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 tubules and around Bowman’s capsule. MUM1⁺ (C) and CD20⁺ (D) B cells demonstrated a similar distribution (original magnification X 400). Three examples of the T:B cell aggregate pattern are provided (E–J). Immunostaining of sequential histological sections revealed well-circumscribed aggregates of CD3⁺ T cells (E, G, I) and CD20⁺ B cells (F, H)(original magnification × 200 upper examples and × 400 lower example) or CD138⁺ plasma cells (400×). In the GC pattern (right), a dense aggregate of lymphocytes consistent with a germinal center was evident on hematoxylin and eosin stained tissue sections (K)(original magnification X 200). On serial sections, CD20⁺ B cells (L) were clustered in the middle of the lymphoid aggregate surrounded by CD3⁺ (M) and CD4⁺ (N) T cells. Scattered CD138⁺ plasma cells (O) were observed in the surrounding parenchyma. Staining for CD21 revealed a dense follicular dendritic cell network characteristic of GCs (P–R) (the CD21 staining corresponding to the previous panels is provided first followed (P) by examples of CD21 staining in two additional biopsies with a GC, Q and R)(original magnification × 400).

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). Double staining for CD20 (red) and Ki-67 (brown)(middle panels) revealed that most proliferating cells in the GC were CD20⁺. In contrast, in the T:B aggregates most Ki-67⁺ cells were CD20^low or CD20⁻. Double staining for Ki-67 (blue) and CD138 (brown)(lower panel) revealed numerous Ki-67⁺CD138⁺ plasmablasts (arrows).

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 in the GC (GC Related) or those that were not represented in the GC (GC Unrelated). The frequency of each expressed clone relative to either the GC or the extra-GC pools of expressed heavy and light chain immunoglobulins are provided.

Analysis of a light chain selected in the GC also revealed evidence of antigen-selected somatic hypermutation. (A) Nucleotide sequences of VL_2-8*01JL_2*01 amplified transcripts were aligned with germline as in Figure 6. (B) Genealogical relationships of sequences are illustrated in a clonal tree with a predicted germline clone at the top. The black closed circle indicates a proposed intermediate clone. (C) R/S ratios of clones in FRW and CDR regions. The p-values were calculated as in Figure 6.

Clonal selection in a Ki-67⁺ foci. (A) Nucleotide sequences of VH_4-34*01D_5-18*01JH_4*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 predicted germline clone at top. (C) R/S ratios of clones in FRW and CDR regions. The p-values were calculated as in Figure 6.

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). In T:B aggregates, CXCL12 (upper right) and CXCL13 (lower left) was often present, while CXCL10 (lower center) and CCL21 (lower right) were found in fewer cases and often with less staining intensity.

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 compared to the diffuse pattern (p=0.00002, Fisher exact test). The extent of interstitial inflammation was expressed as the average percentage of tubulointerstitium cross-sectional area involved with CD45⁺ inflammatory cells. (B) TBM immune complex deposition was more common in biopsies with the T:B aggregate or GC pattern than the diffuse pattern (p=0.00014, Fisher exact test). (C) IgG direct immunofluorescence staining shows no significant TBM staining in a biopsy with diffuse interstitial inflammation (original magnification × 400). (D) Strong granular TBM IgG staining is present in a biopsy with T:B cell aggregates (original magnification × 200). (E) Electron microscopy from the same biopsy as panel D reveals numerous electron dense deposits (arrows) that represent immune complexes within the TBMs (original magnification × 2950).

Evidence of in situ clonal expansion and antigen-selected somatic hypermutation in a intrarenal GC. (A) Nucleotide sequences of VH_3-9*01D_3-10*01JH_3*02 amplified transcripts were aligned with germline. Identical nucleotides are indicated by dashes. Dots indicate unmutated intervening portions of the V-regions. Numbers refer to IMGT amino acid positions. Sequences isolated from the germinal center are marked with an asterisk. (B) Genealogical relationships of sequences are illustrated in a clonal tree with a predicted germline clone at the top. (C) R/S ratios of clones in FWR and CDR regions (including the FWR 5′ to CDR1 which is not shown in “A”). The p-values represent the probability that the observed mutations arose from random chance and had not been selected for affinity to antigen.

Clonal selection in a T:B aggregate. (A) Nucleotide sequences of VH_1-3*01D_{4- 23*01}JH_4*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 predicted germline clone at top. (C) R/S ratios of clones in FRW and CDR regions. The p-values were calculated as in Figure 6.