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.

Keywords: ATM; Genomic stability; HSPCT; Lymphomas; T and B cells.

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

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