A non-leaky Artemis-deficient mouse that accurately models the human severe combined immune deficiency phenotype, including resistance to hematopoietic stem cell transplantation

Abstract

Two Artemis-deficient (mArt(-/-)) mouse models, generated independently on 129/SvJ backgrounds, have the expected T(-)B(-)NK(+) severe combined immune deficiency (SCID) phenotype but fail to mimic the human disease because of CD4(+) T cell leakiness. Moreover, immune reconstitution after hematopoietic stem cell transplantation is achieved more readily in these leaky mouse models than in Artemis-deficient humans. To develop a more clinically relevant animal model, we backcrossed the mArt(-/-) mutation onto the C57Bl/6 (B6) background (99.9%), which resulted in virtually no CD4(+) T cell leakiness compared with 129/SvJ mArt(+/-) mice (0.3% +/- 0.25% vs 19.5% +/- 15.1%, P < .001). The nonleaky mouse also was uniquely resistant to engraftment using allogeneic mismatched hematopoietic stem cells, comparable to what is seen in human Artemis deficiency. The genetic background also influenced Artemis-associated radiation sensitivity, with differing degrees of x-ray hypersensitivity evident in 129/SvJ and B6 backgrounds with both the mArt(-/-) and mArt(+/-) genotypes. Our results indicate that immunogenic and DNA repair phenotypes associated with Artemis deficiency are significantly altered by genetic background, which has important implications for the diagnosis and treatment of SCID. Moreover, the B6 mArt(-/-) mouse provides a more accurate model for the human disease and a more appropriate system for studying human Artemis deficiency and for developing improved transplantation and gene therapy regimens for the treatment of children with SCID.

Figure 1. CD4⁺(A) or CD8⁺(B) T-cells in 129/SvJ and B6 wt and mArt^-/-mice

Each bar represents the mean percent ± standard deviation of CD4⁺ or CD8⁺ T cells gated on CD45⁺ CD3⁺ lymphocytes in blood, spleen, thymus, and lymph node.

Figure 2. Flow cytometric analysis of CD4⁺ and CD8⁺ lymphocytes in a representative leaky 129/SvJ N9 mArt^-/- mouse and non leaky B6 N10 mArt^-/- mouse

WT B6 LN (lymph node) was used as a control. Percent expressed as number of CD4⁺, CD8⁺, or CD4⁺CD8⁺ cells gated on CD45⁺CD3⁺ lymphocytes.

Figure 3. Radiation sensitivity assays

Mouse embryonic fibroblasts (MEFs) were scored for proliferation 48 hours after x-ray exposure based on BrdU containing G1 cells relative to untreated controls. Cell line details are denoted in the legend by cell line number, mouse strain, and Artemis genotype. (A) Radiation sensitivity of GSE-22 transformed mArt^+/+, mArt^+/- and mArt^-/- on 129/SvJ background. (B) Radiation sensitivity of GSE-22 transformed mArt^+/+, mArt^+/- and mArt^-/- on B6 background. (C) Radiation sensitivity of SV40 transformed mArt^+/+, mArt^+/- and mArt^-/- on 129/SvJ background, derived from different animals. (D) Radiation sensitivity of additional mArt^-/- lines from additional animals of noted backgrounds immortalized with GSE-22 (top graph) or SV40 (lower graph).

Figure 4. Immune reconstitution in a representative B6 WT mouse, mArt^-/- mouse, congenic transplanted mArt^-/- mouse, and a congenic transplanted mArt^-/- mouse treated with TBI (3 Gy)

T cell phenotyping was performed on tissues (peripheral blood (PB), spleen, lymph node (LN), and thymus) collected ≥3 months post transplant. B cell phenotyping was done on bone marrow (BM). Percent expressed as number of CD4⁺, CD8⁺, or CD4⁺CD8⁺ cells gated on CD3⁺ lymphocytes; mature B cells (IgM⁺ B220⁺) and immature B cells (IgM^- B220⁺) were gated on lymphocytes.

Figure 5. T and B cell proliferative responses, T cell receptor diversity, and specific IgM response post transplantation

(A) Proliferative responses of lymphocytes to anti-CD3 antibody and LPS. B6 wild type mice (positive controls) and mArt^-/- mice (negative controls) were compared to recipients of congenic HSCT with and without TBI at ≥3 months post transplant. Results (stimulated minus resting) are expressed as counts per minute (cpm) on a log scale. (B) TCR Vβ repertoire analysis. T cells were enriched from either thymus or spleen and prepared as in Materials and Methods. Four representative Vβ regions are shown from a representative B6 WT mouse, a leaky 129/SvJ mArt^-/- mouse, two mArt^-/- recipients of congenic HSCT (451 and 454), and two mArt^-/- recipients of congenic HSCT with TBI (658, 660). (C) Serum IgM-specific antibody response to NP-Ficoll after immunization (minus pre-immune response). The figure shows the average response from recipients (n=4) of congenic HSCT at ≥3 months post transplant. B6 mArt^+/- (n=7) and B6 mArt^-/- (n=4) mice were used as positive and negative controls, respectively. (D) Serum IgM-specific antibody response to NP-KLH after immunization (minus pre-immune response). Average response from recipients (n=3) of congenic HSCT with TBI (3Gy) are compared to B6 mArt^+/- (n=3) and B6 mArt^-/- (n=3) controls.