Answer Problem Set 7
Fall 2003
Problem 1.
A.
Wild type
Tg. #1
Tg.#2
B.
Wild type
Tg.#1
Tg.#2
F1
C.
Bone marrow
blood
Lymph Node
Thymus
Peripheral
Answer Problem Set 7
Fall 2003
D. Bone marrow
Peripheral blood
Lymph node
Thymus
E. Since the Surface bound antigen (BSA) is only present in the liver (i.e. the periphery)
the F1 mouse will still contain B cells displaying the transgenic antibody when we look
in the BM. Once the maturing b cells enter the periphery and come into contact with the
cell surface bound BSA, the Abs on the surface of our B cells will be crosslinked which
kills the cells in the absence of CD40L and leads to deletion in the periphery.
Since this mouse is not transgenic for a specific TCR, the flow profile for CD4 and CD8
is not visibly affected in the thymus and peripheral blood (even though the T cells in this
mouse will be tolerant to BSA).
F.
Transgenic spleen cells:
Tg. #1
Tg. #2
Anti-DNP antibodies
-
Anti- BSA antibodies
+
-
G.
Tg. #1:
No anti-DNP antibodies because Tg.#1 is already transgenic for a particular heavy and
light chain that are specific for BSA. No other ab will be made by B cells in the Tg. #1
mouse.
BSA ab will be made because the #1 Tg. has B cells and T cells specific for BSA which
will be primed in vivo by the DNP immunization, and then restimulated by the
subsequent invitro exposure.
Tg. #2:
Although the Tg. #2 mouse does have DNP specific B cells, they won't be properly
primed because of a lack of BSA specific TCEll help in this mouse, which will be
tolerant (both B and T cells) to BSA because of the high levels in the serum.
So without BSA specific Tcell help in the immunization/priming step, neither BSA or
DNP specific Abs will be made in this mouse.
Answer Problem Set 7
Fall 2003
Problem 2.
Both strains of mice have identical patterns with an IgM specific probe suggesting they
are equivalent in their ability to produce IgM. However, significant differences are seen
with an  probe and an I probe. Strain B has two bands, one of which hybridizes with I
and represents a transcript that has not isotype switched. For strain A, there is only one
form of  specific mRNA and it contains I. Therefore, the defect in Strain A is an in
ability to isotype switch to IgE production.
The defect could be in CD40 or CD40L: an interaction between them is required to
isotype switch.
The defect could be an overproduction of IFN- which blocks DNA rearrangement
(switching).
The defect could not be in IL-4 or its receptor since there is transcription of the
unrearranged gene.
IgE (ng/ml)
1.
2.
3.
B A
B B
TT+T A +BA
4.
TT+T B +BA
TT+T A +BB
TT+T B +BB
T B +BA
5.
6.
7.
The most likely defect would be in CD40L since it is the
T cells and not the N cells from Strain A which are defective. However, it would also be
possible that there is overproduction of IFN- by the defective T cells.
Exposure to antigen is necessary for T cell activation.
only activation T cells express CD40L.
The result would depend upon the nature of the defect.
Most likely it would not be dominant and a functional CD40L gene contributed by the
Strain B male would be expressed and functional on the T cells. Remember there is no
allelic exclusion of gene products like CD40L.
Treatment
Mast cells from Strain A + anti-FcRI
Degranulation
Mast cells from Strain A + Fab from anti-FcRI
Nothing
Mast cells from Strain A + anti-IgE
Nothing
Mast cells from Strain B + anti-FcRI
Degranulation
Mast cells from Strain B + Fab from anti-FcRI
Nothing
Result
Answer Problem Set 7
Fall 2003
Mast cells from Strain B + anti-IgE
Degranulation
Cross-linking of FcRI causes mast cell degranulation. Antibody to FcRI would
crosslink the receptor causing degranulation. Fab is monovalent and therefore unable to
crosslink and cause degranulation. Anti-IgE will crosslink the receptors f they are
occupied by IgE. Strain A is unable to make IgE hence no IgE will be bound to the
receptor and anti-IgE will not cause crosslinking; Strain B makes IgE which will be found
bound to FcRI.
Problem 3.
A selective decrease in IgG1 and IgE is observed. The most likely expanation is a defect
in IL-4 (or IL-4R) or possibly an increase in IFN- because these selectively impact these
isotypes. Since IgG2 and IgG3 remain essentially unchanged it does not appear to be a
general defect such as CD40 or CD40L
The fact that the F1 can mount an immune response shows that B and C have different
defects that can be complemented. Since transfer of T cells into strain B restores an
immune response, there must be a T cell defect in the strain C animals. An excellent
candidate is a defect in CD40L.
Bone majrrow contains the stem cells for both B and T cells. Therefore bone marrow
transfer should make the strain C mice immunocompetent. In this case the mice differ
only in the gene responsible for the defect so there should be no poblem with thymnic
educations of deletion.
The first transcript induced by IL-4 contains the I exon and does not encode a functional
protein. When T cell help ( CD40-CD40L) interaction occurs there is DNA
rearrangement such that VH is joined to C; I is removed from the genome and the
mRNA no longer contains it. T cells for A and B can cause DNA rearrangement but T
cells from C cannot. Again this is consistent with a defect in CD40L.
In this experiment you are analyzing the ability of the B cells from the three strains to
isotype switch in the presence of IL-4 and T cell help. In this case, B cells can respond to
IL-4 and transcribed e containing the Ie transcript. However, they are not able to
rearrange the DNA and isotype switch to IgE. They would therefore appear to be missing
CD40.
Problem 4.
a.
The Strain X mice have undergone an anaphylactic reaction. On first exposure to
Nb there is no antigen specific IgE bound to the mast cells and basophils so there is no
reaction. However, this first exposure to antigen triggers an Nb specific IgE response; this
antigen specific IgE is then bound to the FceRI and the second exposure to Nb triggers the
mast cells and basophils to release their mediators.
b.
1.
2.
3.
4.
Anaphylaxis
Anaphylaxis
Nothing
Nothing
There would be antigen specific IgE in the serum of the Strain X mice which
would bind to FcRI on mast cells and basophils. Upon subsequent challenge with antigen
Answer Problem Set 7
Fall 2003
the mice with this receptor bound IgE would experience an anaphylactic reaction. No or
little antigen specific IgE would be present in the normal mice so no reaction could be
transferred with their serum.
c.
These results are what you would expect given what you know about the
regulation of the IgE response. IL-4 is important in stimulating the germline transcription
necessary for the IgE isotype switch. Treatment with anti-IL-4 therefore inhibits an IgE
response. IFN- inhibits the DNA rearrangement required to isotype switch to IgE.
Treatment with anti-IFN- might then be expected to somewhat increase the IgE response.
d.
All animals, even those not prone to Type I hypersensitivity reactions, have some
circulating IgE and hence some IgE bound to their mast cell and basophil IgERI receptors.
Treatment with anti-IgE crosslinks this IgE and triggers mediator release. This differs
from an antigen specific triggering which requires that the receptor bound IgE recognize
the specific antigen and imposes the requirement for the prior exposure to the antigen.
e.
Nothing would happen when the Fab of the anti-IgE is injected because the Fab
fragment is monovalent and hence would not be able to crosslink the FcRI receptors.
Crosslinking is a prerequisite for mediator release.