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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Jun 27, 2011.
Published in final edited form as:
PMCID: PMC3124090
NIHMSID: NIHMS290185

In situ B cell-mediated immune responses and tubulointerstitial inflammation in human lupus nephritis

Abstract

The most prevalent severe manifestation of systemic lupus erythematosus (SLE) is nephritis which is characterized by immune complex deposition, inflammation, and scarring in both glomeruli and in the tubulointerstitium. Numerous studies indicate that glomerulonephritis results from a systemic break in B cell tolerance resulting in the local deposition of immune complexes containing antibodies reactive with ubiquitous self-antigens. However, the pathogenesis of SLE tubulointerstitial disease is not known. Herein, we demonstrate that in over half of a cohort of 68 lupus nephritis biopsies, the tubulointerstitial infiltrate was organized into either well-circumscribed T:B cell aggregates or germinal centers (GCs) containing follicular dendritic cells. Sampling of the in situ expressed immunoglobulin repertoire revealed that both histological patterns were associated with intrarenal B cell clonal expansion and ongoing somatic hypermutation. However, in the GC histology the proliferating cells were CD138CD20+ centroblasts while in T:B aggregates, they were CD138+CD20low/− plasmablasts. The presence of either GCs or T:B aggregates was strongly associated with tubular basement membrane immune complexes. These data implicate tertiary lymphoid neogenesis in the pathogenesis of lupus tubulointerstitial inflammation.

Keywords: B cells, somatic hypermutation, human, nephritis, systemic lupus erythematosus, tertiary lymphoid neogenesis, plasmablasts

INTRODUCTION

The clinical manifestations of SLE are myriad and range from mild arthralgias and skin rashes to life threatening nephritis (1). Patients with either focal or diffuse proliferative lupus nephritis (LN) have a more rapidly progressive disease and require more aggressive treatment than those with mesangial proliferative or membranous LN (2). Significant morbidity and mortality is a consequence of both the nephritis and the cytotoxic therapeutic regimens used to treat it (3).

Pathologically, LN is characterized by immune complex deposition and inflammation in both glomeruli and the tubulointerstitium that, if left untreated, can result in scarring and irreversible organ failure. Of the pathological manifestations of LN, glomerulonephritis (GN) is both the best studied and the feature most often replicated in murine models of autoimmune disease (46). GN is also the renal manifestation most clearly related to the central pathogenic feature of SLE, systemic loss of B cell tolerance (1, 7, 8).

The presence of serum anti-dsDNA antibodies identify lupus patients at increased risk for GN while increasing titers of anti-dsDNA antibodies often herald renal flares in individual SLE patients (1). Anti-dsDNA antibodies have been isolated from SLE renal biopsies (9, 10) and infusion of some murine and human anti-dsDNA antibodies can induce GN in mice (11, 12). Furthermore, recent immunoelectron microscopic studies have directly demonstrated the presence of anti-dsDNA antibody-containing immune complexes in diseased glomeruli (13, 14). These observations have led to the hypothesis that anti-dsDNA IgG antibodies play a central role in the pathogenesis of lupus GN.

However, tubulointerstitial inflammation (TI) is also a common feature of LN (1517, 22). On renal biopsy, the presence and degree of TI identifies those patients with lupus nephritis at risk for progression to renal failure (15, 16, 22). In contrast, the NIH activity index, which primarily assesses glomerular inflammation, does not correlate with prognosis (15, 18, 19). Furthermore, the presence of tubulointerstitial scarring on renal biopsy is more predictive of subsequent renal failure than glomerular scarring (15, 16, 22). Tubulointerstitial inflammation can occur independently of GN (20, 21) and TI severity does not correlate with titers of anti-dsDNA antibodies (22). These data indicate that TI is an important manifestation of lupus nephritis that might arise from different pathogenic mechanisms from those implicated in GN.

Organ specific inflammation is a defining feature of many autoimmune diseases including Hashimoto’s thyroiditis (23), rheumatoid arthritis (24), Sjogren’s syndrome (25), and multiple sclerosis (26). In these diseases, infiltrating lymphocytes are often highly organized and resemble lymphoid structures found in secondary lymphoid organs during a normal immune response. This feature is referred to as tertiary lymphoid neogenesis (TLN). B cells within these lymphoid structures secrete autoantibodies(27, 28) and are required to locally maintain activated T cells(29). Several reports have noted that infiltrating T cells are a prominent feature of lupus nephritis(3034) while the presence of B cells has been recently noted(3537). However, the significance of these lymphocyte populations in the interstitial infiltrate was unclear.

Herein, we demonstrate that in moderate or severe TI the inflammatory infiltrates are usually organized into structures reminiscent of those observed in secondary lymphoid organs. Most commonly, aggregates of T and B cells containing plasmablasts were observed. However, in some renal biopsies there were germinal center-like structures containing well-organized follicular dendritic cell networks and centroblasts. Both lymphoid structures were functional as both were associated with in situ B cell clonal expansion and somatic hypermutation. These findings implicate organ intrinsic adaptive immune responses in the pathogenesis of lupus tubulointerstitial inflammation.

MATERIALS AND METHODS

Patients and Renal Biopsies

The University of Chicago Medical Center Institutional Review Board approved this study. We reviewed the pathology files at the University of Chicago Medical Center for inpatient renal biopsies consistent with lupus nephritis between 2001 and 2007. Among this group, 68 subjects were found with biopsies containing sufficient material for analysis (six or more glomeruli and a length of ≥ 0.5 cm) and which did not display either Class I or VI nephritis as defined by the 2003 International Society of Nephrology/Renal Pathology Society (ISN/RPS) revised LN classification criteria(38) and who on review of records fulfilled American College of Rheumatology revised criteria for the classification of SLE(39). Each diagnostic biopsy sample consisted of at least three tissue cores that were predominantly divided for light microscopy with smaller portions submitted for immunofluorescence and electron microscopy. Using the NIH system, the activity and chronicity indices were scored at the time of renal biopsy by either one of two renal pathologists (AC, SMM)(40). Control normal renal tissue was obtained from autopsy studies. The clinical charts were then reviewed to collect pertinent clinical data including age, gender, disease manifestations, medication history and serological parameters. Standard procedures were used to process formalin-fixed, paraffin-embedded 2 μm tissue sections for evaluation by light microscopy. For each biopsy, five distributed hematoxylin and eosin stains and three periodic acid-Schiff stains were performed. Standard procedures for direct immunofluorescence microscopy were applied to all cases with fluorescein isothiocyanate (FITC)-conjugated antibodies reactive with the following antigens: IgG, IgA, IgM, C3, C1q, fibrinogen, κ and λ light chains, and albumin (DAKO, Carpinteria, CA). The intensity of immunofluorescence staining was semi-quantitatively scored on a scale of 0 to 4+. All biopsies demonstrated strong immunofluorescence glomerular staining generally in a “full-house” (IgG, IgA, IgM, C3, C1q) pattern, which is characteristic of LN. Standard procedures for electron microscopy were applied to evaluate the renal biopsies using a Philips CM10 electron microscope.

Standard immunohistochemistry was performed on serial paraffin tissue sections using monoclonal antibodies to CD3 (Labvision, Fremont, CA), CD20 (DAKO), CD45 (DAKO), MUM1 (DAKO) and CD138 (DAKO) as primary reagents and appropriate HRP-conjugated secondary antibodies. Isotype controls for each primary reagent, using the matched secondary reagent, are provided in Supplemental Figure 1. Antigens were retrieved by boiling slides for 20 minutes under pressure in 1 mM EDTA (pH8). Double stains were done using the EnVision G/2 Doublestain System (DAKO). The number of positively staining cells was counted without knowledge of the clinical data by one renal pathologist (AC). Biopsies with prominent aggregates of B cells were stained with CD21 (DAKO), Ki-67 (Labvision) and others were stained with CD4 (Labvision), CD8 (Labvision), CXCL10 (R&D Systems, Minneapolis, MN), CXCL12 (R&D), CXCL13 (R&D), CCL21 (R&D), and BAFF (Alexis Biochemicals, San Diego, CA). The interstitial infiltrate was categorized into 3 patterns: 1) diffuse and scattered; 2) T:B cell aggregates; 3) ectopic GC. An arbitrary cut-off of 50 cells composed of both T and B cells satisfied the designation of T:B cell aggregate pattern. The presence of a follicular dendritic cell (CD21+) network was necessary to assign the GC pattern. Of note, the T:B cell aggregate always had areas with diffuse inflammation and the ectopic GC pattern often showed both T:B cell aggregates and diffuse inflammation. Univariate statistical analysis was performed using the Fisher-Exact test and Mann-Whitney test with a p of <0.05 considered statistically significant.

Laser capture microdissection of interstitial CD38+ or Ki-67+ cells

At the time of procurement, renal biopsies were immediately frozen in optimal cutting temperature (OCT, Tissue-Tek, Torrance, CA) media and stored at −80°C. These consisted of ten kidney biopsies from ten patients. Patients A, B, C, D, and F were females (23 to 40 years of age; one Caucasian, three African-American, and one Hispanic) with established diagnoses of SLE from four months to 10 years who were treated with low doses of prednisone prior to kidney biopsy. Patients E, G, H, and J were females (13 to 37 years of age; three African-American, and one Hispanic) with new-onset SLE and no significant immunosuppressive therapy prior to renal biopsy. Patient I is a 28 year old male with hypertension and anabolic steroid use who had acute kidney injury, hyperbilirubinemia, and no evidence of SLE. Eight μm thick frozen tissue sections were placed on positively charged glass slides and immunostained for CD38 (DAKO) or Ki-67, as previously described(41). Briefly, cryosections were fixed in acetone at −20° C for 5 minutes, washed with phosphate buffered saline (PBS) on ice for 30 seconds, treated with 0.1% H2O2 for 30 seconds, and incubated in PBS with 10% normal goat serum for 2 minutes. Tissue sections were incubated with anti-CD38 or anti-Ki67 antibodies for 10 minutes, rinsed in PBS, incubated with horseradish-peroxidase (HRP)-conjugated horse anti-mouse antibodies and 5% goat serum in PBS for 5 min, rinsed in PBS, and incubated in 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA) for 5 minutes. All incubation steps were performed with the glass slide on an ice pack. Finally, the sections were dehydrated in graded alcohols (95% and 100% EtOH) for 30 seconds each and cleared with 2 rinses of xylene for 3 minutes each. Laser capture microdissection (LCM) using the Arcturus Pixcell II (Molecular Devices, Sunnyvale, CA) platform and Capsure® HS LCM caps (Molecular Devices) was performed to isolate CD38 or Ki-67 staining cells. The pulse power was 70 mW, size spot diameter was 15 μm, pulse duration was 5 msec and target voltage was 170 mW. Extraction reservoirs were placed directly on the HS LCM caps.

Synthesis of cDNA

10μl of lysis buffer [0.5× PBS, 10 mM DTT, 20U RNAsin (Promega, Madison, WI) and 1U Prime RNAse Inhibitor (Eppendorf, Hamburg, Germany)] was aliquoted directly into the extraction reservoir of the LCM cap. Caps were immediately placed on dry ice and stored at −70°C. cDNA was synthesized within the extraction reservoir using 25 μl of RT-PCR buffer [375ng random hexamer primer (pd(N)6, Amersham Pharmacia Biotech), 2mM dNTP-Mix (Promega), 10mM DTT, 5% v/v NP-40, 10U RNAsin, 15U Prime RNAse Inhibitor and 125U Superscript II reverse transcriptase (Invitrogen)]. The cap was incubated for 60 minutes at 37°C. RT-PCR reactions were collected by centrifugation at 8,000 rpm for 60s into a 500 μl collection tube and heat-inactivated at 70°C for 15 minutes.

Amplification of Ig Heavy and Light chains

γ, Igλ and Igκ rearrangements were amplified by two rounds of nested PCR in a 40μl volume [50nM primers, 1mM of dNTPs, 1× Coral Load PCR buffer (Qiagen, Valencia, CA), 1.25U HotstarPlus Taq polymerase (Qiagen) and 8μl of cDNA (or 1st round PCR product)]. The IgG specific primers consisted of mixtures complementary to conserved leader and framework regions from heavy and light chain variable regions and conserved 3′ constant light chain regions(4244) and the 3′γ constant region(45). It should be noted that the originally published primer sequences, 3′Sal1-JH1/2 and 3′Sal1-JH4/5 each had a base pair deletion. The correct sequences are: 3′Sal1-JH1/2 5′-TGCGAAGTCGACGCTGAGGAGACAGTGACCAGG-3′; 3′Sal1-JH4/55′-TGCGAAGTCGACGCTGAGGAGACGGTGACCAGG-3′. Targeted sequences were amplified using 50 cycles of PCR under the following conditions: 94°C for 30 sec, 57°C (γ/Igκ) or 60°C (Igλ) for 30 sec; 72°C for 55 sec with a final extension cycle of 72°C for 7 min.

Sequence Analysis

PCR products were cloned into the pCR4-TOPO TA vector (Invitrogen) and plasmid DNA was purified using QIAprep spin mini-prep kits (Qiagen). Multiple bacterial clones were randomly selected for sequencing to ensure proper sampling. The V-QUEST program from IMGT (http://imgt.cines.fr/IMGT_vquest/vquest) was used to identify the V(D)J germline, as well as the CDR and FWR regions. Nucleotide and amino acid mutations in the V-region were identified by alignment with the closest corresponding germline using IMGT. In some cases, the corresponding genomic V segments were amplified and sequenced to confirm that the observed mutations did not represent allelic polymorphisms. Multiple nucleotide changes in a single codon were scored as a single replacement mutation. Related clones were defined by similar CDR3 regions as identified by the junction analysis software provided by IMGT. Sequences that were out of frame or contained mutations that resulted in a non-productive sequence were excluded from analysis (approximately 15% of all sequences, data not shown). For tabulating clonal frequency, a clone was counted if it was obtained from separate LCM picks or if it differed by three or more nucleotides from other cloned sequences within the same LCM “pick”. This latter criterion was used to exclude clones that might appear different due to PCR error. Antigen driven selection was calculated using the JAVA applelet from Lossos et al. (http://www-stat.stanford.edu/immunoglobulin/). Genealogical trees showing the relationships between plasma cells were constructed by analysis of the pattern of somatic mutations.

RESULTS

To examine the possible pathogenic significance of B cells in LN, we identified 68 patients with SLE diagnosed by a rheumatologist who had undergone diagnostic renal biopsies for presumed LN (22). The average age of this cohort was 31 years, 85% were female and 81% were African-American. At the time of biopsy, the average duration of disease was 36 months, the median creatinine was 1.0 mg/dL and 81% had detectable antibodies to dsDNA. Twenty-five percent had only low-dose oral prednisone prior to renal biopsy (20 mg per day or less). Three of the biopsies were ISN/RPS class II, 22 were class III, 33 were class IV, and 10 were class V. Tissue sections from each biopsy were first examined by immunohistochemistry (IHC) with antibodies to CD45, CD3, CD4, CD20 and CD138.

Ectopic lymphoid structures in LN

Three distinct patterns of B cell infiltration were evident (Figure 1). In 48% of biopsies (33/68), the predominant pattern was one of diffuse and scattered lymphocytic (CD45+) infiltration with varying degrees of co-infiltration with CD20+ B lymphocytes or CD138+ plasma cells. Plasma cells and B cells were invariably excluded from glomeruli with only rare circulating CD20+ B cells in glomerular capillaries. In 46% of cases (31/68), there were well-circumscribed aggregates of CD20+ B cells or CD138+ plasma cells with CD3+ T cells in tubulointerstitium. Most of these T cells expressed CD4 (data not shown). Finally, in 6% of biopsies (4/68), structures consistent with GCs were observed. In these structures, CD20+ B cells preferentially occupied the central zone while CD3+CD4+ T cells tended to occur peripherally. Staining with the dendritic cell marker CD21 revealed a central dense reticular network characteristic of follicular dendritic cells (FDCs). CD138+ plasma cells were rare within the central FDC network but common in the surrounding areas. The spatial organization of B cells, T cells, FDCs and tingible body macrophages are all consistent with the observed histological structures being bona fide GCs.

Figure 1
Lymphoid neogenesis in the tubulointerstitium of lupus nephritis. Three distinct patterns of lymphoid involvement were observed in LN. In the diffuse pattern (A–D), CD138+ plasma cells (A, B) were scattered throughout the renal interstitium between ...

Chemokines induce and maintain the spatial organization of immunocytes within secondary lymphoid organs(46). Therefore, we examined if some of these same chemokines were present in SLE renal biopsies (Figure 2). When biopsies with either T:B aggregates or GC phenotype were stained, CXCL12(46) and BAFF(47) was observed in almost all samples tested with prevalences of 95% (18 of 19 biopsies) and 100% (4/4) respectively. Several other chemokines were also commonly expressed. Approximately 70% (12/17) of biopsies with either the T:B aggregate or GC phenotypes had detectable staining for CXCL13,(36) 50% (8/16) for CCL21 and 42% (8/19) for CXCL10. These chemokines were not detectable in normal renal tissue (data not shown). Interestingly, there was little chemokine staining in renal biopsies with a diffuse histological phenotype (n=10, data not shown). Between the T:B aggregates and GC phenotypes there were no clear differences, except that the GC phenotype was associated with more intense chemokine staining. These data suggest that similar factors may organize and maintain the T:B and GC-like histological phenotypes. In contrast, different mechanisms may mediate the diffuse accumulation of plasma cells and B cells in LN.

Figure 2
Chemokine expression in lymphoid neogenesis. Strong BAFF immunohistochemical staining was observed in T:B cell aggregates (upper left; original magnification × 200) and in germinal centers (upper center; original magnification × 200). ...

To begin to determine if the observed histological structures were functional(48), we determined if they were associated with in situ lymphocyte proliferation. Therefore, biopsies manifesting the diffuse, T:B aggregate and GC patterns were stained with antibodies specific for the proliferative marker Ki-67. As demonstrated in Figure 3, the centers of the GC structures contained numerous small proliferating cells. Ki-67+ cells were also commonly observed in T:B aggregates (14 of 22 biopsies, 64%). In contrast, biopsies with a diffuse histology infrequently had Ki-67+ cells (2 of 11 biopsies, 18%). When present, they were usually one or less Ki-67+ cell per high-power field. Ki-67+ tubular epithelial cells were infrequently observed in biopsies from all three histological groups (data not shown).

Figure 3
In situ proliferating centroblasts or plasmablasts in lupus nephritis. Staining for Ki-67 demonstrated that small proliferating cells were commonly observed in T:B aggregates and in GCs but were rare in patients with a diffuse histology (upper panels). ...

To determine if the observed proliferating cells were lymphocytes, we performed two-color IHC with antibodies specific for Ki-67 and either CD20 or CD3. Ki-67+CD4+ positive cells were infrequently observed in all three histological patterns (data not shown). In contrast, numerous Ki-67+CD20+ cells were observed in the GC structures (Figure 2). Ki-67+CD20+ cells were also observed in the T:B aggregates but they were a minor fraction of the Ki-67+ cells (<10%). Most Ki-67+ cells were CD20low or CD20. However, most of these Ki-67+ cells in the T:B aggregates expressed CD138 and were therefore plasmablasts.

In situ lymphocyte organization correlated with both the extent of TI and with specific pathological features (Figure 4). Severe interstitial inflammation (>25% of the interstitium infiltrated by inflammatory cells) was more likely to be found in biopsies with the T:B aggregate (29 of 31 biopsies) and GC (4/4) patterns compared to 16 of 33 biopsies with the diffuse pattern (p=0.00002, Fisher exact test)(Figure 4A). Detectable tubular basement membrane immune complexes (TBMICs)(immunofluorescence) were also infrequent in biopsies manifesting a diffuse pattern (6/33)(Figures 4B and 4C) but were a usual feature of biopsies with either T:B aggregates or GCs (21/35)(Figures 4B). This difference was highly significant (p=0.00014). Electron microscopic analysis of the biopsy in Figure 4D, which had a T:B aggregate histological pattern, revealed that the TBMICs resided within the TBM, characteristic of lupus interstitial nephritis (Figure 4E). Additional IF and EM images demonstrating TBMICs are provided in Supplemental Figure S2.

Figure 4
Increased tubulointerstitial inflammation and immune complex deposition in biopsies with either T:B aggregates or GCs. (A) The T:B cell aggregate and GC histological patterns tend to have increased tubulointerstitial infiltration by inflammatory cells ...

Characterization of in situ immunoglobulin repertoire

The presence of lymphoid-like structures, and aggregates of proliferating plasmablasts, in LN suggested that in situ antigen-driven clonal expansion and somatic hypermutation were occurring. To test this directly, we used laser capture microscopy (LCM) coupled to RT-PCR and sequencing to characterize the in situ heavy and light chain repertoire in renal biopsies from LN patients. We analyzed nine patients. On anti-CD38 antibody stained fresh frozen sections we used LCM to sample a GC from one LN patient (Patient A) and T-B aggregates from four other LN patients (Patients B-E). For comparison, we sampled the expressed immunoglobulin repertoire in three LN patients with a diffuse histology (Patients F-H) and one non-lupus patient with idiopathic interstitial nephritis (Patient I). We also stained sections with anti-Ki67 antibodies and sampled a cluster of proliferating cells in a LN patient with T:B aggregates on biopsy (Patient J).

Immunoglobulin clonal restriction and antigen-driven somatic mutation within the intrarenal germinal center

Patient A was a 27 year-old Caucasian female with a seven-month history of SLE who had been treated with low-dose methotrexate and prednisone. The dsDNA antibody titer at time of biopsy was 1/2560 and anti-Sm antibody was negative. LCM was used to sample 12 separate areas within a GC and another 12 from the surrounding tubulointerstitium. Each LCM pick sampled one to eight visible CD38+ cells. The distribution and frequency of the most commonly expressed immunoglobulin genes identified from either the GC or surrounding parenchyma is provided in Figure 5. A total of 26 γ, 41 λ and 8 κ distinct sequences were identified. As only a few κ sequences were isolated, they were excluded from further analysis. Of the 26 distinct expressed γ V sequences identified, 12 were cloned from the GC and 14 from the surrounding tubulointerstitium. Among the 12 GC γ sequences, four (33%) arose from a single rearrangement (VH3-9*01D3-10*01JH3*02). This rearrangement was also the predominant expressed heavy chain detected in the parenchyma surrounding the GC (Figure 5). Another three (25%) of the GC γ V sequences identified arose from another single rearrangement (VH3-23*01D3-22*01JH3*02). These data indicate that over half (58%) of the γ chain repertoire within the GC arose from two unique recombination events.

Figure 5
Schematic representation showing the most frequently expressed immunoglobulin genes found within a GC and the surrounding parenchyma from patient A. Expressed immunoglobulin genes observed outside the GC were grouped into either those that were also found ...

All of the VH3-9*01D3-10*01JH3*02 encoded sequences were heavily mutated when compared to reported germline segments with all containing a similar core of coding and non-coding mutations (Figure 6A). It was possible that some of the apparent mutations in VH3-9*01D3-10*01JH3*02 represented an unreported allelic polymorphic form of VH3-9. However, these mutations were not observed when the corresponding genomic VH3-9 segment from patient A was amplified from peripheral blood and sequenced (data not shown).

Figure 6
Evidence of in situ clonal expansion and antigen-selected somatic hypermutation in a intrarenal GC. (A) Nucleotide sequences of VH3-9*01D3-10*01JH3*02 amplified transcripts were aligned with germline. Identical nucleotides are indicated by dashes. Dots ...

Comparison of the different VH3-9*01D3-10*01JH3*02 expressed sequences isolated from both the GC and from the surrounding parenchyma suggested that some B cells had sequentially acquired immunoglobulin mutations during clonal expansion. The relative genealogy of these mutations can be demonstrated in a clonal tree (Figure 6B). The accumulation of such hierarchical γ mutations, and the fact that they were identified in closely adjacent B cells, is consistent with in situ clonal expansion and somatic hypermutation(49).

To examine if antigen was selecting for particular somatic mutations within the GC, we analyzed the type and frequency of mutations in the VH3-9*01D3-10*01JH3*02 encoded V regions (containing CDR1 and CDR2). In the absence of antigenic selection, replacement (R) and silent (S) mutations will occur randomly in both the complementarity determining regions (CDRs) and framework regions (FWRs). However, if there is antigen-driven selection, R mutations in the CDRs will be over-represented(50) while R mutations in the FWRs will be under-represented. We used the multinomial method of Lossos et al.(51) to determine the probability that B cells expressing VH3-9*01D3-10*01JH3*02 encoded V regions had undergone antigen-driven selection (Figure 6C). In the CDRs, R mutations were over-represented (R/S=8/1 p=0.028 to 0.035) and under-represented in the FWRs (12/8 to 13/7, p=0.018 to 0.041).

A similar picture was obtained when the λ chain GC repertoire was analyzed. Of the seven sequences obtained from the GC (Figure 5), two were from one unique recombination (VL2-8*01JL2*01)(Figure 7A) and two from another unique recombination (VL1-44*01JL3*02) indicating that over half (57%) of the detectable GC λ clones arose from one of two unique recombination events. Both of these rearrangements were also found in the surrounding parenchyma (Figure 5).

Figure 7
Analysis of a light chain selected in the GC also revealed evidence of antigen-selected somatic hypermutation. (A) Nucleotide sequences of VL2-8*01JL2*01 amplified transcripts were aligned with germline as in Figure 6. (B) Genealogical relationships of ...

Alignment of the different VL2-8*01JL2*01 derived sequences with predicted germline sequences indicated that all had accumulated somatic mutations (Figure 7A). However, the overall number of mutations was less than that observed in the clonally expanded GC γ sequences (Figure 6A). All three mutations observed in CDR1 and CDR2 encoded amino acid replacements, again suggesting selection by antigen. Sequencing of a different, non-selected VL2-8*01 containing λ variable region from patient A confirmed that the observed mutations did not represent allelic variation (data not shown). Assembly of the different VL2-8*01JL2*01 sequences into a clonal tree revealed a mutational hierarchy suggestive of ongoing clonal expansion and somatic hypermutation (Figure 7B). In the most related VL2-8*01JL2*01 sequences (Al4b,c) the distribution of R and S mutations in the CDR (R/S=3/0, p=0.021) was consistent with antigen-driven selection (Figure 7C). A similar trend was seen in the FWRs (R/S=3/1, p=0.155).

Clonal selection in the tubulointerstitium surrounding the GC

Analysis of the surrounding parenchyma provided further evidence of clonal selection. Of the 34 distinct λ sequences identified in the tubulointerstitium, four arose from a single VL2-14*01JL2*01 rearrangement and three from a single VL1-47*01JL2*01 rearrangement. In addition, four distinct interstitial λ sequences arose from two genomic rearrangements identified in GC expressed sequences (Figure 5). Therefore, 32% (11/34) of the identified expressed λ sequences were derived from four rearrangement events. Alignment and further analysis of the VL2-14*01JL2*01 related clones with the predicted corresponding germline segments revealed evidence of sequential somatic hypermutation and antigen-driven clonal selection (Supplemental Figure S3).

Comparison of the repertoire between the GC and surrounding tubulointerstitium revealed important inter-relationships and significant differences. As described above, the repertoire of expressed γ and λ immunoglobulin chains in the GC was well represented in the parenchyma. However, several clonally expanded and/or prevalent parenchymal λ immunoglobulin chains were absent from the GC (Figure 5). These observations suggest that the GC can contribute to the parenchymal repertoire but that the parenchyma does not necessarily contribute to the GC repertoire.

In situ immunoglobulin expression in T:B aggregates

Patient B was a 40 y.o. African-American female with a four-month history of SLE treated with 20 mg/day of oral prednisone. The anti-Sm antibody was positive and the anti-dsDNA antibody titer prior to biopsy was 1/320. LCM was used to obtain 28 independent samples from two different T:B aggregates. A total of 68 distinct γ, 27 λ and 31 κ sequences were identified.

Within the γ population, ten rearrangements were observed more than once (24 of 68 sequences for an overall clonality of 35%). The most common rearrangement was observed four times (4/68, VH1-3*01D4-23-*01JH4*02). Alignment of these cDNA fragments with the predicted germline sequence revealed that the identified sequences had undergone extensive somatic hypermutation (Figure 8A). These clones could also be assembled into a simple clonal tree suggesting ongoing somatic hypermutation (Figure 8B). Analysis of the distribution of mutations indicated selection for replacement mutations in the CDRs and for silent mutations in the FWRs (Figure 8C). These findings are consistent with antigen-driven clonal selection.

Figure 8
Clonal selection in a T:B aggregate. (A) Nucleotide sequences of VH1-3*01D4- 23*01JH4*02 amplified transcripts were aligned with predicted germline sequences as in Figure 6. (B) Genealogical relationships of sequences are illustrated in a clonal tree ...

There was also evidence of clonality when the corresponding light chain sequences were examined. Of the 27 distinct λ sequences, four rearrangements were found more than once with one observed three times (VL1-40*01JL3*02) for an overall clonality of 13/27 or 48%. The frequency and distribution of mutations in the VL1-40*01JL3*02 clones was consistent with extensive somatic hypermutation and antigen-driven clonal selection (Supplemental Figure S4). There was also evidence of clonality in the 31κ sequences with an overall clonality of 11/31 or 35% (data not shown).

Clonality was observed in the immunoglobulin repertoire expressed in the three other T:B aggregate biopsies that were sampled (Patients C–E). A summary of these results is provided in Supplemental Table 1. Clonally related sequences from three of the four biopsies were heavily somatically mutated (Figure 8 and data not shown). However, in one T:B aggregate (Patient C) the observed clonally selected κ chains (9 of 37 distinct sequences) were very similar to the corresponding germline sequences (Supplemental Figure S5). These results indicate that T:B aggregates are associated with moderate clonal restriction. Ongoing somatic hypermutation was observed but was not an invariant feature of the immunoglobulin chains being selected in the T:B aggregates.

In the GC histology, 17 of 41 distinct λ chains used VL2-14*01 and ten used VL1-44*01. For the four patients with a T:B aggregate histology, 33 of 94 were VL2-14*01 and 19 were VL1-44*01. Overall, these two V regions were found in about 55% of identified λ chains. In both the GC and T:B aggregate histologies, the VL2-14*01 and VL1-44*01 segments were primarily found in expanded clonal populations. These results indicate that the overall frequency of specific expressed λ variable segments was similar between the GC and T:B histological patterns.

To determine if the observed restricted expressed repertoires were a specific feature of the GC and T:B aggregates, we sampled the expressed immunoglobulin repertoire in three lupus nephritis patients who had diffuse B cell infiltration on biopsy (Supplemental Table 1, Patients F–H). From patient F, we cloned and compared 37 distinct immunoglobulin heavy chains. Only two arose from the same rearrangement. In the second patient, Patient G, out of 37 sequences three arose from one rearrangement, three from another and two from a third. In patient H, out of 31 sequences three arose from one rearrangement and there were two examples where the same rearrangement was observed twice. A similar degree of clonality was observed in a non-lupus patient (patient I) who had idiopathic acute interstitial nephritis with diffuse B cell infiltration (Supplemental Table 1). In that patient, we identified 34 unique sequences that arose from 29 unique recombination events. Four recombinations were observed twice while none were observed three or more times. Therefore, a modest degree of clonal restriction can be observed in biopsies with a diffuse pattern of B cell infiltration. However, this degree of clonality does not appear to be a specific feature of lupus interstitial nephritis.

In general, we observed more clonal restriction in biopsies with more organized lymphocytic infiltrates. However, there were exceptions as one biopsy with T:B aggregates had a similar degree of clonality (Patient E) as that observed in one of the biopsies manifesting a diffuse morphology (Patient H). These observations indicate that the correlation between the degree of lymphocyte organization and in situ selection is imperfect. It might be expected that evidence of in situ proliferation may more directly identify B cells that are being selected in situ. Therefore, we used LCM to capture an aggregate of Ki-67+ cells in a patient with T:B aggregates on histology (Patient J).

We cloned 18 unique immunoglobulin heavy chains from a single Ki-67+ focus. Of these, 11 arose from two unique recombinations (Patient J). These two expressed heavy chains used the same Vh (VH4-34*01) segment but different D (DH1-1*01 and DH5-18*01) and J (VH4*02 and VH4*02) regions. Analysis of the most common heavy chain recombination (VH4-34*01D5-18-*01JH4*02), constituting 6 of 18 clones, is provided in Figure 9. As can be seen, the aligned sequences contained several common mutations with unique mutations observed in some clones (Figure 9A). These differences, which allowed construction of a clonal tree, suggest ongoing somatic hypermutation (Figure 9B). Interestingly, analysis of the R/S distribution (Figure 9C) in the CDR1 and 2 containing regions revealed no evidence that these mutations had undergone antigen selection. A similar pattern of mutations was observed in those expressed clones arising from the VH4-34*01D1-1-*01JH4*02 recombination (data not shown).

Figure 9
Clonal selection in a Ki-67+ foci. (A) Nucleotide sequences of VH4-34*01D5-18*01JH4*02 amplified transcripts were aligned with predicted germline sequences as in Figure 6. (B) Genealogical relationships of sequences are illustrated in a clonal tree with ...

DISCUSSION

Interstitial inflammation is a prominent feature of human LN that, independently of glomerular involvement, identifies patients at risk for subsequent renal failure (15, 16, 22). Herein, we demonstrate that in over half of our patient cohort the interstitial infiltrate was organized into lymphoid-like structures competent to select for B cells expressing a highly restricted immunoglobulin repertoire. The presence of lymphoid-like structures strongly correlated with detectable TBMICs. These observations suggest that in LN, GCs and T:B aggregates select for cells that locally secrete pathogenic antibodies in the tubulointerstitium.

The two histological patterns, GC and T:B aggregates, appeared to reflect different underlying states of B cell selection. Many aspects of clonal selection in the intrarenal GC were typical of those observed in GCs residing in secondary lymphoid structures (5254). As has been reported for both rodent (55) and human (56) GCs, only a few clones accounted for a majority of the sampled repertoire. Furthermore, most if not all of the observed predominant clones had undergone somatic hypermutation. However, analysis of the frequency and distribution of mutations in non-selected (singly occurring) GC expressed immunoglobulin chains indicated that most had not undergone antigen selection (data not shown). This latter observation is consistent with elegant in vivo imaging studies demonstrating that GCs are open structures that allow B cells to enter freely and scan for antigen (53, 57, 58). Our results are also very similar to those obtained when human lymph nodes from normal volunteers were characterized (56).

In contrast to the proliferating centroblasts observed in the GCs, plasmablasts predominated in those patients with T:B aggregates. These foci of T cells and plasmablasts are reminiscent of the extrafollicular B cell responses that have been recently described in some murine models of autoimmunity (59). In both MRL/Mplpr/lpr mice and in MRL/Mplpr/lpr mice expressing AM14 (a rheumatoid factor antibody), the production of autoantibodies in secondary lymphoid organs preferentially occurs in aggregates of plasmablasts residing outside follicles (6062). Selection and somatic hypermutation might occur in these sites through both T-dependent and TLR-dependent mechanisms (48, 63). However, to our knowledge, we are the first to demonstrate in humans the existence of functional extrafollicular plasmablast aggregates in an organ targeted by an autoimmune disease. Furthermore, our data indicate that such plasmablast foci are a usual feature of LN complicated by severe tubulointerstitial inflammation.

Direct sampling of the proliferating cells in a patient with T:B aggregates revealed a degree of clonal restriction similar to that observed in an intrarenal germinal center. These data suggest that Ki-67 expression directly identifies cells undergoing in situ selection. The Ki-67+ cells occurred within areas of T:B aggregates but not all T:B aggregates contained Ki-67+ cells. This relative discordance may explain the variable clonality observed when aggregates, identified by CD38 staining, were sampled for in situ immunoglobulin expression. These data suggest that there might not be tight correlations between the immunohistological characteristics of lymphocyte aggregates and their function in selection.

In the T:B aggregates, we observed selection for both germline-encoded and highly mutated antibodies. Most antibodies that were mutated had appeared to have undergone antigen-driven selection. A notable exception was the expressed immunoglobulin heavy chain cloned from the foci of Ki-67+ cells. However, current methods of determining antigen driven selection cannot assess antigenic pressure on the CDR3 as several processes determine diversity in this region. Therefore, if antigenic specificity is determined primarily by the CDR3, then selection might not be apparent for the other regions. This possibility might be applicable to VH4-34*01D5-18-*01JH4*02, as the CDR3 contains three arginines which are known to confer DNA binding (64).

Regardless of apparent differences in B cell populations usually selected in each histological type, both GC and T:B histological patterns were strongly associated with TBMICs. This suggests that both lymphoid structures select for cells secreting antibodies that form in situ immune complexes with locally available antigens. It is possible that the selecting antigens are renal specific as such antibodies have been detected in the peripheral serum of patients with lupus nephritis (65). However, it is not clear if these autoantibodies are produced in situ in the kidney or in conventional lymphoid structures.

Our studies have focused on in situ antibody secretion; however, it is likely that resident B cells are also contributing to local inflammation by presenting MHC class II restricted antigens to neighboring T cells (66). The importance of B cells for maintaining systemic pathogenic T cells has been demonstrated in MRL/Mplpr/lpr mice(6769) and in SLE patients treated with rituximab.(70, 71) B cells are also required for maintaining T cell infiltrates in the synovium of patients with rheumatoid arthritis (29). The close proximity of T and B cells in both intrarenal GCs and T:B aggregates, and the attendant clonal expansion of B cells expressing isotype switched antibodies, suggest that these B cells are productively interacting with co-resident T cells.

It is unclear if the available murine models of SLE fully mimic the in situ adaptive immune responses associated with human lupus interstitial nephritis. Lymphocytic infiltrates in the kidneys of both NZB/NZW and MRL/Mplpr/lpr mice contain B cells and/or plasma cells and the antibodies they express display a broad repertoire of specificities (7275). However, it is not known if murine models manifest functional intrarenal GCs or TB aggregates. Rather, available evidence suggests that the NZB/NZW and MRL/Mplpr/lpr mice have diffuse or perivascular intrarenal lymphocytic infiltrations. The in situ organization of B and T cells into lymphoid-like structures could be a unique feature of human LN.

In both GC and T:B aggregates, we observed a high frequency of light chains containing V 2-14*01 and V1-44*01. This could reflect a requirement for these segments to encode certain autoreactive specificities (clonal convergence). However, both V2-14*01 and V1-44*01 are highly represented in the mature B cell peripheral repertoire of both normal individuals and patients with SLE (76). Therefore, the high frequencies noted here may reflect the repertoire of precursor B cell populations.

The different histological patterns of involvement in LN were reminiscent of those observed in the synovial tissue of rheumatoid arthritis patients (24, 77, 78). In both cases, histological features range from diffuse lymphocyte infiltration to fully formed GCs. Sampling the immunoglobulin repertoire in RA synovial GCs(79, 80) and total RA synovium (81, 82) has also revealed evidence of clonal restriction and antigen-driven somatic hypermutation. However, unlike human lupus nephritis, TLN in RA has not been related to specific, prognostically important, pathological features (22).

Dense B cell aggregates are present in half of allograft biopsies and occasional germinal centers (in 11% of biopsies) may be associated with antibody-mediated rejection (83). When kidneys which have been removed for terminal rejection have been examined, tertiary lymphoid neogenesis is a usual feature (84). In contrast, the frequency and significance of TLO in other immune-mediated renal diseases is less clear. Prominent B cell aggregates have been reported in IgA and membranous nephropathy (85, 86). However, GCs have not been reported in either nephropathy and the presence of lymphoid aggregates has not been associated with specific pathological features. A germinal center was reported in one of 16 reported cases of ANCA-associated nephritis (35) while we did not observe any GCs in 32 cases we examined (87). These observations suggest that TLO might be much more common in lupus nephritis than in some other autoimmune nephritides.

In our study, about half of LN renal biopsies had tubulointerstitial infiltrates that were organized into higher order lymphoid structures. However, this is probably an under estimation of the true prevalence of GCs and T:B aggregates in LN. This is because a diagnostic renal biopsy represents only a small fraction of the entire kidney, so sampling error always remains an important consideration. Even with this limitation, our data clearly demonstrate that the detection of lymphoid structures on diagnostic biopsy is highly predictive of specific pathological features.

Based on our studies, we propose that human LN arises from at least two distinct pathogenic processes. The deposition of pro-inflammatory immune complexes in glomeruli likely arises from a breach in systemic tolerance. In contrast, our observations demonstrate that interstitial nephritis is associated with in situ tolerance diatheses. The relative importance of each immunological process is unclear. Furthermore, any interdependence between the two processes is not known. However, observations that interstitial nephritis determines renal survivorship (15, 16, 22) indicate that intrinsic immunological processes contribute to disease severity. Identification of the in situ antigens and factors promoting local B cell selection and expansion in the interstitium should yield important biomarkers and could lead to novel therapeutic strategies in lupus nephritis.

Supplementary Material

supp mat

Acknowledgments

This study was supported by grants from the Lupus Research Institute and the National Institutes of Health (AI082724, AR055646).

We would like to thank Dr. Martin Weigert for his critical evaluation of our work. We would also like to thank Dr. Maria Tretiakova for her expert assistance with the laser capture microdissection, Dr. Linda Wagner-Weiner for identifying patients and Sarah Powers for her careful reading of the manuscript.

Abbreviations

SLE
Systemic lupus erythematosus
GCs
germinal centers
LN
lupus nephritis
GN
glomerulonephritis
LCM
laser capture microdissection
TI
tubulointerstitial inflammation
TLN
tertiary lymphoid neogenesis
TBM
tubular basement membrane
CDR
complementarity determining region

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