Contributions
Abstract: 182
Type: Oral
Abstract Category: Pathology and pathogenesis of MS - MS and infections
Multiple sclerosis (MS) is potentially orchestrated by T-cells directed to autoantigens and/or Epstein-Barr virus (EBV). We determined the phenotype, anatomical location and antigen-specificity of T-cells in paired peripheral blood (PB), cerebrospinal fluid (CSF), normal appearing white matter (NAWM) and white matter brain lesions (BL) of deceased MS patients (n=27).
NAWM and BL biopsies were divided into two parts for (1) snap-freezing for subsequent immunohistochemistry and (2) dispersed for ex vivo T-cell phenotyping by flow cytometry and generation of short-term T-cell lines (TCL). EBV-transformed B-cell lines (autoBLCL) were generated to be used as antigen presenting cells (APC) to enumerate antigen-specific T-cells in TCL. Additionally, one BLCL expressing all MS risk-associated HLA alleles (HLA-A*0301; DRB1*1303 and *1501) was transduced to express seven MS-associated antigens (MSAg: MAG, MBP1, MOG, PLP1, S100B, CNTN2 and NFASC) to detect autoreactive T-cells in HLA-matched TCL. The T-cell receptor (TCR) repertoire of NAWM- and BL-derived TCL was determined by flow cytometry using a panel of TCRVß-specific mAbs.
The T-cell infiltrate in NAWM and BL of MS patients consisted mainly of highly differentiated effector memory CD8 T-cells expressing granzyme B. Compared to paired PB and CSF, CD8 T-cells recovered from both NAWM and BL showed increased expression of markers indicative for ongoing antigenic recognition and activation (CD95L+, CD137+ and ICOS+) and exhaustion (PD1+ and TIM3+), but not of a senescent phenotype (CD57-). The TCRVß repertoire of TCL correlated between paired MS lesions, but not paired NAWM and BL . No MSAg-specific T-cell responses were detected in CSF-, NAWM- and BL-TCL. However, elevated frequencies of autoBLCL-specific CD8 T-cells were detected, mainly in BL-TCL generated from chronic active MS lesions, compared to CSF and NAWM. Notably, autoBLCL-specific CD8 T-cells recovered from two distinct lesions of the same patient expressed an identical TCRVß. Combined TCRVß/CD8 stainings on surplus tissue showed that these autoBLCL-specific CD8+ T-cells were localized in the parenchyma and had a polarized TCR expression suggesting recognition of their cognate antigen locally.
In conclusion, the data implicate that CD8 T-cells in chronic active MS lesions are directed to autoBLCL (potentially EBV proteins), but not to disease-associated autoantigens.
Disclosure: G.P. van Nierop: funded by the Dutch Research Foundation
M.M. van Luijn: funded by the Dutch Research Foundation
S.S. Michels: nothing to disclose
S. Getu: nothing to disclose
M.J. Melief: nothing to disclose
W.J.D. Ouwendijk: funded by Public Health Services grant AG032958 from the National Institutes of Health
R.Q. Hintzen: funded by the Dutch MS Research Foundation
G.M.G.M. Verjans: funded by Public Health Services grant AG032958 from the National Institutes of Health and Niedersachsen-Research Network on Neuroinfectiology of the Ministry of Science and Culture of Lower Saxony, Germany
Abstract: 182
Type: Oral
Abstract Category: Pathology and pathogenesis of MS - MS and infections
Multiple sclerosis (MS) is potentially orchestrated by T-cells directed to autoantigens and/or Epstein-Barr virus (EBV). We determined the phenotype, anatomical location and antigen-specificity of T-cells in paired peripheral blood (PB), cerebrospinal fluid (CSF), normal appearing white matter (NAWM) and white matter brain lesions (BL) of deceased MS patients (n=27).
NAWM and BL biopsies were divided into two parts for (1) snap-freezing for subsequent immunohistochemistry and (2) dispersed for ex vivo T-cell phenotyping by flow cytometry and generation of short-term T-cell lines (TCL). EBV-transformed B-cell lines (autoBLCL) were generated to be used as antigen presenting cells (APC) to enumerate antigen-specific T-cells in TCL. Additionally, one BLCL expressing all MS risk-associated HLA alleles (HLA-A*0301; DRB1*1303 and *1501) was transduced to express seven MS-associated antigens (MSAg: MAG, MBP1, MOG, PLP1, S100B, CNTN2 and NFASC) to detect autoreactive T-cells in HLA-matched TCL. The T-cell receptor (TCR) repertoire of NAWM- and BL-derived TCL was determined by flow cytometry using a panel of TCRVß-specific mAbs.
The T-cell infiltrate in NAWM and BL of MS patients consisted mainly of highly differentiated effector memory CD8 T-cells expressing granzyme B. Compared to paired PB and CSF, CD8 T-cells recovered from both NAWM and BL showed increased expression of markers indicative for ongoing antigenic recognition and activation (CD95L+, CD137+ and ICOS+) and exhaustion (PD1+ and TIM3+), but not of a senescent phenotype (CD57-). The TCRVß repertoire of TCL correlated between paired MS lesions, but not paired NAWM and BL . No MSAg-specific T-cell responses were detected in CSF-, NAWM- and BL-TCL. However, elevated frequencies of autoBLCL-specific CD8 T-cells were detected, mainly in BL-TCL generated from chronic active MS lesions, compared to CSF and NAWM. Notably, autoBLCL-specific CD8 T-cells recovered from two distinct lesions of the same patient expressed an identical TCRVß. Combined TCRVß/CD8 stainings on surplus tissue showed that these autoBLCL-specific CD8+ T-cells were localized in the parenchyma and had a polarized TCR expression suggesting recognition of their cognate antigen locally.
In conclusion, the data implicate that CD8 T-cells in chronic active MS lesions are directed to autoBLCL (potentially EBV proteins), but not to disease-associated autoantigens.
Disclosure: G.P. van Nierop: funded by the Dutch Research Foundation
M.M. van Luijn: funded by the Dutch Research Foundation
S.S. Michels: nothing to disclose
S. Getu: nothing to disclose
M.J. Melief: nothing to disclose
W.J.D. Ouwendijk: funded by Public Health Services grant AG032958 from the National Institutes of Health
R.Q. Hintzen: funded by the Dutch MS Research Foundation
G.M.G.M. Verjans: funded by Public Health Services grant AG032958 from the National Institutes of Health and Niedersachsen-Research Network on Neuroinfectiology of the Ministry of Science and Culture of Lower Saxony, Germany