Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency

Abstract

Background: Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ^null mice is feasible and facilitates the generation of functional T cells conferring protective immunity.

Methods: Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase⁺, CD45.1⁺) and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγ^null recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ^null mice was assessed in a B cell lymphoma model.

Results: Despite the small size of the thymic remnant in NOD-scid IL2rγ^null mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγ^null mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγ^null mice, suggesting that immune dysregulation is not a major concern.

Conclusions: Our findings suggest that intrathymic injection of donor hematopoietic stem and progenitor cells is a safe and effective strategy to establish protective T cell immunity in a mouse model of severe combined immunodeficiency.

Keywords: Cell therapy; Hematopoietic stem cell; NSG mouse; SCID; Thymus.

Fig. 1

Intrathymic injection of hematopoietic stem/progenitor cells induces a functional thymic microenvironment in NSG mice. a NSG recipients were intrathymically injected with 10,000 luciferase-expressing C57BL/6 LSK cells. The whole-body distribution of LSK-derived cells on day 15 after injection was monitored by in vivo BLI. Circles highlight the concentration of injected cells within the thymic area. Pseudocolor images superimposed on conventional photographs are shown. Four representative animals of ten are presented. b NSG recipients were intrathymically injected with 2000 C57BL/6 LSK cells. Thymuses were harvested 1 month after injection and analyzed for thymocyte subsets (DP, CD4 SP, CD8 SP, DN) (upper panel) and DN subsets (DN 1, 2, 3 and 4 subsets defined by expression of CD25 and CD44) (lower panel) by multicolor flow cytometry. Plots for one of three mice are presented. The thymus of an untreated NSG mouse was used as the control. c NSG mice were intrathymically injected with 10,000 luciferase-expressing C57BL/6 LSK cells. The thymus of an untreated NSG mouse was used as the control. Thymuses were harvested at day 60 and analyzed for cytokeratin (CK) 8 (red), CK 14 (green), and DAPI (blue) by immunofluorescence. Images were acquired with an Axio Imager (Zeiss) and processed with Zeiss ZEN imaging software. Scale bar 50 μm. One representative image is shown (n = 4). DP: CD4 and CD8 double-positive cells; DN: CD4 and CD8 double-negative cells; CD4 SP: CD4 single-positive cells; CD8 SP: CD8 single-positive cells

Fig. 2

Intrathymic injection of hematopoietic stem/progenitor cells establishes thymopoiesis in young and aged NSG mice. a NSG mice were intrathymically injected with 10,000 C57BL/6 LSK cells. Thymuses were harvested on day 56 and analyzed by flow cytometry. Left panel shows thymocyte subsets for untreated NOD mice included as a reference group. Center and right panels show frequency and absolute numbers for thymocyte subsets of injected NSG mice. Data combined from two independent experiments are shown. Mean and SEM are presented (n = 8–12). b NSG mice were treated as in (a). Spleens were harvested on day 56 and T cells were analyzed by flow cytometry. Combined data from two independent experiments are shown. Mean and SEM are presented (n = 8–10). c Mice were treated as in (a). Thymuses and spleens were harvested 5 months after injection and analyzed by flow cytometry. Left panel shows frequency of thymocyte subsets; center panel shows absolute numbers of thymocyte subsets, and right panel shows absolute numbers of splenic T cells. Data combined from two independent experiments are shown. Mean and SEM are presented (n = 8–12). d Mice were treated as in (a) for aged mice (6 months old) and young mice (6 weeks old). Thymuses and spleens were harvested at day 56. Panels 1 and 2: absolute numbers of thymocytes and DP thymocytes in young versus aged mice. Panels 3 and 4: absolute numbers of CD4⁺ and CD8⁺ splenic T cells in young versus aged mice. Data combined from two independent experiments are shown. Mean and SEM are presented (n = 8–12)

Fig. 3

The frequency of regulatory T cells is increased in CD4⁺ T cells developing in NSG mice following intrathymic injection of hematopoietic stem/progenitor cells. NSG mice were intrathymically injected with 10,000 C57BL/6 LSK cells. Spleens were harvested on day 56 and CD4⁺ T cell subsets were analyzed by flow cytometry. a Splenic CD25⁺FoxP3⁺ regulatory T cells (frequencies of CD4⁺ T cells) in intrathymically injected NSG mice and NOD control, untreated mice. Combined data from two independent experiments are shown. Mean and SEM are presented (n = 5–10). b The frequencies of Helios⁺ natural regulatory T cells that were generated by intrathymically injected NSG mice. Mean and SEM are presented (n = 9). c The ratio of CD4⁺ effector (CD62L⁻CD44⁺)/regulatory T cells in NOD control mice and intrathymically injected NSG mice. Mean and SEM are presented (n = 5–13)

Fig. 4

T cells derived from intrathymically injected NSG hosts are phenotypically and functionally similar to normal T cells. a Young versus aged NSG recipients were intrathymically injected with 10,000 C57BL/6 LSK cells. Spleens were harvested on day 56 and TCR Vβ families were analyzed by multicolor flow cytometry. Mean and SEM are presented (n = 5). Two normal C57BL/6 mice were included as control. b Female and male NSG hosts were intrathymically injected with 10,000 C57BL/6 LSK cells. Thymuses were harvested 5 months after injection. Thymocyte counts, frequency of thymocyte subsets, and absolute values of splenic T cell subsets were analyzed by multicolor flow cytometry. Mean and SEM are presented (n = 11–13). c NSG mice were treated as described in b. Spleens were harvested 5 months after injection and T cells were analyzed by flow cytometry. Mean and SEM are presented (n = 11–13)

Fig. 5

There is no evidence for major immune dysregulation in NSG mice that were intrathymically injected with hematopoietic stem/progenitor cells. a Female or male NSG recipients were injected intrathymically (IT) or intravenously (IV) with 10,000 C57BL/6 LSK cells. Non-fasting blood glucose readings were acquired from day −1 to day 149 using a glucometer. Mean and SEM are presented (n = 8–10). b Male NSG mice were intrathymically injected with 10,000 C57BL/6 LSK cells. Ears were clipped at 1 month after injection and analyzed histologically by H&E staining (upper panels) and immunohistochemistry (IHC) for CD3 (lower panels). Tissue sections were scanned with a Mirax Micro digital slide scanner (Zeiss) and viewed with Panoramic Viewer software (3DHISTECH Ltd.). Scale bar left panels and top right panel 50 μm; scale bar bottom center panel 100 μm. One representative image is shown (n = 3). An age- and sex-matched untreated NSG mouse was included as control. Bottom right panel: mean and SEM of T cell counts based on CD3 IHC analysis of treated NSG mice are presented. Cell numbers were determined by quantification of the CD3 staining with ImageJ software; measurements were corrected for background signal (based on untreated control NSG mice) (n = 3)

Fig. 6

T cells developing in intrathymically injected NSG hosts display anti-lymphoma activity. a NSG mice were intrathymically injected with 10,000 C57BL/6 LSK cells or PBS. Five weeks after intrathymic injection, A20-TGL mouse lymphoma cells were injected intravenously into all mice. The whole body distribution of A20-TGL luciferase-expressing cells was monitored by in vivo BLI. Pseudocolor images superimposed on conventional photographs are shown (n = 7–8). b Mice in the tumor challenge group were treated as in(a). Spleens were harvested 2 months post tumor challenge. Mice in the no tumor challenge group were treated as follows: NSG mice were intrathymically injected with 10,000 C57BL/6 LSK cells. No A20-TGL cells were administered. Spleens were harvested on day 56 after LSK cell injections. In both groups, splenic CD8⁺ T cells were analyzed for expression of CD62L and CD44 by multicolor flow cytometry. Naïve: CD62L⁺CD44⁻; central memory: CD62L⁺CD44⁺; effector memory: CD62L⁻CD44⁺. One representative dot plot is shown. Mean and SEM are presented (n = 6)