case_images: PMC8093767_01
case_images: Molecular and functional characterization of a novel IL2RG mutation prior to HSCT: (A) Schematic depiction of the IL2RG gene including introns, exons, splice sites and protein domains. (B) IL2RG mutation analysis of cDNA derived from EBV transfected lymphoblastoid cell lines of the patient (day -8 pre HSCT). The patient's IL2RG donor splice site mutation c.115G>C (red line) results in a cryptic donor splice site located at c.115+29 in intron 1. This mutation leads to an insertion of 28 additional nucleotides resulting in a frameshift and premature stop codon at position 48 (highlighted green). (C, D) IL-2RG function analysis in an EBV transfected lymphoblastoid cell line of the patient using flow cytometry. We could demonstrate a reduction of pSTAT5 expression following IL-2 stimulation (red curve, C), which is known to be IL2RG dependent, in the patient's cells compared to those of a healthy control. In C the FACS histograms depicting pSTAT5 expression in IL2-stimulated and unstimulated patient cells overlap. IL2RG independent pSTAT3 expression following IL-6 stimulation (green curve, D) was comparable in patient and healthy control cells. Unstimulated cells are shown in blue (for pSTAT5) and yellow (for pSTAT3). The grey curves depict an isotype control. In D the FACS histograms depicting pSTAT3 expression in unstimulated healthy control cells and the isotype control overlap. The mean fluorescent intensities (MFI) are shown for the patient and the healthy control cells (HC). (E) Lymphocyte proliferation assay prior to HSCT. We can detect severely reduced (*) lymphocyte proliferation following stimulation with PHA, CON A, Tetanus toxin and PWM (dmp = disintegrations per minute of 3H-thymidine incorporation). (F) IL2RG (CD132) expression on B cells using flow cytometry. The mean fluorescent intensities (MFI) are shown for the isotype control and CD132.
case_images: fimmu-12-644687-g001.jpg
case_images: PMC8093767_04_fimmu-12-644687-g001.jpg
case_images: f1
case_images: STAT5 was shown corresponding to the fraction of donor cells within the CD4 population at that time while concomitantly 99% of the CD8 cells were of recipient origin ( Figure 3A  and  Table 1 ). STAT3 phosphorylation following IL-6 stimulation, which is known to be common gamma chain independent, was reexamined in parallel and comparable between patient and healthy control ( Figure 3B ).
case_images: PMC8093767_02
case_images: Molecular and functional characterization of a novel IL2RG mutation prior to HSCT: (A) Schematic depiction of the IL2RG gene including introns, exons, splice sites and protein domains. (B) IL2RG mutation analysis of cDNA derived from EBV transfected lymphoblastoid cell lines of the patient (day -8 pre HSCT). The patient's IL2RG donor splice site mutation c.115G>C (red line) results in a cryptic donor splice site located at c.115+29 in intron 1. This mutation leads to an insertion of 28 additional nucleotides resulting in a frameshift and premature stop codon at position 48 (highlighted green). (C, D) IL-2RG function analysis in an EBV transfected lymphoblastoid cell line of the patient using flow cytometry. We could demonstrate a reduction of pSTAT5 expression following IL-2 stimulation (red curve, C), which is known to be IL2RG dependent, in the patient's cells compared to those of a healthy control. In C the FACS histograms depicting pSTAT5 expression in IL2-stimulated and unstimulated patient cells overlap. IL2RG independent pSTAT3 expression following IL-6 stimulation (green curve, D) was comparable in patient and healthy control cells. Unstimulated cells are shown in blue (for pSTAT5) and yellow (for pSTAT3). The grey curves depict an isotype control. In D the FACS histograms depicting pSTAT3 expression in unstimulated healthy control cells and the isotype control overlap. The mean fluorescent intensities (MFI) are shown for the patient and the healthy control cells (HC). (E) Lymphocyte proliferation assay prior to HSCT. We can detect severely reduced (*) lymphocyte proliferation following stimulation with PHA, CON A, Tetanus toxin and PWM (dmp = disintegrations per minute of 3H-thymidine incorporation). (F) IL2RG (CD132) expression on B cells using flow cytometry. The mean fluorescent intensities (MFI) are shown for the isotype control and CD132.
case_images: fimmu-12-644687-g001.jpg
case_images: PMC8093767_04_fimmu-12-644687-g001.jpg
case_images: f1
case_images: STAT5 was shown corresponding to the fraction of donor cells within the CD4 population at that time while concomitantly 99% of the CD8 cells were of recipient origin ( Figure 3A  and  Table 1 ). STAT3 phosphorylation following IL-6 stimulation, which is known to be common gamma chain independent, was reexamined in parallel and comparable between patient and healthy control ( Figure 3B ).
case_images: PMC8093767_03
case_images: PMC8093767_04
case_images: Molecular and functional characterization of a novel IL2RG mutation prior to HSCT: (A) Schematic depiction of the IL2RG gene including introns, exons, splice sites and protein domains. (B) IL2RG mutation analysis of cDNA derived from EBV transfected lymphoblastoid cell lines of the patient (day -8 pre HSCT). The patient's IL2RG donor splice site mutation c.115G>C (red line) results in a cryptic donor splice site located at c.115+29 in intron 1. This mutation leads to an insertion of 28 additional nucleotides resulting in a frameshift and premature stop codon at position 48 (highlighted green). (C, D) IL-2RG function analysis in an EBV transfected lymphoblastoid cell line of the patient using flow cytometry. We could demonstrate a reduction of pSTAT5 expression following IL-2 stimulation (red curve, C), which is known to be IL2RG dependent, in the patient's cells compared to those of a healthy control. In C the FACS histograms depicting pSTAT5 expression in IL2-stimulated and unstimulated patient cells overlap. IL2RG independent pSTAT3 expression following IL-6 stimulation (green curve, D) was comparable in patient and healthy control cells. Unstimulated cells are shown in blue (for pSTAT5) and yellow (for pSTAT3). The grey curves depict an isotype control. In D the FACS histograms depicting pSTAT3 expression in unstimulated healthy control cells and the isotype control overlap. The mean fluorescent intensities (MFI) are shown for the patient and the healthy control cells (HC). (E) Lymphocyte proliferation assay prior to HSCT. We can detect severely reduced (*) lymphocyte proliferation following stimulation with PHA, CON A, Tetanus toxin and PWM (dmp = disintegrations per minute of 3H-thymidine incorporation). (F) IL2RG (CD132) expression on B cells using flow cytometry. The mean fluorescent intensities (MFI) are shown for the isotype control and CD132.
case_images: fimmu-12-644687-g001.jpg
case_images: PMC8093767_04_fimmu-12-644687-g001.jpg
case_images: f1
case_images: STAT5 was shown corresponding to the fraction of donor cells within the CD4 population at that time while concomitantly 99% of the CD8 cells were of recipient origin ( Figure 3A  and  Table 1 ). STAT3 phosphorylation following IL-6 stimulation, which is known to be common gamma chain independent, was reexamined in parallel and comparable between patient and healthy control ( Figure 3B ).
case_images: Molecular characterization of a second-site IL2RG mutation 45 days post-HSCT. (A) IL2RG mutation analysis of cDNA derived from EBV transfected lymphoblastoid cell lines of the patient (day +45 post-HSCT). Due to a somatic indel mutation in intron 1 (c.115+1_115+7delinsAAGAGGCTTCGGGT), a new cryptic donor splice site within the inserted sequence was created. The transcript showed an insertion of 12 nucleotides, which lead to an inframe insertion of 4 residues (highlighted in blue), restoring the original reading frame. (B) Sequencing of genomic DNA from sorted cell populations (NK cells, CD4-CD8- double negative T cells, CD4+ and CD8+ T cells) of the patient (day +45 post HSCT). Paternal engraftment with wild type IL2RG was found within NK cells and CD4-CD8- double negative T cells. CD4+ and CD8+ T cells carried the somatic indel mutation c.115+1_115+7delinsAAGAGGCTTCGGGT that restored the reading frame (highlighted blue). (C) The distribution of TCR Vbeta elements 1-23 in the patient and an age matched healthy control.
case_images: fimmu-12-644687-g002.jpg
case_images: PMC8093767_04_fimmu-12-644687-g002.jpg
case_images: f2
case_images: Sanger sequencing of the IL2RG region in a cDNA (complementary DNA) library derived from PBMC of the patient on day +45 ( Figure 2A ), as well as in gDNA (genomic DNA) libraries of sorted lymphocytes (1x106 cells/625microl in total), CD4+ (5x103), CD8+(6x104), CD4- CD8- double negative T cells (1.7x103) and NK cells (2x103) was repeated ( Figure 2B ). TCR Vbeta elements in patient's T cells 46 days after HSCT. The majority of the Vbeta elements analyzed showed an oligoclonal skewing. The analysis of TCR Vbeta elements 1-23 revealed the presence of single peaks, demonstrating the existence of predominant clones expressing these particular Vbeta chains ( Figure 2C ). This second-site mutation in CD4+ and CD8+ T cells did not lead to restoration of
case_images: Functional characterization of CD4+ and CD8+ T cells at day +70 post HSCT. (A, B) IL-2RG function analysis in CD4+ and CD8+ T cells of the patient using flow cytometry. (A) We could find decreased pSTAT5 expression following IL-2 stimulation (red curve), which is known to be IL2RG dependent, in CD8+ T cells of the patient compared to those of a healthy control. In the patient pSTAT5 expression could be detected in a small subset of CD4+ T cells only. The mean fluorescent intensities (MFI) are shown for the isotype control, IL-2 stimulated and unstimulated cells (B) IL2RG independent pSTAT3 expression following IL-6 stimulation (green curve) in CD8+ and CD4+ T cells was comparable between patient and healthy control. Unstimulated cell's expression histograms are shown in blue (for pSTAT5) and yellow (for pSTAT3). The grey curves depict an isotype control. The mean fluorescent intensities (MFI) are shown for the isotype control, IL-6 stimulated and unstimulated cells. (C) Lymphocyte proliferation assays +70 days post-HSCT. We can detect reduced (*) lymphocyte proliferation following stimulation with PHA, CON A, Tetanus toxin and PWM (dmp = disintegrations per minute of 3H-thymidine incorporation). (D) IL2RG (CD132) expression on T cells using flow cytometry. The mean fluorescent intensities (MFI) are shown for the isotype control and CD132.
case_images: fimmu-12-644687-g003.jpg
case_images: PMC8093767_04_fimmu-12-644687-g003.jpg
case_images: f3
case_images: STAT5 was shown corresponding to the fraction of donor cells within the CD4 population at that time while concomitantly 99% of the CD8 cells were of recipient origin ( Figure 3A  and  Table 1 ). STAT3 phosphorylation following IL-6 stimulation, which is known to be common gamma chain independent, was reexamined in parallel and comparable between patient and healthy control ( Figure 3B ). Lymphocyte proliferation assays of PBMC's tested in parallel are shown in  Figure 3C  and confirm the impaired functionality of the expanded T cell populations. Nevertheless, the second-site mutation enabled partial restoration of IL2RG expression on the patient's T cells as examined by FACS ( Figure 3D ).
case_images: PMC8093767_05