Enhanced T-cell reconstitution by hematopoietic progenitors expanded ex vivo using the Notch ligand Delta1

Abstract

A physiologic role for Notch signaling in hematopoiesis has been clearly defined in lymphoid differentiation, with evidence suggesting a critical role in T-cell versus B-cell fate decisions. Previously, we demonstrated that activation of endogenous Notch receptors by culture of murine lin(-)Sca-1(+)c-kit(+) (LSK) hematopoietic progenitors with exogenously presented Notch ligand, Delta1(ext-IgG), consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG(1), promoted early T-cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show that culture of LSK precursors with Delta1(ext-IgG) increases the number of progenitors that are able to rapidly repopulate the thymus and accelerate early T-cell reconstitution with a diversified T-cell receptor repertoire. Most of the early T-cell reconstitution originated from cells that expressed lymphoid-associated antigens: B220, Thy1, CD25, and/or IL7Ralpha, whereas the most efficient thymic repopulation on a per cell basis originated from the smaller number of cultured cells that did not express lymphoid-associated antigens. These findings demonstrate the potential of Delta1(ext-IgG)-cultured cells for accelerating early immune reconstitution after hematopoietic cell transplantation.

Figure 1

Density-dependent effect of Delta1^ext-IgG on generation of T-cell progenitors. LSK cells were cultured for 28 days with Delta1^ext-IgG plated at increasing concentrations and were analyzed by FACS to determine the percentage of cells expressing CD44 and CD25. Numbers in right upper corner represent the percent of CD44⁺CD25⁺ cells plus or minus the standard error of the mean (SEM) of 5 separate experiments.

Figure 2

T-cell reconstitution by LSK cells cultured on immobilized Delta1^ext-IgG compared with noncultured LSK cells. (A) Lethally irradiated Ly5.1 C57BL/6 recipients received 5 × 10³ noncultured LSK cells or 1 × 10⁶ Ly5.2 Delta1^ext-IgG-cultured cells along with 1 × 10⁵ Ly5.1 BM cells. FACS analysis and CBCs were performed on blood samples obtained at 3 weeks after HCT to determine the number of Ly5.2⁺ CD3⁺, CD4⁺, CD8⁺, CD19⁺, GR1⁺, and F4/80⁺ cells in mice that received LSK cells cultured on Delta1^ext-IgG (closed bars) compared with noncultured LSK cells (open bar). (B) FACS analysis and CBCs were performed on blood samples obtained at various times after HCT to determine the number of Ly5.2⁺ CD3⁺ cells in mice that received LSK cells cultured on Delta1^ext-IgG (●) compared with noncultured LSK cells (▵). (C-E) FACS analysis was performed on thymuses at 1 week (C), 2 weeks (D), and 3 weeks (E) after HCT to determine the total number of Ly5.1 host and Ly5.2 donor DN1, DN2, DN3, CD4⁺, CD8⁺, or DP cells in mice that received noncultured Ly5.2 LSK cells or Ly5.2 LSK cells cultured on Delta1^ext-IgG. Data represent mean plus or minus SEM of 3 separate experiments (n = 10-15).

Figure 3

Analysis of the TCR Vβ expression by FACS. Lethally irradiated Ly5.1 C57BL/6 recipients received 1 × 10⁶ Ly5.2 Delta1^ext-IgG-cultured cells along with 1 × 10⁵ Ly5.1 BM cells. Blood samples obtained at 3 weeks after HCT were stained with monoclonal antibody against CD3 and TCR Vβ. The percentage of Vβ-positive cells among donor CD3⁺ lymphocytes in mice that received Delta1^ext-IgG-cultured cells (closed bars) and compared with control C57BL/6 mice (open bars) is shown. Data shown are the mean plus or minus SEM of 2 separate experiments (n = 7-10).

Figure 4

T-cell reconstitution in nonirradiated lymphoid cell–deficient mice by LSK cells cultured on immobilized Delta1^ext-IgG compared with noncultured LSK cells. Nonirradiated Ly5.1 Rag^−/− recipients received 1 × 10³ noncultured LSK cells or 1 × 10⁶ Ly5.2 Delta1^ext-IgG-cultured cells. FACS analysis and CBCs were performed on blood samples obtained at various times after HCT to determine the number of Ly5.2⁺ CD3⁺ cells in mice that received LSK cells cultured on Delta (■) compared with noncultured LSK cells (▵). Data represent the mean plus or minus SEM of 3 separate experiments (n = 5-10)

Figure 5

T-cell reconstitution by Delta1^ext-IgG-cultured cells transplanted with increasing doses of whole BM. (A) Lethally irradiated Rag^−/− recipients received 1 × 10⁵, 1 × 10⁶, or 1 × 10⁷ Ly5.1 BM cells augmented with 2.5 × 10⁷ Ly5.2 Delta1^ext-IgG-cultured cells. FACs analysis and CBCs were performed on blood samples obtained at various times after HCT. Panels A to E show the absolute number of cells expressing CD3⁺, CD4⁺, CD8⁺, CD19⁺, and Gr1⁺ cells contained in blood samples from Rag^−/− recipients that received, respectively, 1 × 10⁵, 1 × 10⁶, or 1 × 10⁷ Ly5.1 BM cells augmented with 1 × 10⁷ Ly5.2 Delta1^ext-IgG-cultured cells. Graphs depict engraftment from BM cells (▵) and engraftment from Delta1^ext-IgG-cultured cells (●). Data represent the mean plus or minus SEM of 3 separate experiments (n = 10-15).

Figure 6

T-cell potential of Sca-1⁺c-Kit⁺ cells generated by culturing with Delta1^ext-IgG. (A) Cultured LSK (Ly5.2) cells were stained for Sca-1 and c-Kit and sorted into a fraction containing Sca-1⁺ c-Kit⁺ cells and another fraction containing the rest of the population (Sca-1⁻ cKit⁺, Sca-1⁺c-kit⁻, and Sca-1⁻c-kit⁻) and injected into lethally irradiated C57BL/6 mice with 1 × 10⁵ Ly5.1-competing BM cells. (B) CBCs and FACS analysis were performed on blood samples at 3 weeks after HCT to determine the number of CD3⁺ cells generated in mice receiving either Sca-1⁺ c-Kit⁺ cells (○) or Sca-1⁻cKit⁺, Sca-1⁺c-kit⁻, and Sca-1⁻c-kit⁻ (▿). Data represent the mean plus or minus SEM. (C) Cultured cells were gated for Sca-1– and c-kit–expressing cells and sorted according to the presence or absence of B220, CD25, Thy1, or Il7Rα expression. (D) Lethally irradiated C57BL/6 recipients received 1 × 10⁶ Ly5.2 SK-lymphoid–positive cells and 1 × 10⁵ Ly5.1 BM cells (closed bar) or 1 × 10⁴ Ly5.2 SK-lymphoid–negative cells and 1 × 10⁵ Ly5.1 BM cells (open bar). FACs analysis and CBCs were performed on blood samples obtained at various times after HCT. At 5 weeks, lymphoid (CD3, CD4, CD8, and CD19) and myeloid (GR1 and F4/80) engraftment were compared between the 2 groups. Data represent the mean plus or minus SEM.