3.2 Características Dimensionales y Geométricas
3.2.1 Determinación de las Tolerancias Geométricas
Intraepithélial T cells w ere isolated from m urine gut o f wild type 129/Sv (top plots), w ild type (C B A /C a x 129/Sv) F I (second line plots), heterozygous (third line plots) and hom ozygous (bottom plots) mice. lEL were stained with anti-apTC R , anti-CDSp and anti-C D 8a/Lyt-2.2 or anti-C D 8a/Lyt-2 specific antibodies. apTC R^ T cells w ere gated and analysed by plotting CD8p against CD 8a/Lyt-2 (left-hand side) or C D 8a/L yt-2.2 (right-hand side). The percentages o f positive cell populations are shown in the dot plots.
12 9/S v (CD8.2+/+) CBA/Ca X 129/Sv (CD8.1+/C D8.2+) C I1 + /A 1 ,2 ( C D 8 . 1 + / C D 8 . 2 A 1 . 2 ) 0 . 4 % 3 1 . 5 % 0° 1o’ 1 C 10^ 10 0 . 1 % 5 7 . 5 % 0° 1o' 1C“ 10^ 10 0 . 1 % 5 4 . 0 % : 0 . 4 % 3 2 . 5 % - 1 # " 1 0 . 7 % to" to' 10^ 10" 10^ 0 . 3 % 5 8 . 7 % to" to' 10^ 10" 10^ 4 4 . 1 % 10" 10' 10^ 10" 10^ 1 1.1% 8 . 4 % ^ 10° 1o' 10^ 10" 10"' C l IA 1 ,2 /A 1,2 ( C D 8 . 2 A 1 , 2 / A 1 , 2 ) 2 5 . 1 % 0.1% CO. 00 CO. CO Q O ] 0 . 5 % 2 6 . 0 % 9 . 3 % ‘l M lllll , 1 I 1 llllV CD8a/Lyt-2 CD8a/Lyt-2.2 1 0 ‘ -► 10" 10^
Staining o f lEL from CII^^^'’^Aneo heterozygous m ice w ith anti-CD 8p and anti- C D 8a/L yt-2.2 showed that 11.1% o f the CD8p^ cells appeared to be C D 8 a' and 44.1% were CD8a^. This was not the case w hen the cells w ere stained w ith anti-CD 8p and anti- CD 8a/Lyt-2, w here all the CD8p^ cells are also CD8a^. It is know n that there are no CD8PP hom odim ers and that the p chain can not com e to the cell surface w ithout the a chain. Therefore, we assume that the cells that appear to be C D 8p^C D 8a/L yt-2.2' express the CD 8a/Lyt-2.1 wild type allele on their surface, but not the C D 8a/L yt-2.2 m utated allele. Thus, 78% o f the C D 8ap cells as defined by the w ild type allele also express the m utated allele on their surface. This is consistent w ith the variegation in the expression o f the C D 8a/L yt-2.2 m utated allele that w as observed in peripheral T cells found in lym ph nodes (see figure 36 above).
Expression o f CD8 is dram atically affected in thym ocytes and peripheral T cells by deletion o f CII-1 and CII-2 o f cluster II in the absence o f a second C D 8a allele
Deletion o f D N asel-H SS CII-1 and CII-2 o f cluster II caused a substantial reduction in the percentage o f thym ocytes and peripheral T cells that express C D 8a in CllAi-2/Ai-2^j^gQ m ice com pared to the w ild type control. In order to exam ine the expression o f one m utated allele alone in the absence o f second C D 8a allele, CII'^Af^ m ice w ere generated. For this purpose, a ^^j^g+/Ai,2^ heterozygous m utant
m ouse was crossed to a C D 8a heterozygous null (CD8^^) mouse. The offspring o f this cross w ould have the following genotypes in term s o f CD8 expression: CD8^'^^ (expressing two w ild type alleles), CD8^^ (expressing one w ild type allele), CD8^^Af2 (expressing one m utated allele and one w ild type allele) and CD8'^Af2 (expressing only one m utated allele). A C D8A''2/Ai,2 j^gmozygous m ouse (expressing two m utated alleles)
was also used for the experiment. Thym ocytes and lym ph node cells from these m ice w ere isolated and stained w ith anti-CD4 and anti-CD 8a/Lyt-2. Cells w ere analysed by plotting CD4 against C D 8a/Lyt-2 and gating the different cell populations. The percentages o f cells falling in each gate are shown in the dot plots.
Figure 40 (left-hand side) shows the FACS profiles o f thym ocytes isolated from the m ice described above and stained w ith anti-CD 4 and anti-C D 8a/Lyt-2. The
heterozygous null mouse, that has only one functional allele, expresses C D 8a at lower levels that the w ild type control, but the percentages o f cells falling in each gate are the sam e as in the w ild type control mouse. The hom ozygous m utant mouse, that has two m utated alleles, expresses C D 8a at lower levels than the w ild type m ouse. In addition, in this m ouse the percentages o f DP and CD8 SP cells are reduced, as a result o f the deletion mutation. In the CD8'^^'’^ mouse, that expresses only one m utated allele, the effect is even m ore dramatic. The percentages o f DP and CD8 SP thym ocytes in this m ouse are reduced com pared to the CD8^^'^^^'^ hom ozygous m utant control m ouse and those cells express CD8 at even low er levels com pared to the control. Furtherm ore, the percentage o f cells falling in the CD4 gate is increased, showing a further block in the D N to DP transition o f thym ocytes.
Similarly, in the peripheral T lym phocytes o f CD8"^^*’^ m ice (figure 40, right- hand side) the percentage o f CD8 SP cells that leave the thym us and populate the lym ph nodes is reduced com pared to the hom ozygous control. Those cells express C D 8a at even low er levels than the control mice.
Thus, in CD8'^^'’^ mice (in the absence o f a second C D 8a allele), there are very few cells that get to the DP stage and even fewer cells m anage to pass through selection in the thym us and m igrate to populate peripheral lym phoid organs.