Intrathymic transplantation of bone marrow-derived progenitors provides long-term thymopoiesis

Abstract

The sustained differentiation of T cells in the thymus cannot be maintained by resident intrathymic (IT) precursors and requires that progenitors be replenished from the bone marrow (BM). In patients with severe combined immunodeficiency (SCID) treated by hematopoietic stem cell transplantation, late T-cell differentiation defects are thought to be due to an insufficient entry of donor BM progenitors into the thymus. Indeed, we find that the intravenous injection of BM progenitors into nonconditioned zeta-chain-associated protein kinase 70 (ZAP-70)-deficient mice with SCID supports short- but not long-term thymopoiesis. Remarkably, we now show that the IT administration of these progenitors produces a significant level of donor-derived thymopoiesis for more than 6 months after transplantation. In contrast to physiologic thymopoiesis, long-term donor thymopoiesis was not due to the continued recruitment of progenitors from the BM. Rather, IT transplantation resulted in the unique generation of a large population of early c-Kit(high) donor precursors within the thymus. These ZAP-70-deficient mice that received an IT transplant had a significantly increased prothymocyte niche compared with their untreated counterparts; this phenotype was associated with the generation of a medulla. Thus, IT administration of BM progenitors results in the filling of an expanded precursor niche and may represent a strategy for enhancing T-cell differentiation in patients with SCID.

Figure 1

IT but not intravenous injection of WT BM progenitors into nonconditioned ZAP-70–deficient mice results in long-term thymopoiesis. (A) CD45.2⁺ ZAP-70^−/− mice were injected with lineage negative (lin⁻) BM progenitor cells (2 × 10⁵) isolated from CD45.1⁺ WT mice by either intravenous (IV) or IT routes. Animals were killed 20 to 25 weeks later, and the percentages of CD45.1⁺ cells in the lymph nodes were assessed. Dot plots showing representative CD45.1 and CD3 staining of WT and ZAP-70^−/− mice reconstituted by intravenous and IT administration of BM progenitors are shown (top). The percentages of naive (CD44^intCD62L^hi), central memory (CD44^hiCD62L^hi), and effector (CD44^hiCD62L^lo) T cells in these mice were monitored by assessing CD62L and CD44 expression in gated donor CD3⁺ lymphocytes. Representative dot plots are shown, and the percentages of each population are indicated (bottom). (B) Representative dot plots showing the percentages of CD45.1⁺ cells in the thymi of CD45.1 WT and CD45.2 ZAP-70^−/− mice reconstituted by IV or IT injection of WT CD45.1⁺ progenitors. The accompanying graph shows the absolute numbers of CD45.1⁺ donor thymocytes in ZAP-70^−/− mice reconstituted by intravenously (n = 8) and intrathymically (n = 10) administered progenitors. ***P < .001. (C) CD4/CD8 profiles of thymic donor cells were assessed after gating on CD45.1⁺ thymocytes. Representative dot plots from a control WT mouse and intravenously and intrathymically reconstituted ZAP-70^−/− mice are shown. The percentages of cells in each gate are indicated.

Figure 2

Donor-derived BM progenitors contribute to all stages of thymopoiesis after their IT administration. (A) CD45.1⁺ WT lin⁻ BM cells (2 × 10⁵) were injected intrathymically in ZAP-70^−/− mice. Thymi were harvested between 20 and 25 weeks after injection, and the percentages of donor CD45.1⁺ thymocytes in each thymocyte subset were analyzed. Representative histograms show the percentages of donor TN1, TN2, TN3, TN4, immature single-positive (ISP), CD3⁻ DP, CD3⁺ DP, CD4 SP, as well as CD8 SP thymocytes. (B) Bar graph quantification showing the percentages of CD45.1⁺ donor thymocytes at each maturation stage are presented as means ± SDs (n = 11).

Figure 3

Differentiation of non–T-lineage cells is significantly lower after IT administration of WT BM progenitors. (A) ZAP-70^−/− mice were injected with WT CD45.1⁺ lin⁻ BM progenitor cells (2 × 10⁵) by either intravenous (IV) or IT routes. Animals were killed 20 to 25 weeks later, and donor cells in the spleen were assessed by CD45.1 staining. Within the CD45.1⁺ gate, the relative percentages of granulocyte (Gr1^high) and B-lineage (CD19⁺) donor cells (top) as well as CD4 and CD8 T cells (bottom) in representative WT mice as well as ZAP-70^−/− mice reconstituted by intravenous (IV) and IT administration of donor progenitors are indicated. (B) The graphs show the absolute numbers of LN donor B lymphocytes, granulocytes, and T cells in ZAP-70^−/− mice reconstituted by IV (n = 8) and IT (n = 9) injection of donor progenitors. *P = .01-.015; **P = .008.

Figure 4

Long-term thymic differentiation of donor progenitors in IT-injected mice does not result in the engraftment of BM progenitors with secondary repopulating ability. (A) ZAP-70^−/− mice were injected intravenously or intrathymically with CD45.1⁺ WT lin⁻ BM cells (2 × 10⁵). Bone marrow was harvested 20 to 25 weeks after transplantation, and the absolute numbers of lineage-negative (negative for Ter119, B220, Mac-1, Gr-1, and CD3 staining) CD45.1⁺ cells in the BM were assessed in mice who received an intravenous (IV) transplant (n = 5) and mice that received an IT transplant (n = 4). Absolute numbers of engrafted lin⁻/Sca-1⁺/c-Kit⁺ cells were also determined. *P = .02. (B) Secondary repopulation ability was assessed by transplanting T cell–depleted BM (9 × 10⁶) from these intravenously and intrathymically reconstituted ZAP-70^−/− mice (25 weeks after transplant) into lethally irradiated CD45.2⁺ ZAP-70^−/− mice (9 Gy [900 rad]). As a control, lethally irradiated CD45.2⁺ ZAP-70^−/− mice received a transplant with CD45.1⁺ T cell-depleted BM (9 × 10⁶) from WT mice. The presence of CD45.1⁺ cells in the spleen was assessed 13 weeks later. Representative dot plots showing the percentages of splenic CD45.1⁺ donor B/T lymphocytes are presented. (C) The relative levels of CD45.1⁺ donor cells in the thymi of secondary reconstituted mice are shown in representative dot plots. Results represent data obtained in 3 independent mice.

Figure 5

WT thymic precursors do not sustain long-term thymopoiesis in ZAP-70–deficient hosts. (A) ZAP-70^−/− mice were intrathymically transplanted with DN thymocytes or lineage-negative (lin⁻) BM progenitor cells (2 × 10⁵) isolated from CD45.1⁺ WT mice. Eight weeks later, donor engraftment was assessed by monitoring the percentages of cells expressing the CD45.1 allele. A thymus from a CD45.1⁺ WT mouse is shown as a control. (B) The phenotype of donor cells was assessed by CD4 and CD8 staining of gated CD45.1⁺ thymocytes. A representative dot plot from a control CD45.1⁺ WT mouse is shown. The percentage of cells in each gate is indicated.

Figure 6

IT BM transplantation results in increased WT c-Kit^high thymic precursors and a high progenitor niche occupancy. (A) The phenotype of immature donor thymocytes was assessed in ZAP-70^−/− mice 25 weeks after IT administration of WT CD45.1⁺ BM progenitor cells. CD25/CD44 profiles of donor CD45.1⁺ and recipient CD45.1⁻ TN thymocytes were evaluated to distinguish TN1, TN2, TN3, and TN4 populations, and representative dot plots are shown. Control dot plots of TN thymocytes from CD45/1⁺ WT and ZAP-70^−/− mice not receiving a transplant are shown. (B) The presence of early progenitors within gated CD44⁺/CD25⁻ TN1 thymocytes was assessed by c-Kit staining. (C) Graph shows the absolute numbers of c-Kit^high thymocytes within the TN1 subset of WT, ZAP-70^−/−, and intrathymically reconstituted ZAP-70^−/− mice. The graph in the inset shows the relative percentages of c-Kit^high thymocytes within the donor and recipient populations from ZAP-70^−/− mice that received an IT transplant (n = 5). **P = .008. (D) Hematoxylin and eosin staining of transverse thymus sections from WT, ZAP-70^−/−, and intrathymically reconstituted ZAP-70^−/− mice. Thymi from WT, but not ZAP-70^−/−, mice are normally structured with densely packed cortical regions and less dense medullary regions, whereas thymi from intrathymically reconstituted mice show the formation of an extensive medulla.