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The Journal of Immunology, 2007, 178: 242-252.
Copyright © 2007 by The American Association of Immunologists, Inc.

Cytokine-Dependent Blimp-1 Expression in Activated T Cells Inhibits IL-2 Production1

Dapeng Gong and Thomas R. Malek2

Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL 33101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
After Ag activation of naive T cells in vitro, extensive growth and differentiation into effector cells depend upon IL-2. DNA microarray analysis was used to identify IL-2-dependent molecules regulating this process. In this study, we show that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) is expressed by a cytokine-dependent pathway in activated T lymphocytes. IL-2 production by activated CD4+ and CD8+ T cells inversely correlated with Blimp-1 levels as higher IL-2 production was associated with lower Blimp-1 expression. Furthermore, ectopic expression of Blimp-1 by activated T cells inhibited IL-2 production but enhanced granzyme B and CD25 expression. Collectively, these findings indicate that there is a negative feedback regulatory loop in activated T cells such that IL-2 inhibits its own production through induction of Blimp-1 while promoting an effector cell phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-2 has typically been considered a main driver of T cell immunity based on its essential role as a T cell growth factor in vitro (1, 2). Over the last several years, however, it has become apparent that the major nonredundant role for IL-2 surrounds the production and maintenance of CD4+CD25+Foxp3+ T regulatory cells (3). Moreover, when this T regulatory cell defect is corrected and autoimmunity is absent, mice deficient in responding to IL-2 are competent to generate effective immune responses, although the magnitude of the CD8 response and the production of IFN-{gamma} is greater when IL-2R function is normal (4). Thus, there is sufficient redundancy in vivo to compensate for the lack of IL-2 for T cell immunity.

Even though there are IL-2-independent pathways in vivo, the dominance of IL-2 for in vitro T cell responses should aid in identifying cytokine-dependent molecular pathways that contribute to productive T cell responses after engaging TCR and costimulatory molecules. In this regard, we have previously reported that thymic-specific transgenic expression of IL-2Rbeta in IL-2Rbeta–/– mice (designated Tg–/– in this study) prevented lethal autoimmunity associated with IL-2/IL-2R deficiency by production of T regulatory cells, whereas T cells in the peripheral immune compartment were essentially nonresponsive to IL-2. When naive peripheral Tg–/– T cells were stimulated by anti-CD3 in the presence of APC in vitro, they underwent three to four rounds of proliferation, but did not develop into effector T cells and were markedly defective in T cell growth when restimulated not only by IL-2 and IL-15 but also by other cytokines, notably IL-4 and IL-7 (5). However, the production of effector cells, including their growth potential, was partially rescued by including exogenous IL-4 or IL-12 during the initial culture of Tg–/– T cells with anti-CD3, indicating that other cytokines can substitute for activities normally dominated by IL-2. These findings indicate that T cells activated solely by signal 1 (TCR) and signal 2 (costimulatory molecules) develop a limited nonproductive proliferative response unless they receive a third signal that is readily provided by IL-2, but also other cytokines, which then renders the cells competent to undergo extensive growth and differentiation into effector T cells.

The current study was undertaken to define the types of molecules and their functional contributions as T cells receive this critical early cytokine signal. Our approach was to optimally activate wild-type (WT)3 and Tg–/– C57BL/6 T cells with anti-CD3 and then screen for IL-2 target genes. DNA microarray analysis revealed B lymphocyte-induced maturation protein 1 (Blimp-1) as one such target. Blimp-1, encoded by the Prdm1 gene, is a transcriptional repressor, which is essential for terminal B cell differentiation into plasma cells (6, 7). Although Blimp-1 has been most extensively studied in B cells, there are mounting data that Blimp-1 is an important regulator in many cell types. Blimp-1-deficient mice die during embryogenesis (8) and the Blimp-1 homologues in Xenopus (Xblimp1) and sea urchin (SpKrox1) are required during early development of these two organisms (9, 10). Blimp-1 is required for specification of primordial germ cells during early embryogenesis of the mouse (11, 12, 13) and appears to contribute during differentiation of promyelocytic cells into macrophages or granulocytes (8). Recent studies have implicated Blimp-1 in the regulation of T cell activation and homeostasis (14, 15). In this study, we show that Blimp-1 is expressed by a cytokine-dependent mechanism in activated T lymphocytes and functions to inhibit IL-2 production while enhancing the expression of several molecules expressed by effector cells. These data raise the possibility that Blimp-1 contributes to the maturation of effector T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 and OT-I TCR-transgenic mice were obtained from The Jackson Laboratory or Taconic Farms, respectively. C57BL/6 IL-2Rbeta–/– mice that express thymic-specific transgenic WT IL-2Rbeta has been described previously (16) and are designated Tg–/– in this study. IL-2p-GFP transgenic mice were obtained from Dr. E. V. Rothenberg (California Institute of Technology, Pasadena, CA) (17). All experiments were approved by the University of Miami Institutional Animal Care and Use Committee.

Cell culture

Splenocytes were cultured for 48 h in RPMI 1640 complete medium with anti-CD3 (5% culture supernatant) or LPS (1 or 10 µg/ml; Escherichia coli 026:B6; Sigma-Aldrich) as described previously (16, 18). In some cases cultures contained IL-2, IL-4, IL-12, or IL-15 (10 ng/ml; PeproTech) or anti-IL-2 (25 µg/ml). After activation, B cells, CD4+, CD8+, or total T cells were purified using CD19, CD4, CD8, or Thy1.2 magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol, and then these cells were used for RT-PCR or Western blot analysis. For DNA microarray analysis, the activated CD4+ or CD8+ T cells were further purified by sorting for CD4+CD25high or CD8+CD25high cells using a BD Biosciences Vantage SE sorter. For OT-I TCR transgenic mice, splenocytes were cultured with OVA257–264 peptide (0.1 nM; synthesized by Research Genetics) and IL-2 (19). Three days later cells were harvested, and the activated CD8+ T cells were purified and recultured (1 x 105 cells/ml) with either IL-2 or IL-15 for an additional 2–4 days. For proliferation assay, T cells (2 x 104 cells/well) were cultured in 0.2 ml of complete medium in a 96-well flat-bottom plate with the indicated cytokines for 24 h, and [3H]thymidine was added during the last 4 h of culture. Cells were then harvested on glass-fiber filters and counted in a beta scintillation counter. Data are reported as the means of duplicate or triplicate values that consistently varied by <10% from each other.

DNA microarray preparation and analysis

Total RNA was isolated using TRIzol reagent and sometimes further purified using RNAeasy Mini Kit (Qiagen). Before target production, the quality and quantity of each RNA sample was assessed using a 2100 BioAnalyzer (Agilent). Targets were prepared from 10 µg of total RNA and hybridized to arrays according to the Affymetrix Technical Manual at Expression Analysis using the Mouse Genome 430 2.0 Array for activated CD4 T cells and the U74Av2 array for the activated CD8 T cells. Fluorescent images were detected in a GeneChip Scanner 3000, and expression data were extracted using the MicroArray Suite 5.0 software (Affymetrix). All GeneChips were scaled to a median intensity setting of 500. Each data set was derived from three biologically independent replicate samples and subjected to two-group comparison of multiple samples by Student’s t test analysis.

Abs and FACS analysis

PE-anti-IL 2, PE-anti-CD69, Cy-Chrome-anti-CD8{alpha} (53.6.7), and PE-anti-CD25 (IL-2R{alpha}), PE-anti-IFN-{gamma}, and FITC-anti-human CD2 (hCD2) were purchased from BD Pharmingen. PE-anti-granzyme B was purchased from Caltag Laboratories. FITC-anti-CD4 (GK1.5), FITC-anti-CD8{alpha} (53.6.7), and anti-IL-2-mAb (S4B6) were prepared in our laboratory. FACS analysis was performed using a BD Biosciences FACScan and LSR-1, and CellQuest software as described previously (16). For intracellular staining of IL-2 and IFN-{gamma}, cells (1 x 106/ml) were cultured in RPMI 1640 complete medium with PMA (50 ng/ml), ionomycin (100 ng/ml), and 1 µl of GolgiPlug (containing Brefeldin A; BD Pharmingen) for 4 h. After surface marker staining, cells were permeabilized with Cytofix/Cytoperm (BD Pharmingen), stained with Abs, and analyzed by FACS. The intracellular staining of granzyme B omitted the stimulation step by PMA and ionomycin.

RT-PCR

Total RNA was isolated using the TRIzol reagent (Invitrogen Life Technologies) as described by the manufacturer. RT-PCR was performed as described previously (16). The PCR primers were as follows: Blimp-1, 5'-GCC AAC CAG GAA CTT CTT GTG T-3' and 5'-AGG ATA AAC CAC CCG AGG GT-3'; GAPDH, 5'-TTA GCA CCC CTG GCC AAG G-3' and 5'-CTT ACT CCTTGG AGG CCA TG-3'; c-myc, 5'-GGG CCA GCC CTG AGC CCC CTA GTG C-3' and 5'-ATG GAG ATG AGC CCG ACT CCG ACC-3'; granzyme B, 5'-CGA GAG GAC TTT GTG CTG AC-3' and 5'-ACG TGG AGG TGA ACC ATC C-3'; CCR6, 5'-GGT ACA TTG CCA TCG TCC AG-3' and 5'-GAG GCA GCA ATG CAG GAA AG-3'; IFNi203, 5'-GTC TCC ACC AAC ACC ATC C-3' and 5'-GTC CAT AAA CCA CCA CTG CC-3'; CD19, 5'-ACA GGA CTG GAA GAA GAA G-3' and 5'-ACT GAA TTG AGT GGA GCT G-3'; TCRbeta, 5'-AGG CTA CCC TCG TGT GCT TG-3' and 5'-TGC ACT TGG CAG CGG AAG TG-3'; and IL-2Rbeta, 5'-TGG CCT TGT CCG AAA GGT CA-3' and 5'-CAA GGT CTC TCA CTA CAT TG-3'. PCR cycling conditions were as follows: 94°C, 30 s; 55°C, 30 s; 72°C, 45 s. Unless otherwise indicated, 1 µg of total RNA, treated with RNase-free DNase, was subjected to reverse transcription with oligo(dT) (Promega). The cDNA template was then aliquoted for PCR amplification with the indicated primers. The number of cycles was 16, 20, 24, and 28 for GAPDH; 20, 24, 28, and 32 for granzyme B; and 24, 28, 32, and 36 for the remaining genes.

Western blot analysis

Whole cell lysates were prepared in 20 mM Tris-HCl (pH 7.5)-10% glycerol-150 mM NaCl-1% Nonidet P-40–0.1% SDS-0.5% deoxycholate-2 mM DTT-1 mM PMSF (20). Lysates were subjected to SDS-PAGE on a 10% gel under reducing conditions and Western blotting as described previously (20). The transferred membranes (Millipore) were blocked with 5% milk in PBS plus 0.5% Tween 20, blotted with monoclonal and anti-mouse Blimp-1 Ab (a gift from Dr. K. Calame, Columbia University, New York, NY, or purchased from Novus Biologicals) or polyclonal rabbit anti-mouse beta-actin (Sigma-Aldrich), washed, and then blotted with goat anti-mouse IgG1 ({gamma}1) conjugated to peroxidase (Roche) or goat anti-rabbit conjugated to peroxidase (Jackson ImmunoResearch Laboratories). Films were developed after incubation with ECL Western blotting detection reagents (GE Healthcare BioSciences).

Plasmid and retroviral transduction

Blimp-1 cDNA from LPS-activated B cells was cloned into the pMI retroviral vector (21) (a gift from Dr. Y.-W. He, Duke University, Durham, NC) such that Blimp-1 is placed 5' to the internal ribosome entry site and hCD2, the latter of which serves as a marker to identify and purify transduced cells. Ecotrophic {phi}NXE packaging cell line (from Dr. G. Nolan, Stanford University, Stanford, CA) was used to produce retroviruses (22). To transduce T cells, splenocytes were cultured in RPMI 1640 complete medium with anti-CD3 (5% culture supernatant) for 24 h. Cells were harvested, washed, and resuspended in retrovirus (1 x 106 cells/ml/well) with anti-CD3 in 24-well plates supplemented with 1 µl of polybrene (5 mg/ml; Sigma-Aldrich). Cells were then centrifuged at 2500 rpm for 90 min, after which 1.5 ml of RPMI 1640 complete medium with anti-CD3 and 1.5 µl of polybrene were added into each well. Cells were cultured at 37°C for 24 h and were then subjected to FACS analyses or purified into hCD2+ (transduced cells) and hCD2– (untransduced cells) using hCD2 magnetic beads (Miltenyi Biotec) for proliferation assays, Western blots, or RT-PCR analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of IL-2Rbeta-dependent differentially expressed transcripts in anti-CD3-activated T cells

Splenocytes from WT and Tg–/– C57BL/6 mice were stimulated with anti-CD3 for 48 h because this leads to optimal T cell activation. Total RNA for microarray analysis was then obtained from the activated CD4+ or CD8+ T cells after isolation by initial enrichment through positive selection using anti-CD4 or anti-CD8 magnetic beads followed by cell sorting of CD4+CD25high or CD8+CD25high cells (Fig. 1A). Activated Tg–/– T cells bear lower levels of CD25 because their nonresponsiveness to IL-2 prevents IL-2-dependent CD25 up-regulation during the culture period (23).


Figure 1
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  		FIGURE 1. DNA microarray analysis of IL-2-regulated genes. Spleen cells from WT and Tg–/– mice were stimulated with anti-CD3 for 48 h. Total RNA was extracted from purified activated CD4+ and CD8+ T cells obtained by enrichment using anti-CD4 or anti-CD8 magnetic beads followed by cell sorting of CD4+CD25high or CD8+CD25high T cells. A, Purity of activated CD4+ and CD8+ T cells for probe preparation. Genes differentially increased in WT (B) or Tg–/– (C) T cells representing genes whose expression depends upon IL-2 for induction or down-regulation, respectively. Shown is the raw-fold difference of targets of known or predicted function with differential expression ≥3 (p < 0.05) and intensity signal >100, where there were comparable probe sets for both CD4+ (Affymetrix 430-2.0 array) and CD8+ (Affymetrix U74Av2 array) T cells. Additional genes that fulfill these criteria but where there were not comparable data for both T cell subsets are shown in Table I. D, Semiquantitative RT-PCR of selected targets using mRNA from activated CD8+ T cells.

 
We focused our attention on those gene targets that were most differentially expressed (≥3-fold) and have shown the data for targets of known or predicted function of comparable probes sets (97–100% identical) that were present on the Affymetrix arrays used for activated CD4+ (430–2.0 array) and CD8+ (U74Av2 array) T cells. Other genes identified by this criteria but with probe sets unique to CD4+ or CD8+ T cells are shown in Table I. This analysis revealed 52 gene targets of which 25 were higher in WT T cells (Fig. 1B), i.e., increased by IL-2 responsiveness, and 27 were higher in Tg–/– T cells (Fig. 1C), suggesting that their down-regulation depended upon IL-2. With respect to genes more highly expressed by WT cells (Fig. 1B), one of the most differentially expressed targets was IL-2Rbeta (Il2rb) and it was designated absent in the CD4 probe set, confirming that activated Tg–/– T cells lacked IL-2Rbeta. Furthermore, 9 of these targets, granzyme A and B (Gzma, Gzmb), IFN-{gamma} (Ifng), Blimp-1 (Prdm1), IL-12Rbeta (Il12rb2), IL-2R{alpha} (Il2ra), CIS (Cish), IL-4 (Il4), and P-selectin (Selp), have previously been reported to be up-regulated by IL-2 in lymphoid cells and lines (6, 23, 24, 25, 26, 27, 28). The selective high level of neurogranin (Ngrn, also called RC3), encoding a calcium/calmodulin binding protein, in activated CD8+ Tg–/– T cells (Fig. 1C) has also been observed upon depriving activated T cells of IL-2 thereby promoting apoptosis (29). Semiquantitative RT-PCR analysis confirmed differential expression of selected genes by WT or Tg–/– CD8+ T cells (Fig. 1D), further substantiating the validity of the microarray data.


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  		Table I. Additional differentially expressed genes (≥3-fold) by WT and Tg–/–anti-CD3-activated T cellsa

 
Of the 52 differentially expressed transcripts, more than half (29 of 52) are typically characterized with important functions in the immune system (17 of 52) or encode intracellular proteins that directly regulate transcription (12 of 52). IL-2Rbeta signaling was required for substantial expression of transcripts for granzyme A and B, IFN-{gamma}, IL-12Rbeta, and IL-2R{alpha} by activated CD4+ and CD8+ T cells and for IL-4 and perforin by activated CD4+ or CD8+ T cells, respectively (Fig. 1B and Table I). This result is consistent with an important early role for IL-2 in promoting T effector function. Interestingly, activated Tg–/– CD4+ T cells contained a 5.6-fold higher level of IL-2 transcripts than WT T cells (Table I), raising the possibility that IL-2Rbeta signaling might function to down-regulate IL-2. Given the importance of IL-2 as a T cell growth factor, notably missing from these highly differentially expressed transcripts are those intimately involved in cell cycle progression and/or cell survival. This result may reflect that the contribution of IL-2Rbeta signaling was measured simultaneously with ligation of the TCR and costimulatory molecules, and the latter two stimuli induced significant T cell proliferation (5).

Some identified transcriptional regulators were more highly expressed by activated WT T cells (Fig. 1B) while others were found to be higher in activated Tg–/– T cells (Fig. 1C), suggesting that some of these might be coordinately regulated as Ag-activated T cells receive signals through IL-2Rbeta. Particularly interesting in this regard were Prdm1 (Blimp-1), Idb3 (Id3), and Bcl-6 because each of these was similarly regulated in activated CD4+ and CD8+ T cells. In B cells, Blimp-1 and Bcl-6 are reciprocally regulated such that Blimp-1 drives plasma cell differentiation whereas Bcl-6 favors germinal center reactions (30, 31, 32, 33). Furthermore, Bcl-6 represses Blimp-1 whereas Blimp-1 is a transcriptional repressor of Id3 (34). Until recently (14, 15), Blimp-1 has not been considered a protein expressed by T cells, although its discovery in B lineage cells relied on induction by IL-2 and IL-5 (6). Thus, based on its known cytokine dependence and role in cell fate decisions (7, 35), we sought to further assess the role of Blimp-1 in T cells.

Blimp-1 expression by T lymphocytes

To substantiate the relevance of these microarray results concerning Blimp-1, semiquantitative RT-PCR analysis was first performed using mRNA from purified resting and activated splenic B and CD8+ T lymphocytes (Fig. 2A). This analysis revealed that Blimp-1 mRNA was detected at a relatively low level in both resting B and CD8+ T cells. However, Blimp-1 mRNA expression substantially increased to similar levels after activation of B cells with LPS or CD8+ T cells with anti-CD3 and IL-2. Importantly, the detection of Blimp-1 in T cells cannot be attributed to contaminating B cells as assessed by RT-PCR analysis with primers to CD19 and TCRbeta. Western blot analysis established that Blimp-1 protein was expressed in both CD4+ and CD8+ anti-CD3- activated T cells and that Blimp-1 protein levels in activated T cells were nearly equivalent to those found in LPS-activated B cells (Fig. 2B). Blimp-1 protein was not detected for resting B and T cells, suggesting that the low levels of mRNA observed in resting lymphocytes by RT-PCR might be due to a few contaminating activated cells. Time-course experiments indicated that Blimp-1 protein was first minimally detected 24 h after activation with anti-CD3 for both CD4+ and CD8+ T cells with obvious expression after 48 h in culture (Fig. 2C). When the anti-CD3-activated T cells were further cultured in IL-2, Blimp-1 was still detected in these cells but at a lower level (Fig. 2D, compare lanes 1 and 2). Collectively, these data indicate that Blimp-1 expression by mature T lymphocytes is primarily confined to activated T cells with obvious expression 2 days after induction by anti-CD3 in the presence of APC.


Figure 2
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  		FIGURE 2. Blimp-1 expression by T lymphocytes. The indicated populations of cells from C57BL/6 (A–D) or OT-I TCR-transgenic mice (E) were analyzed for indicated mRNA by RT-PCR using total RNA from 1 x 106 cells (A) or Blimp-1 or beta-actin protein by Western blotting (B–E). For activated B cells, CD19+ B cells were purified and cultured with LPS for 48 h (A and B). For activated T cells, spleen cells were cultured with anti-CD3 (A–D) or OVA257–264 (E) and then purified. In some cases where activated CD8+ T cells were further cultured in cytokines, the anti-CD3 (D) or OVA257–264 (E) activated T cells were purified on day 2 and 3, respectively, washed, and then cultured in the indicated cytokines.

 
When activated TCR transgenic OT-I CD8+ T cells are further cultured in IL-2, the OT-I CTL continue to express properties of activated effector T cells; however, when the OT-I CTL are cultured in IL-15, they express phenotypic properties, i.e., down-regulation of CD69 and CD25 and up-regulation of CD62L and Ly-6C, and functional properties consistent with memory cells (19). When WT CD8+ T cells were activated for 2 days with anti-CD3 (Fig. 2D) or OT-I were cultured for 3 days with OVA peptide and IL-2 (Fig. 2E) and then further cultured with IL-2 or IL-15, the CTL from the IL-2 containing cultures continued to express Blimp-1, whereas Blimp-1 protein was not readily detected in the "memory-like" T cells from the IL-15 containing cultures. Taken together, these data establish that Blimp-1 is expressed by activated T cells and its expression is differentially regulated by IL-2 and IL-15, raising the possibility that Blimp-1 is primarily associated with effector, not memory T cells.

Blimp-1 expression depends upon cytokines

Given the fact that Blimp-1 was expressed rather late after activation of T cells with anti-CD3 raised the question of whether this expression depended upon IL-2. To test this notion, we examined the extent Blimp-1 was expressed by anti-CD3-activated WT vs IL-2-nonresponsive Tg–/– T cells. When compared with control WT T cells, anti-CD3-activated Tg–/– CD8+ T cells expressed substantially less Blimp-1 mRNA (Fig. 1D), and unfractionated Tg–/– T cells did not express Blimp-1 protein (Fig. 3A, lanes 1 and 2). Furthermore, expression of Blimp-1 protein in WT CD4+ and CD8+ T cells was essentially completely blocked when the anti-CD3 activation was performed in the presence of anti-IL-2 (Fig. 3B). These findings indicate that Blimp-1 is not solely induced by signaling through TCR and costimulatory molecules expressed by APC but rather requires IL-2/IL-2R signaling in vitro. This dependency on IL-2 is consistent with the late expression of Blimp-1 protein as assessed in time-course studies. The addition of exogenous IL-2 during anti-CD3 activation of WT T cells increased Blimp-1 expression by 75%, indicating that endogenously produced IL-2 was not sufficient for full induction of this protein (Fig. 3C, lanes 1 and 2). However, the cytokine dependency for Blimp-1 expression is not restricted to IL-2 because inclusion of IL-4 or IL-12 during the culture of Tg–/– T cells with anti-CD3 resulted in obvious expression of Blimp-1 (Fig. 3C, lanes 4–6).


Figure 3
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  		FIGURE 3. Cytokines regulate Blimp-1 expression and IL-2 production. Spleen cells from the indicated mice were cultured with anti-CD3 in the presence of the indicated cytokines or anti-IL-2 for 2 days. The cells were harvested, purified, and total cell extracts were prepared for Western blotting (A–C). Alternatively, the anti-CD3-activated spleen cells were washed and recultured for 4 h with PMA and ionomycin in the presence of GolgiPlug for intracellular FACS analysis for CD4+ and CD8+ T cell subsets (D–F). Blimp-1 and beta-actin protein determined by Western blot analysis. A, Comparison of Blimp-1 expression by naive and anti-CD3-activated WT vs Tg–/– T cells. The effect of anti-IL-2 (B), exogenous IL-2, IL-4, or IL-12 (C) on Blimp-1 expression by anti-CD3-activated WT CD4+ and CD8+ T cells or purified T cells, respectively. Representative histograms (D) of intracellular IL-2 for CD4+ and CD8+ T cells and the percentages of IL-2-producing CD4+ (E) and CD8+ T cells (F), where the data are the mean ± SD of four experiments.

 
IL-2 production inversely correlates with Blimp-1 expression

We were intrigued by the finding that IL-2 mRNA was markedly higher in Tg–/– CD4+ T cells by DNA microarray analysis (Table I), and these cells expressed lower levels of Blimp-1 mRNA and protein (Figs. 1D and 3A). Therefore, we examined whether there was a relationship between the induction of IL-2 and the expression of Blimp-1. For WT-activated CD4+ T cells that expressed readily detectable Blimp-1 when examined immediately after the anti-CD3 culture (Fig. 3B, lane 2), ~20% of the cells produced IL-2, after a separate aliquot of cells was briefly restimulated with PMA and ionomycin, as assessed by intracellular FACS analyses (Fig. 3, D and E). When exogenous IL-2 was added during the culture of WT cells with anti-CD3, Blimp-1 protein levels increased (Fig. 3C, lanes 1 and 2) and the percentage of IL-2-producing cells significantly dropped (Fig. 3, D and E; p = 0.012) after brief restimulation. In contrast, inclusion of anti-IL-2 during the culture of WT CD4 T cells with anti-CD3 resulted in cells that did not detectably express Blimp-1 (Fig. 3B, lane 1), and most (~75%) of these cells produced IL-2 after reculture with PMA and ionomycin (Fig. 3, D and E). As expected, maximal expression of granzyme B and CD25 (IL-2R{alpha}) was dependent upon IL-2 (data not shown), which positively correlated with Blimp-1 levels. Furthermore, PMA and ionomycin restimulation of Tg–/– CD4+ T cells resulted in IL-2 production by the majority (~76%) of Tg–/– CD4+ T cells (Fig. 3, D and E), and these cells did not express Blimp-1 when examined before the restimulation culture (Fig. 3A, lane 2, and C, lane 4). Thus, all of these data relate the extent of IL-2 production with physiological levels of Blimp-1, with higher intracellular IL-2 associated with those T cells with lower Blimp-1. When IL-4 or IL-12 were included with anti-CD3, which led to Blimp-1 expression by Tg–/– T cells (Fig. 3C, lanes 5 and 6), IL-2 production was markedly inhibited (90 and 67%, respectively) upon restimulation by PMA and ionomycin (Fig. 3, D and E). A similar pattern of results was obtained when WT and Tg–/– CD8+ T cells were analyzed for IL-2 production (Fig. 3, D and F). Therefore, these data indicate that the presence of IL-2 down-regulates its own expression and this inhibitory effect correlates with activated T cells that express Blimp-1.

As an initial effort to determine whether there are regulatory sequences associated with the IL-2 gene that are targeted by this negative feedback of IL-2 production, we tested whether a GFP reporter linked to 8.4 kb of 5' upstream DNA sequence of the mouse IL-2 gene was similarly affected. Spleen cells from transgenic mice that expressed this reporter (IL-2p-GFP) show position-independent and copy number-dependent induction of GFP after T cell activation that substantially overlaps, but is not identical, especially for CD8+ T cells, with the activity of the endogenous IL-2 gene (17). When spleen cells from these mice were stimulated with anti-CD3 in the presence or absence of anti-IL-2, there was a substantial increase in the fraction of CD4+ and CD8+ T cells that expressed GFP upon reculture with PMA and ionomycin for those cells cultured with anti-IL-2 (Fig. 4). Because the expression of GFP protein was measured from its induced mRNA transcript, these data indicate that the autoregulatory loop for IL-2 production targets elements within this 8.4-kb fragment adjacent to the mouse IL-2 gene.


Figure 4
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  		FIGURE 4. Anti-IL-2 increases the induction of GFP in IL-2p-GFP mice. Spleen cells from IL-2p-GFP transgenic mice were cultured with anti-CD3 in the absence or presence of anti-IL-2 for 2 days. Activated T cells were restimulated with PMA and ionomycin and assessed for GFP by FACS analysis. Representative histograms of GFP for CD4+ and CD8+ T cells and (A) the percentages of GFP+ CD4+ or CD8+ T cells (B), where the data are the mean ± SD of three experiments.

 
Ectopic expression of Blimp-1 does not regulate T cell growth

Much has been learned about the function of Blimp-1 by ectopic expression of Blimp-1 in B cells. Such studies revealed that Blimp-1 induces growth arrest through the repression of c-myc (20, 36), which in part accounts for the cessation of cell division during plasma cell differentiation. To determine whether Blimp-1 might also regulate T cell growth, we examined the effect of ectopic expression of Blimp-1 on growth arrest and c-myc expression in activated T cells. For these experiments, Blimp-1 cDNA was cloned into the pMI retroviral vector such that Blimp-1 is placed 5' to the internal ribosome entry site and hCD2, the latter of which served as a marker to identify and purify transduced cells. Consistent with previous studies (6), the ectopic expression of Blimp-1 with our construct in the BCL-1 cell line resulted in elevated IgM secretion (data not shown), confirming the activity of Blimp-1 in our vector.

WT and Tg–/– spleen cells were transduced 24 h after activation with anti-CD3, which corresponds to when endogenous Blimp-1 is normally first detected (Fig. 2C). Western blot analysis of purified hCD2+ empty pMI vector (mock) or Blimp-1-transduced cells revealed that the Blimp-1-transduced C57BL/6 or Tg–/– hCD2+ cells expressed ~2-fold more Blimp-1 than mock-transduced hCD2+ WT T cells (Fig. 5A). Although sometimes the transduction efficiencies were lower with the Blimp-1 expression vector, particularly for Tg–/– cells, we still observed similar biological outcomes after ectopic expression of Blimp-1 in the functional experiments described below. Importantly, after the anti-CD3 culture, the mock and Blimp-1-transduced hCD2+ cells exhibited essentially identical cell viability (80–90% alive) for CD8+ and CD4+ T cells, respectively, as assessed by 7-aminoactinomycin D. These data suggest that it is unlikely that ectopic expression of Blimp-1 causes apoptosis of the transduced cells.


Figure 5
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  		FIGURE 5. Ectopic expression of Blimp-1 does not regulate T cell growth. Spleen cells from the indicated mice were activated with anti-CD3 for 24 h. The cells were harvested and transduced with Blimp-1-expressing-vector (Blimp) or control vector (Mock). After an additional 24 h in culture, the transduced cells (hCD2+) were purified. For the hCD2+ cells, Blimp-1 expression was assessed by Western blotting (A), cell growth was examined by further culture in the indicated cytokines (B–E), or c-myc mRNA levels were determined by RT-PCR (F). Cytokine-driven proliferation of hCD2+ Tg–/– (B) or C57BL/6 (C and D) T cells assessed by [3H]thymidine incorporation, or cell growth over time to IL-2 (E) by anti-CD3-activated hCD2+ C57BL/6 T cells. For cell growth, cultures were seeded at 1 x 105 cells/ml. F, c-myc mRNA expression by semiquantitative RT-PCR for hCD2+ Tg–/– T cells where primers for Blimp-1 and GAPDH served as controls.

 
The effect of the ectopic expression of Blimp-1 was then examined for cytokine-dependent growth of purified hCD2+ mock and Blimp-1-transduced T cells. Ectopic expression of Blimp-1 in anti-CD3-activated Tg–/– T cells did not increase the response of these T blasts when recultured with IL-4 or IL-7 (Fig. 5B), which was markedly lower than that induced by WT T blasts (Fig. 5C). Thus, the absence of Blimp-1 in Tg–/– T cells is not solely responsible for the inability of these T cells to undergo substantial clonal expansion to cytokines within the IL-2 family. Furthermore, control and Blimp-1-transduced C57BL/6 T blasts exhibited substantial and indistinguishable proliferative responses to IL-2, IL-4, IL-7, or IL-15 (Fig. 5C). The magnitude of these responses were similar to that typically seen after the culture of untransduced T blasts to these cytokines (5). Analysis of cellular growth to IL-2 in dose-response (Fig. 5D) and time-course (Fig. 5E) experiments indicated essentially identical responses by both groups of transduced T cells. The identical growth rates and thymidine incorporation further support the notion that that Blimp-1 does not adversely affect cell viability. Furthermore, ectopic expression of Blimp-1 did not substantially reduce the level of c-myc mRNA in Tg–/– T cells as assessed by semiquantitative RT-PCR (Fig. 5F). Thus, these data indicate that ectopic expression of Blimp-1 does not exhibit antiproliferative effects in activated T cells. Collectively, these experiments demonstrate that the level of ectopic expression of Blimp-1 achieved by retroviral-mediated transduction does not obviously dysregulate their behavior when assessing growth and cell death.

Ectopic expression of Blimp-1 in the regulation of T cell function

Because Blimp-1 expression in T lymphocytes is restricted to activated effector cells and correlates with inhibition of IL-2 production, we also examined whether ectopic expression of Blimp-1 affected IL-2 production and other molecules associated with activated T cells. For hCD2+ Blimp-1-, but not hCD2+ mock-, transduced WT CD4+ and CD8+ T cells, restimulation with PMA and ionomycin revealed a marked block in IL-2 production as assessed by intracellular FACS analysis (Fig. 6A). The levels of inhibition were ~70 and 65% in CD4+ and CD8+ T cells, respectively. This effect was cell intrinsic to the Blimp-1-transduced cells because IL-2 production was unaffected in the nontransduced hCD2– cells in the same culture (Fig. 6A, compare the upper and lower histograms for hCD2–CD4+ T cells). A similar pattern of results was obtained for Blimp-1-transduced Tg–/– T cells that were restimulated with PMA and ionomycin (Fig. 6B), although the levels of inhibition were less pronounced (~50%). For WT and Tg–/– Blimp-1-transduced T cells, IL-2 production was still seen by hCD2low cells suggesting that blockade of IL-2 production was dependent upon the level of Blimp-1, because we expect higher levels of Blimp-1 to correlate with higher levels of the hCD2 reporter. Consistent with these data, we also found that ectopic expression of Blimp-1 in 2B4 T cell hybridoma also inhibited the induction of IL-2 by anti-CD3 (Fig. 7) or PMA/ionomycin (data not shown). These experiments, therefore, provide direct data to demonstrate that Blimp-1 functions to block IL-2 production by activated T cells.


Figure 6
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  		FIGURE 6. Ectopic expression of Blimp-1 in the regulation of T cell function. Spleen cells from the indicated mice were cultured with anti-CD3 and transduced with the Blimp-1-expression vector (Blimp) or the control vector (Mock). Twenty-four hours after transduction, cells were harvested and either directly examined for expression of CD25, CD69, or intracellular granzyme B or recultured with PMA and ionomycin to assess intracellular IL-2 or IFN-{gamma} production. Representative histograms of IL-2-production by hCD2+ transduced CD4+ and CD8+ T cells from C57BL/6 (A) or Tg–/– mice (B). From 3500 to 4500 events were analyzed for the hCD2+ T cells. The bar graph to the right represents the mean ± SD from four individual experiments (*, p < 0.05; ***, p < 0.001 by Student’s t test). Histograms for expression of granzyme B, CD25, IFN-{gamma}, and CD69 after transduced T cells from C57BL/6 (C) or Tg–/– (D) mice were gated on hCD2+CD4+ or hCD2+CD8+. Mock-transduced cells are shown in the shaded area, whereas Blimp-transduced cells are shown in the open area with a bold line. Data are representative of four experiments.

 

Figure 7
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  		FIGURE 7. Ectopic expression of Blimp-1 inhibits IL-2 production by a T cell hybridoma. The 2B4 T cell hybridoma was cultured and transduced by the control and Blimp-1-expression vectors. After 24 h, transduced cells were washed and recultured in medium supplemented with brefeldin A in the presence of plate-bound anti-CD3 for 4 h. Cells were analyzed for intracellular IL-2 by FACS. Four thousand events were analyzed for the hCD2+ cells. Data are representative of three experiments.

 
FACS analysis of Blimp-1-transduced hCD2+ WT (Fig. 6C) and Tg–/– (Fig. 6D) CD4+ and CD8+ T cells indicated that the expression of granzyme B and CD25 (IL-2R{alpha}) was increased, whereas the expression of the activation marker CD69 and intracellular IFN-{gamma} was largely unaffected, although sometimes slightly enhanced levels of intracellular IFN-{gamma} were seen for Blimp-1-transduced C57BL/6 CD8+ T cells. Quantitative analysis of these results by examining the mean fluorescent intensity revealed that ectopic expression of Blimp-1 in anti-CD3-activated T cells enhanced the level of granzyme B and CD25 ~1.8-fold for both CD4+ and CD8+ T cells with the exception of granzyme B in CD4+ Tg–/– T cells, which minimally changed. When compared with CD8+ T cells, the levels of both granzyme B and CD25 were lower than in CD4+ T cells, and when compared with WT cells, were markedly lower in both CD4+ and CD8+ Tg–/– T cells. This latter result was expected because IL-2 is essential for granzyme B expression and up-regulation of CD25 expression after induction by TCR signaling (23, 24, 37). Although enhancement of these two proteins was seen in Blimp-1-transduced Tg–/– T cells, the overall lower expression relative to WT cells illustrates that Blimp-1 was not sufficient to drive normal expression of these proteins. Increased granzyme B and CD25 by Blimp-1-transduced WT T cells cannot be attributed to enhanced IL-2 production because the levels of these two proteins in the untransduced hCD2– cells in the same cultures were lower and equivalent to that found in control mock-transduced T cells (data not shown) and because increased Blimp-1 is expected to inhibit IL-2 production. Collectively, these data indicate that two molecules whose expression is associated with effector T cells are up-regulated by Blimp-1.

Ectopic expression of Blimp-1 inhibits expression of the IL-2p-GFP reporter

To further investigate the regulation of IL-2 production by Blimp-1, we examined the effect of ectopic expression of Blimp-1 on expression of both IL-2 and the 8.4-kb IL-2p-GFP reporter using T cells from the GFP transgenic mice. As shown for WT T cells, a lower percentage of CD4 and CD8 IL-2-producing T cells was only detected when intracellular IL-2 was assessed for the Blimp-1-transduced cells, as identified by their expression of hCD2 (Fig. 8A). Importantly and in an analogous fashion, a significantly lower percentage of GFP+ cells was also observed for the Blimp-1-transduced hCD2+ T cells (Fig. 8B). This effect was somewhat more striking for hCD2+ Blimp-1-transduced CD4 T cells probably because the IL-2p-GFP reporter is more active than the endogenous IL-2 gene in CD8 T cells (Fig. 8). The inhibitory effect was specific for Blimp-1-transduced cells because the percentage of cells expressing intracellular IL-2 (Fig. 8A) or GFP (Fig. 8B) was essentially identical for hCD2+ and hCD2– cells after mock transduction and for the nontransduced hCD2– cells contained in the cultures after ectopic expression of Blimp-1. Thus, Blimp-1 inhibition of IL-2 production maps to a region within this 8.4-kb upstream DNA sequence of the IL-2 gene.


Figure 8
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  		FIGURE 8. Ectopic expression of Blimp-1 inhibits the IL-2p-GFP reporter. Spleen cells from progeny of two independent founders of IL-2p-GFP transgenic mice were cultured with anti-CD3 and 24 h later transduced with the Blimp-1-expression vector (Blimp) or the control vector (Mock). Twenty-four hours after transduction, cells were harvested and recultured with PMA and ionomycin to assess intracellular IL-2 (A) and GFP (B) expression. hCD2+ represents positively transduced cells, while hCD2– represents nontransduced cells in the same culture. The bar graphs represent the mean ± SD (n = 4) from three independent experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Past work has demonstrated that T cells with a genetic block in responsiveness to IL-2 generated proliferative responses in vitro to TCR and costimulatory signals, but did not develop into effector cells or undergo extensive subsequent expansion to other cytokines within the IL-2 family (5). To begin to ascertain the types of genes regulated by IL-2Rbeta signaling and to begin to evaluate the molecular basis accounting for the aborted T cell activation, DNA microarray analysis was used to identify IL-2-dependent targets in CD4+ and CD8+ T cells. For the most highly IL-2-dependent targets, the major genes identified were those important for immune function followed by transcriptional regulators. Many targets were shared by both CD4+ and CD8+ T cells, indicating that IL-2 controls similar processes for these two major T cell subsets.

We were especially intrigued by the finding that Blimp-1 was the most highly IL-2-dependent transcriptional regulator identified that was shared by both CD4+ and CD8+ T cells. Because we hypothesized that IL-2 controls an important checkpoint for the development of an effector response, we emphasized examining the function of Blimp-1 to begin to assess whether it performs a related role in T cells. Other studies that have performed microarray analysis for IL-2-responsive T cells did not identify Blimp-1 as an IL-2-induced gene (38, 39). These studies stimulated WT T cells through the TCR, and after this initial response and resting in culture medium, they were then triggered by IL-2, conditions that promoted extensive T cell growth. Our approach differed from these studies in that we have examined the early contribution of IL-2 as T cells progress during the response to stimulation through TCR and costimulatory molecules.

Our results indicate that Blimp-1 is expressed in activated T lymphocytes and regulates the expression of several molecules, IL-2, CD25, and granzyme B, important for effector T cells. Activated T cells primarily depend upon IL-2 for Blimp-1 expression because anti-CD3 and costimulatory signals did not induce Blimp-1 in WT T cells in the presence of anti-IL-2 and similarly activated Tg–/– T cells did not express Blimp-1. However, the addition of IL-4 or IL-12 to anti-CD3-activated Tg–/– T cells resulted in Blimp-1 expression, but at a relatively low level. The expression of Blimp-1 by B cells was also regulated by cytokines in that it was up-regulated by IL-2 and IL-5 in the BCL-1 B cell line (6). Although IL-2 is clearly the dominant cytokine that induces Blimp-1 expression by T cells in vitro, the activity of exogenous IL-4 and IL-12 in vitro suggests these two cytokines, IL-2, and perhaps other yet unidentified molecules may serve to induce Blimp-1 during a T cell immune response in vivo. In initial screening of other cytokines, IL-9, IL-21, IFN-{gamma}, IL-1, and IL-6 did not induced Blimp-1 when Tg–/– T cells were cocultured with anti-CD3, but very weak induction was noted by IL-7 (data not shown).

Recent detection of Blimp-1 in T cells primarily measured mRNA by RT-PCR or indirectly using GFP as a reporter that was "knocked-in" the Blimp-1 locus (14, 15). Those reports indicated that Blimp-1 expression is detected in activated T cells 3–6 days after stimulation with plate-bound anti-CD3 and anti-CD28 with IL-2 (14). Our study focused primarily on detection of Blimp-1 protein by Western blot analysis. We similarly found that Blimp-1 expression is most prominent in activated T cells, but our results differed from those experiments in that substantial Blimp-1 was detected by 48 h after activation with anti-CD3, and upon more extended culture in IL-2 the level of Blimp-1 declined. This difference might be due to the culture conditions used to activate the cells because we activated unfractionated spleen cells with soluble anti-CD3, where the presence of APC deliver more potent costimulatory signals (5), and the subsequent culture with IL-2 resulted in considerable cell expansion.

It may be noteworthy that extended culture of Ag-activated OT-I CD8 T cells in IL-2, but not IL-15, maintained expression of Blimp-1 even though both cytokines use the same receptor subunits, IL-2Rbeta and common {gamma} chain, for signal transduction. In these cultures, IL-2 favors CTL whereas IL-15 supports memory-like cells (19), suggesting that Blimp-1 expression is associated with effector T cells. As IL-2 production is suppressed by Blimp-1, the expression of granzyme B and CD25 selectively increased. Thus, the expression, molecular targets, and the activating cytokines of Blimp-1 correlate with T cells as they develop into effector cells. Although ectopic expression of Blimp-1 in anti-CD3-activated T cells increased the levels of granzyme B and CD25, we imagine that this result represents an indirect effect of Blimp-1 because this protein typically functions as a transcriptional repressor.

An important aspect of this study is that we have uncovered a novel autoregulatory loop whereby IL-2 inhibits its own production through induction of Blimp-1. Evidence for this conclusion includes the following: 1) the timing of Blimp-1 expression, which peaks rather late after initial T cell activation and coincides with decreased IL-2 production; 2) the blockade of Blimp-1 expression in WT T cells by anti-IL-2 with a corresponding increase in IL-2 production; 3) the suppression of IL-2 production in T cells after ectopic expression of Blimp-1; 4) the inherently higher production of IL-2 in Blimp-1-negative Tg–/– T cells compared with WT T cells; 5) the increased fraction of cells expressing the IL-2p-GFP reporter for T cells cultured in the presence of anti-IL-2; and 6) the lower fraction of cells expressing the transgenic IL-2p-GFP reporter after ectopic expression of Blimp-1. For these experiments, we have relied on measuring intracellular IL-2 after triggering anti-CD3-activated T cells with PMA and ionomycin. Similarly, past work demonstrated that IL-2 secretion by anti-CD3-activated Tg–/– T cells differed from WT T cells in that it was much higher and uncharacteristically increased between 24 and 48 h (5), indicative of a response that was not appropriately down-regulated, probably because Blimp-1 is not expressed by these activated Tg–/– T cells. Consistent with this notion, Blimp-1-deficient-CD4+ T cells produce substantially more IL-2 than WT CD4+ T cells after activation in vitro (14).

The observation that suppression of IL-2 production by Tg–/– T cells after coculture with IL-4 or IL-12, each of which induces Blimp-1 expression, suggests cytokines other than IL-2 might also induce this inhibitory pathway. Moreover, the blockade of IL-2 production in this situation, where Blimp-1 induction was modest, was much more dramatic than observed after simply ectopic expression of Blimp-1 in anti-CD3-activated Tg–/– T cells. This finding raises the possibility that other cytokine-induced molecules in the activated T cells may cooperate with Blimp-1 in the down-regulation of IL-2. It also remains to be determined whether induction of Blimp-1 is a direct consequence of IL-2, IL-4, or IL-12 signaling or secondary to activation mediated by another cytokine-dependent molecule. Because production of IL-2 is tightly regulated at the level of transcription (40, 41), one simple explanation for our findings is that Blimp-1 may act directly to repress the IL-2 gene. If so, elements within the 8.4-kb 5' upstream sequence of the mouse IL-2 gene are likely to be targeted by Blimp-1 because anti-IL-2, which inhibited Blimp-1 expression, substantially increased GFP expression in CD4+ and CD8+ T cells containing the IL-2p-GFP transgene or conversely increasing Blimp-1 levels by ectopic expression inhibited this reporter. However, we cannot yet rule out that the action of Blimp-1 is more complex and perhaps due to an indirect effect on another gene. A thorough analysis concerning the contribution of Blimp-1 on lL-2 transcription is required to establish the underlying molecular mechanism accounting for the blockade of IL-2 production.

Although our study has not explored the function of Blimp-1 in T cells in vivo, recent work indicated that T-lineage-specific-deletion of Blimp-1 in mice resulted in severe colitis by 4–6 wk of age (14). Because Blimp-1 was deleted in both developing thymocytes and mature peripheral T cells, it is not known whether the accompanying autoimmune inflammatory response in these mice is due to improper T cell development or impaired functional activity of peripheral T cells. The imbalanced homeostasis of Blimp-1-deficient T cells might also be secondary to developmental effects or autoimmunity because we found that ectopic expression of Blimp-1 did not obviously inhibit cytokine-dependent T cell proliferation in vitro. Alternatively, the apparent discrepancy concerning T cell proliferation between the role of Blimp-1 in vivo and our study may simply reflect the in vitro culture conditions because we examined recently activated T cells rather than cells undergoing homeostatic regulation. It also remains possible that the level of ectopic Blimp-1 that we achieved was insufficient to alter proliferative response. Our findings, however, support a direct role for Blimp-1 in regulation of the activation of peripheral T cells whereby its absence might directly contribute to autoimmunity. Because Blimp-1 is critically involved in for the down-regulation of IL-2 production, it is likely that unchecked IL-2 production acts to promote autoreactive effector T cells as one contributing factor to colitis and the inflammatory response detected in Blimp-1-deficient T cells in vivo. However, an imbalance between both autoreactive effector T cells and T regulatory cells might also lead to autoimmunity (3). In this regard, we believe it is noteworthy that the conditional knockout of Blimp-1 in T cells lead to impaired IL-10 production (14) that might attenuate the function of T regulatory cells. Thus, it is likely that both events contribute to the development of colitis in T-lineage-specific-Blimp-1-deficient mice.

Our results demonstrate that the function of Blimp-1 in T cells is not analogous to that in B cells in that Blimp-1 did not arrest T cells proliferation or down-regulate c-myc expression, which are two aspects readily found for Blimp-1-dependent differentiation of B cells into plasma cells (20, 36). Moreover, most direct targets repressed by Blimp-1 in B cells, such as CIITA (42), Pax-5 (43), and Spi-B (32), are B cell-specific and not present in T cells based on our DNA microarray analysis (data not shown). Thus, several of the cellular processes controlled by Blimp-1 in T cells may be distinct from those in B cells. Nevertheless, it seems likely that Blimp-1 function in T cells will overlap with some of its known activities in B cells. Recent work indicates that STAT5 is a critical regulator in B cells to promote B cell survival and germinal center reactions by inducing Bcl-6, which inhibits Blimp-1 expression (30). Our DNA microarray analysis revealed that the two targets genes for Blimp-1 in T cells, granzyme B and CD25, are both STAT5-dependent (24, 37, 44) and that Blimp-1 and Bcl-6 mRNA were reciprocally expressed in WT vs Tg–/– anti-CD3-activated T cells. Direct analysis of effector and memory-like T cells also showed that the expression of Blimp-1 decreased to nearly undetectable levels in memory-like T cells. By contrast, Bcl-6 is heightened in memory T cells (45). Therefore, the interrelationship between STAT5, Bcl-6, and Blimp-1 in T cells may prove to be a relevant starting point to further define the role of Blimp-1 in T cells and its possible relationship for commitment to effector or memory cells.


   Acknowledgments
 
We thank M. A. Yui and E. V. Rothenberg for providing the IL-2p-GFP transgenic mice; M. Lichetenheld and L. Boise for critically reading this manuscript; J. Hyun for help with some of the Western blot analyses; A. Yu, P. Koo, B. Boyter, and L. Zhu for technical assistance; and Sylvester Comprehensive Cancer Center for support of the Transgenic Mouse and FACS facilities.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI40114. Back

2 Address correspondence and reprint requests to Dr. Thomas R. Malek, Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, 1600 Northwest 10th Avenue, Miami, FL 33101. E-mail address: tmalek{at}med.miami.edu Back

3 Abbreviations used in this paper: WT, wild type; hCD2, human CD2. Back

Received for publication April 13, 2006. Accepted for publication September 27, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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