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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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MLV integration clusters in T cell-specific loci.Distribution of MLV integrations (black bars for T cells and red bars for CD34+ hematopoietic progenitor cells), and clusters (black boxes for T cell and red boxes for CD34+ cells), within three RefSeq gene loci (CD40LG, IL2RA, TRAF1) specifically expressed in T cells, as displayed by the UCSC genome browser. H2A.Z, H3K4me1, H3K4me3, H3K9ac and Pol II tracks are those determined by ChIP-seq in the genome of human CD34+/CD133+ HPCs [36] (in red, above the gene) and in human primary T cells [20] (in black, below the gene). Blue boxes represent exons, arrowheads in introns indicate the direction of transcription.
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pone-0015688-g003: MLV integration clusters in T cell-specific loci.Distribution of MLV integrations (black bars for T cells and red bars for CD34+ hematopoietic progenitor cells), and clusters (black boxes for T cell and red boxes for CD34+ cells), within three RefSeq gene loci (CD40LG, IL2RA, TRAF1) specifically expressed in T cells, as displayed by the UCSC genome browser. H2A.Z, H3K4me1, H3K4me3, H3K9ac and Pol II tracks are those determined by ChIP-seq in the genome of human CD34+/CD133+ HPCs [36] (in red, above the gene) and in human primary T cells [20] (in black, below the gene). Blue boxes represent exons, arrowheads in introns indicate the direction of transcription.

Mentions: When analyzed at single-locus resolution, MLV integration clusters in pre-infusion T-cells appear to co-map with promoters and putative regulatory regions of expressed genes. Figure 3 shows three examples of such associations. A ∼50-kb region upstream of the TSS of the CD40LG gene contained 2 clusters (black bars in Figure 3A) and a total of 9 integrations flanking peaks of histone modifications associated with enhancer function (H3K4me1) and upstream of the CD40LG promoter, identified by peaks of H3K4me3, H3K9ac and Pol II. Similarly, the promoter of the T cell-specific IL2RA gene, marked by H3K4me3 H3K9ac and Pol II, is flanked by a cluster of 6 MLV integrations, marked by peaks of H3K4me1 and the H2A.Z variant (Figure 3B). Three clusters containing a total of 15 integrations were identified in less than 40 kb in the TRAF1 locus, upstream, within and downstream of the transcription unit (Figure 3C). Again, the clusters were associated with peaks of H3K4me1, H3K9ac and H2A.Z, identifying putative regulatory regions in the locus. The MLV integration peaks appeared to be cell-specific, since no clusters and very few, sporadic integrations were identified in the same regions in a collection of 8,000 integrations mapped in the genome of human cord blood CD34+ HPCs (Cattoglio et al., submitted), characterized by a different pattern of histone modifications (Figure 3). Conversely, regions heavily targeted by integration clusters in the genome of CD34+ HPCs, such as the LMO2 locus, contained no integration in the T-cell genome (Figure 4A). In loci expressed in both cell types, such as RUNX1, EVI2A/B, and ITGAL, integration clusters were localized in different regions in HPCs (red bars) and T cells (black bars), co-mapping with cell-specific histone modifications associated to promoters and regulatory regions (Figure 4B–D).


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)

MLV integration clusters in T cell-specific loci.Distribution of MLV integrations (black bars for T cells and red bars for CD34+ hematopoietic progenitor cells), and clusters (black boxes for T cell and red boxes for CD34+ cells), within three RefSeq gene loci (CD40LG, IL2RA, TRAF1) specifically expressed in T cells, as displayed by the UCSC genome browser. H2A.Z, H3K4me1, H3K4me3, H3K9ac and Pol II tracks are those determined by ChIP-seq in the genome of human CD34+/CD133+ HPCs [36] (in red, above the gene) and in human primary T cells [20] (in black, below the gene). Blue boxes represent exons, arrowheads in introns indicate the direction of transcription.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008730&req=5

pone-0015688-g003: MLV integration clusters in T cell-specific loci.Distribution of MLV integrations (black bars for T cells and red bars for CD34+ hematopoietic progenitor cells), and clusters (black boxes for T cell and red boxes for CD34+ cells), within three RefSeq gene loci (CD40LG, IL2RA, TRAF1) specifically expressed in T cells, as displayed by the UCSC genome browser. H2A.Z, H3K4me1, H3K4me3, H3K9ac and Pol II tracks are those determined by ChIP-seq in the genome of human CD34+/CD133+ HPCs [36] (in red, above the gene) and in human primary T cells [20] (in black, below the gene). Blue boxes represent exons, arrowheads in introns indicate the direction of transcription.
Mentions: When analyzed at single-locus resolution, MLV integration clusters in pre-infusion T-cells appear to co-map with promoters and putative regulatory regions of expressed genes. Figure 3 shows three examples of such associations. A ∼50-kb region upstream of the TSS of the CD40LG gene contained 2 clusters (black bars in Figure 3A) and a total of 9 integrations flanking peaks of histone modifications associated with enhancer function (H3K4me1) and upstream of the CD40LG promoter, identified by peaks of H3K4me3, H3K9ac and Pol II. Similarly, the promoter of the T cell-specific IL2RA gene, marked by H3K4me3 H3K9ac and Pol II, is flanked by a cluster of 6 MLV integrations, marked by peaks of H3K4me1 and the H2A.Z variant (Figure 3B). Three clusters containing a total of 15 integrations were identified in less than 40 kb in the TRAF1 locus, upstream, within and downstream of the transcription unit (Figure 3C). Again, the clusters were associated with peaks of H3K4me1, H3K9ac and H2A.Z, identifying putative regulatory regions in the locus. The MLV integration peaks appeared to be cell-specific, since no clusters and very few, sporadic integrations were identified in the same regions in a collection of 8,000 integrations mapped in the genome of human cord blood CD34+ HPCs (Cattoglio et al., submitted), characterized by a different pattern of histone modifications (Figure 3). Conversely, regions heavily targeted by integration clusters in the genome of CD34+ HPCs, such as the LMO2 locus, contained no integration in the T-cell genome (Figure 4A). In loci expressed in both cell types, such as RUNX1, EVI2A/B, and ITGAL, integration clusters were localized in different regions in HPCs (red bars) and T cells (black bars), co-mapping with cell-specific histone modifications associated to promoters and regulatory regions (Figure 4B–D).

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