A novel model of SCID-X1 reconstitution reveals predisposition to retrovirus-induced lymphoma but no evidence of gammaC gene oncogenicity

Abstract

The emergence of leukemia following gene transfer to restore common cytokine receptor gamma chain (gammaC) function in X-linked severe combined immunodeficiency (SCID-X1) has raised important questions with respect to gene therapy safety. To explore the risk factors involved, we tested the oncogenic potential of human gammaC in new strains of transgenic mice expressing the gene under the control of the CD2 promoter and locus control region (LCR). These mice demonstrated mildly perturbed T-cell development, with an increased proportion of thymic CD8 cells, but showed no predisposition to tumor development even on highly tumor prone backgrounds or after gamma-retrovirus infection. The human CD2-gammaC transgene rescued T and B-cell development in gammaC(-/-) mice but with an age-related delay, mimicking postnatal reconstitution in SCID-X1 gene therapy subjects. However, we noted that gammaC(-/-) mice are acutely susceptible to murine leukemia virus (MLV) leukemogenesis, and that this trait was not corrected by the gammaC transgene. We conclude that the SCID-X1 phenotype can be corrected safely by stable ectopic expression of gammaC and that the transgene is not significantly oncogenic when expressed in this context. However, an underlying predisposition conferred by the SCID-X1 background appears to collaborate with insertional mutagenesis to increase the risk of tumor development.

Figure 1

Generation and analysis of animals transgenic for human γC. (a) Plasmid construct illustrating the CD2 promoter driven human γC transgene (Sa – Sal1, Xb – XbaI, Pc – PciI, Mu – MluI). (b) Expression of the human γC from the CD2 promoter was confirmed in the transgenic mice by flow cytometry analysis of human γC surface expression in thymocytes from a CD2-hγC transgenic and control mouse. Expression of mouse γC is also shown for comparison. (c) RT-PCR in tissue from spleen (Spl), kidney (Kid), and thymus (Thy) from either a CD2-hγC transgenic animal (lanes indicated by +) or a nontransgenic littermate (lanes indicated by −). Control RNA isolated from PG-13 cells without (ctrl⁻) or with human γC vector (ctrl⁺).

Figure 2

Thymus explanted from transgenic and control animals were analyzed for cell surface marker expression to characterize the cell phenotype. (a) Perturbation of the CD4:CD8 T-cell ratio was observed in the total cell population for the CD2-hγC transgenic mice (1:1) (n = 15) compared with (2:1) in littermate controls (n = 22). Data representative of a transgenic profile and littermate control is shown. Average population values are indicated in Table 1. (b) Transgenic CD8⁺ thymocytes displayed lower expression of HSA (CD24) when compared to nontransgenic controls, illustrating a more mature T-cell phenotype. These histograms were gated on the CD8SP population (quadrant B4 in a). (c) Total thymocyte populations were stained for HSA and TCRβ to show that CD2-hγC transgenic mice appear to have an increased percentage of mature lymphoid cells as represented by TCRβ⁺HSA⁻. Average mean ± s.d. values are given.

Figure 3

Lack of tumor acceleration by human γC in mice. (a) Tumor free survival in two CD2-hγC transgenic lines gCA (n = 45) and gCB (n = 19), and controls (n = 41) after infection with MoMLV. Animals were on a C57/B16xCBA background where wild-type controls were strain and generation matched littermates. In animals succumbing to neoplastic disease there was no significant difference in the rates of lymphoma development between either transgenic line or the controls (gCA versus control, P = 0.32; gCB versus control, P = 0.51). Neither was there a significant difference between the two transgenic lines (gCA versus gCB, P = 0.15). (b) Tumor free survival of CD2-hγC/p53^−/− (n = 17) mice was indistinguishable (P = 0.63) to that observed with p53^−/− littermates without the human γC transgene (n = 15).

Figure 4

Restoration of γC-null lymphoid compartments was determined by organ weight and cell number at necropsy. The ratio of thymus to body weight (a) and thymic cellularity (b) is given for CD2-hγC reconstituted transgenic mice (where cohort numbers for (a) and (b) respectively are n = 18 & n = 3) compared to the strain matched γC^−/− (n = 15 & n = 7) and wild-type (n = 12 & n = 5) controls. *P < 0.05, **P < 0.01. (c) Western blots of thymus extracted from two individual transgenic and control animals showing expression of the transgene only in the CD2-hγC animals. Human γC was functional in the reconstituted animals as confirmed by phosphorylation of STAT5A/B. Loading was controlled with β-actin.

Figure 5

(a) Delayed restoration of T-cell populations in the thymus of γC^−/− mice reconstituted with human γC. Examples of CD4/CD8 profiles are given for CD2-hγC⁺/mγC^−/−, γC^−/− and strain-matched wild-type (mγC⁺) at 8 weeks (top) and 16 weeks (bottom) of age. At 16 weeks, the average percentages ± s.d. for CD4SP were 16.6 ± 8.0, 27.0 ± 16.0 and 17.6 ± 6.8, and for CD4/8DP populations 73.7 ± 11.0, 45.9 ± 20.5 and 74.5 ± 8.8 for the wt (n = 5), null (n = 7) and transgenic (n = 6) animals, respectively. (b) Delayed restoration of the B-cell population in the spleens of γC^−/− mice reconstituted with human γC. The panels show representative examples of viable splenocytes analyzed for immunoglobulin M and CD45R surface expression in three different hγC⁺/mγC^−/− animals in addition to γC^−/− (n = 5 at 8 weeks and n = 7 at 16 weeks) and strain-matched wild-type (mγC⁺, n = 5 at 8 weeks and n = 7 at 16 weeks) control mice. (c) Western blot analysis of human γC in sorted splenic B cells. Purified CD45R⁺ cells were isolated from CD2-hγC⁺/mγC^−/− spleens (n = 4) and analyzed for γC expression. As the antibody crossreacts with murine γC, a positive signal was evident in wild-type mouse spleen, but not in the splenocytes of mγC^−/− animals. β-Actin is shown to control for loading. The hγC⁺ control was the lysate from fibroblast cells (PG13) transfected with the γC vector.

Figure 6

Lymphoma-free survival of MoMLV infected CD2-hγC⁺/mγC^−/− (n = 40), mγC^−/− (n = 11) and strain matched mγC⁺ mice (n = 20). The presence or absence of the CD2-hγC transgene had no significant effect on lymphoma-free survival (P = 0.33), although mγC^−/− were markedly more sensitive to virus-induced lymphoma than wild-type controls (P < 0.001).