The maturation stage of the peripheral G D 4+C D 45R A + subset is different from that of the C D 8+C D45R A +
5.1 Introduction
The results in the previous chapter suggest that although CD45RA+ cells may constitute a thymic lineage, both CD45RA+ and CD45RA- cells are likely to emigrate to the periphery. Furthermore, differences between expression of CD45RA among CD4+ and CD8+ T cells are already apparent. Very few CD4+CD45RA+ cells are seen in the thymus in contrast to CD8+CD45RA+ cells and such a few could not account for the daily emigration to the periphery (discussed in chapter 9 and Lightstone & Marvel, 1990). These data already seemed to contradict the starting hypothesis, namely that expression of CD45RA on T cells represents the first stage along a differentiation pathway from naive T cells expressing CD45RA through activation and conversion to memory cells which have irreversibly lost the CD45RA antigen. This hypothesis had largely arisen from in vitro studies using human cells e.g. (Akbar, etal., 1989; Merkenschlager & Beverley, 1989; Merkenschlager, ef a/., 1988). One possibility previously invoked to account for the paucity of thymic CD45RA+ cells is that CD45RA is expressed on cells just as or immediately after they exit the thymus.
The phenotype of thymic emigrants can be tested directly by labelling intrathymic cells with fluorescein and determining the phenotype of fluorescein labelled cells reaching the periphery (Scollay, et al., 1980). However, such a technique only picks up a small proportion of thymic emigrants and does not provide information about the long term fate of such cells. An alternative approach, and the one taken here, is to study to effects of thymectomy on the phenotype of peripheral T cell subsets. Thymectomy would be predicted to lead to a fall in the size of the peripheral pool of CD45RA+ and not CD45RA' T cells if the former were the only subset to exit the thymus and contribute to the naive T cell pool. However, the changes in the size of this pool are also be dependent on the lifespan and self renewal capacity of the CD45RA+ T cells in the periphery. To address these issues, the percentage of CD45RA+ T cells has been studied from 10 days up to 2 years of age in thymectomized and control mice. Following thymectomy, there is a drop in the proportion of CD45RA+ cells among the CD8+ subset whilst among CD4+ cells the population most affected is the CD45RA". However, there is very little change
with ageing in non-thymectomized mice. Furthermore, in contrast to expectation, CD44, a memory cell marker (Budd, et al., 1987; Butterfield, et a!., 1989), is shown to be expressed by 75% of the CD4+CD45RA+ cells and 20% of CD8+CD45RA+ cells. We also studied expression of CD45RA following activation in vitro. Finally, we looked at the CD45RA phenotype of regenerating T cells.
5.2 Results
5.2 .1 Thymectomy leads to a fall the number of CD8+ C D 4 5 R A + T cells but not CD4+ CD45RA+ cells in the periphery.
To study the effect of thymectomy on the peripheral pool of CD45RA+ cells, B10A and CBA mice were thymectomized at 10 days or 4 weeks of age. Sham thymectomies were undertaken on littermates at the same time. 1 or 2 sham and 2 thymectomized mice were stained at each time point. As shown in figure 5.1 there is a fall in the total number of CD8+ and CD4+ spleen cells following thymectomy though the fall is consistently greater among the CD4+ cells. When the percentage of CD45RA+ cells within each of these subsets is studied, a significant decrease is only found among the CD8+ cells. Among CD8+ cells, only the CD45RA+ population declines significantly. In contrast, the fall in the number of CD4+ cells is largely due to a fall in the number of CD45RA' cells. These changes are not seen at 6 weeks following thymectomy, but are seen from 10 - 12 weeks and became more marked with time (figure 5.1). The changes in the CD45RA subsets following thymectomy are similar in B10A and CBA mice. In CBA mice, thymectomized at 4 weeks of age, only 12% of CD8+ are CD45RA+ 1 year following thymectomy whereas the sham thymectomized animals have a similar percentage to that of nonoperated 2 year old controls (51%). However, the fall in CD8+CD45RA+ cells over and above that expected from ageing is only seen in a proportion of the animals studied one year following thymectomy. It is worth noting however that in mice where a fall in the number of CD8+CD45RA+ cells is seen there is still no fall in the number or percentage of CD4+CD45RA+ cells. As with the B10A mice, there is no difference between the profiles of CD45RA+ cells among CD4+ cells in the sham compared with the thymectomized
Figure 5.1
Effect of thymectomy on numbers of splenic CD4+ and CD8+ cells expressing CD45RA
(A) 30 -, 25 (B) Y 20 - o X 15 J 8 12 weeks
■tLtrw
30 25 20 15 10 - 12 weeks (C) 19 weeks (O I o 5 (D) to I o 5 25 - 20 . 15 _ 10 _ [ Î Ï Î M 19 weeks I:1m
I (E) 30 _ 25 _ 20 _ (F) X 15 a » 10 30 weeks;£i
Total CD8+f a X k
CD8+ CD8+ CD45RA- CD45RA+ 30 -I 30 weeks 25 - (O 20 - o X 15 - a 8 10 - Total CD4+ CD4+ CD4+ CD45RA- CD45RA+B10.A mice thymectomized at 10 days of age 12 weeks (A, B) or 19 w eeks (C, D)previously, 2 mice in each group
CBA mice thymectomized at 4 weeks of age 30 weeks (E, F) previously, 4 mice in each group
Numbers of cells/ spleen expressing: C D 4 and G D 45R A - panels B, D, F CDS and C D 45R A - panels A, 0 , E
□
Sham
Thymectomized
Cell numbers in each population were calculated as the product of the percentage of cells expressing each marker and the total number of cells per spleen
Data are expressed as mean values
mouse (figure 5.2). Thymectomy at 10 days of age gives similar results to those at 4 weeks (figure 5.1 A and 5.1 B).
As shown previously by others (Budd etal., 1987) there is a marked rise in the proportion of CD44+ I cells following thymectomy (figure 5.3). Overall, these data suggest that the peripheral pool of CD8+CD45RA+ T cells is generated and maintained by the exit of CD45RA+ cells from the thymus but this does not appear to be the case for the CD4+CD45RA+ subset. To further investigate the maintenance of CD45RA+ subset in the periphery, the expression of CD45RA among CD4+, CD8+ or Mlg+ cells was determined in ageing mice.
5 .2 .2 Maintenance of the CD45RA+ population among spleen lymphocyte subsets with age in vivo.
The percentage of CD45RA+ cells among the lymphocyte subsets of B10A and CBA spleen cells was determined using two colour flow cytometry. Prior to 1 week of age there are virtually no detectable T cells in the spleen, with most cells expressing no CD4, CD8 or sig. Therefore spleen cells from mice aged 11 days to two years were studied. In almost all experiments, cells from two mice of 6 to 10 weeks of age and 2 mice of another age were stained simultaneously to allow direct comparison of the number of positive cells. The total number of spleen cells rises from 11 days to 1 year of age and then declines by two years (figure 5.4). The percentage of lg+, CD4+ and CD8+ cells increases from 11 days to reach a plateau by 10 weeks following which the values are maintained or decline slightly (figure 5.4). Because the spleen cell numbers and the proportion of GD4+ and CD8+ cells change with time, the data on CD45RA expression in ageing mice are expressed as percentages rather than absolute cell numbers.
CD45RA is expressed on more than 95% of B cells and this does not change with increasing age (figure 5.5). GD45RA is expressed on 71% (sd 1.4%) of GD8+ T cells at three weeks of age. As shown in figure 5.5, there is a decline in the proportion of GD8+GD45RA+ cells up to one year of age (mean 61%, sd 3.7%) but this does not change significantly thereafter.
Figure 5.2
Comparison of expression of CD45RA among CD4+ and CD8+ cells in aged thymectomized and non thymectomized mice
in CD4+
CD45RA