Using Th40:Treg Ratio as a Predictor of Multiple Sclerosis and Other Autoimmune Diseases
Rivera, Erika L.1,2, Vaitaitis, Gisela M.2, Carter, Jessica R.2, Waid, Dan M.2, Wagner, David H2.
1. University of Notre Dame, Notre Dame, IN 46556; 2. Webb‐Waring Center, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
INTRODUCTION
What is Multiple Sclerosis (MS)?
• Autoimmune disease that results in the demyelination
of nerves; impedes transmission of messages1
• Results in debilitating conditions, sometimes
disabilities
• Symptoms: vision problems (tunnel vision);
numbness/weakness of the limbs; coordination
problems, especially in gait; paraparesis1
• Cause: unknown, autoimmunity
• McDonald Criteria: Tool used to diagnose MS2,3
• Main requirement- have 2 attacks or 2 white matter
lesions disseminated in time and space. The
presence of lesions is determined through MRI.4
• Goal is to eliminate other diseases that show
symptoms similar to MS; MS has to be the last
possible explanation
• Based on HLA specificity: certain HLA haplotypes
are associated with disease. In MS, HLA-DR2 and
HLA-DQ6 are associated with the disease5
MATERIALS & METHODS
1. Staining of lymphocytes
• Blood samples were obtained from MS
patients and from individuals without
autoimmune disease. Patient scheduling was
facilitated by the Barbara Davis Center.
• Ficoll separated PBMCs
• Washed with RBC lysis buffer
• Stained with antibodies to CD4, CD40, CD3,
CD25, CD45 r/o and CD8
• Rinsed with PBS
• Fixed in paraformaldehyde 4%
• Part of the fixed cells were permeabilized with
eBioscience Permeabilization Buffer and
internally stained for FXP3.
2. Ran stained samples on flow cytometer
3. Evaluated results from flow cytometer in FlowJo
CONCLUSIONS
•Th40 levels were elevated in T1D. This seems to hold
true in MS patients as well.6
•In T1D the levels of Tregs are lower and appear to have
limited ability to regulate the immunoresponse.6 This
appears to be similar in MS.
•It was noted that the Treg population was also limited in
some of the control patients. This might be due to
exposures to illness or other external factors.8
Figure 2. Comparison of Th40 and Treg cells between
patients and controls. Results show a significant difference
between Th40 levels in MS patients and controls. No
significant difference was noted for Treg levels.
p<0.0001
Th40 & T regulatory (Treg) cells
• Th40: Helper T-cell subpopulation. Defined by
CD3+/CD4+/CD40+
• Elevated in autoimmune diseases, including Type 1
Diabetes (T1D)6
• Treg: help suppress inflammation/immune response by
inhibiting proliferation and cytokine production in
effector cells.7 Defined by CD3+/CD4+/CD25+/CD40• Treg cells usually occur at the same level as Th40 cells
in individuals without autoimmune disease. But in
autoimmunity they are at a lower level and don’t catch
up or regulate to a normal equivalent activity8
RESULTS
Figure 3. Th40:Treg ratio in MS patients. Th40 and Treg
levels of individual patients have been matched by color.
FUTURE DIRECTIONS
• Observe the effects of increasing the number of Treg
cells so that they are at the same level as activated Th40
cells –does it result in the quiescence of Th40 activity
or in any changes in disease course?
• Use Th40:Treg ratio as a predictor of other autoimmune
diseases
• Develop a patient database that will allow for
comparison of other external factors, as well as Th40
and Treg populations. This can assist in the
development of a more concrete picture.
ACKNOWLEDGEMENTS
•
•
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•
•
•
•
•
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NIH Grant for the Colorado Undergraduate Summer Program (CUSP)
Webb-Waring Center
Barbara Davis Center
Dr. Repine, MD- CUSP Director
Dr. Wagner, PhD- CUSP Supervisor
Dan Waid-Administrator
Jessica Carter
Gisela Vaitaitis
Colorado Leaders, Interns, Mentors in Business (CLIMB) Program
PURPOSE
p=0.0097
• Hypothesis: Comparing Th40 to Treg levels, we
expected to see that MS and T1D would have a higher
level of Th40 to Treg, thus limiting the regulation of
Th40 proliferation. This ratio can be used to better
predict autoimmunity in some disease models.
• The development of a test using these ratios to predict
the likelihood of a patient developing MS or other
autoimmune disease would be highly likely and could
help create a better predictive tool.
• Better, earlier diagnosis, hopefully leading to earlier
interventions, and thus limiting symptomology.
• Save patients time and money, allowing for greater
treatment options.
RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
REFERENCES
1. Society, M.S. National MS Society. [cited 2013 July 7]; National Website for National Multiple Sclerosis Foundation]. Available
from: http://www.nationalmssociety.org/index.aspx.
2. Polman, C.H., et al., Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol, 2011. 69(2):
p. 292-302.
Figure 1. Comparison of Th40 cells among different
patients. MS and T1D subjects show elevated Th40 levels
compared to non-autoimmune subjects. The gating is
based off of isotype control samples.
3. Polman, C.H., J.S. Wolinsky, and S.C. Reingold, Multiple sclerosis diagnostic criteria: three years later. Mult Scler, 2005. 11(1): p.
5-12.
Figure 4. Th40:Treg ratio in control subjects. Th40 and
Treg levels of individual subjects have been matched by
color.
4. Fox, R.J. Multiple Sclerosis. 2013 [cited 2013 July 17]; 2000-2011:[Available from:
http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/neurology/multiple_sclerosis/.
5. Wagner, D.H., Jr., Basics of Immunology, S.T.P. Webb-Waring Center, Editor 2012.
6. Waid, D.M., et al., A unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes. Clin Immunol,
2007. 124(2): p. 138-48.
7. Goldsby, R.A., Kindt, T.J., Osborne, B.A., Kuby, J., Immunology. 5th ed. 2003: W. H. Freeman and Company, 41 Madison Avenue,
New York, NY.
8. Wagner, D.H., Jr., Immunology in MS, W.S.S. Program, Editor 2013.
9. Harris, P.A., et al., Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for
providing translational research informatics support. J Biomed Inform, 2009. 42(2): p. 377-81.