Enhanced intrathecal autoBLCL-specific T-cell reactivity, selectively directed towards lytic EBV proteins in two CSF-TCL, suggested a localized T-cell response to EBV in patients with MS. Our data warrant further characterization of the magnitude and breadth of intrathecal EBV-specific T-cell responses in larger patient cohorts.

Toll-like receptors (TLRs) orchestrate immune responses to a wide variety of danger- and pathogen-associated molecular patterns. Compared to the central nervous system (CNS), expression profile and function of TLRs in the human peripheral nervous system (PNS) are ill-defined. We analyzed TLR expression of satellite glial cells (SGCs) and microglia, glial cells predominantly involved in local immune responses in ganglia of the human PNS and normal-appearing white matter (NAWM) of the CNS, respectively. Ex vivo flow cytometry analysis of cell suspensions obtained from human cadaveric trigeminal ganglia (TG) and NAWM showed that both SGCs and microglia expressed TLR1-5, TLR7, and TLR9, although expression levels varied between these cell types. Immunohistochemistry confirmed expression of TLR1-TLR4 and TLR9 by SGCs in situ. Stimulation of TG- and NAWM-derived cell suspensions with ligands of TLR1-TLR6, but not TLR7 and TLR9, induced interleukin 6 (IL-6) secretion. We identified CD45 CD14 SGCs and microglia, but not CD45 leukocytes and CD45 cells as the main source of IL-6 and TNF-α upon stimulation with TLR3 and TLR5 ligands. In conclusion, human TG-resident SGCs express a broad panel of functional TLRs, suggesting their role in initiating and orchestrating inflammation to pathogens in human sensory ganglia.

MS pathology is potentially orchestrated by autoreactive T cells, but the antigens recognized remain unknown. A novel APC/T-cell platform was developed to determine intrathecal CD4+ and CD8 + T-cell responses to candidate MS-associated autoantigens (cMSAg) in clinically isolated syndrome (CIS, n = 7) and MS (n = 6) patients. Human cMSAg encoding open reading frames (n = 8) were cloned into an Epstein-Barr virus (EBV)-based vector to express cMSAg at high levels in EBV-transformed B-cells (BLCLs). Human cMSAg cloned were myelin-associated and -oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, ATP-dependent potassium channel ATP-dependent inwards rectifying potassium channel 4.1, S100 calcium-binding protein B, contactin-2, and neurofascin. Transduced BLCLs were used as autologous APC in functional T-cell assays to determine cMSAg-specific T-cell frequencies in cerebrospinal fluid derived T-cell lines (CSF-TCLs) by intracellular IFN-γ flow cytometry. Whereas all CSF-TCL responded strongly to mitogenic stimulation, no substantial T-cell reactivity to cMSAg was observed. Contrastingly, measles virus fusion protein-specific CD4 + and CD8 + T-cell clones, used as control of the APC/T-cell platform, efficiently recognized transduced BLCL expressing their cognate antigen. The inability to detect substantial T-cell reactivity to eight human endogenously synthesized cMSAg in autologous APC do not support their role as prominent intrathecal T-cell target antigens in CIS and MS patients early after onset of disease. (MOG) and proteolipid protein (PLP), glia-specific proteins like inwards rectifying potassium channel (KIR4.1, ATP-dependent inwards rectifying potassium channel 4.1) and S100 calcium-binding protein (S100B) and neuron-specific proteins like contactin-2 (CNTN2) and neurofascin 155kD isoform (NFASC) [1,2]. The majority of studies detailing cMSAg-specific T cells in MS patients assayed peripheral blood with conflicting outcome [1,2]. Some observed increased auto-reactive T-cell frequencies in MS, while others described equivalent numbers in patients and controls questioning their role in MS [3]. The poor correlation between blood and intrathecal T-cell phenotypes, and TCR repertoires [4] dispute extrapolation of systemic T-cell data to MS immunopathology.Most studies assayed intrathecal cMSAg-specific T-cell responses using autologous PBMC, pulsed with synthetic peptides or recombinant (animal) cMSAg, as APC [2]. This strategy has several limitations [2,3]. First, cMSAg of different species and bacterial-produced antigens have a different amino acid composition and conformation compared with native human cMSAg, respectively, which potentially affect processing and presentation of the cognate epitope [5]. Second, peptide binding is highly HLA allele-specific. Distinct HLA alleles are dominant MS risk or even protective factors [1]. Thus, differences in cMSAg T-cell responses in MS patients and controls may be due to intercohort HLA genotype disparity [1] ensuing mandatory HLA matching of pa...