Medline]
-
Kisielow, P. and von Boehmer, H. 1995. Development and selection of T cells: facts and puzzles. Adv. Immunol. 58:87.[ISI][Medline]
-
Marrack, P. and Kappler, J. 1997. Positive selection of thymocytes bearing
ß T cell receptors. Curr. Opin. Immunol. 9:250.[ISI][Medline]
-
Basson, M. A., Bommhardt, U., Mee, P. J., Tybulewicz, V. L. and Zamoyska, R. 1998. Molecular requirements for lineage commitment in the thymus-antibody-mediated receptor engagements reveal a central role for lck in lineage decisions. Immunol. Rev. 165:181.[ISI][Medline]
-
Davis, C. B., Killeen, N., Crooks, M. E., Raulet, D. and Littman, D. R. 1993. Evidence for a stochastic mechanism in the differentiation of mature subsets of T lymphocytes. Cell 73:237.[ISI][Medline]
-
von Boehmer, H., Aifantis, I., Azogui, O., Feinberg, J., Saint-Ruf, C., Zober, C., Garcia, C. and Buer, J. 1998. Crucial function of the pre-T-cell receptor (TCR) in TCR ß selection, TCR ß allelic exclusion and
ß versus
lineage commitment. Immunol. Rev. 165:111.[ISI][Medline]
-
Singer, A., Bosselut, R. and Bhandoola, A. 1999. Signals involved in CD4/CD8 lineage commitment: current concepts and potential mechanisms. Semin. Immunol. 11:273.[ISI][Medline]
-
Maruo, S., Ahn, H. J., Yu, W. G., Tomura, M., Wysocka, M., Yamamoto, N., Kobayashi, M., Hamaoka, T., Trinchieri, G. and Fujiwara, H. 1997. Establishment of an IL-12-responsive T cell clone: its characterization and utilization in the quantitation of IL-12 activity. J. Leuk. Biol. 61:346.[Abstract]
-
Maruo, S., Toyo-oka, K., Oh-hora, M., Tai, X. G., Iwata, H., Takenaka, H., Yamada, S., Ono, S., Hamaoka, T., Kobayashi, M., Wysocka, M., Trinchieri, G. and Fujiwara, H. 1996. IL-12 produced by antigen-presenting cells induces IL-2-independent proliferation of T helper cell clones. J. Immunol. 156:1748.[Abstract]
-
Miltenyi, S., Muller, W., Weichel, W. and Radbruch, A. 1990. High gradient magnetic cell separation with MACS. Cytometry 11:231.[ISI][Medline]
-
Paliard, X., Malefijt, R. W., de Vries, J. E. and Spits, H. 1988. Interleukin-4 mediates CD8 induction on human CD4+ T-cell clones. Nature 335:642.[ISI][Medline]
-
Yang, L. P., Riley, J. L., Carroll, R. G., June, C. H., Hoxie, J., Patterson, B. K., Ohshima, Y., Hodes, R. J. and Delespesse, G. 1998. Productive infection of neonatal CD8+ T lymphocytes by HIV-1. J. Exp. Med. 187:1139.[Abstract/Free Full Text]
-
Murphy, E. E., Terres, G., Macatonia, S. E., Hsieh, C. S., Mattson, J., Lanier, L., Wysocka, M., Trinchieri, G., Murphy, K. and O'Garra, A. 1994. B7 and interleukin 12 cooperate for proliferation and interferon
production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223.[Abstract]
-
Poussier, P. and Julius, M. 1994. Thymus independent T cell development and selection in the intestinal epithelium. Annu. Rev. Immunol. 12:521.[ISI][Medline]
-
Mosley, R. L., Styre, D. and Klein, J. R. 1990. Differentiation and functional maturation of bone marrow-derived intestinal epithelial T cells expressing membrane T cell receptor in athymic radiation chimeras. J. Immunol. 145:1369.[Abstract/Free Full Text]
-
Bandeira, A., Itohara, S., Bonneville, M., Burlen-Defranoux, O., Mota-Santos, T., Coutinho, A. and Tonegawa, S. 1991. Extrathymic origin of intestinal intraepithelial lymphocytes bearing T-cell antigen receptor
. Proc. Natl Acad. Sci. USA 88:43.[Abstract]
-
Guy-Grand, D., Cerf-Bensussan, N., Malissen, B., Malassis-Seris, M., Briottet, C. and Vassalli, P. 1991. Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J. Exp. Med. 173:471.[Abstract]
-
Bandeira, A., Mota-Santos, T., Itohara, S., Degermann, S., Heusser, C., Tonegawa, S. and Coutinho, A. 1990. Localization of gamma/delta T cells to the intestinal epithelium is independent of normal microbial colonization. J. Exp. Med. 172:239.[Abstract]
-
Malek, T. R., Porter, B. O. and He, Y. W. 1999. Multiple
c-dependent cytokines regulate T-cell development. Immunol. Today 20:71.[ISI][Medline]
-
Maki, K., Sunaga, S., Komagata, Y., Kodaira, Y., Mabuchi, A., Karasuyama, H., Yokomuro, K., Miyazaki, J. I. and Ikuta, K. 1996. Interleukin 7 receptor-deficient mice lack

T cells. Proc. Natl Acad. Sci. USA 93:7172.[Abstract/Free Full Text]
-
He, Y. W. and Malek, T. R. 1996. Interleukin-7 receptor
is essential for the development of 
+ T cells, but not natural killer cells. J. Exp. Med. 184:289.[Abstract]
-
Lodolce, J. P., Boone, D. L., Chai, S., Swain, R. E., Dassopoulos, T., Trettin, S. and Ma, A. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669.[ISI][Medline]
-
Cao, X., Shores, E. W., Hu-Li, J., Anver, M. R., Kelsall, B. L., Russell, S. M., Drago, J., Noguchi, M., Grinberg, A., Bloom, E. T., Paul, W. E., Katz, S. I., Love, P. E. and Leonard, W. J. 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor
chain. Immunity 2:223.[ISI][Medline]
-
DiSanto, J. P., Muller, W., Guy-Grand, D., Fischer, A. and Rajewsky, K. 1995. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor
chain. Proc. Natl Acad. Sci. USA 92:377.[Abstract]
-
Ellmeier, W., Sunshine, M. J., Losos, K., Hatam, F. and Littman, D. R. 1997. An enhancer that directs lineage-specific expression of CD8 in positively selected thymocytes and mature T cells. Immunity 7:537.[ISI][Medline]
-
Hostert, A., Tolaini, M., Roderick, K., Harker, N., Norton, T. and Kioussis, D. 1997. A region in the CD8 gene locus that directs expression to the mature CD8 T cell subset in transgenic mice. Immunity 7:525.[ISI][Medline]
-
Ellmeier, W., Sunshine, M. J., Losos, K. and Littman, D. R. 1998. Multiple developmental stage-specific enhancers regulate CD8 expression in developing thymocytes and in thymus-independent T cells. Immunity 9:485.[ISI][Medline]
-
Hostert, A., Garefalaki, A., Mavria, G., Tolaini, M., Roderick, K., Norton, T., Mee, P. J., Tybulewicz, V. L., Coles, M. and Kioussis, D. 1998. Hierarchical interactions of control elements determine CD8
gene expression in subsets of thymocytes and peripheral T cells. Immunity 9:497.[ISI][Medline]
-
Bacon, C. M., Petricoin, E., III, Ortaldo, J. R., Rees, R. C., Larner, A. C., Johnston, J. A. and O'Shea, J. J. 1995. Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes. Proc. Natl Acad. Sci. USA 92:7307.[Abstract]
-
Jacobson, N. G., Szabo, S. J., Weber-Nordt, R. M., Zhong, Z., Schreiber, R. D., Darnell, J., Jr and Murphy, K. M. 1995. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J. Exp. Med. 181:1755.[Abstract]
-
Lin, J. X., Migone, T. S., Tsang, M., Friedmann, M., Weatherbee, J. A., Zhou, L., Yamauchi, A., Bloom, E. T., Mietz, J., John, S. and Leonard, W. J. 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2:331.[ISI][Medline]
-
Hou, J., Schindler, U., Henzel, W. J., Wong, S. C. and McKnight, S. L. 1995. Identification and purification of human Stat proteins activated in response to interleukin-2. Immunity 2:321.[ISI][Medline]
-
Ahn, H. J., Tomura, M., Yu, W. G., Iwasaki, M., Park, W. R., Hamaoka, T. and Fujiwara, H. 1998. Requirement for distinct Janus kinases and STAT proteins in T cell proliferation versus IFN-
production following IL-12 stimulation. J. Immunol. 161:5893.[Abstract/Free Full Text]
-
Lin, J. X. and Leonard, W. J. 1997. Signaling from the IL-2 receptor to the nucleus. Cytokine Growth Factor Rev. 8:313.[Medline]
-
Nelson, B. H. and Willerford, D. M. 1998. Biology of the interleukin-2 receptor. Adv. Immunol. 70:1.[ISI][Medline]