Journal: Nature Communications

Article Title: Ex vivo-expanded human CD19 + TIM-1 + regulatory B cells suppress immune responses in vivo and are dependent upon the TIM-1/STAT3 axis

doi: 10.1038/s41467-022-30613-z

Figure Lengend Snippet: a Human CD19 + B cells were co-cultured with CD154 + or CD154 − CHO cells for 7 days at different CHO:B-cell ratios and in the presence of IL-2, IL-4 and IL-10. Expansion factor is ratio of the number of live CD19 + B cells at each day to the original number of live CD19 + B cells on day 0 (expansion factor at day 0 = 1). Purity of CD19 + B cells >96% in all experiments. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. b Representative FACS plots and summarised data of IL-10 expression by human CD19 + B cells which were expanded at different CD154 + CHO cell:CD19 + B-cell ratios, are shown. Gating is on fluorescence-minus-one (FMO) controls. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. c Summarised data of IL-10 production by human CD19 + B cells, which were expanded at different CD154 + CHO cell-CD19 + B-cell ratios, are shown. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. d Increased proliferation and % increased IL-10 expression by expBreg following 7-day expansion results in >300-fold expansion of human CD19 + IL-10 + B cells. Expansion factor is the ratio of the number of live CD19 + IL-10 + B cells to the original number of live CD19 + IL-10 + B cells on day 0 (expansion factor at day 0 = 1). Data from experiments performed with cells from different healthy donors ( n = 3) are presented. e Human CD19 + IL-10 + B-cell expansion could be maintained in culture for at least 14 days when CD154 + CHO cells were replaced at day-3 and day-7 within the ex vivo culture system, resulting in almost 900-fold expansion. Data from experiments performed with cells from different healthy donors ( n = 3) are presented. Error bars in each panel represent Mean ± SD ( a – e ). One-way ANOVA with Tukey’s multiple comparisons test ( a – d ) and 2-tailed paired t test ( e ) were used. Source data are provided as a .

Article Snippet: Reagents purchased from R&D Systems include: TGFβ inhibitor SB 431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H -imidazol-2-yl]benzamide), [10 μM/L]; anti-CD154 mAb, clone 40804 [10 μg/ml]; anti-CD40 mAb, clone 82102 [10 μg/ml]; anti-IL-10 mAb, clone 23738 [10 μg/ml]; anti-IL-10Rα mAb, clone 37607 [10 μg/ml]; anti-CD122 mAb, clone 27302 [10 μg/ml]; anti-CD25 mAb, clone 22722 [10 μg/ml]; anti-CD25 mAb, clone 24212 [10 μg/ml]; anti-CD86 mAb, clone 37301 [10 μg/ml]; CD80 mAb, clone 37711 [10 μg/ml]; FAS-L mAb, clone 100419 [10 μg/ml]; PD-1 mAb [10 μg/ml], cat no: AF1086; IL-6 mAb, clone 1936 [10 μg/ml]; IL-6R mAb, clone 17506 [10 μg/ml]; IgG 1κ mAb [10 μg/ml]; IgG 2aκ mAb [10 μg/ml].

Techniques: Cell Culture, Expressing, Fluorescence, Ex Vivo

Journal: Nature Communications

Article Title: Ex vivo-expanded human CD19 + TIM-1 + regulatory B cells suppress immune responses in vivo and are dependent upon the TIM-1/STAT3 axis

doi: 10.1038/s41467-022-30613-z

Figure Lengend Snippet: a Representative histograms of live CD4 + CFSE + T cells and summarised data are shown. Autologous CD4 + CFSE + T cells were cultured with anti-CD3/CD28 beads for 5 days ± expBreg or nCD19 + B cells, at increasing ratios of CD19 + B cells to CD4 + T cells. % Inhibition of CD4 + T-cell proliferation is an expression of Division Index of live CD4 + CFSE + T cells at day 5 relative to that of the Stimulated CD4 + T-cell control. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. Representative FACS plots of CD4 + CFSE + T cells are shown in Supplementary Fig. a. b Representative FACS plots and summarised data of IFNy and TNFa expression by human CD4 + T cells which were cultured with anti-CD3/CD28 beads for 3 days ± expBreg or nCD19 + B cells at increasing ratios of CD19 + B cells to CD4 + T cells, are shown. Gating is on Isotype controls. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. c Representative histograms of live CD4 + VPD + T cells and summarised data are shown when autologous CD4 + VPD + T cells were cultured with anti-CD3/CD28 beads for 5 days ± expBreg that had been expanded for 7 days at increasing ratios of CD154 + CHO cells to CD19 + B cells. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. d Summarised data of % inhibition of CD4 + T-cell proliferation by expBreg within transwell assays. ‘Receiver’ refers to the bottom receiver well of the transwell system whilst ‘Insert’ refers to the transwell insert. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. e Summarised data of % inhibition of CD4 + T-cell proliferation by expBreg when in the presence of blocking CD154 mAb. Blocking CD154 mAb was added between day-1 and day-4 of the 5-day suppression assay. Data from experiments performed with cells from different healthy donors ( n = 3) are presented. Each dot is an individual response. f Summarised data of % inhibition of CD4 + T-cell proliferation by expBreg when in the presence of blocking IL-10 and IL-10R mAbs. Blocking mAbs were added at day-0 of the 5-day suppression assay. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. Error bars in each panel represent Mean ± SD ( a – f ). One-way ANOVA with Tukey’s multiple comparisons test ( b – e ) and 2-tailed paired t test ( a , f ) were used. Source data are provided as a .

Article Snippet: Reagents purchased from R&D Systems include: TGFβ inhibitor SB 431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H -imidazol-2-yl]benzamide), [10 μM/L]; anti-CD154 mAb, clone 40804 [10 μg/ml]; anti-CD40 mAb, clone 82102 [10 μg/ml]; anti-IL-10 mAb, clone 23738 [10 μg/ml]; anti-IL-10Rα mAb, clone 37607 [10 μg/ml]; anti-CD122 mAb, clone 27302 [10 μg/ml]; anti-CD25 mAb, clone 22722 [10 μg/ml]; anti-CD25 mAb, clone 24212 [10 μg/ml]; anti-CD86 mAb, clone 37301 [10 μg/ml]; CD80 mAb, clone 37711 [10 μg/ml]; FAS-L mAb, clone 100419 [10 μg/ml]; PD-1 mAb [10 μg/ml], cat no: AF1086; IL-6 mAb, clone 1936 [10 μg/ml]; IL-6R mAb, clone 17506 [10 μg/ml]; IgG 1κ mAb [10 μg/ml]; IgG 2aκ mAb [10 μg/ml].

Techniques: Cell Culture, Inhibition, Expressing, Blocking Assay, Suppression Assay

Journal: Nature Communications

Article Title: Ex vivo-expanded human CD19 + TIM-1 + regulatory B cells suppress immune responses in vivo and are dependent upon the TIM-1/STAT3 axis

doi: 10.1038/s41467-022-30613-z

Figure Lengend Snippet: a Representative FACS plots of live CD19 + B cells and summarised data demonstrating expression of cell-surface markers by expBreg. Gating is on FMO controls. % Expression is compared between expBreg and autologous non-expanded CD19 + B cells (nCD19 + B cells). Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. Representative FACS plots of nCD19 + B cells are shown in Supplementary Fig. b. b Representative FACS plots of CD19 + expBreg and summarised data demonstrating expression of IL-10 are presented. Gating is on FMO controls. Data from experiments performed with cells from different healthy donors ( n = 3) are presented. c Representative histograms of live CD4 + VPD + T cells and summarised data are presented when autologous CD4 + VPD + T cells and anti-CD3/CD28 beads were co-cultured alone or with CD25 + or CD25 − expBreg. Data from experiments performed with cells from different healthy donors ( n = 3) are presented. Each dot is an individual response. Error bars in each panel represent Mean ± SD ( a – c ) 2-tailed paired t tests ( a , b ) and one-way ANOVA with Tukey’s multiple comparisons tests ( c ) were used. Source data are provided as a .

Article Snippet: Reagents purchased from R&D Systems include: TGFβ inhibitor SB 431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H -imidazol-2-yl]benzamide), [10 μM/L]; anti-CD154 mAb, clone 40804 [10 μg/ml]; anti-CD40 mAb, clone 82102 [10 μg/ml]; anti-IL-10 mAb, clone 23738 [10 μg/ml]; anti-IL-10Rα mAb, clone 37607 [10 μg/ml]; anti-CD122 mAb, clone 27302 [10 μg/ml]; anti-CD25 mAb, clone 22722 [10 μg/ml]; anti-CD25 mAb, clone 24212 [10 μg/ml]; anti-CD86 mAb, clone 37301 [10 μg/ml]; CD80 mAb, clone 37711 [10 μg/ml]; FAS-L mAb, clone 100419 [10 μg/ml]; PD-1 mAb [10 μg/ml], cat no: AF1086; IL-6 mAb, clone 1936 [10 μg/ml]; IL-6R mAb, clone 17506 [10 μg/ml]; IgG 1κ mAb [10 μg/ml]; IgG 2aκ mAb [10 μg/ml].

Techniques: Expressing, Cell Culture

Journal: Nature Communications

Article Title: Ex vivo-expanded human CD19 + TIM-1 + regulatory B cells suppress immune responses in vivo and are dependent upon the TIM-1/STAT3 axis

doi: 10.1038/s41467-022-30613-z

Figure Lengend Snippet: a Representative histograms of CD19 + B cells and summarised data of % STAT3 phosphorylation (pSTAT3) are presented when non-expanded CD19 + B cells (nCD19 + B cells) or expBreg were incubated with media, IL-10 [0.01 μg/ml] or IL-21 [0.05 μg/ml] for 10 min and stained for pSTAT3. Data from experiments performed with cells from different healthy donors ( n = 3) are presented. Each dot is an individual response. b Summarised data of % inhibition of CD4 + T-cell proliferation are presented when autologous CD4 + CFSE + T cells and anti-CD3/CD28 beads were co-cultured with expBreg ± IL-21. expBreg were either pre-incubated with IL-21 [0.05 μg/ml] for the last 48 h of the 7-day expansion co-culture (IL-21-stimulated expBreg) prior to addition to the suppression assay at day-0, or IL-21 [0.05 μg/ml] was directly added at day-0 of the suppression assay ± expBreg (expBreg + hIL-21). Data from experiments performed with cells from different healthy donors ( n = 4) are presented. Each dot is an individual response. Representative histograms of live CD4 + CFSE + T cells are presented in Supplementary Fig. c. c Summarised data of % inhibition of CD4 + T-cell proliferation are presented when autologous CD4 + CFSE + T cells and anti-CD3/CD28 beads were co-cultured with expBreg ± STAT3 inhibitor ± IL-21 [0.05 μg/ml]. expBreg were incubated for 2 h with the STAT3 inhibitor WP 1066 [10 μM] ± hIL-21 [0.05 μg/ml], or DMSO ± hIL-21 [0.05 μg/ml], washed and suppressive function analysed. Data from experiments performed with cells from different healthy donors ( n = 4) are presented. Each dot is an individual response. Representative histograms of live CD4 + CFSE + T cells are presented in Supplementary Fig. a. d Representative FACS plots of live CD19 + expBreg and summarised data are presented which demonstrate expression of TIM-1 in the presence of STAT3 inhibition. Data from experiments performed with cells from different healthy donors ( n = 5) are presented. Each dot is an individual response. e Representative histograms of CD19 + expBreg and summarised data of % STAT3 phosphorylation (pSTAT3) in response to IL-21 stimulation, are presented following gene knockouts or mAb blockade of TIM-1 or CD154 within expBreg. Data from experiments performed with cells from different healthy donors ( n = 4) are presented. Each dot is an individual response. f Representative histograms of live CD4 + VPD + T cells and summarised data of % inhibition of CD4 + T-cell proliferation are presented when autologous CD4 + VPD + T cells and anti-CD3/CD28 beads were co-cultured alone or with mAb-blocked expBreg ± IL-21 [0.05 μg/ml]. expBreg had been pre-incubated with IgG isotype control (Isotype-expBreg) or anti-TIM-1 mAb (TIM-1-blocked-expBreg) for the last 48 h of expansion. Data from experiments performed with cells from different healthy donors ( n = 4) are presented. Each dot is an individual response. Error bars in each panel represent Mean ± SD ( a – f ). 2-tailed paired t tests ( a , d ) and one-way ANOVA with Tukey’s multiple comparisons tests ( b , c , e , f ) were used. Source data are provided as a .

Article Snippet: Reagents purchased from R&D Systems include: TGFβ inhibitor SB 431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H -imidazol-2-yl]benzamide), [10 μM/L]; anti-CD154 mAb, clone 40804 [10 μg/ml]; anti-CD40 mAb, clone 82102 [10 μg/ml]; anti-IL-10 mAb, clone 23738 [10 μg/ml]; anti-IL-10Rα mAb, clone 37607 [10 μg/ml]; anti-CD122 mAb, clone 27302 [10 μg/ml]; anti-CD25 mAb, clone 22722 [10 μg/ml]; anti-CD25 mAb, clone 24212 [10 μg/ml]; anti-CD86 mAb, clone 37301 [10 μg/ml]; CD80 mAb, clone 37711 [10 μg/ml]; FAS-L mAb, clone 100419 [10 μg/ml]; PD-1 mAb [10 μg/ml], cat no: AF1086; IL-6 mAb, clone 1936 [10 μg/ml]; IL-6R mAb, clone 17506 [10 μg/ml]; IgG 1κ mAb [10 μg/ml]; IgG 2aκ mAb [10 μg/ml].

Techniques: Incubation, Staining, Inhibition, Cell Culture, Co-Culture Assay, Suppression Assay, Expressing

Journal: Nature Communications

Article Title: Ex vivo-expanded human CD19 + TIM-1 + regulatory B cells suppress immune responses in vivo and are dependent upon the TIM-1/STAT3 axis

doi: 10.1038/s41467-022-30613-z

Figure Lengend Snippet: a Kaplan–Meier survival curve demonstrating human allograft survival in a humanised mouse model of skin transplantation. Balb/c Rag2 −/− cy −/− mice which were transplanted with human skin allograft are reconstituted with human PBMC alone or ±non-expanded CD19 + B cells or expBreg from a different human donor. n = 6 per group per experiment, one of three independent experiments is shown. Experimental schematic is presented in Supplementary Fig. a. b Summarised data of number of huCD45 + CD4 + T cells and huCD45 + CD20 + B cells of mice. n = 6 per group per experiment, one of three independent experiments is shown. Each dot is an individual mouse response. Gating strategy and representative histograms of live huCD45 + cells are presented in Supplementary Fig. b. c Summarised data of % expression of huCD45 + CD20 + TIM-1 + B cells in mice receiving PBMC + expBreg. n = 6 per group per experiment, one of three independent experiments is shown. Each dot is an individual mouse response. Representative FACS plots of live huCD45 + CD20 + TIM-1 + B cells are presented in Supplementary Fig. d. d Summarised data of serum levels of human IL-10, IL-2 and IL-17a in blood of mice. n = 6 per group per experiment, one of three independent experiments is shown. Each dot is an individual mouse response. e Summarised data of % live huCD45 + CD4 + CD25 + CD127 lo Treg of mice. n = 6 per group per experiment, one of three independent experiments is shown. Each dot is an individual mouse response. Representative FACS plots of live huCD45 + CD4 + CD25 + CD127 lo T cells are presented in Supplementary Fig. f. f Summarised data of % inhibition of CD4 + T-cell proliferation by human CD4 + CD25 + CD127 lo Treg which were generated in the presence or absence of expBreg. Data from experiments performed with cells from different healthy donors ( n = 3) are presented. Each dot is an individual response. Representative FACS plots of live CD4 + CD25 + CD127 lo T cells are shown in Supplementary Fig. g. g Summarised data of % live CD4 + CD25 + CD127 lo human Treg on day-5 of suppression assay when autologous CD4 + T cells were co-cultured with anti-CD3/CD28 beads ± Isotype-expBreg, CD154-blocked-expBreg, IL-10-blocked-expBreg or TIM-blocked-expBreg. Data from experiments performed with cells from different healthy donors ( n = 4) are presented. Each dot is an individual response. In ( a – e ), each experiment used a different HLA-mismatched human donor pair. Error bars in ( b – e ) represent Mean ± SEM. Error bars in ( f , g ) represent Mean ± SD. Log-rank (Mantel–Cox) test ( a ) and one-way ANOVA with Tukey’s multiple comparisons tests ( b – e , g ) were used. Source data are provided as a .

Article Snippet: Reagents purchased from R&D Systems include: TGFβ inhibitor SB 431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1 H -imidazol-2-yl]benzamide), [10 μM/L]; anti-CD154 mAb, clone 40804 [10 μg/ml]; anti-CD40 mAb, clone 82102 [10 μg/ml]; anti-IL-10 mAb, clone 23738 [10 μg/ml]; anti-IL-10Rα mAb, clone 37607 [10 μg/ml]; anti-CD122 mAb, clone 27302 [10 μg/ml]; anti-CD25 mAb, clone 22722 [10 μg/ml]; anti-CD25 mAb, clone 24212 [10 μg/ml]; anti-CD86 mAb, clone 37301 [10 μg/ml]; CD80 mAb, clone 37711 [10 μg/ml]; FAS-L mAb, clone 100419 [10 μg/ml]; PD-1 mAb [10 μg/ml], cat no: AF1086; IL-6 mAb, clone 1936 [10 μg/ml]; IL-6R mAb, clone 17506 [10 μg/ml]; IgG 1κ mAb [10 μg/ml]; IgG 2aκ mAb [10 μg/ml].

Techniques: Transplantation Assay, Expressing, Inhibition, Generated, Suppression Assay, Cell Culture