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The Journal of Immunology, 2001, 166: 241-248.
Copyright © 2001 by The American Association of Immunologists

The Identification of a Novel T Cell Activation State Controlled by a Diabetogenic Gene1

Jodene K. Moore*, Robert I. Scheinman{dagger} and Donald Bellgrau2,*

* Department of Immunology, Barbara Davis Center for Childhood Diabetes, School of Medicine and {dagger} Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cyclin-dependent kinase inhibitor p27kip regulates the cell cycle at the G1-S phase restriction point. S phase entry and cell cycle commitment in peripheral T cells requires p27kip degradation, normally initiated by the receipt of costimulatory signals such as those provided by B7.1 or IL-2. We have previously reported that T cells from BioBreeding (BB)-diabetes-prone (DP) rats exhibit decreased costimulatory requirements for activation and cell cycle entry. In the present study, we find that peripheral T cell subsets from BB-DP rats demonstrate activation-like characteristics, including significantly reduced levels of p27kip as well as increased levels of proliferating cell nuclear Ag (PCNA). Since our previous studies have established that expression of extracellular activation markers are relatively low in unmanipulated peripheral BB-DP T cells; this p27low PCNAhigh phenotype represents a novel activation state. Analyses of T cell subsets from congenic rats demonstrate that this phenotype segregates with the lyp diabetogenic locus and that the p27low PCNAhigh phenotype is T cell specific. This p27low PCNAhigh phenotype is not seen in medullary thymocytes, but appears abruptly in the recent thymic emigrant population, suggesting that the lyp locus does not act directly on cell cycle regulators but rather alters the interaction between T cells and the peripheral environment. These results provide a biochemical basis for costimulation-independent activation and suggest a mechanism whereby a diabetes susceptibility gene contributes to disease development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The BioBreeding (BB)3 rat has provided an excellent animal model of type I diabetes (1). Selective breeding of an outbred colony from which the BB strain was derived (2) led to the development of two sublines, one diabetes prone (DP) and one diabetes resistant (DR). The BB-DP and BB-DR sublines appear to differ in only one disease susceptibility locus (3), mapping to a region of rat chromosome 4 (4, 5). This locus is commonly referred to as lyp, as it produces a profound peripheral T cell lymphopenia (6).

The peripheral T cell pool is diminished by as much as 80% in BB-DP animals. The RT6 differentiation Ag is expressed on the majority of mature T cells postthymically in normal rats while T cells from lymphopenic animals express low or undetectable levels of the RT6 differentiation Ag (7). RT6+ T cells are considered to function as regulators that prevent the development of autoimmunity (1). Indeed, reconstitution of BB-DP rats with RT6+ T cells from non-lymphopenic BB-DR donors protects BB-DP recipients from disease, suggesting that the absence of RT6+ T cells is associated with disease onset (8). Up-regulation of RT6 is associated with the down-regulation of Thy1 (9). In BB-DP rats, the frequency of Thy1+ T cells is markedly increased concurrent with the absence of T cells expressing RT6 Ag (10). One hypothesis stemming from this observation is that RT6+ regulatory T cells never mature and the periphery of the lymphopenic animal is populated by RT6- T cells that are not regulated by RT6+ mature T cells. Consistent with this hypothesis, the BB-DP RT6 allele was shown to be expressed (and thus functional) in F1 crosses, suggesting that the Thy1+ RT6- phenotype is caused by a lyp gene-mediated block in T cell development (11, 12).

It is well established that the deletion of high-affinity, self-reactive T cells occurs in the thymus during the negative selection process (13). However, this process appears to be incomplete, requiring the contribution of peripheral tolerance mechanisms to maintain a T cell repertoire that is devoid of overt autoreactivity (14). Therefore, lyp could exert its effect by interfering with either or both thymic and peripheral tolerance mechanisms. Thymic and peripheral tolerance induction both involve the paradigm commonly described as the two-signal model for T cell activation (15, 16). A basic tenet of the model is that T cells are signaled not only by an interaction through the Ag-specific TCR but also through various costimulatory interactions provided most commonly by costimulatory molecules expressed on APCs (15, 16). It is widely held that signal one (Ag interaction with TCR) without signal two (costimulation) can tolerize peripheral T cells either by anergizing them or causing them to be deleted. In the thymus, signals one and two appear to function differently than they do in the periphery. Evidence indicates that signal one plus two, when provided by thymic APCs to CD4+CD8+ cortical thymocytes (17) and a subset of "single positive" more mature medullary thymocytes (18), initiate anergy or deletion.

We hypothesized that defects in the way T cells respond to these two signals may permit them to escape tolerizing signals, thus promoting the survival of autoreactive T cells. To examine this, we developed a two-signal in vitro activation model to test T cells at various stages in development. Using peripheral lymphocytes, we observed that the provision of signal one alone was sufficient to induce cell cycle progression in T cells from lymphopenic rats while T cells from nonlymphopenic rats remained quiescent (19). Similarly, we found that signal one alone was sufficient to promote the up-regulation of the activation marker OX-40, but that once again, this was limited to T cells from lymphopenic rats. We used the term "promiscuous activation" to describe this costimulation-independent progression into the cell cycle. In the present study, using biochemical approaches, we provide evidence indicating that the cell cycle machinery in unmanipulated T cells derived from BB-DP rats is characteristic of an activated T cell in the absence of in vitro stimulation and arises upon export of thymocytes to the periphery.


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

Specific-pathogen free BB-DP and BB-DR rats were purchased from the University of Massachusetts Medical Center (Worcester, MA). Diabetes-resistant, specific pathogen-free Lewis (LEW), and Sprague Dawley rats were purchased from Charles River Breeding Laboratories (Wilmington, MA). Lymphopenic Fischer (Lyp F344) breeding pairs (4) were kindly provided by Åke Lernmark (R. H. Williams Laboratory, University of Washington, Seattle, WA). Lyp F344 rats were interbred and housed in the Barbara Davis Center animal colony and screened for homo- or heterozygosity at the lymphopenia locus by flow cytometric quantitation of peripheral T cell number and genotypic analysis. Lymphocyte donors were all used at 28–40 days of age. Backcross progeny were weaned at 3 wk of age, marked with ear punches, and a small section of tail was clipped. Genomic DNA was isolated and screened by PCR as described previously (20). Characteristically, lymphopenic animals demonstrated <25% TCR/CD3 double-positive cells and <5% TCR/CD8 double-positive cells (data not shown).

Monoclonal Abs

The following mouse mAbs to Ags on rat lymphoid cells were purchased from PharMingen (San Diego, CA) and used in flow cytometric analysis and in vitro triggering: anti-TCR (clone R73) (21), anti-CD3 (clone G4.18) (22), and anti-CD28 (clone JJ319) (23). Peripheral T cell purification was conducted using the following biotinylated Abs: anti-rat Ig (RIg) (BioSource International, Camarillo, CA), anti-RT1B (clone OX-6; PharMingen) (24), anti-CD11b/c (clone OX-42; PharMingen) (25), and anti-CD90 (Thy1 Ag) (clone OX-7; PharMingen) (9).

mAbs used in immunoblotting included: anti-p27kip (clone 57; Transduction Laboratories, San Diego, CA) (26), antiproliferating cell nulcear Ag (PCNA) (clone 24, Transduction Laboratories) (27), anti-heat shock protein (hsp) 110, (clone 21; Transduction Laboratories) (28), and anti-Zap70 kinase (clone 29; Transduction Laboratories) (29). Secondary HRP-conjugated anti-rabbit Ig Abs (Bio-Rad, Richmond, CA) were used for detection in Western blots.

Peripheral T cell purification

Cervical and mesenteric lymph nodes were harvested from young male rats. Lymph nodes were homogenized to single-cell suspensions using modified tissue grinders as described previously (30). Cells (100 x 106) from normal rat lymph node suspensions were incubated with a mixture of the following biotinylated Abs: 38 µg anti-RIg, 15 µg anti-RT1B, and 10 µg anti-CD11b/c, in a final volume of 3 ml HBSS supplemented with 2% FCS (2% HBSS). Cells plus Ab were then incubated on ice for 30 min, with inversion of tubes to mix contents every 10 min. Cells were washed twice with 2% HBSS to remove unbound Ab, followed by incubation with streptavidin-conjugated magnetic beads (BioSource International) added at a density of 10 beads per target cell. The cell/bead mixture was incubated on ice for 30 min, with inversion every 10 min. Ab-bound B cells and APCs were removed from the suspension by two 10-min incubations on an elemental magnet (BioSource International). Lymphopenic rat T cells were prepared in the same manner with the exception of the Ab mixture, which contained 41 µg anti-RIg, 18 µg anti-RT1B, and 13 µg anti-CD11b/c. The increase in Ab concentration reflected the need to remove a higher percentage of non-T cells from the starting population. The lymphopenic status of the donor also resulted in a reduced T cell yield. In most cases, yields for lymphopenic animals were in the range of 5 x 106 T cells/animal. Nonlymphopenic animals usually provided 10 times that number. Efficiency of APC removal was assessed by FACS analysis of cells stained with anti-TCR and anti-CD3 mAbs. T cell purity was >90% for normal rats and >85% for lymphopenic rats.

Recent thymic emigrant (RTE) purification

Purified peripheral T cells were positively selected for Thy1 (CD90) expression by panning on anti-CD90-coated 6-well plates. Wells were incubated with 20 µg purified anti-Thy1 Ab in PBS. Ab incubation was performed overnight at 37°C, followed by two washes with PBS to remove unbound Ab. A total of 5 x 106 Thy1+ T cells, as assessed by flow cytometric analysis, was suspended in 1 ml of 2% HBSS and incubated in each well for 1 h at 4°C, with shaking every 20 min. Nonadherent and loosely adherent cells were removed from the wells following two washes with 2% HBSS.

Medullary thymocyte isolation

Rat thymi were harvested and homogenized. A total of 100 x 106 cells from thymocyte suspensions was incubated with a mixture of the following biotinylated Abs: 33 µg anti-CD8a, 33 µg anti-CD8b, and 10 µg anti-CD11b/c, in a final volume of 3 ml of 2% HBSS. Cells plus Ab were then processed exactly as described for peripheral T cells. As before, contaminating cells (Ab-bound CD8-positive thymocytes and APC) were removed using an elemental magnet. Characteristically, medullary thymocyte purity, as assessed by flow cytometric quantitation of TCRhigh cells, was >80% for normal and lymphopenic rats, with <7% contamination by TCRlow cortical thymocytes. Removal of TCR-negative cells in the population was accomplished by positive panning in an additional selection step on anti-TCR-coated wells. Wells were incubated overnight with 5 µg/ml anti-TCR in PBS at 37°C, followed by two washes with PBS. Enriched medullary thymocytes suspended in 2% HBSS were incubated on anti-TCR-coated wells for 1 h, with gentle agitation every 20 min, to uniformly disperse cells. Nonadherent cells were removed by two washes with 2% HBSS.

Fibroblast primary cell cultures

Primary fibroblast cell cultures were established from normal and lymphopenic rat fetal skin or adult abdominal fascia. Fetal skin or adult abdominal fascia was removed under sterile conditions and subjected to four washes in sterile DMEM supplemented with 1x penicillin (Pen)/streptavidin (Strep) (Life Technologies, Rockville, MD). Tissue was then minced into 1-mm2 sections and incubated with 25 ml DMEM supplemented with 10% FCS and Pen/Strep in a T-75 flask for 1 wk. Confluent flasks of fibroblasts were washed twice with sterile PBS and then incubated with 3 ml trypsin/EDTA for 5 min at 37°C. Medium-containing serum was added to quench trypsin activity and cells were pelleted by centrifugation. Passage 2 fibroblasts derived from lymphopenic and normal rats were seeded into T-75 flasks at equal numbers and grown to confluence. Medium was changed once, and cells were carried at confluence for an additional week to induce quiescence through contact inhibition.

B cell isolation

Rat spleens were removed, homogenized into single-cell suspensions, washed in HBSS, and resuspended in buffered ammonium chloride. Cells were washed twice in HBSS and resuspended in 2% HBSS. Aliquots of spleen suspension were stained with anti-RT1B-FITC and anti-CD45RA-FITC and subjected to FACS analysis to determine the percentage of B cells within the splenic suspension.

Aliquots of splenocyte suspension containing 5 x 106 B cells in a final volume of 1 ml of 2% HBSS were incubated on 6-well plastic dishes coated with 40 µg/ml rabbit anti-rat Ig and incubated for 60 min at 4°C, with shaking every 20 min to disperse the cells. Nonadherent and loosely adherent cells were removed by two vigorous washes with 2% HBSS. Bound B cells were then washed twice with cold PBS and lysed in 200 ml RIPA lysis buffer in preparation for Western blot analysis. FACS analysis of bound cells demonstrated that this population was >85% RT1B+ and CD45RA+ in both normal and lymphopenic populations.

In vitro T cell triggering

Six-well plates were incubated with anti-TCR Abs (1 µg/ml) in PBS for 12 h at 37°C. Wells were washed twice with PBS and then incubated for 1 h at 37°C with Iscove’s medium supplemented with 2% FCS. Purified pooled T lymphocytes (5 x 106) were resuspended in 1 ml Iscove’s medium supplemented with 5% FCS and were subsequently incubated at 4°C for 1 h. The plates were gently agitated every 15 min during the 4°C incubation to disperse the cells into a uniform monolayer. The supernatant containing nonadherent cells was removed, and the monolayer was washed twice with Iscove’s medium to remove loosely adherent cells. Four milliliters of Iscove’s medium supplemented with 10% FCS, Pen/Strep, glutamine, and 2-ME was added to each well. This medium was additionally supplemented with either nothing, 10 U/ml recombinant human IL-2 (Sigma, St. Louis, MO), or 1 µg/ml anti-CD28. Cells were then incubated at 37°C in 5% CO2 until harvested for flow cytometric or Western blot analysis at 48 h.

Western blot analysis

Cells were isolated as described above and lysed in RIPA lysis buffer (25 mM Tris (pH 7.2), 30 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40) containing the protease and phosphatase inhibitors aprotinin (2 mg/ml), leupeptin (2 mg/ml), sodium fluoride (1 mg/ml), PMSF (1 mM), DTT (1 mM), and sodium orthovanadate (1 mM). Samples were placed on ice for 10 min, then DNA was sheared by passage through 21-gauge needles at 4°C. After an additional 15-min incubation on ice, samples were centrifuged at 15,000 x g for 15 min at 4°C, and the supernatants were collected as whole cell extracts.

Equivalent amounts of protein (10 µg), as measured by the bicinchoninic acid assay (Pierce, Rockford, IL) were resolved by SDS-PAGE on 8% minigels and transferred to a nitrocellulose membrane (15.5 V, 5.5 mA/cm2 for 60 min). Given the lymphopenic status of some donors animals, this necessitated pooling two to three lymphopenic donor equivalents for each Western blot lane. The membranes were blocked overnight in 5% nonfat dry milk/0.1% Tween 20 in TBS (4°C) and then incubated with the primary Ab for 2.5 h (25°C). After multiple 5 min washes in TBS-0.1% Tween 20 for 30 min (25°C), membranes were incubated for 2 h with HRP-conjugated secondary Ab (25°C), washed as before, and binding was visualized by ECL (Amersham, Arlington Heights, IL) and autoradiography. Quantitation of ECL-labeled protein bands on MX-Light ECL film (Kodak, Rochester, NY) was accomplished by digitally scanning films into ImageQuant software (Molecular Dynamics, Sunnyvale, CA). p27kip and PCNA protein relative expression is defined as the ratio between p27kip or PCNA expression and a loading control which consisted of either Zap70 (T cells) or hsp110 (fibroblasts and B cells).

Statistical analysis

All experiments were performed using T cell pools from age-matched rat strains. This was necessary due to the paucity of cell numbers obtained from lymphopenic rats and to the size of the experiments that were conducted. All data were therefore nonparametric. Nonparametric data were analyzed for statistical significance using the Kruskal-Wallis test (31). Analysis of data was performed with JMP statistical software (SAS Institute, Cary, NC). Statistical significance was accepted on the basis of p values <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lymphopenic BB-DP rat peripheral T cells exhibit p27kip levels associated with activated T cells

p27kip acts as the primary regulatory molecule blocking progression through the G1 restriction checkpoint in T cells until activation induces p27kip degradation (32). To compare the status of p27kip in T cells from lymphopenic and nonlymphopenic animals, peripheral T cells from a variety of rat strains were isolated and extracts subjected to Western blot analysis as described in Materials and Methods. Blots were then probed with Abs for p27kip and the T cell-specific Zap70 kinase as a loading control. A representative Western blot of p27kip levels in peripheral T cells is shown in Fig. 1GoA. Inspection of p27kip levels in these strains indicated that T cells derived from LEW, F344, and BB-DR rats expressed increased p27kip protein in comparison to lymphopenic BB-DP peripheral T cells. Densitometric analysis and quantitation of these blots confirmed this finding. Mean p27kip expression levels, normalized to Zap70, for several experiments is summarized in Fig. 1GoB. Lyp BB-DP peripheral T cells exhibited, on average, an 89.0% reduction in p27kip protein expression (p27low phenotype) as compared with T cells from nonlymphopenic strains.



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  		FIGURE 1. Measurement of p27kip protein in various T cell subsets from lymphopenic and non-lymphopenic rat strains. T cell populations were isolated as described in Materials and Methods. A, Representative Western blot of p27kip and Zap70 protein levels. Samples are shown in duplicate from one experiment. Zap70 is used as a control for equal loading. B, Quantitation of relative p27kip expression for peripheral T cells from densitometric analysis of multiple independent experiments. SEM are indicated. Data are normalized to Zap70 expression. Data represents three to five independent experiments. C, Quantitation of relative p27kip expression for the RTE subpopulation of peripheral T cells. Data were quantitated as described for B. SEM are indicated. Data represent three independent experiments. D, Quantitation of relative p27kip expression for medullary thymocytes. Data were quantitated as described for B. SEM are indicated. Data represent three independent experiments.

 
The p27low phenotype is not a generic property of Thy1+ RTEs

One potential explanation for the differences seen in p27kip levels in BB-DP T cells is that these properties are intrinsic to a subpopulation of T cells found in the normal animal, such as the RTE population. Unlike the mouse, where a marker for the RTE population has yet to be identified, in rats, the RTE population has been shown to consist of Thy1+ T cells (9, 33). In normal rats this population is small, but in BB-DP animals, it represents a major portion of the peripheral T cell pool (12). Analysis of our purified T cell preparations from 4- to 6-wk old BB-DP animals indicated that ~60% of the population was Thy1+ (data not shown). To examine p27kip levels in the Thy1+ RTE subset from various rat strains, we purified peripheral T cells by positive selection on anti-Thy1 Ab-coated wells as described in Materials and Methods. Bound Thy1+ T cells were lysed directly in the wells and analyzed by Western blot to determine relative levels of p27kip normalized to Zap70 expression. Fig. 1GoC shows the densitometric quantitation of several independent experiments made from Thy1+ LEW, Fischer (F344), BB-DR, and Lyp BB-DP RTE cells and is quite similar to the data shown in Fig. 1GoB. Inspection of these blots indicated that p27kip levels in Thy1+ RTE from nonlymphopenic strains were roughly equivalent, whereas Thy1+ RTEs from the lymphopenic BB-DP strain exhibited a 78% reduction in p27kip expression. These data indicate that p27kip expression levels in the RTE subset is indistinguishable from that seen in the total peripheral T cell population (compare Fig. 1Go, B and C).

Medullary thymocytes from lymphopenic donors do not express the p27kip low phenotype

We next examined the status of the p27kip phenotype in the medullary Thy1+ thymocyte subsets from both nonlymphopenic F344 and lymphopenic BB-DP rats. Medullary thymocytes (defined as non CD4/8 double positive with high TCR expression) were prepared using magnetic bead depletion as described in Materials and Methods. Following a final purification by incubation on anti-TCR-coated wells, wherein nonadherent cells were removed by washing, the cells that remained bound to the wells were >95% CD3high. Cellular lysates were prepared and examined by Western blot to determine the levels of p27kip protein ex vivo. As before, p27kip levels were quantitated relative to Zap70 expression. An average of several experiments comparing p27kip protein levels in F344 and Lyp BB-DP medullary thymocytes appears in Fig. 1GoD, illustrating that relative p27kip expression levels are equivalent between different rat strains. These data revealed that p27kip protein levels in BB-DP thymocyte subsets from lymphopenic donors closely resembled those in non-lymphopenic donor thymocyte subsets through the medullary thymocyte stage of development and suggest that the p27kip low phenotype is only apparent when cells leave the thymus.

Decreased levels of p27kip in peripheral T cells segregates with lyp

The only difference in diabetogenicity between BB-DR and BB-DP strains is the region of chromosome 4, referred to as lyp. To determine whether lyp alone contributes to this cell cycle phenotype, we obtained Lyp F344 mice from Å. Lernmark; in which the lyp region of chromosome 4 has been backcrossed into the F344 background (4). The frequency and phenotype of peripheral T cells from Lyp F344 rats are not noticeably different from a comparative analysis with T cells from the lymphopenic BB-DP rat; however, Lyp F344 rats do not develop diabetes because they lack other diabetes susceptibility genes including the BB MHC haplotype RT-1u (4). Peripheral T cells were isolated from phenotypically and genotypically confirmed Lyp F344 rats and subjected to Western blot analyses. Blots were probed with anti-Zap70 and anti- p27kip Abs as described above. Normalized p27kip levels from peripheral T cells, RTE, and medullary thymocytes obtained from Lyp F344 animals are shown in Fig. 1Go (B–D). Although p27kip levels in peripheral T cells from non-lymphopenic F344 and BB-DR rats were similar; a clear difference was seen comparing lymphopenic to nonlymphopenic animals. As seen for the BB background, this p27kip low phenotype was evident only in Lyp F344 peripheral T cells. Taken together, these data support the hypothesis that reduced p27kip levels segregates with the lyp locus and manifests only in peripheral T cells.

Downstream components of cell cycle progression correlate with the p27kip phenotype

To more fully characterize the phenotype of T cells derived from animals containing the lyp locus, we examined the status of cell cycle progression at several points downstream from p27kip degradation. An immediate consequence of p27kip degradation is the activation of cyclin-cyclin-dependent kinase (cdk) complexes and the phosphorylation of retinoblastoma (Rb). We measured Rb phosphorylation in freshly isolated, unmanipulated ex vivo lysates from LEW, lymphopenic BB-DP, and lymphopenic F344 rat T cells by Western blotting. As shown in Fig. 2Go, Rb from unstimulated LEW peripheral T cells consistently presented as a single band. In the presence of the lymphopenia locus, either in the BB background or the F344 background, Rb was 2- to 4-fold more abundant and consistently presented as two bands, the higher molecular weight band corresponding to hyperphosphorylated Rb. As shown in Fig. 2Go, the degree of phosphorylation of Rb in lymphopenic animals was variable from preparation to preparation. In multiple experiments, we found that these differences did not consistently segregate with the different genetic backgrounds. Thus, Rb was found to be more abundant and hyperphosphorylated in the presence of the lymphopenia locus and the amount of hyperphosphorylated Rb was not significantly different between F344 and BB genetic backgrounds.



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  		FIGURE 2. Measurement of Rb in lymphopenic and non-lymphopenic rat strains. Representative Western blot of Rb protein from peripheral T cells from LEW, BB-DR, and Lyp BB-DP rats. Zap70 levels are shown as a loading control. Rb protein; pp-Rb, hyperphosphorylated Rb.

 
PCNA is an inducible protein which functions as a requisite processivity factor for DNA polymerase {delta} (34, 35). PCNA expression increases 1000-fold during mid-G1 of the cell cycle and remains high while the cells proliferate following mitogenic stimulation (36, 37, 38, 39). When T cell proliferation ceases, both the biosynthesis and accumulation of PCNA also cease (37). Western blots of lysates derived from LEW, BB-DR, and BB-DP peripheral T cells were probed using an anti-PCNA Ab (Fig. 3GoA). Consistent with our p27kip data, T cells derived from LEW and BB-DR strains had barely detectable PCNA expression while T cells from the p27kip low BB-DP strain expressed large amounts of PCNA. We also compared PCNA expression in RTE as well as total peripheral T cell pools from F344 rats and Lyp F344 rats as shown in Fig. 3GoB. Once again, cells from Lyp F344 rats exhibited significantly higher levels of PCNA than their nonlymphopenic counterparts in both RTE-purified T cells and peripheral T cells.



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  		FIGURE 3. Increased levels of PCNA protein segregate with Lyp and originate at the RTE stage in ontogeny. A, Representative Western blots of PCNA protein from peripheral T cells from LEW, BB-DR, and BB-DP rats. Unstim denotes that the cells were not stimulated before extracts were prepared. B, Representative Western blots of PCNA protein from F344 (left) and Lyp F344 (right) Thy1+ RTEs. Again, Zap70 protein levels are shown to demonstrate equal protein loading.

 
The observation of unphosphorylated Rb and low PCNA expression levels in non-lymphopenic T cells supports the interpretation that the majority of these peripheral T cells are in a resting state. In contrast, hyperphosphorylated Rb and high PCNA levels in ex vivo peripheral T cells from Lyp BB-DP and Lyp F344 rats suggest that these cells are activated.

p27kip degradation and elevated PCNA expression in normal rat T cells requires TCR cross-linking

To confirm that p27kip degradation and PCNA up-regulation are good measures of activation as well as proliferation in rat T cells, we performed the control experiment of following the fate of these proteins after stimulation. Purified LEW peripheral T cells were stimulated in vitro with anti-TCR and anti-CD28 Abs. Lysates were made at 24 h for Western blot analysis of p27 kip levels. Results of Western blot analysis showed that unstimulated LEW peripheral T cells, cultured for 24 h, expressed p27kip levels equivalent to those found in freshly isolated LEW peripheral T cells (Fig. 4GoA). Stimulation of LEW peripheral T cells with the combination of anti-TCR plus anti-CD28 Abs resulted in complete degradation of p27kip protein within 24 h. Fig. 4GoB shows a representative Western blot of PCNA expression in untreated vs in vitro-stimulated LEW peripheral T cells. Unstimulated LEW peripheral T cells cultured for 24 h demonstrated barely detectable levels of PCNA similar to those seen in freshly isolated T cells. LEW peripheral T cells triggered with anti-TCR plus anti-CD28 Abs demonstrated a high level of PCNA expression.



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  		FIGURE 4. Degradation of p27kip and up-regulation of PCNA in normal peripheral T cells requires TCR cross-linking in combination with costimulation. Peripheral T cells from LEW rats were purified and cultured for 24 h as described in Materials and Methods. Cultures were either left untreated (Unstim 24 h) or stimulated with anti-TCR plus anti-CD28 Abs (TCR/CD28 24 h) as described. Cultures were lysed and analyzed by Western blot. A, Representative Western blot of p27kip protein levels following 24-h culture in the absence or presence of stimulation. As before, Zap70 is used as a control for equal loading. B, Representative Western blot of PCNA protein levels following 24-h culture in the absence or presence of stimulation. Zap70 is used as a control for equal loading.

 
Lymphopenic-induced reductions in p27kip are restricted to the T cell lineage

We next determined whether lyp conferred a global change in p27kip expression levels or whether this phenotype was tissue restricted. Primary fibroblast lines were derived from fetal skin or adult abdominal fascia from F344 and lymphopenic F344 rats and rendered quiescent through contact inhibition as described in Materials and Methods. In this series of experiments, p27kip protein levels were normalized to hsp110 levels, as a control for equal protein loading. A representative Western blot of p27kip protein levels in F344 and Lyp F344 fibroblasts is shown in Fig. 5GoA. Inspection of these blots indicated that lymphopenic fibroblasts did not exhibit reduced p27kip levels in comparison to their nonlymphopenic counterparts. Activation of cycling in these cells through serum stimulation resulted in p27kip degradation (data not shown).



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  		FIGURE 5. Primary fibroblasts and splenic B cells from lymphopenic rats demonstrate normal p27kip protein levels. Representative Western blots of p27kip and hsp110 protein levels from tissues derived from F344 and Lyp F344 animals. Data are shown in triplicate. hsp110 protein is shown to demonstrate equal protein loading. No significant differences were detected in the levels of p27kip protein in fibroblasts from lymphopenic vs nonlymphopenic strains. A, Primary fibroblasts derived from adult abdominal fascia. Relative expression for p27kip from F344, 2.20 ± 0.04 and Lyp F344, 2.28 ± 0.12. B, Splenic B cells purified as described in Materials and Methods. Relative expression for p27kip from F344, 1.77 ± 0.19 and Lyp F344, 1.73 ± 0.12.

 
To further investigate the p27kip defect within the hematopoietic lineage of lymphopenic rats, we isolated splenic B cells from F344 and lymphopenic F344 rats by positive panning of RBC-depleted splenocyte suspensions on anti-Ig Ab-coated plastic. p27kip protein levels were normalized against hsp110-loading controls for each sample. A representative Western blot of p27kip protein levels in F344 and Lyp F344 B cells is shown in Fig. 5GoB. Inspection of these blots showed no evidence of quantitative differences in p27kip levels between B cells from nonlymphopenic and lymphopenic rats. The data suggest that the association of lyp and reduced p27kip levels does not extend beyond the T cell lineage in lymphopenic rats.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously described the costimulation-independent cell cycle progression of T cells from lymphopenic BB-DP animals and coined the term "promiscuous activation" to describe this phenomenon (19). In the present study, we examined internal measures of lymphocyte activation and observed that "resting" T cells (isolated from the lymphopenic animal and tested without further in vitro manipulation) expressed low levels of the cdk inhibitor p27kip as well as hyperphosphorylated Rb and high levels of PCNA. These biochemical markers are indicative of a population of actively cycling cells. Conversely, we and others have demonstrated that this population of T cells expresses elevated levels of the QCA Ag, a marker for quiescence (Refs. 10, 40 , and our unpublished data). Our previous studies of OX-40 expression shows a slight elevation, which could be interpreted as evidence for either quiescence or modest activation (19). In contrast, our present study of the intracellular environment of these cells indicates a marked state of activation comparable to high-dose TCR plus CD28 stimulation that only becomes evident upon thymic export. Consistent with our findings, others have reported that the DNA content of peripheral BB-DP T cells is markedly increased, indicating a large population of cycling cells (41). This novel activation state of T cells, expressing surface markers associated with resting cells and an internal biochemistry resembling activated cells, supplies us with a possible mechanism for the promiscuous activation phenotype. Cell cycle progression in T cells from lymphopenic donors may occur with a decreased costimulatory requirement because these cells are primed through the degradation of p27kip.

P27kip functions to inhibit cell cycle progression by inhibiting the ability of the cyclin-activating kinase to phosphorylate cyclin-cdk complexes (42, 43). Cell cycle progression involves the rapid synthesis, assembly, and activation of various cyclin-cdk complexes which then phosphorylate critical substrates (42, 44, 45). One such substrate is Rb, which is phosphorylated by cyclin D-cdk4 and cyclin E-cdk2 complexes (46). Hypophosphorylated Rb binds the transcription factor E2F. Upon cyclin/cdk-mediated phosphorylation of Rb, E2F is released and can induce the expression of new genes necessary for cell cycle progression.

The majority of G0 peripheral T cells isolated from blood or secondary lymphoid organs exist in a quiescent state and express high levels of the p27kip protein (47, 48, 49, 50, 51). TCR triggering alone, in normal T cells, is insufficient to promote G1-S phase progression, in part, due to p27kip-mediated inhibition of cyclin-cdk complexes (52, 53, 54). The second signal, through IL-2R or CD28 ligation, acts in combination with TCR triggering to promote p27kip degradation and cell cycle progression (47, 55). p27kip degradation initiated through CD28 ligation involves signaling through an unidentified IL-2-independent pathway (55), whereas the pathway initiated by IL-2R triggering has been partially elucidated and involves phosphatidylinositol 3-kinase and protein kinase B-mediated signaling (50). Degradation of p27kip involves ubiquitin-mediated targeting of p27kip to the 26S proteosome (56, 57, 58, 59).

What causes this degradation of an essential cell cycle regulator in T cells from lymphopenic donors? Our examination of T cells at various stages of maturation indicated that this novel activation state originates at the RTE stage. Since medullary thymocytes from lymphopenic animals express p27kip levels indistinguishable from normal animals as do fibroblasts and B cells, we must conclude that lyp does not directly affect p27kip levels. Lyp may function by altering the process of thymocyte maturation and/or by altering the nature of the T cell response to peripheral signals. The status of cell cycle regulators such as p27kip, Rb, and PCNA is indistinguishable from normal activated T cells. Thus, both this study as well as the work of others (41) have failed to find evidence of altered thymocyte properties, supporting the hypothesis that lyp functions during the thymocyte/RTE transition to modify T cell responses to peripheral signals. Conversely, thymic organ cultures from BB-DR and BB-DP animals have uncovered more pronounced differences in thymic output, suggesting that lyp alters the thymocyte developmental process (60). Our data show that lyp alone is sufficient to create this peripheral cell cycle phenotype, suggesting that the protein encoded by lyp must play a role in the regulation of T cell maturation and/or peripheral signaling events.

Upon export to the periphery, long-term T cell survival in the absence of conventional antigenic stimulation is dependent on continuous contact with the MHC, a process termed peripheral positive selection (61, 62, 63, 64, 65, 66, 67). Lack of contact with peripheral positive-selecting ligands induces a short life span in peripheral T cell subsets (65, 66, 68). Thus, one possibility is that lyp alters the T cell response to peripheral positive selection.

The lymphopenic environment itself may contribute to the proliferative state of these T cells. Adoptive transfer of murine peripheral T cells into lymphopenic or syngeneic T cell-deficient nude, SCID, or RAG2-/- mice leads to marked proliferation of the donor cells (69, 70), suggesting that T cell "space" may result in a change in the cytokine environment, allowing T cell proliferation under these conditions. Adoptive transfer of large doses of T cells into lymphopenic hosts, on the other hand, does not lead to peripheral expansion or changes in cellular phenotypes (69, 71). This suggests the possibility that the peripheral T cells from lymphopenic donors are activated because they receive clonal expansion signals provided by the lymphopenic environment in much the same way that T cells from nonlymphopenic animals have been shown to clonally expand in a lymphopenic environment.

Our data can be also interpreted as characteristic of previous activation through antigenic exposure, the latter perhaps in the form of an autoantigen. Regardless of the source of the peripheral antigenic stimulus, it is clear from our data that the T cell response to this unidentified stimulus is controlled by the diabetes susceptibility gene lyp and the phenotype that we have described is a consequence of lyp gene involvement in that T cell stimulus.


   Acknowledgments
 
We thank Åke Lernmark for the generous provision of sufficient Lyp F344 rats to establish a colony for this study. In addition, we thank Dr. David Stenger and Dr. Julie Lang for critical readings of this manuscript and many valuable suggestions.


   Footnotes
 
1 This work was supported by grants to D.B. from the National Institutes of Health (AI48805), the Juvenile Diabetes Foundation (198200), and the American Diabetes Association. R.I.S. is supported by grants from the Arthritis Foundation and the American Diabetes Association. Back

2 Address correspondence and reprint requests to Dr. Donald Bellgrau, Barbara Davis Center for Childhood Diabetes, University of Colorado Health Science Center, B, 4200 East Ninth Avenue, Denver, CO 80262. Back

3 Abbreviations used in this paper: BB, BioBreeding rat; DP, diabetes prone; DR, diabetes resistant; LEW, Lewis rat; Lyp F344, lymphopenic Fischer 344 rat; PCNA, proliferating cell nuclear Ag; RTE, recent thymic emigrant; Pen/Strep, penicillin/streptavidin; cdk, cyclin-dependent kinase; Rb, retinoblastoma; hsp, heat shock protein; RIg, rat Ig. Back

Received for publication March 29, 2000. Accepted for publication October 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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