Regulation of IL 2 expression in mitogen-activated murine T lymphocytes

Immunobiol., vol. 174, pp. 300-312 (1987)

Institute of Virology and Immunobiology, University of Wiirzburg, Federal Republic of Germany

Regulation of IL 2 Expression in Mitogen-activated Murine T Lymphocytes R. SWOBODA, E. WECKER, and ANNE LIESE SCHIMPL Received January 6, 1987 . Accepted in Revised Form February 4, 1987

Abstract In mitogen-stimulated mouse spleen cells, the IL 2 gene is only transiently expressed with maximal mRNA steady state levels between 6--14 h post stimulation with Concanavalin A (Con A). This is also reflected by the kinetics of IL 2 release into culture supernatants. Con Astimulated L3T4+ and Lyt2+ T cell subpopulations express the IL2 gene and produce IL2 similarly. The half-life of IL2 mRNA is only 30 min, but can be prolonged significantly by cycloheximide. At later post stimulation times IL 2 gene transcription is reduced, as indicated by the reduced effect of cycloheximide. IL 2 gene expression is not influenced by added IL 2 or IFN-y.

Introduction IL 2 was originally described as a lymphokine that is predominantly or exclusively produced by the UT4+ LytT subset of T cells and as being obligatory for the growth of both the UT4+ LytT and the UT4- Lyt2+ subsets (for review, see reference 1). Despite the fact that some of the tenets of the original IL 2 concept are currently being modified (see »Discussion«), a central role for IL 2 in the regulation of the in vitro immune responses seems firmly established. IL 2 (2, 3) and at least one of the chains of its high affinity receptor (4) have been cloned, and the structure of the genes has been elucidated. In the present study, we investigated the kinetics of IL 2 gene expression at the level of mRNA in mitogen-activated splenic lymphocytes and in freshly isolated T cell subsets. Also, the stability of the mRNA and the influence of various agents on maximal levels of expression and stability were investigated.

Materials and Methods Cell cultures

Spleen cell suspensions were prepared from B6D 2F1, C57Bl/6 or Balb C mice and cultured at 5 X 106/ml in RPM I 1640 supplemented as given in reference 5 and with 5 % fetal calf serum,

Regulation ofIL 2 Expression . 301 5 X 10- 5 M 2-mercaptoethano!' Cells were incubated for the times indicated at 3r in a humidified atmosphere containing 6 % CO 2 in air. Mitogen concentrations used were: Con A, 2.5 Ilg/ml; phorbol myristate acetate, 10 ng/ml; ionomycin, 0.5 11M. When other additives were used, conditions are given below and in the legends to the figures. IL 2 activity was assayed using IL 2-dependent Cl 3 cells. Cells were seeded at 5 X 103/100 III of the abovementioned medium or dilutions of the supernatants to be assayed. After 24 h, the cultures were pulsed with 0.25 IlCi of lH-thymidine (specific activity 2 Ci/mM) for 16 h; cells were harvested, and 3H-thymidine incorporation was monitored.

Isolation of L3T4+ LytT and L3Tr Lytr T cells Spleen cells from Balb C mice were passed over nylon wool as described (6). The resulting cells were treated either with a monoclonal anti-Lyt 2.2 antibody (19/178 (7» or with a monoclonal anti-UT4 antibody (HI29/19 (8) and GK 1.5 (9» at concentrations of 10 Ilg/ml in balanced salt solution (BSS) for 30 min on ice. Cells were washed twice in BSS and subsequently treated for 45 min at 37° with complement (guinea pig serum: low tox rabbit serum = 3: 1) diluted 1: lOin BSS. After treatment, cells were again washed twice in BSS, adjusted to 5 x 106 /ml in RPMI supplemented as above. Fluorescence staining of the two subpopulations showed that contamination by the unwanted T cell population was undetectable after this treatment. Remaining, contaminating non-T cells were insufficient for optimal stimulation with Con A. Therefore, mitomycin-treated accessory cells (50 Ilg mitomycin/ml; 30 min, 3r, washed 4 times with BSS) were added at a ratio of T: accessory cells of 2:1.

Other reagents used Where indicated, cultures received human rIL 2 (a gift of Sandoz, Basel, Switzerland) or murine ry-IFN, a gift of Genentech, via Dr. Albert, Vienna. The specific activities of the cloned lymphokines were 6 x 106 U/mg and 1.3 x 107 U/mg, respectively. They were used at the concentrations given in the legends to the figures. Cycloheximide (Actidion, Serva, Heidelberg, F.R.G.) was used at 20 ltg/ml, actinomycin D (Sigma GmbH, Miinchen, F.R.G.) at 5 ltg/m!.

RNA isolation and Northern blot analyses Cells were harvested at the times indicated, sedimented and total RNA was extracted following the guanidinium isothiocyanate/cesium chloride method (10). RNA samples were denatured, electrophoresed on 1.5 % agarose gels in 2.2 M formaldehyde (11), transferred to nitrocellulose membranes (Schleicher & Schuell, BA83) by diffusion (12) or onto zeta probe membrane (Biorad) by electroblotting (13) in 25 mM sodium phosphate, pH 5.5, at 250 rnA overnight.

Hybridization probes A genomic clone of murine IL 2 was isolated by screening a genomic mouse DNA library in lambda Charon 4 A phages (14) with 3 oligonucleotides corresponding to positions 45-65, 151-171 and 540--560 of the murine IL2 DNA (3). The 0.7 kb Eco RIIHind III fragment (containing 160 bases of the 3rd intron, the fourth exon and 3' sequences extending to position 1013) was subcloned into the gemini vector pGEM-2. This subclone is called Gem II E4R. Essentially, the same results were obtained using other subclones, covering exons 1 and 2.

Hybridization For hybridization, radioactive RNA was synthesized from Gem II E4R using SP6 polymerase (15). The SP6 transcript was separated from unincorporated nucleotides by chromatography through Sephadex G 50, precipitated with t-RNA carrier, and the pellet was dissolved in hybridization buffer. Hybridization buffer was 50 % formamide, 50 mM Na phosphate, pH 6.5,0.8 M NaCl, 1 mM EDTA, 0.1 % SDS, 2.5 x Denhardt's solution, 250 Ilg/ml denatured salmon sperm DNA, 500 ltg/ml yeast RNA, 10 ltg/ml poly A (16). Filters were prehybridized

302 . R. SWOBODA, E. WECKER, and ANNELIESE SCHIMPL 1-4 h at 55°C, the probe was denatured at 90 °C for 5 min, and the radioactive probe was added to the filters. The filters were incubated at 55°C for 12-16 h and washed 3-5 times for 20 min each. Washing solution was 50 mM NaCl, 20 mM Na-phosphate, pH 6.5,1 mM EDTA, 0.1 % SDS. To increase the specificity, filters were finally washed in 2 X SSC (3 times at RT), incubated with 1 !lg/ml RNase A in 2 X SSC for 15 min at RT and in 0.1 X SSC, 0.1 % SDS at 55°C for 30 min (16).

Results

Kinetics of ILl expression after mitogen stimulation of lymphocytes We first investigated the appearance of detectable levels of IL 2 mRN A in spleen cells stimulated with either Con A or a combination of phorbol ester and Ca ionophore. Figure 1 shows the Northern blot analysis of total RNA isolated at various times after stimulation. Panel A, obtained using RNA from Con A-stimulated cells, shows that IL 2 mRNA is virtually undetectable at time zero. A distinct IL 2-specific band becomes visible after approximately 3 h; a maximum plateau level is reached between 6-14 h. At later times, the signal for IL 2 mRNA decreases again, being barely visible when RNA was extracted 24-30 h after stimulation of the cells. Stimulation with PMA and ionomycin induces IL2 RNA more rapidly (panel B). However, the decrease in detectable mRNA levels also starts earlier in these cells. The rapid decline in IL 2 mRNA levels is reflected by the lack of additional IL 2 production at late times after stimulation (Fig. 2).

A

B

1 2 3

4 5 •

7

a

9 10 11 12

13 14 15 1.

- 235

-

1. S

<

IL- 2 >

n 23 5 18S-

Fig. 1. A: Kinetics of IL 2 gene expression in Con A-stimulated spleen cells. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Track No.: h post-stimulation: 0 0.5 1 2 3 4 5 6 7 8 9 10 12 14 16 18 20 B: Kinetics of IL 2 gene expression in PMA and ionomycin stimulated spleen cells. 1 2 3 4 Track No.: h post-stimulation: 4 8 16 24

Regulation of IL 2 Expression . 303

4,0

c-

3,2

2,4 1,6 0,8

o-

n •

I

o

OQ I

~

"'"

co

I

.. ~ I

N

~ I

'" ..

n ~ I

time

(h)

0N

Fig. 2. Kinetics of IL 2 release by Con A -stimulated spleen cells. Culture supernatants were harvested every 4 h and replaced by new media. Supernatants were assayed for increase of )Hthymidine incorporation by an IL 2-dependent T cell line (clone 3). B

A

1 2 3

1234517 ••

Il-2>

-23 5

235 -

- .5

1'5-

Fig. 3. IL 2 gene expression in T cell subpopulations. 1 2 3 A: Track No.: h post-stimulation with Con A: 0 8 8'

< Il- 2

4

o

5

6

8.5

18

7

32

8 9 8 18

spleen cells L3T4 + cells b Lyt2+ cells b , Cycloheximide was present from ~8 h post-stimulation b Mitomycin-treated accessory cells were added to the T cell subpopulations in the ratio 0.5:1 1 2 3 B: Track No.: h post-stimulation with Con A: 8 (AC)' 8 (L3T4 +)' (Lyt2+)' 'Mitomycin-treated nu/nu spleen cells were added as accessory cells in the ratio 0.5:1.

304 . R.

SWOBODA,

E.

WECKER,

and

ANNE LIESE SCHIMPL

Expression of the IL2 gene in L3T4+ Lytr and L3T4- Lytr T cells Figure 3 shows Northern blot analyses of RNAs derived from the UT4 + and Lyt2+ subsets of T cells, isolated at various times after stimulation with Con A. Mitomycin-treated syngeneic splenic non-T cells were added for optimal activation. The data clearly show that similar levels of IL 2 mRNA can be detected after activation of both subsets. IL 2 expression measured at 8 hand 18 h, respectively, is comparable in the two subsets and is higher at the earlier times, similarily as shown for unseparated spleen cells in Figures 1 and 2. Lane 1 of panel B shows that the mitomycin-treated accessory cells alone do not give rise to an IL 2 mRNA signal. Also, Cr 15, cells of a long-term cytotoxic T cell clone, were unable to express IL 2, at least not 8 h after restimulation with Con A.

IL 2 mRNA has a short half-life The decline in detectable IL 2 mRNA levels, starting 12-14 h after Con A stimulation and between 4-8 h after PMA + ionomycin treatment could be due to transient expression of IL2 and/or a very short half-life of IL2 mRNA. To analyse the half-life of the RNAs at the peak of IL 2 expression, Con A-stimulated cells were treated with actinomycin D at 8 hand RNAs were extracted at the time of actinomycin D addition (time 0 in Fig. 4) and 20,35 and 70 min thereafter. The RNAs were then analyzed by Northern cpm

100

~

\

\ \

\

50

20

40

60

80

(min.)

Fig. 4. Half-life of IL 2 mRNA. Spleen cells were stimulated for 8 h, and then they received actinomycin D. The RNA of parallel cultures was analyzed by Northern blot for IL 2 mRNA at the time indicated. Bands containing IL 2 mRNA were cut out from nitrocellulose filters, and their radioactivity was counted. Values represent CPM minus background values determined at 0 h of stimulation.

Regulation of IL 2 Expression . 305

blots for the decline in the intensity of the IL 2 signal. The rate of decline allows to determine the half-life of IL 2 mRNA as being approximately 30 min. During the time of observation, actin mRNA levels stay approximately constant, which is in agreement with a fairly long half-life of this mRNA.

Steady state levels of IL2 mRNA are increased after inhibition of protein synthesis Recently, several cases have been described in which steady state levels of rather short-lived mRNAs were found to be greatly increased after addition of inhibitors of protein synthesis such as cycloheximide. Cases in point are those of c-myc, c-fos and human IL 2 mRNA (27). Figure 5 A shows the effect of cycloheximide on murine IL 2 mRNA levels as compared to those of actin (Fig. 5 B). Lane 3 of panel A shows that cycloheximide per se is unable to induce IL 2 mRNA and that it prevents mRNA induction in Con A -treated spleen cells if present from the onset of the culture (Fig. 5 A, actin>

..........

2 3 4 5 6 7 8 9 10 11 12 13

234567 28S 185 IL- 2>

23S 16 s -

c 1L-2

Fig. 5. Effect of cycloheximide on steady state levels of IL 2 mRNA. A: Track No.: 1 2 3 4 5 h in culture: 0 4 4 4 4 Con A: + + 0-4 0-4 Cycloheximide present (h) B: Track No.: h post Con A-stimulation: Cycloheximide' :

1 0

2 4

3 8

6 8

+

7

8

+

4-8

4 5 6 7 8 9 10 11 12 13 12 16 20 24 30 36 12 16 20 24

+

+ + +

, Cycloheximide was present from 8 h post stimulation until harvest time. The 18S rRNA is here visible, since the filter was not treated with RNase A after hybridization.

306 . R. SWOBODA, E. WECKER, and ANNELIESE SCHIMPL cpm

A \

\ \

\

\

50

20

40

60

A - - - ' - --A

Con A

&--&

ConA with CHI

80

(min,)

Fig. 6. Effect of cycloheximide on half-life of IL 2 mRNA. Experimental conditions see legend to Figure 4. Cycloheximide was added 7.5 h and actinomycin D 8 h post-stimulation with ConA.

lane 5). However, added at later times after Con A activation, e.g. at 8 h (Fig. 5 B, lane 10), it leads to pronounced increases (15-25-fold) in steady state levels of IL2 mRNA, while actin mRNA is not increased. Taken together, these data suggest that cycloheximide treatment per se fails to induce transcription, but has a positive effect in cells which already actively transcribe IL 2 mRNA. In analogy to other systems, it was conceivable that the cycloheximide treatment affects the stability of the mature mRNA itself. Figure 6 shows that this is indeed the case. In this experiment, the half-life of IL2 mRNA in Con A-treated cells is compared to that in cells treated with Con A and cycloheximide. Both cell populations were again treated with actinomycin D to prevent further transcription after 8 h of Con A stimulation. Cycloheximide was added 30 min before actinomycin D. In the absence of cycloheximide, TYz of IL2 mRNA is approximately 30 min; in cycloheximide-treated cells, mRNA levels stay more or less contant over the period of time observed, indicating a TYz of approximately 2h.

Even in the presence of cycloheximide, steady state levels of IL2 mRNA decrease at late times after stimulation In the experiment shown in Figure 7, cycloheximide was added to Con A-stimulated spleen cells 2 h before RNA extraction. This treatment at

Regulation of IL 2 Expression . 307

1234567

- 235 - 16 5 <

IL- 2

Fig. 7. Decrease of cycloheximide effect with increasing time after stimulation. Track No.: 1 2 3 4 5 6 12 14 15 16 17 18 h post-stimulation with Con A' :

7 20

• Cycloheximide was always present during the last 2 h before harvest.

all times studied leads to an increased IL 2 mRNA signal as compared to cells not treated with cycloheximide. However, levels reached after treatment with cycloheximide between 16-18 h (lane 6) or 18-20 h (lane 7) are much lower than those observed after treatment between 10-12 h (lane 1). This observation indicates that the rate of IL 2 gene transcription is lower at 16-20 h than at 10-12 h, and thus, less mRNA is available for stabilization by cycloheximide treatment. Preliminary nuclear run-off data tend to support this interpretation (V. Paetkau, personal communication).

Lack of evidence for an endogenous feed back inhibition by IL2 or IFN-y At the times when IL 2 expression declines, IL 2 and IFN -y ha~e reached maximal levels in the supernatants of mitogen-stimulated cultures and IL 2 receptors are fully expressed. We therefore studied whether IL 2 via its interaction with the receptor or IFN -y was able to modulate IL 2 expression. Figure 8 shows that this was not the case. Neither 100 U/ml of rIFN-y, nor added IL2 (100 Units /ml) affected IL2 mRNA levels measured at 8 and 20 h post-stimulation. IFN-y also was unable to induce IL 2 expression in IL 2-dependent long-term cytotoxic T cells, the Cr1S cells (data not shown).

308 . R. SWOBODA, E. WECKER, and ANNE LIESE SCHIMPL

1234567

235165< IL- 2

Fig. 8. No feedback inhibition of IL2 gene expression by IL2 or IFN-y. Track No.: 1 2 3 4 5 6 h post-stimulation with Con A: 0 8 20 8 20 8 Additions: IFN-y IFN-y IL2

7 20 IL2

Discussion Appropriate stimulation of T lymphocytes leads to the induction of T cell growth factor production (TCGF, IL 2) and to the expression of receptors for IL 2 (1, 2). Within the framework of the IL 2 concept as originally formulated, the L3T4 + Lytr subsets of T cells were considered the sole producers of IL 2 which could then act on both L3T4 + and Lyt2+ subsets. In the meantime, it has been described that: 1) only a subset of L3T4+ cells may produce IL 2 (17), 2) that BSFI may act, albeit at low efficiency, as an alternative growth factor for T cells, especially L3T4+ cells (18), 3) that Lyt2+ cells can grow in the absence of L3T4- cells (19), and 4) that some Lyt2+ clones can make use of alternative pathways for growth (20). In spite of these observations, a central role for IL 2 in controlling the growth of at least many L3T4+ and Lyt2+ T cells has not been questioned. In addition, IL 2 also seems to be able to interact with some non-T cells, at least under certain circumstances (21). In view of the important functions ascribed to IL 2, it was to be presumed that IL 2 expression itself would be under tight control. Using mitogens as stimulants for normal T cells, we show in Figures 1 and 2 that IL 2-specific mRNA is only transiently detectable. Maximal levels are reached at 6-14 h after Con A stimulation and already at 4 h after stimulation with PMA and

Regulation of IL 2 Expression . 309 ionomycin. The latter type of stimulant probably directly acts on the T cells, without the need for accessory cell interaction (22), and therefore may be assumed to rather synchronously induce cells. In both cases, however, steady state IL 2 mRNA levels decrease at later times. The mRNA data clearly show that the decline in IL 2 activity observed late in stimulation is not only due to IL 2 consumption, but to a lack of translatable mRNA. Assays of IL 2 secreted into the supernatants at various intervals bear out these observations. The decrease is observed the sooner the maximum is reached, suggesting an inbuilt program for IL 2 expression. The down-regulation of IL 2 mRNA at late times after stimulation does not seem to depend on an interplay between L3T4+ and Lyt2+ cells. In the separated populations, the kinetics of IL 2 expression are very similar to what one observed in mixed spleen cell cultures. The data in Figure 3 thus allow two conclusions: 1) both T cell subsets freshly isolated from spleen can express IL 2 after mitogen stimulation. This is also borne out by assays for IL2 activity in the supernatants (data not shown); 2) Both subsets show similar kinetics of down-regulation, thus excluding the possibility that, e.g. Lyt2+ suppressor cells could be solely responsible for this phenomenon. Since single-cell assays such as in situ hybridization have so far not been amenable to IL 2 mRN A detection, we do not know whether all cells within the two subpopulations participate in IL 2 synthesis. Indeed, as stated above, this seems unlikely (17). Since we could not detect IL 2 mRNA in long-term cytotoxic T cell lines it would seem that only a subpopulation of Lyt2+ cells, the one that does not turn into cytotoxic cells, produces IL 2 or, alternatively, that pre-cytotoxic cells lose the ability to synthesize IL 2 once they have differentiated into lytic ally competent cells. The former notion of two subpopulations within the Lyt2+ cells seems to be favored by data published by WAGNER et al. (23) and SINGER et al. (24). The phenomenon of IL 2 down-regulation is reminiscent of the regulation of expression of other genes only transiently expressed during activation from Go to the G 1 period of the lymphocyte cell cycle such as myc and fos (25, 26). In these instances, a very short half-life of the respective mRNAs has been described (27). Figures 4-7 show that IL 2 expression behaves very similarly to expression of these genes both with respect to the short half-life of the mature mRNA (ca. 30 min for IL2) and to the possibility of prolonging mRNA half-life by addition of inhibitors of protein synthesis such as cycloheximide (TYz ::::: 2 h). The data on the increased TYz for murine IL 2 shown here are, however, in conflict with observations published by EFRAT and KAEMPFER (28) on human IL 2 expression. These authors also did observe an increase of human IL 2 mRNA levels after treatment of mitogenactivated human T cells with cycloheximide, but in their publications the increased signal is ascribed to an increased flux of pre-mRNA into mRNA rather than an increased TY2. Since we inhibited further transcription and thus pre-mRNA formation by addition of actinomycin D, the high levels of IL 2 mRNA 2 h after additional treatment with cycloheximide are more

310 .

R. SWOBODA, E. WECKER, and ANNELIESE SCHIMPL

consistent with an influence on mRNA stability itself. Our data agree with those of EFRAT and KAEMPFER (28) with respect to the need for protein synthesis to initiate IL 2 mRNA production (Fig. 5 A). Added at the same time with the mitogen, cycloheximide completely prevents IL 2 mRNA appearance at later times. Thus, both EFRAT and KAEMPFER's data on human cells and our own data on the murine system diverge from observations published by KROENKE et al. (29). These authors found that cycloheximide added to human peripheral blood cells at time zero did not prevent human IL 2 expression measured 8 h after stimulation. The conflicting evidence could most easily be reconciled if one assumes that human PBLs contain some T cells which are representative of cells corresponding to those we observe approximately 20 h after mitogen stimulation. In these cells (Fig. 5 B), we detect only very low levels of IL 2 mRNA which, however, are greatly elevated after cycloheximide treatment. T cells present in PBL might contain a proportion of such recently activated T cells. While the effect of protein synthesis inhibition on mRNA stability seems quite clear, the mechanisms underlying this phenomenon are not well understood. Labile nucleases specific for 3' sequences have been invoked (25, 27). However, it seems equally conceivable that RNA degradation (possibly due to the same sequences) can only start once RNA is being or has been translated. The last point our studies addressed concerned possible influences of extrinsic signals such as IL 2 and IFN -yon IL 2 expression. In Figure 8, we demonstrate that neither IL 2 itself nor IFN -y at various concentrations affected levels or times of IL 2 expression, at least not under conditions of optimal stimulation by Con A. This of course does not rule out that under different circumstances, IL 2, IFN -y or other lymphokines endogenous to the cultures might positively or negatively regulate IL 2 expression. At the time, however, it seems equally possible that under physiological conditions, appropriate activation via the antigen receptor leads to a burst or IL 2 synthesis, predominantly in G j • This process might then be repeated as long as receptor-antigen interaction takes place, thus leading to a constant monitoring of the need for further cell divisions. Acknowledgements We thank Misses R. SCHMITT and G. RIESS and Mr. A. ZANT for technical assistance and Dr. M. JANTZEN for the actin clone. This work was supported by the Sonderforschungsbereich 105 of the Deutsche Forschungsgemeinschaft and by grant PTB 038729-8 of the Bundesministerium fiir Forschung und Technologie. References 1. 2.

SMITH, K. A. 1984. Interleukin 2. Ann. Rev. Immunol. 2: 319. YOKOTA, T., N. ARAI, F. LEE, D. RENNICK, T. MOSMANN, and K.-I. ARAJ. 1985. Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: Expression of T-cell growth-factor activity after transfection of monkey cells. Proc. Natl. Acad. Sci. USA 82: 68.

Regulation of IL 2 Expression . 311 3. KASHIMA, N., C. NISHI-TAKAOKA, T. FUJITA, S. TAKI, G. YAMADA, J. HAMURO, and T. TANIGUCHI. 1985. Unique structure of murine interleukin-2 as deduced from cloned cDNAs. Nature 313: 402. 4. MILLER, J., T. R. MALEK, W. J. LEONARD, W. C. GREENE, E. M. SHEVACH, and R. N. GERMAIN. 1985. Nucleotide sequence and expression of a mouse Interleukin 2 receptor cDNA. J. Immunol. 134: 4212. 5. MISHELL, R. I., and R. W. DUTION. 1967. Immunization of dissociated spleen cells from normal mice. J. Exp. Med. 126: 423. 6. JULIUS, M. H., E. SIMPSON, and L. A. HERZENBERG. 1973. A rapid method for the isolation of functional thymusderived murine lymphocytes. Eur. J. Immunol. 3: 645. 7. REILLY, E. B., K. AUDITORE-HARGREAVES, U. HAMMERLING, and P. D. GOTTLIEB. 1980. Lyt-2 and Lyt-3 Alloantigens: Precipitation with monoclonal and conventional antibodies and analysis of one- and two-dimensional polyacrylamide gels. J. Immunol. 125: 2245. 8. PIERRES, A., P. NAQUET, A. VAN AGTHOVEN, F. BEKKHOUCHA, F. DENIZOT, Z. MISHAL, A.-M. SCHMITT-VERHULST, and M. PIERRES. 1984. A Rat Anti-Mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of la-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,r, and T4-, Lyt-2,3+) subsets among Anti-Ia cytolytic T cell clones. J. Immunol. 132: 2775. 9. DIALYNAS, D. P., D. B. WILDE, P. MARRACK, A. PIERRES, K. A. WALL, W. HAVRAN, G. OTTEN, M. R. LOKEN, M. PIERRES, J. KAPPLER, and F. W. FITCH. 1983. Characterization of the murine antigenic determinant, designated UT4a, recognized by monoclonal antibody GK1.5: Expression of UT4a by functional T cell clones appears to correlate primarily with Class II MHC antigen-reactivity. Immunological Rev. 74: 29. 10. MANIATIS, T., E. F. FRITSCH, and J. SAMBROOK. 1982. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. 11. LEHRACH, H., D. DIAMOND, J. M. WOZNEY, and H. BOEDTKER. 1977. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 4743. 12. SOUTHERN, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioI. 98: 503. 13. STELLWAG, E. J., and A. E. DAHLBERG. 1980. Electrophoretic transfer of DNA, RNA and protein onto Diazobenzyloxymethyl (DBM) paper. Nucl. Acids Res. 8: 299. 14. WIRTH, T., K. GLOGGLER, T. BAUMRUKER, M. SCHMIDT, and I. HORAK. 1983. Family of middle repetitive DNA sequences in the mouse genome with structural features of solitary retrovirallong terminal repeats. Proc. Nat!. Acad. Sci. USA 80: 3327. 15. MELTON, D. A., P. A. KRIEG, M. R. REBAGLIATI, T. MANIATIS, K. ZINN, and M. R. GREEN. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor. Nucl. Acids. Res. 12: 7035. 16. Promega Biotec. 1985. Hybridization Conditions for Riboprobe™ System generated Probes. Promega Notes 1: 1. 17. MOSMANN, T. R., H. CHERWINSKI, M. W. BOND, M. A. GIDLIN, and R. L. COFFMAN. 1986. Two types of murine helper T cell clones. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136: 2348. 18. FERNANDEZ-BoTRAN, R., P. H. KRAMMER, T. DIAMANTSTEIN, J. W. UHR, and E. S. VITETTA. 1986. B Cell-Stimulatory Factor 1 (BSF-l) promotes growth of helper T cell lines. J. Exp. Med. 164: 580. 19. VON BOEHMER, H., P. KISSIELOW, W. WEISERSON, and W. HAAS. 1984. LytT T cellindependent functions of Lyt2+ cells stimulated with antigen or concanavalin A. J. Immunol. 133: 59. 20. MOLDWIN, R. L., D. W. LANCKI, K. C. HEROLD, and F. W. FITCH. 1986. An antigen receptor-driven, Interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones. J. Exp. Med. 163: 1566.

312 . R. SWOBODA, E. WECKER, and ANNE LIESE SCHIMPL 21. ZUBLER, R. H., J. W. LOWENTHAL, F. ERARD, N. HASHIMOTO, R. DEVOS, and H. R. MACDoNALD. 1984. Activated B cells express receptors for, and proliferate in response to, pure Interleukin 2. J. Exp. Med. 160: 1170. 22. ERARD, F., M. NABHOLZ, A. DupuY-D'ANGEAC, and H. R. MACDoNALD. 1985. Differential requirements for the induction of Interleukin 2 responsiveness in L3T4 + and Lyt2+ T cell subsets. J. Exp. Med. 162: 1738. 23. HARDT, c., and A. WAGNER. 1986. IL-2 production or cytotoxicity can be induced in either L3T4+ or Lyt2+ murine T cells. Immunobio!. 173: 361. 24. ROSENBERG, A. S., T. MIZUOCHI, and A. SINGER. 1986. Analysis of T cell subsets in rejection of Kb mutant skin allografts differing at class I MHC. Nature 322: 829. 25. KELLY, K., B. H. COCHRAN, C. D. STILES, and P. LEDER. 1983. Cell-specific regulation of the c-myc gene by lymphocytes mitogens and platelet-derived growth factor. Cell 35: 603. 26. REED, J. c., J. D. ALPERS, P. C. NORELL, and R. G. HOOVER. 1986. Sequential expression of proto-oncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc. Nat!' Acad. Sci. USA 83: 3982. 27. SHAW, G., and R. KAMEN. 1986. A conserved AU sequence from the 3' un translated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46: 659. 28. EFRAT, S., and R. KAEMPFER. 1984. Control of biologically active interleukin 2 messenger RNA formation in induced human lymphocytes. Proc. Nat!' Acad. Sci. USA 81: 2601. 29. KRONKE, M., W. J. LEONARD, J. M. DEPPER, and W. C. GREENE. 1985. Sequential expression of genes involved in human T lymphocyte growth and differentiation. J. Exp. Med. 161: 1593. Dr. ANNELIESE SCHIMPL, Institute of Virology and Immunobiology, University of Wiirzburg, Versbacher Str. 7, D-8700 Wiirzburg, Federal Republic of Germany