Th1/Th17 Cytokine Dysregulation during Different Stages of Multiple Sclerosis

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1 Th1/Th17 Cytokine Dysregulation during Different Stages of Multiple Sclerosis Benjamin M. Segal, M.D. Holtom-Garrett Professor of Neurology Director, Multiple Sclerosis Program University of Michigan

2 Disclosures This continuing education activity is managed and accredited by Professional Education Service Group. The material presented in this activity represents the opinion of the faculty. Neither PESG, nor any accrediting organization endorses any commercial products displayed in conjunction with this activity. Commercial support was not received for this activity.

3 Faculty and Staff Disclosures Dr. Segal has been a consultant for the following pharmaceutical companies: TEVA, Biogen, Bristol Myers Squibb and Innate Therapeutics, Inc. He has received non-restricted educational grants from TEVA and Biogen Idec. Professional Education Services Group staff have no financial interest Research Grant Support The research studies presented in this lecture were supported by grants from the National Institutes of Health (Autoimmunity Centers of Excellence) and the Dana Foundation

4 Learning Objectives Learn about the autoimmune theory of MS Learn about T helper cells and their role in neuroinflammation Discuss potential immune biomarkers in RRMS and SPMS

5 The Autoimmune Theory of Multiple Sclerosis There is abnormal neuroinflammation but no consistent evidence for infectious agent in MS lesions The genes associated with susceptibility to MS are immune related Therapeutic efficacy of immunomodulatory agents in decreasing relapse rates and the frequency of gadolinium enhancing lesions (natalizumab, alemtuzamab, fingolimod, rituximab) Similarities between multiple sclerosis and ADEM/ EAE

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7 Are autoreactive T cells more abundant in MS? The literature is inconsistent. Some laboratories have reported comparable frequencies of myelinreactive T cells among PBMC from individuals with MS and healthy controls, while others have reported higher frequencies of myelinreactive T cells in MS. These discrepancies could be explained, in part by methodological differences. In many studies, PBMC or purified T cells were expanded in vitro prior to performing the assay (which could enrich certain subpopulations and bias the TCR repertoire and/ or introduce phenotypic changes) Different T cell assays (measuring distinct properties) are employed across the studies (so that certain T cell subsets, including those with critical pathogenic functions, might not be enumerated) The antigen used to stimulate recall responses varies across the studies

8 A 12 month longitudinal study of myelin-specific cytokine responses and MRI lesion development in RR and SP MS Patient Population 12 RRMS 26 SPMS 39 Healthy Controls Protocol Phlebotomy -every 4 weeks MRI (contrast enhanced) -every 8 weeks EDSS evaluation every -every 12 weeks Immunological Assay IFN and IL-17 Elispots - performed directly ex vivo - Stimuli: whole human MBP Tetanus Toxoid CD3/ CD28

9 Objectives of the Study To determine whether myelin-reactive Th1 and/ or Th17 cells are expanded in individuals with RR or SP MS To assess the diversity of the myelin-reactive T cell repertoire between individuals with MS and to determine whether patterns of cytokine production are stable over time To identify immunological biomarkers that distinguish MS from HC and RRMS from SPMS To identify immunological biomarkers that correlate with degree of disability, disease duration or new lesion formation (radiological and/ or clinical disease activity)

10 What is the relative frequency of MBP-specific Th1 (IFN producing) and Th17 (IL-17 producing) cells in the circulation of individuals with RRMS or SPMS in comparison to age and sex matched healthy controls?

11 Frequency of MBP-specific PBMCs: Personal Average (RRMS v.s. SPMS v.s. Control) IFN IL-17 I------P< I I P< I I------P< I I P< I P-values are adjusted for age, disease duration and EDSS at the first visit.

12 Frequency of MBP-specific PBMCs: Personal Variation (RR v.s. SP+T-SP v.s. Control) IFN IL-17 I------P= I I P< I I------P< I I P< I P-values are adjusted for age, disease duration and EDSS at the first visit.

13 Longitudinal IFN Elispot P values vs. HC RR 2.39e-5 *** SP *** SP+ 2.21e-5 *** Longitudinal IL-17 Elispot P values vs. HC RR *** SP- 2.9e-7 *** SP+ 5.85e-5 ***

14 Is the myelin-reactive Th1/Th17 cell repertoire similar among individuals with MS? Does the pattern of cytokine production by myelinreactive T cells change over time or in association with clinical progression?

15 Distinct patterns of MBP-specific cytokine production among individuals with MS Th1 dominant Th17 dominant IL-17 IFN IL-17 IFN IFN Mixed Healthy control IL IL-17 IFN

16 Cytokine Responses and EDSS/ Disease Duration (SP and RR MS combined) P=0.085 P=0.073 P=0.442 P<0.001

17 Are there immune markers that distinguish between RR and SP MS?

18 MIP-1a (pg/ml) MIP-1a (pg/ml) Serum MIP1 levels are elevated in RRMS MIP-1a * 8 6 MIP-1a RRMS Healthy Control All SP HC RRMS SPMS Patient Group Month * p=0.04 vs. HC

19 6CKine (pg/ml) 6CKine (pg/ml) Serum CCL21 levels are elevated in SPMS 6CKine (CCL21) 6CKine (CCL21) * ** Healthy Control RRMS SP- SP Healthy Control RRMS SP- SP+ All SP Patient Group Month **p=0.0086, *p=.036 vs HC

20 In our study, approximately 35% of individuals with SPMS had one or more gadolinium enhancing lesions on one or more of the six bimonthly MRI scans obtained over a 1 year observation period.

21 The frequency of MBP-specific IL-17 producers rises at the time of lesion formation in individuals with SPMS *

22 Does the subset of SPMS patients with neuroinflammatory lesions differ, fundamentally, from the subset of SPMS patients who are radiologically quiescent? Is the presence of enhancing MRI lesions in SPMS indicative of a more aggressive clinical course? Is the presence of enhancing MRI lesions in SPMS indicative of responsiveness to immunomodulatory agents?

23 Rituximab in patients with primary progressive multiple sclerosis: results of a randomized double-blind placebocontrolled multicenter trial. Subgroup analysis showed time to CDP was delayed in rituximabtreated patients aged <51 years (hazard ratio [HR] = 0.52; p = 0.010), those with gadolinium-enhancing lesions (HR = 0.41; p = 0.007), and those aged <51 years with gadolinium-enhancing lesions (HR = 0.33; p = 0.009) compared with placebo. Hawker K, et al. Ann Neurol Oct;66(4):

24 Eotaxin 3 (pg/ml) Eotaxin 3 is expressed at relatively high levels in the sera of SPMS patients with enhancing lesions Eotaxin 3 (MIP-4a/CCL26) p= Healthy Control RRMS SP- SP+ All SP Patient Group TTEST RR vs HC SP vs HC asp vs HC All MS vs HC RR vs all SP SP vs asp asp vs. RR

25 Eotaxin 3 (pg/ml) Eotaxin 3 is expressed at relatively high levels in the sera of SPMS patients with enhancing lesions Eotaxin 3 (MIP-4a/CCL26) Healthy Control RRMS SP- SP Month

26 ROI Selection in Corpus Callosum Genu Body Splenium

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28 MS enhancing group P04 1st scan 6 th (last) scan

29 Summary PBMCs from MS patients contain a higher frequency of MBPspecific IFN and IL-17 producers than PBMC from healthy controls. This is true of both RR and SP subsets. There are distinct patterns of cytokine production by MBPspecific PBMC among individuals with MS, including Th1 dominant, Th17 dominant and mixed

30 Summary SPMS is not a purely neurodegenerative disease. There is evidence of persistent immune dysregulation during the SP phase of disease. However, the autoimmune response may evolve with disease duration. A subset of SPMS patients exhibit ongoing CNS inflammatory activity. Preliminary data suggests that this subset has a unique immunological signature (ex. Elevated serum Eotaxin- 3). SPMS patients with enhancing MRI lesions suffered more damage (demyelination/ axonopathy, as measured by DTI) than their quiescent counterparts over the course of the 12 month observation period.

31 Conclusions MBP-specific Th1 and Th17 cells are enriched in the peripheral T cell repertoire of individuals with MS (?epiphenomenon or causal relationship)? The pattern of immune dysregulation differs across individuals with MS, suggesting that DMT regimens should be customized based on immune profiling A subset of SPMS patients experience ongoing neuroinflammatory activity and suffer more CNS tissue damage than SPMS patients who are free of neuroinflammation. This subset might be identified by a distinctive immune signature and might be remedial to DMT that are traditionally used in RR MS.

32 Acknowledgements Kevin Carbajal, PhD Heather Grifka Walk Amanda Huber Stephen Lalor Tina Jones Praveen Rao, PhD Judy Rotthoff, RN Julie Rumble, PhD Eileen Scheild, RN Joshua Stoolman Sven Ekholm, MD Ashok Srinivasan, MD Lu Wang, PhD

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34 The frequency of MBP-specific IL-17 producers in the circulation increases with EDSS in SP MS P=0.002

35 The frequency of MBP-specific IFN producers increases with disease duration in RR MS (P<0.001)

36 Correlation (RR +SP MS combined) IFN IL-17 EDSS p=0.442 p<0.001 Disease duration p=0.085 p=0.073

37 Correlation (SPMS Patients) IFN IL-17 EDSS p=0.196 p=0.002 Disease duration p=0.646 p=0.442