- Email: [email protected]
EBNA1 antigen-specific CD8 + T cells in cerebrospinal fluid of patients with multiple sclerosis
EBNA1 antigen-specific CD8 + T cells in cerebrospinal fluid of patients with multiple sclerosis Hebun Erdur, Veronika Scholz, Mathias Streitz, Markus Hammer, Christian Meisel, Constanze Sch¨onemann, Klaus-Peter Wandinger, Berit Rosche PII: DOI: Reference:
S0165-5728(16)30044-3 doi: 10.1016/j.jneuroim.2016.03.010 JNI 476325
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
14 December 2015 12 March 2016 16 March 2016
Please cite this article as: Erdur, Hebun, Scholz, Veronika, Streitz, Mathias, Hammer, Markus, Meisel, Christian, Sch¨onemann, Constanze, Wandinger, Klaus-Peter, Rosche, Berit, EBNA1 antigen-specific CD8 + T cells in cerebrospinal fluid of patients with multiple sclerosis, Journal of Neuroimmunology (2016), doi: 10.1016/j.jneuroim.2016.03.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT EBNA1 antigen-specific CD8+ T cells in cerebrospinal fluid of patients with multiple sclerosis
SC R
IP
Constanze Schönemann4, Klaus-Peter Wandinger5, Berit Rosche1
T
Hebun Erdur1, Veronika Scholz1, Mathias Streitz2, Markus Hammer3, Christian Meisel2,
Department of Neurology, Charité – Universitätsmedizin Berlin
2
Institute of Medical Immunology, Charité – Universitätsmedizin Berlin
3
Department of Nephrology and Internal Intensive Care, Charité – Universitätsmedizin Berlin
4
Institute of Transfusion Medicine, Charité – Universitätsmedizin Berlin
5
Institute of Clinical Chemistry and Department of Neurology, University Medical-Centre
MA
NU
1
Hebun Erdur, MD
CE P
Correspondence:
TE
D
Schleswig-Holstein, Campus Lübeck, Germany
Department of Neurology, Campus Benjamin Franklin
AC
Charité – Universitätsmedizin Berlin Hindenburgdamm 30, 12200 Berlin, Germany Phone: +49-30-84454285, FAX +49-30-84454264 email: [email protected]
1
ACCEPTED MANUSCRIPT Disclosures of all authors' financial relationships B. Rosche has participated in meetings sponsored by Bayer Healthcare, Merck Serono,
T
Biogen Idec, Novartis, Ovamed and Teva Pharmaceuticals and has also received lecture and
SC R
IP
research fees from these companies. The other authors report no disclosures.
Abbreviations: MS = multiple sclerosis, CSF = cerebrospinal fluid, CMV =
AC
CE P
TE
D
MA
NU
Cytomegalovirus, EBV = Epstein Barr virus, EBNA1 = EBV-encoded nuclear antigen-1
2
ACCEPTED MANUSCRIPT Abstract Epidemiological data suggests that Epstein-Barr virus may be involved in the pathogenesis of
T
Multiple Sclerosis (MS). We aimed to determine the frequency of CD8+ T cells specific for
IP
one EBNA1-derived epitope (HPVGEADYFEY) in cerebrospinal fluid (CSF) and blood of
SC R
patients with MS and other inflammatory neurological diseases (OIND). The frequency of specific CD8+ T cells was assessed by HLA-class-I-binding pentamers restricted to HLAB35. The frequency of HPVGEADYFEY-specific CD8+ T cells did neither differ
MA
compared to blood regardless of diagnosis.
NU
significantly in blood nor CSF in MS compared to OIND, but was consistently higher in CSF
AC
CE P
TE
D
Key words: multiple sclerosis, Epstein-Barr virus, cerebrospinal fluid
3
ACCEPTED MANUSCRIPT Introduction Epidemiological and immunological studies suggest a role of Epstein Barr virus (EBV) in the
T
pathogenesis of Multiple Sclerosis (MS). Individuals with a history of infectious
IP
mononucleosis have an increased risk of developing MS (Thacker et al., 2006), while risk of
SC R
MS in EBV-negative individuals is very low (Levin et al., 2010). Immune control of EBVinfection may be dysregulated in MS patients and thus contribute to autoimmunity (Wandinger et al., 2000). However, the exact mechanisms involved in the interplay of EBV-
NU
infection and MS pathogenesis are not well understood (Owens & Bennett, 2012). The
MA
presence of an altered frequency of EBNA-1-specific CD8+ T cells in blood and/or CSF of patients with MS compared with other inflammatory neurological diseases (OIND) could indicate an altered immune response to EBV in MS patients. Studies investigating cellular
TE
D
immune responses to EBNA-1 in the CSF of MS patients are scarce, probably due to restriction of the immunogenic EBNA-1-peptide HPVGEADYFEY to HLA-B35. Thus, these
CE P
analyses can only be conducted in CSF of patients with the HLA haplotype HLA-B35, which is expressed in only approximately 10% of the general population (Müller et al., 2003).
AC
Furthermore, the detection of virus-specific CD8+ T cells in CSF requires rapid analysis of CSF probes after lumbar puncture and is also impaired by the low number of cells in CSF. We aimed (i) to determine the frequency of CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) in blood and CSF of patients with MS. For the purpose of comparison, (ii) we assessed frequencies of CD8+ T cells specific for one CMV-derived epitope (NLVPMVATV of pp65) in blood and CSF, and (iii) corresponding frequencies of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells in patients with OIND.
Methods Patients:
4
ACCEPTED MANUSCRIPT Patients were recruited from the Department of Neurology, Charité – Universitätsmedizin Berlin. Diagnosis of CIS, MS (according to the 2005 McDonald criteria) (Polman et al.,
T
2005), and OIND was made by the attending neurologist. Routine CSF analysis was
IP
conducted for diagnostic purposes and included cell count, IgG, IgM, IgA serum/CSF-
SC R
quotient and oligoclonal bands. Analysis of CSF in MS patients was performed before initiation of therapy with corticosteroids. EBV and CMV IgG and IgM antibody status in serum was determined by ELISA. As diagnostic work-up was not to be delayed by HLA-class
NU
testing, staining of CSF and HLA-testing were done simultaneously. Because of low cell
MA
numbers in CSF, simultaneous staining for specific CD8+ T cells for EBV and CMV was not conducted in all patients. Furthermore, due to logistic reasons, pentamer staining was not performed in all samples. The procedure of staining of CSF and whole blood is described in
TE
D
the supplemental material. Only patients with HLA-A02 or HLA-B35 genotype, positive CMV and/or positive EBV serology were eligible for analysis. The study was approved by the
CE P
local ethics committee and subjects or representatives provided informed consent.
AC
Statistical analysis:
Results of FACS staining were obtained using CellQuest software. These were statistically analysed by means of an independent two-sided t test. Confidence intervals for proportions were calculated using the Wilson score method without continuity correction (Newcombe, 1998). P values < 0.05 were considered significant. SPSS 19 was used for all statistics.
Results: Baseline characteristics A total of 96 patients were screened. An inflammatory neurological disease was diagnosed in 81 patients (of these, CIS was diagnosed in 18 patients, RR-MS in 19, and SP-MS and PP-MS each in one case; 24 patients had a viral inflammatory disease, nine a bacterial and nine 5
ACCEPTED MANUSCRIPT patients other inflammatory diseases). In 15 patients, a non-inflammatory neurological disease was diagnosed.
T
HPVGEADYFEY-specific CD8+ T cells were detected in 11 patients (five with RR-MS, five
IP
with viral or bacterial CNS-inflammation, and one patient with transient diplopia and signs of
SC R
inflammation in CSF). NLVPMVATV-specific CD8+ T cells were present in nine patients (two with CIS, two RR-MS, four with viral or bacterial CNS-inflammation and one patient with primary angiitis of the CNS). In patients with viral inflammatory CNS-disease, herpes
NU
simplex virus (type 1 and 2) and varicella zoster virus were the main causes.
MA
Table 1 lists all patients with positive staining for HPVGEADYFEY- and NLVPMVATVspecific CD8+ T cells and the respective proportions of all CD8+ T cells. Causes of inflammatory disease are also listed in table 1. Figure 1 shows HPVGEADYFEY- and
TE
D
NLVPMVATV-specific CD8+ T cells in blood and CSF of a patient with RR-MS and a
CE P
patient with viral meningitis, respectively.
Frequency of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells in blood and CSF
AC
The frequency of HPVGEADYFEY-specific CD8+ T cells in blood as percent of all CD8+ T cells was not significantly higher in MS patients compared to OIND (MS: mean 0.49% ±0.38% vs. OIND: mean 0.22% ±0.13%; p=0.18). Also, the proportion of NLVPMVATVspecific CD8+ T cells in blood did not differ significantly between CIS/MS and OIND (CIS/MS: mean 1.36% ±1.53% vs. OIND: mean 3.69% ±1.80%; p=0.12). When frequency in CSF was analysed, proportion of HPVGEADYFEY-specific CD8+ T cells did not differ significantly in both patient groups (MS: mean 3.01% ±1.06% vs. OIND: mean 1.59% ±1.11%; p=0.11).
Comparison of distribution of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells
6
ACCEPTED MANUSCRIPT In all patients (9/9, 95% CI 0.70–1.0) with simultaneous pentamer-staining in blood and CSF, HPVGEADYFEY-specific CD8+ T cells were more frequent in CSF than in blood. On the
T
contrary, NLVPMVATV-specific CD8+ T cells were more frequent in blood compared to
IP
CSF in all patients (4/4, 95% CI 0.51–1.0) with simultaneous pentamer-staining in blood and
SC R
CSF.
Discussion:
NU
Our study revealed several novel findings. First, we were able to detect directly CD8+ T cells
MA
specific for one EBNA1-derived epitope (HPVGEADYFEY) in the CSF of patients with MS and OIND by staining with HLA-matched pentamers. Second, we could show that HPVGEADYFEY-specific CD8+ T cells were more frequent in CSF compared to blood
TE
D
regardless of diagnosis. Third, for CMV-specific cells (CD8+ T cells specific to NLVPMVATV of pp65), the opposite was true with a higher frequency in blood compared to
CE P
CSF. Of note, this observation of a different distribution pattern was present in every patient with simultaneous staining of blood and CSF.
AC
Several studies have investigated specific immune responses of CD8+ T cells after stimulation with EBV-peptides. Höllsberg et al. have shown a selective increased cellular response in MS patients upon stimulation with two peptides restricted to HLA-A2 and HLAB7 (Höllsberg et al., 2003), whereas Gronen et al. could not confirm an increased response of CD8+ T cells against the HLA-B7 restricted peptide RPPIFIRRL of EBNA-3A (Gronen et al., 2006). Lünemann et al. showed an increased cellular response of CD4+ T-cells to peptides from the C-terminal region of EBNA-1, but this increase was not significant in CD8+ T cells (Lünemann et al., 2006). In another study, Lünemann et al. showed an increased frequency of IFN-gamma producing T-cells (irrespective of CD4 or CD8 status) in patients with CIS after stimulation with EBNA-1, but this increase was not present after stimulation with HLA-classI restricted peptides from EBNA3a, 3b, 3c, BZLF1, BRLF1, and BMLF1 (Lünemann et al., 7
ACCEPTED MANUSCRIPT 2010). Regarding CD8+ T cells in CSF, Jaquiéry et al. could show an enrichment of EBVspecific CD8+ T cells in patients with early MS, while there was no enrichment of CMV-
T
specific CD8+ T cells in the CSF of these patients (Jaquiéry et al., 2010). Our study adds to
IP
these results by showing that the frequency of HPVGEADYFEY-specific CD8+ T cells in
SC R
MS patients was not significantly higher in blood nor CSF compared with patients with OIND. The comparison of results of different studies is hampered by diverse methodologies and patient cohorts. However, taken together, the available results indicate merely a
NU
selectively increased cellular immune response against EBV in blood and CSF predominantly
MA
in early MS.
To our knowledge, our finding of a different distribution pattern of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells in CSF and blood has not yet been described. This
TE
D
could indicate intrathecal enrichment of HPVGEADYFEY-specific CD8+ T cells. Intrathecal enrichment of CD4+ and CD8+ T cells specific for EBV has been demonstrated in previous
CE P
studies (Wuest et al., 2014, and Lossius et al, 2014). However, the reason for a different distribution and the mechanisms by which this is accomplished remain unclear.
AC
Our study is limited by the low number of included patients with MS which might entail a type 2 error. This was due to HLA-dependence of pentamer-staining and the low frequency of HLA-B35 in the population. Furthermore, blood and cerebrospinal fluid of a large number of patients with MS and appropriate HLA-status are difficult to obtain. The low number of MS patients eligible for analysis in our study should lead to caution when interpreting our study and further research is needed to confirm our results. Also, our results are restricted to patients with the HLA haplotype HLA-B35 and may not be generalized to patients with other HLA-I haplotypes. In conclusion, our study does not indicate a higher frequency of HPVGEADYFEY-specific CD8+ T cells in blood or CSF of patients with MS and the HLA haplotype HLA-B35.
8
ACCEPTED MANUSCRIPT Interestingly, HPVGEADYFEY-specific CD8+ T cells may have a privileged access to CSF
AC
CE P
TE
D
MA
NU
SC R
IP
T
compared to NLVPMVATV-specific (of pp65 of CMV) CD8+ T cells.
9
ACCEPTED MANUSCRIPT References Gronen F., Ruprecht K., Weissbrich B., Klinker E., Kroner A., Hofstetter H.H.,
T
Rieckmann P. 2006. Frequency analysis of HLA-B7-restricted Epstein-Barr virus-
SC R
controls. J Neuroimmunol. 180, 185–192.
IP
specific cytotoxic T lymphocytes in patients with multiple sclerosis and healthy
Höllsberg P., Hansen H.J., Haahr S. 2003. Altered CD8+ T cell responses to selected Epstein–Barr virus immunodominant epitopes in patients with multiple
NU
sclerosis. Clin Exp Immunol. 132, 137–143.
MA
Jaquiéry E., Jilek S., Schluep M., Meylan P., Lysandropoulos A., Pantaleo G., Du Pasquier R.A. 2010. Intrathecal immune responses to EBV in early MS. Eur J Immunol. 40, 878–887.
TE
D
Levin L.I., Munger K.L., O’Reilly E.J., Falk K.I., Ascherio A. 2010. Primary infection with the Epstein-Barr Virus and risk of Multiple Sclerosis. Ann Neurol.
CE P
67, 824–830.
Lossius A., Johansen J.N., Vartdal F., Robins H., Benth J.S., Holmoy T., Olweus J.
AC
2014. High-throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV-reactive CD8 T cells. Eur J Immunol. 44, 3439–52. Lünemann J.D., Edwards N., Muraro P.A., Hayashi S., Cohen J.I., Münz C., Martin R. 2006. Increased frequency and broadened specificity of latent EBV nuclear antigen-1-specific T cells in multiple sclerosis. Brain. 129, 1493–1506. Lünemann J.D., Tintoré M., Messmer B., Strowig T., Rovira A., Perkal H., Caballero E., Münz C., Montalban X., Comabella M. 2010. Elevated Epstein–Barr virus-encoded nuclear antigen-1 immune responses predict conversion to Multiple Sclerosis. Ann Neurol. 67, 159–169.
10
ACCEPTED MANUSCRIPT Müller C.R., Ehninger G., Goldmann S.F. 2003. Gene and haplotype frequencies for the loci HLA-A, HLA-B, and HLA-DR based on over 13.000 German blood
T
donors. Human Immunology. 64, 137–151.
IP
Newcombe R.G. 1998. Two-sided confidence intervals for the single proportion:
SC R
comparison of seven methods. Statist. Med. 17, 857–872.
Owens G.P., Bennett J.L. 2012. Trigger, pathogen, or bystander: the complex nexus linking Epstein-Barr virus and Multiple Sclerosis. Multiple Sclerosis
NU
Journal. 18, 1204–1208.
MA
Polman, C.H., Reingold S.C., Edan G., Filippi M., Hartung H.P., Kappos L., Lublin F.D., Metz L.M., McFarland H.F., O'Connor P.W., Sandberg-Wollheim M., Thompson A.J., Weinshenker B.G., Wolinsky J.S. 2005. Diagnostic criteria for
840–846.
TE
D
Multiple Sclerosis: 2005 revisions to the “McDonald Criteria.” Ann Neurol. 58,
CE P
Thacker E.L., Mirzaei, F., Ascherio, A. 2006. Infectious mononucleosis and risk for Multiple Sclerosis: a meta-analysis. Ann Neurol. 59, 499–503.
AC
Wandinger K.P., Jabs W., Siekhaus A., Bubel S., Trillenberg P., Wagner H., Wessel K., Kirchner H., Hennig H. 2000. Association between clinical disease activity and Epstein–Barr virus reactivation in MS. Neurology. 55, 178–184. Wuest S.C., Mexhitaj I., Chai N.R., Romm E., Scheffel J., Xu B., Lane K., Wu T., Bielekova B. 2014. A complex role of herpes viruses in the disease process of multiple sclerosis. PLoS One. 9, e105434.
11
ACCEPTED MANUSCRIPT Appendix:
IP
T
Table 1. Overview of antigen-specific CD8+ T cells as % of all CD8+ T cells
SC R
Figure 1. Upper row: EBNA1-specific CD8+ T cells in blood (left) and CSF (right) of a patient with RR-MS. Dots in the quadrant (A) represent HPVGEADYFEY-specific CD8+ T cells. Lower row: CMV-specific CD8+ T cells in blood (left) and CSF (right) of a patient with
NU
viral meningitis (HSV type 2). Dots in the quadrant (B) represent NLVPMVATV-specific
AC
CE P
TE
D
MA
CD8+ T cells.
12
ACCEPTED MANUSCRIPT Figure 1.
CSF
NU
SC R
IP
T
blood
CE P
CMV-specific pentamer
AC
CD8
TE
D
MA
EBNA1-specific pentamer
13
ACCEPTED MANUSCRIPT HPVGEADYFEY(EBNA1)-specific CD8+ T cells
RR-MS RR-MS RR-MS RR-MS RR-MS MS patients (mean) Viral meningitis (HSV type 1) Viral meningitis (HSV type 2) Ramsay Hunt syndrome Bacterial meningitis Bacterial meningitis Transient neurological disorder with signs of inflammation in CNS OIND patients (mean) MS vs. OIND patients
0.22% ±0.13% p=0.18
1.59% ±1.11% p=0.11
D
Statistics (ttest)
MA
NU
6 7 8 9 10 11
Frequency of specific CD8+ T cells in CSF in % 1.81 3.83 3.39 ND ND 3.01% ±1.06% 3.01 0.12 1.77 2.3 1.92 0.44
T
1 2 3 4 5
Frequency of specific CD8+ T cells in blood in % 0.71 0.4 1.02 0.28 0.06 0.49% ±0.38% 0.34 0.09 0.42 0.2 0.13 0.13
IP
Diagnosis
SC R
Patient
TE
NLVPMVATV(pp65 of CMV)-specific CD8+ T cells Diagnosis
CE P
Patient
15 16 17 18 19
CIS CIS RR-MS RR-MS CIS/MS patients (mean) Viral meningitis (HSV type 2) Herpes zoster Ramsay Hunt syndrome Bacterial meningitis Primary angiitis of the CNS OIND patients (mean) MS vs. OIND patients
AC
12 13 4 14
Statistics (ttest) Table 1.
14
Frequency of specific CD8+ T cells in blood in % 3.62 0.33 0.51 0.96 1.36% ±1.53% 4.93 ND 4.52 ND 1.63 3.69% ±1.80% p=0.12
Frequency of specific CD8+ T cells in CSF in % 1.56 ND ND ND 1.99 2.1 0.54 0.19 0.73
ACCEPTED MANUSCRIPT Highlights
CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) were detected in
IP
T
blood and CSF of MS patients.
Frequency of HPVGEADYFEY-specific CD8+ T cells did not differ significantly in blood
SC R
nor CSF in MS compared to patients with other inflammatory central nervous system diseases.
AC
CE P
TE
D
MA
compared to blood regardless of diagnosis.
NU
Frequency of HPVGEADYFEY-specific CD8+ T cells was consistently higher in CSF
15
S0165-5728(16)30044-3 doi: 10.1016/j.jneuroim.2016.03.010 JNI 476325
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
14 December 2015 12 March 2016 16 March 2016
Please cite this article as: Erdur, Hebun, Scholz, Veronika, Streitz, Mathias, Hammer, Markus, Meisel, Christian, Sch¨onemann, Constanze, Wandinger, Klaus-Peter, Rosche, Berit, EBNA1 antigen-specific CD8 + T cells in cerebrospinal fluid of patients with multiple sclerosis, Journal of Neuroimmunology (2016), doi: 10.1016/j.jneuroim.2016.03.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT EBNA1 antigen-specific CD8+ T cells in cerebrospinal fluid of patients with multiple sclerosis
SC R
IP
Constanze Schönemann4, Klaus-Peter Wandinger5, Berit Rosche1
T
Hebun Erdur1, Veronika Scholz1, Mathias Streitz2, Markus Hammer3, Christian Meisel2,
Department of Neurology, Charité – Universitätsmedizin Berlin
2
Institute of Medical Immunology, Charité – Universitätsmedizin Berlin
3
Department of Nephrology and Internal Intensive Care, Charité – Universitätsmedizin Berlin
4
Institute of Transfusion Medicine, Charité – Universitätsmedizin Berlin
5
Institute of Clinical Chemistry and Department of Neurology, University Medical-Centre
MA
NU
1
Hebun Erdur, MD
CE P
Correspondence:
TE
D
Schleswig-Holstein, Campus Lübeck, Germany
Department of Neurology, Campus Benjamin Franklin
AC
Charité – Universitätsmedizin Berlin Hindenburgdamm 30, 12200 Berlin, Germany Phone: +49-30-84454285, FAX +49-30-84454264 email: [email protected]
1
ACCEPTED MANUSCRIPT Disclosures of all authors' financial relationships B. Rosche has participated in meetings sponsored by Bayer Healthcare, Merck Serono,
T
Biogen Idec, Novartis, Ovamed and Teva Pharmaceuticals and has also received lecture and
SC R
IP
research fees from these companies. The other authors report no disclosures.
Abbreviations: MS = multiple sclerosis, CSF = cerebrospinal fluid, CMV =
AC
CE P
TE
D
MA
NU
Cytomegalovirus, EBV = Epstein Barr virus, EBNA1 = EBV-encoded nuclear antigen-1
2
ACCEPTED MANUSCRIPT Abstract Epidemiological data suggests that Epstein-Barr virus may be involved in the pathogenesis of
T
Multiple Sclerosis (MS). We aimed to determine the frequency of CD8+ T cells specific for
IP
one EBNA1-derived epitope (HPVGEADYFEY) in cerebrospinal fluid (CSF) and blood of
SC R
patients with MS and other inflammatory neurological diseases (OIND). The frequency of specific CD8+ T cells was assessed by HLA-class-I-binding pentamers restricted to HLAB35. The frequency of HPVGEADYFEY-specific CD8+ T cells did neither differ
MA
compared to blood regardless of diagnosis.
NU
significantly in blood nor CSF in MS compared to OIND, but was consistently higher in CSF
AC
CE P
TE
D
Key words: multiple sclerosis, Epstein-Barr virus, cerebrospinal fluid
3
ACCEPTED MANUSCRIPT Introduction Epidemiological and immunological studies suggest a role of Epstein Barr virus (EBV) in the
T
pathogenesis of Multiple Sclerosis (MS). Individuals with a history of infectious
IP
mononucleosis have an increased risk of developing MS (Thacker et al., 2006), while risk of
SC R
MS in EBV-negative individuals is very low (Levin et al., 2010). Immune control of EBVinfection may be dysregulated in MS patients and thus contribute to autoimmunity (Wandinger et al., 2000). However, the exact mechanisms involved in the interplay of EBV-
NU
infection and MS pathogenesis are not well understood (Owens & Bennett, 2012). The
MA
presence of an altered frequency of EBNA-1-specific CD8+ T cells in blood and/or CSF of patients with MS compared with other inflammatory neurological diseases (OIND) could indicate an altered immune response to EBV in MS patients. Studies investigating cellular
TE
D
immune responses to EBNA-1 in the CSF of MS patients are scarce, probably due to restriction of the immunogenic EBNA-1-peptide HPVGEADYFEY to HLA-B35. Thus, these
CE P
analyses can only be conducted in CSF of patients with the HLA haplotype HLA-B35, which is expressed in only approximately 10% of the general population (Müller et al., 2003).
AC
Furthermore, the detection of virus-specific CD8+ T cells in CSF requires rapid analysis of CSF probes after lumbar puncture and is also impaired by the low number of cells in CSF. We aimed (i) to determine the frequency of CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) in blood and CSF of patients with MS. For the purpose of comparison, (ii) we assessed frequencies of CD8+ T cells specific for one CMV-derived epitope (NLVPMVATV of pp65) in blood and CSF, and (iii) corresponding frequencies of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells in patients with OIND.
Methods Patients:
4
ACCEPTED MANUSCRIPT Patients were recruited from the Department of Neurology, Charité – Universitätsmedizin Berlin. Diagnosis of CIS, MS (according to the 2005 McDonald criteria) (Polman et al.,
T
2005), and OIND was made by the attending neurologist. Routine CSF analysis was
IP
conducted for diagnostic purposes and included cell count, IgG, IgM, IgA serum/CSF-
SC R
quotient and oligoclonal bands. Analysis of CSF in MS patients was performed before initiation of therapy with corticosteroids. EBV and CMV IgG and IgM antibody status in serum was determined by ELISA. As diagnostic work-up was not to be delayed by HLA-class
NU
testing, staining of CSF and HLA-testing were done simultaneously. Because of low cell
MA
numbers in CSF, simultaneous staining for specific CD8+ T cells for EBV and CMV was not conducted in all patients. Furthermore, due to logistic reasons, pentamer staining was not performed in all samples. The procedure of staining of CSF and whole blood is described in
TE
D
the supplemental material. Only patients with HLA-A02 or HLA-B35 genotype, positive CMV and/or positive EBV serology were eligible for analysis. The study was approved by the
CE P
local ethics committee and subjects or representatives provided informed consent.
AC
Statistical analysis:
Results of FACS staining were obtained using CellQuest software. These were statistically analysed by means of an independent two-sided t test. Confidence intervals for proportions were calculated using the Wilson score method without continuity correction (Newcombe, 1998). P values < 0.05 were considered significant. SPSS 19 was used for all statistics.
Results: Baseline characteristics A total of 96 patients were screened. An inflammatory neurological disease was diagnosed in 81 patients (of these, CIS was diagnosed in 18 patients, RR-MS in 19, and SP-MS and PP-MS each in one case; 24 patients had a viral inflammatory disease, nine a bacterial and nine 5
ACCEPTED MANUSCRIPT patients other inflammatory diseases). In 15 patients, a non-inflammatory neurological disease was diagnosed.
T
HPVGEADYFEY-specific CD8+ T cells were detected in 11 patients (five with RR-MS, five
IP
with viral or bacterial CNS-inflammation, and one patient with transient diplopia and signs of
SC R
inflammation in CSF). NLVPMVATV-specific CD8+ T cells were present in nine patients (two with CIS, two RR-MS, four with viral or bacterial CNS-inflammation and one patient with primary angiitis of the CNS). In patients with viral inflammatory CNS-disease, herpes
NU
simplex virus (type 1 and 2) and varicella zoster virus were the main causes.
MA
Table 1 lists all patients with positive staining for HPVGEADYFEY- and NLVPMVATVspecific CD8+ T cells and the respective proportions of all CD8+ T cells. Causes of inflammatory disease are also listed in table 1. Figure 1 shows HPVGEADYFEY- and
TE
D
NLVPMVATV-specific CD8+ T cells in blood and CSF of a patient with RR-MS and a
CE P
patient with viral meningitis, respectively.
Frequency of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells in blood and CSF
AC
The frequency of HPVGEADYFEY-specific CD8+ T cells in blood as percent of all CD8+ T cells was not significantly higher in MS patients compared to OIND (MS: mean 0.49% ±0.38% vs. OIND: mean 0.22% ±0.13%; p=0.18). Also, the proportion of NLVPMVATVspecific CD8+ T cells in blood did not differ significantly between CIS/MS and OIND (CIS/MS: mean 1.36% ±1.53% vs. OIND: mean 3.69% ±1.80%; p=0.12). When frequency in CSF was analysed, proportion of HPVGEADYFEY-specific CD8+ T cells did not differ significantly in both patient groups (MS: mean 3.01% ±1.06% vs. OIND: mean 1.59% ±1.11%; p=0.11).
Comparison of distribution of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells
6
ACCEPTED MANUSCRIPT In all patients (9/9, 95% CI 0.70–1.0) with simultaneous pentamer-staining in blood and CSF, HPVGEADYFEY-specific CD8+ T cells were more frequent in CSF than in blood. On the
T
contrary, NLVPMVATV-specific CD8+ T cells were more frequent in blood compared to
IP
CSF in all patients (4/4, 95% CI 0.51–1.0) with simultaneous pentamer-staining in blood and
SC R
CSF.
Discussion:
NU
Our study revealed several novel findings. First, we were able to detect directly CD8+ T cells
MA
specific for one EBNA1-derived epitope (HPVGEADYFEY) in the CSF of patients with MS and OIND by staining with HLA-matched pentamers. Second, we could show that HPVGEADYFEY-specific CD8+ T cells were more frequent in CSF compared to blood
TE
D
regardless of diagnosis. Third, for CMV-specific cells (CD8+ T cells specific to NLVPMVATV of pp65), the opposite was true with a higher frequency in blood compared to
CE P
CSF. Of note, this observation of a different distribution pattern was present in every patient with simultaneous staining of blood and CSF.
AC
Several studies have investigated specific immune responses of CD8+ T cells after stimulation with EBV-peptides. Höllsberg et al. have shown a selective increased cellular response in MS patients upon stimulation with two peptides restricted to HLA-A2 and HLAB7 (Höllsberg et al., 2003), whereas Gronen et al. could not confirm an increased response of CD8+ T cells against the HLA-B7 restricted peptide RPPIFIRRL of EBNA-3A (Gronen et al., 2006). Lünemann et al. showed an increased cellular response of CD4+ T-cells to peptides from the C-terminal region of EBNA-1, but this increase was not significant in CD8+ T cells (Lünemann et al., 2006). In another study, Lünemann et al. showed an increased frequency of IFN-gamma producing T-cells (irrespective of CD4 or CD8 status) in patients with CIS after stimulation with EBNA-1, but this increase was not present after stimulation with HLA-classI restricted peptides from EBNA3a, 3b, 3c, BZLF1, BRLF1, and BMLF1 (Lünemann et al., 7
ACCEPTED MANUSCRIPT 2010). Regarding CD8+ T cells in CSF, Jaquiéry et al. could show an enrichment of EBVspecific CD8+ T cells in patients with early MS, while there was no enrichment of CMV-
T
specific CD8+ T cells in the CSF of these patients (Jaquiéry et al., 2010). Our study adds to
IP
these results by showing that the frequency of HPVGEADYFEY-specific CD8+ T cells in
SC R
MS patients was not significantly higher in blood nor CSF compared with patients with OIND. The comparison of results of different studies is hampered by diverse methodologies and patient cohorts. However, taken together, the available results indicate merely a
NU
selectively increased cellular immune response against EBV in blood and CSF predominantly
MA
in early MS.
To our knowledge, our finding of a different distribution pattern of HPVGEADYFEY- and NLVPMVATV-specific CD8+ T cells in CSF and blood has not yet been described. This
TE
D
could indicate intrathecal enrichment of HPVGEADYFEY-specific CD8+ T cells. Intrathecal enrichment of CD4+ and CD8+ T cells specific for EBV has been demonstrated in previous
CE P
studies (Wuest et al., 2014, and Lossius et al, 2014). However, the reason for a different distribution and the mechanisms by which this is accomplished remain unclear.
AC
Our study is limited by the low number of included patients with MS which might entail a type 2 error. This was due to HLA-dependence of pentamer-staining and the low frequency of HLA-B35 in the population. Furthermore, blood and cerebrospinal fluid of a large number of patients with MS and appropriate HLA-status are difficult to obtain. The low number of MS patients eligible for analysis in our study should lead to caution when interpreting our study and further research is needed to confirm our results. Also, our results are restricted to patients with the HLA haplotype HLA-B35 and may not be generalized to patients with other HLA-I haplotypes. In conclusion, our study does not indicate a higher frequency of HPVGEADYFEY-specific CD8+ T cells in blood or CSF of patients with MS and the HLA haplotype HLA-B35.
8
ACCEPTED MANUSCRIPT Interestingly, HPVGEADYFEY-specific CD8+ T cells may have a privileged access to CSF
AC
CE P
TE
D
MA
NU
SC R
IP
T
compared to NLVPMVATV-specific (of pp65 of CMV) CD8+ T cells.
9
ACCEPTED MANUSCRIPT References Gronen F., Ruprecht K., Weissbrich B., Klinker E., Kroner A., Hofstetter H.H.,
T
Rieckmann P. 2006. Frequency analysis of HLA-B7-restricted Epstein-Barr virus-
SC R
controls. J Neuroimmunol. 180, 185–192.
IP
specific cytotoxic T lymphocytes in patients with multiple sclerosis and healthy
Höllsberg P., Hansen H.J., Haahr S. 2003. Altered CD8+ T cell responses to selected Epstein–Barr virus immunodominant epitopes in patients with multiple
NU
sclerosis. Clin Exp Immunol. 132, 137–143.
MA
Jaquiéry E., Jilek S., Schluep M., Meylan P., Lysandropoulos A., Pantaleo G., Du Pasquier R.A. 2010. Intrathecal immune responses to EBV in early MS. Eur J Immunol. 40, 878–887.
TE
D
Levin L.I., Munger K.L., O’Reilly E.J., Falk K.I., Ascherio A. 2010. Primary infection with the Epstein-Barr Virus and risk of Multiple Sclerosis. Ann Neurol.
CE P
67, 824–830.
Lossius A., Johansen J.N., Vartdal F., Robins H., Benth J.S., Holmoy T., Olweus J.
AC
2014. High-throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV-reactive CD8 T cells. Eur J Immunol. 44, 3439–52. Lünemann J.D., Edwards N., Muraro P.A., Hayashi S., Cohen J.I., Münz C., Martin R. 2006. Increased frequency and broadened specificity of latent EBV nuclear antigen-1-specific T cells in multiple sclerosis. Brain. 129, 1493–1506. Lünemann J.D., Tintoré M., Messmer B., Strowig T., Rovira A., Perkal H., Caballero E., Münz C., Montalban X., Comabella M. 2010. Elevated Epstein–Barr virus-encoded nuclear antigen-1 immune responses predict conversion to Multiple Sclerosis. Ann Neurol. 67, 159–169.
10
ACCEPTED MANUSCRIPT Müller C.R., Ehninger G., Goldmann S.F. 2003. Gene and haplotype frequencies for the loci HLA-A, HLA-B, and HLA-DR based on over 13.000 German blood
T
donors. Human Immunology. 64, 137–151.
IP
Newcombe R.G. 1998. Two-sided confidence intervals for the single proportion:
SC R
comparison of seven methods. Statist. Med. 17, 857–872.
Owens G.P., Bennett J.L. 2012. Trigger, pathogen, or bystander: the complex nexus linking Epstein-Barr virus and Multiple Sclerosis. Multiple Sclerosis
NU
Journal. 18, 1204–1208.
MA
Polman, C.H., Reingold S.C., Edan G., Filippi M., Hartung H.P., Kappos L., Lublin F.D., Metz L.M., McFarland H.F., O'Connor P.W., Sandberg-Wollheim M., Thompson A.J., Weinshenker B.G., Wolinsky J.S. 2005. Diagnostic criteria for
840–846.
TE
D
Multiple Sclerosis: 2005 revisions to the “McDonald Criteria.” Ann Neurol. 58,
CE P
Thacker E.L., Mirzaei, F., Ascherio, A. 2006. Infectious mononucleosis and risk for Multiple Sclerosis: a meta-analysis. Ann Neurol. 59, 499–503.
AC
Wandinger K.P., Jabs W., Siekhaus A., Bubel S., Trillenberg P., Wagner H., Wessel K., Kirchner H., Hennig H. 2000. Association between clinical disease activity and Epstein–Barr virus reactivation in MS. Neurology. 55, 178–184. Wuest S.C., Mexhitaj I., Chai N.R., Romm E., Scheffel J., Xu B., Lane K., Wu T., Bielekova B. 2014. A complex role of herpes viruses in the disease process of multiple sclerosis. PLoS One. 9, e105434.
11
ACCEPTED MANUSCRIPT Appendix:
IP
T
Table 1. Overview of antigen-specific CD8+ T cells as % of all CD8+ T cells
SC R
Figure 1. Upper row: EBNA1-specific CD8+ T cells in blood (left) and CSF (right) of a patient with RR-MS. Dots in the quadrant (A) represent HPVGEADYFEY-specific CD8+ T cells. Lower row: CMV-specific CD8+ T cells in blood (left) and CSF (right) of a patient with
NU
viral meningitis (HSV type 2). Dots in the quadrant (B) represent NLVPMVATV-specific
AC
CE P
TE
D
MA
CD8+ T cells.
12
ACCEPTED MANUSCRIPT Figure 1.
CSF
NU
SC R
IP
T
blood
CE P
CMV-specific pentamer
AC
CD8
TE
D
MA
EBNA1-specific pentamer
13
ACCEPTED MANUSCRIPT HPVGEADYFEY(EBNA1)-specific CD8+ T cells
RR-MS RR-MS RR-MS RR-MS RR-MS MS patients (mean) Viral meningitis (HSV type 1) Viral meningitis (HSV type 2) Ramsay Hunt syndrome Bacterial meningitis Bacterial meningitis Transient neurological disorder with signs of inflammation in CNS OIND patients (mean) MS vs. OIND patients
0.22% ±0.13% p=0.18
1.59% ±1.11% p=0.11
D
Statistics (ttest)
MA
NU
6 7 8 9 10 11
Frequency of specific CD8+ T cells in CSF in % 1.81 3.83 3.39 ND ND 3.01% ±1.06% 3.01 0.12 1.77 2.3 1.92 0.44
T
1 2 3 4 5
Frequency of specific CD8+ T cells in blood in % 0.71 0.4 1.02 0.28 0.06 0.49% ±0.38% 0.34 0.09 0.42 0.2 0.13 0.13
IP
Diagnosis
SC R
Patient
TE
NLVPMVATV(pp65 of CMV)-specific CD8+ T cells Diagnosis
CE P
Patient
15 16 17 18 19
CIS CIS RR-MS RR-MS CIS/MS patients (mean) Viral meningitis (HSV type 2) Herpes zoster Ramsay Hunt syndrome Bacterial meningitis Primary angiitis of the CNS OIND patients (mean) MS vs. OIND patients
AC
12 13 4 14
Statistics (ttest) Table 1.
14
Frequency of specific CD8+ T cells in blood in % 3.62 0.33 0.51 0.96 1.36% ±1.53% 4.93 ND 4.52 ND 1.63 3.69% ±1.80% p=0.12
Frequency of specific CD8+ T cells in CSF in % 1.56 ND ND ND 1.99 2.1 0.54 0.19 0.73
ACCEPTED MANUSCRIPT Highlights
CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) were detected in
IP
T
blood and CSF of MS patients.
Frequency of HPVGEADYFEY-specific CD8+ T cells did not differ significantly in blood
SC R
nor CSF in MS compared to patients with other inflammatory central nervous system diseases.
AC
CE P
TE
D
MA
compared to blood regardless of diagnosis.
NU
Frequency of HPVGEADYFEY-specific CD8+ T cells was consistently higher in CSF
15