Identification of circulating nonclassic human leukocyte antigen G (HLA-G)-dimer molecules in cerebrospinal fluids from MS stable patients.

Objective: HLA-G is a non-classical HLA class I antigen expressed as membrane bound and soluble (sHLA-G) isoformswith a restricted tissue distribution and anti-inflammatory functions due to binding affinity for immune inhibitory receptors (ILT-2, ILT-4, KIR2DL4). sHLA-G levels in cerebrospinal fluid (CSF) were demonstrated to be more elevated in multiple sclerosis (MS) patients than in controls, and in MS patients without magnetic resonance imaging (MRI) evidence of disease activity. Recently, it was discovered that HLA-G can exist as a dimer with a substantially increase binding to inhibitory receptors. Methods: The presence of HLA-G dimers and monomers was evaluated in CSF samples from 52 MS patients subdivided according to MRI in active (N=18) and stable (N=34) disease, 50 patients with other inflammatory (OIND) and 50 patients with non-inflammatory (OIND) neurological diseases. The samples were analyzed by ELISA test and Western Blot assay in native and denaturating conditions by means of two HLA-G specific monoclonal antibodies (ELISA and immunoprecipitation: MEM-G9, G233; Exbio, Praha). Results: The ELISA and Western blot results reported increased sHLA-G CSF levels in MS patients in comparison with OIND and NIND patients (pb0.0001) and higher CSF concentrations in stable than in active MS patients (pb0.001). The native Western blot analysis revealed both the HLA-G monomeric (39 kDa) and dimeric (78 kDa) conformations in CSF samples from MS patients with a stable disease, in NIND and OIND patients. NIND and MS stable patients presented a prevalence of the dimeric isoform. The Western blot under reducing conditions showed also a faint band at 53 kDa. On the contrary, CSF samples from MS patients with an active disease presented only the HLA-G monomeric conformation. The different HLA-G structures are recognized by both the anti-HLA-G antibodies G233 and MEM-G9 in ELISA and Western blot assays. Conclusions: These data demonstrate that HLA-G dimeric molecules are mainly present in CSF samples from stable MS and NIND patients, while CSF from active MS patients present only monomeric HLA-G conformation. The presence of dimeric HLA-G structure in CSF samples fromstable MS disease status support the implication of this structure in immune and inflammation control in central nervous system. In fact, this conformation is characterized by an increased binding avidity than HLA-G monomers, which translates into augmented signaling through ILT-2 receptor.