T cell receptor BV gene usage in interstitial cellular infiltrates in active Heymann nephritis
Huiling Wu,
Geoff Y. Zhang and
John F. Knight
Centre for Kidney Research, Royal Alexandra Hospital for Children, Westmead, New South Wales, Australia
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Abstract
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Background. Infiltration of the kidney by mononuclear cells is a prominent feature of active Heymann nephritis (HN). These cells could be present as a part of generalized inflammatory response, or could be proliferating in response to specific antigens. To examine these questions, we have analysed the T cell receptor (TCR) BV repertoire of T cells infiltrating the renal interstitium at regular time intervals throughout the course of the disease.
Methods. HN was induced in Lewis rats by immunization with renal tubular antigen (Fx1A) in complete Freund's adjuvant (CFA). Kidneys were collected 8 and 12 weeks after immunization. Renal tissue was homogenized and RNA extracted. RTPCR and sequencing were used to characterize expression of TCR BV genes.
Results. Preferential expression of TCR BV2 and BV16 gene families was seen at 8 weeks. By 12 weeks the diversity of the TCR BV gene repertoire had increased and was highly heterogeneous. Sequence analysis of BV2, and BV16 RTPCR products from 8 week HN kidneys revealed conserved usage of CDR3 regions, and an over-representation of arginine residues in the CDR3 regions at a frequency of between 60 and 100% of clones sequenced in most of BV2 and BV16 subfamilies.
Conclusion. The preferential usage of CDR3 region sequences in TCR BV2 and BV 16 families indicates clonal expansion of individual T cells in HN kidneys at 8 weeks. The conserved usage of arginine residues in the CDR3 regions may indicate recognition of select antigenic epitopes. By 12 weeks, the diverse TCR BV repertoire in the kidney may be due to epitope spreading or may represent a non-specific inflammatory response in the late phase of the disease.
Keywords: amino acid sequence; CDR3 region; Heymann nephritis; T cell; T cell receptor
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Introduction
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Active Heymann nephritis (HN) is an experimental rat model of autoimmune-mediated glomerulonephritis (GN) similar to membranous GN in humans [1,2]. Active HN is commonly induced by immunizing inbred rats with a crude renal tubular antigen (RAT/Fx1A) emulsified in complete Freund's adjuvant (CFA) [3,4]. The pathogenesis of HN is thought to be through the binding of anti-Fx1A autoantibodies to autoantigens expressed on glomerular epithelial cells, leading to the formation of subepithelial immune deposits (IDs) along the glomerular basement membrane with the activation of complement, resulting in severe glomerular injury and proteinuria [5].
Previous studies on the role of T cells in the mediation of HN have proven that the production of anti-Fx1A autoantibodies in the development of HN is dependent upon CD4+ T helper (Th) cells [6]. Blocking these CD4+ cells by using anti-CD4 mAb totally abolishes the proteinuria, glomerular Ig deposition and mononuclear cell infiltrates in the kidney [7]. Although T cells have an established role in the production of anti-Fx1A autoantibodies in HN, the pathogenic mechanism of T cells in the mediation of glomerular and interstitial injury is still uncertain. Previous studies on active HN have also shown that the disease is associated with tubulointerstitial inflammation, characterized by tubular injury and interstitial mononuclear cell infiltration [8]. These infiltrates include
ßTCR bearing CD4 and CD8 T cell populations [7,9]. Furthermore, anti-CD8 mAb therapy to temporarily deplete CD8+ T cells reduces the level of proteinuria as well as glomerular and interstitial mononuclear cell infiltrates, but has no effect on autoantibody titres or glomerular Ig deposition. More recent studies examining mononuclear infiltrates and cytokine mRNA expression in the kidney have demonstrated that the progressive development of infiltrates of activated T cells is coincident with the development of proteinuria [10]. It is unknown whether the T cells seen in the glomerular and renal interstitium are proliferating in response to renal antigens or whether they represent a non-specific inflammatory response as a result of Ig deposition in glomeruli.
To address these questions, we have analysed the TCR BV repertoire of T cells infiltrating the renal cortex throughout the course of HN in order to examine the nature of T cell infiltration into the kidney with active HN, and in an attempt to identify potential targets for immunotherapeutic intervention.
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Subjects and methods
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Experimental animals
Inbred male Lewis rats were obtained from the Animal Resources Centre in Perth, Australia. Rats weighing 180200 g at the age of 8 weeks were used in all experiments. The rats were maintained on standard chow and water, and housed in the animal facility at the Children's Medical Research Institute (Sydney, NSW, Australia). Outbred male Sprague-Dawley rats were used for producing Fx1A.
Antigen preparation
Fx1A was prepared as described previously [4,11]. Briefly, kidney cortices from rats were dissected and pushed through a 150 mesh steel sieve. Filtrates were centrifuged twice at 400 g for 10 min. The supernatant, which contained the tubular fraction, was washed three times with distilled water and ultracentrifuged at 78 680 g for 45 min. The sediment was lyophilized and stored at -80°C.
Induction of active HN
Each group of five Lewis rats was immunized subcutaneously (s.c.) into each of their hind footpads with 100 µl of Fx1A emulsified in CFA, which contained a total of 15 mg Fx1A, 1 mg mycobacterium tuberculosis HRa37 (Difco, Detroit, MI, USA), 100 µl of incomplete Freund's adjuvant (IFA) (Sigma, St Louis, MO, USA) and 100 µl of PBS. A booster dose of 7.5 mg of Fx1A in 50 µl of IFA and 50 µl PBS was given s.c. at the back of the neck 2 weeks later. The five control rats were immunized with the appropriate emulsion prepared without Fx1A.
Autoantibody determination
Autoantibody titres were determined by ELISA at 4, 8, 10 and 12 weeks post-Fx1A/CFA challenge by using the standard ELISA procedure described previously [7]. Briefly, all wells of an Immulon 1 ELISA microtiter plate (Dynatch Laboratories, Alexandria, VA, USA) were coated with 4 µg solubilized Fx1A in 100 µl of coating buffer and reacted sequentially with test sera, alkaline phosphatase conjugated goat anti-rat IgG (Zymed laboratories, Inc., San Francisco, CA, USA) and substrate solution (0.5% p-nitrophenyl phosphate (Sigma) in carbonate buffer, pH 9.6. Absorbance was read at 405 nm on an ELISA reader (Dynatech Laboratories). Normal Lewis rat serum was used as the negative control. A dilution series of a known, strongly positive serum from active HN (provided by Dr Mark Penny) and a dilution of the test samples were assayed in triplicates.
Urinary protein estimation
Twenty-four urine samples were collected in metabolic cages at 4, 6, 8 and 12 weeks post-Fx1A/CFA inoculation. Urine protein concentrations were determined by colorimetric assay (Biorad, Oakland, CA, USA).
Organ harvest
At two time points after immunization, the rats were sacrificed and the kidneys were perfused with phosphate-buffered saline (PBS) via abdominal aorta puncture to eliminate peripheral blood contamination. Each kidney was divided in half: one portion was snap frozen and used for RTPCR analysis, while the other portion was further divided into two pieces. One piece was placed in formalin and another was embedded in OCT and snap frozen for histological examination. The spleen from each animal was also harvested and frozen for use in PCR experiments.
The diseased rats were divided into two groups according to the following clinical criteria. Group 1: early HN. Kidneys were collected 8 weeks after immunization from rats who had <100 mg/day of proteinuria with a minimal number of mononuclear cells infiltrating the renal cortex. Group 2: late HN. Kidneys were collected 12 weeks after immunization from rats who had >300 mg/day of proteinuria with focal mononuclear cells infiltrating the renal cortex.
Histological examination and immunofluorescence
Paraffin sections were stained with periodic acid-Schiff's reagent (PAS) by standard methods. The frozen sections were incubated at humidified room temperature for 30 min with fluorescein isothiocyanate (FITC)-labelled goat anti-rat IgG (Zymed Laboratories, Inc.).
RTPCR for the TCR BV repertoire
RTPCR for the TCR BV repertoire was performed as described previously [12,13]. Total RNA was extracted from the snap frozen kidney or spleen tissue using RNAzolTM B (Cinna/Biotecx, Houston, TX, USA) according to the manufacturer's recommendations. cDNA was synthesized using random hexamer primers (Promega, Madison, WI, USA) and a M-MuLV reverse transcriptase kit (Gibco-BRL, Drand Island, NY, USA) following standard protocol. Oligonucleotide primers used for rat TCR Vß120 were those described previously [14]. The TCR BC oligonucleotide primer (5'-tgtttgtctgcgatctctgc-3') for TCR BV was designed using PCRprim software. The PCR profile used was 32 cycles for spleen and 36 cycles for kidney for 1 min each at 95, 60 and 72°C for TCR Vß. PCR amplification was performed in triplicate for TCR BV gene usage in all experiment samples in cDNA dilutions chosen so that the PCR amplification for the experimental sample fell upon the steep part of the standard curves.
The specificity of each PCR product was verified by separate hybridization with tris (2,2'-bipyridine) ruthenium (II) chelate (TBR)-labelled sequence-specific oligonucleotide probes. The sequence of probe specific for TCR Vß PCR products was 5'-aggtctccttgtttgagcca-3'. The electrochemiluminescent signal of the hybridized probe was detected with a QPCR 5000 system (Perkin Elmer) as described [12,13,15]. The relative luminosity of each Vß family member was expressed as a percentage of the total luminosity detected in all of the Vß regions for a given sample.
Cloning and sequencing of TCR BV RTPCR products
TCR BV PCR products were purified by the PCR Prep DNA purification system and then cloned into the pGEM-T vector system (Promega). DNA plasmid preparations were made from individual colonies using the Wizard plus SV miniprep kit (Promega). DNA sequencing was performed in both directions by the dideoxy chain determination method using the dye-labelled dideoxynucleotides as described by the manufacture (Perkin Elmer), and run on a Perkin-Elmer automated sequencing apparatus. Oligonucleotide primers used for DNA sequencing were the same as those used in RTPCR.
Statistical analysis
The paired t-test was used to determine whether there was a significant difference between the BV percentage in paired kidney and spleen samples.
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Results
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Induction of active HN
All rats in the two groups immunized with rat Fx1A/CFA developed active HN with evidence of autoantibody production, glomerular Ig deposition and abnormal 24-h urine protein, while controls did not (Table 1
). There was typical granular staining for rat IgG along glomerular capillary loops at 8 and 12 weeks after immunization; however, no significant difference in intensity of this deposition between the two groups was found. The protein excretion in the 24-h urine collection after immunization was 74±19.8 and 466.3±85.4 mg/day at 8 and 12 weeks, respectively. In contrast, the urinary protein excretion of all control rats immunized with CFA alone remained in the normal range (16.2±3.12 mg/day). The urinary protein excretion of 10 age- and sex-matched unimmunized Lewis rats was 16.1±1.3 mg/day.
Histological examination of renal tissue with PAS staining showed mononuclear cell infiltrates in the kidneys taken at 8 and 12 weeks. This infiltrate was predominantly seen in the periglomerular and renal interstitium. Only very few mononuclear cells were found in glomeruli. As shown in Figure 1
, a minimal number of mononuclear cell infiltrates was detected in a representative kidney of an HN rat at 8 weeks, while focal mononuclear cell infiltrates were present in a representative kidney of an HN rat with heavy proteinuria at 12 weeks. No infiltrates were identified at any time in kidneys from control rats.

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Fig. 1. A minimal number of mononuclear cell infiltrates in the renal interstitium with PAS staining (left) was shown at 8 weeks, while focal areas of mononuclear cell infiltrates in renal interstitium (right) were shown at 12 weeks (magnification: x200).
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Analysis of the splenic TCR BV repertoire in rats
We first performed RTPCR for TCR BV genes with splenic RNA isolated from normal Lewis rats (Figure 2
). Figure 2A
shows a representative 2% agarose gel of the peripheral TCR BV repertoire from splenic RNA of a normal rat. Figure 2B
shows the relative expression of each BV gene family as a percentage of the total of all BV segments combined from five normal rats from different shipments, measured by the QPCR system. Spleens from normal Lewis rats expressed all 20 known TCR BV segments. Certain BV genes, including BV2, 4, 5, 13 and 15, were expressed at the highest levels of individual percentages, whereas BV1, 6, 8.6 and 11 were expressed in the least abundance and the rest were present in intermediate percentages.

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Fig. 2. Peripheral TCR BV gene repertoire in normal Lewis rats. (A) Representative TCR BV RTPCR products from splenic RNA of a normal rat were separated on 2% agarose gel and were of the expected size, based on molecular weight markers in the first lane. (B) Relative quantity of each individual BV RTPCR product expressed as a percentage of the total of all BV genes combined (n=5), measured by the QPCR system.
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Splenic TCR BV usages in rats after immunization with Fx1A/CFA (the disease group) and after immunization with CFA alone (the control group) were then analysed to determine whether immunization shapes the peripheral TCR BV repertoire. No significant differences between the two groups (P>0.05) were found at 12 weeks post-immunization (Figure 3
). There was no significant difference in the peripheral BV gene repertoire between normal rats and immunized rats.

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Fig. 3. Splenic TCR BV gene repertoire from immunized rats. Relative quantity of each individual BV RTPCR product expressed as a percentage of the total of all BV genes combined. Open bars and black bars represent the TCR repertoire from rats immunized with CFA alone (n=5) and from rats immunized with FxA1/FCA, respectively.
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TCR BV repertoire in the kidney
To examine the nature of T cell infiltration into the kidney with active HN, we examined the TCR repertoire of infiltrating T cells in kidneys at 12 weeks post-immunization. The analysis of TCR Vß usage in kidneys revealed that all 20 TCR BV families were expressed in the kidneys (Figure 4
). TCR BV2, BV13, BV15 and BV16 were present in higher individual percentages, but there was no statistically significant difference in TCR BV-relative expression in the kidneys compared with splenic tissues. This result revealed that the TCR BV gene repertoire detected in the kidneys with the advanced HN were quite diverse and suggested a polyclonal response.

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Fig. 4. TCR BV repertoire from kidneys of rats with advanced disease at 12 weeks after immunization. The bars in the graph represent the relative quantity of TCR BV RTPCR products from spleen (grey bars) and kidneys (black bars) of five rats with HN. Relative quantities of each individual BV RTPCR product are expressed as a percentage of the total of all BV genes combined (P>0.05).
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A more restricted pattern was seen in T cells infiltrating the kidney in the early phase of HN. The analysis of TCR BV usage at 8 weeks revealed that the five kidneys preferentially expressed BV2 and BV16 in most kidneys with early HN as compared with spleen (P<0.05), but other members of the BV family were present as well (Figure 5
). This result indicates that TCR BV gene usage in kidneys with minimal cell infiltrates early in the course of HN is less heterogeneous than that in the late phase of HN.

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Fig. 5. TCR BV RTPCR products from five kidneys of rats with a minimal number of mononuclear cells in the interstitium at 8 weeks after immunization. The bars in the graph represent the relative quantity of TCR BV RTPCR products from spleen (grey bars) and kidney (black bars) of five rats with HN. The relative quantity of each individual BV RTPCR product is expressed as a percentage of the total of all BV genes combined. *P<0.05; **P<0.01.
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Sequence analysis of Vß gene products in the kidney in the early course of disease
To examine amino acid sequences of ß chain CDR3 regions of TCRs from T cells infiltrating the kidneys, we next cloned and sequenced individual RTPCR products of BV2 and BV16 from two kidneys with a minimal number of cell infiltrates in the kidney cortex. The deduced amino acid sequences were shown in Table 2
. Analysis of the CDR3 region sequences of each individual BV RTPCR product revealed some evidence for clonal expansion of individual T cells (bold numbers in Table 2
). Four out of 10 clones derived from BV2 from kidney 1 shared an identical CDR3 sequence at the nucleotide level, while four out of 10 clones derived from BV2 from kidney 2 had a common CDR3 sequence. Similarly, four out of 10 clones derived from BV16 from kidney 1 shared identical CDR3 sequences, whereas five out of 10 clones from RTPCR BV16 from kidney 2 had a common CDR3 sequence. However, the same clones found in kidney 1 were not found in kidney 2. Interestingly, analysis of CDR3 sequences showed over-representation of arginine (R) residues encoded by N-region (non-germline) addition/deletion in the sequence from two kidneys (Table 2
). Ten of 10 sequences (100%) from clones encoding BV2 RTPCR products from kidney 1 showed arginine in their CDR3 regions, while 60% of sequences from Vß 16 RTPCR products contained arginine in their CDR3 regions, also in this kidney. Sequences of BV2 and BV16 RTPCR products from kidney 2 also had an arginine in the CDR3 regions at a frequency of 6070%. In contrast, BV2 and BV16 CDR3 sequences from the spleen of a rat with early HN were more heterogeneous (Table 3
). These results indicate that some BV2 and BV16 bearing T cells infiltrating kidney contain conserved amino acids, such as arginine in the CDR3 region.
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Table 2. TCR ß chain CDR3 sequences of RTPCR products from two kidneys with a minimal number of T cell infiltrates at 8 weeks
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Discussion
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In this study we have analysed the TCR BV repertoire from T cells infiltrating the renal cortex in active HN. The results show that the TCR repertoire detected in kidneys with HN is heterogeneous. A diverse TCR BV repertoire could be generated by a polyclonal T cell response to a number of antigenic epitopes in autoantigens. In active HN, the major pathogenic autoantigens are megalin (gp330) and the receptor-associated protein (RAP). Two pathogenic epitopes were mapped to 14 amino acids [16] in RAP and 46 amino acids in the second ligand-binding domain (LBD II) of megalin [17]. Recent findings provide further evidence for the presence of multiple pathogenic epitopes in HN, in that all four ligand-binding domains in megalin contain pathogenic epitopes [18]. The presence of autoreactive pathogenic T cells recognizing multiple antigens and several epitopes derived from each antigen may provide a reasonable explanation for the detection of a heterogenous TCR repertoire during active disease [14,19].
Analysis of the TCR repertoire in the kidneys in the early course of the disease showed preferential expression of TCR BV2 and BV16 genes, and revealed conserved usage of CDR3 regions in some of those BV segments, suggesting clonal expansion of individual T cells in kidneys in the early course of HN. Interestingly, sequence analysis of these BV RTPCR products demonstrated over-representation of positively charged arginine residues in the CDR3 regions of infiltrating T cells from the two kidneys examined. These findings are similar to studies on the TCR BV repertoire of T cells infiltrating the kidneys in a murine model of autoimmune interstitial nephritis and in Sjogren's syndrome (SS) patients with interstitial nephritis [20,21]. In these studies, a positively charged residue (arginine) in the CDR3 region was found, suggesting that some of the TCR sequences obtained from diseased kidneys might be derived from T cells recognizing a common target antigen expressed on renal tubular basement membrane. Lewis rats develop the tubulointerstitial lesion and cell-mediated reactivity against kidney-specific antigens when they are immunized with syngeneic kidney homogenates [22]. In active HN, antibody to Fx1A could gain access to proximal tubular brush border from the glomerular filtrate and mediate downstream injury of these tubular structures [23]. Tubular cells exposed to filtered antibodies could themselves become nephritogenic through expression of inflammatory mediates and markers, and tubular injury by antibody may release endogenous tubular antigens [9,24] or tubular intrinsic antigens. Over-representation of arginine residues in the CDR3 regions of T cells infiltrating the kidneys in early HN suggests that infiltrating T cells might recognize limited epitopes of an antigen or a set of antigens associated with renal tubular injury.
Our results show increasing heterogeneity of the TCR repertoire in the late course of HN. One possible explanation is the immunological notion of epitope spreading [25,26]. Initial autoreactive T cells recognize a single immunodominant epitope and use a restricted TCR BV gene repertoire. After antigen recognition, activated T cells initiate inflammation, leading to exposure of cryptic antigens and then to influx of numerous T cells with specificities different from the original restricted cells. It is also possible that Ig deposition in the glomeruli with the activation of complement and resultant severe glomerular injury and proteinuria may attract non-specific inflammatory T cells into the kidney or trigger a secondary T cell immune response against intrinsic antigens. The secondary T cell immune responses may modulate a wide range of immune events through up-regulation of costimulatory molecules and the release of cytokines, leading to the further broadening of the initially selective T cell response. This is consistent with the observation of increased interstitial cytokine mRNA expression over time during the course of active HN for Th1-, Th2- and macrophage-derived cytokines in HN [10]. By 12 weeks, the diverse TCR BV repertoire in the HN kidney may be due to epitope spreading or may represent a non-specific inflammatory response in the late phase of the disease.
In conclusion, preferential expression of TCR BV2 and BV16, and the conserved usage of CDR3 regions in some of those Vß segments suggest clonal expansion of individual T cells in the kidneys in HN. Over-representation of arginine residues in the CDR3 regions of T cells infiltrating the kidneys might contribute to recognition of limited epitopes of an antigen or a set of antigens in the kidney of this HN model.
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Acknowledgments
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We thank Dr Mark Penny (Liverpool Hospital) for his assistance with establishing the active HN model, and the Children's Medical Research Institute (CMRI) for providing animal care. The assistance of Prof. Paul Roy and Dr Takashi Ando with interpretation of kidney sections was greatly appreciated. This work was supported by grants from the Australian Kidney Foundation and the New Children's Hospital Fund. H.W. was the recipient of an Overseas Postgraduate Research Scholarship from the University of Sydney.
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Notes
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Correspondence and offprint requests to: Dr Huiling Wu, Centre for Kidney Research, Royal Alexandra Hospital for Children, Looked Bag 4001, Westmead, NSW 2145, Australia. 
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Received for publication: 23. 3.00
Revision received 21. 2.01.