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The Journal of Immunology, 2003, 170: 2356-2366.
Copyright © 2003 by The American Association of Immunologists

Growth Factor Independence-1B Expression Leads to Defects in T Cell Activation, IL-7 Receptor {alpha} Expression, and T Cell Lineage Commitment1

Loretta L. Doan2,*,{dagger}, Mary Kate Kitay2,*, Qing Yu{ddagger}, Alfred Singer{ddagger}, Sabine Herblot, Trang Hoang, Susan E. Bear||, Herbert C. Morse, III§, Philip N. Tsichlis# and H. Leighton Grimes3,*,{dagger}

* Institute for Cellular Therapeutics and Department of Surgery, University of Louisville School of Medicine, Louisville, KY 40202; {dagger} Department of Biochemistry and Molecular Biology, {ddagger} Experimental Immunology Branch, National Cancer Institute, and § Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; Clinical Research Institute of Montreal, Quebec, Canada; || Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107; and # Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Although recent studies have characterized the effects of proximal TCR signaling on T cell differentiation, the downstream integration of these signals remains largely unknown. The growth factor independence-1 (GFI1) and GFI1B transcriptional repressors may regulate cytokine signaling pathways to affect lymphocyte growth and survival. In this study, we show that Gfi1 expression is induced upon induction of the T cell program. Gfi1B expression is low and dynamic during T cell development, but is terminated in mature thymocytes. Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional consequences of constitutive expression. GFI1 potentiates response to TCR stimulation and IL-2, whereas GFI1B-transgenic T cells are defective in T cell activation. Moreover, GFI1B-transgenic thymocytes display reduced expression of the late-activation marker IL-7R{alpha}, and a decrease in CD4-8+ single-positive T cells that can be mitigated by transgenic expression of BCL2 or GFI1. These data show that GFI1 and GFI1B are functionally unique, and implicate a role for GFI1 in the integration of activation and survival signals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Early thymocyte precursors are CD4-CD8- (double negative; DN)4 and are signaled to differentiate first into CD4+CD8+ (double positive; DP) thymocytes. Further differentiation of DP thymocytes into mature CD4+8- single-positive (CD4 SP) or CD4-8+ (CD8 SP) T cells is triggered by the engagement of TCRs on the immature TCRhighCD4+CD8+ thymocytes by self-peptide/MHC complex on thymic epithelial cells (1, 2). Negative selection eliminates immature DP thymocytes through clonal deletion of those T cells that have high affinity for self-peptide and thus are potentially autoreactive (3). Positive selection occurs when low-affinity TCR-ligand interactions trigger a signal for survival and results in termination of one or the other CD4 or CD8 coreceptor molecule. The choice of which coreceptor to extinguish is referred to as lineage commitment.

The growth factor independence-1 (GFI1) and GFI1B proteins are closely related nuclear oncoproteins that may regulate cytokine pathways. Gfi1 was originally identified as the gene up-regulated by insertion of Moloney murine leukemia virus in a thymic lymphoma that was selected for its ability to grow in the absence of the T cell cytokine IL-2 (4). Forced expression of GFI1 in the IL-2-dependent parental cell line potentiates the outgrowth of IL-2-independent cell lines, without inducing IL-2 (4, 5). Gfi1B was identified by low stringency hybridization screening with a cDNA probe encoding the zinc-finger region of Gfi1 (6). GFI1 and GFI1B are 97% homologous in the carboxyl-terminal 165 aa that code for six Cys-His zinc fingers. An amino-terminal 20-aa snail and Gfi1 (SNAG) domain, responsible for nuclear localization and transcriptional repressor function, is also highly conserved (5). In contrast, the 236 intervening amino acids between the GFI1 SNAG and zinc-finger domains bear no homology to the corresponding 145 aa of GFI1B. Both proteins bind to virtually identical DNA consensus sequences and function as transcriptional repressors in a SNAG-dependent manner (5, 6). GFI1 is mildly antiapoptotic and inhibits growth arrest of IL-2-dependent T cell lines under conditions of limiting IL-2 (5, 7), while GFI1B inhibits both IL-6-induced differentiation and growth arrest of M1 myelomonocytic cells (6). Mice deleted for GFI1 have altered inflammatory responses and differentiation in the myeloid lineage (8), while mouse embryos deleted for GFI1B die in utero due to a lack of definitive erythropoiesis (9).

Gfi1 and Gfi1B are differentially expressed in lymphoid compartments. Northern analysis reveals that Gfi1 is expressed in the bone marrow and thymus, with low-level expression in the spleen, whereas Gfi1B is expressed in the bone marrow and spleen, with low-level expression in the thymus (6). Both Gfi1 and Gfi1B show regulated expression during T cell development, but Gfi1B expression is terminated in mature thymocytes. Gfi1 message is not expressed in G0 splenic T cells, but is induced upon T cell activation (4, 10). Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional basis for differential expression of these factors. Transgenic expression of GFI1 potentiates T cell activation. In contrast, ectopic expression of GFI1B in T cells results in defective T cell activation, lower numbers of peripheral T cells, a reduction in IL-7R{alpha} expression, and a developmental block to CD8 SP T cells. The block to CD8 SP development is mitigated by forced expression of BCL2 or GFI1. These data indicate that GFI1 and GFI1B are not redundant for T cell activation function, and implicate integration of activation and survival signaling in CD8 lineage commitment.


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

The generation of the CD2-GFI1-transgenic mice has been described (10). The lck-Gfi1-transgenic mouse (line 3A) was generated by cloning the rat Gfi1 cDNA into the BamHI site of the TLC vector (11). This vector contains a 3.2-kb fragment of the mouse lck proximal promoter and a 2.2-kb fragment of the human growth hormone (GH) gene, which provides exons and introns for splicing and polyadenylation sequences. A 2.2-kb fragment of the 3' locus control region of the human CD2 gene is located downstream of the GH to obtain copy-number- and insertion-site-independent levels of expression. The GFI1B-transgenic mice (lines 5B and 5C) were constructed in an identical manner to the GFI1 transgenic except the construct contained the cDNA for mouse Gfi1B inserted into the BamHI site. The GFI1 or the GFI1B transgene (1–5 ng/µl) was microinjected into C57BL/6J (The Jackson Laboratory, Bar Harbor, ME) eggs according to standard methodology in the Laboratory of Immunopathology (National Institute of Allergy and Infectious Diseases, National Institutes of Health).

Recombination-activating gene (RAG)2-/-/HY mice (Taconic Farms, Germantown, NY), and Eµ-BCL2-25-transgenic mice (The Jackson Laboratory) (12) were purchased from commercial vendors. All mice were on a C57BL/6 J background, bred in the Baxter Barrier animal care facility at the University of Louisville School of Medicine, and housed under specific pathogen-free conditions. GFI1- and GFI1B-transgenic daughters who were heterozygous for the RAG mutation, as well as the HY TCR transgenes were then backcrossed with their RAG2-/-, HY TCR+ fathers. Colonies were expanded by intercrossing of littermates. BCL2/GFI1B and CD2-GFI1/GFI1B bitransgenics were generated in a similar manner. All animal work performed was reviewed and approved by the University of Louisville Institutional Animal Care and Use Committee.

Antibodies

Abs with the following specificities were used for cell stimulations: CD3{epsilon} (145.2C11) and CD28 (37.51). Abs with the following specificities were used for staining of thymocytes and splenocytes: CD4 (RM4-5 and GK1.5), IL-7R{alpha} (A7R34; eBiosciences, San Diego, CA), CD8{alpha} (53-6.7), CD3{epsilon} (145-2C11), TCR{alpha}{beta} (H57-597), TCR V{beta}8 (F32), CD24{alpha} (M1/69), CD69 (H1.2F3), and CD25 (PC61). Abs were purchased from BD PharMingen (San Diego, CA) unless otherwise noted. Intranuclear staining was performed using anti-GIF1B goat polyclonal IgG (sc-8559), anti-GFI1 goat polyclonal IgG (sc-8558), normal goat IgG (sc-2028) control, and secondary bovine anti-goat IgG-FITC (sc-2348), all from Santa Cruz Biotechnology (Santa Cruz, CA).

Preparation of cell suspensions

Thymuses from 4- to 6-wk-old mice were removed and disrupted between frosted ends of glass slides and washed twice with medium 199 (Life Technologies, Gaithersburg, MD). Cells were obtained from spleens by perfusion with 10 ml of medium 199. Both thymocyte and splenocyte cell debris was depleted by passage through Nytex nylon-mesh screens. Splenocytes were treated with ammonium chloride-potassium bicarbonate solution (150 mM NH4Cl and 10 mM KHCO3) to lyse RBCs. For experiments requiring isolation of T cells, splenic cell preps were depleted of other cell types by the use of T cell enrichment columns (R&D Systems, Minneapolis, MN). All cells were counted with a Coulter Counter model Z2 (Coulter, Miami, FL), and viability was assayed by trypan blue exclusion.

Flow cytometric analysis

Cell surface staining was performed by incubating 1 x 106 cells with mAbs at varying concentrations in FACS medium (HBSS with 0.1% BSA, 0.1% sodium azide, and 0.036% sodium bicarbonate) for 20 min on ice. Stained cells were washed twice with FACS medium and fixed in 1% formaldehyde (Polysciences, Warrington, PA). For intranuclear staining, cells were fixed in 2% formaldehyde in PBS and permeabilized and stained in PBS plus 5% FBS and 0.5% Triton X-100. Flow cytometry was performed on a FACSCalibur, FACSVantageSE, or FACStar flow cytometer using standard CellQuest acquisition. Data were analyzed using CellQuest (BD Biosciences, Mountain View, CA) and FlowJo (Tree Star, San Carlos, CA) software. The absolute cell numbers of gated cells per thymus or spleen were calculated by multiplying the percentage of each population with the total number of cells per thymus or spleen respectively.

Northern, Western, and RT-PCR analyses

RT-PCR analyses were performed as previously published (13, 14). The sequences of the Gfi1-specific primers were 5'-CACACCTTCATCCACACAGG-3' and 5'-GATGAGCTTTGCACACTGGA-3', and the probe was 5'-TACCGTGAGGATGTCTTCCC-3'. The sequences of the Gfi1B-specific primers were 5'-AGCACAGAGTCTCCCTTGGA-3' and 5'-CAAAGGTTTTGCCACAGACA-3', and the probe was 5'-ACCCCTCATGGGCTAGAAGT-3'. The Gfi1B pattern was confirmed with the primers 5'-GAGCAGCATACTCACGTCCA-3' and 5'-TTCATGTCCGACTTCTGGTG-3', and the probe was 5'-CAAAGCCTTCAAGCGTTCAT-3'.

Western blotting with Abs against GFI1 (sc-6357), GFI1B (sc-8559; Santa Cruz Biotechnology), GFI1 and GFI1B (sc-6357), p27 (554069; BD PharMingen), and IFN regulatory factor (IRF)1 Ab (sc-640) was performed as follows. Single-cell suspensions of primary thymocytes were lysed at a concentration of 10–20 x 106 cells/100 µl SDS lysis buffer (25 mM Tris (pH 7.5), 10% glycerol, and 2% SDS) supplemented with protease inhibitors complete (Roche, Basel, Switzerland) and 2 mM PMSF. Protein concentration was determined using the BCA protein assay reagent (Pierce, Rockford IL), and 75 µg of cellular extract was run on a 10% SDS-polyacrylamide gel, transferred to Immobilon-polyvinylidene fluoride (Millipore, Bedford, MA), and blocked for 1 h at room temperature in blocking buffer (5% milk, 20 mM Tris (pH 7.3), 6.85 mM NaCl, 0.1% (v/v) Tween 20, and 0.5 g/L MgCl2). Membranes were incubated overnight at 4°C in primary Ab diluted in 5% protease-free BSA (Fisher Biotech, Pittsburgh, PA), and then HRP-conjugated secondary Ab (Amersham, Piscataway, NJ) for 1 h at room temperature. Blots were developed using ECL reagents (Amersham). For Western analysis of sorted thymocytes and purified T cells, 1 x 106 cells were resuspended in 15 µl of lysis buffer (50 mM HEPES (pH 7.8), 450 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1% Nonidet P-40 (15), protease inhibitors complete (Roche), and 2 mM PMSF), then sonicated using a Misonix (Farmingdale, NY) sonic dismembrator with microprobe tip. Loading buffer (4x) was added, and the lysates were boiled. The entire contents of the lysate were loaded onto a denaturing SDS-polyacrylamide gel, and Western blotting was performed as described above.

Cell stimulation and proliferation

Single-cell suspensions of spleen cells in RPMI 1640 (Life Technologies) supplemented with 5% FBS, L-glutamine, penicillin, streptomycin, gentamicin, 2% HEPES (all from Life Technologies) and 0.1% 2-ME (Sigma-Aldrich, St. Louis, MO) were plated in 96-well round-bottom plates (Corning, Corning, NY) at a density of 1 x 105/well in 100 µl. Stimuli were added as indicated at a range of concentrations to assess dose dependency. The stimuli were low-endotoxin, no-azide anti-CD3 (145.2C11) and anti-CD28 (37.51) (both Abs from BD PharMingen), and recombinant human IL-2 (Chiron, Emeryville, CA). Cells were cultured for 48 or 72 h, then pulsed with [3H]thymidine (1 µCi; Amersham), and harvested 18 h later using a TOMTEC-Harvester 96/Model Mach II (Wallac, Akron, OH). Proliferation was determined by measuring radioactivity (Wallac 1205-SP2 betaplate counter).

Coreceptor reversal

Purified DP thymocytes (>96%) were obtained by panning with IgM anti-CD8 (83-12-5)-coated plates. DP thymocytes (5 x 106/ml) were first placed into signaling cultures and stimulated for 12–18 h with a combination of phorbal-12-myristate-13-acetate (0.6 ng/ml) and ionomycin (0.6 µg/ml) (P+I; Calbiochem, La Jolla, CA) (16). At the conclusion of signaling culture, cells were harvested, washed, and placed into nonstimulatory recovery cultures for an additional 12–16 h. Cells were stained for CD4 and CD8 expression, and CD4+CD8- cells were obtained by electronic sorting of the stained cells. The purified CD4+8- cells were further cultured in postrecovery cultures in the presence or absence of 6 ng/ml recombinant mouse IL-7 (Calbiochem) overnight, after which they were harvested and stained for CD4 and CD8 expression. All cultures were performed at 37°C in 5% CO2 humidified air atmosphere in RPMI 1640 supplemented with 5 x 10-5 M 2-ME and 10% FCS that had been depleted of endogenous steroids by pretreatment with 0.5% Norit A charcoal and 0.05% dextran for 30 min at 56°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gfi1 and Gfi1B are regulated during T lymphopoiesis

We examined the steady-state mRNA levels of Gfi1 and Gfi1B during T lymphopoiesis (Fig. 1A). Thymocyte populations were sorted, RNA was extracted, and RT-PCR was performed to detect ribosomal S16 expression (13, 14). The products of the reaction were analyzed by Southern blot with a radiolabeled S16-specific oligonucleotide probe. The signal was quantified by phosphor imager, and the samples were normalized to obtain equivalent S16 signal from each template. Subsequent analysis of Gfi1 expression in the S16-normalized cDNA templates revealed low-level signal in CD4-CD8-CD44+CD25- cells (DN1) and 10-fold greater levels in CD4-CD8-CD44+CD25+ (DN2) thymocytes. The transition between DN1 and DN2 corresponds to T lymphocyte lineage commitment. Signal intensity from the Gfi1 RT-PCR product gradually increases to double the DN2 levels at the CD4+CD8+TCRhigh stage, which contains cells that have been recently positively selected and are about to undergo lineage commitment. Gfi1 RT-PCR product levels then decrease 10-fold in CD4 and CD8 SP thymocytes. In striking contrast, the signal levels of probed and quantified Gfi1B RT-PCR product were low, but increased at stages corresponding to TCR {beta}-chain selection (DN3), and positive selection (DP TCRhigh). In the thymus, Gfi1 expression is gradually induced upon induction of the T cell differentiation program, whereas low-level Gfi1B expression correlates with positive selection events.



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  		FIGURE 1. Expression of endogenous GFI1 and GFI1B. A, Blotted and probed RT-PCR analyses of Gfi1 and Gfi1B in reverse-transcribed thymocyte-subset cDNA templates. RT-PCR was first performed for S16, and the products of the reaction were blotted, probed, and quantified. Next, the cDNA templates were normalized to obtain equivalent signal from the blotted and probed S16 products. Finally, primer/probe pairs for Gfi1 and Gfi1B were applied to the normalized cDNA templates to determine the Gfi1 and Gfi1B RT-PCR product levels in thymocyte subsets relative to S16. B, Western blot analysis of sorted thymocyte subsets. A single-thymocyte suspension from 4-wk-old WT mice was stained and sorted as indicated. Sorted cells (1 x 106) of each subpopulation were lysed, and the total lysate was probed with anti-GFI1 (top panel) or anti-p27 (bottom panel) as a loading control. In contrast to the expression pattern of Gfi1 message, GFI1 protein levels are moderate in DP thymocytes and intermediate CD4+CD8low cells, but increase in mature CD4 and CD8 SP cells. C, Flow cytometric analysis of GFI1 expression in thymocyte subsets. Permeabilization of surface-stained, formaldehyde-fixed thymocytes and subsequent incubation with Abs to GFI1, followed by a secondary Ab conjugated to FITC, yielded an expression profile for GFI1 similar to that observed by standard Western blotting techniques. Specifically, the level of GFI1 protein increases in SP thymocytes. The pattern of GFI1 expression in DN thymocytes has been published (15 ). D, Flow cytometric analysis of GFI1B expression in thymocyte subsets. Intranuclear staining of surface-stained, formaldehyde-fixed thymocytes revealed expression of GFI1B in the relatively rare CD4-CD8-CD44- populations of cells. The average change in MFI is indicated below each graph, and is defined as follows: {Delta}MFI = (MFIGFI1B/MFIIgG) x 100 (±SEM). GFI1B-transgenic CD4-CD8-CD44- thymocytes have a higher MFI in comparison to WT cells, whereas RAG2-/- thymocytes, which lack the capacity for TCR signaling (42 ), do not express detectable levels of GFI1B. Although available reagents for detection of GFI1B are not ideal, and the protein is likely expressed at very low levels in normal cells, the observed shift is significant and reproducible.

 
GFI1 and GFI1B are expressed in normal thymocytes

To examine the level of GFI1 in thymocyte subsets, we performed Western analysis on one million sorted DP, CD8 SP, CD4 SP, and CD4+CD8low thymocytes (Fig. 1B). The level of GFI1 does not differ between the bulk of DP thymocytes and those poised to make a lineage commitment step (CD4+CD8low); however, the level of GFI1 is dramatically higher in SP thymocytes. To confirm these data, we examined thymocyte expression of GFI1 by intranuclear staining and flow cytometry. Like the Western blot data, flow cytometric analyses revealed higher levels of GFI1 protein in SP thymocytes (Fig. 1C). The up-regulation of Gfi1 message in DP thymocytes, with subsequent increase in GFI1 protein in SP cells, suggests that GFI1 may play a role in the transition between these two developmental stages.

RT-PCR analyses revealed restricted expression of Gfi1B in relatively rare thymocyte subsets (Fig. 1A). Not surprisingly, Western analysis and intranuclear stains for GFI1B failed to reveal GFI1B expression in bulk thymocytes (data not shown). Therefore, we focused on a flow-cytometric analysis of the relatively rare CD44- DN3 and DN4 thymocytes that appear to express the highest levels of Gfi1B message (Fig. 1A). We first examined the DN3 and DN4 cells from GFI1B-transgenic mice (detailed below, Fig. 1D). A comparative 66% shift in mean fluorescence intensity (MFI) ({Delta}MFI = MFIGFI1B/MFIIgG x 100) between the control IgG antisera and GFI1B-reactive antisera indicates that GFI1B protein is present (Fig. 1D). In a similar manner, analysis of the DN3 and DN4 cells from nontransgenic littermates revealed a 41% shift in MFI between control and GFI1B-specific antisera stains (Fig. 1D).

The RT-PCR data (Fig. 1A) indicate that Gfi1B expression correlates with positive selection events. To explore this correlation, we examined thymocytes from RAG2-/- mice, which are arrested at the DN3 stage because they lack the pre-TCR selection signal that follows RAG-mediated rearrangement of the TCR {beta}-chain. Flow-cytometric analyses reveal no difference in MFI between control and GFI1B-reactive antisera in RAG2-/- thymocytes (Fig. 1D). These data indicate that signals from the pre-TCR may be required for GFI1B expression.

Transgenic expression of GFI1 and GFI1B

GFI1 and GFI1B bind the same DNA sequence and repress transcription in a manner dependent on the SNAG repressor domain (5, 6), suggesting the possibility that these factors are redundant. High-level transgenic expression of GFI1 in the thymus results in a block to T cell development at a stage corresponding to selection of cells after successful formation of the TCR {beta}-chain (15). Given the thymic phenotype of GFI1 overexpression, we constructed transgenic mice expressing GFI1 and GFI1B in developing and mature T cells (Fig. 2A). Transgene-specific Northern analysis revealed that GFI1-transgenic founders had moderate expression while GFI1B-transgenic founders had higher levels of expression; representative lines are shown (Fig. 2B). Western analysis of total thymocytes and of column-enriched splenic T cells indicates that the transgenic GFI1B protein is expressed in both the thymus and the periphery (Fig. 2C). Finally, flow cytometric analysis of the GFI1B-transgenic thymocyte populations revealed transgenic GFI1B expression in DN, DP, CD8 SP, and CD4 SP thymocytes (Fig. 2D).



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  		FIGURE 2. Expression of transgenic GFI1 and GFI1B. A, Schematic representation of the GFI1- and GFI1B-transgene constructs. The rat cDNA of Gfi1 (4 ) and mouse cDNA of Gfi1B (6 ) were placed under the control of the lck proximal promoter and 2.2-kb human CD2 enhancer, with human growth hormone (GH) gene (11 ). B, Northern blot analysis of total thymus or spleen RNA from representative GFI1- (line 3A) and GFI1B- (line 5B) transgenic lines hybridized with transgene-specific probes. Total RNA from transgenic animals revealed moderate levels of transgenic Gfi1 in both thymus and spleen, while transgenic Gfi1B is expressed at higher levels. C, Western blot analysis of transgenic GFI1B in thymic or column-purified splenic T cell whole-cell protein extracts. Cell extracts from two control (WT) and two GFI1B-transgenic mice were analyzed by Western blot with an Ab specific for the last 20 aa of GFI1 (cross-reactive to GFI1B). Densitometric analysis revealed that transgenic GFI1B is expressed at 5-fold higher levels than is endogenous GFI1 in nontransgenic littermate thymocytes. Lysates probed with antiserum against IRF-1 act as a control for loading. D, Flow cytometric analysis of GFI1B expression in GFI1B-transgenic thymocyte subsets. Transgenic expression of GFI1B is observed in all thymocyte subsets, although the level of expression is somewhat lower in CD8 SP cells.

 
To explore the subcellular localization of transgenic GFI1 and GFI1B, the cytoplasm of permeabilized and stained thymocytes was removed using Nonidet P-40. Stripping the cytoplasm resulted in a loss of signal for the mitochondria-localized BCL2, but did not alter the {Delta}MFI of thymocytes stained for GFI1 or GFI1B (data not shown), indicating that the targets of the respective antisera are nuclear. Given that these antisera are specific for GFI1 and GFI1B in Western analysis, our flow cytometric data indicate that transgenic GFI1 and GFI1B are nuclear in T cells.

Transgenic expression of GFI1 enhances T cell response to CD3 cross-linking and IL-2

GFI1 was previously shown to confer IL-2 independence to rat T cell lymphomas (4, 7). IL-2 is a critical T cell cytokine during activation, and though peripheral lymphocytes do not express detectable levels of Gfi1, activation signals induce Gfi1 within 30 min (10). Moreover, transgenic expression of GFI1 was previously shown to mildly increase [3H]thymidine uptake given a fixed amount of CD3-cross-linking Ab (10). To further examine the effect of GFI1 on T cell activation potential, spleen cells from 4- to 6-wk-old mice were stimulated by titration of a CD3-cross-linking Ab. T cells from mice expressing GFI1 from either the Lck promoter-driven transgene (Fig. 2A) or a previously published CD2 promoter-driven transgene (10) proliferated at a higher rate than T cells from control mice as evidenced by enhanced [3H]thymidine uptake (Fig. 3A) and by increased numbers of cells in each cellular division as evidenced by CSFE staining (data not shown). To dissect the response of GFI1-transgenic splenic T cells to stimulation, we limited the amount of CD3 Ab, and titrated IL-2. Again, we found that cells from GFI1-transgenic mice proliferated more vigorously in response to stimulation than cells from nontransgenic littermates (Fig. 3B). A flow cytometric analysis of splenocytes from control and GFI1-transgenic mice showed equivalent absolute cell numbers of total splenocytes and T cell subsets as delineated by the markers CD4, CD8, TCR{beta}, and CD3{epsilon} (Fig. 3C), or CD62 ligand and CD44 (data not shown). Therefore, GFI1 potentiates the response to CD3 and IL-2 stimulation.



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  		FIGURE 3. GFI1 potentiates, whereas GFI1B inhibits, T cell activation. A and B, Spleen cells from 4- to 6-wk-old GFI1-transgenic mice (line 3A or a CD2 promoter-driven GFI1-transgenic (10 )) and nontransgenic littermate control mice were isolated and stimulated with either a dose titration of plate-bound Ab to CD3{epsilon} (A) or a low level of anti-CD3{epsilon} (0.06 µg/ml) and increasing amounts of human rIL-2 (B) and cultured for 48 h. [3H]Thymidine (1 µCi) was added, and plates were incubated for 18 h. Proliferation was measured as cpm of [3H]thymidine incorporation. Error bars indicate SEM. C, Spleen cells isolated from 4- to 6-wk-old GFI1-transgenic mice, as well as littermate controls, were stained with Abs to CD4 and CD8, and then analyzed by flow cytometry. A representative FACS profile is shown. No difference was observed in total number of splenocytes or in splenic T cell numbers between either GFI1 transgenics of either line and littermate controls. D, Spleen cells from 4- to 6-wk-old GFI1B-transgenic mice (lines 5B and 5C) were activated as in A for either 48 h (left panel) or 72 h (right panel). [3H]Thymidine (1 µCi) was added, and plates were incubated for 18 h. Proliferation was measured as cpm of [3H]thymidine incorporation. E, Spleen cells isolated from 4- to 6-wk-old GFI1B-transgenic line 5B, as well as littermate controls, were stained with Abs to CD8 and CD4 and analyzed by flow cytometry. A representative FACS profile is shown. Absolute spleen cell numbers from all mice examined were determined and are expressed in the table below as cell numbers x106 ± SEM in the table below.

 
GFI1B-transgenic mice display peripheral T lymphopenia and a profound defect in activation after CD3 cross-linking

We next looked at the response of GFI1B-transgenic splenic T cells to stimulation with anti-CD3{epsilon} and found that they neither died (data not shown) nor proliferated substantially. Spleen cells from 4- to 6-wk-old mice from two GFI1B-transgenic lines (5B or 5C; Fig. 3D) were stimulated by titration of a CD3-cross-linking Ab. T cells from GFI1B-transgenic mice proliferated at a substantially lower rate than T cells from control mice as evidenced by [3H]thymidine uptake, even when the activated cells were given an additional 24-h incubation (Fig. 3D; 48 vs 72 h).

An unanticipated explanation for this observation came from the finding that spleen cells from GFI1B-transgenic mice show significant reduction in the numbers of CD4 and CD8 T cells that could respond to CD3 stimulation (Fig. 3E). Mature CD4 cells were reduced to 32% of wild-type (WT) levels, while mature CD8 cells were reduced to 46% of WT levels (Fig. 3E). However, neither the TCR-expression level on splenic T cells (data not shown) nor the total number of splenocytes (Fig. 3E) was significantly reduced in GFI1B-transgenic mice. Because both the GFI1B-transgenic lines gave equivalent data, we focused on the 5B line for further studies.

The unresponsiveness of GFI1B lymphocyte populations to TCR-mediated activation signals could be due to either T lymphopenia or a defect in signaling. To determine the mechanism, T cells were purified by negative selection, normalized to CD3{epsilon}+ T cell numbers, and stimulated simultaneously with both anti-CD3{epsilon} and anti-CD28 in a coreceptor activation assay. Costimulated GFI1B-transgenic cells showed a marked inability to proliferate as compared with cells from nontransgenic littermates (Fig. 4A). In addition, GFI1B transgenics demonstrated decreased proportions of cells expressing activation markers CD69 and CD25 (IL-2R {alpha}-chain) as compared with WT cells (Fig. 4B), as well as a decrease in fluorescence intensity of these markers on positive cells (data not shown). Therefore, GFI1B-transgenic T cells are profoundly impaired in response to activation signals because of an intrinsic signaling defect, and not because of the overall reduction in CD3{epsilon}+ T cell numbers. These data are diametrically opposed to our findings for GFI1-transgenic mice.



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  		FIGURE 4. The GFI1B-induced inhibition of activation is cell autonomous and includes defective up-regulation of early activation markers. A, Four-wk-old GFI1B-transgenic and control splenic T lymphocytes were purified by negative selection, normalized for numbers of CD3+ T cells, and then costimulated with Abs to CD3{epsilon} and CD28. Proliferation was measured by [3H]thymidine incorporation, and the stimulation index was calculated. A representative of three experiments is shown. B, Splenocytes from GFI1B-transgenic ({circ} and {diamond}, black background) and littermate controls (• and {diamondsuit}, white background) were stimulated with Abs to CD3 for 24 h then stained with Abs to CD4, CD8, CD25 ({circ} and •), and CD69 ({diamond} and {diamondsuit}), and analyzed by flow cytometry. CD4+ or CD8+ events were gated, and the percentages of CD25+ or CD69+ cells are depicted. Fewer GFI1B-transgenic splenocytes exhibit induction of either CD25 or CD69. Bars = mean.

 
Transgenic GFI1B expression alters thymocyte lineage commitment and maturation

To determine the cause of peripheral T lymphopenia, we examined the thymus. Thymocytes were stained with Abs against CD4, CD8, and TCR{beta} and analyzed by flow cytometry. In GFI1B-transgenic mice, the number of CD4 SP cells was considerably enhanced while there was a severe reduction in the development of CD8 SP T cells (Fig. 5A). The CD8 SP compartment contains both mature and immature intermediate single-positive (ISP) cells (CD8 ISP). CD8 ISP cells, in contrast to CD8 SP cells, do not have high-level TCR expression. Therefore, the analysis was repeated through a TCRint-high gate, which would include CD4 SP and CD8 SP thymocytes and their immediate precursors (17). The ratio of CD4+ to CD8+ cells was increased in the TCRint-high population from a normal ratio of 7:1 to a ratio of 32:1 in the GFI1B-transgenic mice (Fig. 5B). Therefore, few mature CD8 SP cells are generated in the GFI1B-transgenic mice. Moreover, the lower numbers of CD8 SP cells provide a potential explanation for peripheral CD8 lymphopenia.



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  		FIGURE 5. Transgenic expression of GFI1B in the thymus results in enhanced numbers of mature CD4+ thymocytes and decreased CD8+ populations. A and B, Thymocytes from GFI1B-transgenic and control littermates were stained with Abs to TCR{beta}, CD4, and CD8, and analyzed by flow cytometry. The average number of total thymocytes x 106 ± SEM is expressed above the plots, with percentage of cells in each gate in A. In B, cells are gated on TCRint-high events, and the CD4 vs CD8 FACS profiles are depicted. Thymocyte subsets are expressed as absolute cell numbers x 106 per thymus ± SEM. C, Thymocytes from 4-wk-old GFI1B-transgenic and control littermates were stained with Abs to CD24{alpha}, CD4, and CD8, and analyzed by flow cytometry. FACS profiles depicted are gated on CD4+ events. Absolute thymocyte cell numbers from all mice examined were determined and are expressed as cell numbers x106 ± SEM.

 
We next examined the CD4 SP population. As CD4 SP thymocytes mature, they down-regulate CD24/heat-stable Ag. Therefore, the most mature CD4 SP thymocytes express low levels of CD24 while less mature CD4 SP thymocytes express higher levels of this marker (17, 18, 19). We found that the number of immature CD4 SP thymocytes (CD24high) was relatively normal in GFI1B-transgenic mice; however, the number of mature CD4 SP thymocytes was dramatically increased by 5-fold (Fig. 5C). These data are supported by the surface expression of TCR{beta}, Qa-2, and CD62 ligand (data not shown), which also indicate that GFI1B-transgenic mice have greater numbers of phenotypically mature CD4 SP thymocytes. We have also examined the effects of the GFI1B transgene on a class II MHC-null background to find that no CD4 SP cells were generated (data not shown). These data exclude the possibility that the increased CD4 SP cells are due to redirection of MHC class I-restricted CD8 SP development into the CD4 lineage. Altered signaling during activation in GFI1B-transgenic thymocytes may lead to the accumulation of cells that should not normally be selected (defective negative selection) or the accumulation of cells unable to mature and egress.

The development of CD8 SP T cells in GFI1B-transgenic mice is not rescued by expression of the HY class I-restricted transgene

It is unlikely that GFI1B represses CD8 or MHC class I expression because thymocytes from GFI1B-transgenic mice reveal normal surface expression of CD8{alpha} and CD8{beta} in the DP fraction, and are not class I deficient (data not shown). To determine whether the GFI1B block to CD8 development involved an alteration in repertoire selection, GFI1B-transgenic mice were mated to RAG2-/-/HY mice, and resulting progeny were backcrossed to generate GFI1B/RAG2-/-/HY mice. The HY transgene encodes a class I-restricted TCR that selects large numbers of V{beta}8+ thymocytes into the CD8+ T cell lineage in female mice (20). Because the Rag2 gene product is necessary for TCR rearrangement, all RAG2-/-/HY/GFI1B-transgenic thymocytes express only the HY TCR as evidenced by V{beta}8 staining (data not shown). Positive selection of CD8+ T cells by the HY TCR was severely reduced in the RAG2-/-/HY/GFI1B female mice as compared with controls (Fig. 6). Therefore, the critical defect in GFI1B-transgenic mice is not simply an inability to form a class I-restricted TCR.



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  		FIGURE 6. Expression of GFI1B inhibits the development of female HY-transgenic CD8-SP T cells. Thymocytes from 4-wk-old female RAG2-/-/HY and RAG2-/-/HY/GFI1B mice were examined for the expression of CD4, CD8, and TCR V{beta}8 (used in the HY transgene) by flow cytometry. Representative FACS plots are shown with the total number of RAG2-/-/HY and RAG2-/-/HY/GFI1B thymocytes above the plots. Thymocyte subsets are expressed as absolute cell numbers x 106 per thymus ± SEM.

 
GFI1B expression overcomes a block to DP development imposed by autoreactive TCR signaling

To determine whether GFI1B-transgenic thymocytes display defective TCR signaling and/or activation in vivo, we next examined the male RAG2-/-/HY/GFI1B mouse thymus. The HY TCR recognizes the male HY Ag when presented by H-2Db. Male HY-transgenic thymocytes are blocked at the DN stage by autoreactive HY TCR signaling that mimics negative selection (21). Mutant mice with defective intracellular signaling overcome this block and accumulate V{beta}8+ DP cells (22, 23, 24). GFI1B-transgenic peripheral T cells are defective in T cell activation (Figs. 3D and 4A). In agreement with these data, GFI1B-transgenic thymocytes overcome the block to development imposed by the autoreactive HY transgene, as shown by a modest 4-fold accumulation of CD8+TCR{beta}+ cells (11.1 vs 47.9%) and a doubling of total thymus cellularity (Fig. 7, A and B). Therefore, our in vitro (Figs. 3 and 4) and in vivo (Fig. 7) data support a model in which ectopic expression of GFI1B leads to defective T cell activation.



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  		FIGURE 7. Expression of GFI1B overcomes a block to development in male HY-transgenic T cells. Representative FACS plots showing expression of CD4 and CD8 (A) or CD8 and TCR V{beta}8 (B) in male HY-transgenic mice. Male RAG2-/-/HY/GFI1B mice show a 4-fold increase in the number of CD4+CD8+ and TCR V{beta}8+ thymocytes when compared with RAG2-/- HY littermates. The total numbers of RAG2-/-/HY and RAG2-/-/HY/GFI1B thymocytes are shown above the plots.

 
GFI1B impairs CD8 SP development in vitro and decreases IL-7R{alpha} expression

We have previously described an in vitro model system in which DP thymocytes can be signaled to differentiate into CD8 SP T cells (16). In this experimental system, signaled DP thymocytes initially terminate CD8 transcription, differentiate into CD4+8- intermediate thymocytes, and up-regulate surface expression of IL-7R. In the presence of IL-7, CD4+8- intermediate thymocytes terminate CD4 transcription and reinitiate CD8 transcription (events referred to as coreceptor reversal) and ultimately differentiate into CD8 SP T cells (16). Consequently, we assessed the ability of signaled DP thymocytes from WT and GFI1B-transgenic mice to differentiate in vitro into CD8 SP T cells (Fig. 8A). We isolated WT and GFI1B-transgenic DP thymocytes (Fig. 8A, D0) and stimulated them with P+I as previously described (16). Signaled DP thymocytes from both WT and GFI1B-transgenic mice were induced to differentiate into CD4+8- intermediate cells (Fig. 8A, D2). Notably, in vitro-generated intermediate CD4+8- cells from GFI1B-transgenic mice expressed lower surface levels of IL-7R{alpha} compared with cells from WT mice (Fig. 8A, D2). We then added IL-7 to both populations of in vitro-generated intermediate CD4+8- thymocytes, and, after 24 h, pronase stripped the cells to remove pre-existing CD4/CD8 surface proteins so that we could determine the CD4/CD8 proteins that the cells were actively synthesizing. Addition of IL-7 (Fig. 8A, D3) followed by pronase stripping and re-expression culture revealed that 69.3% of WT cells had undergone coreceptor reversal and differentiated into CD8 SP T cells. In contrast, only 21.0% of GFI1B-transgenic cells had undergone coreceptor reversal to become CD8 SP T cells (Fig. 7A, D4). Thus, GFI1B-transgenic thymocytes at the CD4+8- intermediate stage of development are quantitatively deficient in their ability to undergo coreceptor reversal in response to IL-7 and are impaired in their ability to undergo in vitro differentiation into CD8 SP T cells.



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  		FIGURE 8. GFI1B impairs coreceptor reversal. A, D0, Purified CD4+CD8+ cells were obtained from pooled thymocytes of four 4-wk-old GFI1B-transgenic or four control littermates by panning with anti-CD8 Ab, and subsequently stimulated with P+I for 16 h at 37°C. Stimulated cells were stripped of their surface coreceptors with pronase protease, cultured in complete medium overnight, and stained for CD4 and CD8 expression, and purified CD4+8- cells were obtained by cell sorting. D2, Control (dashed line) and GFI1B-transgenic (solid line) cells were analyzed by flow cytometry for IL-7R{alpha} expression. Purified CD4+8- cells (2 x 106) were cultured overnight in the presence of IL-7. Flow analysis of samples of each culture indicated CD4 and CD8 surface levels before (D3) and after pronase protease treatment and culture in complete medium overnight (D4). B, Four-week-old GFI1B-transgenic (solid line) and normal littermate controls (dashed line) were stained for CD4, CD8, and IL-7R{alpha}, and analyzed by flow cytometry. Representative histograms comparing the IL-7R{alpha} expression in thymocyte subpopulations are shown.

 
To determine whether the lower in vitro expression pattern of IL-7R{alpha} on GFI1B-transgenic thymocytes was a possible explanation for the defective CD8 SP development in vivo, we next examined the expression of IL-7R{alpha} in unmanipulated thymocytes. Cells were stained for CD4, CD8, and IL-7R{alpha}, then gated on thymocyte subpopulations and analyzed for IL-7R{alpha} expression (Fig. 8B). The level of IL-7R{alpha} on DN thymocytes was not altered by the presence of the GFI1B transgene. However, in both CD4 SP and CD8 SP thymocytes, in which IL-7R{alpha} expression is regulated by TCR signaling, GFI1B-transgenic mice had lower levels of IL-7R{alpha} than did WT littermate controls (Fig. 8B). Thus, GFI1B-transgenic thymocytes are defective in both T cell activation and the expression of activation-induced genes such as CD25, CD69, and IL-7R{alpha}. The lower level of IL-7R{alpha} on GFI1B-transgenic thymocytes provides a potential explanation for altered CD8 SP development in the GFI1B-transgenic mice. Moreover, examination of peripheral T cell subsets revealed lower levels of IL-7R{alpha} (data not shown). Because IL-7 has been shown to regulate the survival of naive CD4 T cells (25, 26, 27, 28), the lack of IL-7R{alpha} expression is a possible explanation for peripheral CD4 T cell lymphopenia.

Bitransgenic BCL2/GFI1B mice generate CD8 SP thymocytes.

GFI1B-transgenic thymocytes express lower levels of IL-7R{alpha} (Fig. 8). IL-7 maintains the expression of endogenous BCL2 in T-lineage cells (29). BCL2 does not support thymic positive selection in the absence of MHC (30, 31, 32); however, transgenic expression of BCL2 can substitute for survival signals induced by cytokines such as IL-7 (32). GFI1B-transgenic mice were mated to Eµ-BCL2–25-transgenic mice, in which the human BCL2 transgene is expressed mainly in T-lineage cells (12). As expected by the presence of the BCL2 transgene, total thymocyte cellularity was increased in bitransgenic mice (12); however, we found that BCL2/GFI1B bitransgenics show increased numbers of TCRint-high CD8 SP thymocytes and a normal ratio of CD4 SP to CD8 SP thymocytes (Fig. 9). Although BCL2 may have pleiotropic effects on T cell development (31), the ability of BCL2 to rescue CD8 SP development in GFI1B-transgenic mice is consistent with the role of BCL2 as a downstream target of IL-7.



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  		FIGURE 9. Transgenic BCL2 restores CD8 SP thymocytes in GFI1B-transgenic mice. Thymocytes from GFI1B and BCL2/GFI1B-bitransgenic littermates were stained with Abs to TCR{beta}, CD4, and CD8, and analyzed by flow cytometry. Cells are gated on TCRint-high events, and representative CD4 vs CD8 FACS profiles are depicted. A scatter plot of CD8 SP cells in BCL2-transgenic, GFI1B-transgenic, and BCL2/GFI1B-bitransgenic mice reveals the increase in the CD8 SP population in the bitransgenic animals (in comparison to GFI1B-transgenic mice). Each diamond represents the percentage of TCRint-high CD8 SP cells per thymus of an individual mouse (five to seven mice per group). The black bar is the mean value for each group.

 
Bitransgenic GFI1/GFI1B mice generate CD8 SP thymocytes

We next determined whether GFI1 could alter the defects engendered by GFI1B expression. GFI1 enhances T cell activation, whereas GFI1B impairs this process (Fig. 3). Because GFI1 and GFI1B bind to the same DNA sequence, it is possible that some of the defects in GFI1B-transgenic thymocytes are the result of an imbalance between DNA-bound GFI1B vs GFI1. In fact, 6-wk-old GFI1/GFI1B-bitransgenic mice generate twice as many TCRint-high CD8 SP thymocytes than do littermate GFI1B transgenics (Fig. 10, p = 0.0003). The lower level of expression of the GFI1 transgene in comparison to the GFI1B transgene may explain the modest ability of GFI1 transgene to compete with transgenic GFI1B. Nevertheless, GFI1 expression increases the number of GFI1B-transgenic CD8 SP cells.



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  		FIGURE 10. Transgenic GFI1 increases the generation of CD8 SP thymocytes in GFI1B-transgenic mice. Thymocytes from 6-wk-old GFI1B and GFI1/GFI1B-bitransgenic littermates were stained with Abs to TCR{beta}, CD4, and CD8, and analyzed by flow cytometry. Cells are gated on TCRint-high events, and representative CD4 vs CD8 FACS profiles are depicted. A scatter plot of CD8 SP cells in GFI1-transgenic, GFI1B-transgenic, and GFI1/GFI1B-bitransgenic mice shows the partial restoration of CD8 SP thymocytes observed in bitransgenic animals (in comparison to GFI1B-transgenic mice). Each diamond represents the percentage of TCRint-high CD8 SP cells per thymus of an individual mouse (four to six mice per group). The black bar is the mean value for each group. The increase is statistically significant with a p value of 0.0003 as determined using two-tailed Student’s t test.

 
Transgenic expression of GFI1 does not alter other GFI1B-induced defects. The GFI1B-induced abnormal generation of phenotypically mature CD4 SP thymocytes remains in the GFI1/GFI1B-bitransgenic thymus (Fig. 10). Moreover, peripheral T lymphopenia in GFI1B transgenics is not altered by BCL2 or GFI1. It is known that BCL2 transgene-induced elevation in thymocyte numbers does not alter peripheral T cell numbers (29). Likewise, we find that neither BCL2 nor GFI1 increases the number of peripheral CD8 SP T cells in the GFI1B-transgenic spleen. GFI1, like BCL2, appears to ameliorate only the GFI1B effect on the production of CD8 SP cells. The inability of either the BCL2 or GFI1 transgene to cancel the GFI1B-induced alteration in the expression of IL-7R{alpha} (data not shown) indicate that both BCL2 and GFI1 must act to increase CD8 SP generation by a mechanism that is independent of the induction of IL-7 signaling. However, it is formally possible that GFI1, like BCL2, acts downstream of IL-7 signaling and that transgenic expression of either protein mimics the effects of IL-7.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The phenotypes observed in GFI1- and GFI1B-transgenic mice can be understood in the context of the normal expression pattern of either factor. GFI1 is induced at the transition between DP and SP cells. GFI1 may thus play a role in the activation-induced developmental steps between DP and SP thymocytes. GFI1B is induced at steps in thymocyte development in which thymocytes are activated. In fact, RAG2-/- cells that lack the ability to activate do not express GFI1B (Fig. 1). GFI1 and GFI1B have opposite effects on T cell activation. Peripheral GFI1-transgenic T cells activate better than do those from WT littermates, whereas GFI1B-transgenic T cells are impaired in activation. Both phenotypes are cell autonomous, as they occur in purified T cells from either transgenic (Fig. 4, and data not shown).

The impairment of T cell activation function in GFI1B-transgenic T cells appears to be linked to an inability to signal properly after TCR engagement and costimulation. First, purified GFI1B-transgenic T cells do not activate given CD28- and CD3-cross-linking Abs (Fig. 4A). Second, the TCR signaling-dependent RAS/mitogen-activated protein kinase-induced expression of early activation markers CD25 and CD69 (33) is impaired in GFI1B-transgenic T cells (Fig. 4B). Finally, in male HY-transgenic mice, GFI1B expression rescues the generation of DP thymocytes that are normally deleted due to strong autoreactive TCR signals (21). Similar results have been obtained in male HY-transgenic mice that are rendered defective in TCR signaling by deletion of intracellular proteins that participate in the TCR-signaling cascade (22, 23, 24). However, the GFI1B-induced defect in T cell activation cannot be restricted to proximal signaling molecules in the TCR- and costimulatory-signaling pathways, because GFI1B-induced impairment of CD8 SP cell formation is also observed after in vitro drug-stimulated activation that bypasses the need for proximal TCR- and costimulatory-signaling events (Fig. 8). The ability of GFI1B-transgenic thymocytes to respond to cytokine signaling may also be affected. Although activated GFI1B-transgenic thymocytes fail to induce IL-7R{alpha} expression, transgenic expression of IL-7R{alpha} (34) did not change the GFI1B-induced phenotypes (data not shown), whereas expression of the IL-7 downstream effector BCL2 increased CD8 generation. Thus, GFI1B expression in T cells induces cell-autonomous defects in intracellular signaling that may impair T cell activation at multiple steps.

GFI1B may serve as a negative regulator of GFI1-enhanced activation. Both GFI1 and GFI1B are expected to repress genes to alter the kinetics or activation potential of intracellular signals. The induction of GFI1B in activated thymocytes could result in competition between GFI1 and GFI1B for DNA binding at specific promoters. Alternatively, GFI1 and GFI1B may regulate different promoters. Both scenarios appear to be relevant to the GFI1B-induced T cell defects. Because transgenic GFI1 expression doubles the generation of GFI1B-transgenic CD8 SP cells, GFI1 and GFI1B may compete at promoters to regulate genes important for CD8 development. Target genes responsible for this phenotype may be properly regulated by GFI1, but improperly regulated by GFI1B in cells about to undergo lineage commitment. Interestingly, transgenic BCL2 or GFI1 rescued CD8 SP development, but did not affect GFI1B-induced changes in CD4 SP development, the expression of IL-7R{alpha}, or peripheral T lymphopenia. Target genes responsible for the latter GFI1B-induced phenotypes should be independent of GFI1 regulation and instead uniquely regulated by GFI1B. GFI1 and GFI1B differ in amino acid sequence in the region between the SNAG repressor domain and the zinc-finger DNA-binding domain. These dissimilar regions may mediate interaction with other proteins (such as other transcription factors or adapter proteins), leading to differential regulation of target promoters by GFI1 and GFI1B. The normal role of GFI1B in the thymus may be to regulate the extent of GFI1-mediated thymocyte activation during development. Our data are the first to demonstrate that GFI1 and GFI1B are not redundant for T cell activation functions, providing a potential biological explanation for the termination of GFI1B expression in mature T cells.

IL-7 is critical for the survival and proliferation of immature thymocytes into mature CD8 SP cells (16, 32, 35). IL-7 is constitutively present in the thymus; however, IL-7R{alpha} expression is not induced until TCR stimulation ceases (16). Thus, whether TCR signaling continues or ceases determines the ability to differentiate into CD8 SP T cells. This new perspective on lineage commitment is referred to as the kinetic signaling model. In accordance with this model, GFI1B-transgenic thymocytes, in which GFI1 function is compromised, fail to integrate activation signals, resulting in impaired expression of activation-induced survival genes such as IL-7R{alpha} (16). Models of lineage commitment which require quantitative or temporal differences in TCR signals to direct T cell development (36, 37) would predict that the reduced activation in GFI1B-transgenic T cells should lead to improved CD8 SP development (38). Transgenic expression of GFI1B in the thymus instead results in a severe decrease in the generation of CD8 SP cells. A similar phenotype is exhibited by mice made deficient for IFN {alpha}{beta} signaling by knocking out the transcription factor IRF-1 (39). These mice are devoid of CD8 SP T cells even though they contain normal numbers of CD4 SP T cells. We have established that the GFI1B-transgenic thymus defects are not due to GFI1B repression of IRF-1 (Fig. 2B), CD8, or MHC class I (data not shown), the absence of a functional TCR rearrangement (Fig. 6), or a TCR signal-independent redirection of CD8 cells to a CD4 lineage choice (data not shown). However, in agreement with the kinetic signaling model, a signal-independent survival cue from the downstream target of IL-7, BCL2, increases CD8 SP generation in the context of GFI1B expression. Therefore, the defect in generation of CD8 SP cells in GFI1B-transgenic mice appears to be linked to an inability to transduce a postactivation signal-dependent survival cue.

GFI1 functions to inhibit T cell death induced by specific stimuli. Specifically, GFI1 increases survival of explanted thymocytes (40) and decreases apoptosis induced by TCR ligation (41). In GFI1B-transgenic T cells, GFI1 expression does not correct GFI1B-impaired expression of IL-7R{alpha}; however, like BCL2, transgenic GFI1 increases CD8 SP generation in the GFI1B-transgenic thymus. The salient function of GFI1 in CD8 SP generation may be GFI1 mediation of cytokine-induced survival signaling that is impeded in GFI1B-transgenic thymocytes. Taken together, these data are the first to suggest a role for GFI1 in the integration of activation signals from the TCR with survival signals from cytokines such as IL-7.


   Acknowledgments
 
We thank Jennifer Punt, Gordon Ross, and Thomas Mitchell for critically reviewing the manuscript. We gratefully acknowledge the following individuals: Avedis Kazanjian, Amy Barber Shonk, Rachel Rivoli, Natalie Claudio, and Yajuan Jiang for excellent technical assistance; Barry Udis and Michael Tanner for expert flow cytometry; and Michael Alexander for initial T cell activation assays. We also thank the members of the Institute for Cellular Therapeutics for helpful discussions, and we give special thanks to Tarik Moroy and Holger Karsunky for the CD2-GFI1 mice, and Thomas Malek for the generous gift of IL-7R{alpha}-transgenic mice.


   Footnotes
 
1 This work was supported by Hope Street Kids, the Jennifer Sacco Memorial Fund, the University of Louisville School of Medicine Grant-in-Aid, a University of Louisville Research Initiation Grant, and in part by the Commonwealth of Kentucky Research Challenge Trust Fund and the Jewish Hospital Foundation. L.L.D. is supported by a National Science Foundation Graduate Research Fellowship. This work was supported in part by PHS CA56110 (to P.N.T.). Back

2 L.L.D. and M.K.K. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. H. Leighton Grimes, Institute for Cellular Therapeutics, University of Louisville, Baxter Biomedical Research Building, Suite 404-F, 570 South Preston Street, Louisville, KY 40202-1760. E-mail address: lee.grimes{at}louisville.edu Back

4 Abbreviations used in this paper: DN, CD4-CD8- (double negative); DP, CD4+CD8+ (double positive); CD4 SP, CD4+8- single positive; CD8 SP, CD4-8+ single positive; GFI1, growth factor independence-1; MFI, mean fluorescence intensity; ISP, intermediate single positive; P+I, phorbal-12-myristate-13-acetate and ionomycin; SNAG, snail and Gfi1 repressor domain; GH, human growth hormone; RAG, recombination-activating gene; IRF, IFN regulatory factor; WT, wild type. Back

Received for publication April 30, 2002. Accepted for publication December 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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