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High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.

Cattoglio C, Maruggi G, Bartholomae C, Malani N, Pellin D, Cocchiarella F, Magnani Z, Ciceri F, Ambrosi A, von Kalle C, Bushman FD, Bonini C, Schmidt M, Mavilio F, Recchia A - PLoS ONE (2010)

Bottom Line: Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion.Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing.The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

View Article: PubMed Central - PubMed

Affiliation: IIT Unit of Molecular Neuroscience, Istituto Scientifico H. San Raffaele, Milan, Italy.

ABSTRACT
The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

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Association between histone modifications and retroviral integrations in T cells.Each row in the heat map corresponds to a different DNA-bound protein (p300, PCAF, CBP, Pol II, H2A.Z, CTCF) or histone post-translational modification (acetylation, ac, and methylation, me), according to the ChIP-seq databases from Barski et al. [20] and Wang et al. [21]. Chromatin features were annotated in an interval of ±500 bp around MLV and HIV vector integrations, as whole datasets or split into the three classes reported in Figure 1a. Color shades from blue to red are indicative of the direction and the strength of the association between epigenetic features and integrations, as calculated by statistical comparison against matched random controls using the ROC area method [23]. ROC values between 0 and 0.5 (blue shades) reflect a negative correlation compared to random, 0.5 (grey) means no correlation, values above 0.5 (red shades) indicate a positive correlation. Results of statistical tests comparing data sets to each other and to random can be found in the Supplementary statistical analysis S1.
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pone-0015688-g002: Association between histone modifications and retroviral integrations in T cells.Each row in the heat map corresponds to a different DNA-bound protein (p300, PCAF, CBP, Pol II, H2A.Z, CTCF) or histone post-translational modification (acetylation, ac, and methylation, me), according to the ChIP-seq databases from Barski et al. [20] and Wang et al. [21]. Chromatin features were annotated in an interval of ±500 bp around MLV and HIV vector integrations, as whole datasets or split into the three classes reported in Figure 1a. Color shades from blue to red are indicative of the direction and the strength of the association between epigenetic features and integrations, as calculated by statistical comparison against matched random controls using the ROC area method [23]. ROC values between 0 and 0.5 (blue shades) reflect a negative correlation compared to random, 0.5 (grey) means no correlation, values above 0.5 (red shades) indicate a positive correlation. Results of statistical tests comparing data sets to each other and to random can be found in the Supplementary statistical analysis S1.

Mentions: To gain insight on the chromatin conformation of the genomic regions targeted by MLV in T cells, we annotated the 8,277 integration sites in pre-infusion T cells with 41 types of histone modifications (methylations and acetylations) or chromatin-bound proteins mapped genome-wide in human T cells [20], [21]. As a comparison, we re-analyzed 7,782 insertions of an HIV-derived lentiviral vector in pre-infusion T-cells [22]. The analysis was carried out in a window of 1 kb (±500 bp) around each insertion site, using matched random controls (see Methods for definition) as background. The direction and strength of the correlations between epigenetic features and retroviral integrations were quantified using the Receiver Operation Characteristic (ROC) method as previously described [23], allowing the associations to be graphically expressed as heat maps. Integrations were analyzed as a whole or broken down into TSS-proximal, intragenic and intergenic groups. As shown in Figure 2, histone modifications marking active transcription units and/or enhancers (all acetylations and H3K4 methylations) showed the strongest association (red shades in the heat map) with MLV integration sites. The association was particularly evident for, but not limited to, TSS-proximal insertions. A similar scenario was found for the binding of the p300 and CBP histone acetyl transferases, Pol II, the insulator-binding protein CTCF and the H2A.Z histone variant. A weaker enrichment of transcription-associated histone modifications was generally observed around HIV insertion sites, with the exception of those extending throughout the body of active genes (H4K12ac, H2BK5me1, H3K27me1, H3K36me3, and H4K20me1). H2A.Z was under-represented around HIV insertion sites compared to matched random controls. Both MLV and HIV integrations were negatively associated (blue shades in the heat map) with marks linked to transcriptional repression and/or heterochromatin (H3K27 and H3K9 mono- and di-methylations). Finally, little or no correlation with retroviral integration was scored for histone modifications that have no evident bias towards active or inactive genes (H3K79 methylations, H3R/H4R methylations and H4K20me1) (Figure 2 and Supplementary Statistical Analysis S1).


High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.

Cattoglio C, Maruggi G, Bartholomae C, Malani N, Pellin D, Cocchiarella F, Magnani Z, Ciceri F, Ambrosi A, von Kalle C, Bushman FD, Bonini C, Schmidt M, Mavilio F, Recchia A - PLoS ONE (2010)

Association between histone modifications and retroviral integrations in T cells.Each row in the heat map corresponds to a different DNA-bound protein (p300, PCAF, CBP, Pol II, H2A.Z, CTCF) or histone post-translational modification (acetylation, ac, and methylation, me), according to the ChIP-seq databases from Barski et al. [20] and Wang et al. [21]. Chromatin features were annotated in an interval of ±500 bp around MLV and HIV vector integrations, as whole datasets or split into the three classes reported in Figure 1a. Color shades from blue to red are indicative of the direction and the strength of the association between epigenetic features and integrations, as calculated by statistical comparison against matched random controls using the ROC area method [23]. ROC values between 0 and 0.5 (blue shades) reflect a negative correlation compared to random, 0.5 (grey) means no correlation, values above 0.5 (red shades) indicate a positive correlation. Results of statistical tests comparing data sets to each other and to random can be found in the Supplementary statistical analysis S1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008730&req=5

pone-0015688-g002: Association between histone modifications and retroviral integrations in T cells.Each row in the heat map corresponds to a different DNA-bound protein (p300, PCAF, CBP, Pol II, H2A.Z, CTCF) or histone post-translational modification (acetylation, ac, and methylation, me), according to the ChIP-seq databases from Barski et al. [20] and Wang et al. [21]. Chromatin features were annotated in an interval of ±500 bp around MLV and HIV vector integrations, as whole datasets or split into the three classes reported in Figure 1a. Color shades from blue to red are indicative of the direction and the strength of the association between epigenetic features and integrations, as calculated by statistical comparison against matched random controls using the ROC area method [23]. ROC values between 0 and 0.5 (blue shades) reflect a negative correlation compared to random, 0.5 (grey) means no correlation, values above 0.5 (red shades) indicate a positive correlation. Results of statistical tests comparing data sets to each other and to random can be found in the Supplementary statistical analysis S1.
Mentions: To gain insight on the chromatin conformation of the genomic regions targeted by MLV in T cells, we annotated the 8,277 integration sites in pre-infusion T cells with 41 types of histone modifications (methylations and acetylations) or chromatin-bound proteins mapped genome-wide in human T cells [20], [21]. As a comparison, we re-analyzed 7,782 insertions of an HIV-derived lentiviral vector in pre-infusion T-cells [22]. The analysis was carried out in a window of 1 kb (±500 bp) around each insertion site, using matched random controls (see Methods for definition) as background. The direction and strength of the correlations between epigenetic features and retroviral integrations were quantified using the Receiver Operation Characteristic (ROC) method as previously described [23], allowing the associations to be graphically expressed as heat maps. Integrations were analyzed as a whole or broken down into TSS-proximal, intragenic and intergenic groups. As shown in Figure 2, histone modifications marking active transcription units and/or enhancers (all acetylations and H3K4 methylations) showed the strongest association (red shades in the heat map) with MLV integration sites. The association was particularly evident for, but not limited to, TSS-proximal insertions. A similar scenario was found for the binding of the p300 and CBP histone acetyl transferases, Pol II, the insulator-binding protein CTCF and the H2A.Z histone variant. A weaker enrichment of transcription-associated histone modifications was generally observed around HIV insertion sites, with the exception of those extending throughout the body of active genes (H4K12ac, H2BK5me1, H3K27me1, H3K36me3, and H4K20me1). H2A.Z was under-represented around HIV insertion sites compared to matched random controls. Both MLV and HIV integrations were negatively associated (blue shades in the heat map) with marks linked to transcriptional repression and/or heterochromatin (H3K27 and H3K9 mono- and di-methylations). Finally, little or no correlation with retroviral integration was scored for histone modifications that have no evident bias towards active or inactive genes (H3K79 methylations, H3R/H4R methylations and H4K20me1) (Figure 2 and Supplementary Statistical Analysis S1).

Bottom Line: Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion.Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing.The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

View Article: PubMed Central - PubMed

Affiliation: IIT Unit of Molecular Neuroscience, Istituto Scientifico H. San Raffaele, Milan, Italy.

ABSTRACT
The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

Show MeSH
Related in: MedlinePlus