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DNA Vaccination Against Specific Pathogenic TCRs Reduces Proteinuria in Active Heymann Nephritis by Inducing Specific Autoantibodies

Centre for Kidney Research, The Children’s Hospital at Westmead, Sydney, Australia

	Abstract

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We have previously identified potential pathogenic T cells within glomeruli that use TCR encoding V5, V7, and V13 in combination with J2.6 in Heymann nephritis (HN), a rat autoimmune disease model of human membranous nephritis. Vaccination of Lewis rats with naked DNA encoding these pathogenic TCRs significantly protected against HN. Proteinuria was reduced at 6, 8, 10, and 12 wk after immunization with Fx1A (p < 0.001). Glomerular infiltrates of macrophages and CD8⁺ T cells (p < 0.005) and glomerular IFN- mRNA expression (p < 0.01) were also significantly decreased. DNA vaccination (DV) causes a loss of clonality of T cells in the HN glomeruli. T lymphocytes with surface binding of Abs were found in DNA vaccinated rats. These CD3⁺/IgG⁺ T cells expressed V5 and V13 that the DV encoded. Furthermore, FACS shows that these CD3⁺/IgG⁺ cells were CD8⁺ T cells. Analysis of cytokine mRNA expression showed that IL-10 and IFN- mRNA were not detected in these CD3⁺/IgG⁺ T cells. These results suggest that TCR DNA vaccination produces specific autoantibodies bound to the TCRs encoded by the vaccine, resulting in blocking activation of the specific T cells. In this study, we have shown that treatment with TCR-based DV, targeting previously identified pathogenic V families, protects against HN, and that the mechanism may involve the production of specific anti-TCR Abs.

	Introduction

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Human membranous nephritis is a major cause of end-stage renal disease in the adult population (1). Active Heymann nephritis (HN)³ is a model of membranous nephritis (2, 3) induced in the Lewis rat by immunization with a crude renal tubular Ag (RAT/Fx1A) (4). It is associated with immune deposits in glomeruli, and infiltration of both glomeruli and interstitium by mononuclear cells. The pathogenesis of HN is thought to be through the binding of anti-Fx1A autoantibodies to the autoantigen (gp330 or Megalin) expressed on glomerular epithelial cells, resulting in severe glomerular injury and proteinuria (5). However, studies on the role of T cells in the etiology of HN have shown that blocking CD4⁺ T cells by using anti-CD4 mAb before immunization totally prevents proteinuria, glomerular Ig deposition, and mononuclear cell infiltrates in the kidney in this model (6). More recently, studies have shown that the appearance of activated T cells, principally Th1 and cytotoxic effector T cells and macrophages within the glomeruli at 8 wk, is coincidental with the development of proteinuria (7). This result implies a role for glomerular infiltrating T cells in the pathogenesis of HN. Furthermore, permanent depletion of these CD8⁺ T cells abolishes proteinuria and reduces glomerular infiltrates (8). These findings suggest that T cells,and more specifically CTLs, may mediate glomerular injury.

TCR CDR3 spectratyping is a powerful tool for identifying autoimmune disease-inducing T cells whose mechanism of action is poorly understood (9). We have recently demonstrated clonal overexpansion of T cells using TCR encoding V5, V7, and V13 in combination with J2.6 (10) within glomeruli in this HN model. This was done using TCR spectratyping analysis and subsequent CDR3 region sequencing of spectratype-derived T cell clones. These overexpressed clones potentially represent pathogenic T cells. Vaccination with DNA encoding the V region of an autoreactive TCR has been shown to provide an effective means to prevent autoimmune diseases such as EAE by generating suppressor T cells (11). This study tested the effect of DNA vaccination targeting three specific TCR V chains in HN. We sought to confirm the pathogenic nature of the subset of T cells bearing this restricted TCR repertoire and investigate the therapeutic use of DNA vaccination in HN. The results of these studies are outlined below.

	Materials and Methods

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Experimental animals and induction of active HN

Inbred male Lewis rats were obtained from the Animal Resources Centre in Perth, Australia. Rats weighing 180–200 g at the age of 6 wk were used in all experiments. Outbred male Sprague-Dawley rats were used for producing Fx1A. Fx1A was prepared as described (12, 13). Each group of six Lewis rats was immunized s.c. into each of their hind footpads with 100 µl Fx1A emulsified in CFA, which contained a total of 15 mg of Fx1A, 1 mg Mycobacterium tuberculosis HRa37 (Difco, Detroit, MI), 100 µl IFA (Sigma-Aldrich, St. Louis, MO), and 100 ml of PBS. The control rats were immunized with the appropriate emulsion prepared without Fx1A.

DNA vaccination

RNA was isolated from the HN kidney as described (10) and reverse transcribed into cDNA. cDNA was amplified by PCR with primers specific for either V5 (5'-AAGATGTCTGGGGTGGTCCAGTCT-3'), V7 (5'-AGCATGTACTGGTATCGACAAGACCCAGATC-3'), or V13 (5'-AGAATGACTGGAGTCACCCAGTCCC-3) and a reverse J2.6 primer (5'-CGTCATAAAACCGTGAGCTTGGTGC-3). cDNA was also amplified with a V8.2 primer as described (9) and a C primer to generate a control vaccine. All forward primers were designed to include an in-frame ATG. PCR products were cloned into the pTarget T plasmid DNA (Promega, Madison, WI) according to the manufacturer’s instructions. Plasmid DNA from colonies with an insert was sequenced to confirm the insertion of the correct gene with the ATG in frame. Large-scale preparation of plasmid DNA was performed using the Mega prep kit (Qiagen, Hilden, Germany).

For DNA vaccination, animals were pretreated with 0.75% bupivacaine (1 µl/g bodyweight; Sigma-Aldrich) by injection into the tibialis anterior muscle one week before the first vaccination. This is known to enhance the efficiency of DNA vaccination. DNA (300 µg) was injected into the same site three times at weekly intervals. Two weeks after the last vaccination, rats were challenged with Fx1A in CFA.

Autoantibody determination

Anti-Fx1A Ab titers (total Ig) were determined by ELISA as described (6). Triplicate sample ODs were read at 405 nm, corrected for a control sample of known strongly positive serum OD. IgG subclass were assessed by the same method using alkaline phosphatase-conjugated mouse mAb to rat IgG1 and IgG2a (BD PharMingen, San Diego, CA).

Urinary protein estimation

Twenty-four hour urine samples were collected in metabolic cages at 4, 6, 8, 10, and 12 wk post-Fx1A/CFA inoculation. Urine protein concentrations were determined by colorimetric assay (Bio-Rad, Hercules, CA).

Immunoperoxidase staining

Glomeruli were isolated as previously described (10). Isolated glomeruli were enzymatically permeabilized for immunoperoxidase staining by incubation at 37°C with a solution of 0.1 mg/ml collagenase D and 2 mg/ml trypsin inhibitor in PBS. The slides were then washed in PBS for 15 min and stained with mAbs, followed by HRP-labeled goat anti-mouse IgG (BD PharMingen, San Diego, CA). The slides were incubated with 3,3-diaminobenzidine tetrahydrochloride to produce a dark brown end-product. The mAbs used were W3/25 for CD4⁺ T cells and some macrophages, MRCOX-8 for CD8⁺ T cells, ED-1 for macrophages and some dendritic cells, and MRCOX-12, which recognizes rat Ig-L chains (all purchased from Serotec, Oxford, UK). Stained cells in each isolated glomerular specimen were counted in individual glomeruli throughout all focal planes and the results were expressed as cells per glomerulus with 50–100 glomeruli counted per sample.

RT-PCR for TCR V repertoire

RNA was extracted from isolated glomeruli as described (10) and reverse transcribed to cDNA (14). Oligonucleotide primers used for the rat TCR V1–20 were those described (15). The PCR profile consisted of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min for 40 cycles in a DNA thermal cycler (PerkinElmer, Wellesley, MA). Aliquots (1 ul) of the PCR products were re-amplified for six cycles with a nested 5'-FAM-labeled C primer.

CDR3 spectractyping

Each labeled PCR product was loaded on POP4 sequencing gels together with size standard (Genescan-500 ROX) on an Applied Biosystems (Foster City, CA) 373A DNA sequencer. Data were processed using the Genescan Analysis 2.1 Software (Applied Biosystems) which records the fluorescence intensity in each peak.

Semiquantitive RT-PCR for cytokine genes

Semiquantitive RT-PCR for cytokine genes was performed as previously described (14). Briefly, oligonucleotide primers used for rat cytokine genes IFN- and IL-10 were those described (7, 16, 17). GAPDH primers served as the internal control. The PCR profile used was one minute each at 95, 55, and 72°C for 30 cycles for GAPDH, 35 or 40 cycles for IFN-, and 40 cycles for IL-10. RT-PCR fragments of cytokine genes were electrophoresed on a 2% agarose gel. Signals were quantitated by a Molecular Analyst (Bio-Rad).

Flow cytometry analysis and cell sorting

Mononuclear cells extracted from blood were stained with goat anti-rat IgG Ab conjugated with FITC (Zymed Laboratories, San Francisco, CA) and anti-rat CD3 mAb (clone G4.18) conjugated with PE (BD PharMingen San Diego, CA), or with anti-rat IgG conjugated with PE and anti-rat CD8a (clone OX 8), or anti-rat CD4 (clone OX35) conjugated with FITC (BD PharMingen). All samples were analyzed on a FACScan analyser (BD Biosciences, Mountain View, CA). IgG⁺CD3⁺ cells were then sorted using a FACSCaliber (BD Biosciences).

Statistical analysis

Urine protein estimations, anti-Fx1A Ab levels for each group were expressed as mean ± SD. Comparison between groups and among the groups was made by ANOVA. Paired Student t tests were used to determine whether changes in the levels of cytokine gene expression were significantly different. A value of p < 0.05 was considered significant.

	Results

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DNA vaccination protects against HN

Rats were divided into four groups: group 1(DV): DV with TCR V5, 7, and 13 in HN; group 2 (DV control): DNA vaccination with TCR V8 used as a control vaccine in HN; group 3 (HN): HN; group 4 (CFA): immunization with CFA only as a HN control. DNA vaccination encoding TCR V5, V7, and V13 significantly reduced proteinuria at 6, 8, 10, and 12 wk post Fx1A immunization (Fig. 1) compared with HN and DV controls. There was no significant difference in proteinuria between the HN and DV control groups. Control rats immunized with only CFA never developed proteinuria.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. DV encoding TCR V5, V7, and V13 with J2.6 significantly reduced proteinuria at 6, 8, 10, and 12 wk post Fx1A immunization compared with the control group (*, p < 0.001). HN: Heymann nephritis without DNA vaccination (DV) (). DV: Heymann nephritis with DV with three TCR Vs (). CFA: CFA alone controls without Fx1A (). DVctrl: Heymann nephritis with DV with V8 as control group for DV (). All results were expressed as mean ± SD. n = 6 per group.

View larger version (58K): [in this window] [in a new window]   FIGURE 2. Immunoperoxidase staining of isolated whole glomeruli 12 wk postimmunization with Fx1A/FCA. A, DV reduced macrophage and CD8+ T cell infiltrates in glomeruli at 12 wk (**, p < 0.005). All results were expressed as mean ± SEM of cells per glomerular. n = 6 per group. B, A representative immunoperoxidase staining of isolated whole glomeruli from different treatment groups. No change in deposition of Ig was seen in glomeruli in HN compared with DV and DV control groups.

DNA vaccination reduces IFN- mRNA expression in HN glomeruli

To test whether DNA vaccination alters T cell function in the glomeruli, we examined cytokine profiles in isolated glomeruli at 12 wk post–Fx1A/CFA immunization by semiquantitative RT-PCR. The results demonstrated lower mRNA expression for IFN- (p < 0.01) in the DV group when compared with HN and DV controls (Fig. 3) with levels not different from CFA controls.

View larger version (45K): [in this window] [in a new window]   FIGURE 3. Semiquantitative RT-PCR analysis of cytokine expression in glomeruli from 12 wk postimmunization with Fx1A/FCA. GAPDH gene served as an internal control. All results are expressed as mean ratio of cytokine gene densitometry score to GAPDH densitometry score ± SD. n = 6 per group. IFN- expression was decreased in DV rats as compared with HN rats (*, p < 0.01).

CDR3 spectratyping is a well described method used as a measure of oligoclonality of T cells. A normal splenic sample of a single V family gives a Gaussian distribution of 6–11 peaks each separated by three nucleotides. Oligoclonal T cells give fewer peaks in a restricted distribution. Single clones give a single peak. Our previous study demonstrated oligoclonal expansion of T cells in HN glomeruli (10) using CDR3 spectratyping and subsequent sequencing of the CDR3 regions of spectratype-derived T cell clones. To test whether DNA vaccination would change a pattern of TCR CDR3 spectratypes in glomeruli, we examine CDR3 spectratypes of TCR V5, 7, and 13 in glomeruli in DNA vaccinated rats. HN glomeruli showed marked restriction with a single peak in CDR3 spectratypes of V 5, 7, and 13 as we previously reported (10). The glomeruli isolated from DNA vaccinated rats showed diverse CDR3 lengths with more peaks in their CDR3 spectratypes (Fig. 4) which matched the CFA control glomeruli. This result indicates that DNA vaccination may reduce the expansion of pathogenic T cell clones in the glomeruli, revealing underlying background TCR diversity.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. CDR3 spectratypes from a representative animal from DV group compared with those from HN, CFA group, and spleen. The HN glomeruli showed marked restriction of three TCR Vs. DV glomeruli showed diverse CDR3 lengths with more peaks in their CDR3 spectratypes similar to CFA control glomeruli.

There was no difference in the level of serum anti-Fx1A Abs or in the levels of IgG1 in DV, HN, and DV control groups; however, IgG2a was suppressed by DNA vaccination early in the course of HN (Fig. 5).

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Comparison of Ig responses in four groups. n = 6 per group. Anti-Fx1A Ab levels expressed as percentage binding of known positive serum (anti-Fx1A titer 1:250) measures by ELISA and expressed as mean ± SD. There was no differences in the level of serum anti-Fx1A Abs or in the levels of IgG1 in DV, HN, and DV control groups. However, IgG2a was suppressed by DNA vaccination early in the course of HN (*, p < 0.05).

We analyzed the mechanism underlying the protection induced by DNA vaccination. To test whether DNA vaccination with TCR V5, 7, and 13 would delete T cells expressing V5, 7, and 13 by producing autoantibodies, we analyzed the TCR V repertoire by RT-PCR in DNA vaccinated rats. We found that DNA vaccinated rats still expressed TCR V5, 7, and 13 in both peripheral blood and isolated glomeruli. No significant changes in TCR repertoire were found in peripheral blood between DNA vaccinated and unvaccinated rats. We then asked whether DNA vaccination produces autoantibodies that bind the TCR. We sought to identify these T cells to see whether they were deleted or functionally impaired. We stained mononuclear cells from DNA vaccinated and unvaccinated rats with PE conjugated anti-CD3 Ab and FITC-conjugated anti-IgG. A small population of CD3⁺IgG⁺ cells were found in DNA vaccinated rats, in both control TCR vaccination and pathogenic TCR vaccination, but not in unvaccinated HN rats (Fig. 6). We also found that those CD3⁺IgG⁺ cells were CD8⁺ T cells by staining mononuclear cells from DNA vaccinated and unvaccinated rats with FITC-conjugated anti-CD8 Ab and PE-conjugated anti-rat IgG (Fig. 7). Furthermore, on analysis of their TCR V repertoire, sorted CD3⁺IgG⁺ T cells almost exclusively expressed TCR V5 and V13, two of the three V families encoded in the DNA vaccination compared with diverse TCR repertoire seen in sorted CD3⁺ T cells (Fig. 8). Analysis of cytokine mRNA expression in the CD3⁺/IgG⁺ T cells showed that IL-10 and IFN- mRNA were not detected (Fig. 9). These results suggest that TCR DNA vaccination produced specific autoantibodies that bound to the TCRs encoded by the DNA vaccination, resulting in activation blockade of these T cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Identification of IgG+CD3+ cells in peripheral lymphocytes in DNA vaccination rats by FACS. Freshly isolated lymphocytes from HN rats without DV (HN), rats with pathogenic TCR DV, and control TCR DV were stained with anti rat IgG (FITC) and anti-rat CD3 (PE). The number in brackets represent the percentage of IgG+CD3+ cells among total CD3+ T cells. n = 3 per group. One representative experiment is shown.

View larger version (52K): [in this window] [in a new window]   FIGURE 7. Identification of IgG+CD8+ T cells in peripheral lymphocytes in DNA vaccination rats by FACS. Freshly isolated lymphocytes from HN rats without DV (HN) and rats with DV were stained with anti rat IgG (PE) and anti-rat CD8 (FITC) or anti-rat CD4 (FITC). n = 3 per group. One representative experiment is shown.

View larger version (66K): [in this window] [in a new window]   FIGURE 8. TCR repertoire analysis of sorted IgG+CD3+ T cells from peripheral lymphocytes in rats with DNA vaccination with V5, 7, and 13. These CD3+/IgG+ T cells expressed V5 and V13 that the DV encoded. TCR gene usage of the sorted IgG+CD3+ T cells from three individual animals in the top three panels restricted to V5 and V13 in contrast to the diversity of V genes seen in the sorted CD3+ T cells in the bottom panel. Lanes 1–20 represent each TCR V family and the last lane represents a negative control for RT-PCR.

View larger version (39K): [in this window] [in a new window]   FIGURE 9. Expression of cytokine mRNA by sorted IgG+CD3+ T cells from peripheral lymphocytes in rats with DNA vaccination with V5, 7, and 13 by semiquantitative RT-PCR. IFN- and IL-10 were not detected in the sorted IgG+CD3+ T cells. GAPDH gene served as an internal control. Lanes 1–3 represent PCR products from sorted IgG+CD3+ T cells from peripheral lymphocytes in vaccinated rats (n = 3). Lanes 4 and 5 represent RT-PCR products from ConA stimulated T cells and Th2 cell lines we described previously, which served as positive controls for cytokine genes. Lane 6 represents a negative control for RT-PCR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In HN, previous studies by Penny et al. (7) demonstrated that the onset of proteinuria is associated with glomerular infiltration of CD8⁺ Tc1 cells and macrophages. Furthermore, permanent depletion of these CD8⁺ T cells prevents the onset of proteinuria, suggesting that Tc1 cytotoxic cells may mediate glomerular injury (8). A recent study has shown that IL-4 therapy prevents the development of proteinuria in active HN by inhibition of cytotoxic CD8⁺ T cells (20). In this study of IL-4 therapy, HN rats treated with rIL-4 had reduced infiltrating CD8⁺ cells in glomeruli and reduced mRNA level for IFN- but unchanged CD4⁺ T cells infiltration in glomeruli compared with HN controls. In our study, DNA vaccination reduced the glomerular infiltrates of CD8⁺ T cells and macrophages, but accumulation of glomerular CD4⁺ T cells was similar to HN controls. IFN- mRNA expression was significantly reduced in glomeruli of DNA vaccinated rats compared with HN rats, consistent with reduced infiltrates of CD8⁺ T cells, and suggesting that the major source of IFN- in HN is CD8⁺ T cells. Furthermore, CDR3 spectratype patterns in glomeruli with DNA vaccinated rats were similar to those seen in CFA controls, compared with an oligoclonal restricted TCR repertoire in HN glomeruli. This supports that specific DNA vaccination reduced the oligoclonal pathogenic T cells in glomeruli exposing the underlying diverse pattern of TCR repertoires normally found. Taken together, these results suggest that DNA vaccination suppressed the cytotoxic CD8⁺ T cell response, which is necessary for induction of proteinuria in this model (8). As mentioned above, IL-4 treatment inhibits CD8 T cells while leaving CD4 T cell infiltrates unchanged in HN but significantly reducing proteinuria (20). It is possible that suppression of CD8⁺ T cell activation in HN glomeruli by specific DNA vaccination results in a reduced number of CD8⁺ T cells infiltrating the glomeruli but leaves CD4⁺ T cells unchanged.

We have demonstrated that TCR DNA vaccination produced specific Abs to the TCRs that the DNA vaccination encoded. The Ab coated cells were seen using flow cytometry. The small number of cells coated with Abs possibly reflects the combination of the three V families with J2.6, that is one of 12 possible Js. Thus, Abs are generated to only the pathogenic subsets of the T cell population. These specific Abs did not lead to the depletion of V5, V7, and V13 T cells. These results are consistent with the observations made in transplantation models and the EAE model (11). Anti-TCR V13-specific DNA vaccination prolonged heart allograft survival in adult rats (21) and DNA vaccination against an immunoregulatory CD8⁺ T cell clone bearing a V18-D1-J2.7 TCR abolished DST-induced allograft tolerance without the depletion of the clone (22). In the EAE model, encephalitogenic T cell lines and clones mainly used a restricted TCR gene V8.2 (18) (19). Vaccination with DNA encoding the V8.2 region of an autoreactive TCR protected mice from EAE (11). This protection was attributed to a shift from a Th1 to Th2 cytokine profile in the V8.2 target cells rather than due to their depletion. However, suppressive effects were obtained from other studies with no signs of a Th2 bias (23), suggesting that Th2 deviation is not essential for the protective effect of DNA vaccination. We have examined the cytokine profile in HN glomeruli. DNA vaccination reduced glomerular IFN- mRNA expression but did not elevate levels of Th2 cytokine mRNA. We also analyzed the cytokine profile of CD3⁺IgG⁺ cells from DNA vaccinated rats. CD3⁺IgG⁺ cells, which were CD8⁺ T cells, had little IFN- and IL-10 mRNA expression. It is interesting to note that CD8⁺ T cells were significantly decreased in number in the glomeruli of the DNA vaccinated rats. We speculate that autoantibodies produced following DNA vaccination bind specific TCRs and block the activation of these T cells. Our results suggest that this specifically targeted pathogenic subset of the T cell population either did not infiltrate the glomeruli or failed to expand and were unable to express pathogenic cytokines. This would appear to demonstrate the specific suppressive effects of DNA vaccination. The interaction of the TCR with Ag in the context of MHC has been greatly advanced by the recent crystallographic evidence of TCR structure and mutational analysis of TCR activation. It is possible that Abs binding to the V-chain can directly limit the normal activation of these T cells preventing clonal expansion and cytokine production (24). Furthermore, data on the effect of V Abs in blocking CD8 function has been shown in antitumor specific CTL clones, which is consistent with our data (25).

In conclusion, we have shown that immunotherapy with TCR-based DNA vaccination protects against HN. This is based on our previous identification of pathogenic V TCRs by TCR CDR3 spectratyping analysis and subsequent CDR3 region sequencing of spectratype-derived T cell clones. The mechanism for this appears to be the induction of V specific Abs. DNA vaccination targeting the T cell receptors of pathogenic T cells is an attractive therapeutic alternative to broad immunosuppression in the treatment of glomerulonephritis.

	Acknowledgments

 
We thank Mary Sartor for her excellent technical assistance in cell sorting and the staff of the animal house at the Children’s Medical Research Institute for assistance with animal care.

	Footnotes

 
¹ This work was supported by a grant from the National Health and Medical Research Council of Australia (Grant No.249414).

² Address correspondence and reprint requests to Dr. Huiling Wu, Centre for Kidney Research, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead NSW 2145, Australia, E-mail address: huilingw{at}med.usyd.edu.au

³ Abbreviations used this paper: HN, Heymann nephritis; DV, DNA vaccination; EAE, experimental autoimmune encephalomyelitis.

Received for publication March 3, 2003. Accepted for publication August 8, 2003.

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