Transcript Identification and Initial Characterization of a Mouse with an Inverted T Cell Ratio Andrew Johnson, Brady Evans and Sophia Sarafova Biology Department, Davidson.

Identification and Initial Characterization of a Mouse with an
Inverted T Cell Ratio
Andrew Johnson, Brady Evans and Sophia Sarafova
Biology Department, Davidson College, Davidson NC
0,2
0
4(24) 6 (59) 8 (13) 10 (27) 12 (27) 14 (7) 16 (20) 18 (17) 20 (7) 22 (6) 24 (25) 26 (5) 28 (6) 40 (3)
Age in wks (sample size)
Conclusions
The Mut phenotype (CD4:CD8 < 1) is generally not
expressed at birth, but develops over the first six months of
life
 The depressed T cell ratio is caused by a reduced number of
CD4 T cells, not by an inflated population of CD8 T cells
 There does not seem to be a correlation between relative
levels of thymic production of mature CD4 and CD8 T cells
and the peripheral ratio
50
13
0
20
15
Count
10
5
50
Count
25
38
10e4
13
0
20
10e1
10e2
10e3
10e4
Green Fluorescence (GRN-HLog)
1 Thymus – 75.65 x 106 cells
10e0
3c
71%
Fig. 5d: B6 (red line) and Mut (blue line) cells gated for CD8 were overlaid
and analyzed for CD44.
M1
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
10e4
CD44 Expression in B6 and Mut LN cells
Plot3: Gated by : CD4 SP cells
10e0
5a
M1
M2
10e0
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
10e4
0.227
Plot6: Gated by : CD8 SP cells
M1
0.98%
10e1
10e2
10e3
Green Fluorescence (GRN-HLog)
Mut. 2 Thymus – 177.1 x
106
10e4
cells
10e0
6 cells
B6 LNPlot2:
– 63.5
x 10
Gated
by : Liv
e Cells
10e4
4.52%
17%
5b
75%
M1
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
10e4
21.77%
M1
25.91%
M2
10e0
CD8
Figure 3a-c: The thymuses of a B6 (3a) and two mice
determined to be mutants (3b and 3c) were removed and
cell counts were determined using a hemacytometer with
0.4% trypan blue. CD4 and CD8 percentages are referring
to the percent of live cells in CD4 or CD8 SP stages. Qa-2
percentages are referring to the percent of Qa-2+ cells
within the CD4SP/CD8SP populations. The ratio given is
that of the %CD4+Qa-2+:%CD8+Qa-2+.
5c
Mut 1Plot2:
LN –Gated
39 x by
10: 6Livcells
e Cells
10e4
Plot2: Gated by : Liv e Cells
10e1
10e2
10e3
Green Fluorescence (GRN-HLog)
10e4
31.23%
M1
17.74%
M2
10e0
5d
Plot3: Gated by : CD4 Cells
15.84%
10e1
10e2
10e3
Green Fluorescence (GRN-HLog)
260
0,4
Count
25
38
10e4
Yellow Fluorescence (YLW-HLog)
10e1
10e2
10e3
M2
Mut.
Fig. 5c: B6 (red line) and Mut (blue line) cells gated for CD4 were overlaid
and analyzed for CD44.
48.34%
M1
32.91%
58.08%
M1
10e0
CD44
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
10e4
10e0
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
Future Directions
 Compare the survival rate of B6 and Mut LN cells in culture
 Explore the possible significance of the increased population of CD8CD44hi cells
 Compare proliferation rates in response to activation in vivo using CFSE staining
Acknowledgements
We would like to thank Amy Becton for her maintenance of the mouse colonies, the NIH for their
contribution of reagents, and the Davidson College Department of Biology for funding.
10e4
Plot6: Gated by : CD8 Cells
195
0,6
Mut.2
Count
130
0,8
0.268
65
1
Mut. 1
0
1,2
10e4
10e0
1,4
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
Yellow Fluorescence (YLW-HLog)
10e1
10e2
10e3
1,6
B6
Mut.2
Figure 5a-b: Inguinal, axillary, brachial and superficial cerivical lymph nodes
were removed from a B6 (5a) and a Mut mouse (5b). Cells were stained for
CD4 and CD8.
10e0
Figure 2: To
follow up, the PBL
of 246 mice from
the colony
expressing the
apparent mutation
were screened for
CD4:CD8 ratio
and separated by
age at date of
screening.
M1
280
1,8
Mut.1
210
Figures. 1a-b : Percent CD4 and CD8 cells in PBL: During initial screenings, 56 mice from the
mutant colony were bled and stained for CD4 and CD8. Those displaying ratios below 1 were
deemed mutant, while those with ratios above 1 were deemed wild type. A significant
difference was noted in the number of CD4 cells between strains (p< 4 x 10-5).
Avg. CD4:CD8 ratio
1.21%
0
WT
10
19%
Plot6: Gated by : CD8 SP cells
10e0
Mut.
B6
70
CD8
CD8
0
0
CD4
CD4
5
Yellow Fluorescence (YLW-HLog)
10e1
10e2
10e3
0
CD8
CD4
2
15
CD4
Count
140
2
10e0
10e0
4
20
40
4
2
10e0
6
M1
10e4
6
4.81%
30
25
Plot3: Gated by : CD4 SP cells
15
CD8
10e4
cells
Count
10
8
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
5
WT
10e0
0
8
M1
30
CD4
106
10e4
Count
20
10
0.207
58%
10
Mut.
10e4
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0
10
10e1
10e2
10e3
Red Fluorescence (RED-HLog)
Plot6: Gated by : CD8 SP cells
4b
20
12
4a
M1
15
12
10e1
10e2
10e3
Green Fluorescence (GRN-HLog)
Count
10
14
12%
M2
5
14
0.85%
Plot2: Gated by : Liv e Cells
10e0
% of PBL
18
16
10e0
B6 Thymus – 107.95 x
20
16
M1
CD8
3b
Plot3: Gated by : CD4 SP cells
Figures 4a-b: Percent of mature CD4, CD8 cells in the thymus and lymph
nodes: Comparing the percent of thymic cells that are mature, Qa-2+ CD4 or
CD8 cells (4a) to peripheral ratios (4b) demonstrates that there does not
seem to be a correlation between thymic population size and peripheral
levels.
0
1b
5.9%
Yellow Fluorescence (YLW-HLog)
10e1
10e2
10e3
1a
18
Plot2: Gated by : Liv e Cells
10e0
Yellow Fluorescence (YLW-HLog)
10e1
10e2
10e3
20
3a
CD4
The peripheral CD4:CD8 T cell ratio is tightly regulated, and while it decreases
with age, it is generally maintained above 1 in mice. In a routine experiment, we
noticed a mouse displaying a CD4:CD8 T cell ratio well below the norm, at
about 0.5. Further screening of the colony revealed that 1/3 to 1/4 of the mice
display this unusual phenotype. Initial characterization of the periphery indicates
that the nearly inverted CD4:CD8 T cell ratio was the result of a decrease in the
number of CD4 T cells and not from a significantly altered number of CD8 cells
(Fig. 1, Fig. 2). It has also become apparent that this phenotype usually does
not become manifest for the first few months of life (Fig. 3). For our research
purposes, we have defined any mouse in this colony that develops a CD4:CD8
T cell ratio less than 1 as mutant (Mut). Using a B6 control, we demonstrate that
there are no obvious defects in the production or emigration of CD4 cells from
the thymus (Fig. 4). Interestingly, we have also noted that the Mut mice display
a larger population of CD8CD44hi cells than their B6 counterparts (Fig. 5).
Qa-2+ Thymocytes Ratio
0
Abstract
CD4 and CD8 SP cells in
B6 and Mut thymus cells
 Adoptive transfer experiments to test the ability of Mut cells to undergo homeostatic
proliferation
 Development of true-breeding strains within the Mut mouse colony
10e4