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Blood, 1 February 2006, Vol. 107, No. 3, pp. 987-993. Prepublished online as a Blood First Edition Paper on October 6, 2005; DOI 10.1182/blood-2005-07-2834.
IMMUNOBIOLOGY Modulation of STAT1 protein levels: a mechanism shaping CD8 T-cell responses in vivoFrom the Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, RI.
Type 1 interferons (IFNs) are induced in vivo, administered therapeutically, and potential targets for amelioration of autoimmune diseases. The cytokines mediate profound antiproliferative effects. Signal transducer and activator of transcription 1 (STAT1)-dependent signaling pathways are required for inhibition of proliferation, and viral infections can elicit high levels of type 1 IFNs as well as total STAT1 protein expression. Thus, a mechanism must be in place to help antigen-specific T cells overcome IFN-induced inhibition of proliferation. The studies reported here demonstrate that total CD8 T-cell proliferation in the presence of IFNs, ex vivo in response to cytokines and in vivo during viral infection, is inhibited through a STAT1-dependent mechanism. In contrast, major proportions of antigen-specific CD8, but not CD4, T cells are rendered less sensitive to this inhibition, express lower endogenous levels of total STAT1, and are selectively proliferating in the presence of type 1 IFN, at key times after viral challenge. Taken together, these novel results show that differential STAT1 expression is used by the immune system to modify cytokine-mediated effects on T-cell expansion and have implications for the consequences of therapeutic intervention in cytokine function.
Type 1 IFNs (IFN  and IFN ) mediate a wide range of biologic effects, including antiviral and antimicrobial defense, immunoregulation, proliferation inhibition, and survival of activated T cells.1 They are induced during a variety of infections, most notably certain viral infections; are used therapeutically to treat cancers, viral infections, and multiple sclerosis1-4; and are being developed as vaccine adjuvants.5 There is a growing interest in neutralizing their in vivo functions to help control certain autoimmune diseases, including systemic lupus.6 Gene expression analyses using cell lines have shown that hundreds of genes can be differentially regulated by IFN-/,7 and certain biologic effects attributed to these cytokines are paradoxical.8 Thus, there must be sophisticated mechanisms controlling the downstream consequences of exposure to the cytokines.
Receptor binding of IFN
The studies presented here were undertaken to evaluate IFN effects on T-cell proliferation and to investigate how the antigen-specific cells required for defense can escape the growth inhibitory effects mediated by the cytokines in vivo. Responses to lymphocytic choriomeningitis virus (LCMV) infections of mice were characterized because the conditions are associated with well-defined and extended production of type 1 IFNs, as well as striking expansion of antigen-specific CD8 T cells.19-21 Moreover, previous work from our group has shown that at the times overlapping with IFN
Mice and in vivo manipulations Specific pathogen-free wild-type C57BL/6 mice were purchased from Taconic Laboratory Animals and Services (Germantown, NY). STAT1-deficient mice initially on the 129 background22 were crossed onto the C57BL/6 background for more than 5 generations, and colonies were established in the animal care facilities at Brown University. Experimental groups were age matched, and all mice used in experiments were 8 to 12 weeks of age. Animals obtained outside of Brown University were housed for at least 1 week before use. Handling of mice and experimental procedures were in accordance with institutional guidelines for animal care and use. Experiments were initiated on day 0, with mice either not infected or infected intraperitoneally with 2 x 104 plaque-forming units (PFUs) LCMV Armstrong strain, clone E350. Where indicated, LCMV was measured in a viral plaque assay with vero cells as described.23 Splenic leukocyte preparations and CD8 T-cell enrichment On indicated days following initiation of experiments, mice were killed and spleens were harvested. Leukocytes were prepared as previously described.23 In certain experiments, CD8 T cells were isolated by negative selection using magnetic-activated cell sorting (MACS) enrichment kits and the program DepleteS on the autoMACS instrument (Milteny Biotec, Auburn, CA). The purity of enriched samples was greater than 90%. CFSE proliferation analysis
Leukocytes were resuspended to 1 x 107 cells/mL in PBS containing 5% FBS, and CFSE labeling was performed as described24 with some modifications. Briefly, CFSE was rapidly mixed with the cells to a final concentration of 5 µM and incubated for 5 minutes at room temperature. After 2 washes with 10 volumes of PBS-5% FBS, the CFSE-labeled leukocytes were incubated in the absence or presence of cytokines. In all experiments, the concentration of cells was 1 x 106 cells in 200 µL RPMI-10% FBS. Cells were incubated in 96-well plates at 37°C for the times indicated. A final concentration of 20 ng/mL IL-2, IL-7, and IL-15 was used. rhIL-2 was from Cetus Corporation (Berkeley, CA); rmIL-7 and rmIL-15 were purchased from R&D Systems (Minneapolis, MN). Human IFN Flow cytometric analyses Studies evaluating in vivo BrdU incorporation by CD8 T cells were adapted from the BrdU Flow Kit protocol (BD Biosciences). Briefly, 1 mg BrdU was injected intraperitoneally into mice 2 hours prior to harvest. Splenic leukocytes were isolated as described, surface labeled with PerCP-conjugated CD8 antibody and APC-conjugated H-2Db tetramer binding the immunodominant LCMV peptide GP33-41 (KAVYNFATC) (Beckman Coulter, Immunomics, San Diego, CA), or APC-conjugated CD4 antibody followed by intracellular staining with fluorescein isothiocyanate (FITC)-labeled anti-BrdU and/or phycoerythrin (PE) anti-STAT1. For intracellular STAT1 labeling, a custom-made PE-conjugated STAT1 antibody was used (BD Biosciences), using the solutions and protocol from the BrdU Flow kit to detect nuclear and cytoplasmic STAT1. The approach was developed as a modification of reported staining of human monocytes.25 Studies of cells isolated from the STAT-/- mice demonstrated specificity of staining. Isotype control antibodies were used in all the analyses. The mean fluorescence intensities of the isotype and STAT1-/- controls were lower than the first log of intensity with narrow uniform peaks. IFN evaluation
Serum was obtained from mice anesthetized with isoflurane (Abbott Laboratories, Abbott Park, IL) as previously described.26 Active IFN Cell lysates and Western blot analysis
Total splenic leukocytes, enriched CD8 T cells, and non-CD8 cells, as well as CD8 T cells sorted for high or low CFSE staining were lysed in a solution containing 50 mM Tris HCl pH 7.5, 0.3M NaCl, 0.5% Triton X-100, 2 mM EDTA, 0.4 mM Na3VO4, and a cocktail of protease inhibitors (Roche Diagnostics, Indianapolis, IN). Protein (30 µg) was separated on SDS-PAGE gels (Gradipore 4%-20% LongLife MicroGels; Gradipore, Hawthorne, NY), following the Gradipore protocol for blotting. Monoclonal anti-STAT1 antibody (clone 1) was purchased from BD Transduction Laboratories (Lexington, KY); rabbit polyclonal anti-STAT4 antibody (C-20) obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and rabbit polyclonal anti- Real-time PCR
RNA from sorted CD8 T cells was extracted with the RNAeasy kit from Qiagen (Valencia, CA), using on column digestion with DNAse I (Qiagen). One-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed with Quantitect Probe RT-PCR kit (Qiagen) following the amplification parameters given in the manufacturer protocol. Sequences of STAT1 primers and FAM-labeled probe were as follows: forward primer, 5'-AAGCGAACTGGATACATCA; reverse primer, 5'-CCGGGACATCTCATCAAAC; and probe, 5'-CAGACCACAGACAACC. Fold changes were calculated relative to the level of
Role for STAT1 in IFN-mediated inhibition of CD8 T-cell proliferation
The STAT1 effects on CD8 T-lymphocyte proliferation were evaluated in vitro with responses to exogenous growth factors. Splenic leukocytes derived from STAT1-competent or -deficient mice were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE) and incubated with IL-2, IL-7, or IL-15. All of these cytokines use the common cytokine-receptor To examine the STAT1-dependent effects on proliferation in vivo, the CD8 T-lymphocyte proliferation elicited in response to LCMV infection was examined. For these experiments, the thymidine analog, BrdU, was injected into STAT1-competent and -deficient mice. This approach marks populations replicating their DNA. Uninfected (day 0) mice or mice infected with LCMV for 1, 2, 3, or 4 days received injections of BrdU 2 hours prior to killing (Figure 1B). These periods of infection overlap with peak type 1 IFN induction. Although overall splenic cell yields were not dramatically altered in wild-type as compared with STAT1-deficient mice and both groups responded to infection with the production of type 1 IFNs, CD8 T cells had deregulated proliferation in STAT1-deficient mice (Figure 1B). In contrast to the minimal expansion of, and low BrdU incorporation by, wild-type CD8 T lymphocytes, there was a greater than 2-fold expansion, and approximately 10% of these cells in the STAT1-deficient mice were labeled with BrdU. The expanding subsets were not specific for the LCMV immunodominant epitopes because they did not bind class I MHC molecules presenting these determinants (data not shown). Thus, STAT1 is critical for inhibiting nonspecific CD8 expansion at early times after challenge in vivo. Differential regulation of STAT1 protein levels in responding CD8 T cells The aforementioned results demonstrate that high type 1 IFNs and STAT1 protein levels can be beneficial in limiting nonspecific T-cell proliferation. However, antigen-specific T cells need to be expanded under these conditions. In uninfected spleens, approximately 10% of the leukocytes are CD8 T cells having a diverse, nonspecific repertoire for antigen. By day 5 after infection, the CD8 T cells comprise up to 15% of the spleen and a low frequency of these can be identified as virus specific. The subset can represent greater than 50% of the leukocytes and is primarily virus specific by day 8 after infection.21,31,32 The day-5 and -6 time points are key because they overlap with periods of CD8 T-cell effector function, declining viral burdens, and type 1 IFN responses.21 Previous studies from our group have demonstrated that STAT1 levels are induced in total splenic leukocytes at days 2 to 5 after LCMV infection.18 To extend this characterization to particular cell subsets, STAT1 levels were determined by Western blot analysis of proteins extracted from CD8 T cells, as compared with non-CD8 and total splenic leukocyte populations isolated from uninfected (day 0) or day 3, 5, or 8 LCMV-infected mice. Remarkably, although STAT1 protein was modulated in all the populations tested (Figure 2), being low on days 0 and 8 and induced to high levels on days 3 and 5 after infection, the kinetics of induction were narrower in CD8 T cells, reaching its peak at day 3 and lower at day 5. The virus (4.7 ± 0.1 log PFU/g on day 5, and 4.2 ± 0.1 log PFU/g on day 6) and type 1 IFNs (50.5 pg/mL serum on day 5 and below detection on day 6 for this experiment [see also Pien et al21]) were still detectable at these times. Hence, STAT1 levels are limited in CD8 T cells at times when elevated expression of the transcription factor is maintained in other subsets, and a subset of the population is being activated in the presence of virus and type 1 IFN to mediate viral clearance.
Differential STAT1 expression in proliferating CD8 T cells
Two different approaches were developed to selectively evaluate STAT1 levels in CD8 T cells induced to proliferate during the infection. The first took advantage of the observation that splenic leukocytes harvested on day 5 after LCMV challenge continue to develop, without addition of exogenous factors, during in vitro culture.33 Cells obtained from uninfected, day 5, or day 8 LCMV-infected mice were labeled with CFSE and evaluated for proliferation at days 1, 2, 3, 4, and 5 after culture. Extensive ex vivo proliferation could only be demonstrated with the cells isolated on day 5 (Figure 3; data not shown). The CD4 T-cell subset had only modest proliferation under these conditions, but the CD8 T-cell subset continued to proliferate such that greater than 30% of the cells had diluted out the CFSE by day 3 after culture, and the recovery of this subset almost doubled. Interestingly, the addition of exogenous IFN
To directly examine STAT1 protein levels within individual cells immediately after isolation, a variety of antibodies were screened for their ability to fluorescently label total STAT1 protein intracellularly. The approach required optimizing conditions for cytoplasmic and nuclear staining of total STAT1 using a phycoerythrin-conjugated anti-mouse STAT1 antibody. Isotype control antibodies and cells derived from STAT1-/- mice were used as negative controls to demonstrate specificity of staining (see "Materials and methods"). The results of staining for STAT1 protein in total leukocyte populations complemented the results of the Western blot analysis and extended them to evaluate expression within individual cells. Low but detectable levels of STAT1 were expressed with relative uniformity in populations from uninfected mice, expression was dramatically elevated with greater than 87% of cells expressing high levels on days 5 and 6 after infection, and decreased on days 7 and 8 (Figure 5). As expected, the CD8 but not the CD4 T-cell subset was expanding in vivo starting on day 6 after infection (Figure 5). Gating for analysis of the CD4 T-cell population demonstrated that the majority of these cells had STAT1 levels similar to those observed in total populations (Figure 5). In contrast, the CD8 T-cell population also had STAT1 being induced but a subset had lower expression on day 6 as compared with total or CD4 T cells, that is, 47% versus 11% and 10%. Moreover, the levels of expression were lower in a proportion of the T cells throughout the peak induction of STAT1 with bright staining at only 53% on day 6, 26% on day 7, and 15% on day 8 as compared with approximately 90%, 70%, and 40% to 60% of the total and CD4 T-cell populations (Figure 5). Thus, 2 different populations of CD8 T cells were developing, one with induced STAT1 levels equivalent to those achieved in total and CD4 T-cell populations and another with lower STAT1 levels.
To further explore the subset of the proliferating cells in vivo, splenic leukocytes were harvested from uninfected or LCMV-infected mice who received injections of 5-bromo-2'-deoxyuridine (BrdU) 2 hours prior to killing, and fluorescently labeled with anti-CD8 or anti-CD4, anti-BrdU, and anti-STAT1.31 As expected, the proportions of CD4 T cells were not dramatically increasing on days 5, 6, 7, or 8 after infection (Figure 6A). Only 12% of these cells were incorporating the DNA precursor, BrdU, on day 6, but they were contained within the CD4 T-cell subset having relatively low levels of STAT1. Analysis of the expanding CD8 T cells demonstrated that greater than 40% of these were incorporating BrdU on day 6 after infection, and they were preferentially represented in the higher proportion of STAT1-low cells (Figure 6B). Thus, as compared with total cells and CD4 T cells, CD8 T cells are expanded to higher proportions and numbers during infection, have higher proportions of the low STAT1subset, and the populations replicating their DNA during the 2 hours immediately prior to harvest are found in the low STAT1 subset. Differential STAT1 expression in antigen-specific CD8 T cells To examine STAT1 levels within the antigen-specific CD8 T-cell subset, a fluorescently labeled MHC class I Db tetramer conjugated to the LCMV peptide GP33-41 was also used for staining. This LCMV peptide is an immunodominant viral determinant, and Db GP33-41 is bound by T-cell receptors specific for the complex.31 Splenic leukocytes harvested from uninfected or LCMV-infected mice who had received injections of BrdU 2 hours prior to being killed were stained with anti-CD8, anti-BrdU, anti-STAT1, and Db GP33-41. The antigen-specific CD8 T-cell subset was preferentially found in the low STAT1 subset on day 6 after infection, representing about 10% of the CD8 T cells (Figure 6B). Analysis of the Db GP33-41-positive and BrdU-positive or -negative subsets showed that all of the antigen-specific cells were low for STAT1 (Figure 6C). In contrast, analysis of the Db GP33-41-negative subset showed that STAT1-low and -high populations were present (Figure 6C). These results suggest that the greater higher proportions of the STAT1-low, BrdU-positive cells, that is, 40%, contained antigen-specific cells for other immunodominant viral peptides31 or cells in cycle having down-regulated antigen receptors.34 Although the proportions cannot be precisely quantitated, these data conclusively show that the in vivo-proliferating, antigen-specific CD8 T cells display a phenotype characterized by low STAT1 expression.
The data presented here show that STAT1 is an important factor in regulating inappropriate CD8 T-cell proliferation in vivo at early times during viral infection. Moreover, they conclusively demonstrate that different cell types are induced to express a range of total STAT1 levels during the course of responses to viral infections. Although all leukocytes have elevated expression of the transcription factor, a subset of dividing, antigen-specific cells has lower levels and reduced sensitivity to IFN-mediated antiproliferative effects. The dichotomy of the magnitude of expression allows antigen-specific CD8 T-cell expansion, even in the presence of type 1 IFN, to defend against infection. Thus, a fine-tuning of cytokine effects by regulation of total levels of intracellular signaling molecules is defined.
The modulation of STAT1 protein levels following viral infection within antigen-specific CD8 T cells is reported here for the first time. The studies show that the ability to proliferate in the presence of type 1 IFNs is a consequence of less total protein rather than protein absence. Although earlier studies with STAT1-deficient cells suggested that the presence or absence of the molecule might be the factor determining the consequences of cytokine exposure,8 the in vivo titration of the levels observed here provides a more biologically conservative mechanism, with the potential to maintain a cell's ability to access antiviral gene targets for defense while limiting the antiproliferative effects of IFN exposure. The titration of expression is more than about 2 logs of fluorescence intensity when evaluated within individual cells by FACS analysis, and intensity changes with the kinetics of infection in all populations (see Figure 5). Consistent with the observed dynamic increases and decreases in expression, careful timing was required to demonstrate the difference, with ex vivo cells expanded in culture, by Western blot analysis (see Figure 4). This may explain why earlier attempts by others have failed to detect reduced STAT1 within total proliferating CD4 T-cell populations by Western blot analysis.35 Alternatively, because the proportions of CD4 T cells with low STAT1 levels were much smaller (Figures 5, 6), the difference in STAT1 levels may provide a mechanism preferentially favoring antigen-specific CD8 T-cell proliferation in the context of high type 1 IFN induction. Current studies in our laboratory are extending the characterization of STAT1 levels to other cell types and advancing the understanding of the pathways regulating STAT1 expression in antigen-specific CD8 T cells.
The results add to and extend earlier work from our group demonstrating that type 1 IFNs promote IFN- The results have profound implications concerning the consequences of therapeutic intervention to regulate IFN levels and functions.2-6 They suggest that treatments with IFNs to protect against viral infections and cancer may differentially regulate immune function depending on the cellular levels of total STAT1 protein. If the cytokines are administered under conditions whereby all of the T cells respond with high expression of STAT1, the treatments may interfere with the expansion of antigen-specific cells for defense. However, neutralization of endogenous IFN function to protect against autoimmune diseases may result in conditions promoting the activation and expansion of nonspecific cells as well as limit the antimicrobial defense mechanisms activated by the cytokine. Clearly, learning how to control the levels of STAT1 within cells and using this during cytokine manipulation may help promote appropriate immune responses and allow access to the antimicrobial defense functions as needed.
We thank Ken Nguyen for thoughtful insights and suggestions, and Kathryn Doiron for technical assistance.
Submitted July 18, 2005; accepted September 11, 2005.
Prepublished online as Blood First Edition Paper, October 6, 2005; DOI 10.1182/blood-2005-07-2834.
Supported by the National Institutes of Health (grants RO1-AI55677, RO1-CA41268, and F31-GM20760), the Rhode Island Foundation, and a National Defense Science and Engineering Graduate Fellowship.
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Christine A. Biron, Box G-B629, 69 Brown St, Brown University, Providence, RI 02912; e-mail: christine_biron{at}brown.edu.
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Abstract
Introduction
-activated sequence elements. Other STAT family members have been implicated in the signaling activated by type 1 IFNs,
Rn (an indicator of the signal generated by the PCR) against cycle numbers. The CT values, indicating the earliest cycle for detection, are given in the legends. 
) and CD8 (
) T cells. The bars denote deviations of the means. The experiment has been repeated more than 2 times, obtaining similar results.
) CD8 T cells is shown. The ratio of STAT expression was calculated using amplification efficiencies (1.93 for STAT1 and 1.99 for 
