Activation-Induced Cytidine Deaminase Expression in Follicular Dendritic Cell Networks and Interfollicular Large B Cells Supports Functionality of Ectopic Lymphoid Neogenesis in Autoimmune Sialoadenitis and MALT Lymphoma in Sjögren’s Syndrome

Patients and samples

All patients fulfilled the revised criteria of the American-European Consensus Group (22) for either primary (pSS) of secondary Sjogren’s syndrome (sSS). Demographic and main laboratory characteristics are described in Table I⇓. Biopsies from mSG of 10 patients with nonspecific chronic sialoadenitis (NSCS) (8 females and 2 males, mean age 56.5; range 33 to 71; mean disease duration 72-mo range (24–120 mo)), were chosen as control group. Normal human lymph nodes (LNs) obtained from patients undergoing vascular surgery were used for comparison. Procedures were performed after informed consent approved by the hospital Ethics Committee (REC 05/Q0702/1).

Formaldehyde-fixed, paraffin-embedded tissue samples were obtained from mSG biopsies of 22 SS patients with focus score ≥1 (23). In addition, parotid samples of 5 SS patients) with lympho-epithelial sialoadenitis (LESA) and 18 SS patients with low-grade MALT lymphoma (SS-MALT-L) were selected from the biopsy bank of the Department of Oral Pathology at Guy’s Hospital. Histologically, MALT-L were diagnosed by the presence of sheets or halos of monocytoid B cells, most often infiltrating epimyoepithelial islands. The existence of clonal populations was confirmed by VDJ PCR for the H chain Ig genes. Of the 18 patients with SS-MALT-L, previous (average 3.3 years, range 1–9) mSG biopsies collected between 1989 and 2002 were available from eight patients.

Fresh parotid samples were obtained from 2 patients with SS and LESA and 3 patients with SS and MALT-L undergoing diagnostic procedures (2 parotidectomy, 3 parotid tail biopsies) whereas control glands were obtained from 2 patients with squamous cell carcinoma and 1 normal parotid. Freshly collected parotid glands were part embedded in paraffin for immunohistochemistry (IHC) and part dissected in multiple pieces and stored in a 10:1 v/v of RNALater (Ambion) at −80°C for TaqMan RT-PCR analysis.

Immunohistochemistry

A list of primary and secondary Abs used is reported in Table II⇓.

Grading analysis, histological characterization of lymphoid proliferation and AID expression in mSG of SS patients

The cellular infiltrate and degree of lymphoid organization were assessed by immunohistochemical staining of sequential sections of mSGs with Abs to CD3, CD20, CD21, and peripheral node addressin (Meca-79) as previously reported (11, 12). Briefly after de-waxing and re-hydration formalin-fixed, paraffin-embedded 3-μm thick sections underwent appropriate Ag retrieval with Target retrieval solution or proteinase K (DakoCytomation). Double staining for CD3 and CD20 was used to analyze T/B cell segregation using the Dako EnVision Doublestain system (DakoCytomation) as previously reported (11, 12).

Based on the results of the double staining, a scoring system based on the size and degree of organization of infiltrates was used (11, 12). Cellular aggregates with periductal lymphocytes numbering between 10 and 50 were defined as grade 1 aggregates (G1), grade 2 aggregates (G2) comprised >50 periductal lymphocytes whereas grade 3 aggregates (G3) were characterized by >50 periductal lymphocytes plus the presence of GC-LSs. In addition, on the basis of the CD3/CD20 double staining, lymphocytic G2 foci were classified as 1) nonsegregated (NS-G2), when no clear compartmentalization of T and B cells in discrete areas was recognized and 2) segregated (S-G2), when the foci displayed separated T and B cell-rich areas. The presence of GC-LSs was determined by the presence of T and B lymphocytes and CD21⁺FDC networks on sequentially stained sections (2, 11, 12). To minimize any bias due to the cutting level, some follicles were cut and stained every 50 μm. Staining for AID was performed on sequential sections of the above to correlate the degree of follicular organization with AID expression. After Ag unmasking using Target retrieval solution (pH 6; DakoCytomation) and incubation with protein block solution (DakoCytomation) endogenous biotin was blocked with the avidin-biotin blocking system (DakoCytomation). Primary Ab EK2-5G9 was incubated at an appropriate dilution for 1 h at room temperature and biotinylated rabbit anti-rat Ig was used as a secondary Ab. Following a 1-h incubation and three washes, HRP or AP-streptavidin-biotin complex (DakoCytomation) was added to the section and incubated for 30 min. After further washes color reaction was developed with diaminobenzidine (HRP, DAB+; DakoCytomation) or Vector red (for AP; Vector Laboratories). Double staining for AID and the cellular markers CD20 and CD3 was performed to confirm the cellular source of AID⁺ cells. Aggregates were considered positive for AID when at least three positive cells were stained.

Quantitative Taqman real-time PCR

Total RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen), with on column DNase I digestion to avoid genomic DNA contamination. First-strand cDNA was synthesized from 500 ng of total RNA with a Thermoscript RT-PCR System for first-strand cDNA synthesis (Invitrogen Life Technologies). For quantitative Taqman real-time evaluation of AID, CD21 long isoform (CD21L, specific for FDC (24)) primers and probes were obtained from Applied Biosystems (Table II⇑). Samples were run in triplicate at 20 ng of cDNA/well, detected using the ABI PRISM 7900HT instrument and results analyzed using the ABI PRISM 7900HT sequence detection system version 2.1. Relative quantification was assessed using the comparative C_T (threshold cycle) method with cDNA from normal human lymph nodes used for calibration.

Histological characterization of the lymphoid proliferation in SS patients with MALT-Ls and relationship with AID expression

To characterize B cell subpopulations within SS parotids with LESA and SS-MALT-L, in addition to double staining for CD3/CD20 (T/B cell segregation), parotid sections were double stained for CD20/IgD and CD21/bcl-2 and single staining for bcl-6 to identify, together with morphological analysis, follicular, GC and marginal zone-like B cells. AID expression was then evaluated on sequential sections.

Statistical analysis

Differences in quantitative variables were analyzed by the Mann-Whitney U test. χ² test with Yates’ correction when required or Fisher’s exact test when appropriate were used to evaluate associations of qualitative variables in the different groups. Spearman’s rank correlation was performed to correlate expression levels of AID and CD21L mRNA in SS parotids. All the statistical analyses were performed using GraphPad Prism version 3.03 for Windows. A value of p < 0.05 was considered statistically significant.

Histomorphological grading, T-B cell compartmentalization and formation of FDC networks in SS-mSGs and SS-parotids

In SS-mSGs the total number of aggregates (mean ± SEM) at three consecutive cutting levels (50 μm a part was 254 ± 5; the majority were G2 aggregates (60.2%; 153.3 ± 5.5), a smaller percentage were G1 (33.5%; 85.5 ± 3.5) and fewer were G3 foci (6.3%; 16.5 ± 2.4). These graded aggregates were further analyzed by double staining with anti-CD3/CD20 and anti-CD21/bcl-2 Abs for the presence of T/B cell segregation and FDC networks. As we have previously reported (11), G1 aggregates were populated by a predominance of CD3⁺ lymphocytes with fewer B cells (Fig. 1⇓A) whereas G2 and G3 foci were characterized by increased number of B cells. In addition, although most G2 aggregates demonstrated no obvious compartmentalization in B and T cell areas (Fig. 1⇓B), 32.4% of the G2 showed a clear segregation of B and T cells in discrete areas (Fig. 1⇓C). All G3 foci displayed features of mature secondary lymphoid organ follicles with a central duct surrounded by B cells and CD21⁺ FDC networks forming GC-LS bordered by T lymphocytes (Fig. 1⇓D).

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FIGURE 1.

Characterization of the cellular infiltrates and degree of lymphoid organization in mSG from patients with SS. Sequential paraffin-embedded sections of SS-mSG patients were double stained for T (CD3, brown) and B lymphocytes (CD20, purple) (A–D) and single stained for CD21 (purple) to detect CD21⁺ FDC networks (B–D insets). Periductal inflammatory foci were classified into four histologic groups according to the size and the degree of lymphoid organization based on the evidence of compartmentalization of T and B cells in discrete areas and presence of networks of CD21⁺ FDCs to identify GC-LSs. Representative examples of grade 1 (G1; <50 periductal lymphocytes) (A), nonsegregated grade 2 (NS-G2; >50 periductal lymphocytes with no evidence of T/B cell segregation) (B), segregated grade 2 (S-G2; >50 periductal lymphocytes with evidence of T/B cell segregation (C), and grade 3 (G3; >50 periductal lymphocytes, with presence of CD21⁺ FDC network as marker of GC-LSs) (D) are shown (original magnification, ×100). E, Prevalence of salivary gland biopsies with CD21⁺FDC networks characterizing G3 aggregates with GC-LS in SS-mSG, SS parotids and SS-MALT-Ls as compared with NSCS.

In SS-mSG the presence of G1 and G2 aggregates was found in 100% of patients whereas G3 foci were found in 8 of 22 patients (36.3%, Fig. 1⇑E). Conversely, in the NSCS cases evaluated, G1 aggregates were detected in 70% of the patients (SS vs NSCS, p = ns), G2 aggregates were detected in 10% (p < 0.001) whereas, as previously reported (11, 12), no G3 aggregates were detected in NSCS patients (p < 0.04, Fig. 1⇑E).

In comparison with SS-mSGs, SS-parotids and SS-MALTs displayed a higher prevalence of G3 aggregates (100% of the sample analyzed, p > 0.02 and p > 0.002 for SS-parotids and SS-MALT-Ls vs mSG, respectively, Fig. 1⇑E) with larger and more numerous CD21⁺GC-LSs.

AID expression is invariably associated with the presence of FDC networks in ectopic germinal centers in SS mSGs and parotids

Sequential section analysis in SS-mSGs identified the precise and exclusive colocalization of AID expressing cells within GC-LSs characterized by CD21⁺FDC networks even in the absence of a morphologically defined GC (Fig. 2⇓, A, D, and G). AID was mainly localized in the cytoplasm with a minority of B cells also expressing nuclear AID, in line with previous reports in secondary lymphoid organs (9, 10). The number of AID⁺ cells within GC-LSs in SS-mSGs was generally lower as compared with SS-parotids, where larger GCs were observed (Fig. 2⇓, B, E, and H). Importantly, within GC-LSs, B cells were consistently negative for bcl-2 (Fig. 2⇓, D and E) and positive for bcl-6 (not shown) confirming that AID⁺ cells belong to the GC B cell population as seen in lymph nodes (Fig. 2⇓F). The distribution of AID⁺ cells within GC-LSs in SS-mSGs and SS-parotids also closely resembled that seen in GCs of secondary lymphoid organs (Fig. 2⇓, C, F, and I).

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FIGURE 2.

AID is invariably expressed within GC-LSs in mSG and parotids in patients with SS. Sequential paraffin-embedded sections from SS patients were double stained for CD3 (brown) and CD20 (purple) (A–C), for bcl-2 (brown) and CD21 (purple) (D–F) and single stained for AID (purple) (G–I). Representative examples of SS mSG (A, D, G) and parotids (B, E, H) are shown together with a mature lymphoid follicle in a lymph node (C, F, I) for comparison. Presence of CD21⁺FDC network (D) is invariably associated with AID expression (G) that can be observed even in relatively small periductal aggregates and in the absence of clear T/B cell segregation (A) in SS-mSG. AID expression is more prominent (H) in larger GC-LS (E) in SS-parotids and similar to the expression pattern observed in secondary lymphoid organs (I) (original magnification, ×200 in A, D, G and ×100 in B, E, H and C, F, I). J, Prevalence of AID expression in mSG of patients with NSCS, SS-mSG, SS-parotids, and SS-MALT-L parotid biopsies from SS patients. K, Prevalence of AID expression in periductal inflammatory foci characterized by increasing degrees of lymphoid organization (G1 to G3) in mSGs of SS suggests that the presence of CD21⁺FDC networks sustains AID expression. ∗, scattered AID-positive B cells with the appearance of interfollicular large B cells observed in inflammatory foci in close association with numerous GC-LSs in a labial SG biopsy from a single SS patient.

We then quantified the prevalence of AID⁺ aggregates in the salivary glands of the different populations studied. Positive staining for AID was observed in 8 of 22 SS-mSG biopsies but not in NSCS (p < 0.0001, Fig. 2⇑J) and exclusively in samples where CD21⁺ FDC networks were present. In SS-mSGs, AID was not observed either in G1 or G2 foci, either segregated or not, suggesting that the presence of FDC networks is required for AID expression in B cells in ectopic lymphoid neogenesis. Conversely, all but one G3 foci (p < 0.0001 vs G1 and G2) stained positively for AID, suggesting that the presence of FDC networks is sufficient to support AID expression (Fig. 2⇑K).

AID expression also identifies IF large B cells surrounding GCs in mSGs and parotids of SS

In addition to GC B cells within FDC networks, a second pattern of AID expression was observed in mSGs and parotids of SS patients. As shown in Fig. 3⇓, numerous large B cells often with dendritic morphology strongly expressed AID, mainly in the cytoplasm and to a lesser extent in the nucleus, and were localized in close association with CD21⁺AID⁺GCs in SS-mSGs (Fig. 3⇓, A and D) and SS-parotids (Fig. 3⇓, B and E). Importantly, this population was exclusively observed in SGs where G3 foci with CD21+ FDC networks were present. Interestingly, in SS-mSGs characterized by numerous G3 foci, IF B cells were also found in adjacent smaller aggregates lacking GC-LSs, possibly related to the ability of these cells to migrate within the glands. This pattern of expression is highly similar to that observed in secondary lymphoid organs (Fig. 3⇓, C and F) where AID⁺ IF large B cells are mainly localized within the T cell area surrounding follicular structures, as previously reported (9, 10). Double staining of IF B cells with CD20 confirmed that they belonged to the B cell population (not shown). The number of GC-associated IF large B cells was significantly higher in SS-parotids as compared with SS-mSGs (Fig. 3⇓G), consistent with the larger size of GCs in SS parotids.

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FIGURE 3.

AID expression in SS-mSG and SS-parotids also identifies cells resembling the recently described population of IF large B cells. Sequential paraffin-embedded sections from SS patients were double stained for bcl-2 (brown) and CD21 (purple) (A–C) and single stained for AID (brown) (D–F). Representative examples of SS mSG (A–D) and parotids (B–E) are shown with a tertiary lymphoid follicle in a lymph node (C–F) for comparison. AID⁺ cells with large cytoplasm and sometimes dendritic morphology (arrowheads, D–F) were frequently and exclusively observed in SS salivary glands characterized by the presence of G3 foci with GC-LSs, often in close proximity with CD21⁺FDC networks (D, E). The morphology and pattern of distribution are highly reminiscent of the population of IF large B cells recently described in secondary lymphoid organs as shown in lymph node (F). The number of IF large B cells within G3 foci and surrounding FDC networks was significantly higher in SS parotids as compared with SS mSG (G) (original magnification, ×200 in A–D and ×100 in B–E and C–F).

Prevalence of AID⁺ FDC networks in SS-mSG and circulating autoantibodies in patients with SS

A clear tendency, although not statistically significant, toward increased prevalence of anti-Ro and anti-La Abs was observed comparing SS patients who displayed AID⁺ inflammatory infiltrates characterized by GCs within mSGs to SS patients without AID⁺GC in mSGs (75% vs 35.8% p = 0.07), RF (87.5% vs 57%), and ANA (87.5% vs 64.3%). These data strengthen the functional importance of AID expression in an ectopic setting.

AID mRNA transcripts levels are markedly over-expressed in SS-parotids with LESA and MALT-L and closely correlate with CD21L mRNA

To quantitatively analyze the expression levels of AID in SS-parotids and SS-MALT-Ls compared with secondary lymphoid organs and control parotids, we performed quantitative Taqman real-time PCR. AID mRNA transcripts were strongly expressed in all fragments from both SS-parotids and SS-MALT-Ls (Fig. 4⇓A). Interestingly, AID levels in all SS parotids were similar or higher in comparison to normal lymph nodes, suggesting that the SS parotid ectopic lymphoid infiltrate is characterized by the induction of a molecular program with the capacity to support a locally active humoral immune response. Expression of AID was not detected in any fragments obtained from control parotids (Fig. 4⇓, A and B). Importantly, in the parotids examined, AID transcripts closely correlated (Spearman r = 0.88, p > 0.0001, Fig. 4⇓C) with the expression levels of CD21L, an isoform of CD21 that is specifically expressed in FDCs (24). This evidence further substantiates that AID expression in SS parotids is dependent on CD21⁺FDCs.

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FIGURE 4.

AID mRNA in SS parotid glands with LESA and MALT-L closely correlated with CD21L and is expressed at similar levels to secondary lymphoid organs. A, Quantitative Taqman real-time PCR was used to measure parotid AID mRNA levels in reactive parotids and MALT-L samples from patients with SS and in control parotids. After normalization for the endogenous control (human β-actin) results were expressed as relative quantification to AID mRNA expression in normal lymph nodes which was set as one. B, Representative examples of the PCR products (10 μl for AID and 1 μl for β-actin) were run in 1.8% TBE-agarose gel to ensure the presence of a single specific amplification product and confirm specificity of TaqMan real-time PCR. Amplicons of the expected length (99 bp for AID, top gel and 171 bp for β-actin, bottom gel) were observed in all SS parotids with no AID mRNA expression detectable in controls. C, Linear regression showing strong positive correlation between expression levels of AID and CD21L mRNA in SS parotid samples with LESA and MALT-L. See text for statistical analysis.

AID protein is exclusively expressed within residual GCs and IF large B cells in SS- MALT-L

We then characterized the different B cell subpopulations responsible for aberrant AID expression in SS parotids with LESA and MALT-L. Two distinct areas of cellular organization with peculiar histological features could be identified within SS-MALT-Ls (25). Numerous follicular structures, characterized by T/B cell segregation (Fig. 5⇓, A and B) and FDC networks (Fig. 5⇓, C and D), were detected within 100% of the samples analyzed (see Fig. 1⇑E). These areas in SS-MALT-Ls were defined as the reactive component of the lymphoid proliferation and were mainly inhabited by GC B cells within FDC networks surrounded by CD20⁺/IgD⁺/bcl-2⁺ follicular B (FoB) cells (Fig. 5⇓, G and H), Conversely, MZ-like B cells expressed a different phenotype (CD20⁺/IgD⁻/bcl-2⁺) (17, 25) with peculiar histological features (monocytoid-like morphology with pale nuclei, evident nucleoli and frequent mitotic figures) and were distributed in dense sheets within the parenchyma and often infiltrated and subverted the ducts. The areas inhabited by this cell population were defined as the malignant component of the MALT-L, based on the morphological features, surface Ags and L chain restriction analysis.

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FIGURE 5.

AID expression in parotid MALT-L in patients with SS retains its association with residual GC-LSs but is not detectable in MZ-like B cells infiltrating the ducts. Representative example of a low-grade MALT-L arising in a parotid gland of a patients with SS. Sequential paraffin-embedded sections were double stained for CD3 (brown) and CD20 (purple) (A, B), for bcl-2 (brown) and CD21 (purple) (C, D), single stained for AID (brown) (E, F) and double stained for IgD (brown) and CD20 (purple) (G, H) to define, together with the morphological analysis, the residual reactive and malignant components of the MALT-L. AID was exclusively expressed in residual GCs characterized by T/B segregation (A, B) and CD21⁺ FDC networks (C, D) and surrounded by CD20⁺/IgD⁺/bcl-2⁺ follicular B cells (G, H). Conversely, AID was not detectable among the population of CD20⁺/IgD⁻/bcl-2⁺ MZ-like B cells infiltrating residual ductal structures (G, H) (original magnification, ×40 in A, C, E, G and ×200 in B, D, F, H.

Notably, in SS-MALT-L parotids AID was exclusively expressed within the reactive but not the malignant areas (Fig. 5⇑, E and F). Notably, as shown in Fig. 5⇑, AID was only detected in areas characterized by clear T/B cell segregation with CD21⁺ FDC networks forming GCs (Fig. 5⇑, C and D), surrounded by CD20⁺/IgD⁺/bcl-2⁺ FoB cells (Fig. 5⇑, G and H). AID⁺ B cells resembling IF large B cells were also detected in some but not all SS MALT-L, most often in association with the T cell area. GCs in SS-MALT-Ls were most often closely associated with ductal structures and CD21⁺ FDC networks sometimes surrounded a central duct. Conversely, the dense sheet of malignant MZ-like CD20⁺/IgD⁻/bcl-2⁺ B cells that completely infiltrated residual duct were consistently negative for AID expression in all the samples analyzed (Fig. 5⇑, E and F).

Prevalence of ectopic germinal centers in SS mSG biopsies is increased in patients who subsequently develop parotid MALT-L

Analysis of serial biopsies within parotid glands has been previously used to demonstrate clonal relationship between the prelymphomatous stage and subsequent malignant transformation of B cells (18). It has been suggested that this may relate to the chronic Ag-driven B cell proliferation and the consequent genetic instability associated with SHM (19). The relationship between the presence of a similar phenomenon in mSG of patients with SS and the development of MALT-L is unknown. Therefore, we retrospectively analyzed the prevalence of GC-LSs in mSG biopsies of 8 SS patients who later developed parotid MALT-L. We showed that the prevalence of GC-LSs in previous mSG biopsies was significantly increased in these patients compared with the general SS population studied (75% vs 33.3% p = 0.04). Although larger and longitudinal studies are needed, this supports the possibility that the presence of GC-LSs in mSG biopsies associated with chronic B cell stimulation may identify a subgroup of patients at an increased risk of developing lymphoma.