Thymus-autonomous T cell development in the absence of progenitor import

Abstract

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

Figure 1.

T cell development in thymus grafts in the absence of donor HSC engraftment. (A) Newborn wild-type thymi were transplanted into Rag2^−/−γ_c^−/−Kit^W/Wv recipients (n = 57), and analyzed 9–11 wk later. One thymus graft representative of sustained T cell development (left column), and the endogenous thymus of the same recipient (middle column) are shown. Rag1^−/− recipients (n = 3) were transplanted as controls and analyzed at 6 wk (right column). Dot plots display cells pregated for thymus donor (CD45.1⁺) or host origin (CD45.1^-), and stained for CD4 and CD8 expression. Numbers indicate percentages of cells in each quadrant. (B) CD45.1⁺ thymus grafts implanted into CD45.1⁻ Rag2^−/−γ_c^−/−Kit^W/Wv (n = 57) or Rag1^−/− (n = 7) recipients were analyzed 9–11 wk later by flow cytometry for the percentage of DP thymocytes (left), for the percentage of CD45.1⁺ donor cells per total cells (middle), and for absolute numbers (right) of CD45.1⁺ donor-derived thymocytes within each graft. Each symbol represents an individual lobe. (C–F) CD45.1⁺ wild-type mice (B6/Ly5.1), nontransplanted Rag2^−/−γ_c^−/−Kit^W/Wv mice, and a representative Rag2^−/−γ_c^−/−Kit^W/Wv recipient (n = 57) at 9–11 wk after thymus transplantation were analyzed for the presence of HSCs (Lin⁻Sca1⁺Kit⁺; C; gated on Lin⁻), T cells (CD3⁺CD19⁻), B cells (CD3⁻CD19⁺; D; total splenocytes), NK cells (CD3⁻NK1.1⁺), NKT cells (CD3⁺NK1.1⁺; E; gated on CD19⁻ splenocytes), and myeloid cells (CD11b⁺ cells expressing varying levels of Gr1; F; gated on CD4⁻CD8⁻CD3⁻CD19⁻Ter119⁻ splenocytes). Percentages shown in F next to the corresponding FACS plots refer to percentage of donor (CD45.1⁺) or host (CD45.2⁺) cells. Numbers indicate percentages of cells in each gate.

Figure 2.

Sustained T cell development and T cell export visualized by RAG2p-GFP reporter. (A) Newborn thymi from RAG2p-GFP reporter mice were transplanted into Rag2^−/−γ_c^−/−Kit^W/Wv recipients. RAG2p-GFP control thymus, 2 RAG2p-GFP grafts 10 wk after transplantation (graft A and graft B are two lobes from the same thymus that were separated at the time of transplantation), and the endogenous thymus of the Rag2^−/−γ_c^−/−Kit^W/Wv host are shown. Expression of CD4 and CD8 was analyzed in CD45.1⁺ donor cells, gated for GFP⁺ and GFP⁻. Plots are representative of one out of four experiments, each with four to seven recipients. (B) CD45.1⁺CD3⁺ cells from peripheral blood of a B6/Ly5.1 control and from a representative Rag2^−/−γ_c^−/−Kit^W/Wv host bearing RAG2p-GFP grafts are shown. A total of 19 recipient mice were analyzed at the indicated time points in weeks (w) for the presence of GFP⁺CD4⁺ (top row) and GFP⁺CD8⁺ (bottom row) recent thymic emigrants. Numbers indicate percentages of cells in each quadrant.

Figure 3.

Intrathymic differentiation in the absence of continuous progenitor import. (A and B) 9 wk after thymus transplantation, Rag2^−/−γ_c^−/−Kit^W/Wv recipients and C57BL/6 control mice received 1 mg BrdU and thymocytes were analyzed 2 h or 5 d later. Dot plots in A show expression of CD4 and CD8 by BrdU⁺ cells and represent examples of individual thymus grafts. Numbers indicate percentages of cells in each region. (B) Percentages of BrdU⁺ cells within each indicated population (DN, DP, CD4, and CD8) were analyzed 2 h and 5 d after BrdU injection. Results from one experiment are shown, where each dot is an individual C57BL/6 thymus (B6) or an individual thymus graft (G). The 2-h pulse labeling was done in 3 independent experiments, in which 3, 4, or 8 grafts with DP were analyzed. The 5-d chase experiment was performed once, and 9 grafts with DP were analyzed. (C) B6/Ly5.1 control thymus, and a representative graft after 10 wk in a Rag2^−/−γ_c^−/−Kit^W/Wv host, were analyzed for the indicated markers. Cells stained for CD44 and CD25 were pregated as Lin⁻CD45.1⁺ donor cells, and cells stained for CD44 and Kit were pregated as Lin⁻CD25⁻CD45.1⁺ donor cells. Numbers indicate percentages of cells in each gate. A total of 3 independent experiments were performed, in each of which 3–10 grafts with cellularities >10⁶ cells were analyzed.

Figure 4.

TCR diversity under conditions of thymus autonomy. TCR α and β chain analysis was performed on cDNA of total thymocytes derived from control mice (T; n = 2) or from grafts (G; n = 4). (A and B) Frequencies of V-J pairings are represented as heat maps for TCR β (A) and TCR α (B) chains. Each graph displays the number of times (in percentage; color in the bottom of panel B) defined V and J segments were paired in the TCR rearrangements identified. (C and D) The quantitative contribution of individual CDR3 clonotypes is represented as retrieval frequency (percentage of all TCR sequences) for β (C) and for α (D) chains, and was determined by counting identical CDR3 nucleotide sequences. The 10 most frequent CDR3 clonotypes are shown as colored bars, gray bars represent the remaining TCR sequences. Grafts were analyzed 10 wk after transplantation.

Figure 5.

Competition between thymus-intrinsic and -extrinsic T cell progenitors is regulated by cytokine receptors. (A) Newborn thymus lobes were grafted into the indicated recipients. After 9 wk, cells of donor thymus and host origin were analyzed for CD4 and CD8 expression. Numbers are percentages of cells in each quadrant. Numbers of analyzed recipients are displayed in B. (B) Summary of the proportions of persistent (filled bars) versus exhausted (open bars) T cell development in thymus grafts. Exhausted refers to recipients in which both grafted lobes contained only circulating SP T cells but no DP thymocytes, whereas persistent indicates recipients with ongoing T cell development (presence of DP and SP cells) in at least one lobe. Number (n) indicates the total number of recipients. Data in B were obtained 9 to 11 wk after transplantation.