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The Journal of Immunology, 2003, 170: 4612-4622.
Copyright © 2003 by The American Association of Immunologists

TGF-{beta}1 Regulates Lymphocyte Homeostasis by Preventing Activation and Subsequent Apoptosis of Peripheral Lymphocytes1

Ramireddy Bommireddy*, Vijay Saxena{dagger}, Ilona Ormsby*, Moying Yin*, Gregory P. Boivin{ddagger}, George F. Babcock§,¶, Ram R. Singh{dagger} and Thomas Doetschman2,*

Departments of * Molecular Genetics, Biochemistry and Microbiology, {dagger} Internal Medicine, {ddagger} Pathology and Laboratory Medicine, and § Surgery, University of Cincinnati College of Medicine, Cincinnati, OH 45267; and Shriners Hospital for Children, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TGF-{beta}1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-{beta}1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-{beta}1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1-/- mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-{gamma} and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1-/- T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1-/- T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4+ T cells have elevated intracellular Ca2+ levels. However, upon subsequent stimulation in vitro, increases in Ca2+ levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1-/- splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca2+ levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-{beta}1 is a pleiotropic polypeptide growth regulatory factor that functions during embryogenesis in preimplantation, and yolk sac and tooth development (1, 2, 3). In the cardiovascular system it is required for cardiac hypertrophy (4) and platelet function (5). In cancer it functions by regulating genetic stability (6), inhibiting carcinogen-induced tumorigenesis (7), enhancing progression to invasive spindle carcinomas (8), and maintaining mucosal tissue integrity in the large intestine (9, 10). In the immune system TGF-{beta}1 plays a critical role in immune regulation (11, 12). It regulates inflammation (13, 14), induction of self-tolerance and oral-tolerance (15, 16, 17), and autoimmunity (18). TGF-{beta}1 has been shown to induce Ag-specific unresponsiveness in naive T cells (19), and TGF-{beta}1-secreting Th3-type regulatory T cells and CD8+ suppressor T cells can maintain immune homeostasis and inhibit autoimmune disorders (20, 21, 22, 23). TGF-{beta}1 is secreted upon cross-linking of CTLA-4, suggesting that TGF-{beta}1 is one of the factors involved in CTLA-4-mediated lymphocyte homeostasis (24). In vitro studies have shown that TGF-{beta}1 can act as a positive as well as a negative regulator of lymphocyte proliferation and apoptosis (25, 26).

Tgfb1 knockout mice develop severe inflammatory lesions in multiple organs and die within 3 wk after birth (13, 14). Anti-LFA-1 Ab (27) and fibronectin peptides (28) rescue these mice from inflammatory disorders, suggesting that either extravasation or T cell activation is affected. T lymphocytes are the primary effectors in the immunopathology of these mice as Tgfb1-/- scid (27), Tgfb1-/- Rag2-/- (9), Tgfb1-/- Rag1-/- (4), and Tgfb1-/- athymic nude mice (our unpublished observations) survive months longer than do immunocompetent Tgfb1-/- mice. Studies using mice expressing dominant negative TGF-{beta} receptor type II transgenes under T cell-specific promoters have revealed that T cells are hyper-responsive, acquire a memory phenotype, induce generation of autoantibodies, and produce inflammatory lesions in lung and colon tissues (29). However, another study found that only CD8+ T cells are hyper-responsive in dominant negative TGF-{beta} receptor type II transgenic mice (30). Also, TGF-{beta}1 can inhibit differentiation to Th2 cells by inhibiting IL-4 induction (31). Finally, Tgfb1-/- mice on an MHC class I-deficient background ({beta}2-microglobulin-null mice) live much longer than Tgfb1-/- mice on a MHC class II-deficient background, suggesting that abnormal expression and presentation of self-Ags by MHC class I molecules to CD8+ T cells may also contribute to the inflammatory phenotype (32). T cells from the spleens of Tgfb1-/- mice do not respond to Con A mitogenic stimulation, whereas purified splenic CD4+ T cells and CD4+ T cells depleted of CD4+CD25+ regulatory T cells do respond to anti-CD3 and anti-CD28 Ab-induced mitogenic stimulation, suggesting that Tgfb1-/- T cells respond to mitogenic stimulus (24, 33, 34).

One of the major difficulties in determining what causes the inflammatory disease in Tgfb1-/- mice is the possibility that the inflammatory disorder may have secondary effects that are independent of or synergistic with the absence of TGF-{beta}1. For example, in Tgfb1 knockout mice the production of inflammatory cytokines increases (13), the percentage of CD4 single-positive thymocytes increases 7-fold, and hyperproliferation leads to enlarged spleens and lymph nodes (35). However, since these characteristics are common to inflammatory stress in general, it is not clear how TGF-{beta}1-deficient lymphocytes would behave in the absence of inflammatory stress. To determine how Tgfb1-/- lymphocytes respond to mitogens we have combined PMA and ionomycin to stimulate splenocytes in a TCR- and APC-independent manner (36), and we have stimulated them in a receptor-mediated manner using mitogens such as Con A, anti-CD3, and anti-CD28 (37). Our results indicate that splenocytes from Tgfb1-/- mice are hyporesponsive to Con A and anti-CD3, as judged by their reduced proliferative response and IL-2 production. TCR stimulation also causes diminished intracellular Ca2+ ([Ca2+]i)3 elevation in mutant T cells. Stimulation with PMA and ionomycin, however, rescues these cells from anergy. Hence, the lack of optimal [Ca2+]i elevation by receptor-mediated mitogenic stimulus is the primary cause of the hyporesponsiveness of Tgfb1-/- T cells.


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

Tgfb1-/- mice (129xCF-1) were generated as previously described (13). These mice were bred onto BALB/c (N2-N3), C3H, and 129 (N4–N5) backgrounds. More extensive backcrossing was not performed because it leads to a precipitous decline in births of Tgfb1-/- animals (1). Mice were housed in a specific pathogen-free mouse facility in which mice did not have any serologic titers to common viral pathogens and mycoplasma at University of Cincinnati Medical Center.

Reagents and Abs

RPMI 1640 medium, AIM-V medium, Dulbecco’s PBS and HBSS were purchased from Life Technologies (Gaithersburg, MD). RBC lysing buffer, trypan blue, Con A, ionomycin, PMA, propidium iodide, and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). Indo-1/AM was purchased from Calbiochem (La Jolla, CA). Pluronic F-127 was purchased from Molecular Probes (Eugene, OR). Paraformaldehyde was purchased from Electron Microscopy Sciences (Washington, PA). [Methyl-3H]thymidine (sp. act., 6.7 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA). Falcon flat-bottom 96-well Microtest Primaria tissue culture plates and 24-well Multiwell tissue culture plates were purchased from BD Biosciences (Franklin Lakes, NJ). PMA (10 µM) and ionomycin (100 µM) stocks were prepared in DMSO, aliquoted, and stored at -80°C.

Purified anti-mouse CD3{epsilon}, anti-mouse CD28, anti-mouse CD16/CD32 (Fc{gamma}III/II receptor), anti-mouse IL-2, anti-mouse IL-4, anti-mouse IL-10, and anti-mouse IFN-{gamma} Abs and FITC-anti-mouse CD3{epsilon}, FITC-anti-mouse CD4 (L3T4), CyChrome-anti-mouse CD4, R-PE-anti-mouse CD8{alpha} (Ly-2), R-PE-anti-mouse CD19, R-PE-anti-mouse CD69, R-PE-anti-mouse CD122, and fluorochrome-conjugated isotype control Abs were purchased from BD PharMingen (San Diego, CA). R-PE-anti-mouse CD11a (LFA-1) was purchased from BioDesign (Saco, ME).

PCR genotyping

Genotypes of the newborn pups from heterozygous matings were determined by PCR amplification of tail DNA and size fractionation on agarose gels (9).

Inflammation index

After the thymus and spleen were removed for in vitro studies, Tgfb1 null mice were fixed in 10% neutral-buffered formalin. Tissue sections were scored for inflammatory lesions with a severity scale from 0–4. Although inflammation from 25–30 organs was measured, only 8–12 organs were found to be severely affected (13, 35). The sum of scores for all organs was divided by the number of organs analyzed to generate an inflammation index. Mice severely affected by multifocal inflammation had inflammation indices ranging from 1.0–2.0 (3 wk old), whereas wild-type mice had no inflammation.

Preparation of thymocytes and splenocytes

Newborn pups and young mice (for most of the studies <2-wk-old mice were used) were sacrificed by isoflurane overdose, and thymus and spleen were dissected out aseptically into a petri dish containing RPMI 1640 medium. Tissue was mechanically separated into individual cells with a syringe and 22-gauge needle. Cells were centrifuged at 1000 rpm for 5 min, and RBCs were depleted with RBC lysing buffer, and the cells were washed once with RPMI 1640 medium. Cells were suspended in Aim-V serum-free medium. Viable cells were counted using trypan blue dye exclusion as an indicator. Cells were resuspended at 2 x 106/ml in Aim-V serum-free medium for all in vitro culture studies. Thymocytes and splenocytes were prepared in Dulbecco’s PBS or HBSS for staining and flow cytometry.

Phenotype analysis of splenocytes

One million splenocytes from each mouse were stained on ice for 30 min for CD3, CD4, CD8, and LFA-1 expression using fluorochrome-conjugated mAbs (BD PharMingen) in the presence of 10% FBS and Fc blocking Ab. After the cells were washed once, they were resuspended in HBSS-containing 0.09% NaN3 and analyzed in EPICS XL flow (Beckman Coulter, Fullerton, CA), FACSCalibur, or BD-LSR flow cytometers (BD Biosciences, San Jose, CA). Side and forward angle light scattering was used to electronically gate the cells of choice and to exclude debris. Ten thousand events within the gate region were collected for each sample. Fluorochrome-conjugated isotype-matched Abs were used as controls for nonspecific binding. Cells stained with each Ab individually were used to set compensation networks for each fluorochrome. In all experiments control cells were taken from either Tgfb1+/+ littermates or Tgfb1+/- littermates when wild-type littermates were not available. Data were analyzed using System II or CellQuest software (BD Biosciences, San Jose, CA).

Assessment of proliferation by tritiated thymidine incorporation

Splenocytes (2 x 105) were cultured in 200 µl of Aim-V serum-free medium in the presence of various concentrations of mitogens in triplicates in flat-bottom, 96-well tissue culture plates. After 3 days of in vitro culture at 37°C and 5% CO2, cultures were pulsed with 0.5 µCi of tritiated thymidine for a period of 14–16 h, cells were harvested onto glass-fiber filters, and radioactivity was counted in a Beckman scintillation counter. Data are represented as the mean disintegrations per minute ± SD from triplicate cultures. Background incorporation of unstimulated cultures was always <3000 dpm. Optimization of cell number, mitogen concentration, and serum replacements was conducted in preliminary studies. The optimum concentrations were: Con A, 0.5 µg/ml; anti-CD3, 0.5 µg/ml; PMA, 1 nM; and ionomycin, 250 nM in Aim-V serum-free medium supplemented with L-glutamine, streptomycin sulfate, gentamicin sulfate, and human serum albumin.

[Ca2+]i measurements by flow cytometry

Splenocytes (1 x 107/ml) from 2-wk-old mice in cell loading medium (CLM: HBSS, containing Ca2+ and Mg2+, and 1% FBS) were loaded with 10 µM Indo-1/AM (stock solution, 2 mg/ml dissolved in Pluronic F-127) for 40 min at 30°C (38). Cells were washed three times with CLM, resuspended in CLM, surface-stained with azide-free fluorochrome-conjugated anti-CD4 and anti-CD19 for 20 min on ice, washed once with CLM, and left on ice until analyzed. Cells were prewarmed at 37°C for 10 min before collection for Ca2+ measurements. Cells were collected for 30 s before adding the agonist. For each analysis cells were collected for 512 s at a flow rate of 300–400 events/s in a BD-LSR flow cytometer machine equipped with a helium-cadmium UV laser. Ratiometric analyses of Ca2+-bound Indo-1 (FL5)/free Indo-1 (FL4) were performed using CellQuest (BD Biosciences) and FlowJo (Tree Star, San Carlos, CA) software programs. [Ca2+]i levels before and after stimulation in CD4+ (T cells) and CD19+ (B cells) splenocytes were analyzed after gating on these cells. Based on the concentration of [Ca2+]i levels after acquiring each sample for 512 s, cells were further grouped into low (M1), intermediate (M2), medium high (M3), and high (M4) [Ca2+]i cells (39, 73).

Cytokine analysis

Splenocytes and thymocytes (1 x 106/ml) in Aim-V serum-free medium were cultured in 24-well plates at 37°C and 5% CO2 for 2–3 days. Supernatants were collected and frozen until cytokines were analyzed by sandwich ELISA. Cells were harvested, washed, and stained for cell cycle analysis (see below).

Cell cycle analysis

Splenocytes were washed once in chilled PBS, fixed in 70% ethanol at -20°C at least for 2 h, washed again with PBS, and incubated with 1 ml of PI staining cocktail (PBS containing 0.1% Triton X-100, 0.2 mg/ml RNase, and 20 µg/ml propidium iodide) for 30 min at room temperature. Cells were analyzed by flow cytometry, and the data were analyzed using System II and Flow Jo software programs.

Statistical analysis

The significance of the differences between wild-type and mutant mouse responses was calculated using Student’s t or Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tgfb1-/- splenocytes are hyporesponsive to mitogenic stimulation

We have recently shown that Tgfb1-/- thymocytes are hyper-responsive to mitogenic stimulation (73). Here, we show that splenocytes from Tgfb1 mutant mice have a markedly decreased proliferative response to Con A compared with wild-type splenocytes (Fig. 1A). As expected, thymocytes from the same Tgfb1 null mice (d14) are hyper-responsive to Con A (0.25–1.0 µg/ml) stimulation (Fig. 1B). These data suggest that while immature Tgfb1-/- thymocytes are hyper-responsive, mature, peripheral T cells display an anergic phenotype. At higher concentrations of Con A (2.0 µg/ml and above), mitogenic responses are lower in both Tgfb1+/+ and Tgfb1-/- mice, but the decrease is more striking in the mutants (data not shown). Similar hyporesponsiveness is also observed in Tgfb1-/- T cells when stimulated with anti-CD3 Ab (Fig. 2A).



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  		FIGURE 1. Tgfb1-/- splenocytes are hyporesponsive to mitogenic stimulation. Splenocytes (A) and thymocytes (B) from d14 mice were cultured in the presence of Con A and pulsed with [3H]thymidine as described in Materials and Methods. Data are presented as the mean ± SD of triplicate determinations (dpm). Similar responses were observed in most mice tested (p < 0.002; see Table I).

 


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  		FIGURE 2. Tgfb1-/- splenocytes respond to receptor-independent mitogenic stimulation. Splenocytes from d13 mice were cultured in the presence of anti-CD3 (0.5 µg/ml), anti-CD3 (0.5 µg/ml) plus anti-CD28 (0.25 µg/ml), anti-CD3 (0.5 µg/ml) plus PMA (1 nM), anti-CD3 (0.5 µg/ml) plus ionomycin (125 nM) (A), or PMA plus ionomycin (B) and pulsed with [3H]thymidine as described in Materials and Methods. Data are presented as the mean ± SD of triplicate determinations (dpm). Similar responses were observed in most mice tested (anti-CD3, p < 0.02; 1 nM PMA plus 125 nM ionomycin (Iono), p < 0.07; 1 nM PMA plus 250 nM Iono, p < 0.4; 0.5 nM PMA plus 250 nM Iono, p < 0.7; see Table I). Experiments using various concentrations of anti-CD28 (5–500 ng/ml), PMA (0.5–1.0 nM), and Iono (125–250 nM) were repeated three to five times with similar results.

 
Treatment with PMA and ionomycin, but not anti-CD28 and PMA, rescues Tgfb1-/- T cells from proliferative hyporesponsiveness

To analyze whether the hyporesponsive phenotype of Tgfb1-/- splenic T cells is due to a defect in T cell signaling at the membrane receptor level or to events downstream of the receptor, we stimulated T cells with various mitogens. Mitogens, such as Con A and anti-CD3, activate T cells in a receptor-mediated and APC-dependant manner, whereas PMA and ionomycin synergistically activate T cells in a receptor-independent and APC-independent manner. PMA activates protein kinase C (PKC), but does not elevate [Ca2+]i. Anti-CD3 stimulation of T cells activates PKC ({alpha}, {beta}, {delta}, and {epsilon} isozymes) in young donors, whereas only PKC{beta} is activated in older people (40). However, PKC{phi} has been shown to be important for NF-{kappa}B activation in mature, but not immature, T cells (41). Ionomycin elevates [Ca2+]i, but [Ca2+]i alone does not activate PKC in the absence of PMA or Con A. Addition of the Ca2+-mobilizing agent ionomycin along with either Con A or PMA synergistically activates T cell proliferation.

We first examined the proliferative response of Tgfb1-/- splenic T cells to anti-CD3 stimulation in the presence of comitogens such as anti-CD28, PMA, or ionomycin. Our results show that mutant cells still do not respond to anti-CD3, even in the presence of anti-CD28, PMA, or ionomycin (Fig. 2A). Activation of the PKC pathway by PMA also does not rescue the hyporesponsive phenotype of splenic T cells to anti-CD3 (Fig. 2A). Stimulation with PMA and ionomycin, however, reverses the hyporesponsiveness of mutant cells (Fig. 2B). Activation of the calcineurin pathway by ionomycin synergizes with Con A to partially rescue mutant cells from the hyporesponsive phenotype (Fig. 3). Stimulation of splenocytes with ionomycin alone does not result in any mitogenic response, as the amount of tritiated thymidine uptake is the same as that observed with medium alone (~500 ± 100 dpm). As in the case of anti-CD3 stimulation, activation of the PKC pathway by PMA also does not rescue the hyporesponsive phenotype of splenic T cells to Con A (Fig. 3). The observed differences between Con A plus ionomycin (Fig. 3) and anti-CD3 plus ionomycin (Fig. 2A) could result from Con A being a polyclonal T cell activator that cross-links surface receptors, whereas anti-CD3{epsilon} (clone 2C11) Ab binds to CD3{epsilon} on T cells and is not as potent as Con A. The synergistic effect of ionomycin in the presence of Con A on Tgfb1-/- splenocytes, albeit partial, is consistent. These results suggest that the hyporesponsiveness of Tgfb1-/- splenocytes is not due to their being unresponsive to mitogenic stimulation, but, rather, to an altered threshold of activation. We have recently shown that thymocytes in the mutant mice respond well to anti-CD3 plus anti-CD28 and to anti-CD3 plus PMA (73). The lack of mutant splenocyte response to Con A and anti-CD3 could be due to inefficient costimulation and/or signaling by receptors on T cells, which, in turn, could be due to a defect in one or more of the T cell activation pathways, such as PKC, calcineurin, and mitogen-activated protein kinase.



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  		FIGURE 3. Ionomycin partially rescues Tgfb1-/- T cells from proliferative hyporesponsiveness. Splenocytes from d18 mice were cultured in the presence of Con A (0.5 µg/ml), Con A (0.5 µg/ml) plus ionomycin (125 nM), Con A (0.5 µg/ml) plus PMA (1 nM), or 1 nM PMA plus 250 nM ionomycin and pulsed with [3H]thymidine as described in Materials and Methods. Data are presented as the mean ± SD of triplicate determinations (dpm) from one of three similar experiments. [3H]thymidine uptake in ionomycin-treated cultures was the same as that in cultures in medium alone, and there was no difference between wild-type and mutant mice (in wild-type mouse, 522 ± 61 with medium alone and 406 ± 54 with 125 nM ionomycin; in the mutant mice, 604 ± 60 with medium alone and 582 ± 29 with 125 nM ionomycin, with similar amounts of uptake in the other mutant mouse).

 
As shown in Table I, splenocytes from most mutant mice of different ages respond poorly or not at all to Con A or anti-CD3 stimulation, but respond well to 1 nM PMA and 125 nM ionomycin, suggesting that Tgfb1-/- splenic T cells retain the ability to respond to mitogenic stimulation. Note that at optimal doses of PMA plus ionomycin (1 and 250 nM, respectively) there is no significant difference in the proliferative response between Tgfb1+/+ and Tgfb1-/- splenocytes. That no correlation between the degree of inflammation and the extent of mitogenic response is found (Table I) indicates that our results are independent of the general state of inflammation in the mutant animal.


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  		Table I. PMA plus ionomycin induces mitogenesis of splenocytes in Tgfb1 null micea

 
Analyses of over two dozen mutant mice revealed that splenocytes from ~20% of the animals do not respond to any stimulation, including PMA and ionomycin. T cell proliferation data from such animals are shown in Table II. To further investigate this nonresponsive phenotype, we enumerated CD3+ T cells by flow cytometry on splenocytes. We found that the nonresponder mutant animals have a marked reduction in CD3+ T cells (Table II). This paucity of T cells in the spleens of Tgfb1-/- mice may be due to their recruitment into nonlymphoid organs such as liver, lung, and heart, where they cause a lethal inflammatory disease (13, 42). However, the near-normal response to receptor-mediated mitogenic stimulation in some of the responder mice (~10% of the mice tested; see Table I) is probably due to the presence of near-normal percentages of CD3+ T cells, as the percentage of CD3+ cells in the spleen of a responder Tgfb1-/- mouse is slightly greater than that in the spleen of a Tgfb1+/+ mouse (28.5 vs 23.3%).


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  		Table II. Splenocyte mitogenic response to Con A, anti-CD3, and PMA plus ionomycina

 
Splenocytes from B cell- and TGF-{beta}1-deficient mice (Igh6-/-Tgfb1-/-) also fail to respond to Con A and anti-CD3, but respond well to PMA and ionomycin (Fig. 4A; data for anti-CD3 not shown), suggesting that B cell proliferation does not account for the response of TGF-{beta}1-deficient splenocytes to PMA and ionomycin (Figs. 2B and 3). The lack of response of nude mouse splenocytes to stimulation by PMA and ionomycin (Fig. 4B) also suggests that T cells are the primary responders under these culture conditions. Note that the maximum uptake of tritiated thymidine upon PMA plus ionomycin stimulation is <3000 dpm in Fig. 4B.



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  		FIGURE 4. B cells do not affect hyporesponsiveness of T cells to mitogenic stimulation in Tgfb1-/- mice. Splenocytes from d35 mice with no mature B cells (Igh6-/- Tgfb1-/- mice live 1–2 wk longer than immunocompetent Tgfb1-/- mice; A), or splenocytes from 2-mo-old athymic nude mice with no mature T cells (Tgfb1-/- athymic nude mice live 3–4 mo longer; B) were cultured in the presence of Con A, PMA (P) plus ionomycin (I), or medium alone and pulsed with [3H]thymidine as described in Materials and Methods. Note the maximum uptake of tritiated thymidine upon PMA plus ionomycin stimulation is <3000 dpm in B. Data are presented as the mean ± SD of triplicate determinations (dpm) from one of three similar experiments.

 
The proliferative hyporesponsiveness to Con A or anti-CD3 in Tgfb1-/- mice could be due to decreased production of cytokines such as IL-2. Activation with PMA and ionomycin, however, may cause sufficient IL-2 production.

Decreased IL-2 production by Tgfb1-/- splenocytes

To determine whether TGF-{beta}1 deficiency affects early events such as cytokine production by lymphocytes, we measured cytokine levels in splenocyte cultures upon Con A or anti-CD3 stimulation (Fig. 5A). Basal levels of IL-2 in unstimulated cells (medium only) were very low to undetectable in both wild-type and mutant splenocytes. Upon Con A or anti-CD3 stimulation IL-2 levels increased in wild-type splenocytes compared with those cultured in medium alone (p = 0.02), but not in mutant splenocytes. In fact, IL-2 levels were barely detectable in anti-CD3-stimulated splenocytes from most mutant animals (wild-type vs mutant, p = 0.03). Importantly, such reduced or absent IL-2 responses upon TCR cross-linking or mitogenic stimulation can be rescued by PMA plus ionomycin stimulation of mutant splenocytes, resulting in significantly higher levels of IL-2 in Tgfb1-/- splenocytes (PMA plus ionomycin vs Con A, p = 0.02; PMA plus ionomycin vs anti-CD3, p = 0.02). These data demonstrate a consistent correlation between the degree of IL-2 production and the proliferative response. Namely, if IL-2 production is intermediate, as after anti-CD3 or Con A stimulation, the mitogenic response is also intermediate (Table III). If IL-2 production is high, as after PMA plus ionomycin treatment, the mitogenic response is high compared with that to receptor-mediated mitogenic stimulation. These data suggest that treatment with PMA and ionomycin rescues splenocytes from proliferative hyporesponsiveness by strongly inducing IL-2 production.



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  		FIGURE 5. Decreased IL-2 and increased IFN-{gamma} production by Tgfb1-/- splenocytes. Splenocytes from 2-wk-old mice were cultured for 2–3 days in the presence of Con A, anti-CD3, or PMA plus ionomycin. Culture supernatants were assayed for IL-2 (A) or IFN-{gamma} (B). Data are represented as picograms per milliliter (A, IL-2) or nanograms per milliliter (B, IFN-{gamma}) of cytokine(s) for individual Tgfb1+/+ (•) or Tgfb1-/- ({circ}) mice. The mean value for each group is shown by vertical bars.

 

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  		Table III. Con A stimulates IFN-{gamma} production by Tgfb1-/- splenocytesa

 
T cells from Tgfb1-/- mice produce increased amounts of IFN-{gamma}

Since Tgfb1-/- thymocytes are hyper-responsive (Fig. 1B), it is possible that hyperactive thymocytes, upon maturation and export to the periphery, recognize self-Ags and undergo activation in vivo. Such in vivo-activated T cells should secrete higher amounts of cytokines. Indeed, thymocytes from Tgfb1-/- (1- to 2-wk-old) mice, upon PMA plus ionomycin stimulation, produce significantly higher amounts of IFN-{gamma} than do wild-type thymocytes (mean ± SE, 1.25 ± 0.66 in Tgfb1+/+ vs 9.69 ± 5.49 ng/ml in Tgfb1-/- thymocyte cultures stimulated with 1 nM PMA and 250 nM ionomycin; p = 0.04; n = 7).

While Con A- or anti-CD3-stimulated splenocytes from 2-wk-old (day 15) Tgfb1-/- mice do not respond well to ex vivo mitogenic stimulation (Figs. 1A and 2A, and Table I) and do not secrete IL-2 (Fig. 5A), they produce large amounts of IFN-{gamma} (Fig. 5B). Basal levels of IFN-{gamma} in unstimulated cells (medium only) are very low (mean values in Tgfb1+/+ and Tgfb1-/- are 0.24 and 0.26 ng/ml, respectively). This increase in IFN-{gamma} production by mutant splenocytes upon stimulation is noteworthy considering the decreased numbers of CD3+ T cells and their hypoproliferative response in most Tgfb1-/- mice (Table III). The increase in IFN-{gamma} production is not necessarily due to the polarization of T cells toward Th1, since IL-2, the other Th1 cytokine, is decreased (Fig. 5A), as are the Th2 cytokines, IL-4 and IL-10. Furthermore, the increase in IFN-{gamma} production is more dramatic when Tgfb1-/- splenocytes are stimulated with 1 nM PMA and 125 nM ionomycin (10.5-fold compared with wild-type splenocytes, p = 0.05; n = 5; Fig. 5B). PMA plus ionomycin stimulation also induces the production of other cytokines, IL-4 and IL-10, in Tgfb1-/- splenocytes compared with that induced by Con A or anti-CD3, although the levels are not significantly different from those in wild-type cells (data not shown). These data suggest that the increased IFN-{gamma} production by Tgfb1-/- T cells reflects their prior activation in vivo.

Splenocyte [Ca2+]i levels are elevated in Tgfb1-/- mice

TCR engagement triggers a series of events, including immediate-early events such as [Ca2+]i flux, early events such as cytokine release, and late events such as proliferative response. The results presented in Figs. 1–5 demonstrate impairments in proliferation and cytokine production in Tgfb1-/- T cells. These changes in late as well as early post-TCR engagement events could result from impairments in immediate-early events, which in themselves may be influenced by TGF-{beta}1. To examine this we measured basal [Ca2+]i levels and changes in [Ca2+]i levels upon anti-CD3 stimulation using flow cytometry. Our results revealed that splenic T cells, but not B cells, have elevated basal [Ca2+]i levels in 2-wk-old mice (Fig. 6A). Analysis of changes in [Ca2+]i levels after stimulation of Tgfb1-/- splenocytes with anti-CD3 suggests that elevation of cytosolic Ca2+ levels in CD4+ T cells is significantly reduced compared with that in cells from control mice (Fig. 6B). This defect in elevation of [Ca2+]i levels is restricted to T cells, as stimulation of B cells (CD19+) with anti-IgM does not produce any difference in elevation of [Ca2+]i levels in the same preparation of cells (Fig. 6C). This abnormality in [Ca2+]i mobilization is associated with down-regulation of CD3 receptors on splenic T cells (Fig. 6D; note the decrease in mean fluorescence intensity (MFI) of CD3 on mutant splenocytes). These observations suggest that CD3 receptor down-regulation and [Ca2+]i elevation are responsible for the reduced [Ca2+]i changes and decreased mitogenic response observed in these T cells.



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  		FIGURE 6. Tgfb1-/- CD4+ splenic T cells have higher [Ca2+]i levels and do not respond well upon TCR stimulation. The fluorescence ratio of Ca2+-bound Indo-1 (FL5)/free Indo-1 (FL4) was measured as described in Materials and Methods. A, Overlays of FL5/FL4 vs cell count histograms of unstimulated CD4+ splenocytes from Tgfb1+/- ({blacksquare}) and Tgfb1-/- ({square}) mice. Percentages of cells in M2 gate are shown in A. B, Anti-CD3 (20 µg/ml)-stimulated changes in [Ca2+]i levels in CD4+ splenocytes (T cells; red, Tgfb1+/-; blue, Tgfb1-/-). C, Anti-IgM (20 µg/ml)-stimulated changes in [Ca2+]i levels in CD19+ splenocytes (B cells; red, Tgfb1+/-; blue, Tgfb1-/-). D, CD3 expression on splenocytes of Tgfb1+/- ({blacksquare}) and Tgfb1-/- ({square}) mice. The percentage of CD3+ cells and the MFI in arbitrary units of CD3 expression are shown. The genotypes of Tgfb1+/- (+/-) and Tgfb1-/- (-/-) mice are shown. FITC-anti-CD4 and R-PE-anti-CD19 were used to gate T and B cells, respectively. FITC-anti-CD3 was used to stain for the surface expression of CD3 on splenocytes. The data represent one of two similar experiments using d15 mice.

 
Tgfb1-/- T cells do not undergo mitogenesis

To determine the fate of stimulated splenic T cells, cell cycle analysis was performed. The percentage of cells in S and G2-M phase is only 6% and is comparable in wild-type and mutant mice, suggesting that there is no spontaneous mitosis in the absence of mitogens in mutant mice (Fig. 7, top panels). The percentages of stimulated wild-type cells in S and G2-M phase is 21% (Con A) and 15% (anti-CD3) compared with 6% in medium alone, whereas there is no increase in S and G2-M phase populations in mutant splenocytes (Fig. 7, middle and lower panels). This suggests that while wild-type splenocytes undergo mitogenesis, mutant splenocytes do not. Additionally, there is an increase in the percentage of cells in sub-G0 phase in mutant compared with wild-type splenocytes (Con A, 64 vs 44%; anti-CD3, 81 vs 53%; Fig. 7, middle and lower panels), suggesting that stimulated mutant splenocytes undergo apoptosis instead of mitosis, probably due to prior activation (see below).



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  		FIGURE 7. Splenocytes in Tgfb1-/- mice die upon stimulation with Con A or anti-CD3. Splenocytes from 2-wk-old mice were cultured in the presence of medium only (top panels), Con A (1 µg/ml; middle panels), or anti-CD3 (1 µg/ml; lower panels) for 2 days and stained with propidium iodide for cell cycle analysis as described in Materials and Methods. The percentages of cells in each phase of the cell cycle are shown in the histograms. Data represent one of two similar experiments.

 
Tgfb1-/- splenic T cells exhibit evidence of prior activation in vivo

In Figs. 1–7, we analyzed the responses of Tgfb1-/- splenocytes upon mitogenic stimulation. To investigate the phenotypes of Tgfb1-/- splenocytes without any ex vivo stimulation, we analyzed the expression of CD3, CD4, CD8, LFA-1, CD69, and CD122 (IL-2R {beta}-chain) and forward scatter (represents the size of the cell) by flow cytometry in cells from mutant and wild-type spleens. First, we found that splenic CD3+ T cells were enlarged in mutant mice. The increased size was more pronounced in CD4+ T cells than in CD8+ T cells (Fig. 8A). Second, since TCR down-modulation is a feature of activated T cells (43, 44), we examined the expression of CD3 on splenic T cells. Results from d16 wild-type and Tgfb1-/- mice show comparable numbers of total splenocytes (52 million in wild-type vs 47 million in mutant cells) and comparable percentages of CD3+ T cells (18.6 vs 15.4%; Fig. 8B). Strikingly, the MFI of CD3 was decreased in total spleen cells (Fig. 8B, upper left panel) and CD4+ (Fig. 8B, upper middle panel) and CD8+ (Fig. 8B, upper right panel) T cells in Tgfb1-/- splenocytes compared with wild-type splenocytes. The down-regulation of CD3 expression in mutant cells was more pronounced in mutant CD8+ cells (Fig. 8B, upper right panel). Furthermore, mutant splenic T cells exhibited a marked down-regulation of CD8 expression. For mice that had comparable percentages of CD4+ and CD8+ cells, the relative MFIs of CD8 were 1710 and 614 in wild-type and mutant mice (Fig. 8B, right lower panel). Given that most Tgfb1-/- mice had about a 3-fold reduction in splenic CD4+ and CD8+ T cells (data not shown), the down-regulation of CD3 and CD8 was more profound in most mutant animals than that observed in the mutant mouse described in Fig. 8. Such TCR and CD8 down-regulation in TGF-{beta}1-deficient splenic T cells is consistent with the previous observation of decreased inflammation in MHC class I-deficient Tgfb1-/- mice (32), suggesting that enhanced presentation of self-Ags by MHC class I molecules to CD8+ T cells may contribute to their continual activation and the development of an inflammatory phenotype.



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  		FIGURE 8. T cells in the spleens of Tgfb1-/- mice are activated. Splenocytes from d16 mice were stained with fluorochrome-labeled anti-CD3, anti-CD4, anti-CD8, anti-CD11a (LFA-1), CD69, and CD122 Abs as described in Materials and Methods. Forward scatter analysis of T cells (A) and expression of T cell markers such as CD3, CD4, and CD8 (B) or of activation markers such as CD11a, CD69, and CD122 (C) are shown as overlay histograms ({blacksquare}, Tgfb1+/+; {square}, Tgfb1-/-). Numbers inside the histograms represent the percentage of cells positive for the indicated surface Ags (CD3, CD69, and CD122) or the MFI for CD8 and CD11a. Data are representative of two to six mice. FITC-anti-CD3, R-PE-anti-CD8, CyChrome-anti-CD4, R-PE-anti-CD69, R-PE-anti-CD122, and R-PE-anti-CD11a were used to detect the surface expression of CD3, CD8, CD4, CD69, CD122, and LFA-1, respectively.

 
Finally, we analyzed the expression of the activation markers LFA-1 (CD11a), CD69, and CD122 on splenic T cells. As shown in Fig. 8C, CD11a expression was markedly increased in mutant splenocytes; the MFI of CD11a was >2-fold higher in Tgfb1-/- CD3+ splenocytes than in Tgfb1+/+ CD3+ splenocytes (Fig. 8C, upper left panel). The expression of CD69 and CD122 was also increased in mutant CD3+ splenocytes relative to that in wild-type cells (Fig. 8C, upper right and lower left panels). CD122 expression was particularly elevated in mutant CD4+ T cells (23.4% in mutant vs 5.9% in wild-type; Fig. 8C, lower right panel).

Taken together, the increased cell volume, down-regulation of CD3, increased [Ca2+]i levels, increased production of IFN-{gamma}, and up-regulation of LFA-1, CD69, and CD122 by Tgfb1-/- splenocytes suggest that T cells in mutant mice undergo constant activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tgfb1-/- mice develop severe inflammatory lesions in multiple organs and die around 3 wk of age. Backcrossing these mice onto immunodeficient backgrounds has revealed that T cells are the primary effectors in the inflammatory phenotype of Tgfb1-/- mice. It is clear that there is a positive relationship between age and the degree of inflammation in Tgfb1-/- mice (35) (Table I), and that many phenotypes, such as epithelial cell proliferation and ICAM and MHC class II up-regulation, are eliminated if the inflammation is curtailed by genetic combination of Tgfb1-/- and scid mice (27). The present study demonstrates that although thymocytes (thymic T cells) are hyper-responsive, mature peripheral T cells (splenic T cells) are hyporesponsive to receptor-mediated mitogenic stimulation, as judged by their proliferative response and IL-2 production. This suggests that T cells become activated in vivo in response to self-Ag presentation and display an anergic phenotype when stimulated ex vivo with Con A and anti-CD3 that activate T cells through TCR-mediated signaling events. Treatment with PMA plus ionomycin, a mitogenic regimen that activates lymphocytes in a receptor-independent and APC-independent manner, is able to stimulate splenic T cells to proliferate as well as to produce IL-2. This anergic phenotype could result from the hyper-responsiveness of thymocytes that mature and migrate to the periphery, where they become activated in response to self-Ag presentation and recognition.

[Ca2+]i measurements before and after stimulation confirm that the free [Ca2+]i levels in Tgfb1-/- splenic T cells are elevated and that Ca2+ release is impaired upon stimulation with anti-CD3. However, splenic B cells in these mice are not affected by the absence of TGF-{beta}1, suggesting that T cells are specifically affected. This is consistent with our observations that T cells, but not B cells, are the primary effectors in inflammatory disease, since Tgfb1-/- athymic nude mice also live much longer than immunocompetent Tgfb1-/- mice. FK506-binding protein-12 (FKBP12) is known to be released from association with TGF-{beta} receptor I upon ligand binding (45) and to recruit calcineurin to the inositol trisphosphate receptor complex (46), where together they are required for fully functional inositol trisphosphate receptor activity (47, 48). That FKBP12 can function in TGF-{beta} signaling is strongly suggested by the facts that Fkbp12 knockout mice have cardiomyocyte defects that involve abnormal Ca2+ channel function (49), and Fkbp12 and Tgfb2 knockout mice have similar morphological defects in the developing heart (49, 50). However, there is considerable debate regarding the role of FKBP12 in TGF-{beta} signaling, since many members of FKBP are found to be expressed ubiquitously. Therefore, it is still unclear which isoform of FKBP is involved in TGF-{beta} signaling in T cells (51, 52, 53, 54). In the absence of TGF-{beta}1 there could be disequilibria of TGF-{beta} type I receptor-associated FKBPs, resulting in leaky Ca2+ release channels.

TGF-{beta}1 is known to have multiple suppressive actions on immune cells. Based on the data presented here we hypothesize that TGF-{beta}1 acts by preventing activation and autoimmune disorders by regulating Ca2+ signaling and homeostasis. It is known that alteration of signal threshold levels due to mutations affecting the TCR complex and its downstream effectors leads to altered thymic selection and hyper-responsiveness (38, 55, 56, 57). It is therefore possible that in the absence of TGF-{beta}1, the hyper-responsive, self-reactive T cells that escape thymic negative selection are exported to the periphery. Escape to the periphery of a few autoreactive thymocytes with a lowered threshold for activation could result in enough expansion to cause autoimmune disease (58). It is also known that a few autoreactive cells always escape to the periphery, but that their activation is inhibited by TGF-{beta}1-secreting regulatory T cells (58). Thymocytes and mature naive T cells, but not memory T cells, must recognize self-MHC molecules in the thymus and periphery for their survival (36, 59, 60). However, recognition of self-MHC molecules by TCR alone can lead to activation if there are disturbances in the signaling environment (55, 56). It is known that activated T cells exhibit enhanced adhesion to endothelial cells (61), that TGF-{beta}1 inhibits the adhesion of neutrophils and T cells to endothelial cells (62, 63), and that TGF-{beta}1 affects platelet integrin activity (5). These facts are consistent with splenocytes from Tgfb1-/- mice exhibiting enhanced adhesiveness to endothelial cells (28). Since LFA-1 is up-regulated on CD3+ T cells in Tgfb1-/- mice, and since anti-LFA-1 treatment rescues these mice from inflammatory autoimmune disease, it is reasonable to speculate that Tgfb1-/- T cells adhere to endothelial cells and extravasate into peripheral organs more rapidly in Tgfb1-/- mice.

Regulatory and suppressor T cells develop in the thymus in the first week after birth. Thymectomy of neonatal mice in the first week of life, but not on the day of birth or 7 days after birth, results in the development of autoimmune disease, which can be reversed by adoptive transfer of either thymocytes from neonates or lymphocytes from spleen and lymph nodes of adult mice (58, 64). Suppressor and regulatory T cells secrete TGF-{beta}1 (21, 22). Since TGF-{beta}1 is produced in the thymus and is secreted by both suppressor and regulatory T cells, depletion of TGF-{beta}1 with Ab reverses the regulatory function of CD4+CD25+ T cells and makes them proliferate in response to mitogenic stimuli (65). However, another recent study by Shevach and his co-workers (66) found that CD4+CD25+ T cells are able to inhibit Th cell responses even in the absence of functional TGF-{beta}1 induction, suggesting that regulatory T cells can use other pathways in addition to TGF-{beta}1. Recent studies by Karlsson and co-workers (33) showed that the disruption of Tgfbr2 in bone marrow cells by an inducible knockout approach is sufficient to cause autoimmune disease. This demonstrated that TGF-{beta} functions in a cell autonomous fashion, but the underlying mechanism was not clear. Our work suggests that the mechanism underlying autoimmunity in TGF-{beta}RII-deficient mice was the blockage of TGF-{beta}1-induced signaling resulting in Ca2+ deregulation and T cell autoreactivity.

Autoreactive T cells are deleted in the thymus by negative selection. In this process T cells that recognize self-Ags with a low enough affinity so that they are not activated will survive negative selection and be exported to the periphery. Autoimmune disease can occur if there is up-regulation of self-MHC molecules and presentation of self-Ags, up-regulation of costimulatory molecules, or a lowering of the threshold level of T cell activation due to increased or decreased production of stimulatory or inhibitory cytokines (67). Naive T cells need to interact with self-MHC molecules for their survival in the periphery. Normally, interaction of TCR with self-MHC per se is not sufficient to activate a naive T cell in the periphery (60). However, since TGF-{beta}1 is a negative regulator of T cells, TGF-{beta}1-deficient T cells that exhibit a lower threshold level of activation may become activated upon engagement with self-MHC molecules. Naive T cells upon stimulation with mitogens such as Con A or anti-CD3 produce cytokines such as IL-2, IL-4, and IL-10 and subsequently proliferate. However, T cells in Tgfb1-/- mice are activated in vivo as seen by their down-regulation of surface CD3 expression and up-regulation of LFA-1, CD69, and CD122. Upon in vitro stimulation these cells do not produce enough IL-2 to proliferate, so they undergo apoptosis rather than divide. Only receptor-independent stimulation with PMA and ionomycin induces enough IL-2 production to induce Tgfb1-/- cells to proliferate. Recent studies using transgenic T cells showed that T cells desensitized with self-reactive peptides also exhibit decreased production of IL-2, decreased Ca2+ flux, and nonresponsiveness to anti-CD3 stimulation (68). It is also known that upon restimulation CD8+ T cells become unresponsive, whereas CD4+ T cells undergo activation-induced cell death (69). Consequently, splenocytes from TGF-{beta}1-deficient mice have abnormally high [Ca2+]i levels, resulting in self-activation in vivo and anergy ex vivo.

TGF-{beta}1 mediates its inhibitory effects on many cell types, including lymphocytes, through Caenorhabditis elegans Sma and Drosophila Mad proteins (SMAD)2 and SMAD3. However, there is evidence suggesting that TGF-{beta}1 can mediate some of its effects, such as inducing fibronectin expression, through SMAD-independent mechanisms (70). Consequently, Smad3-null thymocytes and mature T cells were found to be normal in their mitogenic response, although resistant to TGF-{beta}1-mediated suppression of their mitogenic response (71, 72). Since SMAD3-deficient mice live much longer than TGF-{beta}1-deficient mice due to less severe forms of inflammation, and since SMAD3-deficient thymocytes are not hyper-responsive to mitogenic stimulation, as we have shown TGF-{beta}1-deficient thymocytes to be, we can conclude that regulation of [Ca2+]i levels by TGF-{beta}1 is SMAD3 independent.

In conclusion, TGF-{beta}1 is critical for maintaining immune homeostasis and prevention of autoimmunity. Naive T cells must interact with self-MHC to survive in the periphery. Self-activation is normally inhibited because [Ca2+]i levels are not sufficient for synergy with costimulatory pathways. TGF-{beta}1-deficient T cells have spontaneously high [Ca2+]i levels, which alters the threshold level for activation upon TCR/MHC interaction. Upon self-Ag recognition they become activated, cause an autoimmune-type inflammatory response, and become anergic to ex vivo stimulation.


   Acknowledgments
 
We thank Sandi Engle for the suggestion of using the inflammation index. We thank Wen Yun Sun for expert assistance with PCR genotyping, and James Cornelius for expert assistance with flow cytometry. Sandy Schwemberger’s help with flow cytometry and analysis of Ca2+ measurements is greatly appreciated.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants HD26471, ES05652, ES06096, and CA84291 (to T.D.) and AR47322 and AR47363P&F (to R.R.S.) and by a grant from Shriners of North America (to G.F.B.). Back

2 Address correspondence and reprint requests Dr. Thomas Doetschman, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524. E-mail address: thomas.doetschman{at}uc.edu Back

3 Abbreviations used in this paper: [Ca2+]i, intracellular Ca2+; CLM, cell loading medium; FKBP, FK506-binding protein; MFI, mean fluorescence intensity; PKC, protein kinase C; SMAD, Caenorhabditis elegans Sma and Drosophila Mad proteins. Back

Received for publication November 19, 2002. Accepted for publication February 24, 2003.


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