 -cell apoptosis in T-cell mediated autoimmune diabetes

1. -cell apoptosis in T-cell mediated autoimmune diabetes Kurrer, M.O., S.V. Pakala, H.L. Hanson, and J. D. Katz. 1997. -cell apoptosis in t-cell mediated autoimmune diabetes. Proceedings of the National Academy of Science 94: 213-218. Presented by Merlande M. Dieujuste

2. Insulin dependent diabetes mellitus (IDDM) • One of the 4 major types of diabetic disorders • ß-cells in the islets of the pancreas are destroyed by a T-cell mediated autoimmune attack • Presently no cure exists • If left untreated, other conditions can result

3. Nonobese diabetic (NOD) mice are the models of choice • Pathogenesis of Type 1 Diabetes similar in nonobese diabetic (NOD) mice and humans • Female NOD mice develop insulitis by 4 weeks of age, diabetes at approximately 15 weeks of age • NOD mice helped recognize ß-cell destruction as the cause of IDDM

4. Rational-Finding the mode of ß-cell death • In order to determine how ß-cells die, the following was initiated: • Complete studies in NOD strain mice • Analyze ß-cell destruction in situ, meaning within the tissues of the pancreas • Create a model in which the T-cells are specific to the destruction of the islet cells

5. BDC2.5 T-cell receptor (TCR) transgenic mice crossed onto NOD.scid mice • Diabetogenic T-cell clone derived from an NOD mice • T-cell receptor genes rearranged in the CD4+ diabetogenic T-cell clone • Genes transferred into a transgenic mouse strain • BDC2.5 TCR transgenic mice created, then crossed onto NOD.scid mice • Mice were killed by cervical dislocation or CO2 ashyxiation

6. Flow Cytometry, Accessing Diabetes • Three color flow cytometry used to analyze the thymus, the spleen and the lymph nodes for sole production of CD4+ T-cells • Diabetes was assessed by measuring venous blood through a glucose meter • Diabetes dated from first reading • Considered diabetic after two measurements > 250mg/dl

7. Preparing pancreas sections • The pancreas were fixed and embedded in paraffin • Sections were deparaffinized and stained with hemotoxylin and eosin (H&E stain) • Terminal dideoxynucleotidetransferase (TdT)-mediated X-dUTP nick and end labeling (TUNEL) stain utilized to detect apoptosis • ß-cells were detected through immunohistochemistry staining for insulin

8. Analyzing pancreas sections • An average of 100 islets per pancreas were analyzed • Sections were analyzed in five levels and insulitits was graded on scale range 0-3 • Level of insulitis determined for each islet, then divided by the total number of islets examined

9. T-cell Transfer • Spleen and mesenteric lymph node cells from BDC2.5 TCR transgenic mice were cultured with mouse islet cells • Transferred to a second flask and supplemented with recombinant IL-2 • Cells analyzed by flow cytometry, then transferred intravenously into the mice • Spleens and mesenteric lymph nodes from diabetic BDC2.5/NOD.scid mice under went same process

10. Experiment One-Selection of BDC2.5 mice in NOD. Scid mice • BDC2.5 TCR transgenic mice crossed onto NOD.scid mice • Elimination of all T and B lymphocytes except CD4+ T cells • Three-color flow cytometry performed on thymus and spleen cells from TCR/NOD.scid transgenic, TCR transgenic, and negative littermates

11. Transgenic TCR expression selects CD4+ T cells in thymus

12. Analyzing CD4, CD8, and V4 (TCR-) TCR expression

13. Lack of CD8+ T cells in the spleen –top row

14. Lack of CD8+ T cells in the spleen- middle row

15. Lack of CD8+ T cells in the spleen- bottom row

16. Result- Positive selection of the BDC2.5 TCR in NOD.scid mice • BDC2.5 TCR were able to bind and express only CD4+ T-cells • V4 was expressed only on CD4+ CD8- thymus cells • Normal T lymphocyte production favored the CD4+ subset • Normal levels of BDC2.5 TCR and CD4 co-receptor present

17. Experiment 2- Progression to Insulitis and diabetes • Pancreas sections from 3-6 mice of each time group were analyzed • Presence of diabetes determined using a glucose meter • 80-100 islets cells scored for insulitis based on the following criteria: • 0= no insulitis • 1= peri- and mild insulitis • 2= moderate insulitis • 3= severe insulitis

18. Scoring insulitis in BDC2.5/NOD.scid mice

19. Rapid progression of insulitis • BDC2.5/NOD.scid mice had an advanced and acute development of insulitis • Accelerated insulitis commenced between 14-16 days • CD4+ T-cells, F4/80+ macrophage, RB6+ neutrophils, and natural killer cells present in the infiltrates

20. Incidence of diabetes in BDC2.5/NOD.scid mice

21. Rapid progression to diabetes • Several BDC2.5/NOD.scid mice became diabetic at 22 days old • All BDC2.5/NOD mice diabetic by 30 days old, and died from complications by approximately 33 days • Diabetes development was not accompanied by CD8+ T cells • BDC2.5 TCR+, CD4+ T cells accelerated progression of diabetes

22. Experiment 3- Transferring insulitis and diabetes to NOD.scid mice • Observe whether or not insulitis and diabetes can be transferred efficiently • CD4+ T cells from severely diabetic BDC2.5/NOD.scid into 16 adult NOD.scid mice • Insulitis assessed and scored from H&E stained sections of pancreas • Incidence of diabetes assessed using a glucose meter

23. Evidence of insulitis in NOD.scid recipient

24. Incidence of Diabetes in NOD.scid recipient

25. Result- Diabetes and insulitis accelerated in NOD.scid recipient • Severe and destructive insulitis by day 9-10 • All NOD.scid recipients had diabetes by day 11 (blood glucose less than or equal to 500)

26. Experiment 4- Study and analyze -cell death in vivo • Rapid progression from insulitis to diabetes in BDC2.5/NOD.scid and NOD.scid mice • -cell death analyzed in pancreas from BDC2.5/NOD.scid mice (21-28 days) and NOD.scid mice (6-9 days) • Evidence of insulitis, necrosis, and apoptosis determined through immunohistochemistry

27. -cell apoptosis in BDC2.5/NOD.scid mice

28. Quantifying -cell apoptosis • Analyzed 31 NOD.scid recipients of direct transfer and in-vitro activated T cells • Randomized groups of recipients were killed at daily intervals • Analyzed pancreas for insulitis and apoptosis using TUNEL procedure, anti-insulin reagents and hemotoxylin

29. Insulitic islets versus Total islets

30. Result- Level of insulitis correlates with presence of apoptotic β-cells • Direct transfer and in-vitro-activated T cells gave similar results • Pancreas did not show signs of either insulitis or apoptosis after 3-4 days of transfer • One mouse had moderate insulitis 5 days after transfer • 12% of islets infiltrated by leukocytes showed one or more apoptotic β-cells • Severity of insulitis rose 7-10 days after transfer

31. Apoptotic -cells are found in insulitic islets cells • Of 4500 islets examined, 1917 were insulitic • 233 islets had the occurrence of -cell apoptosis

32. Overall Conclusion • BDC2.5 TCR transgenes crossed onto the NOD.scid mice is a simple and productive model • Sole presence of -cell specific CD4+ T-cells increases rate of diabetes onset • In-situ analysis allowed determination that -cells die by apoptosis • Apoptosis specific to islets cells with insulitis • Most likely occurs due through Fas/Fas-L or TNF-/TNF R pathways or by the IL-1/inducible nitric oxide synthase pathway

33. ANY QUESTIONS??????