Journal of Immunology 1998, 161: 4489-4492 Center for Pathology, Center of Immunology, Washington University School of M

1. Journal of Immunology 1998, 161: 4489-4492 Center for Pathology, Center of Immunology, Washington University School of Medicine, St.Louis, MO

2. Goal of Paper • To show importance of Residues 56 and 57 of the B chain in MHC 2 with respect to maturation of T-cells and development of Diabetes Mellitus

3. Abbreviations • mAb: Monoclonal Antibody • Ag: antigen • I-Ag7PD: Strain mutant of MHC 2 where positions 56 and 57 are now PD in B chain region from H and S. • IDDM: Insulin Dependant Diabetes Mellitus • NOD: Non-Obese Diabetic • APC: Antigen Presenting Cell • TCR: T-cell Receptor

4. IDDM AKA: Type 1 or juvenile Diabetes Characterized by: • Immune response to B-Islet cells of pancreas as foreign • B-islet cells are insulin producers and secrete insulin from the pancreas • Results in Hyperglycemia and other pathologies Pancreas

5. NOD The non-obese diabetic NOD mouse is an inbred strain of type I, insulin dependent diabetes mellitus (IDDM). The NOD mouse was developed by the Shionogi Research Laboratories in Japan in 1974 from a cataract prone subline, CTS of an outbred ICR mouse (Makino S. et al., 1980, Kolb H, 1987, Tochino Y., 1987). In the sixth generation of inbreeding of the CTS strain, the NOD and NON mice were separated. The NOD strain was established in 1980 after 20 generations of inbreeding.http://www.m-b.dk/Datasheets/nod.htm • Insulin Dependence • No excessive weight gain even though body signals to eat more • Giving NKT-enriched thymocytes mice did not develop diabetes (Data Not Shown)

6. NOD continued • Albino, A B c D Adh-3a,  Akp-1b, Akp-2b, Car-2a , Es-1b, Es-2b, Es-3c, Es-5b, Es-9a, Gpd-1b, Gpi-1a, Got-1b, Hbbs Id, h-1a, Lap-1b, Ldr-5f, Ldr-1a, Ldr-2a, Mod-1b, Mup-1a, Pep-3b, Pgm-1a, Trfb • The NOD mouse is considered to be a recombinant of the H-2d and H-2b, referred to as H-2g . • The class I MHC of related strains: NON: H-2d, CTS: H-2d, ILI: H-2d, BALB/c: H-2d, C57BL/6J: H-2b.Which mouse strain to choose as control strain in an experiment must depend on the actual experimental design. It is advisable to consult references dealing with subjects related to the subjects of the actual experiments.

7. BDC2.5 Strain • Strain DescriptionNOD.Cg-Tg(TcraBDC2.5)1Doi Tg(TcrbBDC2.5)2Doi Rag1tm1Mom/DoiJ mice carry both rearranged TCR alpha and beta genes from the cytotoxic CD4+ T cell clone BDC-2.5 and a disrupted Rag1 mutation. The Rag1tm1Mom mutation prevents recombination of endogenous TCR and Ig so that mature T cells in these mice express only the BDC2.5 TCR. On the NOD background, mice carrying the transgenes and homozygous for the Rag1 mutation develop diabetes extremely early (mean age of 25 days), while mice heterozygous (or wildtype) for the Rag1 allele and carrying the BDC2.5 transgenes have a reduced incidence of diabetes relative to NOD/LtJ controls (12% incidence at age 30 weeks). (Katz et al 1993, Gonzalez et al 2001, Mombaerts et al 1992) The Jackson Laboratory

8. CB17.SCID • Strain Description: • Mice homozygous for the Prkdcscid mutation lack both T and B cells due to a defect in V(D)J recombination. Therefore, they easily accept foreign tissue transplants, including human tumors, making them effective models for testing new cancer treatments and as hosts for human immune system tissues (i.e., SCID-hu). SCID mice are also useful for examining the relationship between immunity and disease.  In the C.B-17-scid strain the scid mutation is carried on an inbred strain that is congenic to the BALB/c mouse except for the immunoglobulin heavy chain allele obtained from the C57BL/Ka strain. The C.B-17-scid does exhibit extremely low levels of Ig in ~20% of the mice at 12 weeks of age. The incidence of Ig will increase as the mice age. Despite this "leakiness" C.B-17-scid's do not mount an antibody response to challenge by immunogenic material.  

10. I-Ag7PD: Strain mutant of MHC 2 where positions 56 and 57 are now PD in B chain region from H and S. Aspartic Acid (D) Proline (P) Histadine (H) Serine (S)

11. Figure 1 • Shows expression of MHC-2 molecules in the different mouse strains with 3 different Ab assays • Found that the distribution of Ab-Ag to be different in different strains. • Specifically: • Cross strain expressed same levels of MHC-2 as wt. • PD and wt differ in only the B chain • Lower affinity of Ab to B chain in mutant over wt, possible lower expression

12. Figure 2 a CPM/Culture • Antigen ovalbumin (OVA) analysis shows T-cells can bind to MHC-2 complexes • Makes sense Ovalbumin is NOT HUMAN

13. Figure 2 b CPM/Culture • BALB/c + OVA had no response by T-cells • PD mutant had low levels of activity

14. Figure 2 c • Shows that the PD mutant did present “Ag” to T-cell specific to PD mutant MHC-2, therefore acts as functional presentor

15. Figure 3 • Experiment shows that B-cells of pancreas presented as Ag on F1 but not on PD mutant using diabetogenic T-cells from NOD- BDC2.5 mice

16. TCR bearing cells from BDC2.5 SCID mice transfected into lethally irradiated transgenic mice with specificity to B-cell Ag Allowed 8 wks for bone marrow to mature in deficient animals Found that Large number of mature single positive CD4 T cells in F1, but none in BALB/c, as expected Found same levels of mature CD4 T cells in PD, showing (+) selection by PD MHC-2 After 3 mo, none of the chimeric mice showed signs of Diabetes Figure 4

17. Figure 5 • In vitro proliferation assay shows that PD mutant able to induce B-islet Ag in an MHC restricted fashion, showing peripheral T-cell functionality

18. Conclusions • Demonstrated that TCR interact with PD mutant in thymus and induces positive selection of T-cells • No interaction with TCR and PD when islets presented as Ag • Protection of mice not caused by negative selection of diabetogenic T-cells in thymus • TCR-MHC-Ag recognition has great plasticity

19. Issues with Paper • Shows that PD mutant had either poor recognition of T-cells or poor binding of protein Ag • Never determined the islet specificity activation of T-cells and Ag presentation