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The Journal of Immunology, 2005, 175: 5857-5865.
Copyright © 2005 by The American Association of Immunologists

A Defect in Deletion of Nucleosome-Specific Autoimmune T Cells in Lupus-Prone Thymus: Role of Thymic Dendritic Cells1

Marissa A. Michaels, Hee-Kap Kang, Arunan Kaliyaperumal, Ebenezar Satyaraj, Yan Shi and Syamal K. Datta2

Division of Rheumatology, Departments of Medicine and Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H471–94. In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the {alpha}{beta}TCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is widely believed that defects in peripheral tolerance cause lupus autoimmunity (1, 2). Studies with transgenic (Tg)3 TCRs rendered "autoreactive" against model Ags that were artificially expressed as neo-self Ags, or with TCRs against superantigens, have indicated that negative selection is intact in the thymus of lupus-prone mice (3, 4, 5, 6, 7), and indeed lupus-prone mouse strains do not develop global autoimmunity. However, the rules for positive or negative selection for major endogenous autoantigens like nucleosomes that are routinely released from large numbers of apoptotic cells in generative lymphoid organs (8, 9) might be different, especially in the complex background of lupus susceptibility. Even in the normal thymus, it is not known whether nucleosomes released from apoptotic thymocytes are processed and presented to developing T cells, because apoptotic cell disposal is silent (10, 11, 12, 13). Paradoxically, nucleosomes, the major products of apoptosis, are also the major immunogens inciting lupus autoimmunity. Th cells that primarily drive the production of pathogenic anti-DNA autoantibodies in lupus are primed to nucleosomal histone peptides from early life (14, 15, 16). To study central (and peripheral) regulation of nucleosome-specific T cells in normal and lupus-prone mice, we have made new lines of Tg mice bearing nucleosome-specific TCR of a pathogenic lupus Th clone.


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

NZB, SWR, and B10. D2 mice were purchased from The Jackson Laboratory. Lupus-prone (SWR x NZB)F1 (SNF1) hybrids (17) and MHC-matched (SWR x B10. D2)F1 (SBF1) hybrids were bred at our animal facility. Derivation of Tg mice is described in Results. All studies were approved by the Animal Care and Use Committee of Northwestern University.

Nucleosomes and peptides

Nucleosomes were prepared as described (14). The histone H4 peptide, H471–94, recognized by the Tg TCR, and a control peptide H2B59–73, were synthesized by F-moc chemistry (Mimotopes), purified by HPLC using a gradient of water and acetonitrile, and analyzed by mass spectrometry for purity.

Flow cytometry

For three-color staining of surface markers on thymocytes or splenic T cells, the appropriate combination of Abs (BD Pharmingen) conjugated to either FITC, PE, or biotin was used. Biotinylated Abs were revealed by streptavidin-CyChrome (Cy5). Abs against CD4, CD8, and CD69; pan {alpha}{beta}TCR (H57–597); TCRs V{beta}4, V{beta}8, V{beta}17, and TCRs V{alpha}2, V{alpha}3.2, V{alpha}8, V{alpha}11.1/11.2 were used. Annexin VFITC staining was done per the manufacturer (BD Biosciences). Four-color stainings for regulatory T cells (Treg) were done with anti-CD4-allophycocyanin, anti-CD8-biotin-Cy, anti-CD25-FITC, and anti-CD62L-PE. Usually 100,000 events were acquired in FACScan and analyzed by CellQuest software (BD Biosciences) after gating around live cells by forward and side scatter. In some experiments, cells were stained with propidium iodide (PI; BD Biosciences) just before analysis to detect or exclude dead cells.

For analyzing surface markers on DC ex vivo, whole thymocyte populations were prepared by collagenase-DNase digestion followed by washes in EDTA-containing buffer to prevent clumping as described (18, 19). To avoid unnecessary manipulations, further purifications were not done, but CD11c-FITC-positive cells were gated and analyzed after two-color staining of thymocytes. Abs to CD11c conjugated to FITC, MHC class II (M5/114.15.2)-PE, CD80-PE, CD86-PE, CD40-PE, CD8{alpha}-PE, CD11b-PE, B220 (CD45R)-allophycocyanin, CD4-PE, and isotype controls were from BD Biosciences, and anti-CD205-PE was from Cedarlane Laboratories.

Peptide induced deletion of thymocytes in vitro

Thymic organ culture. Pieces of thymus (usually three pieces per lobe) from 4-wk-old mice were cultured on nitrocellulose membranes placed on gel foam in the presence of control H2B59–73 or the H471–94 peptide, or anti-CD3 Ab, as described (20). After 3 days, thymocytes were harvested for flow cytometry.

Thymic reaggregation culture. The thymus was disrupted into single cell suspensions and 2.5 x 106 cells were cultured with 2.5 x 106 autologous, splenic B cells + macrophage (M{phi}) (irradiated 3000 rad) in 2 ml of medium/well in 24-well plates (Falcon; BD Biosciences), in the presence of titrated amounts of peptides or anti-CD3 Ab for various periods. Thymocytes were then stained for CD4 (Cychrome) and CD8 (PE), and annexin V (FITC), as described above. Percent specific apoptosis by flow cytometry was calculated as (the percentage of experimental apoptosis – the percentage of spontaneous apoptosis)/(100 – the percentage of spontaneous apoptosis) x 100 (21).

Presentation of autoepitopes by thymic APC (dendritic cell (DC) and non-DC) to T cell hybridomas specific for nucleosomes and its H471–94 peptide

Thymic DC were prepared by collagenase-DNase digestion, as described (18, 19), followed by two rounds of purification using CD11c-immunomagnetic beads from Miltenyi Biotec. Purity of DC preparation (80–90%) was confirmed by flow cytometry with dual staining for CD11c and MHC class II. The non-DC cells from the Miltenyi column flow-through fractions were depleted of lymphoid cells by anti-Thy1.2 and C' (15) to yield non-DC thymic APC (M{phi} and epithelial cells).

Graded numbers of each type of thymic APC from the normal SWR or SBF1, or the autoimmune SNF1 backgrounds (wild type (WT)) were cocultured with highly sensitive T cell hybridomas 102 and 210, which are specific for H471–94 peptide. These hybridomas, derived from WT SNF1 mice, can produce cytokines in response to the nucleosomal peptides eluted from class II (I-Ad or I-Aq) in attomole concentrations (15).

In some experiments, the thymic APC subsets (DC or non-DC) were preactivated by LPS (100 ng/ml) for 18 h before coculture with the T cell hybridomas.

In addition, graded numbers of APC subsets, prepulsed with the cognate H471–94 peptide or nucleosomes for 4, 6, or 18 h, were also cultured with the indicator hybridomas to detect any subtle differences in processing and presentation of exogenously added autoantigens.

In other experiments, mice were injected with anti-CD3 Ab in vivo (50 µg of 145–24C11 mAb i.p./mouse). After thymic apoptosis had peaked 40 h later (22), thymic DC were prepared from the injected mice and cocultured with the H471–94 peptide-specific T cell hybridomas.

TCR down-regulation assay with splenic T cells and APC

The splenic CD4+ T cells were isolated, as described (15, 23). Splenic B cell plus macrophage (B + M{phi}) APC were prepared by treating splenocytes with anti-Thy1.2 (TIB99) and rabbit complement and irradiated (3000 rad). Fresh splenic, CD4+ T cells (2.5 x 106/well) were cocultured in triplicate with irradiated B + M{phi} (2.5 x 106 cells) and different concentrations of "control" or "test" peptide, or anti-CD3 (2 µg/ml) in a 2 ml final volume for 18 h in 24-well plates (Falcon; BD Biosciences). Staining to detect down-regulation of TCR, along with staining for other surface markers were done as described (15, 24). Cells were stained with anti-CD4-PE, anti-{alpha}{beta}TCR-FITC (H57–597), and biotinylated anti-CD69 or anti-CD25, then gated CD4+ cells were analyzed by flow cytometry.

Serum autoantibody quantitation

IgG class autoantibodies to ssDNA, dsDNA, histone, and nucleosome (histone-DNA complex) were measured by ELISA (14, 15).

Monitoring development of lupus nephritis

The mice were monitored every 2 wk for proteinuria using albustix until they were moribund. Grading of glomerulonephritis was done, as described (23, 25, 26).

Isolation of Treg cells from spleen

As described (27), CD4+CD25+ T cells were purified by the mouse Treg cell isolation kit according to the manufacturer (Miltenyi Biotec). Purity of isolated cell subsets was >90% by flow cytometry.

ELISPOT assay

As described (27), ELISPOT assay plates (Cellular Technology) were coated with capture Ab against IFN-{gamma} (BD Pharmingen). Splenic T cells (1 x 106/well) from the mice were cocultured for 24 h with syngeneic APC (3000 rad radiated B cells, macrophages, and DC, 5 x 105/well) that were prepulsed with graded doses of H471–94 peptide or nucleosomes. In other assays, to detect Treg, CD4+CD25+ or CD4+CD25– T cells (0.3, 0.6, 1.25, or 2.5 x 105 cells/well) from transgenic or WT SNF1 mice were cocultured with target T cells and irradiated (3000 rad) splenic APC from 4-mo-old WT SNF1 mice in the presence of various concentrations of nucleosomes or PBS alone as control. Cells from both types of assays were removed after 24 h of incubation and the reactions were visualized by addition of the individual anti-cytokine Ab-biotin and subsequent AP-conjugated streptavidin. Cytokine-expressing cells were detected by Immunospot scanning and analysis (Cellular Technology).

Statistical analysis

The two-tailed t test for paired samples was performed. Results are expressed as mean ± SD. For nephritis incidence, {chi}2 and log rank tests were used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
New lines of Tg mice bearing a pathogenic lupus TCR

For TCR transgene constructs, we selected the disease-relevant lupus Th clone 3A, which is capable of inducing pathogenic autoantibodies in vitro and accelerating the development of lupus nephritis in vivo (14, 26, 28, 29). Clone 3A responds to nucleosome and specifically a peptide in its histone H4, H471–94, but it also cross-reacts with H416–39 peptide despite expressing a single pair of functional TCR{alpha}{beta} (26, 30, 31, 32). Both H471–94 and H416–39 peptides are major epitopes that are spontaneously recognized by autoimmune T and B cells of lupus-prone SNF1 and (NZB x NZW) (BWF1) mice, as well as lupus patients in vivo, and the peptides can induce lupus nephritis in SNF1 mice upon immunization and suppress established disease upon tolerization (16, 30, 33, 34, 35). Clone 3A expresses TCR V{beta}4 and V{alpha}19 (26, 32), but designation changed with fluctuation in TCR nomenclature (31). According to the latest (<http://imgt.cines.fr>), the V{alpha} chain of 3A TCR would be called TRAV5D-4, and the V{beta} chain TRBV2. We will use the original designation to prevent confusion, because commercial anti-TCR Abs also have retained old names. We isolated the functionally rearranged TCR {alpha}- and {beta}-chain genes along with their promoters and the flanking regulatory sequences from the genomic DNA of 3A. We inserted our constructs into M. Davis’s TCR shuttle vectors (31). The lupus TCR gene constructs were expressed upon transfection of EL4 (American Type Culture Collection) or the TCR-negative 4G4 T cell lymphoma lines. The transfected TCR genes of clone 3A reconstituted recognition of nucleosomes and H471–94 peptide (31). The 3A TCR, although derived from SNF1 mice and educated by I-Ad/q, could recognize nucleosomes or the H471–94 peptide presented by almost all I-A molecules, and human HLA-DR as well (31). This type of MHC-dependent, but unrestricted, recognition is a feature of nucleosome-responsive T cells and interestingly, is also found in the case of "universal" epitopes derived from certain virulent pathogens, such as, tetanus or malaria (36, 37). Remarkably, in transfection studies with TCR constructs, the 3A TCR’s {alpha}-chain was sufficient to confer this promiscuous recognition of nucleosomal epitope even when paired with a TCR {beta}-chain of irrelevant specificity, but possessing charged residues in its CDR3 (31). The 3A TCR{alpha} and TCR{beta} transgene constructs were injected separately into fertilized eggs of SWR mice. The normal SWR is the parental strain of SNF1 mice from which Th clone 3A was derived, and SWR has the appropriate MHC class II (I-Aq) for thymic selection of clone 3A’s TCR. The SWR eggs were good for transgene microinjection, as expected (38). Inheritence of the 3A TCR {alpha}-chain (V{alpha}19.1-J{alpha}41) and {beta}-chain (V{beta}4-D{beta}-J{beta}2.6) transgene in DNA and expression of mRNA were detected in tail clippings and blood cells by PCR, using primers as described (31). Expression of 3A Tg TCR’s V{beta}4 in peripheral blood T cells was also determined by flow cytometry. The {alpha}Tg SWR mice with high-level expression of Tg TCR{alpha}, and the {beta}Tg SWR with 80–90% of CD4+ T cells expressing V{beta}4 were then used for further analysis, and for breeding among their respective littermates to produce mice homozygous for TCR {alpha}Tg and {beta}Tg. The {alpha}Tg and {beta}Tg SWR lines of mice were intercrossed to derive {alpha}{beta}Tg SWR mice. Each of the Tg SWR lines were then crossed with NZB to derive {alpha}, {beta}, and {alpha}{beta}Tg mice in the lupus-prone SNF1 background. Although processing and presentation of nucleosomal peptides are equivalent in I-Aq (SWR) or I-Ad (NZB) bearing APC (31), we crossed a nonautoimmune strain, B10. D2, which is MHC-matched with NZB (I-Ad, I-Ed), with each of the Tg SWR lines to derive another set of Tg mice bearing 3A’s TCR in a normal background, which we call Tg SBF1. The {alpha}{beta}Tg SWR mice analyzed herein were heterozygous for the TCR transgene like the Tg SNF1 and Tg SBF1 mice. Female animals, that were screened to be positive for expression of Tg TCR {alpha}- and/or {beta}-chains by PCR, and V{beta}4 by flow cytometry (depending on the line), were analyzed further.

Nucleosome-specific thymocytes bearing lupus Tg-TCR {alpha} alone or {alpha}{beta} are deleted in normal but not in lupus-prone background

In the normal SWR or SBF1 backgrounds, introduction of the lupus TCR transgene led to a marked, spontaneous deletion of thymocytes in vivo (Fig. 1, Table I). As compared with WT SWR and SBF1, total thymocyte numbers were reduced 3- to 5-fold (60–80%) in {alpha} and {alpha}{beta} Tg SWR and 2.5- to 3-fold (60–64%) in SBF1 lines (p < 0.001). Deletion in the {alpha}Tg SWR and {alpha}Tg SBF1 lines, where the lupus TCR {alpha}-chain would pair with any endogenous TCR {beta}-chain, is consistent with previous transfection studies showing that the {alpha}-chain was sufficient for conferring nucleosome specificity even when paired with an irrelevant TCR {beta}-chain (31). Reduction in thymocyte numbers in {alpha}Tg lines was proportionate in the subpopulations: double positive (DP), CD4 single positive (SP),and CD8 SP. In the case of {beta}Tg SWR mice, a 58% reduction in total thymocytes occurred, but that was not the case in the SBF1 normal background. Abnormally premature expression of a functionally rearranged TCR {beta}-chain could have diminished positive selection and reduction in thymocyte numbers even without causing deletion. In the {alpha}{beta}Tg SWR mice where both chains for nucleosome specificity were supplied ready-made, the reduction in SP thymocytes was more marked, but even then pronounced skewing toward CD4 SP cells (CD4 SP 29-fold more than CD8 SP) was consistent with class II (I-A) restriction of the original 3A TCR (31). Similar results were found in {alpha}{beta}Tg SBF1. In marked contrast to the SWR and SBF1 backgrounds, no reduction in Tg thymocyte numbers was detected in the lupus-prone SNF1 background, as compared with WT SNF1, even in animals with high-level of expression of Tg-TCR. Moreover, no CD4 SP skewing was detected in the {alpha}{beta}Tg SNF1 lines. Flow cytometry of thymocytes after annexin V + PI staining for spontaneous apoptosis were consistent with above results (Fig. 1B), showing a marked increase in brightly staining annexin V+ cells in TCR {alpha} or {alpha}{beta} Tg lines of the normal backgrounds (p < 0.01 to <0.001). This assay (Fig. 1B) shows an apparently large fraction of apoptotic cells, because it requires several hours of handling of thymocyte single-cell suspensions in vitro, which interferes with engulfment of preapoptotic thymocytes. Nevertheless, the differences are striking. In contrast, normal lines bearing the transgenic TCR {beta}-chain only did not show such an increase in apoptotic cells (data not shown).



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  		FIGURE 1. Deletion of lupus TCR Tg-bearing thymocytes occurs in the normal SWR and SBF1 backgrounds, but not in the lupus-prone SNF1 background. A, CD4- and CD8-stained dot plots representing eight 2-mo-old mice of each Tg line are shown. Total thymocytes numbers are shown on top of each panel and the percentage of each population (except double negatives) is shown in quadrants. All mice were high expressors of the Tg, as in Fig. 2A. Overall density of the dot plots are adjusted approximately in proportion to the relative differences in total thymocyte counts in Table I. B, Spontaneous ongoing apoptosis in freshly isolated thymocytes occurs at higher rates in Tg mice in normal as compared with lupus background. Thymocytes were stained with CD4 and PI + annexin V. CD4+ gated thymocytes that include both CD4+ SP and CD4+CD8+ DP cells are shown. The numbers under respective Tg lines represent the percentage of annexin bright cells that are positive for annexin, as well as annexin and PI (right quadrants). Background isotype and fluorochrome controls were done separately for each line’s thymocytes. Representative of eight animals of each line tested.

 

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  		Table I. Total number of thymocytes and their subsets

 
Dual TCR-expressing T cells–down-regulation of Tg TCR with up-regulation of endogenous TCRs affects apoptosis induced by H471–94, but not by anti-CD3 or radiation

In thymocytes, and mature peripheral T cells, down-regulation of the Tg-TCR and compensatory up-regulation of endogenous TCR {alpha} and {beta} expression occurred to a variable extent, but this defensive measure was more frequent (p < 0.001) in the {alpha}{beta}Tg SNF1 mice (Fig. 2, A–C). At the time of initial screening (3–4 wk age), in the {alpha}{beta}Tg SWR and SBF1 mice, 12.67 and 11.59% of CD4 T cells in peripheral blood had down-regulated the V{beta}4 transgenic TCR, respectively, and the down-regulation was slight (intermediate expressors, as in Fig. 2A) in all of them, whereas, at that age, 81.08% of peripheral blood CD4+ T cells from {alpha}{beta}Tg SNF1 mice had down-regulated the Tg TCR, and among those 37% showed marked down-regulation (Fig. 2A). In thymic organ cultures, where thymic APC presented exogenously added peptides, apoptosis of Tg thymocytes occurred in the Tg thymic organ cultures, after incubation with H471–94 peptide indicating that the transgenic TCR pair was being functionally expressed (data not shown). Similarly, in thymic reaggregation cultures (TRC), where irradiated splenic APC were used, only 0.001 µg of H471–94 peptide was required for inducing marked apoptosis of Tg thymocytes, equivalent to or even better than anti-CD3 induced apoptosis, as long as they expressed high levels of Tg-TCR, as detected by PCR and V{beta}4 staining. Herein, to detect significant apoptosis induced by addition of peptide in TRC using Tg SWR or SBF1 thymocytes, we used thymocytes from very young mice (3–4 wk). Remarkably, in age-matched {alpha}{beta}Tg SNF1 lines with Tg TCR down-regulation, up to 10,000-fold more (10 µg) of the H471–94 peptide was required to cause apoptosis in TRC, but the amount of anti-CD3 required to induce apoptosis remained unchanged, as endogenous TCR had taken over (Fig. 2, D and E). The dose-dependent specificity of the results with cognate H471–94 peptide indicate that the Tg lines, as typed by PCR and V{beta}4 expression were indeed expressing the functional lupus Tg TCR on their surface. Injection of anti-CD3 Ab in vivo (22) also caused equivalent degree of deletion of thymocytes in lupus (SNF1) and normal (SBF1) mice (Table II). Furthermore, exposure of {alpha}{beta}Tg thymocytes from SWR, SBF1 and SNF1 background mice to radiation of 600–1500 rad (39) produced equivalent % of apoptotic cells. After exposure to 600 rad, 84.2 and 85.86% of SBF1 and SNF1 thymocytes, respectively, were apoptotic by annexin V staining. These results indicate that the defect in deletion in lupus background was not due to any unusual resistance of their thymocytes to apoptosis.



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  		FIGURE 2. Tg TCR is down-regulated and endogenous TCRs are up-regulated in Tg mice correlating with sensitivity to deletion by cognate peptide. A, Degrees of spontaneous down-regulation of Tg TCR (V{beta}4) in peripheral blood CD4 T cells in different {alpha}{beta}Tg SNF1 animals. High expressors of Tg TCR (left) have V{beta}4 mean fluorescence intensity (MFI) of 216, intermediate expressors (middle) with MFI of 97, and markedly lowered expressors (right) with MFI of 56. Representative of peripheral blood screening of 177 Tg mice. B, High-level (left half–four panels) or down-regulated (right half–4 panels) expressors of TCR transgene: thymocyte profile of Tg lines that inherited both the TCR {alpha} and {beta} transgenes by PCR, and expressed either high level (left) or down-regulated V{beta}4 (right) by FACS screening (as in A). Three-color staining of thymocytes was done with Ab to CD4, CD8, and either mAb to V{beta}4 (for transgenic TCR) or pan-{alpha}{beta}TCR. Upper panels, Histogram overlays showing V{beta}4 or all-{alpha}{beta}TCR expression in gated CD4+CD8+ DP thymocytes. Lower panels, Dot plots of gated CD4+ SP thymocytes. Representative of eight separate experiments. C, Endogenous TCR expression in CD4+ thymocytes (includes SP and DP) of {alpha}{beta}Tg SNF1. Three-color stainings with anti-CD4, a mixture of commercially available Abs to endogenous TCR V{alpha}, and either anti-V{beta}4 or V{beta}8 were done. The percentage of dual TCR+ cells indicated in upper right quadrants. Representative of six independent experiments. D, Thymocytes (DP) from high Tg-TCR expressing, {alpha}{beta}Tg SWR mice (middle) are 10,000-fold more sensitive to deletion than those from {alpha}{beta}Tg SNF1 animals with down-regulated Tg-TCR (right) after incubation with H471–94 peptide in vitro (0.001 vs 10 µg). Three-color staining of cells with Ab to CD4, CD8, and annexin V were done after TRCs (TRC, on left) of thymocytes using irradiated, autologous splenic APC for presenting peptides, or anti-CD3 Ab. Histogram overlays of gated DP cells from TRC (left panel) are shown in the middle and right panels. "Control" peptide incubations were done with H2B59–73 (0.001 µg in middle and 10 µg in right panel). Color code: middle panel (red = anti-CD3, blue = H471–94 0.001 µg, black = control peptide); right panel (red = anti-CD3, green = H471–94 10 µg, black = H471–94 0.001 µg, purple = control peptide). Thymocytes from 3- to 4-wk-old animals were used, before the reduction in thymocyte numbers had peaked. Representative of five separate experiments. E, H471–94 peptide induced deletion in TRC (14 h) was markedly different in {alpha}{beta}Tg SWR vs {alpha}{beta}Tg SNF1, but anti-CD3 Ab induced deletion was equivalent. Data from six high ({alpha}{beta}Tg SWR) and six intermediate expressors ({alpha}{beta}Tg SNF1) of the transgene are shown.

 

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  		Table II. Effect of anti-CD3 Ab injection on thymocyte counts

 
Natural display of nucleosomal peptide or related epitope/s by thymic DC and their surface phenotypes.

Spontaneous (natural) display of the cognate peptide for the Tg TCR or related epitope/s was markedly greater in thymic DC from WT normal SWR and SBF1 mice (p < 0.01 to <0.001) than the DC from autoimmune SNF1 (WT), as determined by ability of the thymic DCs by themselves to stimulate highly sensitive T cell hybridomas specific for the H471–94 peptide (Fig. 3A). These hybridomas, derived from SNF1 mice, can produce IL-2 in response to peptide eluted from class II (I-Ad or I-Aq) in attomole concentrations (15). Non-DC thymic APCs, containing thymic epithelial cells and macrophages, from the normal strains also displayed the epitope/s, but 2- to 5-fold less than thymic DCs, and again, this epitope display was undetectable in the non-DC APC of SNF1 thymus (data not shown). Preactivation of the thymic APC subsets by LPS (100 ng/ml) for 18 h before coculture with the T cell hybridomas did not change the results for spontaneous display (data not shown). In contrast, no differences were found in processing and presentation of exogenous nucleosomes or H471–94 peptide that were deliberately fed to the normal or lupus thymic DCs to stimulate the indicator hybridomas (Fig. 3B). After anti-CD3 Ab injection that causes massive thymocyte apoptosis in vivo (Table II), some increase in spontaneous stimulation of the T cell hybridomas by thymic DC from the SNF1 mice did occur, but it was still much lower (p < 0.01 to <0.001) than SWR thymic DC (Fig. 3C).



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  		FIGURE 3. Stimulation of H471–94 specific T cell hybridomas by thymic DC. A, IL-2 production response of T hybridomas cultured with thymic DC without addition of peptide. T hybridoma 102 (left) and 210 (right). Background values of any IL-2 production by the hybridoma cells cultured alone were subtracted. A and B represent seven independent experiments. B, Response of T hybridoma 102 cultured with thymic DC and graded doses of H471–94 peptide (left) or nucleosomes (right), added deliberately. Background values of cultures without any added peptide were subtracted. C, Response of T hybridoma 102 to thymic DC from animals undergoing anti-CD3 induced thymic apoptosis in vivo. No autoantigens were added in these cultures. Represents three separate experiments. D, Surface phenotype of thymic DC. Molecules important for Ag presentation are shown. The lower limits of the M1 marker in the histograms coincide with the upper limits of isotype controls in dot blots. Therefore, the dull-positive cells on the left of the marker are not included in the analysis in Table III, and the percentages and fluorescence intensities of the dull-positive DCs were similar between the two strains. Representative of four separate experiments.

 
The yield of thymic DCs upon purification were very similar among 4- to 6-wk-old SWR, SBF1, and SNF1 mice, respectively, being 0.51 ± 0.09%, 0.51 ± 0.13%, and 0.54 ± 0.1% of total thymocytes. To characterize the DCs further, surface markers of CD11c+ cells in total thymocyte populations were studied in steady state without any further manipulations. In contrast to SNF1, the normal SWR thymic DCs had a small but consistent and significant (p < 0.01) increase in cells staining more intensely for MHC class II, CD80, CD86 and B220 (Table III and Fig. 3D). The positive staining of the DCs for CD11b and CD4 (Table III) could be partly due to pick up from exogenous sources, as reported (18), but they were similar between the two strains.


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  		Table III. Surface markers of CD11c+-gated cells (DC) in the thymus

 
Peripheral T cells in the Tg mice

We have followed the expression of the transgenic TCR {beta} with anti-V{beta}4 mAb and also by RT-PCR, using peripheral blood lymphocytes, but the transgenic TCR{alpha} expression could only be detected by RT-PCR with specific primers (31). Although an Ab for the Tg TCR{alpha}-chain or its clonotype could not be generated for staining, the induction of apoptosis of Tg thymocytes specifically by addition of the nucleosomal H471–94 peptide, but not by control peptide/s, clearly indicated that the Tg TCR was expressed on the surface of thymocytes in the Tg mice (along with endogenous TCRs). Similarly, on presentation of the H471–94 peptide, but not control peptide, peripheral T cells from the Tg mice in all backgrounds up-regulate CD69 and down-regulate their surface TCR, as detected by a pan-{alpha}{beta}TCR specific mAb (Fig. 4A). Similar approaches have been used in other transgenic systems to track transgenic TCR bearing T cells (40). Introduction of the TCR {alpha} or {alpha}{beta} Tg into the normal SBF1 background markedly increased the response of splenic T cells to H471–94 peptide or nucleosomes as compared with WT SBF1 (Fig. 4C; p < 0.001). However, in the SNF1 lupus background, where spontaneous priming to these autoepitopes occur from early life (30), only the {alpha}Tg SNF1 mice showed responses comparable to the WT SNF1, but paradoxically the autoreactive responses of splenic T cells from the {alpha}{beta}Tg SNF1 mice were markedly suppressed (Fig. 4B; p < 0.01).



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  		FIGURE 4. A, Different degrees of TCR down-regulation induced in peripheral T cells by cognate peptide. CD4 T cells were incubated with irradiated APC and either control peptide (H2B59–73), anti-CD3 Ab, or H471–94 peptide at 1, 10, and 100 µg/ml. Three-color staining with Abs to CD4, CD69, and pan-{alpha}{beta}TCR were done. Numbers represent the percentage of cells in each quadrant. The {alpha}{beta}Tg SWR mouse in this example was an intermediate expressor of Tg TCR (see Fig. 2A); but T cells of the {alpha}{beta}Tg high expressors behaved like that from the {alpha}Tg SWR mouse. Representative of five separate experiments. B, IFN-{gamma} responses of T cells to autoantigens are markedly lower in TCR {alpha}{beta}Tg SNF1 mice as compared with {alpha}Tg and WT SNF1 mice in ELISPOT assay. T cells from 5-mo-old Tg or WT SNF1 mice were challenged with cognate peptide H471–94 or nucleosomes in vitro. Baseline IFN-{gamma} spots in T cells plus APC alone were 5 ± 3 spots per 1 x 106 T cells (*, p < 0.01). C, IFN-{gamma} responses of T cells to autoantigens are markedly increased in both {alpha}Tg and {alpha}{beta}Tg SBF1 as compared with WT SBF1 mice in ELISPOT. T cells from 5-mo-old Tg or WT SBF1 mice were challenged with the autoantigens in vitro. Baseline IFN-{gamma} spots in T cells plus APC alone were 8 ± 3 spots per 1 x 106 T cells.

 
Natural history of lupus

Despite substantial deletion in normal backgrounds, autoreactive cells escape to the periphery. However, all Tg SWR and SBF1 lines of mice have a normal life span and do not develop lupus nephritis. This is expected, because the normal strains lack peripheral lupus-susceptibility traits, which are numerous, such as anti-DNA B cell hyperactivity, hyperactive DC, or lowered threshold of activation in T cells (1, 41). The autoantibody levels in {alpha} or {alpha}{beta}Tg SBF1 mice were similar to background levels found in WT SWR or SBF1 (data not shown). By contrast, the {alpha}Tg SNF1 mice produced autoantibodies and developed lupus nephritis, at an incidence comparable to WT SNF1 (Fig. 5). However, the {beta} and {alpha}{beta}Tg SNF1 mice produced much lower levels of autoantibodies and had markedly decreased incidence of lupus nephritis (p < 0.001; Fig. 5).



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  		FIGURE 5. Natural history of lupus in the transgenic mice. A, Serum IgG autoantibody levels at different ages. Serum samples from {beta}Tg SNF1 mice were not available at the 11–14 mo age. * means p < 0.001. B, Cumulative incidence of severe lupus nephritis (grade of 3+ or above) in TCR transgenic, female mice in the lupus-prone SNF1 background. Number of animals ranged from 13 to 15 per line.

 
Potent Treg are induced in {alpha}{beta}Tg SNF1 mice in the periphery

The reason for the low incidence of autoantibodies and nephritis in the {beta}Tg SNF1 mice could be because the {beta}Tg TCR in combination with any of a multitude of endogenous TCR {alpha}-chains would not be autoreactive. Indeed, there was no increase in response to cognate peptide or nucleosomes in {beta}Tg SNF1 mice (data not shown), as in the case of {beta}Tg SWR (Fig. 4A). However, it was surprising why the {alpha}{beta}Tg SNF1 mice had markedly diminished lupus disease. Down-regulation of transgenic TCR, with up-regulation of endogenous TCR might have diminished autoreactivity of the transgenic T cells in these mice. The other possibility could be an increase in Treg cells and/or their activity. The percentage of CD4+CD25+ Treg cells in CD4+ SP thymocytes and splenocytes from 6- to 7-wk-old animals were actually less in the {alpha}{beta}Tg SNF1 mice, as compared with the {alpha}Tg SNF1 or WT SNF1 mice (Table IV), and the percentage of CD62Lhigh cells among the CD4+25+ T cells were similar among the three lines, ranging between 50 and 68% (not shown). However, functionally, the peripheral Treg cells from spleens of {alpha}{beta}Tg SNF1 mice were much more potent (Fig. 6). As shown previously, pathogenic autoantibody-inducing T cells of WT SNF1 lupus mice spontaneously recognize nucleosomes from apoptotic cells and the autoimmune response is mainly Th1 type (14, 30). Splenic CD4+25+ T cells from the {alpha}{beta}Tg SNF1 mice herein, directly suppressed the IFN-{gamma} response of WT SNF1 lupus T cells to nucleosomes presented by APC, up to 36-fold more than CD4+CD25+ T cells obtained from age-matched WT SNF1, and 4.6-fold more than those obtained from {alpha}Tg SNF1 mice (p < 0.01, Fig. 6). Indeed, the CD4+CD25+ T cells from WT SNF1 mice had no suppressive activity, but actually augmented the response of the target T cells to the autoantigen (Fig. 6).


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  		Table IV. CD4+CD25+ Treg cells in thymus and spleen

 


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  		FIGURE 6. Treg induction in Tg SNF1 mice. CD4+CD25+ Treg cells from {alpha}{beta}Tg SNF1 mice markedly suppressed the IFN-{gamma} responses of unmanipulated WT SNF1 lupus T cells to nucleosomes presented by APC in ELISPOT assay (ratio of Treg:lupus Th = 1:4), as compared with those from {alpha}Tg or WT SNF1 mice. CD4+CD25+ T cells from WT SNF1 mice actually increased IFN-{gamma} response in coculture with lupus T and APC. Results are expressed in mean ± SEM from three experiments (three mice per group). Baseline number of IFN-{gamma} spots produced by WT SNF1’s target T cells plus APC cultures with 0.1 µg/ml nucleosomes, were 677 ± 46 spots per 1 x 106 T cells, and the cultures without autoantigens were 5 ± 3 spots per 1 x 106 T cells. The purity of each subset of T cells was >90%. Percent suppression of IFN-{gamma} response is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A basic question is why does T cell tolerance break down in lupus, particularly to nucleosome, a ubiquitous and natural product of apoptosis that is especially prevalent in the thymus? Obviously many peripheral factors contribute to this outcome (1, 41). However, the transgenic mice bearing a prototypic, nucleosome-specific, lupus TCR from pathogenic Th clone 3A that we derived, show for the first time a central defect in the lupus-prone thymus that impairs its ability to delete autoimmune T cells specific for a major, disease-relevant epitope in nucleosomes. Our experimental system also shows for the first time that peptide epitopes from nucleosomes, which are the major products of ongoing apoptosis in the thymus, are indeed processed and presented naturally to developing thymocytes, and the rules for thymic selection against this ubiquitous and special autoantigen might be different from model Ags or superantigens. In addition, our transgenic mice reveal various defensive mechanisms (deletion, down-regulation of autoreactive TCR and up-regulation of endogenous TCR {alpha} and {beta}) observed previously in other systems (40, 42, 43, 44, 45) that come into play when facing an artificially large population of T cells specific for an ubiquitous autoantigen released from apoptotic cells. We did not breed our Tg into Rag–/– background, because B cells are needed for autoantibody production and development of lupus, along with many other lupus susceptibility traits (46, 47, 48, 49).

The amount, location, and timing of nucleosomal epitopes expressed in thymic ontogeny may determine the anatomy of apoptosis (9, 50). Almost all DP thymocytes are severely deleted in mice bearing a class I restricted Tg TCR reacting against a male-specific Ag that is probably displayed by all cells in the thymus (i.e., class I positive), (43). In contrast, our Tg TCR is class II restricted and probably sees the nucleosomal peptide/s (or related Ags) expressed by class II-positive thymic APC at a different stage of differentiation (50, 51). For instance, in the {alpha}{beta}Tg SWR mice, CD4 SP cells are relatively more depleted (~8-fold, or 88%) than DP thymocytes (2.5-fold, 60%) (Fig. 1, Table I), suggesting that deletion is occurring at the medulla or corticomedullary junction at the transition of DP to CD4+ SP stage. Even with ongoing deletion, CD4 SP skewing was marked in {alpha}{beta}Tg SWR, with a ~53-fold decrease in CD8 SP cells, as compared with an 8-fold decrease in CD4 SP cells, and a similar trend was seen in {alpha}{beta}Tg SBF1 (Table I). In contrast, no deletion or CD4 SP skewing was found in the Tg SNF1 mice indicating some deficiency in expression of the nucleosomal H4 epitope in the lupus-prone SNF1 thymus.

One possibility is that nucleosomes might not be presented in sufficiently high levels in the thymus of lupus-prone SNF1 mice due to lack of appropriate processing and display of the epitope H471–94 for which the Tg TCR is specific. Although apoptotic cells are normally removed by phagocytosis before release of any immunogenic nuclear material (10, 11), marked deletion was consistently and clearly evident in the same Tg thymocytes in normal SWR or SBF1 background, indicating that H471–94 or similar epitope/s was processed from nucleosomes and presented to developing thymocytes in normal mice. Using highly sensitive T cell hybridomas as readouts, the natural display of nucleosome-related peptides were strikingly different among the thymic APC of normal vs lupus strains. A small but significant diminution in expression of class II and costimulatory molecules in the thymic DC of lupus-prone SNF1 mice could have decreased the efficiency of presentation of endogenous nucleosomal epitopes. Histological abnormalities have been detected in the thymic stromal cells of NZB mice (52), which might be somehow connected to this nucleosome-specfic tolerance defect in the SNF1 progeny’s thymic APC function. Nevertheless, thymocytes of the {alpha}{beta}Tg SNF1 mice were positively selected, therefore, some epitope/s related to H471–94 peptide must have been presented in the SNF1 thymus. Although, the presentation level might not have been sufficiently high, or of the right quality to cause thymocyte deletion in vivo, or be detectable by the indicator hybridomas, it was sufficient to cause down-regulation of Tg TCR on thymocytes and peripheral T cells in the SNF1 background. Indeed we show that the cause of the defect in deletion resides in thymic DC, and not in the thymic lymphoid cells, because Tg line thymocytes in normal or lupus backgrounds were equally susceptible to deletion by irradiation or exposure to anti-CD3 Ab in vivo or in vitro. The resistance to apoptosis in SNF1 thymocytes by the cognate H471–94 peptide was a consequence of down-regulation of their Tg-TCR, along with up-regulation of endogenous TCRs. In contrast to the thymic DC, peripherally, DCs are activated in lupus (53), further demonstrating the complex mixture of different susceptibility traits in this disease.

In the periphery, the incidence of lupus nephritis was decreased in the {beta}Tg SNF1 mice, probably because the {beta} TCR from clone 3A could combine with any endogenous TCR {alpha}-chain to make it nonautoimmune. The incidence of lupus nephritis in {alpha}Tg SNF1 mice was similar to WT SNF1, but that in the {alpha}{beta}Tg SNF1 mice was again markedly decreased (Fig. 5). One would expect that lupus nephritis would be accelerated further in SNF1 mice bearing a pathogenic TCR transgene. However, thymic development is artificially skewed in transgenic TCR mice. Most probably, in the transgenic SNF1 mice, premature and global expression of a functionally rearranged pathogenic TCR from the very beginning of thymic ontogeny, sets in motion several layers of protective mechanisms even in the lupus prone thymus, such as down-regulation of the pathogenic TCR, and subsequently in its periphery, and induction of Treg cells as well. Unlike other autoreactive systems using model Ags (54), we did not find an increase in Treg cells in the thymus of {alpha}{beta}Tg SNF1 mice, but potent Treg cells were induced in the periphery of these mice, as in other artificially autoreactive systems (55). Perhaps, the Tg T cells with down-regulated Tg TCR, but up-regulated endogenous TCR are a fertile source of Treg cells, as in other systems (56). As opposed to the Tg SNF1, in the WT SNF1 thymus, the spontaneous expression of a pathogenic TCR like 3A, might occur at a much lower frequency, and in due course of time after successive steps of TCR{beta} followed by {alpha}-chain gene rearrangements. The natural sequence of events in the wild type thymus could allow a relatively low frequency of thymocytes bearing 3A-like TCR to develop; and the selective defect in deletion in the lupus-prone thymus would permit them to sneak through. By contrast, in the transgenic animals, 3A TCR is expressed from the earliest stage of development and in all thymocytes, when the expression of other accessory molecules and signaling thresholds involved in positive and negative selection might be different.

Certain novel findings relevant to the etiology of lupus have emerged from these studies, despite the complexity of analyzing transgenic T cells with a naturally arising, pathogenic specificity for an endogenous ubiquitous autoantigen in the background of spontaneous lupus susceptibility. In mice prone to develop spontaneous systemic lupus erythematosus, there is a defect in central tolerance that appears to be selective for nucleosome autoantigens and it is caused by inefficient presentation of the relevant epitopes by thymic DC, which is distinct from defects localized to thymic lymphoid cells found in the NOD mouse model of diabetes (57, 58).


   Acknowledgments
 
We thank Dr. Lynn Doglio of our Tg mouse core facility for injection of SWR oocytes with our constructs and initial help in deriving the founder lines.


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


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

1 This work was supported by National Institutes of Health Grants RO1-AI41985 and R37-AR39157 (to S.K.D.). Back

2 Address correspondence and reprint requests to Dr. Syamal K. Datta, Division of Rheumatology, Northwestern University Feinberg School of Medicine, 240 East Huron Street, McGaw 2300, Chicago, IL 60611. E-mail address: skd257{at}northwestern.edu Back

3 Abbreviations used in this paper: Tg, transgenic; Treg, regulatory T cell; M{phi}, macrophage; DC, dendritic cell; PI, propidium iodide; WT, wild type; DP, double positive; SP, single positive; TRC, thymic reaggregation culture. Back

Received for publication April 29, 2005. Accepted for publication August 18, 2005.


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