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Journal of Immunology

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The NOD mouse was developed by the Shionogi Research Laboratories in Japan in ... Ag on F1 but not on PD mutant using diabetogenic T-cells from NOD- BDC2.5 mice ... – PowerPoint PPT presentation

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Title: Journal of Immunology


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/Datashe
    ets/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.  

9
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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
  • 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

Figure 1
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
Figure 4
  • 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

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
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