Defective Selection of Thymic Regulatory T Cells Accompanies Autoimmunity and Pulmonary Infiltrates in Tcra-Deficient Mice Double Transgenic for Human La/Sjögren’s Syndrome-B and Human La-Specific TCR

Autoantibodies directed to the RNA-binding Ags Ro/Sjögren’s syndrome (SS)-A and La/SS-B occur frequently in patients with the autoimmune disorders systemic lupus erythematosus (SLE), SS, and neonatal lupus erythematosus. Ro and La Abs are among the earliest specificities detected before SLE diagnosis (1), are heritable traits in SLE patients (2), and closely associate with a type I IFN gene signature in SS (3) and type I IFN activity in SLE (4). Serum La Abs typically co-occur with Ro Abs and are used as disease classification markers (5). A lower incidence of both dsDNA Abs and renal disease has been observed in SLE patients positive for both anti-Ro and anti-La compared with SLE patients having anti-Ro alone (5). SS patients suffer from lymphocytic infiltration of the exocrine glands, resulting in glandular dysfunction and dryness of the eyes and mouth. Primary SS patients with precipitating levels of Abs to La and Ro are more likely to suffer from extraglandular manifestations of the disease (6), including pulmonary function abnormalities (7).

The high serum concentration (8), class-switched and somatically mutated nature (9), and association with particular HLA class II alleles (10) of human anti-La Abs in SLE and SS suggest that they are Th cell dependent. In previous studies, we demonstrated incomplete immunologic tolerance to human La/SS-B (hLa) in healthy mice expressing a hLa transgene (11). This tolerance occurred in CD4⁺ T cells, whereas no tolerance was detectable in hLa-specific B cells from mice expressing hLa (12). Prior studies uncovered evidence of Ag-specific suppressor T lymphocytes specific for the small nuclear ribonucleoprotein A Ag (13) in the periphery of healthy mice. However, thymic mechanisms of CD4⁺ T cell tolerance to La or other RNA-binding Ags targeted in systemic autoimmunity have not been examined.

TCR transgenic (Tg) mice, in which the phenomenon of allelic exclusion narrows the T cell repertoire to one consisting primarily of a single T cell specificity, have been invaluable for identifying cellular mechanisms of development and tolerance in CD4⁺ T lymphocytes specific for model Ags, illustrating both thymic clonal deletion (14–16) and thymic regulatory T cell (tTreg) selection (17–19). The outcome of T cell development depends upon TCR affinity and the concentration and level of presentation of the selecting antigenic epitope (20–22). Controlling zygosity of MHC alleles that restrict selecting T cell epitopes has been successfully used in TCR Tg models as a strategy to alter Ag presentation levels, resulting in altered T cell developmental outcomes (23). Loss of CD4⁺ T cell tolerance has been observed in TCR Tg mice specific for myelin basic protein (24, 25) and pancreatic islet Ag (26), but disease phenotypes were observed only in mice also deficient in Rag or endogenous Tcr genes (24, 25), or those that were SCID mutant (26). Loss of tolerance and concomitant disease in all of these models was mediated by defective CD4⁺ T cell–mediated suppression and/or defective tTreg development (27).

In this study, we investigated cellular mechanisms of CD4⁺ T cell tolerance and the consequences of its loss for a representative ubiquitous, RNA-binding nuclear Ag, La. We report the creation of TCR Tg mice specific for an immunodominant T cell epitope of the hLa Ag restricted by murine I-E^k and the outcome of crossing of these animals to previously described mice (11) expressing physiologic levels of nuclear hLa Ag, in the presence and absence of endogenous Tcra deficiency. We demonstrate that thymic deletion and tTreg selection are consequences of hLa neo–self-Ag expression and that anti-La autoimmunity induced by endogenous Tcra deficiency results in pulmonary pathology. These results suggest that self-Ag–specific T cells contribute to rheumatic lung disease.

Line 3 hLa Tg mice (11) backcrossed to C57BL/6J (B6) >12 generations were crossed to B6 mice congenic for H-2^k (B6.AK-H2^k/FlaEgJ; The Jackson Laboratory, Bar Harbor, ME) to generate hLa^+/−H-2^k/k or hLa^+/−H-2^k/b mice. B6 Tcra^−/− mice (B6.129S2-Tcra^tm1Mom/J; The Jackson Laboratory) were used to generate genotypes on a Tcra^−/− background.

Constructs to generate 3B5.8 TCR^+/− Tg mice (C57BL/6-Tg(Tcr3B5.8)Adf) were created by cloning the Tcra (TRAV12-2/J17 [Vα8.4/Jα17]) and Tcrb (TRBV4/D1/J2-1 [Vβ10/D1/Jβ2.1]) cDNAs from an I-E^k–restricted hybridoma [3B5.8, generated as described previously (28)] specific for the hLa67-76 peptide VIVEALSKSK were cloned into human CD2 minigene-driven T cell expression constructs (29) using adapter primers containing XmaI restriction enzyme sites (β-chain: [975 bp] 3B5.8 BETA V: 5′-ccc ggg atg ggc tgt agg ctc cta agc tgt gtg gcc ttc-3′ and TCRBC: 5′-tcc cgg gtc agg aat ttt ttt tga cca tgg cca tc-3′; α-chain: [870 bp] 3B5.8 α V: 5′-ccc ggg atg aac atg cat cct gtc acc tgc tca gtt c-3′ and CD2 TCR C: 5′-atg gag ctt ggg acc cgg gct ctg tca gtc-3′). SalI- and NotI-cleaved and purified α- and β-chain constructs were comicroinjected in equimolar amounts into fertilized ova of B6 mice and Tg mice generated.

hLa Tg status was verified by PCR of genomic DNA as described previously (11, 12). Genomic DNA of 3B5.8 TCR Tg mice was typed using the same primer sets listed earlier. Tcra wild type and mutant alleles were typed by PCR as recommended by The Jackson Laboratory. H-2 haplotypes were inferred by flow cytometry of PBLs using mAbs directed to I-E^k (clone 14-4-4S) and I-A^b (clone 25-9-17).

Animals were maintained under pathogen-free barrier conditions in the Oklahoma Medical Research Foundation (OMRF) Laboratory Animal Resource Center. Mice deficient in endogenous Tcra genes were maintained on water or Napa Nectar (Systems Engineering, Napa, CA) containing 0.2 mg/ml trimethoprim and 1.0 mg/ml sulfamethoxazole to prevent opportunistic infections and rectal prolapses. All studies were approved by the OMRF Institutional Animal Care and Use Committee. Unless otherwise indicated, mice were 7–9 wk old at the time of evaluation.

Single-cell suspensions of whole thymi or spleens from individual mice were prepared using 70-μm nylon mesh, treated with 0.14 M NH₄Cl₂/17 mM Tris, pH 7.2 to lyse RBCs, washed, and quantified by hemocytometer using trypan blue exclusion. Lungs were minced, incubated for 30 min at 37°C with 250 U/ml DNaseI (Sigma-Aldrich) and 1 mg/ml Collagenase D (Roche) in DMEM, sieved through 40-μm nylon mesh, washed, and cells quantified as described earlier. Cells (1–2 × 10⁶/organ) were stained for multicolor flow cytometry, events collected on an LSRII cytometer (BD Biosciences), and analyzed using FACSDiva (BD Biosciences) or FlowJo (TreeStar, Ashland, OR) software. Surface markers included anti–CD8a-biotin or -PerCP (clone 53-6.7), –CD25-allophycocyanin or -biotin (PC61), –Vβ10-PE (B21.5), CD69-biotin (H1.2F3), –CD44-biotin (IM7), –CD4-allophycocyanin-Cy7 or –allophycocyanin Alexa750 (RM4-5), –CD62L-allophycocyanin (MEL-14), Vα2-FITC (B20.1), Vα3.2-FITC (RR3-16), Vα8.3-FITC (B21.14), and Vα11.1/11.2-FITC (RR8-1). Biotinylated Abs were detected using streptavidin-PE-Texas Red (CalTag/Invitrogen). Intracellular Foxp3 staining was performed using anti–Foxp3-FITC or -allophycocyanin (clone FJK-16s; eBioscience) according to the manufacturer’s protocol. Lymphocyte populations were identified by forward and side scatter. Absolute cell numbers and mean fluorescence intensity (MFI) data were compared using t test with Welch’s correction as necessary or by one-way ANOVA with Tukey’s multiple-comparison posttest. Comparisons of percentage data were evaluated using the Mann–Whitney U test.

Microcultures containing nylon wool–enriched (30) 3B5.8 TCR Tg splenic T cells in a 1:1 ratio with gamma-irradiated (2200 rad) splenocytes (APC) from sex- and H-2–matched B6 mice were incubated in triplicate with dilutions of specific (hLa61–84 peptide, endotoxin-depleted 6xHis-tagged hLa or mouse La recombinant proteins) or nonspecific (HEL 46–61 peptide or endotoxin-depleted 6xHis-HEL) control Ags in complete T cell medium (MEM with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, and 2 mM Na-pyruvate, 0.1 mM nonessential amino acids, and 50 μM 2-ME). Cultures were incubated for 72 h at 37°C in 10% CO₂ and pulsed with [³H]thymidine at 1 μCurie/well during the last 18 h. Cultures were harvested onto glass fiber filters and counted by liquid scintillation.

Pooled thymi (eight mice per genotype) were minced in 15 mg/ml Liberase TH and 1.3 mg/ml DNase, then disrupted using two cycles of the “m_spleen_02” program on a gentleMACS Dissociator (Milentyi Biotec) with a 15-min incubation at 37°C between programs. Cells were washed with RPMI/20% FCS/10 mM Hepes, resuspended in RPMI/10% FCS/10 mM Hepes and passed through a 40-μm filter by gravity. Thymocytes were depleted with biotinylated mAbs to CD3 (clone 145-2C11), CD5 (53-7.3), CD25 (7D4), and erythroid cells (Ter¹¹⁹) followed by Dynabeads Biotin Binder (Invitrogen) using the manufacturer’s instructions. The depleted fractions were labeled with mAbs CD11c-PerCp-Cy5.5 (N418), CD45-allophycocyanin-Cy7 (30-F11), CD11b-PE-Cy7 (M1/70), Ly-51–Alexa 647 (6C3), CD3-BV605 (17A2), and UEA-1–biotin (Vector Laboratories)/SA-PE-Cy5. For constitutive Ag presentation experiments, thymic dendritic cells (DCs) were sorted as FITC autofluorescence⁻PI⁻CD3⁻CD45⁺CD11b⁻CD11c⁺, and medullary thymic epithelial cells (mTECs) as PI⁻CD3⁻CD45⁻UEA-1⁺ on an Ly-51 × UEA-1 dot plot as gated in Williams et al. (31) on a MoFlo sorter (Cytomation). For evaluation of MHC expression in separate experiments, anti–I-E^kβ–PE (clone 17-3-3; Santa Cruz Biotechnology) was added to the sort mixture and large data files were collected. Alternatively, mTECs were isolated as described earlier, restained with anti–I-E^kβ, and resorted by MoFlo. Data were analyzed by FlowJo version 10.

IgG Abs to rAgs were measured by ELISA as previously described (28).

Graded numbers of thymic DCs were cultured in triplicate, and 1200 mTECs per well were cultured in duplicate with 5 × 10⁴ 3B5.8 T hybridoma cells in 0.2 ml complete T cell medium in round-bottom 96-well tissue culture plates. After 24 h of culture, cell supernatants from individual wells were collected and evaluated for concentrations of IL-2 by ELISA (IL-2 ELISA Set; BD Biosciences).

Single lung lobes of individual mice were inflated with HBSS, fixed in 10% buffered formalin, embedded in paraffin, and sectioned (ten 6-μm sections collected at 100-μm intervals per lobe). Slides were stained with H&E and evaluated by a board-certified veterinary pathologist (S.D.K.). Lymphoid aggregates were scored as mild (0.5–1.0), moderate (1.5–2.0), marked (2.5–3.0), or severe (3.5–4.0). Intraalveolar macrophages were scored as the average number per five high-power fields evaluated. Statistical differences among groups were evaluated by one-way ANOVA with Dunn’s multiple comparison posttest.

B6 mice Tg for both TCRα (Trav12-2/J17; Vα8.4/Jα17) and TCRβ (Trbv4/D1/J2-1; Vβ10/D1/Jβ2.1) chain cDNAs isolated from an I-E^k–restricted, hLa aa 67–76–specific T cell hybridoma (3B5.8) were crossed with B6 mice congenic for H-2^k to introduce I-E^k. Flow cytometric analysis showed that 99% of CD4⁺ PBLs from H-2^k/b 3B5.8 TCR Tg mice expressed Vβ10, a 10-fold increase over non-Tg littermates (Fig. 1A). The cell-surface expression level of Vβ10 on 3B5.8 TCR Tg CD4⁺ T cells was indistinguishable from that of non-Tg mice (Fig. 1A), indicating a physiologically normal expression level of the Tg TCR. Splenic T cells from H-2^k/b or H-2^k/k 3B5.8 TCR Tg mice proliferated in a dose–response manner to recombinant human La (hLa) protein, but not to recombinant mouse La or nonspecific control Ag, whereas responses from non-Tg littermate mice were near background levels (Fig. 1B). Thus, CD4⁺ T cells expressing a functional 3B5.8 TCR predominate in H-2^k-positive 3B5.8 TCR Tg mice.

The effect of H-2 on thymic selection of Vβ10⁺ T cells in 3B5.8 TCR Tg mice was next evaluated. No significant differences in total thymic cellularity of 3B5.8 TCR Tg mice of H-2^k/k, H-2^k/b, or H-2^b/b haplotypes were observed (k/k: n = 10: 9.3 ± 1.3 × 10⁷; k/b, n = 8: 12.0 ± 1.8 × 10⁷; b/b, n = 3: 11.8 ± 0.6 × 10⁷). However, fractions of CD4⁺CD8⁻ (CD4 single-positive [SP]) Vβ10⁺ cells were reduced in H-2^b/b 3B5.8 TCR Tg mice compared with wild type, non-Tg mice of H-2^k/b (Fig. 2A, upper panels) and H-2^k/k haplotypes (data not shown). In contrast, substantially increased percentages of Vβ10⁺ CD4SP thymocytes were observed in both H-2^k/b and H-2^k/k 3B5.8 Tg mice compared with non-Tg littermate mice of the same H-2 haplotype (Fig. 2A, middle panels). Drastically increased numbers of Vβ10-expressing CD4SP cells in H-2^k/b and H-2^k/k 3B5.8 Tg mice indicated that there was efficient positive selection of 3B5.8 T cells by H-2^k. Conversely, Vβ10⁺ CD4SP did not develop in H-2^b/b 3B5.8 Tg mice (Fig. 2B) despite their abundance at the CD4⁺CD8⁺ double-positive (DP) stage (Fig. 2C). Similar numbers of Vβ10⁺ CD4SP were observed in H-2^k/b and H-2^k/k 3B5.8 TCR Tg mice (Fig. 2B), indicating equivalent positive selection of the Tg T cells by these two haplotypes. Numbers of Vβ10⁺ DP thymocytes were also significantly elevated in H-2^k/b and H-2^k/k 3B5.8 TCR Tg mice compared with non-Tg littermates of the same H-2 (Fig. 2C).

No significant differences in the fractions or absolute numbers of Vβ10⁺CD4⁺ splenocytes were observed between 3B5.8 TCR Tg mice of the H-2^k/b (n = 8, 2.23 ± 0.25 × 10⁷) or H-2^k/k haplotypes (n = 10, 1.77 ± 0.21 × 10⁷). In contrast, 14- to 18-fold fewer Vβ10⁺CD4⁺ splenocytes were observed in 3B5.8 TCR Tg H-2^b/b mice (n = 3, 1.22 ± 0.38 × 10⁶).

Therefore, CD4⁺ T cells bearing the 3B5.8 TCR are positively selected with similar efficiency by H-2^k/k and H-2^k/b mice but are not selected in H-2^b/b mice.

To determine the effect of H-2 haplotype on thymic development of hLa-specific T cells in the presence of the hLa neo–self-Ag, percentages and numbers of Vβ10⁺ CD4SP and DP thymocytes from 3B5.8 TCR Tg mice were compared with those of mice expressing both 3B5.8 and hLa Tgs. The hLa Tg mice express nuclear hLa ubiquitously at levels similar to the natural mouse La Ag (11). 3B5.8/hLa double-Tg total thymocyte numbers were significantly reduced compared with that of 3B5.8 single-Tg mice on both H-2^k/k (2.01 ± 0.39 × 10⁷ versus 9.35 ± 1.35 × 10⁷; p < 0.0001) and H-2^k/b (5.71 ± 0.80 × 10⁷ versus 12.0 × 10⁷; p = 0.0018) backgrounds. Significantly reduced percentages (Fig. 2A, lower panels) and absolute numbers (Fig. 2B) of 3B5.8 thymocytes were observed at the CD4SP stage in double-Tg mice compared with 3B5.8 single-Tg littermates in both the H-2^k/b and H-2^k/k models, consistent with thymic clonal deletion. Deletion was also detected at the DP stage (Fig. 2C), although it was less efficient in H-2^k/b compared with H-2^k/k double-Tg mice, because there was only a 2-fold decrease in H-2^k/b DP Vβ10⁺ thymocytes compared with a 6-fold decrease in H-2^k/k 3B5.8/hLa double-Tg versus H-2 matched 3B5.8 single-Tg mice. Vβ10 MFI on Vβ10⁺ CD4SP thymocytes of both H-2^k/k and H-2^k/b double-Tg mice was reduced compared with that of 3B5.8 single-Tg mice of the same haplotype (data not shown), suggesting encounter with specific I-E^k/hLa MHC/peptide complexes in vivo.

These trends were also apparent in the periphery, because splenic numbers of CD4⁺CD8⁻Vβ10⁺ 3B5.8 T cells were significantly reduced in 3B5.8/hLa double-Tg mice of both H-2 haplotypes (3B5.8 versus 3B5.8/hLa: H-2^k/b, 2.23 ± 0.25 × 10⁷ versus 8.96 ± 1.06 × 10⁵, p < 0.0001; H-2^k/k, 1.77 ± 0.20 × 10⁷ versus 6.09 ± 0.86 × 10⁵, p < 0.0001).

To assess the development of tTregs, we assessed CD25 and Foxp3 expression in thymic Vβ10⁺ cells. Cells coexpressing CD25 and Foxp3 in the spleen, which may include a combination of peripherally expanded tTregs and de novo–differentiated peripheral Tregs, were also measured. Percentages (Fig. 3A) and absolute numbers (Fig. 3B) of CD4SP Vβ10⁺CD25⁺Foxp3⁺ thymocytes were significantly increased in H-2^k/b, 3B5.8/hLa double-Tg mice compared with 3B5.8 single-Tg littermates. This phenomenon was observed only in H-2^k/b mice, and not H-2^k/k mice (Fig. 3B). Percentages of Vβ10⁺ splenocytes with a Treg phenotype were drastically increased in 3B5.8/hLa double-Tg mice compared with 3B5.8 single-Tg littermates (Fig. 3A); however, their absolute numbers were not increased because spleens of double-Tg mice were significantly smaller than that of 3B5.8 single-Tg mice. Development of tTregs in response to nuclear hLa expression could be induced by a mechanism of endogenous Vα TCR inclusion. To evaluate this possibility, we used a mixture of available mAbs specific for five different endogenous TCR Vαs to assess tTregs. Although a small number of thymic CD4SP of hLa/3B5.8 double-Tg mice expressed these endogenous Vα, the Vα-included cells did not develop into tTregs (Supplemental Fig. 1). Sorted Vβ10⁺CD4⁺CD25⁺ splenocytes from H-2^k/b 3B5.8/hLa double-Tg mice were evaluated for their capacity to suppress proliferation of Vβ10⁺CD4⁺ splenic effector T cells (Teffs) sorted from 3B5.8 single-Tg mice in the presence of irradiated APCs and hLa 61–84 peptide, which contains the 3B5.8 epitope. The CD25⁺ population isolated from 3B5.8/hLa double-Tg mice had mild but detectable suppressive activity and was anergic to peptide stimulation (Supplemental Fig. 2A).

Thus, in the presence of the hLa neo–self-Ag, CD4⁺ T cells expressing the 3B5.8 hLa-specific TCR are strongly deleted in H-2^k/k mice, are less efficiently deleted in H-2^k/b mice, and a portion of the surviving cells in the H-2^k/b model are positively selected as tTregs.

Because T cells bearing the 3B5.8 receptor are not selected by H-2^b/b, differences in efficiency of negative selection and capacity to positively select natural Tregs in H-2^k/b versus H-2^k/k mice are likely due to differences in MHC class II presentation of the hLa epitope recognized by the 3B5.8 TCR. To investigate this, we isolated thymic DCs and mTECs from pooled thymi of groups of mice of H-2^k/k, H-2^k/b, and H-2^b/b haplotype, and evaluated expression of I-E^k. Both thymic DCs and mTECs from H-2^k/k mice expressed higher cell-surface levels of I-E^k than mice of H-2^k/b haplotype, as expected (Fig. 4A). To determine whether these thymic APCs constitutively present the 3B5.8 T cell epitope, we isolated thymic DCs and mTECs from groups of hLa Tg and non-Tg littermate mice of haplotype H-2^k/k or H-2^k/b and tested them for the capacity to stimulate the 3B5.8 hybridoma in vitro. Thymic DCs from both H-2^k/k and H-2^k/b hLa Tg mice stimulated IL-2 release from the 3B5.8 hybridoma in a dose–response fashion, whereas thymic DCs from non-Tg mice did not (Fig. 4B, left panel). Thymic DCs from H-2^k/k hLa Tg mice stimulated greater IL-2 release compared with thymic DCs from H-2^k/b hLa Tg mice (Fig. 4B, left panel). Although only small numbers of mTEC could be isolated from any group of mice (median 2500, range 1200–5000 cells for 7 independent groups of 8 mice each), constitutive expression of the 3B5.8 epitope by mTECs from hLa Tg H-2^k/k mice was suggested by induction of a miniscule but detectable amount of IL-2 from the 3B5.8 T cell hybridoma (Fig. 4B, right panel). Increased expression of cell-surface I-E^k and elevated constitutive expression of the 3B5.8 T cell epitope were also evident in peripheral APCs (Supplemental Fig. 3). Thus, the 3B5.8 epitope is presented less effectively in thymi of H-2^k/b mice compared with H-2^k/k mice, and this inefficient presentation occurs concurrently with positive selection of tTregs and less effective thymic clonal deletion.

Defective Treg selection and/or function and autoimmune pathology have been demonstrated in other TCR Tg mouse models of CD4⁺ autoimmunity if the mice were deficient in endogenous Tcr genes, Rag genes, or were Scid mutant. Thymic T cell development was evaluated in Tcra-deficient H-2^k/b 3B5.8/hLa neo–self-Ag double-Tg mice to determine whether similar Treg defects would be observed. Total thymocyte numbers were reduced in Tcra^−/− double- (3.77 ± 0.77 × 10⁷, n = 11) compared with single-Tg mice (12.84 ± 1.27, n = 11; p < 0.0001). Efficient thymic clonal deletion of CD4⁺Vβ10⁺ thymocytes was suggested by their significantly reduced numbers in Tcra^−/− 3B5.8/hLa double-Tg mice (Fig. 5A). In contrast with and unlike Tcra-intact double-Tg mice, increased absolute numbers of CD4⁺Vβ10⁺Foxp3⁺CD25⁺ thymocytes were not observed in Tcra^−/− double-Tg mice, indicating a lack of positive selection of tTregs as expected (Fig. 5B).

Absolute numbers of Foxp3⁺CD25⁺ T cells in spleens of Tcra^−/− 3B5.8xhLa double-Tg mice were not elevated compared with 3B5.8 single-Tg littermates (data not shown); however, some peripheral CD4⁺Vβ10⁺Foxp3⁺CD25⁺ cells were still detectable. To determine whether these cells had suppressive activity, we sorted and cocultured splenic CD4⁺Vβ10⁺CD25⁺ T cells of Tcra^−/− 3B5.8/hLa double-Tg mice with CD4⁺Vβ10⁺CD25⁻ 3B5.8 Teffs from Tcra^−/− 3B5.8 single-Tg mice in the presence of irradiated, T cell–deficient APCs and hLa61–84 peptide, and T cell proliferation was measured. Proliferation of 3B5.8 Teffs was not reduced by coculture with CD4⁺Vβ10⁺CD25⁺ T cells of Tcra^−/− 3B5.8/hLa double-Tg mice (Supplemental Fig. 2B), indicating an absence of detectable suppressive activity. Therefore, selection of tTregs is deficient in Tcra^−/− 3B5.8/hLa double-Tg mice, and suppressive activity of Tregs found in the periphery of Tcra^−/− 3B5.8/hLa double-Tg mice, which may include expanded tTregs and peripherally differentiated Tregs, is defective.

Although the absolute numbers of splenic Thy1.2⁺CD4⁺Vβ10⁺ T cells were severely reduced in Tcra-deficient double-Tg mice compared with their single-Tg littermates (3B5.8 Tg: 218.7 ± 31.9 × 10⁵ versus 4.4 ± 0.5 × 10⁵, p < 0.0001), evaluation of these cells for expression of CD44 and CD62L revealed conversion to the effector/memory phenotype (Fig. 6A). These cells also expressed higher levels of CD69 in double-Tg mice (MFI 821 ± 116) compared with 3B5.8 single-Tg littermates (418 ± 48; p = 0.01).

To determine whether deletion of the endogenous Tcra locus in 3B5.8 TCR/hLa double-Tg mice results in serologic autoimmunity, we evaluated serum samples from Tcra^−/− 3B5.8 single-Tg and Tcra^−/− 3B5.8/hLa double-Tg mice of various ages for Abs to recombinant 6xhis-hLa/SS-B Ag. Strikingly, in contrast with the endogenous Tcra-intact double-Tg model, all Tcra^−/− 3B5.8/hLa double-Tg mice developed high-titer serum IgG Abs to recombinant hLa Ag, but not to an irrelevant foreign Ag from Bacillus anthracis (6xhis-PA) that is immunogenic in H-2^kmice (32) (Fig. 6B). Anti-hLa Abs were prominent by 6 wk of age in all tested Tcra^−/− double-Tg mice and were substantially increased in the same animals by 8 wk of age. Separate litters of Tcra^−/− double-Tg mice aged to either 12–15 or 20–28 wk had sustained, high-titer anti-hLa autoantibodies similar to those of 8-wk-old mice. No Abs to recombinant mouse Ro60-MBP Ag were detectable by ELISA or Western blot (data not shown), indicating a lack of intermolecular B cell epitope spreading in this model.

Multiple organs of 16- to 22-wk-old mice were evaluated for evidence of autoimmune pathology. Interstitial, often peribronchial, lymphocytic accumulations were consistently observed in lung tissue of Tcra^−/− 3B5.8/hLa double-Tg mice, but not their 3B5.8 single-Tg or non-Tg littermates (Fig. 7A, 7B). Numbers of intra-alveolar macrophages were slightly increased in 3B5.8/hLa double-Tg mice, but this did not significantly differ from non-Tg Tcra^−/− mice of the same age and H-2 haplotype (Fig. 7B). Small lymphocytic infiltrations were occasionally but inconsistently observed surrounding the hepatic portal vein in 3B5.8/hLa double-Tg mice, but lymphocytic infiltration of salivary gland tissue was notably absent (data not shown). Flow cytometric analysis of lung cell infiltrates revealed drastically increased fractions of CD4⁺Vβ10⁺ T cells of effector memory phenotype (Fig. 7C) expressing elevated cell-surface levels of CD69 (3B5.8/hLa Tg: 2101 ± 276 versus 3B5.8 Tg: 871 ± 122; p = 0.003) in double-Tg mice compared with 3B5.8 single-Tg littermates. As also observed in spleens (data not shown), some CD4⁺ T cells from the lungs of 3B5.8/hLa double-Tg mice downregulated cell-surface Vβ10 (Fig. 7D, left panel). Notably, among CD4⁺ T lymphocytes isolated from spleen (data not shown) and lung tissues (Fig. 7D, right panel) of Tcra^−/− double-Tg mice, only those Vβ10⁺ cells expressing the hLa TCR were activated, as evidenced by their selective upregulation of CD69. Moreover, CD4⁺Vβ10⁺ T cells isolated from lungs of 3B5.8 single-Tg mice did not express elevated CD69, indicating that CD4⁺ T cell activation in lungs was induced by the hLa neo–self-Ag and not other bystander mechanism(s) (Fig. 7D, right panel). The presence of these infiltrates did not affect the survival of Tcra^−/− 3B5.8xhLa double-Tg mice compared with Tcra^−/− 3B5.8 single-Tg or hLa single-Tg littermates (data not shown).

Therefore, deficiency of endogenous Tcra genes in mice double Tg for the hLa-specific 3B5.8 TCR and the hLa neo–self-Ag results in defective selection of tTregs, defective suppressive activity of Tregs isolated from the periphery, serologic anti-La autoimmunity, and accumulation of activated, effector memory T cells in lung tissue.

This study highlights thymic mechanisms of immunologic tolerance to the class of ubiquitous, RNA-binding self-Ags that are frequent targets of systemic autoimmunity in rheumatic diseases and demonstrates pathologic consequences of CD4⁺ T cell autoimmunity to La. Using the first TCR Tg mouse model specific for a clinically relevant, RNA-binding nuclear Ag, we show that thymic clonal deletion and tTreg differentiation are induced by nuclear expression of the La Ag and are thus mechanisms of normal thymic tolerance to this class of Ag. A previous study documented evidence for nuclear ribonucleoprotein A–specific suppressor T cells in the periphery of healthy mice, but did not determine whether tTregs, which contribute substantially to the peripheral Treg pool, are positively selected (13). Thus, this study is the first demonstration, to our knowledge, of tTreg-positive selection in response to an RNA-binding nuclear Ag.

Thymic DCs isolated from H-2^k/k and H-2^k/b hLa Tg mice constitutively presented the hLa epitope recognized by the hLa-specific 3B5.8 TCR. This is in stark contrast with an earlier study concluding that thymic DCs were incapable of presenting nuclear Ags (33). In that study, bone marrow–derived cells from thymus did not constitutively present nuclear β-galactosidase, nor was cross-presentation of the Ag by thymic DCs detected. In this respect, our findings are similar to those of Datta and colleagues (34), who clearly showed constitutive presentation of nucleosomal histone peptide by thymic DCs in a healthy mouse model of nucleosome-induced thymic clonal deletion. Features that may promote efficient thymic DC presentation of nuclear Ags may include altered direct presentation pathways secondary to the nucleic acid binding character of the nuclear La and nucleosome Ags and/or the true ubiquitous expression of these Ags, which could surpass a necessary threshold of cellular material available for cross-presentation by thymic DCs.

In addition, positive selection of tTregs in lower peptide-presenting H-2^k/b, but not higher peptide-presenting H-2^k/k 3B5.8/hLa double-Tg, mice provides clear support for the avidity model of Treg selection (35, 36). Specifically, positive selection of CD4SP, in the absence of expression of the hLa neo–self-Ag, was equivalent in the two models, thus establishing the avidity model of tTreg selection without the potential factor of altered tuning (37) at early stages of development that has confounded other studies (38). Although endogenous Vα inclusion in tTregs from 3B5.8/hLa double-Tg mice was not detected using available Vα Abs, the possibility that hLa expression promoted inclusion of a different endogenous Vα as a mechanism further promoting tTreg differentiation cannot be ruled out. Expression of dual TCRs using the Tg Vβ10 could theoretically reduce cell-surface expression levels of the La-specific TCR and promote tTregs by lowering Ag-specific TCR signaling.

Pathologic consequences of La-specific autoimmunity, if any, have been difficult to ascertain in human rheumatic disease, because this antigenic specificity nearly always co-occurs with humoral autoimmunity directed to the Ro Ag. Prior studies have not reported pathology after immunization of normal mice with human or mouse La proteins or peptides in adjuvant (39, 40). This may be the result of incomprehensive evaluation of tissue pathology and/or the presence of effective tolerance mechanisms that control infiltration of organs by Teffs. Elimination of endogenous TCR gene rearrangements or endogenous TCR loci in other TCR/neo–self-Ag double-Tg mouse models resulted in loss of tolerance and disease secondary to failed immunosuppression by CD4⁺ T cells. Thus, we used deletion of endogenous Tcra genes as a tool to impair tTreg development and/or other forms of tolerance in 3B5.8xhLa double-Tg mice to observe the effect of anti-La autoimmunity in the CD4⁺ T cell compartment. Elimination of endogenous Tcra genes precluded positive selection of tTregs in 3B5.8/hLa double-Tg mice as expected. Although elimination of endogenous Tcra genes impaired tTreg-positive selection in hLa Tg mice, other mechanisms in addition to loss of tTregs may contribute to or be responsible for induction of autoimmunity in Tcra^−/− hLa/3B5.8 TCR double-Tg mice, including potential cellular or functional loss of other thymic-derived suppressor populations we did not measure or peripherally generated Tregs. Regardless of the mechanism, however, this maneuver resulted in early, specific, and highly penetrant serologic and cellular autoimmunity to the La Ag.

Intermolecular epitope spreading to Ro was not observed in autoimmune Tcra^−/− 3B5.8/hLa double-Tg mice. This could be a consequence of poor physical interaction between hLa and mouse Ro Ags. Using mouse cells transfected with an hLa expression construct, an hLa-specific mAb could coimmunoprecipitate mouse Ro protein, but the efficiency of Ro pulldown was significantly less than could be observed in human cells (41).

The Tcra-deficient 3B5.8 TCR/hLa double-Tg mouse model provided us with a unique opportunity to observe potential pathologic consequences of La-specific cellular and humoral autoimmunity in the absence of autoimmunity to Ro. Although the pathologic consequences after immunization of mice with La Ag have not previously been reported, we reasoned that defective selection of tTregs and/or defective CD4-mediated immunosuppression, as had been demonstrated in other models, might promote pathology because of unchecked T cell autoreactivity. Because immunization of BALB/c mice with peptides of 60 kDa Ro induced serologic epitope spreading to the La Ag and focal lymphocytic infiltrates of salivary glands and other features of SS (42), observation of exocrinopathy was a potential outcome. The La autoimmune mice displayed no inflammation of salivary glands; however, peribronchial accumulations of activated, effector/memory CD4⁺ T lymphocytes expressing the La TCR were consistently observed in the lungs. An essentially identical T cell phenotype was observed among CD4⁺ T lymphocytes of the spleen, reflecting systemic autoimmunity expected for this ubiquitous Ag. This activated phenotype was restricted to hLa-specific T cells because there was no T cell activation or pathology in Tcra^−/− 3B5.8 TCR single-Tg mice or activation of cells from Tcra^−/− 3B5.8 TCR/hLa double-Tg mice that had downregulated Vβ10. Peng et al. (43) observed that although nonspecific bystander activation of T cells could induce low levels of polyreactive autoantibodies, only activation by bona-fide self-Ag could induce cellular infiltration of target organs on the MRL^lpr/lpr lupus background. Detection of autoreactivity only in the presence of cognate Ag, high-titer hLa autoantibodies, and lack of responses to Ro60 Ag in this report indicate that the responses were not polyreactive. Together, these findings suggest that the pulmonary pathology observed in this model is a consequence of autoimmunity rather than nonspecific mechanisms of La-specific T cell activation secondary to homeostatic expansion or other bystander mechanisms.

We recently observed both IgG anti-La autoantibodies and similar peribronchial lymphocytic infiltrates in the B6.Sle1.Yaa model of lupus-like autoimmunity (44). Interestingly, similar peribronchial accumulations of CD4⁺ T lymphocytes have been observed in primary SS (45) and were assumed to occur secondary to exocrinopathy. Our study suggests that such pathology can occur in the absence of prior exocrinopathy and may be associated with systemic autoantigen-specific T lymphocytes. In support of this, there is a case report of pulmonary pseudolymphoma preceding SS by 2 years (46). Lymphoid interstitial lung disease in SS is diverse and can occur as follicular bronchiolitis, as observed in our model, to more diffuse lymphocytic interstitial pneumonia (LIP) most prominent in relation to bronchioles with usually mild fibrosis, to pseudolymphoma with nodular infiltrates (47, 48). Interestingly, restrictive pulmonary disease in SS was associated with Abs to Ro and La (7), and a highly significant association was observed between anti-La Abs in SS and internal organ manifestations including in lungs and liver (49).

This study demonstrates that thymic clonal deletion and tTreg differentiation are cellular mechanisms of CD4⁺ T cell tolerance to a clinically relevant, ubiquitous, nuclear Ag, provides new support for the avidity model of tTreg selection, and identifies a novel link between La-specific cellular autoimmunity and lymphoid interstitial lung disease.

We thank Jacob Bass and Diana Hamilton of the OMRF Flow Cytometry Core Facility, Beverly Hurt of the OMRF Graphics Resource Center, and Kathryn Bryant for assistance. We are grateful to D. Kioussis for providing the CD2 minigene expression construct and to James McCluskey and Tom Gordon for providing human La Tg mice and vectors encoding recombinant La proteins used in this study.

The authors have no financial conflicts of interest.