Journal: iScience

Article Title: A distinct TCR + CD19 + population in the peritoneal cavity: B1a cells as precursors of extrathymic T cells

doi: 10.1016/j.isci.2026.115505

Figure Lengend Snippet: Adoptively transferred B1a cells differentiate into DP cells and extrathymic T cell lineages (A) Two weeks after the adoptive transfer of B1a cells into muMT mice, the frequencies of TCR + CD19 + DP cells and B1a cells were assessed in the PEC, spleen, liver, small intestine, and thymus by flow cytometry. (B) Four weeks after the adoptive transfer of CD45.2 + B1a cells into CD45.1 + recipients, flow cytometric analysis was performed to evaluate the frequencies of CD45.2 + extrathymic T cells, DP cells, and B1a cells in the indicated tissues.

Article Snippet: CD45.1 + CD3 + T cells were isolated from mouse spleens, and CD45.2 + CD19 + B cells were isolated from the peritoneal cavity using magnetic-activated cell sorting (MACS) with anti-biotin microbeads (130-097-046; Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Adoptive Transfer Assay, Flow Cytometry

Journal: Stem Cell Reviews and Reports

Article Title: Differentiation Epitopes Define Hematopoietic Stem Cells and Change with Cell Cycle Passage

doi: 10.1007/s12015-022-10374-4

Figure Lengend Snippet: Stem cell potential in primary sorted Lineage positive subpopulations. (A) Engraftment of primary sorted Lin + subpopulations. 1 × 10 6 CD45.1 donor-derived WBM, erythroid (Ter119 + ), B-lymphoid (B220 + ), 2 × 10 6 myeloid (GR1 + and CD11b + pooled antibodies), or 70,000 T-lymphoid (CD3 + , CD4 + and CD8 + pooled antibodies) Lin + subsets were infused with 3 × 10 5 CD45.2 WBM cells into lethally irradiated CD45.2 mice and peripheral blood chimerism was determined at 1, 3 and 6 months post-transplant. Data are represented as average % donor chimerism ± SEM ( n = 8 mice/group). (B) Multi-lineage analysis of donor-derived peripheral blood cells at 6 months post-transplant for each donor Lin + subpopulation

Article Snippet: Peripheral blood (via tail vein bleeds) and marrow chimerism and lineage specificity were determined by flow cytometry (BD LSR II™ flow cytometer, BD Biosciences) using fluorescently tagged antibodies against CD45.1, CD45.2, B220, and CD3 (BD Biosciences) for lymphoid percentage of donor and host peripheral blood cells, and against CD45.1, CD45.2, CD11b, and GR-1 (BD Biosciences) for myeloid percentage of donor and host peripheral blood cells.

Techniques: Derivative Assay, Irradiation

Journal: Frontiers in Immunology

Article Title: Mobilized Multipotent Hematopoietic Progenitors Promote Expansion and Survival of Allogeneic Tregs and Protect Against Graft Versus Host Disease

doi: 10.3389/fimmu.2020.607180

Figure Lengend Snippet: G-CSF-mobilized MPP can suppress MLR by enhancing Foxp3 + Treg proliferation. (A) CFSE dilution was assessed in B6 CD4 + CD25 − T cell (50,000/well) activated by BALB/c DC (10,000/well) cultured alone (red) or with splenic mobMPP (blue) at a 1:1 ratio (50,000 CD4 + CD25 − T:50,000 mobMPP/well). Percentage of divided T cells were 43.5± 3.17% in control, versus 41.23± 8.9% in coculture with mobMPP ( p = 0.80). Data from 3 independent experiments (n = 3) were compared using Student’s unpaired t -test (ns, not significant). (B) Total B6 CD4 + T cells (containing both CD25 − and CD25 + CD4 + T-cells) activated by BALB/c dendritic cells were cultured alone (red), with splenic mobilized MPP at a 1:1 ratio (light blue), or with non-mobilized bone marrow MPP at a 1:1 ratio (green). Percentages of divided T cells were 64.88± 10.48% in control, versus 63 ± 1.30% and 70.43 ± 17.19% in coculture with mobMPP ( p = 0.87) or bone marrow MPP ( p = 0.73), respectively. Data were compared using one-way ANOVA with Tukey’s multiple comparison test (ns, not significant). (C) Total B6 CD4 + T cells (containing both CD25 − and CD25 + CD4 + T-cells) CFSE dilution is similar when B6 CD4 + T cells activated by BALB/c DC were cultured alone or with mobMPP at 1:1 ratio (50,000 CD4 + T cells:50,000 mobMPP/well). A higher number of MobMPP in the well (1:2 ratio (50,000 CD4 + T cells: 100,000 mobMPP/well), or 1:3 ratio (50,000 CD4 + T cells: 150,000 mobMPP/well) does not impact total B6 CD4 + T cell proliferation (one experiment, n = 1). (D) B6 CD4 + CD25 − CD45.1 T cells activated by BALB/c DC were cultured alone (red) or with CD45.2 Tregs at a 1:1 ratio (dark blue), or with CD45.2 Tregs and CD45.2 mob-MPP at a 1:1:1 ratio (light blue) (n = 3). Percentages of CD45.1 divided T cells were respectively 71.54 ± 5.27% in control, 48.52 ± 0.86% in the presence of Tregs, ( p = 0.0125, *) and 35.49 ± 1.66% with both Tregs and mobMPP, (** p = 0.0028). The percentage of divided T cells was higher when both Tregs and mobMPP were present, rather than Tregs alone (** p = 0.0023). Data were compared using Student’s unpaired t -test (ns, not significant). (E) B6 Treg cells activated with allogeneic BALB/c DC were cultured alone (red) or with mobMPP (blue) (n = 11). The number of Tregs in proliferation (≥ division 1) is lower when Tregs are cultured alone (3918 ± 905 vs. 28792 ± 6659, ** p = 0.0014). Percentages of Tregs in proliferation (≥ division 1) are also lower when Tregs are cultured alone (64.28 ± 25.66% vs. 87.40 ± 9.26%, * p = 0.0108, using Student’s unpaired t -test).

Article Snippet: Thereafter, donor natural Tregs were analyzed at days 8 and 30 post-transplantation, in lymph nodes and spleen using mAbs against CD45.1, CD3, CD4, CD25 (eBioscience).

Techniques: Cell Culture, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Mobilized Multipotent Hematopoietic Progenitors Promote Expansion and Survival of Allogeneic Tregs and Protect Against Graft Versus Host Disease

doi: 10.3389/fimmu.2020.607180

Figure Lengend Snippet: G-CSF mobilized MPP prevent GVHD by enhancing Foxp3 + Tregs in vivo . (A) Lethally irradiated (6 Gy) BALB/cJ recipients received 1 × 10 7 B6 T cell-depleted BM cells alone (black, n = 4) or together with 2 × 10 6 purified B6 T cells (allogeneic control, red, n = 7). In a third group 0.35 × 10 6 B6-derived mobMPP were co-transferred with the two other populations (blue, n = 7) on day 0. Less than 40% of mice having received co-transferred mobMPP died over 80 days whereas median survival was 40 days without mobMPP (* * p = 0.004). Data shown represent pooled results from 2 independent experiments. Results were compared with Kaplan-Meier survival curves. (B) Mean weight loss of BALB/cJ recipients at day 40 post-HSCT (median survival group) was: -10.57 ± 2.28% in mice co-injected with mobMPP (n = 6) vs. -27.44% ± 3.60% in allogeneic controls (n = 4). Mean clinical GVHD scores in recipients at day 40 post-HSCT was: 0.8 ± 0.19 in mice co-injected with mobMPP (n = 6) vs. 3.06 ± 1.1 in allogeneic controls (n = 4). Data were compared using Student’s unpaired t -test (** p ≤ 0.01). (C) Lethally irradiated (6 Gy) BALB/cJ recipients received 1 × 10 7 B6 T-cell depleted BM cells alone (black, n = 4), with 2 × 10 6 purified CD25 − B6 T cells (allogeneic control, red, n = 7) or with additional 2 × 10 6 purified CD25 − B6 T cells and 0.35 × 10 6 B6-derived mobMPP (blue, n = 7). Median survival in mice having received allogeneic HSCT plus CD25 − T cells was 30 days vs. 33 days in mice with co-transferred mobMPP ( p = 0.52). Results were compared with Kaplan-Meier survival curves (ns, not significant). (D) Percentage of Foxp3 + Tregs in allogeneic CD45.1 + CD4 + B6 T cells were studied in control BALB/c mice (red) or in BALB/c mice transplanted with mobMPP (blue) (n = 3). The percentage of Tregs in the CD45.1 + CD4 + T cells in mesenteric lymph nodes were 3.84 ± 0.71% vs. 5.76 ± 0.67%, (* p =0.0274), respectively. The percentage of Tregs in the CD45.1 + CD4 + T cells in inguinal lymph nodes were 3.64 ± 0.28 vs. 7.31 ± 0.29%, (**** p < 0.0001, using Student’s unpaired t-test). (E) Representative confocal fluorescence micrograph of inguinal lymph nodes. BALB/c mice were sacrificed at day 15 post-allogeneic HSC and inguinal lymph nodes were harvested for confocal microscopy. Top: inguinal lymph node from control allografted BALB/c mice. Below: inguinal lymph node from allografted BALB/c mice with CD45.1 mobMPP (DAPI, blue; Foxp3, green; CD45.1 red; merged images show Foxp3/CD45.1 colocalization, indicated by *). Bar: 0.2 μm.

Article Snippet: Thereafter, donor natural Tregs were analyzed at days 8 and 30 post-transplantation, in lymph nodes and spleen using mAbs against CD45.1, CD3, CD4, CD25 (eBioscience).

Techniques: In Vivo, Irradiation, Purification, Control, Derivative Assay, Injection, Fluorescence, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Mobilized Multipotent Hematopoietic Progenitors Promote Expansion and Survival of Allogeneic Tregs and Protect Against Graft Versus Host Disease

doi: 10.3389/fimmu.2020.607180

Figure Lengend Snippet: G-CSF mobilized MPP induce tolerance. (A) Steady state B6 CD4 + CD45.1 + T cells activated by BALB/c (left) or C3H (right) DC were cultured alone (purple) or with Tregs in 10:1 ratio (50,000 T cells: 5,000 Tregs). Tregs were sorted from the spleen of allografted control BALB/c mice (red), or from allografted BALB/c mice with mobMPP (light blue) (n = 3). CFSE dilution within CD45.1 + T cells were compared. (One representative experiment out of 3). (B) Left: Steady state B6 CD4 + CD25 low CD45.1 + T cells activated by BALB/c DC were analyzed for CFSE dilution (purple) and compared to CFSE dilution of allografted B6 CD4 + CD25 low CD45.1 + T cells in BALB/c mice with (light blue) or without (red) adoptive transfer of mobMPP. Percentage of steady state B6 CD4 + CD25 low CD45.1 + T cells in division is 70.57 ± 6.81% (purple) vs. 41.37 ± 1.03% (red), vs. 70.57 ± 6.81% (light blue), respectively ** p ≤ 0.005 by one-way ANOVA with Tukey’s multiple comparisons test. Right: Steady state B6 CD4 + CD25 low CD45.1 + T cells activated by C3H dendritic cells were analyzed for CFSE dilution (purple) and compared to CFSE dilution of allografted B6 CD4 + CD25 low CD45.1 + T cells in C3H mice with (light blue) or without (red) adoptive transfer of mobMPP. Percentage of steady state B6 CD4 + CD45.1 T cells in division is 76.60 ± 7.18% (purple) vs. 42.40 ± 7.35% (red), vs. 33.37. ± 5.69% (light blue), respectively, n = 3, (* p = 0.0275, ** p = 0.0097; ns, not significant, by one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Thereafter, donor natural Tregs were analyzed at days 8 and 30 post-transplantation, in lymph nodes and spleen using mAbs against CD45.1, CD3, CD4, CD25 (eBioscience).

Techniques: Cell Culture, Control, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: Mobilized Multipotent Hematopoietic Progenitors Promote Expansion and Survival of Allogeneic Tregs and Protect Against Graft Versus Host Disease

doi: 10.3389/fimmu.2020.607180

Figure Lengend Snippet: Human mobilized MPP share features of immature myeloid cells endowed with naïve Treg proliferation properties. (A) Human G-CSF mobilized peripheral blood stem cells (PBSC) were analyzed by flow cytometry. We first distinguished the CD34 + fraction from the CD34 − fraction of cells. In the gated CD38 + CD34 + fraction, a population of cells that lacked CD90 and CD45RA, homogeneously expressed CD13 and CD33 and was identified and termed mobMPP. Mobilized hematopoietic stem cells (mobHSC) were distinguished as CD34 + CD38 − CD45RA − CD90 − . Below, in the same PBSC, naïve Tregs defined as CD3 + CD4 + CD45RA + CD25 + were analyzed by flow cytometry. All gates were based on isotype control. (B) Anti-CD3 and -CD28 activated naïve Treg cell proliferation after 4 days of culture alone (red) with mobMPP (light blue) or with mobHSC (orange) at a 1:1 ratio (n = 3). Percentages of divided naïve Tregs were in control = 29.17 ± 8.58% vs. 60.77 ± 2.847% with mobMPP vs. 29.67 ± 9.06% with mobHSC, respectively. Data were compared using one-way ANOVA with Tukey’s multiple comparison test (ns, not significant, * p = 0.0371). (C) Anti-CD3 and -CD28 activated CD4 + CD25 − T cell proliferation after 4 days of culture alone (red) with mobMPP (light blue) or with mobHSC (orange) at a 1:1 ratio (n = 3). Percentages of divided T cells were in control = 78.03 ± 8.945 vs. 82.57 ± 7.583% with mobMPP, vs. 40.70 ± 9.815% with mobHSC respectively. Data were compared using one-way ANOVA with Tukey’s multiple comparison test (ns, not significant, * p =0.0289). (D) Naïve Treg cells activated by anti-CD3 and anti-CD28 were cultured alone or with mobMPP. After 4 days of culture, intra-nuclear Foxp3 was measured.

Article Snippet: Thereafter, donor natural Tregs were analyzed at days 8 and 30 post-transplantation, in lymph nodes and spleen using mAbs against CD45.1, CD3, CD4, CD25 (eBioscience).

Techniques: Flow Cytometry, Control, Comparison, Cell Culture