Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice

Abstract

In the absence of thymopoiesis, T lymphocytes are nevertheless present, mainly in the gut epithelium. Ontogeny of the extrathymic pathway and the extent of its involvement in euthymic mice are controversial. These questions have been addressed by assessing the expression of recombinase activating gene (RAG) through the use of green fluorescent protein RAG2 transgenic mouse models. In athymic mice, T lymphopoiesis occurs mainly in the mesenteric lymph node and less in the Peyer's patches. Ontogenic steps of this lymphopoiesis resemble those of thymopoiesis, but with an apparent bias toward gamma delta T cell production and with a paucity of oligoclonal alpha beta T cells possibly resulting from a deficit in positive selection. Whether in athymic or euthymic mice, neither T intraepithelial lymphocytes (IEL) nor cryptopatch cells (reported to contain precursors of IEL) displayed fluorescence indicating recent RAG protein synthesis. Newly made T cells migrate from the mesenteric node into the thoracic duct lymph to reach the gut mucosa. In euthymic mice, this extrathymic pathway is totally repressed, except in conditions of severe lymphocytic depletion. Thus, in normal animals, all gut T IEL, including CD8 alpha alpha(+) cells, are of thymic origin, CD8 alpha alpha(+) TCR alpha beta(+) IEL being the likely progeny of double negative NK1-1(-) thymocytes, which show polyclonal V alpha and V beta repertoires.

Figure 1.

GFP^H lymphocytes in tissue sections from nude or euthymic mice. (a) MLN, nude mouse: clusters of fluorescent GFP^H cells located in some medullary cords (empty spaces are lymphatic vessels of the medulla). (b) PP, nude mouse: rare GFP^H cells located in an interfollicular T area near lymphatic vessels. (c) Cryptopatch, nude mouse: lack of GFP^H cells in the patch and adjacent epithelium (∼10 cryptopatches were explored in various mice). (d) MLN, euthymic mouse: absence of GFP^H cells in the medulla. ×70.

Figure 2.

Analysis of GFP expression during T lymphopoiesis in athymic mice and thymopoiesis. (a–d) MLN (a and b) and thymus (c and d). (a and c) Comparative features of DP and SP cells analyzed for GFP (middle histograms) and TCRβ (right histograms) expressions. Note that DP cells express less TCRβ chains than SP and that there is a small difference between thymus and MLN DP cells in this respect. (b and d) TN CD25^H cells (after gating out CD3⁺, CD4⁺, CD8⁺, and CD19⁺ cells) analyzed for GFP content of CD44⁺ DN2 (middle histograms) and CD44⁻ DN3 cells (right histograms). All other TN cells were GFP⁻ and most of the MLN CD25^L cells had the phenotype of cryptopatch cells (reference 8). Lack or rarity of DN4 cells (CD44⁻ CD25⁻) results from gating out CD3 and coreceptors bearing cells with potent antibodies binding even cells with trace amounts of these molecules, as is the case of most DN4 cells. The percentages of c-Kit⁺ and IL7-Rα⁺ in CD25⁺ TN MLN cells and thymocytes are shown in the right panels. (e) Thymus: γδ⁺ cells analyzed for GFP content. (f–h) LP lymphocytes analyzed for GFP content. (f) DP cells are GFP^H and (g) TN CD25^H cells (after gating out of cells as described above) are GFP^H. (h) TN c-Kit⁺ CD25^+/− cells, belonging to a population of CD44⁺ Thy-1^+/− IL7-R⁺ CD4^+/− cells (not depicted), i.e., with the phenotype of cryptopatch cells (references and 20), are GFP⁻.

Figure 3.

Exploration of the memory phenotype and of the TCRβ repertoire of MLN αβ⁺ CD4⁺ cells of nude and euthymic mice. (a) MLN CD4⁺ cells from a nude mouse express the phenotype of memory cells, in contrast with comparable cells from an euthymic mouse. Cells were triply labeled with anti-TCRβ, anti-CD4, and anti-CD45RB mAbs. (b) The TCRβ repertoire of MLN DP cells from a nude mouse shows the gaussian-like profile characteristic of polyclonal populations (top), whereas that of SP CD4⁺ cells is oligoclonal (middle), in contrast with that of comparable cells from an euthymic mouse (bottom).

Figure 4.

Analysis of lymphopoiesis in various Tx or euthymic mice. (a) MLN cells, mouse Tx at birth (compare with Fig. 2 a). (b) MLN cells, 7-wk-old euthymic mouse, sham Tx (match of mouse with adult Tx shown in c). Presence of some DP with GFP and TCRβ expression similar to that of SP CD4⁺ cells (right panels). Note that many CD4⁺ cells are GFP^L. (c) MLN, 7-wk-old mouse 2 wk after thymectomy. All DP and SP cells are GFP⁻ (compare with b). (d and e) MLN cells from 8-wk-old Tg TCRβ^−/− mice, Tx (d) or sham Tx (e) 2 wk earlier, used for easy study of γδ⁺ cells. GFP^L cells are absent in the Tx mouse. (f) MLN cells of lethally irradiated non-Tg mouse reconstituted 18 d earlier with bone marrow cells from a nude Tg mouse. DP cells are GFP^H. Note that MLN cell recovery was 10-fold less than that from normal mice. (g) Lymphocytes isolated from the LP of a Tx RAGγc^−/− mutant mouse reconstituted 36 d earlier with nude Tg mouse bone marrow cells (note that some CD4⁺ GFP⁻ cells, originating from the graft, are detectable). (h) MLN cells from a 14-mo-old mouse (cell recovery was ∼20-fold less than in younger mice). DP cells with GFP expression comparable to that of DP thymocytes.

Figure 5.

GFP^L γδ¹ T cells in nude mice; comparison between MLN, circulating cells, and IEL. γδ⁺ T from MLN (a), thoracic duct lymphocytes (TDL; b), and IEL (c) analyzed for GFP expression.

Figure 6.

Analyses of DN αβ¹ thymocytes. Studies of (a) GFP content and (b) NK1-1 expression (in this last case, cells were from a B6 mouse because the Tg mice do not express NK1.1). (c) TCRα and β repertoires of DN NK1.1⁻ thymocytes: polyclonal repertoires expressing chains different from that peculiar to NKT cells.