Intrathecal CD4⁺ and CD8⁺ T‐cell responses to endogenously synthesized candidate disease‐associated human autoantigens in multiple sclerosis patients

Abstract

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...

Phenotypic and functional characterization of T cells in white matter lesions of multiple sclerosis patients

“…An allogeneic BLCL (BLCL-GR; HLA-A*01;03, -B*07;27, -C*02;07, -DRB1*13;15, -DQB1*06;06), carrying the major MS-associated HLA class I and II risk alleles (HLA-A*03, -DRB1*15 and -DRB1*13) [ 49 ], was transduced to express the seven following human cMSAg constitutively: (1) oligodendrocyte-specific proteins [myelin-associated glycoprotein (MAG), myelin basic protein isoform 1 (MBP1) and myelin oligodendrocyte glycoprotein (MOG)]; (2) neuron-specific proteins [contactin-2 (CNTN2) and 155-kDa isoform of neurofascin (NFASC)] and (3) glia-specific proteins [inwards rectifying potassium channel (KIR4.1) and S100 calcium-binding protein B (S100B)] [ 42 ]. Stable cMSAg-expressing BLCL-GR lines, validated by flow cytometry using cMSAg-specific mAb, were used as allogeneic APC to detect cMSAg-specific CD4 + and CD8 + T cells simultaneously in TCL of HLA-matched MS patients (Online Resource 1) [ 42 ]. To validate this BLCL platform, we also transduced BLCL-GR to express measles virus fusion protein (MVF) and assayed their APC function by incubation with MVF-specific CD4 + (4-F99) and CD8 + (2-F40) T-cell clones (TCC) as described previously [ 43 ].…”

Memory B Cells are Major Targets for Effective Immunotherapy in Relapsing Multiple Sclerosis

“…Although the etiology of MS is thought to be autoimmune, no consistent T or B cell autoantigen has been described, even after analysis of sequences of infiltrating B cells (Blauth et al, 2015, van Nierop et al, 2016). Whilst T and B cell autoimmunity is clearly present in people with MS (Agahozo et al, 2016, Blauth et al, 2015, van Noort et al, 2010), this may be secondary to damage rather than being the primary driver of the disease.…”

Programmed death 1 is highly expressed on CD8+CD57+ T cells in patients with stable multiple sclerosis and inhibits their cytotoxic response to Epstein–Barr virus

“…In addition, in inflamed cerebral meninges of post‐mortem MS brain tissue, infiltrates of effector and cytotoxic CD8 + T cells and innate immune responses characterized by interferon‐ α production have been detected in proximity to EBV‐infected plasma cells . Therefore, the observed increase of CD8 + T cells specific for EBV lytic antigens in the peripheral blood and cerebrospinal fluid (CSF) of patients with MS during relapses suggests a close association between replication of EBV in the CNS and inflammatory disease activity . Also, the evidence for active replication of EBV in patients with MS indicates that the virus has the capacity to escape immune control by cytotoxic lymphocytes .…”

“…8,10 Therefore, the observed increase of CD8 + T cells specific for EBV lytic antigens in the peripheral blood and cerebrospinal fluid (CSF) of patients with MS during relapses suggests a close association between replication of EBV in the CNS and inflammatory disease activity. [11][12][13] Also, the evidence for active replication of EBV in patients with MS indicates that the virus has the capacity to escape immune control by cytotoxic lymphocytes. 11 However, the results from other studies examining the involvement of EBV infection in the MS pathological process diverged.…”