OP0300 Enrichment of T follicular-helper cells (TFH) and exclusion of t follicular-regulatory cells (TFR) from ectoPIC germinal centers in salivary glands of sjogren's syndrome patients
Authors: E Pontarini, W Murray-Brown, C Croia, E Astorri, D Lucchesi, N Lepse, N Sutcliffe, A Tappuni, C Pitzalis, M Bombardieri
Journal: Annals of the Rheumatic Diseases (2017)

<h3>Background</h3> B/T cell aggregates in the salivary glands (SG) of Sjögren9s syndrome (SS) can give rise to ectopic lymphoid structures (ELS) forming ectopic germinal centers (GCs), which has been linked to the development of MALT lymphoma (MALT-L). T follicular-helper cells (Tfh) and T follicular-regulatory cells (Tfr) are specialized CD4+ T-cells that positively and negatively regulate, respectively, the magnitude of the GCs response and the onset of autoimmunity. <h3>Objectives</h3> To characterize the infiltration of Tfh and Tfr in the SG infiltrates of patients with SS in the context of the presence/absence of ectopic GCs and in subjects with MALT-L. <h3>Methods</h3> SG biopsies with matching histology and RNA from 37 SS and 38 non-specific chronic sialadenitis (NSCS) patients were stratified as ELS-/ELS+ based on CD3/CD20/CD21/CD138 immunostaining (IHC). Histological samples and mRNA from 12 parotid MALT-L were also studied. Gene expression was measured by Taqman rt-PCR. Multicolor immunofluorescence/confocal microscopy for CD3, CD4, CD45RO, ICOS, PD1, BCL6 and FoxP3 was used to identify Tfh and Tfr. <h3>Results</h3> Tfh (CD4<sup>+</sup>CD45RO<sup>+</sup>PD1<sup>+</sup>ICOS<sup>+</sup>FoxP3<sup>-</sup>) cells and Tfr (CD4<sup>+</sup>CD45RO<sup>+</sup>PD1<sup>+</sup>ICOS<sup>+</sup>FoxP3<sup>+</sup>) cells were strongly enriched in ELS+ vs ELS- SS samples. The Tfh:Tfr ratio in ELS+ SG was approximately 2:1. Interestingly, while in tonsils Tfr were routinely detected within GCs, in ELS+ SG Tfr were predominantly excluded from the B cell follicles and accumulated in the T cell rich areas at the periphery of the lymphoid aggregates. Conversely, Tfh densely infiltrated the B cell rich areas and, within ectopic GCs, acquired BCL6. Furthermore, Tfh infiltration closely correlated with SG IL-21 mRNA expression, which in turn was strongly correlated with CD3, CD20 and CD138 IHC scores and with CXCL13, LTb, BAFF, AID and Pax5 gene expression. Finally, MALT-L samples displayed 10-fold higher IL-21 mRNA and twice as much PD1<sup>+</sup>ICOS<sup>+</sup>BCL6<sup>+</sup> Tfh-cells/field compared to ELS+ SS samples. <h3>Conclusions</h3> Within the SG of SS patients Tfh cells closely segregate with lesional IL-21 expression, localize within ELS and are strongly enriched during MALT-L development. Conversely, although Tfr cells are also recruited to ELS+ SG in SS patients, we consistently demonstrated follicular exclusion of this subset from ectopic GCs. This suggests that Tfr in SS SG fail to exert their physiological immunoregulatory properties in controlling the magnitude of the GCs response and B cell autoreactivity, as observed in tonsils. <h3>Acknowledgements</h3> This work was supported by project grants from the Medical Research Council (MR/N003063/1 to MB) and Arthritis Research UK (grant 20089 to MB). <h3>Disclosure of Interest</h3> E. Pontarini: None declared, W. Murray-Brown: None declared, C. Croia: None declared, E. Astorri: None declared, D. Lucchesi: None declared, N. Lepse: None declared, N. Sutcliffe: None declared, A. Tappuni: None declared, C. Pitzalis: None declared, M. Bombardieri Consultant for: Amgen/Medimmune, GSK and UCB