Figure 1.
T-cell compartments, proteasome assembly, and function are restored posttransplant, enabling intracellular protein homeostasis and downregulation of the type-1 interferon response. (A-B) TCRb repertoire was determined by high-throughput sequencing of sorted T-cell subsets (T conventional, CD8+ T cells, Treg, T follicular helper cells). Representative hierarchical tree maps show TRB repertoire diversity in patients with truncating POMP mutations. Each dot represents a unique CDR-3, and the size of each dot corresponds to the frequency of that CDR-3 in the total population of sequences obtained. Shannon’s H entropy index measures the diversity of the repertoire, taking into account the clonal size distribution in the overall repertoire. Gini-Simpson index (Simpson_1-D) measures inequality of a given repertoire so that the lower the Simpson_1-D, the more unequal is the distribution of individual clonotypes in the sample results for patients A and B. (C) Whole-cell lysates from pre- and post-SCT peripheral blood mononuclear cell (PBMC) from PRAID patients A and B were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis prior to western blotting using antibodies specific for various proteasome components and/or subunits, including USP14, Rpt1, Rpt2, Rpt3, Rpt4, Rpt5, Rpt6, PA28-α, β1i/LMP2/PSMB9, β5i/LMP7/PSMB10, β5/PSMB5, β1/PSMB6, and POMP, as indicated. Equal protein loading was ensured by probing the membrane with a monoclonal anti-GAPDH antibody. (D) PBMC derived from PRAID patients A and B before and after SCT were subjected to nondenaturing protein extraction to generate protein lysates, which were subsequently run on 3% to 12% native–polyacrylamide gel electrophoresis with proteasome bands being detected by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide. Two exposure times are shown. (E) Protein lysates were tested for their chymotrypsin-, caspase-, and trypsin-like activities by incubating the samples with 0.1 mM of the Suc-LLVY-AMC, Suc-LLE-AMC, and Bz-GGR-AMC fluorogenic substrates, respectively. Fluorescence initiated by AMC release was measured every 15 minutes for the first 2 hours and every 30 minutes for the last 2 hours. Indicated on the y-axis are the raw fluorescence values. (F) Protein lysates derived from patients PBMC pre- and post-SCT were probed for the amounts of K-48–linked ubiquitinated proteins. (G-H) The expression of interferon type-I–inducible genes was evaluated in patient-derived PBMC pre- and posttransplant, and a subset of these was reevaluated posttransplant. The expression of all interferon type-I–regulated gene levels evaluated decreased posttransplant; results are expressed as fold change with respect to control gene expression. Results pretransplant are representative of 3 independent repeats with 3 replicates each. Results posttransplant are only representative of 2 independent repeats with 3 replicates each. Samples were compared using Student t test. Ind., individual; OD, optical density; Pat., patient; SCT, stem cell transplant.

T-cell compartments, proteasome assembly, and function are restored posttransplant, enabling intracellular protein homeostasis and downregulation of the type-1 interferon response. (A-B) TCRb repertoire was determined by high-throughput sequencing of sorted T-cell subsets (T conventional, CD8+ T cells, Treg, T follicular helper cells). Representative hierarchical tree maps show TRB repertoire diversity in patients with truncating POMP mutations. Each dot represents a unique CDR-3, and the size of each dot corresponds to the frequency of that CDR-3 in the total population of sequences obtained. Shannon’s H entropy index measures the diversity of the repertoire, taking into account the clonal size distribution in the overall repertoire. Gini-Simpson index (Simpson_1-D) measures inequality of a given repertoire so that the lower the Simpson_1-D, the more unequal is the distribution of individual clonotypes in the sample results for patients A and B. (C) Whole-cell lysates from pre- and post-SCT peripheral blood mononuclear cell (PBMC) from PRAID patients A and B were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis prior to western blotting using antibodies specific for various proteasome components and/or subunits, including USP14, Rpt1, Rpt2, Rpt3, Rpt4, Rpt5, Rpt6, PA28-α, β1i/LMP2/PSMB9, β5i/LMP7/PSMB10, β5/PSMB5, β1/PSMB6, and POMP, as indicated. Equal protein loading was ensured by probing the membrane with a monoclonal anti-GAPDH antibody. (D) PBMC derived from PRAID patients A and B before and after SCT were subjected to nondenaturing protein extraction to generate protein lysates, which were subsequently run on 3% to 12% native–polyacrylamide gel electrophoresis with proteasome bands being detected by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide. Two exposure times are shown. (E) Protein lysates were tested for their chymotrypsin-, caspase-, and trypsin-like activities by incubating the samples with 0.1 mM of the Suc-LLVY-AMC, Suc-LLE-AMC, and Bz-GGR-AMC fluorogenic substrates, respectively. Fluorescence initiated by AMC release was measured every 15 minutes for the first 2 hours and every 30 minutes for the last 2 hours. Indicated on the y-axis are the raw fluorescence values. (F) Protein lysates derived from patients PBMC pre- and post-SCT were probed for the amounts of K-48–linked ubiquitinated proteins. (G-H) The expression of interferon type-I–inducible genes was evaluated in patient-derived PBMC pre- and posttransplant, and a subset of these was reevaluated posttransplant. The expression of all interferon type-I–regulated gene levels evaluated decreased posttransplant; results are expressed as fold change with respect to control gene expression. Results pretransplant are representative of 3 independent repeats with 3 replicates each. Results posttransplant are only representative of 2 independent repeats with 3 replicates each. Samples were compared using Student t test. Ind., individual; OD, optical density; Pat., patient; SCT, stem cell transplant.

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