The role of the thymus and recent thymic migrants in the maintenance of the adult peripheral lymphocyte pool

Abstract

The thymus is essential for the initial seeding of T cells to the periphery, but its role in maintaining the adult T cell pool remains poorly defined. We investigated whether changes to the rate of T cell export could form part of the mechanism(s) controlling the homeostatic regulation of the size and composition of the peripheral T cell pool. Using neonatal thymi grafted under the kidney capsule, we found that irrespective of whether the pool was oversupplied (by thymic grafts) or undersupplied (due to neonatal thymectomy), the thymic export rate was constant from both the host and graft thymus, and the periphery remained constant in size. Recent thymic emigrants (RTE) were also tracked to determine the extent of their acceptance into the T cell pool of a normal mouse. As a population, RTE are phenotypically mature, but were distinct from resident T cells in the periphery, being released in a CD4/CD8 ratio approximately twice that of established peripheral T cells. This export ratio is similar to that of T cells in the mature thymic compartment, but soon after entry into the periphery, the ratio falls, indicating separate thymic and peripheral regulation of the CD4/CD8 ratio. RTE may also be preferentially incorporated into the periphery, causing displacement of resident T cells, thus maintaining the size of the peripheral pool. Although not vital for the maintenance of a functional T cell pool, the acceptance of RTE in a "full" peripheral pool would ensure that the T cell receptor repertoire is kept diverse and that the T cell population encompasses a broad range of naive as well as memory T cells.

Figure 1

Ly5.2⁺ thymic lobes grafted beneath the kidney capsule of Ly5.1 congenic mice are progressively reconstituted by host-derived cells. Fig. 1 shows graft- and host-derived thymocytes at markedly different stages of maturity. Despite the different profiles of thymocytes of graft and host origin, the profile of the total thymocytes in the graft remains normal. The progression of Ly5.2⁺ cells through the various thymus compartments is shown by the increasingly mature phenotype over the first 3 wk, coupled with the decreasing proportion of total thymocytes they represent. By week 4, the grafted thymus has been reconstituted completely by host-derived stem cells, indicative of the absence of long-term resident thymocytes. Profiles are typical examples of results. 5–10 mice were used at each time point.

Figure 2

The size of the peripheral T cell pool does not increase when an additional thymus is grafted beneath the kidney capsule. Examination of lymphoid organs 8 wk after engraftment of two neonatal thymic lobes revealed no significant difference between the size of the thymus, spleen, or lymph nodes of grafted animals and those of nongrafted littermates. Results are from 16 grafted and 9 nongrafted animals. Error bars represent 1 SD from the mean.

Figure 3

Thymic export rates, as measured by intrathymic FITC injection, are not reduced in thymic-grafted mice. T cell emigration rates from the mature (8-wk-old) thymus of normal mice was compared with that of thymic-grafted mice. Despite no significant difference in the size of the peripheral T cell pools of grafted and nongrafted mice, the rate of T cell export remained constant. Both in situ and grafted thymi, irrespective of the presence or absence of the other, exported T cells at the rate of ∼0.7% of total thymocytes per day. This illustrated that grafted thymi were exporting cells in a proportion similar to that of an in situ thymus, and that no change to export rates from either thymus was induced by an increase in T cell supply to the peripheral pool. Each group contained 4 mice (total of 20). Error bars represent 1 SD from the mean.

Figure 4

Homeostatic control of total lymphocyte numbers can be overcome. Nine neonatal thymic lobes were grafted beneath the kidney capsule of 6-wk-old mice. Mice were killed 8 wk after grafting, and lymphocyte numbers and phenotypes were evaluated. The total lymphocyte pool increased by 15% over age-matched controls, due to a 30% increase in the T cell pool. B cell numbers were not affected by grafting. Four mice were used in each group (total of eight). Error bars represent 1 SD from the mean.

Figure 5

The CD4/CD8 ratio of RTE is distinct from that of peripheral T cells. For RTE detected in the periphery after intrathymic FITC injection, the CD4/CD8 ratio was 3.5, unlike the typical 1.5:1 ratio observed in the periphery. No difference was seen between the CD4/CD8 ratio of RTE from in situ thymi and those of grafted thymi. Using grafted Ly5.2 congenic thymi, migrant cells were examined 1 and 3 wk after export, and the CD4/CD8 ratio was found to vary between lymphoid organs. At both week 1 and week 3 after grafting, the spleen and lymph nodes carried recently exported cells that were still of a ratio higher than that of the resident cells but lower than that of the RTE. Significantly, cells in the blood exported within 3 wk of assay had a ratio lower than that of surrounding residents. For the overall pool and the RTE, 45 mice were used for spleen and lymph node results, 18 for blood. Five mice were used at the 1-wk time point, and 10 at the 3-wk time point. Error bars represent 1 SD from the mean.

Figure 6

The T cell export rate was correlated with the age of the graft by FITC injection at five time points up to 4 wk after grafting and again at 8 wk. The export rate rose steadily in the first 2 wk after grafting, and remained at 0.6–0.8 × 10⁶ cells/d until the final time point. A transient but consistent peak began at day 14 of 1.25 × 10⁶ cells/d, which may be associated with the extremely rapid growth of the graft at this time (see also Fig. 1). Each of the six time points represents the mean export rate from groups of three to seven mice. Error bars represent 1 SD from the mean.

Figure 7

T cells exported to a full periphery are accepted into the T cell pool, presumably at the expense of longer-term resident cells. The number of cells of graft origin (Ly5.2 ⁺) present in the peripheral pool at various time points (A) was compared with the number estimated to have been exported from the time of grafting (B), calculated using previously determined migration rates. In the 21 d after grafting, the number of graft origin cells found in the periphery matched closely the number estimated to have been exported over the same period at each of five time points. This suggests that RTE accumulated in the full periphery at the expense of resident T cells. Beyond 3 wk, the export of Ly5.2⁺ cells fell progressively due to reconstitution by host precursor cells. From this time, the peripheral Ly5.2⁺ population decayed, due in part to no new Ly5.2⁺ cells being exported. Each of the seven time points represents the mean of 3–10 mice. Error bars represent 1 SD from the mean.