Journal: bioRxiv

Article Title: MYD88 mutations in clonal hematopoiesis promote inflammation and hematopoietic stem cell expansion

doi: 10.1101/2025.06.19.660202

Figure Lengend Snippet: (A) Genotyping analysis of Myd88 WT ;Rosa-CreERT2 and MyD88 L252P ;RosaCreERT2 homozygous mice following 4-OHT induced recombiation. ( B ) Proportions of donor-derived CD45.2 CD11b+Gr1lo and CD11b+Grhi cells in the PB of Myd88 WT and Myd88 L252P on left panel. Error bars represent SEM (n = 11-13 per group). (C) Representative flow cytometry gating of donor-derived CD45.2 CD11b+Gr1lo and CD11b+Grhi cells in the PB of Myd88 WT or Myd88 L252P . Error bars represent SEM. Significance was determine with a Student’s t-test for two groups or ANOVA for multiple groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Representative flow cytometry gating of donor-derived CD45.1 and CD45.2 LK (Lin-cKit+Sca1-), LSK (Lin-ckit+Sca1+), LT-HSC (LSK CD150+CD48-), ST-HSC (LSK CD150-CD48-), MPP (LSK CD150-CD48+), CMP (LK CD34+16/32-), MEP (LK CD34-CD16/32-), GMP (LK CD34+CD16/32+), CLP (Lin-ckit-Sca1loCD127+CD135+) from the BM of mice competitively engafted with Myd88 WT or Myd88 L252P BM cells. (E) Representative images of spleens and BM sections (Hematoxylin and Eosin) from recipient mice transplanted with Myd88 WT and Myd88 L252P BM cells.

Article Snippet: For flow cytometric analysis of lineage positive cells or stem/progenitor HSCs, PB or BM samples were processed in 1xRBC lysis for 15 minutes, followed by incubation in the following antibodies, DAPI (D1306, ThermoFisher Scientific), 7AAD (00-6993-50, eBiosciences), CD11b-PE-Cy7 (25-0112-81, eBiosciences), Gr1-eFluor450 (48-5931-82, eBiosciences), CD3-PE (12-0031-83, eBiosciences), B220-APC (17-0452-82, eBiosciences), CD45.1-Brilliant Violet 510 (110741, BioLegend), CD48-APC (11-0481-85, eBiosciences), CD117-APC-Cy7 (135135, BioLegend), Ly-6A/E(Sca-1)-PE (12-5981-82, eBiosciences), CD135-PE-Cy5 (135312, BioLegend), CD150-PerCpCy5.5 (115922, BioLegend), CD127-Brilliant Violet 605 (35041, BioLegend) and CD45.2-APC-eFluor780 (47-0454-82, eBiosciences) or CD45.2-eFluor450 (48-0454-82, eBiosciences).

Techniques: Derivative Assay, Flow Cytometry

Journal: bioRxiv

Article Title: MYD88 mutations in clonal hematopoiesis promote inflammation and hematopoietic stem cell expansion

doi: 10.1101/2025.06.19.660202

Figure Lengend Snippet: ( A ) 4-OHT (1 μM) treated cKit+ enriched BM cells (n = 1000) from Myd88 WT and Myd88 L252P mice were plated in methylcellulose and assessed for colony formation (n = 3 per group from biological replicates): erythroid progenitor cells (BFU-E), granulocyte-macrophage progenitors (CFU-G/M/GM), and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells (CFU-GEMM). ( B ) Serial colony replating potential of cKit-enriched BM cells from MYD88 WT and MYD88 L252P mice in methylcellulose. Error bars represent the SEM (n = 3 per group from biological replicates). ( C ) Representative images of colonies from panel B. (C) Experimental overview of competitive BM transplants (cBMT). ( E ) Summary of donor-derived Myd88 WT and Myd88 L252P PB proportions (CD45.2) from the cBMT recipient mice at the indicated time points (n = 14-15 mice per group). ( F ) Representative flow cytometry plots of donor-derived CD45.1 (WT) or CD45.2 (Myd88 WT or Myd88 L252P ) PB cells from primary cBMTs. ( G ) Proportions of donor-derived CD45.2 populations from the PB of primary cBMTs: myeloid (CD11b + ), T (CD3 + ), and B (B220 + ) cells at the indicated time points (n= 11-13 mice per group). Error bars represent SEM (n = 11-13 per group). ( H ) Proportions of donor-derived CD45.2 populations from the BM of primary cBMTs at 16 weeks (n = 7-10 mice per group): LK (Lin - cKit + Sca1 - ), LSK (Lin - ckit + Sca1 + ), LT-HSC (LSK CD150 + CD48 - ), ST-HSC (LSK CD150 - CD48 - ), MPP (LSK CD150 - CD48 + ), CMP (LK CD34 + 16/32 - ), MEP (LK CD34 - CD16/32 - ), GMP (LK CD34 + CD16/32 + ), CLP (Lin - ckit - Sca1 lo CD127 + CD135 + ). ( I ) Experimental overview of non-competitive BM transplants (BMT). ( J ) Complete PB counts of MYD88 WT and MYD88 L252P at the indicated time points post BM transplantation and tamoxifen administration. Error bars represent SEM (n = 7-8 mice per group). ( K ) Kaplan-Meier survival curves for recipient mice transplanted with MYD88 WT (n = 12) and MYD88 L252P BM cells (n = 13 mice per group). Data from 2 independent biological replicates. ( L ) Weight of spleen isolated from recipient mice transplanted with Myd88 WT and Myd88 L252P BM cells. Error bars represent SEM (n = 7 mice per group). ( M ) Representative images of spleen sections (Hematoxylin and Eosin), PB smears (Wright-Giemsa), and lungs (Hematoxylin and Eosin) from recipient mice transplanted with Myd88 WT and Myd88 L252P BM cells. White arrows denote infiltrates of monocytes or neutrophils in the spleen. Scale bars of H&E and Wright-Giemsa images represent 50 μm. Error bars represent SEM. Significance was determined with a Student’s t-test for two groups or ANOVA for multiple groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: For flow cytometric analysis of lineage positive cells or stem/progenitor HSCs, PB or BM samples were processed in 1xRBC lysis for 15 minutes, followed by incubation in the following antibodies, DAPI (D1306, ThermoFisher Scientific), 7AAD (00-6993-50, eBiosciences), CD11b-PE-Cy7 (25-0112-81, eBiosciences), Gr1-eFluor450 (48-5931-82, eBiosciences), CD3-PE (12-0031-83, eBiosciences), B220-APC (17-0452-82, eBiosciences), CD45.1-Brilliant Violet 510 (110741, BioLegend), CD48-APC (11-0481-85, eBiosciences), CD117-APC-Cy7 (135135, BioLegend), Ly-6A/E(Sca-1)-PE (12-5981-82, eBiosciences), CD135-PE-Cy5 (135312, BioLegend), CD150-PerCpCy5.5 (115922, BioLegend), CD127-Brilliant Violet 605 (35041, BioLegend) and CD45.2-APC-eFluor780 (47-0454-82, eBiosciences) or CD45.2-eFluor450 (48-0454-82, eBiosciences).

Techniques: Derivative Assay, Flow Cytometry, Transplantation Assay, Isolation

Journal: bioRxiv

Article Title: MYD88 mutations in clonal hematopoiesis promote inflammation and hematopoietic stem cell expansion

doi: 10.1101/2025.06.19.660202

Figure Lengend Snippet: (A) Quantitative RT-PCR (RT-PCR) analysis of the indicated cytokines genes in Myd88 WT and Myd88 L252P cKit+ BM cells incubated with 4-OHT to induce recombination for 72 hours followed by treatment with vehicle (DMSO) or 1 μM IRAK1/4-inhibitor (IRAK1/4-inh; NCGC-1481) for 24 hours (n = 3 per group from biological replicates). (B) cKit+ enriched BM cells incubated with 4-OHT (1 μM) were serially plated in methylcellulose and assessed for colony formation in the presence of vehicle (DMSO) or 1 μM IRAK1/4-inh (NCGC-1481) (n = 3 per group from biological replicates). (C) Experimental overview of competitive BM transplants (cBMT) following administration of the IRAK1/4-inh (NCGC-1481). Chimeric mice were administered daily with either vehicle control (PBS) or IRAK1/4-inh (30 mg/kg) via intraperitoneal (IP) injection for 6 weeks. FACS analysis of PB was performed pre- and post-administration at weeks 0, 2, 4, and 6. (D) Percent change (related to pre-treatment) in PB chimerism of individual donor-derived CD45.2 cells following administration of vehicle or IRAK1/4-inh at the indicated time points (n = 10 for Myd88 WT ; n = 26 for Myd88 L252P ). Data from 2 independent biological replicates. (E) Proportions of PB donor-derived myeloid (CD11b), T (CD3), and B (B220) cells within the CD45.2 populations of Myd88 WT (n = 10) and Myd88 L252P (n = 26) chimeric mice following treatment with vehicle or IRAK1/4-inh at the indicated time points. Data from 2 independent biological replicates. Error bars represent SEM. Significance was determined with a Student’s t-test for two groups or ANOVA for multiple groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: For flow cytometric analysis of lineage positive cells or stem/progenitor HSCs, PB or BM samples were processed in 1xRBC lysis for 15 minutes, followed by incubation in the following antibodies, DAPI (D1306, ThermoFisher Scientific), 7AAD (00-6993-50, eBiosciences), CD11b-PE-Cy7 (25-0112-81, eBiosciences), Gr1-eFluor450 (48-5931-82, eBiosciences), CD3-PE (12-0031-83, eBiosciences), B220-APC (17-0452-82, eBiosciences), CD45.1-Brilliant Violet 510 (110741, BioLegend), CD48-APC (11-0481-85, eBiosciences), CD117-APC-Cy7 (135135, BioLegend), Ly-6A/E(Sca-1)-PE (12-5981-82, eBiosciences), CD135-PE-Cy5 (135312, BioLegend), CD150-PerCpCy5.5 (115922, BioLegend), CD127-Brilliant Violet 605 (35041, BioLegend) and CD45.2-APC-eFluor780 (47-0454-82, eBiosciences) or CD45.2-eFluor450 (48-0454-82, eBiosciences).

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Incubation, Control, Injection, Derivative Assay