A central role for thymic emigrants in peripheral T cell homeostasis

Abstract

After initial seeding by thymic emigrants, homeostatic regulation of the T cell pool has been thought to occur entirely within the periphery. Here we report that the degree of thymic emigration directly affects the number and the CD4/CD8 ratio of peripheral T cells. We demonstrate that the increase in T cell pool size caused by the engraftment of 2, 6, or 9 thymic lobes correlates almost exactly with the number of emigrants exported from those grafts in the previous 3 weeks, regardless of how long the graft has been in place. The extent of the increase supports the concept of a 3-week period after thymic export in which emigrant T cells are exempt from peripheral T cell homeostasis. This apparent exclusion of recent thymic emigrants from the niche-based regulation of peripheral T cell numbers ensures repertoire turnover throughout adult life and provides the basis of a direct and previously unrecognized role for the thymus in the regulation of peripheral T cell homeostasis.

Figure 1

(A) Thymic grafting causes a significant increase in T cell pool size proportional to the number of grafted lobes. The T cell pool size of mice grafted with two, six, or nine thymic lobes was significantly higher than those of normal mice (two lobes, P < 0.001; six or nine lobes, P < 0.0001; unpaired Student’s t test) and significantly different from each other (P < .0001; ANOVA test). (B) The increase in T cell pool size caused by thymic grafting is consistent with separate regulation of thymic emigrants. Estimations of the extent of the increases caused by thymic grafting based on the number of grafted lobes were made by using a modified theory of peripheral T cell homeostasis. This theory proposes that emigrant T cells survive outside the restrictions of homeostatic regulation for up to 3 weeks postthymic export. The resultant estimates closely matched the pool sizes of mice engrafted with two, six, and nine thymic lobes.

Figure 2

Changes in T cell pool size are the direct result of changed thymic export rates. Six-week-old mice were grafted with six thymic lobes, and CD4, CD8, and overall T cell pool sizes were compared with those of nongrafted controls after a further 8 or 16 weeks. Although a significant difference was seen between grafted and nongrafted mice (P < .0001, unpaired Student’s t test), no difference was observed between mice grafted for 8 or 16 weeks. A minimum of six mice were used per group, with error bars representing 1 SD from the mean.

Figure 3

The CD4/CD8 ratio of an emigrant T cell population falls with age. Donor-derived T cells from thymic grafts were detected in Ly5.1 recipient mice by using anti-Ly5.2, anti-CD4, and anti-CD8. Mean results from a minimum of five mice are indicated at each time point, with separate analysis performed for spleen, lymph nodes, and blood. Error bars represent 1 SD from the mean.

Figure 4

The CD4/CD8 ratio of the overall T cell pool of young mice (14 weeks) is significantly higher than that of aged mice (14 months). Peripheral T cells from the spleen and lymph nodes of young and old, nongrafted mice were labeled with anti-CD4 and anti-CD8, and the CD4/CD8 ratios were compared. Five mice were analyzed in each group. Error bars represent 1 SD from the mean.

Figure 5

(A) The increase in CD44hi cells among emigrant T cell populations postexport is gradual and does not indicate repertoire skewing. Donor-derived T cells from congenic thymic grafts were detected in the spleen, lymph nodes, and blood of Ly5.1 recipient mice by using anti-Ly5.2 in conjunction with anti-CD44, anti-CD4, and anti-CD8. A represents the mean proportion of CD44hi-expressing emigrant cells derived from recipient lymph nodes. Between 3 and 10 mice were assessed per group, with error bars representing 1 SD from the mean. B shows the Vβ profile of T lymphocytes (a similar distribution was observed for CD4 and CD8 subsets) derived from the lymph nodes of a mouse grafted for 9 weeks. This profile is representative of profiles from the spleen and lymph nodes from four different mice grafted with two thymic lobes for between 3 and 9 weeks.

Figure 6

T cells from thymic-grafted mice are not enriched for apoptotic cells. Lymphocytes from mice grafted with nine thymic lobes for 6 weeks were isolated from spleen, lymph nodes, and blood and stained with anti-Ly5.2, anti-CD4, and anti-CD8 mAbs and biotinylated annexin V as an indicator of pending apoptosis. Thymocytes cultured overnight were used as a positive control for annexin V binding. Data are representative of profiles from four grafted and four nongrafted mice.