Hematopoietic stem/progenitor cell transplantation recovers immune defects and prevents lymphomas in Atm-deficient mice

Abstract

Background

Ataxia-telangiectasia (A-T) is a rare autosomal recessive multi-system and life-shortening disease, characterized by progressive cerebellar neurodegeneration, immunodeficiency, radiation sensitivity and cancer predisposition, with high incidence of leukemia and lymphoma. A-T is caused by mutations in the gene encoding for ATM protein that has a major role in maintaining the integrity of the genome. Because there are no cures for A-T, we aimed to tackle immunodeficiency and prevent cancer onset/progression by transplantation therapy.

Methods

Enriched hematopoietic stem/progenitor cells (HSPCs), collected from bone marrow of wild-type mice, were transplanted in the caudal vein of 1 month old conditioned Atm^−/− mice.

Results

Genomic analyses showed that transplanted Atm positive cells were found in lymphoid organs. B cells isolated from spleen of transplanted mice were able to undergo class switching recombination. Thymocytes were capable to correctly differentiate and consequently an increase of helper T cells and TCRβ^hi expressing cells was observed. Protein analysis of isolated T and B cells from transplanted mice, revealed that they expressed Atm and responded to DNA damage by initiating an Atm-dependent phosphorylation cascade. Indeed, aberrant metaphases were reduced in transplanted Atm-deficient mice. Six months after transplantation, Atm^−/− mice showed signs of aging, but they maintained the rescue of T cells maturation, showed DNA damage response, and prevented thymoma.

Conclusion

We can conclude that wild-type enriched HSPCs transplantation into young Atm-deficient mice can ameliorate A-T hematopoietic phenotypes and prevent tumor of hematopoietic origin.

To the Editor,

Ataxia-Telangiectasia (A-T) is a rare recessive autosomal disorder, commonly referred as genome instability syndrome and as primary immunodeficiency (PID). A-T patients suffer of neurodegeneration, aging, sterility, radiosensitivity and cancer predisposition. The cause of the disease are mutations in the ATM (ataxia telangiectasia mutated) gene that encodes for a serine/threonine kinase of the phosphatidylinositol-3-kinase related kinase (PI3KK) family, majorly involved in cell cycle regulation and DNA repair mechanisms [1]. Malignancies of hematological origin, specifically affecting B and T cell lineages, are frequent in A-T patients, with a high incidence of 21.7% by the age of 15 years [2,3,4]. The most common forms are Acute Lymphocytic Leukemias (ALL), non-Hodgkin’s and Hodgkin’s lymphomas. In older patients are observed Chronic T-Cell Leukemias (CCL) and solid tumors, including gastric, colon, pancreas, breast, liver, esophageal and basal cell carcinomas, ovarian dysgerminoma and uterine leiomyoma [3].

Currently, there is no cure for the disease and A-T patients die before their forties while treatments are only symptomatic and supportive [5,6,7]. Bone marrow heterologous transplantation is one of the therapeutic options that could tackle immunodeficiency, as it is for other PIDs, but due to the extreme radiosensitivity of the patients and non-myeloablative conditioning side effects, its use has been restricted and the outcomes are highly unpredictable [8]. Gene therapy strategies could be then a favourable alternative to the allo-transplantation, since correcting patients’ own cells will allow autologous transplantation, avoiding most of side effects such as graft vs host disease, tissue rejection, infections, and the need of Human Leukocyte Antigen (HLA)-identical related donors [9]. In this scenario, it becomes urgent to identify the critical hematopoietic cell population to be eventually engineered with Atm and transplanted to counteract immunodeficiency and hematopoietic cancer predisposition of A-T disease.

Previous works demonstrated the feasibility of bone marrow transplantation in Atm-deficient mice and the amelioration of the phenotype [10,11,12,13] and we have already proved that global Atm reactivation can revert A-T in transgenic mouse models [14]. In this study, we expand on prior research describing the transplantation and engraftment of wild-type hematopoietic stem/progenitor cells (HSPCs) into Atm^−/− mice as well as their effect on immunological markers, genomic stability and DNA damage response. Using magnetic beads, we selected Lineage negative and c-Kit positive (Lin⁻c-Kit⁺, LK) cells from bone marrow, that are known to be hematopoietic stem and progenitor cells [15] and so B and T cell progenitors. The final enriched LK population was around 3% of harvested cells and c-Kit⁺ cells both Sca-1⁺ and Sca-1⁻ reached around 80% of purification (Suppl. Figure 1a). This population was maintained 24 h after culture with the significant increase of c-Kit⁺Sca-1⁺ subpopulation (Suppl. Figure 1a), already known to be capable of reconstituting hematopoiesis in lethally irradiate wild-type mice [15]. A further characterization revealed that also the proportion of CD150⁺ hematopoietic stem cells (HSCs) was maintained, whereas a significant increase was observed in the CD48⁺ hematopoietic progenitor cells (HPCs) subset (Suppl. Figure 1b).

Before transplantation, we verified the efficacy of the conditioning approach on Atm^−/− mice [11,12,13], and extremely decreased T cells percentage was observed in peripheral blood for up to 7 weeks (Suppl. Figure 1c).

Then, we transplanted 3 to 5 million LK-enriched cells from wild-type male donors into conditioned Atm^−/− female recipients and analyzed the LK-transplanted female mice (Atm^LKT) as shown in the flow chart reported in Suppl. Figure 1d.

We found substantial improvements in CD3 positive T cells and CD4 single-positive helper T cells in transplanted mice compared to Atm^−/− animals in peripheral blood 4 and 7 weeks after transplantation (Fig. 1a,b). We discovered that thymus size, which is generally hypoplastic in Atm^−/− mice, gained weight 7 weeks after transplantation (Fig. 1c). Flow cytometry analysis demonstrated significant increase in CD4 single-positive helper T cells and TCRβ^hi thymocytes in transplanted animals, both of which reached levels comparable to wild-type mice (Fig. 1d, e). Furthermore, if Atm^−/− animals normally had reduced numbers of IgG1 positive B cells owing to class switch defects, B cells from 7 weeks transplanted mice showed an increase in IgG1 positive cells, equivalent to wild-type mice (Fig. 1f). We finally observed that male transplanted LK cells indeed reached lymphoid organs in Atm^−/− females as indicated by the presence of the Sry male marker and the wild-type Atm coding genes (Fig. 1g). Following DNA damage, Atm protein could phosphorylate Atm targets including the transcription factor KAP1, the cell cycle checkpoint proteins Chk1 and Chk2, and the DNA double-strand breaks marker H2AX (Fig. 1h).

Fig. 1

Restoration of T cells in Atm^−/− systemic blood and thymus, and B-cells in spleen after Atm^+/+ LK cells transplantation. a Histograms of total, helper and cytotoxic T cells (CD3, CD4, CD8) in peripheral blood 4 weeks after transplantation. b Histograms of total, helper and cytotoxic T cells (CD3, CD4, CD8) in peripheral blood 7 weeks after transplantation. c Thymus and spleen picture and relative weight bar charts. The ratio between organ weight (g) and body weight (g) is reported. d Representative flow cytometry panels of CD4 and CD8 T cells and relative histogram of mean values of CD4 and CD8 single positive T cells are shown. e Histogram of mean values of TCRβ^hi expressing cells analyzed by flow cytometry. f Representative flow cytometry dot-plot of B cells class switching cultured in LPS and IL4 for 96 h and relative histogram of mean values of IgG1 expressing cells. g Representative genomic PCR analysis of mouse tissues collected 7 weeks after LKT. The Atm 200 bp band identifies the wild-type sequence of Atm gene, whereas the Atm 400 bp band identifies the knockout sequence of Atm gene. The Sry 100 bp band identifies male cells in the female background and 18 s rRNA gene is used as a housekeeping sequence. h Western blot analysis of Atm expression and DNA damage response in isolated thymocytes and splenocytes. Neocarzinostatin (NCS) was used as DNA damage inducer. Phosphorylation of KAP1, Chk1, Chk2 (upper band) and H2AX (γH2AX) indicate DNA damage response. Tubulin was used as loading control. N = 3 mice of each group. *P ≤ 0.05 and ** P ≤ 0.01

We determined that 1 × 10⁶ Atm^+/+ LK-enriched cells were sufficient for effective T cell reconstitution in blood, Atm expression and DNA damage response in thymocytes of transplanted Atm^−/− mice (Atm^{−/−LKTLow}), 7 weeks after transplantation (Suppl. Figure 2a–c). To further elucidate the relevance of LK cells in the reconstitution of the immune system of Atm-deficient mice, Lineage negative and c-Kit depleted (Lin⁻c-Kit⁻, LK-) cells were transplanted (Suppl. Figure 3). Notably none of the phenotypes described above were rescued by transplantation of Atm^+/+ LK cells, despite they reached the lymphoid organs (Suppl. Figure 3a–h), supporting previous works on the hematopoietic function of c-Kit⁺Sca-1⁺ cells [15].

Long-term study, 6 months post-transplantation, revealed an increase of lifespans and fur whitening in transplanted Atm^−/− mice (Atm^{−/− LKTLong}; Fig. 2a, b), when compared to non-transplanted Atm^−/− mice that usually die at 3–4 months before showing signs of aging in the fur. Further studies will be conducted to address if Atm^{−/− LKTLong} mice present more aging features. Notably, improvement of the immune systems in blood and thymus with tumor prevention (Fig. 2c–e), decreased chromosome breaks of cultured B cells (Fig. 2f), reduced trans-rearrangement rates in thymocytes (Fig. 2g), and correct DNA damage response in vitro (Fig. 2h) were observed in transplanted mice compared to untreated Atm^−/− animals.

Fig. 2

Extended life span and genomic instability prevention after long-term Atm^+/+ LK transplantation. a Kaplan–Meier survival curve Long-rank Mantel-Cox test for Atm^+/+, Atm^−/− and Atm^{−/−LKTLong} mice. The red dotted line indicates Atm^−/− mice median survival (95 days; Atm^+/+ N = 31; Atm^−/− N = 25; Atm^{−/−LKTLong} N = 6 **** P ≤ 0.0001). b Picture of 7 months old mice. Atm^{−/− LKTLong} mice present different levels of white/grey fur. c Histogram of flow cytometry analysis of blood from 7 months old Atm^{−/−LKTLong} mice, relative to Atm^+/+ littermate and 2–4 months old Atm^−/− tumor-free mice. d Representative thymus and spleen pictures of 7 months old mice after LKT relative to thymi from Atm^+/+ littermate and 4 months old Atm^−/− tumor-free mice. e Histogram of flow cytometry analysis of isolated thymocytes from 7 months old Atm^{−/−LKTLong} mice, relative to Atm^+/+ littermate and 2–4 months old Atm^−/− tumor-free mice. f Representative image of FISH assay in B cell metaphases and histogram of defective B cell metaphases. In the inset example of an aberrant chromosome. In red the PNA-bio telomere probe and in blue the DAPI DNA staining. Scale bar = 2 μm. g Pictures of PCR for gamma receptor rearrangement (γ) and gamma-beta receptors trans-rearrangement (trans) using as template genomic DNA (500 ng, 100 ng, 10 ng, 1 ng) prepared from thymocytes. The ratio among trans bands and γ is reported for 10 ng dilution PCR product. Examples of short-term (Atm^−/−LKT, 7 weeks) and long-term, (Atm^{−/−LKTLong}, 7 months) LK transplantation are reported. Histogram of fold change among trans and γ ratios in thymus of Atm^−/− and Atm^{−/−LKTLong} mice; the weakest bands among different DNA amount were considered. h Representative western blot of Atm expression and DNA damage response following NCS treatment in isolated thymocytes of long-term transplanted mice. Phosphorylation of KAP1, Chk1, Chk2 (upper band) and H2AX (γH2AX) indicate DNA damage response. Tubulin was used as loading control. N = 3 mice of each group. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

In conclusion, our work shows the potential of Atm^+/+ LK cell transplantation to counteract immune deficiency and genomic instability at least till 7 months of age, trough restoration of Non-Homologous End Joining and Homologous recombination repair, respectively, in a mouse model of the A-T disease. Moreover, this study offers the opportunity to investigate gene therapy applications in induced Pluripotent Stem (iPS) cells or HSPCs of A-T patients followed by autologous transplantation.