Journal of Immunology

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

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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

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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

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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
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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)

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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.

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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
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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.  

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(No Transcript)
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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)
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• 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
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Figure 2 a
CPM/Culture

• Antigen ovalbumin (OVA) analysis shows T-cells
  can bind to MHC-2 complexes
• Makes sense Ovalbumin is NOT HUMAN

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Figure 2 b
CPM/Culture

• BALB/c OVA had no response by T-cells
• PD mutant had low levels of activity

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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

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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

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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

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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

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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

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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