Characterization of ectopic germinal centers in Sjögren's syndrome (SS) and non-SS non-sicca patients.

Recently, we have published a study in which shown that focal lymphocytic sialadenitis (FLS), an important criterion for Sjögren syndrome (SS) diagnosis only when a focus score (FS) ≥1 is detected, can also be observed when assessing intraoral biopsies containing minor salivary gland (mSG) parenchyma from healthy asymptomatic individuals (Silva et al., 2022). In this study, the frequency of germinal centers (GCs) comparing primary SS (pSS) and non-SS non-sicca patients was similar. By immunohistochemical analysis (IHC), all GCs were Bcl-6 and CD23 positive, with variable expression of CD10. However, some foci (without GCs) were also CD23 positive (Silva et al., 2022). Our results, supported by previous study (Nakshbandi et al., 2020), indicate that Bcl-6 is a reliable marker for GC detection in these cases. In SS patients, the presence of GCs when assessing FLS seems to be associated with more severe disease, including higher levels rheumatoid factor, anti-SSA/B antibodies, proinflammatory mediators, higher FS, reduced saliva production (Carubbi et al., 2019; Risselada et al., 2013), as well as predictor of MALT lymphoma development (Sène et al., 2018; Theander et al., 2011), with some studies not supporting this latter (Haacke et al., 2017, 2019). In our Oral Histopathology Laboratory (FORP/USP), evaluating the D2-40 and CD57 immunoexpression in palatine tonsil specimens (diagnosed as follicular hyperplasia), we have frequently observed a strong expression of D2-40 within GCs, evidencing follicular dendritic cell (FDC) networks (Marsee et al., 2009; Yu et al., 2007), as well as a homogeneous distribution of strongly stained CD57+ cells inside the GCs, characterizing T-follicular helper (TFH) cells (Sattarzadeh et al., 2015) (Figure 1a). Thus, considering the importance of GC detection, the lack of specificity of routinely used GC markers, as well as the similar frequency of GCs assessing FLS in SS and non-SS non-sicca patients, in this study, we have focused comparative analysis of GCs, using D2-40 and CD57, in correlation with Bcl-6 and Ki-67 immunomarkers, aiming to highlight differences with diagnostic implications in these two populations. This study was approved by the Ethics Committee in Human Research from FMRP/USP (statement number: 68748117.0.0000.5440). The clinicopathological features of the patients included in this study are shown in Table 1. Ten of 13 cases (pSS) and five of 11 cases (non-SS non-sicca) were published previously by us (Silva et al., 2022). The SS diagnosis followed strict criteria of American College of Rheumatology/European League Against Rheumatism (Shiboski et al., 2017). The six cases of non-SS non-sicca (not previously published) were selected after careful microscopical analysis of 500 oral mucosal biopsies (350 females and 150 males; mean age, 50 years), clinically and microscopically corresponding to inflammatory/reactive lesions, which also presented, at the periphery of the specimen and away from the main lesion, typical FLS features (Silva et al., 2022). Similar with these cases, the presence of GCs have been reported in chronic sialadenitis—not otherwise specified (Geyer et al., 2010), as well as inflammatory fibrous hyperplasia (Rangel et al., 2008). It is possible that immune mechanisms associated with chronic stimuli (e.g., sialoliths and trauma) are promoting the formation of GCs through a greater degree of lymphocyte organization. The antibodies used were Bcl-6 (Clone LN22, 1:500 dilution, Leica Biosystems), D2-40 (Clone M3619, 1:500 dilution, DakoCytomation), CD57 (Clone TB01, 1:600 dilution, DakoCytomation), and Ki-67 (Clone ab16667, 1:500 dilution, Abcam). GC histomorphology, Bcl-6 positivity, as well as inclusion and exclusion criteria, were previously defined (Silva et al., 2022). For D2-40, immunoreactivity was graded semiquantitatively according to the percentage of positivity of FDCs per focus: 0, <5%; 1+, 5%–25%; 2+ > 25%–50%; and 3+, >50%. The intensity of staining was graded as weak (1+), moderate (2+), or strong (3+). For CD57, the scoring system was: 0, no positive cells; 1+, 1–5 positive cells; 2+, >5–10 positive cells per focus; and 3+, >10 positive cells. For Ki-67, the scoring system was: 1+, <10% positive cells; 2+, 10%–35% positive cells; and 3+, >35% positive cells clustered ones within a focus. Analysis of variance was applied to assess differences between groups. For categorical data, the G-test and chi-square test was applied. A probability (p) value <0.05 was considered statistically significant (IBM-SPSS 20.0 statistics). Our results showed the area and largest diameter of GCs, as well as FLS with FS≥1, were significantly higher in pSS than non-SS non-sicca group. On IHC, 45/121 and 28/66 foci in pSS and non-SS non-sicca group, respectively, showed Bcl-6 positivity characterizing (also by histomorphology) GCs. Almost all Bcl-6+ foci were also D2-40 and CD57 positive (Table 1 and Figure 1b–e). The known heterogeneous distribution of FDCs in the dark and light zones of GCs, associated with differential regulation of FDC markers in these compartments (Allen & Cyster, 2008), may explain the presence of scarce Bcl-6+/D2-40- foci in our study. However, D2-40 and CD57 expression was not restricted to GCs. In fact, D2-40+/Bcl6- foci (39% vs. 23%, p = 0.062) and CD57+/Bcl-6- foci (11% vs. 4%, p = 0.24) were detected more frequently in pSS than non-SS non-sicca group. Regarding percentage of positivity and intensity of D2-40, it was greater in pSS than non-SS non-sicca group (both, p = 0.0001). Considering the frequency of CD57+ cells, it was also greater in pSS than non-SS non-sicca group (p < 0.001; Figure 1b–e). In this study, only a cluster of ≥5 Bcl-6+ cells, showing a nuclear staining pattern with adequate morphology and organization, was considered positive and characterizing a GC (Haacke et al., 2017; Nakshbandi et al., 2020; Silva et al., 2022). All these GCs expressed Ki-67 and frequently exhibited a polarized positivity, typical of reactive/benign nature of the lymphoid proliferation (Almeida et al., 2020). D2-40 antibody recognizes a mucin-type transmembrane glycoprotein, which is expressed in GC FDCs networks, including FDC tumors, as well as in lymphatic endothelium, kidney podocytes, mesothelium, and pulmonary alveolar cells. Moreover, regarding immune cells, macrophages and Th17 cells can express D2-40 (Marsee et al., 2009; Yu et al., 2007). In this study, D2-40 expression also was observed in myoepithelial cells, lymphatic vessels, and basal cell layer of surface epithelium. The D2-40+/Bcl-6- foci probably represent primary lymphoid follicles or isolated FDCs. CD57 antibody recognizes a 110 kDa protein on a subset of NK and T cells, and neural tissues. Inside GCs, CD57+ TFH cells provide key signals to B cells for GC development, modulating immunoglobulin synthesis. Moreover, TFH cells can also express Bcl-6 (Crotty, 2021). In this study, CD57+ cells were observed inside (TFH cells) and outside (NK or T cells) GCs, besides highlighting isolated neural bundles. Inside GCs, Bcl-6+ cells morphologically resembling lymphocytes favored TFH cells (Crotty, 2021). In summary, our results show histomorphological (area and diameter) and immunophenotypical (D2-40 and CD57 expression profile) differences, suggesting distinctive cellular and/or molecular interactions in the pathogenesis of GCs when comparatively evaluating FLS in pSS and non-SS non-sicca patients. Evânio Vilela Silva: Conceptualization; methodology; writing – original draft; investigation; writing – review and editing; data curation; formal analysis. Bruno Augusto Benevenuto de Andrade: Investigation; conceptualization; methodology; formal analysis; writing – review and editing; writing – original draft. Heitor Albergoni Silveira: Data curation; writing – original draft; formal analysis; methodology; investigation. Eduardo Melani Rocha: Formal analysis; writing – review and editing; writing – original draft; investigation; methodology; conceptualization. Fernando Chahud: Investigation; formal analysis; methodology; conceptualization. Mariângela Ottoboni Brunaldi: Methodology; investigation; writing – review and editing; conceptualization. Andreia Bufalino: Funding acquisition; methodology; formal analysis; writing – review and editing; data curation; supervision. Jorge Esquiche León: Methodology; funding acquisition; conceptualization; visualization; formal analysis; writing – review and editing; writing – original draft; data curation; project administration. The authors are grateful to the entire staff of FMRP/USP, FORP/USP, FOAr/UNESP (Araraquara) and UFRJ, as well as the financial support provided by FAPESP, CAPES and CNPq (Brazil). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. Jorge Esquiche León have received research Grants (2015/22586-2, 2016/11419-0, 2016/14636-2, 2022/07479-9, and 2022/12760-9) from State of São Paulo Research Foundation (FAPESP) and research grant (304241/2021-0) from National Council for Scientific and Technological Development (CNPq). The authors have no conflict of interest in this article. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.