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Negative Regulation of CD8⁺ T Cell Function by the IFN-Induced and Double-Stranded RNA-Activated Kinase PKR¹

Suzanne Kadereit^*, Hui Xu, Tara M. Engeman, Yi-Li Yang, Robert L. Fairchild and Bryan R. G. Williams^2,*

Departments of ^* Cancer Biology and Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; and Institute of Molecular Biology, University of Zurich, Honggerberg, Zurich, Switzerland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The IFN-induced and dsRNA-activated kinase (PKR) mediates the antiviral and antiproliferative effects of IFN- and IFN-. Despite these findings, Pkr^-/- mice have no overt immunological phenotype. Here we tested the role of PKR in cellular immunity by determining the induction and elicitation of contact hypersensitivity in Pkr^-/- mice, a model of T cell-mediated immunity. When compared with wild type, the magnitude of contact hypersensitivity responses in Pkr^-/- mice were 2-fold higher and of extended duration. This was also observed when naive recipients of immune CD8⁺ T cells from sensitized Pkr^-/- and CD4⁺ T cells from sensitized wild-type Pkr^+/+ or Pkr^-/- mice were challenged with hapten, indicating a regulatory defect intrinsic to the CD8⁺ T cell population. Isolated lymph node T cells from Pkr^-/- mice were hyperproliferative during Con A-mediated stimulation. These results implicate PKR for the first time in the growth control of mature T lymphocytes and give insight into the negative regulation of CD8⁺ T cell-mediated immune responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The IFN type I-induced and dsRNA-activated kinase (PKR)³ is a well-characterized component of IFN-regulated antiviral and antiproliferative responses (1, 2, 3, 4, 5, 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 phosphorylation, all major transcription factors in the immune system (7, 8, 9, 10, 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 immune function. Furthermore, PKR plays a positive role in TNF- and Fas-mediated apoptosis, and may thus play a role in peripheral tolerance (15, 16, 17, 18, 19, 20, 21, 22).

Unlike IRF-1 or Fas mutant mice, Pkr^-/- mice have no overt immunological phenotype. However, because they exhibit a signaling defect in response to IFN-, we tested the ability of Pkr^-/- mice to mount cellular immune responses by analyzing Ag-specific 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 sensitizing 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, 26, 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, 28, 29). These studies are supported by results from this laboratory demonstrating that most of the IFN- producing cells induced by epicutaneous sensitization with DNFB or oxazolone are hapten-specific CD8⁺ T cells (30, 31, 32, 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, 28, 29, 30, 31, 32, 33).

Here we report that PKR plays a negative role in the regulation of CHS, as Pkr^-/- mice react with exaggerated and sustained cutaneous 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

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Mice

Pkr^-/- and wild-type mice (Pkr^+/+) on a 129 SvEv background are described 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 25 µl of 0.25% DNFB (Sigma, St. Louis, MO) on the shaved abdomen and 5 µl on the footpads. Sensitized and unsensitized control animals were challenged on day +5 by applying 10 µl 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 increase of each group of three sensitized or nonsensitized mice (i.e., six ears) ± SEM.

Mice were depleted of CD4⁺ or CD8⁺ T cells by injection with 100 µg anti-CD4 mAb GK1.5 or anti-CD8 mAb YTS 169 on three consecutive days before DNFB sensitization as previously described (30, 31, 32, 33). Treated sentinel mice were used to evaluate the efficiency of CD4⁺ or CD8⁺ T cell depletion by Ab staining and flow cytometry analysis of spleen and lymph node cells (LNC) and was always >95% when compared with cells from control, rat IgG-treated mice.

Transfer of CHS

CHS was passively transferred by LNC from sensitized mice. Donor Pkr^-/- and Pkr^+/+ were sensitized with DNFB on days 0 and +1, and on day +4 skin draining LNC suspensions were prepared. For in vitro depletion of CD4⁺ T cells, 1 x 10⁷ LNC/ml were incubated with 10 µg/ml of GK1.5 Ab and depleted with magnetic anti-rat IgG-beads (Dynal, Lake Success, NY). A total of 2.5 x 10⁷ cells 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 sensitized Pkr^-/- and Pkr^+/+ 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 x 10⁷ cell 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 precoated with 30 µl/well anti-CD3 mAb 145.2C11 (25 µg/ml) for 90 min at 37°C. As a negative control, wells were coated with an Ab to a Vß not expressed by 129 SvEv T cells, anti-Vß17a mAb KJ23a. The wells were washed and 2 x 10⁵ LNC were delivered to each well in 200 µl complete RPMI 1640 medium and cultured in a 7% CO₂ incubator 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, 31, 32, 33).

Flow cytometry

A total of 2.5 x 10⁵ LNC from immune mice 4 days after sensitization were cultured in 200 µl of complete RPMI 1640 (Life Technologies, Grand Island, NY) on plate-bound anti-CD3 Ab (30, 31, 32, 33) for 72 h and stained with FITC-labeled anti-CD8, PE-labeled anti-CD4, or biotin-labeled anti-Fas Abs (BD Pharmingen, San Diego, CA), followed by cyanin-labeled anti-biotin Ab (Becton-Dickinson, San Jose, CA). Cells were analyzed by flow cytometry using CellQuest software (Becton-Dickinson), and Fas-expression was plotted vs either the PE or the FITC-positive T cell population.

Evaluation of cell death

A total of 2 x 10⁶/ml LNC was grown in complete RPMI 1640 for 48 h in the presence of 2 µg/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 x 10⁴ CD8⁺ 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 x 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 1 µCi [³H]thymidine and 16 h later were harvested onto fiber filter mats, and the amount of ³H incorporation was determined by liquid scintillation counting.

In experiments testing the proliferation of DNFB-immune T cells, cocultures of 2 x 10⁵ CD4⁺ or CD8⁺ T cells and 10⁶ DNFB-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 100 µg/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 1 µCi/well [³H]thymidine and harvested 18–20 h later and ³H incorporation was determined as above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased CHS in Pkr^-/- mice

After sensitization and challenge with DNFB, the magnitude and duration of the inflammatory immune response (i.e., ear swelling) in wild-type Pkr^+/+ and Pkr^-/- was compared. In the absence of prior sensitization, hapten challenge resulted in a slight swelling in both sets of mice (Fig. 1A). After hapten sensitization and challenge, the ear swelling response in wild-type mice was of limited magnitude and duration. By contrast, sensitized Pkr^-/- mice responded 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-regulation of the CHS response in the Pkr^-/- mice.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Increased CHS response in Pkr-/- mice. A, Mice were challenged on both ears without prior sensitization () or sensitized on the abdominal skin and challenged on both ear (•), and cutaneous swelling was measured over time. B, Before sensitization and challenge, one group was depleted of CD4+ T cells by Ab treatment (•). The mean increase in ear thickness following DNFB challenge is shown in units of 10-4 inches ± SEM. Results are representative of two independent experiments.

Normal IFN-, but increased IL-4, production by CD4⁺ T cell from Pkr^-/- mice

The exaggerated CHS response observed in Pkr^-/- mice suggested 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 sensitized Pkr^-/- mice by culture on anti-CD3 mAb-coated wells, there was no significant increase in IFN- production (Fig. 2). Moreover, we observed 2-fold increased production of the cytokine IL-4 by immune CD4⁺ T cell from Pkr^-/- 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 sensitized Pkr^+/+ and Pkr^-/- mice on anti-Vß17a-coated wells produced nondetectable levels of the cytokines (data not shown). It was also possible that the different ear swelling responses observed in Pkr^+/+ and Pkr^-/- mice to DNFB sensitization and challenge were due to temporal or sustained differences in IFN- production. However, no differences in anti-CD3 mAb stimulated IFN- production by CD8⁺ T cells from DNFB-sensitized Pkr^+/+ and Pkr^-/- mice were observed when culture supernatants were removed at various times and tested for IFN- production (Fig. 3).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Abnormal cytokine profile of isolated LNC from Pkr-/- mice. Pkr+/+ and Pkr-/- 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 x 105 immune cell aliquots or 2 x 105 LNC from unsensitized naive Pkr-/- mice were cultured on anti-CD3 or anti-Vß17a mAb-coated wells for 48 h. Supernatants were tested by sandwich ELISA for IFN- and IL-4 production. Cell culture on control, anti-Vß17a, mAb-coated wells did not stimulate detectable IFN- production by cells from any of the test groups. Results are representative of two independent experiments.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Comparison of IFN- production by DNFB-immune CD8+ T cells from Pkr+/+ and Pkr-/- mice. Pkr+/+ () and Pkr-/- () mice were depleted of CD4+ T cells by injection of anti-CD4 mAb on 3 consecutive days before sensitization with 0.25% DNFB on days 0 and +1. On day +4, LNC suspensions from sensitized and unsensitized () Pkr-/- mice were prepared and 2 x 105 cell aliquots were cultured on anti-CD3 or anti-Vß17a 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-Vß17a, mAb-coated wells did not stimulate detectable IFN- production by cells from any of the test groups. Results are representative of two independent experiments.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Hapten-stimulated proliferation of immune CD4+ and CD8+ T cells from Pkr+/+ and Pkr-/- mice. Pkr+/+ and Pkr-/- 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 x 105 immune cell aliquots or 2 x 105 LNC from unsensitized naive Pkr-/- mice were cultured with 106 DNFB-labeled or unlabeled Pkr-/- spleen cells. After 48 h, cultures were pulsed with 1 µCi [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.

CHS can be elicited in naive mice by adoptive transfer of CD8⁺ T cells from sensitized mice (29, 30, 31, 32). To further compare the immune function of CD8⁺ T cells from Pkr^+/+ and Pkr^-/- mice, LNC were prepared from sensitized wild-type Pkr^+/+ and Pkr^-/-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 recipients by immune CD8⁺ T cells from wild-type mice (Fig. 5A). In contrast, CD8⁺ T cells from sensitized Pkr^-/- mice were able to confer exaggerated and sustained swelling to naive wild-type mice. When immune CD8⁺ T cells mice were cotransferred with immune CD4⁺ T cells from either Pkr^+/+ or Pkr^-/- mice to naive wild-type mice the responses to hapten challenge were again exaggerated (Fig. 5C). In contrast, when immune CD8⁺ T cells from wild-type mice were cotransferred with immune CD4⁺ T cells from either Pkr^+/+ or Pkr^-/- mice, responses were again of short duration (Fig. 5B). These results indicated that the excessive CHS response observed in Pkr^-/- mice is intrinsic to the effector CD8⁺ T cell population and not to the regulatory CD4⁺ T cell population.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Transfer of increased CHS to naive mice by immune CD8+ T cells from Pkr-/- mice. Pkr+/+ and Pkr-/- 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-depleted Pkr+/+ (•) and Pkr-/- () mice, 2 x 107 LNC aliquots were transferred to naive wild-type Pkr+/+ mice, and the recipients were immediately challenged with 0.2% DNFB. B, A total of 1 x 107 LNC aliquots from CD4+ T cell-depleted wild-type mice and 1 x 107 cell aliquots from anti-CD8+ T cell-depleted Pkr+/+ () or Pkr-/- () mice were cotransferred to naive wild-type Pkr+/+ mice and the recipients were immediately challenged with 0.2% DNFB. As a negative control, 2 x 107 cells from naive Pkr-/- mice were transferred to Pkr+/+ recipients. C, A total of 1 x 107 LNC aliquots from CD4+ T cell-depleted Pkr-/- mice and 1 x 107 cell aliquots from anti-CD8+ T cell-depleted Pkr+/+ () or Pkr-/- () mice were cotransferred to naive wild-type Pkr+/+ 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 10-4 in ± SEM. Results are representative of two independent experiments.

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-regulation of Fas on T cells from sensitized Pkr^-/- 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 stimulated primed CD8⁺ T cells from Pkr^-/- mice (Fig. 6B).

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Normal Fas-up-regulation and AICD in T cells from Pkr-/- 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 from Pkr-/- mice () and Pkr+/+ mice () were grown on medium (control) or plate-bound -CD3 Ab (anti-CD3) and cell death assessed by PI incorporation and subsequent analysis by FACS. Results are representative of four independent experiments.

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 immune 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 proliferation. Although T cells from wild-type mice cease to proliferate after 48 h in 10 U/ml of IL-2, probably due to IL-2 withdrawal-induced apoptosis, T cells from Pkr^-/- mice continue to 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.

View larger version (16K): [in this window] [in a new window]   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 mean 3H incorporation of triplicate cultures ± SD. Results are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We show here that Pkr^-/- mice display an exaggerated inflammatory response to Ag challenge. Although CHS is largely an IFN--mediated response, IFN- production by immune CD8⁺ T cells from Pkr^-/- mice was not significantly increased. Rather we observed elevated IL-4 production by CD4⁺ T cells from Pkr^-/- mice. Although the role of IL-4 in CHS is controversial, it is necessary for the second phase of the effector stage, as IL-4-deleted mice show decreased response after 48 h (34). Therefore, increased 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 transferred 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-type Pkr^+/+ 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 from Pkr^-/- 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 from Pkr^-/- mice, suggesting that after primary stimulation, 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 interesting 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 be down-regulated in their ability to proliferate shortly after primary activation, limiting the number of available effector cells.

	Acknowledgments

 
We thank Sandy Der for valuable discussions during the course of this study, Natalia Lerner for expert technical assistance with animal care, and Sherrie Vidmar for assistance in preparing this manuscript.

	Footnotes

 
¹ This 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.

² Address correspondence and reprint requests to Dr. Bryan R. G. Williams, Department of Cancer Biology NN10, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195.

³ Abbreviations used in this paper: PKR, dsRNA-activated kinase; AICD, activation-induced cell death; CHS, contact hypersensitivity; DNFB, 2,4-dinitro-1-fluorobenzene; IRF-1, IFN regulatory factor 1; LNC, lymph node cells; PI, propidium iodide.

Received for publication October 13, 2000. Accepted for publication September 26, 2000.

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