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The Journal of Immunology, 2003, 171: 2840-2847.
Copyright © 2003 by The American Association of Immunologists

A Failure to Repair Self-Proteins Leads to T Cell Hyperproliferation and Autoantibody Production 1

Hester A. Doyle, Renelle J. Gee and Mark J. Mamula2

Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is clear that many factors can perturb T cell homeostasis that is critical in the maintenance of immune tolerance. Defects in the molecules that regulate homeostasis can lead to autoimmune pathology. This simple immunologic concept is complicated by the fact that many self-proteins undergo spontaneous posttranslational modifications that affect their biological functions. This is the case in the spontaneous conversion of aspartyl residues to isoaspartyl residues, a modification occurring at physiological pH and under conditions of cell stress and aging. We have examined the effect of isoaspartyl modifications on the effector functions of T lymphocytes in vivo using mice lacking the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT). PCMT-/- CD4+ T cells exhibit increased proliferation in response to mitogen and Ag receptor stimulation as compared with wild-type CD4+ T cells. Hyperproliferation is marked by increased phosphorylation of members of both the TCR and CD28 signaling pathways. Wild-type mice reconstituted with PCMT-/- bone marrow develop high titers of anti-DNA autoantibodies and kidney pathology typical of that found in systemic lupus erythematosus. These observations, coupled with the fact that humans have polymorphisms in the pcmt gene, suggest that isoaspartyl self-proteins may alter the maintenance of peripheral immune tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcell homeostasis is maintained through the integration of a wide variety of mechanisms. These include low affinity interactions with self-Ag, homeostatic proliferation, and subsequent differentiation into memory T cells (1, 2). The disruption of many individual components in these pathways often results in increased T cell proliferation (3), a lowered threshold of Ag activation (4), and a loss of immune tolerance as evidenced by systemic autoimmune pathology (3, 4, 5).

Many studies in which signaling and cell cycle molecules have been genetically deleted have pinpointed critical molecules within T cells that are needed for the maintenance of T cell homeostasis (6, 7, 8). However, normal cellular events, such as cell stress and aging, have the potential to induce posttranslational modifications to cellular proteins that can potentially alter the biological activity of the protein(s) in which they are found (9). Alterations in these molecules, whether by deletion or by modification, often result in the development of autoimmune pathology. One posttranslational modification that increases in aged or stressed cells is the spontaneous conversion of an aspartyl residue to an isoaspartyl residue (10). The present study examines the effect of intracellular isoaspartyl peptides on cellular immune functions in vivo using a mouse model in which the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT) 3 was disrupted (11). Mice lacking PCMT have increased isoaspartyl content in their cells with the mortality of animals occurring at 4–6 wk of age (11). Isoaspartyl formation is a nonenzymatic reaction that takes place under physiological conditions and is enhanced in aged or stressed cells (10, 12, 13, 14, 15, 16). In some cases, the presence of isoaspartyl residues can also interfere with the biological function of the protein in which they form (12, 17, 18). All organisms examined to date, both prokaryotic and eukaryotic, have evolved the highly conserved PCMT repair enzyme that catalyzes the conversion of an isoaspartyl residue back to the normal aspartyl residue (19).

The rationale for these studies focuses on how spontaneously arising posttranslational modifications may alter the normal course of immune tolerance. We have found that the presence of isoaspartyl residues in T cells amplifies their response to mitogen and receptor-mediated stimulation via altered phosphorylation of mitogen-activated protein kinase (MAPK) family members. The in vivo biological effects of these modifications were examined by the reconstitution of normal mice with bone marrow from hyperresponsive PCMT-/- mice. Wild-type mice bearing PCMT-/- lymphocytes generate strong autoimmunity indicating an inability of these cells to maintain immune tolerance, even when selected in a normal thymic and peripheral environment. These studies demonstrate that the spontaneous generation of isoaspartyl residues within lymphocytes may be a focal point from which autoimmunity arises.


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

PCMT+/- mice were a generous gift of Dr. S. Clarke (University of California, Los Angeles, CA) and were generated by inserting a neo cassette into exon one of the pcmt gene (11). PCMT-/- mice were obtained by intercrossing PCMT+/- C57BL/6 mice in the Yale Animal Resource Center (New Haven, CT). Mice were screened for the presence of the neo cassette and the absence of the pcmt gene by PCR analysis of tail DNA. Primer sequences were: pcmt forward 5'-gcagcgacggcagtaacagc-3', pcmt reverse 5'-cgcatcgagcgagcacgtactcgg-3', neo forward 5'-gcacgaggaagcggtcagcccattc-3', and neo reverse 5'-cgcatcgagcgagcacgtactcgg-3'. PCMT-/- mice were used at 4–6 wk of age. C57BL/6 mice (National Cancer Institute, Frederick, MD) and B6.SJL-ptprcaPep3B/Boy/J (The Jackson Laboratory, Bar Harbor, ME) were used at 4–6 wk of age. Unless otherwise stated, wild-type mice are age-matched littermates.

Measurement of isoaspartyl proteins in cells

Spleen, lymph node, and thymus cells were resuspended in immunoprecipitation buffer (10 mM Tris, pH 8.0, 500 mM NaCl, and 0.1% Nonidet P-40) and sonicated. Cell lysates were centrifuged 15,000 x g for 10 min and the total protein concentration of the supernatants was determined using the DC Protein Assay per manufacturer’s instructions (Bio-Rad, Hercules, CA). Isoaspartyl content of spleen, lymph node, and thymus cell lysates was measured using a PCMT vapor diffusion assay (ISOQUANT Protein Deamidation Detection kit; Promega, Madison, WI). Briefly, 2 µg of cell lysate were incubated with 1 µCi S-adenosyl-L-[methyl-3H]methionine (Amersham Life Science, Piscataway, NJ) and PCMT in sodium phosphate buffer at 30°C for 30 min. The methyl-transfer reaction was terminated with a basic pH stop solution. Fifty microliters of the reaction mixture were spotted onto a sponge insert attached to a scintillation vial cap and placed on a scintillant-filled vial for 1 h at 37°C. The sponge-containing caps were removed and replaced with new caps, and radioactivity was measured with a scintillation counter (Beckman Coulter, Fullerton, CA). PBS served as the negative control and the isoaspartyl {delta} sleep-inducing peptide served as the positive control.

B cell proliferation assays

B cells were isolated from spleen by negative selection using the MACS system (Miltenyi Biotec, Auburn, CA). The resulting cell population was 80–85% pure as determined by flow cytometry. Cells were resuspended in Click’s medium + 5% FCS supplemented with 2 mM L-glutamine, 0.1 mM 2-ME, and antibiotics (100 U/ml penicillin/streptomycin, 50 µg/ml gentamicin) then plated at 1 x 105 cells/well in a 0.2-ml volume in 96-well flat-bottom microtiter plates with either 1 µg/ml LPS (Sigma-Aldrich, St. Louis, MO), 40 µg/ml anti-IgM Ab (Jackson ImmunoResearch Laboratories, West Grove, PA), 2.5 µg/ml anti-CD40 Ab (clone HM 40-3; BD PharMingen, San Diego, CA) plus 20 ng/ml IL-4 (R&D Systems, Minneapolis, MN), or medium alone. Cells were incubated for 48 h at 37°C, 5% CO2, after which cells were pulsed with 1 µCi [3H]thymidine (ICN Chemicals, Irvine, CA) for 18 h before being harvested onto filters with a semiautomatic cell harvester. Radioactivity was counted with a Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD).

Mitogen and anti-CD3{epsilon} mAb stimulation

Spleen cells were suspended in Click’s medium + 5% FCS supplemented with 2 mM L-glutamine, 0.1 mM 2-ME, and antibiotics. Cells were plated at 5 x 105 cells/well in a 0.2-ml volume in 96-well flat-bottom microtiter plates with or without 2 µg/ml Con A (Sigma-Aldrich). For anti-CD3 mAb stimulation of splenocytes, wells of a 96-well U-bottom microtiter plate were coated with or without 30 µl of a 2.5 µg/ml concentration of anti-CD3{epsilon} mAb (clone 145-2C11, hamster IgG; BD PharMingen) in PBS and incubated at 37°C for 1 h. Wells were washed three times with PBS before the addition of 2.5 x 105 cells/well in a 0.2-ml volume. After incubating for 2–3 days at 37°C, 5% CO2, cells were pulsed with 1 µCi [3H]thymidine/well (ICN Chemicals) for 18 h and harvested onto filters with a semiautomatic cell harvester. Radioactivity was counted with a Betaplate liquid scintillation counter (Wallac). CD3/CD28 stimulation was done by coating 96-well flat-bottom plates with 0.5 µg of anti-CD3 mAb overnight at 4°C. Wells were washed, and 0.1 µg of anti-CD28 mAb (clone 37.51; BD PharMingen) was added to the appropriate wells before the addition of 1 x 105 purified CD4+ T cells. CD4+ T cells were obtained by negative selection of spleen and lymph node cells using the MACS system (Miltenyi Biotec). T cell purity was 80% as determined by FACS analysis.

T lymphocyte proliferation assays

Wild-type and PCMT-/- mice were immunized s.c. with 50 µg of OVA (Sigma-Aldrich) emulsified 1:1 in CFA (Sigma-Aldrich) at the base of the tail and hind footpad. Ten days later, draining lymph node T lymphocytes were isolated by negative selection using a mixture of Abs against B220 (TIB 146), Mac-1 (TIB 128), and anti-I-Ab (Y3-JP) for 1 h on ice. Cells were washed and incubated with sheep anti-mouse/rat Ig-coated magnetic beads (PerSeptive Biosystems, Framingham, MA) at a ratio of 5:1 (beads:cells) for 1 h at 4°C. Enriched T cells (82–88% pure) were separated from non-T cells with a magnet. T lymphocytes (1 x 105) were resuspended in Click’s medium + 5% FCS supplemented with L-glutamine, 2-ME, antibiotics, and cultured with irradiated (2500 rad) C57BL/6 spleen cells (5 x 105) with or without Ag. Purified protein derivative of Mycobacterium tuberculosis (M. tuberculosis H37 RA; Difco, Detroit, MI) served as a positive control. After 3 days, cells were pulsed and harvested as described above.

Abs and flow cytometry

All Abs were purchased from BD PharMingen and included: FITC anti-CD4 (RM4-5, rat IgG2a), PE anti-CD62L (MEL-14, rat IgG2a), CyChrome CD44 (IM-7, rat IgG2b), FITC anti-CD8 (53-6.7, rat IgG2a), PE anti-CD4 (H129.19, rat IgG2b), CyChrome B220 (RA3-6B2, rat IgG2a), FITC anti-I-Ab (AF6-120.1, mouse IgG2a), PE anti-CD11b (M1/70, IgG2b), FITC anti-CD45.1 (A20, mouse IgG2a), and FITC anti-CD45.2 (104, mouse IgG2a). Cell surface staining with optimal concentrations of fluorochrome-conjugated mAb was performed on 5–10 x 105 cells in 0.2 ml of PBS + 1% BSA + 0.1% NaN3 for 30 min at 4°C. Cells were washed three times and fixed in PBS + 1% paraformaldehyde. Samples were analyzed on a FACSCalibur (BD Immunocytometry Systems, Mountain View, CA).

Determination of activation-induced cell death (AICD)

CD4+ T cells from PCMT-/- or wild-type mice were purified by negative selection using the MACS system (Miltenyi Biotec), plated at 4 x 106 cells/well of a six-well plate in the presence of 2.5 µg/ml Con A and incubated 2 days at 37°C, 5% CO2. Cells were washed extensively and dead cells were removed by Ficoll gradient separation (Amersham Pharmacia, Piscataway, NJ). Cells were plated in wells coated with anti-CD3 mAb plus 50 U/ml IL-2 and incubated for 48 h before staining for apoptosis. Cells were washed two times with binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and 1 x 105 cells incubated with 5 µl each Annexin VPE and 7-amino-actinomycin D (7-AAD) for 15 min at room temperature in the dark. Four-hundred microliters of binding buffer were added to each sample and analyzed by FACS within 1 h. Cells undergoing apoptosis stain annexin V+ 7-AAD-. Nonstimulated cells served as negative controls for apoptosis.

PMA/ionomycin stimulation of PCMT-/- lymphocytes

Lymphocytes from PCMT-/- or wild-type mice were purified as described above (see mitogen and anti-CD3 mAb stimulation) and incubated in wells containing PMA (10 ng/ml) plus ionomycin (500 ng/ml; Sigma-Aldrich) at 5 x 105 cells/well. The cultures were incubated for 48 h before the addition of 1 µCi [3H]thymidine per well. Cultures were incubated an additional 18 h, then harvested as described above.

Cyclosporin A (CsA) inhibition of PCMT-/- lymphocyte proliferation and IL-2 production

Lymphocytes from PCMT-/- or wild-type mice were purified as described above (see mitogen and anti-CD3 mAb stimulation). Lymphocytes were cultured for 1 h at 37°C with 0.5 µg/ml CsA (Sigma-Aldrich). Cells were washed once with complete Click’s medium and plated in anti-CD3 mAb-plated wells (10 µg/ml) at 5 x 105 cells/well. The cells were incubated for 24 h, at which time 110 µl of culture supernatant were removed from each well and frozen at -80°C until tested for the concentration of IL-2. The cells were then incubated at additional 24 h, at which time 1 µCi [3H]thymidine was added to the wells, incubated an additional 18 h, then harvested as described above.

The amount of IL-2 in the culture supernatants was determined by incubating 100 µl of supernatant with 5 x 103 CTLL cells. A standard curve was prepared from a known concentration of IL-2. Cells were incubated at 37°C for 24 h then pulsed with 1 µCi [3H]thymidine. Cells were harvested 12 h later as described above.

Phosphoprotein analysis of TCR-stimulated PCMT-/- lymph node cells

Lymph node cells were stimulated with plate-bound anti-CD3 mAb (10 µg/ml) + anti-CD28 mAb (5 µg/ml) for 2 min in Click’s medium without FCS. The reaction was stopped by putting the cells on ice, the cells were spun down, and the pellet was homogenized in ice-cold lysis buffer (20 mM MOPS (pH 7.0), 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 40 mM {beta}-glycerophosphate (pH 7.4), 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF, 3 mM benzamidine, 5 µM pepstatin A, 10 µM leupeptin, 0.5% Nonidet P-40). The homogenate was centrifuged at 14,000 rpm for 15 min and the resulting supernatant was removed and immediately assayed for protein concentration by the Bradford assay (Bio-Rad). The final concentration of protein was adjusted to 0.5 mg/ml in SDS-PAGE sample buffer. Three-hundred fifty micrograms of protein were subjected to the Kinetworks Phospho-site Screen (KPSS-1.1) and analysis of phosphoproteins done per the manufacturer’s specifications (Kinexus Bioinformatics, Vancouver, British Columbia, Canada). Results were expressed in both a gel format and as densitometry readings. According to the manufacturer’s protocol, changes >25% are considered significant. Percentage change in band intensity was calculated as ((PCMT-/- trace quantity - wild-type trace quantity)/PCMT-/- trace quantity) x 100.

Generation of PCMT-/- bone marrow chimeras

Bone marrow was isolated aseptically from PCMT-/- or wild-type littermates, washed in PBS, and resuspended in PBS + 1 mM HEPES at a concentration of 1 x 108 cells/ml. B6.SJL-ptprcaPep3B/BoyJ/J (CD45.1) congenic mice were sublethally irradiated (750 rad) using a 137Cs irradiator. Mice were rested several hours before i.v. injection of 20 x 106 cells. Four to six weeks after bone marrow transfer, bone marrow reconstitution was confirmed by FACS analysis of peripheral blood cells for the CD45.2 marker as described above.

Indirect immunofluorescence antinuclear Abs (ANA)

Indirect immunofluorescence assays for ANA were performed using commercially available substrates (Quidel, San Diego, CA). Briefly, 30 µl of a 1/100 dilution of mouse sera were placed on slides coated with human epithelial cells (HEp-2) and incubated for 2 h at room temperature. Slides were washed 10 min in PBS, and then individual wells were incubated with 30 µl of a 1/50 dilution of FITC anti-mouse IgG Ab (Sigma-Aldrich) and incubated in the dark for 2 h. Slides were washed for 10 min in PBS, and examined by fluorescence microscopy. Serum from a single MRL lpr/lpr mouse positive for ANA served as the positive control.

Anti-dsDNA Ab ELISA

Anti-dsDNA autoantibody was examined using a commercially available ELISA (Sanofi-Pasteur Diagnostics, Chaska, MN). Briefly, DNA-coated wells were blocked with PBS + 5% BSA for 1 h at room temperature, then washed three times with PBS. Fifty microliters of 1/100 dilutions of mouse sera were added to each well and incubated 2 h at room temperature. Wells were washed three times with PBS, followed by the addition of 50 µl of a 1/1000 dilution of goat anti-mouse IgG alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL) for 2 h at room temperature. Wells were washed five times with PBS and 50 µl of p-nitrophenylphosphate substrate (Sigma-Aldrich) were added to each well. The plates were read at 405 nm on a spectrophotometric ELISA reader at various time points. Experimental sera were normalized to a single MRL lpr/lpr positive control serum used in every assay.

Kidney pathology

Kidneys from PCMT-/- and wild-type bone marrow reconstituted mice were collected at 7–9 mo post-bone marrow transfer and immediately immersed in 10% formalin (Fisher, Pittsburgh, PA). Thin sections and H&E staining were performed by the Yale Dermatopathology Laboratory. Blinded samples were examined for pathology at x20 magnification.

Statistical analysis

All statistics were performed using a Student’s t test. A value of p < 0.05 was regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isoaspartyl residues are generated by the spontaneous deamidation of asparagine or isomerization of aspartic acid (Fig. 1). The resulting unstable succinimide intermediate is spontaneously hydrolyzed under physiological conditions at either carbonyl group, giving a mixture of normal aspartyl protein (30% product) or isoaspartyl residues (70% product). The latter modification is the result of the peptide bond though the side chain {beta} carbonyl, leaving the {alpha} carbonyl as the free carbon (Fig. 1) (20).



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  		FIGURE 1. Structure and formation of isoaspartyl residues. Isoaspartyl residue formation occurs at physiologic temperature and pH via the spontaneous intramolecular deamidation at Asn-X linkages or by the isomerization of Asp-X linkages. Hydrolysis of the cyclic imide intermediate results in the formation of the isoaspartyl form of protein (~70–80% of the product) and of the normal aspartyl form (~20–30% of the end product). The isoaspartyl form results in the peptide bond occurring though the {beta}-carbonyl rather than the normal {alpha}-carbonyl group.

 
Isoaspartyl cell content in lymphoid organs

Mice lacking PCMT were used to define the effect of isoaspartyl modifications on self-proteins in cellular immune functions. PCMT-/- mice are unable to repair posttranslational modifications resulting in enhanced isoaspartyl modifications in tissues (11). All groups of mice examined had virtually identical distributions of cell types (B and T cell subsets) in the thymus, lymph node, and spleen (data not shown). An analysis of B and T cell activation states by flow cytometry also revealed no distinct differences among any of the mouse groups. Taken together, these observations suggest that no significant defects or differences exist in central T cell selection or in the development of T cell subsets in the periphery of PCMT-/- mice as compared with wild-type mice.

We next quantitated the isoaspartyl content of cell lysates from spleen, lymph node, and thymus from wild-type and PCMT-/- mice using a vapor diffusion assay that specifically identifies isoaspartyl posttranslational modifications (see Materials and Methods). As illustrated in Fig. 2, PCMT-/- cell lysates had significantly increased levels of intracellular isoaspartyl modifications in the spleen (p < 0.004), lymph node (p < 0.001), and thymus (p < 0.020) as compared with wild-type mice. Because it has been observed that isoaspartyl modifications may alter the biological functions of proteins, we next examined whether fundamental responses of immune cells were altered when cells were unable to repair these modifications.



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  		FIGURE 2. Isoaspartyl content is elevated in proteins from PCMT-/- mice. The specific isoaspartyl content of cell lysates from each organ was determined using a vapor diffusion assay (see Materials and Methods). The results represent the difference between the experimental counts and background cpm. Background was 864 cpm and the {delta} sleep-inducing peptide positive control was 11,638 cpm. Error bars represent the SD. *, Significant difference (p < 0.01) as compared with wild-type cells.

 
PCMT-/- mice have increased T cell proliferative responses to mitogen or receptor-mediated stimulation

Initial analyses of immune function were performed in PCMT-/- mice to determine the effect of isoaspartyl accumulation on B and T lymphocyte functions. The most striking differences between PCMT-/- and wild-type mice were observed in the T cell compartment. There was a significant increase in PCMT-/- splenocyte proliferation in response to Con A as compared with cells from wild-type animals (p < 0.002; Fig. 3A). Consistent with the above observation, anti-CD3 mAb receptor-mediated stimulation of T cells also resulted in a significant increase in proliferative signaling in PCMT-/- splenocytes as compared with wild-type splenocytes (p < 0.001; Fig. 3A). Anti-CD3 mAb stimulation of purified PCMT-/- CD4+ T cells in the presence of anti-CD28 mAb further confirmed that the hyperproliferation was indeed in the T cell compartment (Fig. 3B). The addition of anti-CD28 mAb to the cultures enhanced proliferation in all groups, including in PCMT-/- lymphocytes (Fig. 3B).



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  		FIGURE 3. Lymphocytes from PCMT-/- mice hyperproliferate in response to mitogen and TCR stimulation. A, Splenocytes from PCMT-/- and wild-type mice were incubated with or without Con A or plate-bound anti-CD3 mAb 2–3 days before harvesting. B, Purified CD4+ T cells from PCMT-/- and wild-type mice were incubated with plate-bound anti-CD3 mAb with or without anti-CD28 mAb for 72 h before harvesting. Error bars represent the SD (absence of bars indicates error too small to be observed in this figure). *, Significant difference (p < 0.01) as compared with wild-type cells.

 
An analysis of B lymphocyte functions revealed no apparent differences between PCMT-/- mice and wild-type mice with respect to peripheral B cell numbers, activation phenotypes, or distribution (data not shown). As indicated in Fig. 4, B cell responses to mitogen and receptor-mediated stimulation were nearly identical between PCMT-/- and wild-type mice.



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  		FIGURE 4. Normal B cell functions are found in PCMT-/- mice. Purified B cells from PCMT-/- or wild-type mice were incubated with or without LPS, anti-IgM Ab, or anti-CD40 mAb + IL-4 for 2 days before harvesting. Error bars represent the SD. Results are representative of three experiments with five to seven mice per group.

 
Having demonstrated that PCMT-/- splenocytes and CD4+ T cells were hyperresponsive to mitogen and receptor-mediated stimulation in vitro, we next determined whether T cells in lymph nodes from PCMT-/- mice would respond in a similar manner in vivo to a nominal foreign Ag. For these studies, PCMT-/- and control littermate mice were immunized with OVA-CFA for 10 days followed by an analysis of recall responses of purified T cells stimulated with OVA Ag. As seen in Fig. 5, T cells from PCMT-/- mice responded with up to 4-fold greater proliferation as compared with T cells from wild-type mice. The enhanced T cell response of PCMT-/- mice was not due to differences in APC function, because no differences were found in the ability of PCMT-/- splenocytes to present OVA as compared with wild-type splenocytes (data not shown).



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  		FIGURE 5. PCMT-/- T cells hyperproliferate in response to OVA restimulation. PCMT-/- and wild-type mice were immunized with 50 µg of OVA emulsified in CFA. Ten days later, purified T cells were isolated from draining lymph nodes and cocultured with irradiated syngeneic splenocytes plus titrating amounts of OVA. Purified protein derivative of M. tuberculosis controls ranged from 3310 to 8261 cpm.

 
A hyperproliferative phenotype of T cells may cause abnormal thymic selection to occur or allow abnormally activated or memory T cell populations to arise over time in vivo. Flow cytometry analysis revealed that there were no significant differences in CD4 or CD8 populations in the spleen or lymph node of PCMT-/- mice as compared with wild-type littermates, including the surface levels of CD3 (data not shown). Furthermore, the majority of CD4+ and CD8+ cells in both organs was of a naive phenotype (CD62LhighCD44low) and resembled those populations found in age-matched wild-type mice (78 vs 78%). Thymic selection appeared fundamentally normal by the similarity of double- and single-positive cells in the thymus of PCMT-/- mice compared with those seen in wild-type littermates (CD4+CD8+; 87% in both PCMT-/- and wild-type mice). Finally, APCs (CD11b+) and resident B cell populations (B220+) in the spleen were equivalent in numbers and distribution between all mouse groups (data not shown). Additionally, there were no detectable differences in the expression of MHC class II on either the B220+ or CD11b+ positive populations.

We next examined the possibility that the increased hyperproliferation in PCMT-/- T cells reflected a decrease in apoptosis of these cells after AICD. To address this possibility, CD4+ T cells from PCMT-/- and wild-type mice were induced to undergo AICD followed by staining with annexin V and 7-AAD. Cells undergoing apoptosis are found to be annexin V+ 7-AAD-. CD4+ T cells from both PCMT-/- mice and wild-type mice exhibited similar apoptotic populations upon stimulation with anti-CD3 mAb (Table I). We can conclude from these studies that abnormal proliferative functions of T cells bearing increased posttranslational modifications are not due to any defects in the death pathway of cells.


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  		Table I. Percentage of cells undergoing apoptosis after anti-CD3 mAb stimulation

 
Analysis of TCR signaling events

The hyperproliferative phenotype of PCMT-/- T cells upon TCR stimulation suggests certain signaling molecules are affected by the accumulation of isoaspartic acid residues within the cell. In characterizing where this defect might be within the TCR signaling pathway, we stimulated PCMT-/- T cells with PMA and ionomycin. PMA + ionomycin bypasses the proximal TCR signaling events by activating protein kinase C (PKC). Along with the Ca2+ ionophore ionomycin, PMA is a strong activator of T cell proliferation. As seen in Fig. 6, PCMT-/- T cells stimulated with PMA/ionomycin proliferated to the same extent as wild-type T cells. These results suggest that the PKC pathway in PCMT-/- T cells functions in a similar manner as wild-type mice, and that any defects in T cell signaling are downstream of PKC activation.



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  		FIGURE 6. PCMT-/- T cells do not hyperproliferate in response to PMA + ionomycin and are inhibited by CsA. A, PCMT-/- and wild-type T cells were stimulated with PMA + ionomycin for 48 h before harvesting. B, PCMT-/- and wild-type T lymphocytes were pretreated 1 h with CsA, washed, and then incubated for 48 h in the presence of plate-bound anti-CD3 mAb before harvesting. C, IL-2 production by anti-CD3 mAb stimulated PCMT-/- or wild-type T cells pretreated with CsA. Cells were pretreated 1 h with CsA, washed, and then incubated for 24 h in the presence of plate-bound anti-CD3 mAb. IL-2 was measured using the IL-2-dependent cell line CTLL. Error bars represent the SD. Results are representative of three experiments using two to three mice per group.

 
We next examined the affect of CsA on the proliferation and IL-2 production of PCMT-/- mice. CsA inhibits calcineurin, a molecule that removes the phosphate group from cytoplasmic NFAT. The removal of this phosphate group allows for the nuclear translocation of NFAT, and thus the transcription of the IL-2 gene. PCMT-/- T cells pretreated with CsA before anti-CD3 mAb stimulation showed a dramatic decrease in both proliferation and in IL-2 production. There was no statistical difference between PCMT-/- T cells and wild-type T cells with regard to proliferation or IL-2 production. This suggests that IL-2 signaling in these two groups of cells is similar.

Increased phosphorylation of signaling molecules in PCMT-/- lymph node cells after TCR/CD28 stimulation

The hyperproliferative phenotype of PCMT-/- T cells upon TCR stimulation suggests certain signaling molecules are affected by the accumulation of isoaspartic acid residues within the cell. Cell lysates prepared from anti-CD3/CD28 mAb-stimulated lymph node cells from PCMT-/- and wild-type mice were analyzed for differences in the phosphorylation of proteins involved in TCR signaling. As illustrated (Table II and Fig. 7), a number of signaling proteins were hyperphosphorylated in PCMT-/- lymph nodes as compared with wild-type lymph nodes. CD3 ligation resulted in the hyperphosphorylation of MAPK pathway members MAPK/extracellular-regulated kinase 1/2 (MEK1/2), extracellular signal-regulated kinase (ERK)1/2, and 90-kDa ribosomal S6 kinase (RSK)1. There was no difference in the phosphorylation status of two other members of the MAPK pathway, c-Jun N-terminal kinase and p38 (data not shown). PKC{alpha}, also activated upon TCR/CD28 stimulation, was hyperphosphorylated in PCMT-/- lymph node cells. CD28 stimulation has been shown to induce protein kinase B{alpha} (PKB{alpha}) phosphorylation (21) and indeed PKB{alpha}, as well as one of its substrates, glycogen synthase kinase 3{alpha} (GSK3{alpha}), was hyperphosphorylated in anti-CD3/CD28 mAb-stimulated PCMT-/- cells. This is in addition to another CD28-regulated molecule, p70 S6 kinase, which is also hyperphosphorylated in PCMT-/- lymph node cells. Together, these data suggest that no single molecule, but rather a combination of signaling molecules, contributes to the hyperproliferative phenotype in PCMT-/- T lymphocytes.


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  		Table II. Differential phosphorylation of proteinsa

 


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  		FIGURE 7. Increased phosphorylation of signaling molecules in anti-CD3/CD28 mAb-stimulated lymph node cells. Lymph node cells from either PCMT-/- mice or wild-type littermates were stimulated with anti-CD3/CD28 mAb for 2 min at 37°C. Cell lysates were then prepared for analysis of phosphoproteins by immunoblot.

 
Mice reconstituted with PCMT-/- bone marrow develop Abs to self-Ags

It has been observed in humans with systemic lupus erythematosus (SLE) and in murine models of SLE that T cells exhibit hyperresponsiveness to antigenic stimulation in a manner much like that found in PCMT-/- T cells (22, 23, 24, 25). Human and murine SLE is marked by the appearance of autoantibodies to a variety of intracellular macromolecules including nucleosomes, dsDNA, and ribonucleoproteins. We next examined whether this abnormal T cell phenotype could lead to other autoimmune phenomenon in vivo.

Because PCMT-/- mice only survive ~6 wk, long-term studies of immune responses were performed in irradiated wild-type mice receiving PCMT-/- bone marrow. For these studies, CD45.1-bearing congenic mice were reconstituted with CD45.2-marked PCMT-/- bone marrow, allowing the fate of transferred cells to be tracked. We first examined whether serum Abs from bone marrow-reconstituted mice would bind intracellular proteins in indirect immunofluorescence assays. Sera from mice reconstituted with wild-type littermate bone marrow failed to exhibit any detectable ANAs by indirect immunofluorescence (Fig. 8A). In contrast, sera from mice reconstituted with PCMT-/- bone marrow showed positive staining of nuclear Ags with some staining of the nucleoli (representative pattern in Fig. 8B). In attempts to identify specific intranuclear components bound by autoimmune sera in PCMT-/--reconstituted mice, ELISA was performed with dsDNA as the antigenic substrate. As shown in Table III, mice reconstituted with PCMT-/- bone marrow generated elevated levels of anti-DNA autoantibodies as compared with mice receiving wild-type bone marrow.



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  		FIGURE 8. Indirect immunofluorescence using sera from congenic mice reconstituted with either wild-type littermate or PCMT-/- bone marrow. Sera were collected from congenic mice reconstituted with (A) wild-type or (B) PCMT-/- bone marrow and examined for the presence of ANAs on HEp-2 cell substrates. C, Positive control serum from MRL lpr/lpr mouse.

 

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  		Table III. Autoreactivity in wild-type mice reconstituted with PCMT-/- bone marrowa

 
Kidney pathology is observed in wild-type mice receiving PCMT-/- bone marrow

After ANAs were found in recipients of PCMT-/- bone marrow, we next examined pathology in kidney sections of host animals. Some recipients of PCMT-/- bone marrow exhibited severe renal pathology as found in Fig. 9A. The pathology was marked by perivascular cellular infiltration, infiltration of myointimal regions with lymphocytes, focal areas of necrosis, and endovasculitis. In contrast, recipients of wild-type bone marrow were unremarkable and primarily normal in renal architecture (Fig. 9B).



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  		FIGURE 9. Histopathology of kidney sections from bone marrow recipient mice. Kidney sections were obtained from congenic C57BL/6 mice 7–9 mo after receiving PCMT-/- syngeneic bone marrow (A) or wild-type bone marrow (B).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies presented here were initiated after an understanding that isoaspartyl modifications are enhanced in aging and stressed cells. For example, cells that have been aged in vitro and cells that have been stimulated by mitogens accumulate high levels of intracellular isoaspartylated proteins (15, 16, 26, 27). The formation of isoaspartyl modifications is one of the single major mechanisms for altering the biological functions of cellular proteins under physiologic conditions of temperature and pH (28). For example, isoaspartyl modifications have been observed in age-damaged calmodulin at Asp/Asn-Gly sequences in the Ca2+ binding domain (9). Nearly 80% of its biological activity is destroyed by this modification that can then be restored by repair with PCMT (17). Isoaspartyl and/or deamidated residues have also been found to reduce some properties of CD4 in binding to gp120 of HIV (29) and of the biological activity of murine IL-1 (30).

Eukaryotes have evolved the enzyme, PCMT, to selectively methylate the {alpha}-carboxyl residue of isoaspartic acids in attempts to repair these deleterious modifications. Highly conserved PCMT enzyme functions have also been identified in plant cells, fungi, insects, and bacteria (31). It is also clear that recombinant proteins possess isoaspartyl modifications that alter their biological functions, a concern that may influence their use in living hosts. The endoplasmic retention signal found on PCMT suggests that isoaspartyl repair functions are initiated as newly synthesized proteins exit the endoplasmic reticulum. Despite these efforts for cellular repair of isoaspartyl modifications, the less-than-perfect efficiency of PCMT allows modified proteins to escape into the cellular milieu particularly under conditions of aging and cellular stresses such as heat shock.

The consequences of isoaspartyl accumulation in cellular proteins, and thus cell function, are best followed in the PCMT-/- mouse. Although there is an increase in isoaspartyl residues in cell lysates from all the lymphoid organs we examined, the most dramatic effect of these modifications appears to be in the T cell compartment. The accumulation of isoaspartyl residues has no apparent affect on lymphoid development, as there were normal numbers and subset distributions of cells within the spleen, lymph nodes, and thymus. This is similar to other studies where abnormal hyperproliferating T cell responses were observed without alterations in T cell subsets, cell numbers or resting vs active phenotypes (32).

T cell stimulation by either mitogen or TCR was clearly amplified in PCMT-/- mice as compared with wild-type mice. Perturbations that result in hyperproliferative T cell phenotypes frequently lead to autoimmune syndromes, autoantibody production, and/or autoimmune pathology. Some examples of this can be found in genetic knockout models of p21 (33, 34), Mgat5 (6), Gadd45 (8), E2F2 (4), and TGF{beta}RII (44) as well as observations with lprfas defect (25, 35), all of which lead to T cell hyperproliferation and autoimmunity.

The enhanced T cell proliferation in response to Ag was not accompanied by an accumulation of activated lymphocytes in vivo or by decreased levels of apoptosis. Signaling through the PKC pathway also seems intact in PCMT-/- lymphocytes, as demonstrated by a similar level of proliferation of PCMT-/- T lymphocytes and wild-type lymphocytes in response to PMA + ionomycin treatment. The regulation of calcium flux also seems to be intact in PCMT-/- mice in that again, PCMT-/- T lymphocytes show a marked reduction of proliferation and IL-2 production after CsA treatment in a manner similar to wild-type T lymphocytes. However, increased proliferation of PCMT-/- lymphocytes was reflected by increased phosphorylation of several signaling molecules in both the TCR and CD28 pathways. Several members of the MAPK pathway, MEK1/2, ERK1/2, and RSK1, are affected in PCMT-/- lymphocytes. Prior studies have similarly demonstrated that the hyperactivation of MEK and ERK contributes to the hyperproliferation of hemopoietic cells (36).

Another molecule involved in TCR/CD28 signaling, PKC{alpha}, was also hyperphosphorylated and has been linked to increased proliferation in thymocytes, as well as other cell types (37, 38). Upon CD28 ligation, PKB{alpha} and one of its substrates, GSK3{alpha}, were hyperphosphorylated in PCMT-/- lymph node cells. Phosphorylated GSK3{alpha} is inactive and unable to regulate NFAT removal from the nucleus (39, 40), thereby promoting cytokine gene expression. Finally, another molecule involved in CD28 signaling, p70 S6 kinase (41), was overexpressed in PCMT-/- lymph node cells. The above signaling data demonstrates that the defects in PCMT-/- T lymphocytes reside in several TCR/CD28 signaling proteins proximal to PKC as well as in the Ras pathway as described above.

Normal mice reconstituted with PCMT-/- bone marrow exhibiting hyperproliferative T cells also developed anti-DNA autoantibodies over time. Although PCMT-/- bone marrow was selected on a "normal" unmodified repertoire of self-peptides, it is likely that altered thresholds of T cell activation in these mice may affect thymic selection. Once in the periphery, T cells originating from PCMT-/- bone marrow may have a lowered threshold for activation such that self-peptides normally maintaining peripheral tolerance are now able to induce T cell activation. It is clear from our studies that much lower amounts of a nominal foreign Ag are needed to induce proliferation in PCMT-/- mice as compared with wild-type T cells (Figs. 3 and 5).

The data presented here demonstrate that an accumulation of isoaspartyl residues in cells alters the effector function of lymphocytes leading to autoantibody production. The use of PCMT-/- cells allowed us to artificially provoke intracellular modifications that would otherwise accumulate spontaneously in aged and stressed cells. Recent studies (42, 43) have identified humans with polymorphisms in the PCMT gene that will now warrant an examination of these modifications in the context of human autoimmune syndromes. Thus, isoaspartyl formation, and its deleterious effect on lymphocyte function, may be one triggering event by which spontaneous autoimmunity arises.


   Acknowledgments
 
We thank Dr. Steven Clarke for the PCMT+/- mice, Jon Lowenson for technical advice, and Chris DeVry for the PCMT/neo PCR protocol. ISOQUANT Protein Deamidation Detection kits were a kind gift from Promaga.


   Footnotes
 
1 This study was supported by National Institutes of Health Grants AR-47759 (to H.A.D.) and AI-36529 and AI-48120 (to M.J.M.), as well as grants from the Arthritis Foundation (Robert Wood Johnson, Jr. Biomedical Science Award) and the Ethel F. Donaghue Women’s Investigator Program at Yale University. Back

2 Address correspondence and reprint requests to Dr. Mark J. Mamula, Yale University School of Medicine, P.O. Box 208031, 300 Cedar Street, New Haven, CT 06520-8031. E-mail address: mark.mamula{at}yale.edu Back

3 Abbreviations used in this paper: PCMT, protein carboxyl methyltransferase; MAPK, mitogen-activated protein kinase; 7-AAD, 7-amino-actinomycin; CsA, cyclosporin A; ANA, antinuclear Ab; AICD, activation-induced cell death; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; MEK, MAPK/extracellular-regulated kinase; RSK, 90-kDa ribosomal S6 kinase; PKB, protein kinase B; GSK, glycogen synthase kinase; SLE, systemic lupus erythematosus. Back

Received for publication September 11, 2002. Accepted for publication July 16, 2003.


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