Expression of Toll-Like Receptors on Peripheral Blood Cells After Allogeneic Stem Cell Transplantation: Results of a Prospective Study,
Authors: Skert Cristina , Fogli Manuela , Perucca Simone , Fiorentini Simona , Garrafa Emirena , Filì Carla , Peli Annalisa , Colombi Chiara , Bergonzi Cesare , Malagola Michele , Turra Alessandro , Caruso Arnaldo , Russo Domenico 
Published: October 2019

Abstract
Abstract 4071

Introduction.
Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, and in the pathogenesis of autoimmune and graft-versus-host (GVHD) diseases. Pattern recognition receptors such as Toll-like receptors (TLRs) play a key role in the cross-talk between innate and adaptive immune system. TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs), such as common protein, carbohydrate or DNA/RNA pattern motifs. TLRs are also receptors for endogenous ligands and damaged tissue, suggesting that both pathogen-derived molecules and products of damaged tissue can trigger signals which are responsible for the regulation of innate and adaptive immune responses. Extracellular ligands are recognized by surface TLRs (TLR1,TLR2,TLR4,TLR5, and TLR6). Intracellular TLRs (TLR3,TLR7,TLR8 and TLR9) bind mainly to foreign nucleic acids and sometimes detect self DNA/RNA.

Aim of the study.
Very little is known about expression and function of TLRs in vivo in patients who underwent allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to the onset of acute GVHD.

Methods.
The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and at the onset of GVHD. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls.

Results.
We analyzed 17 healthy donors and 34 patients. Median age was 46 years (range, 22–64) and 22 patients were male. Acute GVHD developed in 19 patients (12 with grade >=2). Clinical and transplant characteristics did not differ in patients with and without GVHD. Lymphocytes and monocytes of patients with acute GVHD showed higher levels of TLR5 (3,5±2,3 vs1,9±1,6 p=0,03; 25,8±25,9 vs 9,0±5,0 p=0,02) and a decreased expression of TLR1 (2,5±2,8 vs 4,3±2,8 p=0,02; 21,4±21,9 vs 54,9±37,4 p=0,005) and TLR9 (63,8±30,4 vs 111,1±62,9 p=0,03; 85,3±73,9 vs 164,2±90,6 p=0,01). IFN-gamma relative induction post-stimulation of TLR2,3,4 and 9 was significantly decreased in patients with acute GVHD (p< 0,04).

Conclusions.
TLRs show a different profile of expression in patients with acute GVHD in comparison with patients without it. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. In particular, a decreased expression of TLR-9 (receptor of hypomethylated DNA) on lymphocytes and monocytes can promote TLR-7 activation, inducing type I interferons and other pro-inflammatory cytokines. TLR-1 and −5, which are ligands for bacterial cell wall, could also be involved in the pathogenesis of GVHD. Moreover, acute GVHD negatively correlates with IFN-gamma production upon TLR2,3,4 and 9 activation. The assessment of a larger number of patients could be useful to understand the complex interplay among pathogens, self or non-self DNA and RNA, and the immune system.

Disclosures:
No relevant conflicts of interest to declare.