University of Birmingham Birmingham, United Kingdom
SABA NAYAR1, Jason D. turner1, Saba Asam1, Serena Colfrancesco2, Onorina Berardicurti3, Valentina Iannizzotto1, Charlotte G Smith1, Joe Flint1, Ana Teodósio1, David H Gardner1, Professor Simon Bowman4, Wan-Fai Ng5, Adam Croft1, Andrew Filer1, Benjamin A. Fisher1, Christopher Buckley6 and Francesca Barone7, 1University of Birmingham, Birmingham, United Kingdom, 2University of Rome, Rome, Italy, 3Rheumatology Unit, Pescara Hospital, Pescara, Italy, 4University Hospitals Birmingham, Birmingham, United Kingdom, 5Newcastle University, Newcastle, United Kingdom, 6University of Oxford, Oxford, United Kingdom, 7University of Birmingham, Needham, MA
Background/Purpose: Tertiary lymphoid structures (TLS) are accumulations of lymphoid cells that share similar cellular compartments, organization and function when compared to secondary lymphoid organs (SLOs) such as tonsil. TLS germinal centres (GC) provide a local hub for maturation and proliferation of auto-reactive B-cells and expansion of malignant B-cell clones. TLS that form within salivary glands (SGs) of Sjögren's syndrome (SjS) patients are clearly associated with poor disease outcome, autoantibody production and lymphoma development. The phenotypical and functional features supporting TLS pathogenic properties are largely unclear.
Methods: Frozen SGs were obtained from SjS patients and selected for presence of germinal centre (GC)+ TLS. SG and tonsil were stained, microdissected. Isolated RNA from SG TLS and tonsil GC were used for RNAseq using ClonTech SMARTseq v4 kit. Nanostring GeoMx Digital Spatial Profiler (DSP) and Multiplex IHC (Lunaphore Comet) were used for proteomic validation.
Results: Transcriptomic analysis revealed 3836 significantly differentially expressed genes in SG TLS as compared to tonsil GC. Predominant expression of the lymphoid chemokines/cytokines (CCL19, CCL21, CXCL13 and BAFF) and costimulatory molecules (CD40, ICOS, ICOSL, PD1, PDL1, PDL2) was identified in both TLS and tonsil GCs. Interestingly, CCL19, BAFF, PDL2 expression was higher in SjS TLS than tonsil GCs. Conversely, CCL21 expression was greater in tonsil GCs. Gene expression for CXCL12 was enriched in TLS GC as compared to tonsil, suggesting a potential association of this molecule with the highly inflammatory ectopic setting of TLS. Importantly, TLS GC showed significantly increased expression of STING1, IFNG and TNF along with other cytokines (IL33, IL23). Inflammatory genes encoding for the chemokines CXCL10, CXCL9 and their receptor CXCR3, were detected in TLS GC, but absent in tonsil GCs, suggesting that TLS GCs are defined by a different pro-inflammatory profile signature compared to tonsil GC. Transcriptomic and proteomic analysis of TLS GC unveiled an altered cell-proliferation/apoptosis profile with downregulation of BCL6 and AICDA, the enzymes responsible for B cell affinity maturation. Moreover, spatial proteomic analysis on Geomx and multiplex IHC revealed differences in abundance of immune cell populations. The TLS GC showed presence of GranzymeB+ CD8 T cells along with T follicular and peripheral helper cells. Furthermore, high expression of CD27, CD127, GITR and TNFSF9 was enriched in TLS GC.
Conclusion: Our study provides the first comprehensive transcriptomic and proteomic overview of the mechanisms regulating the local pathogenic microenvironment in TLS and demonstrate that these differences diverge from SLOs. We demonstrated that, although characterised by similar anatomical organization, TLS pathogenic cytokines, and cellular signature is associated with low levels of bcl6, and AID, aberrant apoptosis and costimulation, compared to GC in tonsil. Our findings unveil impaired regulation of the B cell cycle, responsible for the survival of autoreactive, poorly selected B cell clones as a key feature of SS.
Disclosures: S. NAYAR, None; J. turner, None; S. Asam, None; S. Colfrancesco, None; O. Berardicurti, None; V. Iannizzotto, None; C. Smith, None; J. Flint, None; A. Teodósio, None; D. Gardner, None; P. Bowman, Novartis, AstraZeneca, AbbVie/Abbott, Galapagos; W. Ng, None; A. Croft, None; A. Filer, None; B. Fisher, Servier, Galapagos, Janssen, Novartis, Bristol-Myers Squibb(BMS); C. Buckley, None; F. Barone, Candel Therapeutics.
