Negative Regulation of CD8 ⴙ T Cell Function by the IFN-Induced and Double-Stranded RNA-Activated Kinase PKR 1
Suzanne Kadereit,* Hui Xu,
†Tara M. Engeman,
†Yi-Li Yang,
‡Robert L. Fairchild,
†and Bryan R. G. Williams
2*
The IFN-induced and dsRNA-activated kinase (PKR) mediates the antiviral and antiproliferative effects of IFN-␣and IFN-␥.
Despite these findings Pkrⴚ/ⴚmice have no overt immunological phenotype. Here we tested the role of PKR in cellular immunity by determining the induction and elicitation of contact hypersensitivity inPkrⴚ/ⴚmice, a model of T cell-mediated immunity.
When compared with wild type, the magnitude of contact hypersensitivity responses inPkrⴚ/ⴚmice were 2-fold higher and of extended duration. This was also observed when naive recipients of immune CD8ⴙT cells from sensitizedPkrⴚ/ⴚand CD4ⴙT cells from sensitized wild-typePkrⴙ/ⴙorPkrⴚ/ⴚmice were challenged with hapten, indicating a regulatory defect intrinsic to the CD8ⴙT cell population. Isolated lymph node T cells fromPkrⴚ/ⴚmice were hyperproliferative during Con A-mediated stimu- lation. These results implicate PKR for the firs time in the growth control of mature T lymphocytes and give insight into the negative regulation of CD8ⴙT cell-mediated immune responses. The Journal of Immunology,2001, 165: 6896–6901.
T
he IFN type I-induced and dsRNA-activated kinase (PKR)3 is a well-characterized component of IFN-regu- lated antiviral and antiproliferative responses (1– 6).There is emerging evidence suggesting that PKR may also play a role in the regulation of the immune response. PKR activates the transcription factors NF-B, IFN regulatory factor-1 (IRF-1), and activating transcription factor-2 and regulates STAT1 serine phos- phorylation, all major transcription factors in the immune system (7–11). PKR has been suggested to mediate the surface expression of CD4, and its activity is regulated by IL-3 and IFN-␥(8, 12, 13).
PKR gene-deleted mice (Pkr⫺/⫺) show increased susceptibility to virus infection and are not protected against viral infection by IFN-␥ (14), suggesting a defect in IFN-␥-mediated cellular im- mune function. Furthermore, PKR plays a positive role in TNF-␣ and Fas-mediated apoptosis, and may thus play a role in peripheral tolerance (15–22).
Unlike IRF-1 or Fas mutant mice,Pkr⫺/⫺mice have no overt immunological phenotype. However, because they exhibit a sig- naling defect in response to IFN-␥, we tested the ability ofPkr⫺/⫺
mice to mount cellular immune responses by analyzing Ag-spe- cific and IFN-␥-mediated responses in contact hypersensitivity (CHS). CHS is a T cell-mediated response in sensitized individuals to epicutaneously applied hapten (23, 24). Challenge with the sen-
sitizing hapten results in an inflammatory response characterized by edema and swelling of the skin. After 48 h, the response is rapidly down-regulated and the swelling regresses. Results from clinical and experimental studies have supported a role for CD4⫹ and CD8⫹T cells as the effector cells of the response (25–27). Use of either Ab-mediated depletion of CD4⫹vs CD8⫹T cells or mice with targeted deletions of class II MHC genes have implicated CD8⫹T cells as the primary effector cells in CHS responses to the model haptens 2,4-dinitro-1-fluorobenzene (DNFB) and oxazolone (27–29). These studies are supported by results from this labora- tory demonstrating that most of the IFN-␥producing cells induced by epicutaneous sensitization with DNFB or oxazolone are hapten- specific CD8⫹ T cells (30 –33). In contrast, the hapten-specific CD4⫹T cell compartment is largely skewed to IL-4, IL-5, and IL-10 producing cells. Hapten-primed CD4⫹T cells are essential for down-regulation of CHS, because sensitized mice devoid of CD4⫹T cells respond to hapten challenge with exaggerated and sustained swelling of the skin (27–33).
Here we report that PKR plays a negative role in the regulation of CHS, asPkr⫺/⫺mice react with exaggerated and sustained cu- taneous swelling. We further show that the defect is intrinsic to the CD8⫹T cell population and that PKR plays a role in the negative regulation of T cell proliferation. Our results implicate, for the first time, PKR in the negative control of mature T lymphocytes.
Materials and Methods
Mice
Pkr⫺/⫺and wild-type mice (Pkr⫹/⫹) on a 129 SvEv background are de- scribed elsewhere (14). Adult female mice of 8 –10 wk old were used throughout these studies.
Sensitization and elicitation of CHS
Groups of three mice were sensitized by two daily paintings (days 0 and
⫹1) with 25l of 0.25% DNFB (Sigma, St. Louis, MO) on the shaved abdomen and 5 l on the footpads. Sensitized and unsensitized control animals were challenged on day⫹5 by applying 10l of 0.2% DNFB to each side of each ear. Ear swelling was measured in a blinded manner 24 h after challenge with an engineer’s micrometer (Mitutoyo Precision USA, Elk Grove Village, IL). The magnitude of ear swelling is given as the mean Departments of *Cancer Biology and†Immunology, Lerner Research Institute, Cleve-
land Clinic Foundation, Cleveland, OH 44195; and‡Institute of Molecular Biology, University of Zurich, Honggerberg, Zurich, Switzerland
Received for publication October 13, 2000. Accepted for publication September 26, 2000.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants RO1 AI 34039 (to B.R.G.W.) and RO1 AR 44673 (to R.L.F.) from the National Institutes of Health.
2Address correspondence and reprint requests to Dr. Bryan R. G. Williams, Depart- ment of Cancer Biology NN10, Lerner Research Institute, Cleveland Clinic Founda- tion, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: williab@ccf.org
3Abbreviations used in this paper: PKR, dsRNA-activated kinase; AICD, activation- induced cell death; CHS, contact hypersensitivity; DNFB, 2,4-dinitro-1-fluoroben- zene; IRF-1, IFN regulatory factor 1; LNC, lymph node cells; PI, propidium iodide.
Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-123200
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⬎ control, rat IgG-treated mice.
Transfer of CHS
CHS was passively transferred by LNC from sensitized mice. Donor Pkr⫺/⫺andPkr⫹/⫹were sensitized with DNFB on days 0 and⫹1, and on day⫹4 skin draining LNC suspensions were prepared. For in vitro deple- tion of CD4⫹T cells, 1⫻107LNC/ml were incubated with 10g/ml of GK1.5 Ab and depleted with magnetic anti-rat IgG-beads (Dynal, Lake Success, NY). A total of 2.5⫻107cells were injected into the retro-orbital sinus of naive wild-type (Pkr⫹/⫹) recipient mice, the mice were challenged immediately on the ears with 0.2% DNFB, and the CHS response was measured as above.
CHS was also passively transferred using CD4⫹T cell- and CD8⫹T cell-enriched LNC populations from DNFB sensitizedPkr⫺/⫺andPkr⫹/⫹
mice. To prepare the donors, mice were depleted of CD4⫹or CD8⫹T cells before hapten sensitization. On day⫹4, LNC suspensions were prepared and 1⫻107cell aliquots of the CD4⫹and CD8⫹T cell populations were cotransferred i.v. to naive wild-type recipients for hapten challenge as above.
Cytokine ELISA
Mice were depleted of CD4⫹or CD8⫹T cells by injection of specific Ab on 3 consecutive days before sensitization with DNFB. On day⫹4, LNC from DNFB-sensitized, and as a negative control from unsensitized, mice were prepared and stimulated to produce cytokines by culture on anti-CD3 mAb-coated wells. The 96-well U-bottom tissue culture plates were pre- coated with 30l/well anti-CD3 mAb 145.2C11 (25g/ml) for 90 min at 37°C. As a negative control, wells were coated with an Ab to a Vnot expressed by 129 SvEv T cells, anti-V17a mAb KJ23a. The wells were washed and 2⫻105LNC were delivered to each well in 200l complete RPMI 1640 medium and cultured in a 7% CO2incubator at 37°C. After 48 h, the culture supernatants were harvested and assayed for IFN-␥and IL-4 production by sandwich ELISA as previously described (30 –33).
Flow cytometry
A total of 2.5⫻105LNC from immune mice 4 days after sensitization were cultured in 200l of complete RPMI 1640 (Life Technologies, Grand
Evaluation of cell death
A total of 2⫻106/ml LNC was grown in complete RPMI 1640 for 48 h in the presence of 2 g/ml of Con A (Sigma-Aldrich, St. Louis, MO), washed with 10 mg/ml␣-methylmannoside and cultured in 50 IU/ml of IL-2 (Life Technologies) to predispose the T cells to activation-induced cell death (AICD) as previously described (21). Cells (⬎98% T cells) were depleted of CD4⫹T cells as described above and 5⫻104CD8⫹T cells grown in triplicate in the presence or absence of plate-bound anti-CD3 Ab.
From 48 to 120 h, triplicates were pooled and stained with FITC-labeled anti-CD8 Ab and propidium iodide (PI, Sigma). Cells were analyzed by flow cytometry, plotting PI vs FITC-positive CD8⫹T cell population.
Proliferation assay
Total LNC were stimulated with Con A and IL-2 as above and 5⫻104 cells grown in triplicate in medium in the presence of 10 or 50 IU/ml of IL-2. At various times, cultures were pulsed with 1Ci [3H]thymidine and 16 h later were harvested onto fiber filter mats, and the amount of3H incorporation was determined by liquid scintillation counting.
In experiments testing the proliferation of DNFB-immune T cells, co- cultures of 2⫻105CD4⫹or CD8⫹T cells and 106DNFB-labeled, or as a negative control unlabeled, stimulator spleen cells were established in triplicate in the wells of 96-well U-bottom culture plates. Spleen cells from Pkr⫺/⫺mice were labeled with 100g/ml DNFB for 30 min at 37°C and given 2000 rad ␥-irradiation before use as stimulator cells. After 48 h, cultures were pulsed with 1Ci/well [3H]thymidine and harvested 18 –20 h later and3H incorporation was determined as above.
Results
Increased CHS inPkr⫺/⫺mice
After sensitization and challenge with DNFB, the magnitude and duration of the inflammatory immune response (i.e., ear swelling) in wild-typePkr⫹/⫹andPkr⫺/⫺was compared. In the absence of prior sensitization, hapten challenge resulted in a slight swelling in
FIGURE 1. Increased CHS response inPkr⫺/⫺mice.A, Mice were challenged on both ears without prior sensitization (f) or sensitized on the abdominal skin and challenged on both ear (F), and cutaneous swelling was measured over time.B, Before sensitization and challenge, one group was depleted of CD4⫹T cells by Ab treatment (F). The mean increase in ear thickness following DNFB challenge is shown in units of 10⫺4inches⫾SEM. Results are representative of two independent experiments.
both sets of mice (Fig. 1A). After hapten sensitization and chal- lenge, the ear swelling response in wild-type mice was of limited magnitude and duration. By contrast, sensitizedPkr⫺/⫺mice re- sponded to challenge with exaggerated and sustained swelling (Fig. 1A), approaching the level of unregulated response observed in animals depleted of CD4⫹ T cells before sensitization with DNFB (Fig. 1B). These results suggest a defect in the down-reg- ulation of the CHS response in thePkr⫺/⫺mice.
Normal IFN-␥, but increased IL-4, production by CD4⫹T cell fromPkr⫺/⫺mice
The exaggerated CHS response observed in Pkr⫺/⫺ mice sug- gested that these mice produce normal, if not elevated, levels of IFN-␥, one of the mediators of CHS. However, following in vitro re-stimulation of immune CD8⫹T cells from sensitizedPkr⫺/⫺
mice by culture on anti-CD3 mAb-coated wells, there was no sig- nificant increase in IFN-␥production (Fig. 2). Moreover, we ob- served 2-fold increased production of the cytokine IL-4 by im- mune CD4⫹ T cell fromPkr⫺/⫺ mice. As previously reported, culture of T cells from naive mice on anti-CD3 mAb-coated wells produced low to nondetectable levels of IFN-␥and IL-4 (Fig. 2)
and culture of immune T cells from sensitizedPkr⫹/⫹andPkr⫺/⫺
mice on anti-V17a-coated wells produced nondetectable levels of the cytokines (data not shown). It was also possible that the dif- ferent ear swelling responses observed inPkr⫹/⫹andPkr⫺/⫺mice to DNFB sensitization and challenge were due to temporal or sus- tained differences in IFN-␥production. However, no differences in anti-CD3 mAb stimulated IFN-␥production by CD8⫹T cells from DNFB-sensitizedPkr⫹/⫹and Pkr⫺/⫺mice were observed when culture supernatants were removed at various times and tested for IFN-␥production (Fig. 3).
To further examine hapten reactivity of immune CD4⫹ and CD8⫹T cells from sensitizedPkr⫹/⫹andPkr⫺/⫺mice, prolifer- ation induced during culture with DNP-labeled stimulator cells was compared. For both CD4⫹and CD8⫹T cells, slightly higher proliferative responses were consistently observed in cocultures of DNP-spleen cells and T cells from sensitizedPkr⫺/⫺mice when compared with T cells from sensitized wild-type mice (Fig. 4).
There was no difference in proliferation of immune T cells from Pkr⫹/⫹ and Pkr⫺/⫺ mice during culture with unlabeled spleen FIGURE 2. Abnormal cytokine profile of isolated LNC fromPkr⫺/⫺mice.Pkr⫹/⫹andPkr⫺/⫺mice were sensitized with 0.25% DNFB on days 0 and
⫹1. On day⫹4, LNC suspensions from sensitized mice were prepared and were separated into CD4⫹and CD8⫹T cell populations, and 2⫻105immune cell aliquots or 2⫻105LNC from unsensitized naivePkr⫺/⫺mice were cultured on anti-CD3 or anti-V17a mAb-coated wells for 48 h. Supernatants were tested by sandwich ELISA for IFN-␥and IL-4 production. Cell culture on control, anti-V17a, mAb-coated wells did not stimulate detectable IFN-␥ production by cells from any of the test groups. Results are representative of two independent experiments.
FIGURE 3. Comparison of IFN-␥production by DNFB-immune CD8⫹ T cells fromPkr⫹/⫹andPkr⫺/⫺mice.Pkr⫹/⫹(䡺) andPkr⫺/⫺(〫) mice were depleted of CD4⫹T cells by injection of anti-CD4 mAb on 3 con- secutive days before sensitization with 0.25% DNFB on days 0 and⫹1. On day⫹4, LNC suspensions from sensitized and unsensitized (E)Pkr⫺/⫺
mice were prepared and 2⫻105cell aliquots were cultured on anti-CD3 or anti-V17a mAb-coated wells. Beginning 48 h after culture initiation and every 24 h thereafter, culture supernatants were removed and analyzed by sandwich ELISA for levels of IFN-␥production. Cell culture on control, anti-V17a, mAb-coated wells did not stimulate detectable IFN-␥produc- tion by cells from any of the test groups. Results are representative of two independent experiments.
FIGURE 4. Hapten-stimulated proliferation of immune CD4⫹ and CD8⫹T cells from Pkr⫹/⫹andPkr⫺/⫺mice.Pkr⫹/⫹andPkr⫺/⫺mice were depleted of CD4⫹or CD8⫹T cells by injection of anti-CD4 or anti- CD8 mAb on 3 consecutive days before sensitization with 0.25% DNFB on days 0 and⫹1. On day⫹4, LNC suspensions from sensitized mice were prepared and 2⫻105immune cell aliquots or 2⫻105LNC from unsen- sitized naivePkr⫺/⫺mice were cultured with 106DNFB-labeled or unla- beledPkr⫺/⫺ spleen cells. After 48 h, cultures were pulsed with 1Ci [3H]thymidine for 18 h and [3H] incorporation was determined by liquid scintillation counting. The data represent the mean 3H incorporation of triplicate cultures⫾SD. Results are representative of three independent experiments.
cells. Low levels of proliferation were observed in cocultures of LNC from unsensitized mice and DNP-labeled or unlabeled cells.
CD8⫹T cells from sensitizedPkr⫺/⫺mice transfer exaggerated CHS responses to naive mice
CHS can be elicited in naive mice by adoptive transfer of CD8⫹T cells from sensitized mice (29 –32). To further compare the im- mune function of CD8⫹T cells fromPkr⫹/⫹and Pkr⫺/⫺mice, LNC were prepared from sensitized wild-typePkr⫹/⫹andPkr⫺/
⫺mice, depleted of CD4⫹T cells and injected into naive wild-type mice. The recipient mice were ear challenged with DNFB and the ear swelling response was measured 24 h later. As expected, we found transfer of normal CHS responses to naive wild-type recip- ients by immune CD8⫹T cells from wild-type mice (Fig. 5A). In contrast, CD8⫹T cells from sensitizedPkr⫺/⫺mice were able to confer exaggerated and sustained swelling to naive wild-type mice.
When immune CD8⫹ T cells mice were cotransferred with im- mune CD4⫹T cells from eitherPkr⫹/⫹orPkr⫺/⫺mice to naive wild-type mice the responses to hapten challenge were again ex- aggerated (Fig. 5C). In contrast, when immune CD8⫹T cells from wild-type mice were cotransferred with immune CD4⫹ T cells from eitherPkr⫹/⫹orPkr⫺/⫺mice, responses were again of short duration (Fig. 5B). These results indicated that the excessive CHS response observed inPkr⫺/⫺mice is intrinsic to the effector CD8⫹ T cell population and not to the regulatory CD4⫹ T cell population.
Fas up-regulation and AICD are normal in activated T cells fromPkr⫺/⫺mice
Activated peripheral CD4⫹and CD8⫹T cells are eliminated by Fas or TNF-␣-mediated apoptosis during immune responses. Both apoptotic pathways are deficient in mouse embryo fibroblasts from Pkr⫺/⫺mice, the defect in the Fas-mediated pathway being due to a failure to up-regulate Fas (17). Therefore, we tested the up-reg- ulation of Fas on T cells from sensitizedPkr⫺/⫺mice following in vitro stimulation, as well as AICD, under conditions which have been described to induce Fas and TNF-␣-mediated apoptosis (21).
We detected no deficiency in Fas up-regulation on the surface of the T cells (Fig. 6A) and found no difference in cell death of stim- ulated primed CD8⫹T cells fromPkr⫺/⫺mice (Fig. 6B).
Hyperproliferation of T cells fromPkr⫺/⫺mice
PKR has been shown to have a strong anti-proliferative effect, and constitutive overexpression of wild-type PKR is growth inhibitory (4). Accordingly, we hypothesized that hyperproliferation of im- mune effector CD8⫹T cells could induce exaggerated CHS, by continuous proliferation and cytokine secretion. To test this, lymph node T cells were activated with Con A for 48 h, grown in the presence of IL-2 (10 or 50 U/ml) for 48 h, and analyzed for pro- liferation. Although T cells from wild-type mice cease to prolif- erate after 48 h in 10 U/ml of IL-2, probably due to IL-2 with- drawal-induced apoptosis, T cells fromPkr⫺/⫺mice continue to FIGURE 5. Transfer of increased CHS to naive mice by immune CD8⫹T cells fromPkr⫺/⫺mice.Pkr⫹/⫹andPkr⫺/⫺mice were depleted of CD4⫹ or CD8⫹T cells by injection of anti-CD4 or anti-CD8 mAb on 3 consecutive days before sensitization with 0.25% DNFB on days 0 and⫹1.A, On day
⫹4, LNC suspensions were prepared from CD4⫹T cell-depletedPkr⫹/⫹(F) andPkr⫺/⫺(E) mice, 2⫻107LNC aliquots were transferred to naive wild-typePkr⫹/⫹mice, and the recipients were immediately challenged with 0.2% DNFB.B, A total of 1⫻107LNC aliquots from CD4⫹T cell-depleted wild-type mice and 1⫻107cell aliquots from anti-CD8⫹T cell-depletedPkr⫹/⫹(䡺) orPkr⫺/⫺(〫) mice were cotransferred to naive wild-typePkr⫹/⫹
mice and the recipients were immediately challenged with 0.2% DNFB. As a negative control, 2⫻107cells from naivePkr⫺/⫺mice were transferred to Pkr⫹/⫹recipients.C, A total of 1⫻107LNC aliquots from CD4⫹T cell-depletedPkr⫺/⫺mice and 1⫻107cell aliquots from anti-CD8⫹T cell-depleted Pkr⫹/⫹(䡺) orPkr⫺/⫺(〫) mice were cotransferred to naive wild-typePkr⫹/⫹mice and the recipients were immediately challenged with 0.2% DNFB. The thickness of challenged ears was measured every 24 h after challenge as indicated. The mean increase in ear thickness following DNFB challenge is shown in units of 10⫺4in⫾SEM. Results are representative of two independent experiments.
proliferate (Fig. 7). In the presence of 50 U/ml of IL-2, wild-type T cells cease to proliferate after 96 h, whereas Pkr⫺/⫺T cells continue to proliferate, indicating that PKR may play a role in the regulation of primary T cell proliferation.
Discussion
PKR has been shown to play a role in mediating the antiviral and antiproliferative effects of IFN. Deficiency in IFN-␥signaling has been linked to a role for PKR in the activation of the transcription factors NF-B and IRF-1. However, unlike IRF-1 and NF-B gene-deleted mice,Pkr⫺/⫺mice appear healthy and have normal CD4⫹and CD8⫹ T cell counts. Here we describe the immune response ofPkr⫺/⫺mice during CHS,which is an IFN-␥-mediated cellular immune response.
We show here thatPkr⫺/⫺mice display an exaggerated inflam- matory response to Ag challenge. Although CHS is largely an IFN-␥-mediated response, IFN-␥production by immune CD8⫹T cells fromPkr⫺/⫺mice was not significantly increased. Rather we observed elevated IL-4 production by CD4⫹T cells fromPkr⫺/⫺
mice. Although the role of IL-4 in CHS is controversial, it is nec-
essary for the second phase of the effector stage, as IL-4-deleted mice show decreased response after 48 h (34). Therefore, in- creased production of IL-4 by immunized CD4⫹T cells may play a partial role in the excessive CHS we observe in Pkr⫺/⫺mice.
Moreover, we showed here that exaggerated CHS can be trans- ferred to naive animals after adoptive transfer of immune CD8⫹T cells from Pkr⫺/⫺ mice and that this transfer is not negatively regulated by CD4⫹T cells from sensitized wild-typePkr⫹/⫹or Pkr⫺/⫺mice.
It is probable that effector CD8⫹T cells in CHS are eliminated by AICD through TNF-␣or Fas-mediated apoptosis. However, we found no defect in Fas up-regulation on CD8⫹T cells fromPkr⫺/⫺
mice after TCR stimulation, nor any defect in AICD after primary stimulation followed by secondary stimulation or IL-2 withdrawal.
On the contrary, we observed increased proliferation of stimulated T cells fromPkr⫺/⫺mice, suggesting that after primary stimula- tion, these cells are not down-regulated, but continue to grow.
PKR has been shown to have a strong anti-proliferative effect, mediated through inhibition of protein synthesis (3). Results shown in this report suggest for the first time a role for PKR in the negative growth control of T cells. This could result from direct inhibition of protein synthesis or may be indirect, through a defect resulting in increased production of IL-4, which has a growth- promoting role for activated T cells (35). In this regard, it is in- teresting to note here, that CD4⫹T cells from IRF-1-deleted mice also produce increased levels of IL-4 in the context of an infection (36). Because the PKR activator dsRNA is produced during viral replication, it is possible that in the context of viral infection, PKR plays a role in IL-4 regulation, upstream of IRF-1.
Treatment of cells with IFN-␥results in activation of PKR (8) and may thus be the activator of PKR in the context of CHS.
Although CHS is a noninfectious model, our results suggest that PKR may play a critical role in the down-regulation of the immune response. Taken together, our results shed light on the negative regulation of CHS effector cells, suggesting that these cells are not only eliminated by AICD after secondary activation, but may also FIGURE 6. Normal Fas-up-regulation and AICD
in T cells fromPkr⫺/⫺mice.A, Isolated LNC from sensitized mice were stimulated with plate-bound anti- CD3 Ab and expression of Fas receptor measured after 72 h on CD4⫹or CD8⫹T cells. Indicated are mean channel fluorescence values.B, Con A- and IL-2-stimulated CD8⫹ T cells fromPkr⫺/⫺ mice (䡺) andPkr⫹/⫹mice (f) were grown on medium (control) or plate-bound␣-CD3 Ab (anti-CD3) and cell death assessed by PI incorporation and subse- quent analysis by FACS. Results are representative of four independent experiments.
FIGURE 7. . Hyperproliferation of LNC from Pkr⫺/⫺ mice. Isolated LNC were stimulated with Con A and IL-2 for primary stimulation and grown on medium with IL-2 over a period of 120 h, and proliferation was assessed by [3H]thymidine incorporation. The data represent the mean3H incorporation of triplicate cultures⫾ SD. Results are representative of three independent experiments.
Sherrie Vidmar for assistance in preparing this manuscript.
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