Identification of a human recent thymic emigrant phenotype

Abstract

The ability to measure human thymic output would be an invaluable tool for the study of the development of the naive T cell repertoire, as well as naive T cell regeneration after intensive cytotoxic chemotherapy or effective antiretroviral therapy of progressive HIV infection. We and others have demonstrated previously that quantification of T cell receptor rearrangement excision circles (TREC) within peripheral T cell populations provides insight into the frequency of recent thymic emigrants (RTE) and, therefore, into thymic function. However, measurement of RTE by this approach is complicated by the fact that TREC levels also are determined by turnover within the naive T cell compartment. Here, we report a phenotypic approach to RTE measurement. We demonstrate that alphaE integrin (CD103) expression is up-regulated very late in thymic development on a subset of CD8(+)/CD4(-) thymocytes and also defines a distinct subset of naive CD8(+) T cells in the periphery. The latter subset is differentiated from circulating CD103(+) mucosa-associated memory T cells by its naive T cell phenotype (CD45RO(-), CD62L(bright), CD27(bright), CD11a(dim), CD95(dim)) and its high concentration of TREC. Indeed, sorted CD103(+) naive CD8(+) cells display higher levels of TREC than their CD103(-) naive counterparts, and these cells demonstrate an age-related decline in frequency that is enhanced significantly by thymectomy. The thymic dependence of this subset and the cells' relatively evanescent presence in the periphery suggest that these cells are a population of RTE and that quantification of their frequency in peripheral blood provides an estimate of the level of ongoing thymopoiesis.

Figures

Figure 1

Multiparameter flow cytometric analysis of CD103 expression among thymocytes and peripheral blood CD8+ T cells. (A) Thymocytes were examined for their correlated expression of CD3, CD4, CD8, and CD103. Five thousand events, gated on viable cells, are shown in each plot. CD103+ events are colored red (with the percentage of these cells given in the upper right corner of the left plot), whereas CD103− events are colored green. The boxed area in the right plot delineates mature CD3bright, CD8 single positive thymocytes with the percentage of CD103+ within this population indicated. (B and C) Mononuclear cells from umbilical cord blood (B) and adult blood (C) were examined for their correlated expression of (i) CD8, CD103, CD45RO, and CD27 and (ii) CD8, CD103, CD45RO, and CD62L. Five thousand (B) or 10,000 (C) events, gated on viable CD8+ small lymphocytes, are shown in each plot. In these analyses, the CD8 vs. CD103 profiles and the percent positive for the delineated subsets essentially were identical for the two staining combinations. In B, CD103+ events are colored red (with the percentage of these cells given in the upper right corner of the left plot), whereas CD103− events are colored green. Note that cord blood CD8+ T cells are almost entirely naïve in phenotype (CD27bright, CD62Lbright, CD45RO−). In C, CD103+ events falling within the naïve cell cluster (putative RTE) are enlarged and colored red, whereas all other CD103+ events (memory) are colored blue (with the respective percentage of these two populations given in the upper right corner of the left plot in the same color).

Figure 2

CD8 TCR excision circles per 105 CD8+ T cells in peripheral blood (CD8 TREC) as a function of age in euthymic (○) and remotely (>3-year) athymic (●) subjects. Age 0 samples refer to umbilical cord blood.

Figure 3

Quantification of CD103-defined subsets among mature CD8+ thymocytes and peripheral blood CD8+ naïve T cells. (A) Percentage (log scale) of CD8+/CD4−/CD3+ thymocytes expressing CD103 in thymi removed from subjects ranging in age from 6 weeks to 20 years. (B) Percentage (log scale) of CD8 T cells expressing a naïve phenotype (CD62L+, CD45RO−) and CD103 from the peripheral blood of euthymic from birth to 73 years of age (○) and from remotely (>3-year) athymic individuals from ages 20 to 73 years (●). (C) Percentage (linear scale) of total naïve T cells (CD27+, CD62L+, CD45RO−) with the CD8+ subset from peripheral blood from the same individuals as shown in B.

Figure 4

Relative percentage of CD103+ naïve and total naïve T cells within the CD8+ subset and the relative number of CD8 TREC per 105 CD8+ T cells in two subjects at the time of (solid bars) and 6 months after (shaded bars) thymectomy.