f6 ( a ) Confocal analysis showing the effects of PLD inhibition ( FIPI , 750 nM ) on the levels of endogenous LC3 . CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum ( normal medium , Nm ) or in the absence of nutrients ( starvation , St ) . Insets denote high magnification of the selected areas . Scale bar , 10 μm . ( b ) Quantification of the average number , size and surface area of LC3 - positive compartments per cell after FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 180 - 285 cells for Nm ; n = 449 - 519 cells for St ) . * * P0 . 01 . ( c ) Western blot analysis showing levels of LC3 - I and LC3 - II in control - and FIPI - treated CHO cells grown in normal medium ( Nm ) , after nutrient starvation ( St ) or after nutrient starvation in the presence of 50 nM bafilomycin ( St + B ) . Treatments were for 3 h . Right panel : quantification of protein signal intensities showing LC3 - II protein levels in extracts from nutrient - starved and bafilomycin - treated CHO cells following FIPI treatment . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 4 ) . ( d ) Western blot analysis of the effects of PLD inhibition on the levels of total Tau , phosphorylated Tau ( p - Tau ) and detergent - insoluble Tau . Organotypic brain slices from hTau mice were treated with either DMSO ( D ) or 750nM FIPI ( F ) and harvested after 5 days of treatment . Left panel : western blot analysis showing immunoreactivities for total human Tau ( CP27 ) , hyperphosphorylated tau ( p - Tau , ser202 / ser205 , CP13 ) , p62 and tubulin from the supernatant fraction and aggregated tau ( CP27 ) and p62 from the sarkosyl - insoluble fraction . Two representative pairs are displayed . Right panel , quantification of total proteins ( Tau , p - Tau , p62 and tubulin ) and sarkosyl - insoluble aggregates of tau ( insoluble Tau ) and p62 ( insoluble p62 ) . Black bars , DMSO ( vehicle ) ; grey bars , FIPI treatment . Values denote means ± s . e . m . ( n = 7 ) . * * P0 . 01 ; * , P0 . 05 . For the statistical analysis of FIPI - treated samples , tau and p62 protein levels were compared with tubulin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f6(a) Confocal analysis showing the effects of PLD inhibition (FIPI, 750 nM) on the levels of endogenous LC3. CHO cells were treated with PLD inhibitor FIPI or DMSO for 3 h in the presence of serum (normal medium, Nm) or in the absence of nutrients (starvation, St). Insets denote high magnification of the selected areas. Scale bar, 10 μm. (b) Quantification of the average number, size and surface area of LC3-positive compartments per cell after FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=180-285 cells for Nm; n=449-519 cells for St). **P0.01.(c) Western blot analysis showing levels of LC3-I and LC3-II in control- and FIPI-treated CHO cells grown in normal medium (Nm), after nutrient starvation (St) or after nutrient starvation in the presence of 50 nM bafilomycin (St+B). Treatments were for 3 h. Right panel: quantification of protein signal intensities showing LC3-II protein levels in extracts from nutrient-starved and bafilomycin-treated CHO cells following FIPI treatment. Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=4).(d) Western blot analysis of the effects of PLD inhibition on the levels of total Tau, phosphorylated Tau (p-Tau) and detergent-insoluble Tau. Organotypic brain slices from hTau mice were treated with either DMSO (D) or 750nM FIPI (F) and harvested after 5 days of treatment. Left panel: western blot analysis showing immunoreactivities for total human Tau (CP27), hyperphosphorylated tau (p-Tau, ser202/ser205, CP13), p62 and tubulin from the supernatant fraction and aggregated tau (CP27) and p62 from the sarkosyl-insoluble fraction. Two representative pairs are displayed. Right panel, quantification of total proteins (Tau, p-Tau, p62 and tubulin) and sarkosyl-insoluble aggregates of tau (insoluble Tau) and p62 (insoluble p62). Black bars, DMSO (vehicle); grey bars, FIPI treatment. Values denote means±s.e.m. (n=7). **P0.01; *, P0.05. For the statistical analysis of FIPI-treated samples, tau and p62 protein levels were compared with tubulin.


f7a ) Western blot analysis showing reduced levels of LC3 - II in KO MEFs . ( b ) Quantification of protein signal intensities from western blots showing LC3 - II levels in MEFs extracts after normalization to the control protein GAPDH . In a and b , MEFs were grown in normal medium ( Nm ) , treated for 2 h with 50 nM bafilomycin in normal medium ( B ) , or grown in nutrient - deprived medium in the absence ( St ) or presence of 50 nM bafilomycin ( St + B ) . Values denote means ± s . e . m . ( n = 4 ) . ( c ) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3 - positive compartment in KO MEFs . The DAPI staining is shown in blue . Cells were deprived of nutrients for 2 h in the absence ( St , upper panels ) or presence of bafilomycin ( St + B , lower panels ) . Scale bar , 5 μm . ( d ) Quantification of the average size ( top panel ) and number of LC3 - positive compartments per cell ( bottom panel ) in Pld1 WT and KO MEFs starved for 2 h . Values denote means ± s . e . m . ( n = 6 , number of cells = 26 - 45 for all the conditions ) . * * P0 . 01 ; * P0 . 05 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f7a) Western blot analysis showing reduced levels of LC3-II in KO MEFs. (b) Quantification of protein signal intensities from western blots showing LC3-II levels in MEFs extracts after normalization to the control protein GAPDH. In a and b, MEFs were grown in normal medium (Nm), treated for 2 h with 50 nM bafilomycin in normal medium (B), or grown in nutrient-deprived medium in the absence (St) or presence of 50 nM bafilomycin (St+B). Values denote means±s.e.m. (n=4).(c) Immunostaining of endogenous LC3 in Pld1 WT and KO MEFs showing a reduced number and size of the LC3-positive compartment in KO MEFs. The DAPI staining is shown in blue. Cells were deprived of nutrients for 2 h in the absence (St, upper panels) or presence of bafilomycin (St+B, lower panels). Scale bar, 5 μm. (d) Quantification of the average size (top panel) and number of LC3-positive compartments per cell (bottom panel) in Pld1 WT and KO MEFs starved for 2 h. Values denote means±s.e.m. (n=6, number of cells=26-45 for all the conditions). **P0.01; *P0.05.


f8 ( a ) Immunofluorescent detection of endogenous LC3 ( red ) in liver sections from fed or starved Pld1 WT and KO mice . Sections were counterstained with DAPI . Scale bar , 5 μm . ( b ) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice . Histograms show the average number , size and surface area of LC3 - positive compartments . Values denote means ± s . e . m . ( ( n = 25 - 28 ) , 4 mice per genotype ) . * * P0 . 01 . ( c ) Electron microscopic analysis showing a reduction in the size and number of autophagosome / amphisome - like structures ( AP ) in PLD1 - deficient mice after food restriction . Representative electron micrographs from animals analysed in a showing APs ( arrows ) and autophagolysosomes / lysosomes ( AL , arrowheads ) in liver sections from starved WT and KO mice . Insets show high magnification of the selected areas . Scale bar , 1 μm . ( d ) Morphometric analysis of the number ( per 100 μm2 cytoplasmic area ) and relative surface area ( % cytoplasmic area ) of APs and ALs . Values denote means ± s . e . m . ( n = 36 electron micrograms , from a total of 3 , 600 μm2 cytoplasmic area per genotype ) . * * P0 . 01 . ( c ) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1 - deficient mice after food restriction . Values denote means ± s . e . m . ( n = 60 ) . * * P0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f8(a) Immunofluorescent detection of endogenous LC3 (red) in liver sections from fed or starved Pld1 WT and KO mice. Sections were counterstained with DAPI. Scale bar, 5 μm. (b) Quantitative analysis of endogenous LC3 immunofluorescence in liver sections derived from fed or starved Pld1 WT and KO mice. Histograms show the average number, size and surface area of LC3-positive compartments. Values denote means±s.e.m. ((n=25-28), 4 mice per genotype). **P0.01.(c) Electron microscopic analysis showing a reduction in the size and number of autophagosome/amphisome-like structures (AP) in PLD1-deficient mice after food restriction. Representative electron micrographs from animals analysed in a showing APs (arrows) and autophagolysosomes/lysosomes (AL, arrowheads) in liver sections from starved WT and KO mice. Insets show high magnification of the selected areas. Scale bar, 1 μm. (d) Morphometric analysis of the number (per 100 μm2 cytoplasmic area) and relative surface area (% cytoplasmic area) of APs and ALs. Values denote means±s.e.m. (n=36 electron micrograms, from a total of 3,600 μm2 cytoplasmic area per genotype). **P0.01. (c) Morphometric analysis of the diameter of APs and ALs in the liver of PLD1-deficient mice after food restriction. Values denote means±s.e.m. (n=60). **P0.01.


Figure 1Real - time qRT - PCR analysis of C / EBPβ mRNA in CD4 + naïve T cells inactivated ( naive ) or cultured for 24 hr under conditions as indicated with DMSO or ATRA ( all - trans retinoic acid ) . Data are representative of two independent experiments with consistent results and normalized with β - actin ( mean and s . e . m . of quadruplicates ) Immunoblot analysis of C / EBPβ in the nuclear fraction of naïve CD4 + T cells cultured for 2 d under conditions as indicated . The immunoblot was quantified using Image JFlow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean value from 8 ( C are shown on the right Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gateFlow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean values fro 6 ( D are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gateFlow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPζ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean values fro E 7 ( F are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Real-time qRT-PCR analysis of C/EBPβ mRNA in CD4+ naïve T cells inactivated (naive) or cultured for 24 hr under conditions as indicated with DMSO or ATRA (all-trans retinoic acid). Data are representative of two independent experiments with consistent results and normalized with β-actin (mean and s.e.m. of quadruplicates)Immunoblot analysis of C/EBPβ in the nuclear fraction of naïve CD4+ T cells cultured for 2 d under conditions as indicated. The immunoblot was quantified using Image JFlow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean value from 8(C are shown on the right Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gateFlow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro 6(D are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gateFlow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPζ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean values fro E 7(F are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Figure 2Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the upper panel and pooled data from 4 ( A are shown in the bottom . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gate . Error bars represent mean ± s . e . mFlow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the upper panel and pooled data fro ) , 3 ( B are shown in the bottom . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gate . Error bars represent mean ± s . e . mReal - time qRT - PCR analysis of FACS - sorted GFP + cells from CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d in the conditions as indicated . Data are representative of two independent experiments with consistent results and normalized with β - actin ( mean ± s . e . m . of triplicates ) Body weight of RAG - 2 - / - mice after transfer with 5 x 105 CD4 + CD45RBhigh cells alone ( circles ) or with 2 x 105 FACS - sorted CD4 + GFP + cells transduced with control retrovirus ( MigRI ) ( squares ) or retrovirus encoding C / EBPβ ( triangles ) and cultured for 2 d in the presence of TGF - β and culture supernatant . Each time point contains three ( circles ) or four ( squares and triangles ) mice in each group . Error bars represent mean ± s . dHematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm Histological scores Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m ) Comparison of the spleen weight Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the upper panel and pooled data from 4 (A are shown in the bottom. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate. Error bars represent mean ± s.e.mFlow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the upper panel and pooled data fro ), 3 (B are shown in the bottom. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate. Error bars represent mean ± s.e.mReal-time qRT-PCR analysis of FACS-sorted GFP+ cells from CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d in the conditions as indicated. Data are representative of two independent experiments with consistent results and normalized with β-actin (mean ± s.e.m. of triplicates)Body weight of RAG-2-/- mice after transfer with 5 x 105 CD4+CD45RBhigh cells alone (circles) or with 2 x 105 FACS-sorted CD4+GFP+ cells transduced with control retrovirus (MigRI) (squares) or retrovirus encoding C/EBPβ (triangles) and cultured for 2 d in the presence of TGF-β and culture supernatant. Each time point contains three (circles) or four (squares and triangles) mice in each group. Error bars represent mean ± s.dHematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm Histological scores Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)Comparison of the spleen weight Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)


Figure 3Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb / c mice that received RAG2 - / - DO11 . 10 CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding A - C / EBP or C / EBPβ . ( A ) The recipients were immunized via intravenous injection of OVA323 - 339 peptide ( 20 ug ) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization . ( B ) The recipients were fed 1 % OVA solution in drinking water for 5 consecutive days . Representative experiments are shown in the left panel and pooled data from 4 ( A ) or 3 ( B ) independent experiments with mean values are shown on the right . Dot plots are gated for CD4 + KJ . 1 . 26 + GFP + ( top ) or CD4 + KJ . 1 . 26 + GFP - ( bottom ) and numbers indicate percent Foxp3 + cells in the gate . Statistical analysis was performed using one - way ANOVA . ( * p < 0 . 05 , * * p < 0 . 01 ; ns , not

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Flow cytometry of intracellular Foxp3 staining of mesenteric lymph node cells of Balb/c mice that received RAG2-/-DO11.10 CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding A-C/EBP or C/EBPβ. (A) The recipients were immunized via intravenous injection of OVA323-339 peptide (20 ug) 1 and 3 d after transfer of transduced cells and analyzed 5 d after the first immunization. (B) The recipients were fed 1% OVA solution in drinking water for 5 consecutive days. Representative experiments are shown in the left panel and pooled data from 4 (A) or 3 (B) independent experiments with mean values are shown on the right. Dot plots are gated for CD4+KJ.1.26+GFP+ (top) or CD4+KJ.1.26+GFP- (bottom) and numbers indicate percent Foxp3+ cells in the gate. Statistical analysis was performed using one-way ANOVA. (*p<0.05, **p<0.01; ns, not significant


Figure 4EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl - or methyl - CRE sequence in the TSDRChIP - qPCR assay of C / EBPβ on the Foxp3 TSDR in naïve CD4 + T cells cultured for 2 d under conditions as indicated . Data are representative of two independent experiments with consistent results ( mean ± s . e . m . of triplicates ) Flow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data from 4 ( C with mean values are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the gateFlow cytometry of intracellular Foxp3 staining in CD4 + naïve T cells transduced with control retrovirus ( MigRI ) or retrovirus encoding C / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data fro 5 ( D are shown on the right . Dot plots are gated for CD4 + GFP + and numbers indicate percent Foxp3 + cells in the

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4EMSA of nuclear extracts of Jurkat cells transfected with indicated expression vectors and assessed with a probe covering the putative unmethyl- or methyl-CRE sequence in the TSDRChIP-qPCR assay of C/EBPβ on the Foxp3 TSDR in naïve CD4+ T cells cultured for 2 d under conditions as indicated. Data are representative of two independent experiments with consistent results (mean ± s.e.m. of triplicates)Flow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data from 4 (C with mean values are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gateFlow cytometry of intracellular Foxp3 staining in CD4+ naïve T cells transduced with control retrovirus (MigRI) or retrovirus encoding C/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data fro 5 (D are shown on the right. Dot plots are gated for CD4+GFP+ and numbers indicate percent Foxp3+ cells in the gate


Figure 5CD4 + naïve T cells from Foxp3EGFP mice were transduced with control retrovirus ( MIN ) or retrovirus encoding C / EBPβ ( MIN - C / EBPβ ) and stimulated with anti - CD3 and anti - CD28 for 2 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . GFP + ( Foxp3 + ) hNGFR + T cells were sorted to high purity ( 0 hr ) and restimulated with anti - CD3 and anti - CD28 for 2 d in the presence of IL - 4 , IL - 6 , IL - 12 or none . Foxp3 expression was assessed based on GFP expression . Data are representative of two independent experiments with consistent resultsFlow cytometry of CFSE - labeled CD45 . 1 + CD4 + T cells stimulated 3 d alone or together with CD45 . 2 + GFP ( Foxp3 ) + NGFR + - sorted CD4 + naïve T cells from Foxp3EGFP mice transduced with control retrovirus ( Min ) or retrovirus encoding C / EBPβ and cultured for 3 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . Histograms are gated for CD45 . 1 + . The ratios shown are responder to suppressor . Data are representative of two independent experiments with consistent resultsEAE disease course of RAG2 - / - mice that received CD45 . 1 + 2D2CD4 + naive T cells alone or together with CD4 + GFP / Foxp3 + iTreg cells from CD45 . 1 - 2D2CD4 + GFP ( Foxp3 ) + naïve T cells transduced with either control retrovirus ( MIN ) or retrovirus encoding C / EBPβ , cultured in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs and sorted based on GFP / Foxp3 expression . The recipients were immunized with myelin oligodendrocyte glycoprotein peptide ( 100 ug ) 1 d after transfer . Data show mean ± s . e . m of the EAE clinical score of 9 ( day 1 to day 11 ) , 5 ( day 12 to day 16 ) or 4 ( day 17 to day 20 ) mice of each groupThe percentage of IL - 17 - producing cells in CD45 . 1 + Vβ11 + CD4 + T cells in the spinal cords 11 days after transfer Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . d ) The absolute number of IL - 17A + CD45 . 1 + Vβ11 + CD4 + T cells in the spinal cords 11 days after transfer . Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . d ) Flow cytometry of Foxp3 staining of splenic cells . Histograms are gated for CD45 . 1 - CD4 + Vβ11 + . Data are representative of two independent experiments with consistent resultsBody weight of RAG - 2 - / - mice after transfer with 5 x 105 CD45 . 1 + CD4 + CD45RBhigh cells alone ( circles ) or with 2 x 105 FACS - sorted CD45 . 1 - CD4 + GFP ( Foxp3 ) + NGFR + cells transduced with control retrovirus ( Min ) ( filled rectangles ) or retrovirus encoding C / EBPβ ( empty rectangles ) and cultured for 2 d in the presence of TGF - β , anti - IFN - γ and anti - IL - 4 Abs . Each time point contains three mice in each group . Error bars represent mean ± s . e . mHematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm ) Hematoxylin and eosin staining of the distal colons of mice ( Scale bars , 200μm ) Histological scores . Each symbol represents an individual mouse . Small horizontal lines indicate the mean ( error bars , s . e . m ) The percentage of Foxp3 + cells in CD45 . 1 - CD4 + T cells in colonic lamina propria 11 weeks after

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 5CD4+ naïve T cells from Foxp3EGFP mice were transduced with control retrovirus (MIN) or retrovirus encoding C/EBPβ (MIN-C/EBPβ) and stimulated with anti-CD3 and anti-CD28 for 2 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. GFP+(Foxp3+) hNGFR+ T cells were sorted to high purity (0 hr) and restimulated with anti-CD3 and anti-CD28 for 2 d in the presence of IL-4, IL-6, IL-12 or none. Foxp3 expression was assessed based on GFP expression. Data are representative of two independent experiments with consistent resultsFlow cytometry of CFSE-labeled CD45.1+CD4+ T cells stimulated 3 d alone or together with CD45.2+GFP(Foxp3)+NGFR+-sorted CD4+ naïve T cells from Foxp3EGFP mice transduced with control retrovirus (Min) or retrovirus encoding C/EBPβ and cultured for 3 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. Histograms are gated for CD45.1+. The ratios shown are responder to suppressor. Data are representative of two independent experiments with consistent resultsEAE disease course of RAG2-/- mice that received CD45.1+2D2CD4+ naive T cells alone or together with CD4+GFP/Foxp3+ iTreg cells from CD45.1-2D2CD4+GFP(Foxp3)+ naïve T cells transduced with either control retrovirus (MIN) or retrovirus encoding C/EBPβ, cultured in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs and sorted based on GFP/Foxp3 expression. The recipients were immunized with myelin oligodendrocyte glycoprotein peptide (100 ug) 1 d after transfer. Data show mean ± s.e.m of the EAE clinical score of 9 (day 1 to day 11), 5 (day 12 to day 16) or 4 (day 17 to day 20) mice of each groupThe percentage of IL-17-producing cells in CD45.1+Vβ11+ CD4+ T cells in the spinal cords 11 days after transfer Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.d)The absolute number of IL-17A+CD45.1+Vβ11+CD4+ T cells in the spinal cords 11 days after transfer. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.d)Flow cytometry of Foxp3 staining of splenic cells. Histograms are gated for CD45.1-CD4+Vβ11+. Data are representative of two independent experiments with consistent resultsBody weight of RAG-2-/- mice after transfer with 5 x 105 CD45.1+CD4+CD45RBhigh cells alone (circles) or with 2 x 105 FACS-sorted CD45.1-CD4+GFP(Foxp3)+NGFR+ cells transduced with control retrovirus (Min)(filled rectangles) or retrovirus encoding C/EBPβ (empty rectangles) and cultured for 2 d in the presence of TGF-β, anti-IFN-γ and anti-IL-4 Abs. Each time point contains three mice in each group. Error bars represent mean ± s.e.mHematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm)Hematoxylin and eosin staining of the distal colons of mice (Scale bars, 200μm) Histological scores. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (error bars, s.e.m)The percentage of Foxp3+ cells in CD45.1-CD4+ T cells in colonic lamina propria 11 weeks after transfer


Figure 6Flow cytometry of intracellular FOXP3 staining ( right ) or isotype control ( left ) in human CD4 + CD25 - CD45RA + naïve T cells transduced with control retrovirus ( Min ) or retrovirus encoding hC / EBPβ and cultured for 2 d under conditions as indicated . Representative experiments are shown in the left panel and pooled data with mean ± s . e . m are shown on the right . Histograms are gated for CD4 + NGFR + . Statistical analysis was performed using unpaired two - tailed t test . ( * p < 0 . 05 ; ns , not significant ) Flow cytometry of CFSE - labeled human CD4 + CD25 - T cells ( responder ) stimulated 4 d alone or together with NGFR + - sorted human CD4 + CD25 - CD45RA + naïve T cells ( suppressor ) transduced with control retrovirus ( Min ) or retrovirus encoding hC / EBPβ and cultured for 5 d in the presence of hTGF - β , anti - hIFN - γ and anti - hIL - 4 Abs . Histograms are gated for CFSE + . The ratio of responder to suppressor is 1 : 0 . 2 ( top ) or 1 : 0 . 4 ( bottom ) . Data are representative of two independent experiments with consistent

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Figure 6Flow cytometry of intracellular FOXP3 staining (right) or isotype control (left) in human CD4+CD25-CD45RA+ naïve T cells transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 2 d under conditions as indicated. Representative experiments are shown in the left panel and pooled data with mean±s.e.m are shown on the right. Histograms are gated for CD4+NGFR+. Statistical analysis was performed using unpaired two-tailed t test. (*p<0.05; ns, not significant)Flow cytometry of CFSE-labeled human CD4+CD25- T cells (responder) stimulated 4 d alone or together with NGFR+-sorted human CD4+CD25-CD45RA+ naïve T cells (suppressor) transduced with control retrovirus (Min) or retrovirus encoding hC/EBPβ and cultured for 5 d in the presence of hTGF-β, anti-hIFN-γ and anti-hIL-4 Abs. Histograms are gated for CFSE+. The ratio of responder to suppressor is 1:0.2 (top) or 1:0.4(bottom). Data are representative of two independent experiments with consistent results


Figure 2L ) Example images of β - Gal + traced cells post - irradiation and at homeostasis . Scale bar = 100um . M ) The number of fully traced β - Gal + crypt - villus axes / cm in irradiated and non - irradiated mice . Lineage tracing events were quantified from small intestines divided into four equal segments ( S1 - 4 , with S1 being the most proximal ) , stained with X - Gal , embedded in paraffin , cut into 5um thick sections , and counterstained with neutral red . Data are expressed as mean ± SEM . N = 3 mice / group , P - value generated by multiple student ' s t - tests . N ) The number of β - Gal + fully traced crypt - villus axes ( i . e . lineage tracing events ) normalized to the number of pre - injury crypt marked β - Gal + cells ( quantified from d0 control mice ) . S1 + S2 = the two most proximal intestinal segments . Data are expressed as mean ± SEM . N = 4 mice / group , P - value generated by two - tailed student ' s t - test .

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Figure 2L) Example images of β-Gal+ traced cells post-irradiation and at homeostasis. Scale bar= 100um. M) The number of fully traced β-Gal+ crypt-villus axes/cm in irradiated and non-irradiated mice. Lineage tracing events were quantified from small intestines divided into four equal segments (S1-4, with S1 being the most proximal), stained with X-Gal, embedded in paraffin, cut into 5um thick sections, and counterstained with neutral red. Data are expressed as mean ± SEM. N=3 mice/group, P-value generated by multiple student's t-tests. N) The number of β-Gal+ fully traced crypt-villus axes (i.e. lineage tracing events) normalized to the number of pre-injury crypt marked β-Gal+ cells (quantified from d0 control mice). S1 + S2 = the two most proximal intestinal segments. Data are expressed as mean ± SEM. N=4 mice/group, P-value generated by two-tailed student's t-test.


Figure 3D ) Organoid formation efficiency of different aged LRC populations . N = 3 mice / group , N = 3 wells quantified per mouse . Data are expressed as mean ± SEM . N = 3 mice / group , E ) Images of organoids quantified in ( D ) .

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Figure 3D) Organoid formation efficiency of different aged LRC populations. N=3 mice/group, N=3 wells quantified per mouse. Data are expressed as mean ± SEM. N=3 mice/group,E) Images of organoids quantified in (D).


Figure 4C ) Western blot demonstrating LC3 levels from equal numbers of sorted CytoIDlow and CytoIDhigh cells . 200 , 000 cells were loaded per lane . E ) Representative images of organoids formed from sorted CytoIDlow , CytoIDhigh , and Lgr5 - GFPhigh cells . F ) Quantification of the organoid - formation efficiency in ( E ) . N = 4 mice / group , N = 3 wells quantified per mouse . P - Values displayed on graph . Data are expressed as mean ± SEM . P value generated by ratio paired two - tailed t - test . G ) Effect of bafilomycin on organoid formation efficiency in CytoIDlow and CytoIDhigh cells . Cells were plated in media containing different concentrations of bafilomycin ( 0 , 2 . 5 , 5 , 10 , or 25 ng / mL ) on day 0 , media was changed on day 3 to media that did not contain bafilomycin and organoids were imaged on day 5 after plating . N = 4 mice / group , N = 4 wells quantified per mouse . H ) Representative images of organoids quantified in ( G ) .

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Figure 4C) Western blot demonstrating LC3 levels from equal numbers of sorted CytoIDlow and CytoIDhigh cells. 200,000 cells were loaded per lane.E) Representative images of organoids formed from sorted CytoIDlow, CytoIDhigh, and Lgr5-GFPhigh cells. F) Quantification of the organoid-formation efficiency in (E). N=4 mice/group, N=3 wells quantified per mouse. P-Values displayed on graph. Data are expressed as mean ± SEM. P value generated by ratio paired two-tailed t-test.G) Effect of bafilomycin on organoid formation efficiency in CytoIDlow and CytoIDhigh cells. Cells were plated in media containing different concentrations of bafilomycin (0, 2.5, 5, 10, or 25 ng/mL) on day 0, media was changed on day 3 to media that did not contain bafilomycin and organoids were imaged on day 5 after plating. N=4 mice/group, N=4 wells quantified per mouse.H) Representative images of organoids quantified in (G).


Figure 6A ) Representative plots demonstrating the distribution of cells with and without CytoID puncta within sorted CytoID low , high , Chga + , and c - Kit + cells . B ) Quantification of the percentage of cells with and without puncta within CytoID low , high , Chga + , and c - Kit + cells . N = 3 mice / group ( biological replicates ) . C ) Representative images and quantification of organoid - formation efficiency of CytoID low and high cKit + and Chga + cells . Scale bars = 500um . N = 3 mice / group ( biological replicates ) , N = 4 wells quantified per mouse ( technical replicates ) . D ) Immunofluorescent images of crypts from CAG - LC3 - GFP - RFP and Lgr5 - GFP mice 6h post - irradiation . Sections were stained for γH2AX , E - cadherin , and either LC3 - RFP or Lgr5 - GFP respectively . E ) Quantification of the number of γH2AX foci in cells with RFP + puncta , without autophagic puncta , or in Lgr5 + CBCs . N = 3 mice / group , 40 - 80 cells quantified / mouse .

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Figure 6A) Representative plots demonstrating the distribution of cells with and without CytoID puncta within sorted CytoID low, high, Chga+, and c-Kit+ cells.B) Quantification of the percentage of cells with and without puncta within CytoID low, high, Chga+, and c-Kit+ cells. N=3 mice/group (biological replicates).C) Representative images and quantification of organoid-formation efficiency of CytoID low and high cKit+ and Chga+ cells. Scale bars = 500um. N=3 mice/group (biological replicates), N=4 wells quantified per mouse (technical replicates).D) Immunofluorescent images of crypts from CAG-LC3-GFP-RFP and Lgr5-GFP mice 6h post-irradiation. Sections were stained for γH2AX, E-cadherin, and either LC3-RFP or Lgr5-GFP respectively. E) Quantification of the number of γH2AX foci in cells with RFP+ puncta, without autophagic puncta, or in Lgr5+ CBCs. N=3 mice/group, 40-80 cells quantified/mouse.


Figure 1 ( A ) Serum IgG ELISA reactivity with MTB ( H37Rv ) whole cell lysate , purified cell membrane fraction and secreted culture filtrate proteins ( CFP ) for TB patients and healthy donors ( HD ) . ELISA graphs ( left ; TB : black lines ; n = 25 ; HD : red dotted lines ; n = 2 ) and area under the curve ( AUC ) values for all tested samples ( right ; TB : n = 25 ; HD : n = 17 ) are shown . Median is shown . ( B ) AUC values for the IgA serum ELISA response against MTB whole cell lysate , purified cell membrane fraction and secreted culture filtrate proteins ( CFP ) of TB patients ( n = 25 ) and healthy donors ( HD ; n = 9 ) . Median is shown . ( C ) Flow cytometry gating strategy and frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) for one representative TB patient ( TB24 ) and HD ( left ) . Frequency of circulating CD19 + CD27 + + CD38 + plasmablasts of all CD19 + B cells in peripheral blood of TB patients ( n = 24 ) and HD ( n = 8 ) . Dashed line indicates threshold for detectable plasmablast populations ( right ) . Median and SEM are shown . ( D ) Expression of CD19 and activation markers ( CD86 , CD84 and CD24 ) in plasmablasts and memory B cells as measured by flow cytometry . ( E ) Plasmablast response of patient TB7 ( top ) and IgG serum response against MTB cell lysate of patient TB7 , TB19 and TB39 ( bottom ) and one HD at the indicated time points before and after treatment onset . ( F ) Dots indicate AUC values for the anti - MTB cell lysate IgG serum ELISA response ( y - axis ) versus frequency of circulating plasmablasts ( x - axis ) for individual TB patients .

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Figure 1(A) Serum IgG ELISA reactivity with MTB (H37Rv) whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) for TB patients and healthy donors (HD). ELISA graphs (left; TB: black lines; n=25; HD: red dotted lines; n=2) and area under the curve (AUC) values for all tested samples (right; TB: n=25; HD: n=17) are shown. Median is shown.(B) AUC values for the IgA serum ELISA response against MTB whole cell lysate, purified cell membrane fraction and secreted culture filtrate proteins (CFP) of TB patients (n=25) and healthy donors (HD; n=9). Median is shown.(C) Flow cytometry gating strategy and frequency of circulating plasmablasts (CD19+CD27++CD38+) for one representative TB patient (TB24) and HD (left). Frequency of circulating CD19+CD27++CD38+plasmablasts of all CD19+ B cells in peripheral blood of TB patients (n=24) and HD (n=8). Dashed line indicates threshold for detectable plasmablast populations (right). Median and SEM are shown.(D) Expression of CD19 and activation markers (CD86, CD84 and CD24) in plasmablasts and memory B cells as measured by flow cytometry.(E) Plasmablast response of patient TB7 (top) and IgG serum response against MTB cell lysate of patient TB7, TB19 and TB39 (bottom) and one HD at the indicated time points before and after treatment onset.(F) Dots indicate AUC values for the anti-MTB cell lysate IgG serum ELISA response (y-axis) versus frequency of circulating plasmablasts (x-axis) for individual TB patients.


Figure 2 ( A ) Gating strategy , phenotype and frequency of circulating plasmablasts ( CD19 + CD27 + + CD38 + ) isolated by flow cytometric cell sorting from 3 TB patients ( TB7 , TB24 , TB33 ) in comparison to one representative HD . Boxes indicate sort gates . The plasmablast frequency is indicated . ( B ) Absolute number of somatic hypermutations ( SHM ) in the IGHV , IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7 , TB24 and TB33 . The absolute number of sequences analyzed is indicated below the graph . Geometric means with SEM are indicated in grey . SHM means of historic data from sorted CD27 + IgA + or CD27 + IgG + cells from peripheral blood of HD are indicated in red for comparison ( Berkowska et al , 2015a ; Tiller et al , 2007 ) . ( C ) Isotype distribution of plasmablast and memory B cells from TB7 , TB24 and TB33 . PB , plasmablasts ( CD19 + CD27 + + CD38 + ) ; M , memory B cells ( CD19 + CD27 + ) . ( D ) Gating strategy , phenotype and frequency of HBHA - reactive memory B cells ( CD19 + CD27 + HBHA + ) in peripheral blood of one representative TB patient ( TB29 ) , health - care worker ( HCW ) 2 and HD , respectively . ( E ) Dots indicate frequency of HBHA - reactive memory B cells out of all CD27 + memory B cells in individual TB patients ( n = 23 ) and HCWs ( n = 7 ) . Mean and SEM are indicated . P - value was determined using Wilcoxon - Mann - Whitney test ; * * * * p < 0 . 0001 . ( F ) Dots indicate the frequency of resting memory B cells ( CD19 + CD27 + CD10 - ) out of all B cells in peripheral blood of individual TB patients compared to HD . ( G ) Anti - HBHA serum IgG ELISA reactivity for TB patients and HCW ( black lines ) compared to two representative HD ( red lines ) . Dashed line indicates the threshold OD405nm for positive reactivity ( left ) . Asterisks indicate serum responses of donors selected for single sorting of HBHA - reactive memory B cells . Data are representative of two independent experiments . ( H ) Absolute number of somatic hypermutations ( SHM ) in the IGHV ( IGHA , IGHG and IGHM ) , IGKV or IGLV segments of sorted anti - HBHAmemory cells from TB patients and HCWs . Geometric means with SEM are indicated in grey . For comparison , red lines indicate the historic SHM means for randomly sorted CD27 + IgA + , CD27 + IgG + or CD27 + IgM + cells from peripheral blood of HDs ( Berkowska et al , 2015a ; Tiller et al , 2007 ; Tsuiji et al , 2006 ) . ( I ) Number and size of clonally expanded B cell clusters among HBHA - reactive memory B cells from two TB patients ( TB35 , TB29 ) and HCWs ( HCW1 , HCW 2 ) . Cells in clusters are indicated in grey , single cells are indicated in white . No B cell clusters were shared between donors . ( J ) IgA , IgG and IgM isotype distribution of single - cell - sorted HBHA - reactive memory cells . Mean and SEM are indicated .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Gating strategy, phenotype and frequency of circulating plasmablasts (CD19+CD27++CD38+) isolated by flow cytometric cell sorting from 3 TB patients (TB7, TB24, TB33) in comparison to one representative HD. Boxes indicate sort gates. The plasmablast frequency is indicated.(B) Absolute number of somatic hypermutations (SHM) in the IGHV, IGKV and IGLV segments of IgA and IgG plasmablast antibody genes sequenced from TB7, TB24 and TB33. The absolute number of sequences analyzed is indicated below the graph. Geometric means with SEM are indicated in grey. SHM means of historic data from sorted CD27+IgA+ or CD27+IgG+ cells from peripheral blood of HD are indicated in red for comparison (Berkowska et al, 2015a; Tiller et al, 2007).(C) Isotype distribution of plasmablast and memory B cells from TB7, TB24 and TB33. PB, plasmablasts (CD19+CD27++CD38+); M, memory B cells (CD19+CD27+).(D) Gating strategy, phenotype and frequency of HBHA-reactive memory B cells (CD19+CD27+HBHA+) in peripheral blood of one representative TB patient (TB29), health-care worker (HCW) 2 and HD, respectively.(E) Dots indicate frequency of HBHA-reactive memory B cells out of all CD27+ memory B cells in individual TB patients (n=23) and HCWs (n=7). Mean and SEM are indicated. P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.(F) Dots indicate the frequency of resting memory B cells (CD19+CD27+CD10-) out of all B cells in peripheral blood of individual TB patients compared to HD.(G) Anti-HBHA serum IgG ELISA reactivity for TB patients and HCW (black lines) compared to two representative HD (red lines). Dashed line indicates the threshold OD405nm for positive reactivity (left). Asterisks indicate serum responses of donors selected for single sorting of HBHA-reactive memory B cells. Data are representative of two independent experiments.(H) Absolute number of somatic hypermutations (SHM) in the IGHV (IGHA, IGHG and IGHM), IGKV or IGLV segments of sorted anti-HBHAmemory cells from TB patients and HCWs. Geometric means with SEM are indicated in grey. For comparison, red lines indicate the historic SHM means for randomly sorted CD27+IgA+, CD27+IgG+ or CD27+IgM+ cells from peripheral blood of HDs (Berkowska et al, 2015a; Tiller et al, 2007; Tsuiji et al, 2006).(I) Number and size of clonally expanded B cell clusters among HBHA-reactive memory B cells from two TB patients (TB35, TB29) and HCWs (HCW1, HCW 2). Cells in clusters are indicated in grey, single cells are indicated in white. No B cell clusters were shared between donors.(J) IgA, IgG and IgM isotype distribution of single-cell-sorted HBHA-reactive memory cells. Mean and SEM are indicated.


Figure 3 ( A , B ) Representative ELISA graphs show reactivity of antibodies from patient TB7 to ( A ) MTB whole cell lysate or ( B ) MTB bacteria ( left ) . Dashed red line indicates the threshold OD405nm for positive reactivity . Green line indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) . AUC ( area under curve ) values indicate the reactivity to ( A ) MTB cell lysate or ( B ) whole MTB bacteria for antibodies from TB7 , TB24 , TB33 . Total numbers of analyzed antibodies are indicated ( right ) . ( D ) ELISA graph shows reactivity to the MTB cell membrane fraction for MTB - reactive ( n = 26 ) and non - reactive ( n = 14 ) antibodies . ( E ) Reactivity to whole BCG bacteria as determined by flow cytometry for three representative MTB cell lysate - reactive antibodies and one non - reactive antibody ( isotype control , mGO53 ) .

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Figure 3(A, B) Representative ELISA graphs show reactivity of antibodies from patient TB7 to (A) MTB whole cell lysate or (B) MTB bacteria (left). Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)). AUC (area under curve) values indicate the reactivity to (A) MTB cell lysate or (B) whole MTB bacteria for antibodies from TB7, TB24, TB33. Total numbers of analyzed antibodies are indicated (right).(D) ELISA graph shows reactivity to the MTB cell membrane fraction for MTB-reactive (n=26) and non-reactive (n=14) antibodies.(E) Reactivity to whole BCG bacteria as determined by flow cytometry for three representative MTB cell lysate-reactive antibodies and one non-reactive antibody (isotype control, mGO53).


Figure 4 ( A ) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot . The negative isotype control ( mGO53 ) and a commercially available anti - LAM antibody ( positive control ) are shown for comparison . ( B ) Anti - BCG specificity of the LAM - reactive antibody TB24PB037 ( black ) compared to E . coli as determined by ELISA with whole bacteria ( left ) and flow cytometry ( right ) . ( C ) Anti - LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations . The mean ± SD of the absorbance was calculated . ( D ) Antibody TB24PB037 binds to MTB - LAM but not to LAM from M . smegmatis . ELISA performed as described in ( C ) . ( E ) Representative HBHA ELISA for antibodies cloned from HBHA - reactive memory B cells of patient TB35 and HCW1 . Dashed red line indicates the threshold OD405nm for positive reactivity . Green line indicates negative control antibody ( mGO53 ; ( Wardemann et al , 2003 ) ) . ( F ) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity . ( G ) Fluorescence microscopy shows BCG - and MTB - reactivity of representative anti - HBHA antibodies compared to a non - reactive isotype control antibody . Scale bar 10 μm ( top / bottom ) .

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Figure 4(A) Binding of antibodies TB24PB037 and TB33PB123 to LAM purified from MTB as determined by Western Blot. The negative isotype control (mGO53) and a commercially available anti-LAM antibody (positive control) are shown for comparison.(B) Anti-BCG specificity of the LAM-reactive antibody TB24PB037 (black) compared to E.coli as determined by ELISA with whole bacteria (left) and flow cytometry (right).(C) Anti-LAMELISA for TB24PB037 or negative control antibody at the indicated concentrations. The mean ±SD of the absorbance was calculated.(D) Antibody TB24PB037 binds to MTB-LAM but not to LAM from M. smegmatis. ELISA performed as described in (C).(E) Representative HBHA ELISA for antibodies cloned from HBHA-reactive memory B cells of patient TB35 and HCW1. Dashed red line indicates the threshold OD405nm for positive reactivity. Green line indicates negative control antibody (mGO53; (Wardemann et al, 2003)).(F) Reactivity to HBHA was confirmed by Western Blot for a selected set of antibodies with HBHA ELISA reactivity.(G) Fluorescence microscopy shows BCG- and MTB-reactivity of representative anti-HBHA antibodies compared to a non-reactive isotype control antibody. Scale bar 10 μm (top/bottom).


Figure 5Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( A ) Representative results for individual MTB - reactive plasmablast antibodies . Mean and SEM are indicated . Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( B ) Direct comparison of all tested MTB - reactive IgA ( n = 12 ) and IgG ( n = 26 ) antibodies . Mean and SEM are indicated . Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( C ) Representative results for individual HBHA - reactive memory B cell antibodies from TB patients and HCW . Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( D ) Same as in ( B ) but with HBHA - reactive antibodies as shown in ( C ) . ( D ) Direct comparison all tested HBHA - reactive IgA ( n = 6 ) and IgG ( n = 7 ) antibodies . Relative bacterial counts ( CFU ) compared to the medium control ( 100 % ) in human lung epithelial A549 cells 1 h after infection with MTB , pre - incubated with recombinant MTB - reactive IgA1 , IgA2 or IgG1 antibodies and non - reactive isotype control as indicated . ( E ) Direct comparison of the indicated MTB - reactive antibodies expressed as IgA and IgG . Mean and SEM are indicated . P - values were determined using non - parameric t - test , * p < 0 . 1 ; * * p < 0 . 01 , ns : p > 0 . 5 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (A) Representative results for individual MTB-reactive plasmablast antibodies. Mean and SEM are indicated.Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (B) Direct comparison of all tested MTB-reactive IgA (n=12) and IgG (n=26) antibodies. Mean and SEM are indicated.Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (C) Representative results for individual HBHA-reactive memory B cell antibodies from TB patients and HCW.Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (D) Same as in (B) but with HBHA-reactive antibodies as shown in (C). (D) Direct comparison all tested HBHA-reactive IgA (n=6) and IgG (n=7) antibodies.Relative bacterial counts (CFU) compared to the medium control (100%) in human lung epithelial A549 cells 1 h after infection with MTB, pre-incubated with recombinant MTB-reactive IgA1, IgA2 or IgG1 antibodies and non-reactive isotype control as indicated. (E) Direct comparison of the indicated MTB-reactive antibodies expressed as IgA and IgG. Mean and SEM are indicated. P-values were determined using non-parameric t-test, * p<0.1; **p<0.01, ns: p>0.5.


Figure 6 ( A ) Human lung epithelial A549 cells and primary bronchial epithelial cells ( hAECB ) lack FcγR ( CD16 , CD64 and CD32 ) and FcαR ( CD89 ) cell surface expression as analyzed by flow cytometry . Human THP1 cells serve as positive control . ( B ) The neonatal FcR ( FcRn ) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting . Data are representative of at least two independent experiments . ( C ) Absolute bacterial counts ( CFUs ) in human THP1 macrophages 1 h after infection with MTB pre - incubated with the LAM - reactive IgA1 ( left ) or IgG1 ( right ) antibody TB24PB037 or the respective isotype control antibody as indicated . Mean and SEM are indicated .

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Figure 6(A) Human lung epithelial A549 cells and primary bronchial epithelial cells (hAECB) lack FcγR (CD16, CD64 and CD32) and FcαR (CD89) cell surface expression as analyzed by flow cytometry. Human THP1 cells serve as positive control.(B) The neonatal FcR (FcRn) is expressed in both A549 cells and human THP1 cells as determined by Western Blotting. Data are representative of at least two independent experiments.(C) Absolute bacterial counts (CFUs) in human THP1 macrophages 1 h after infection with MTB pre-incubated with the LAM-reactive IgA1 (left) or IgG1 (right) antibody TB24PB037 or the respective isotype control antibody as indicated. Mean and SEM are indicated.


Figure 7 ( A ) Absolute bacterial counts ( CFUs ) in human lung epithelial A549 cells 1 h after infection with MTB pre - incubated with purified serum IgA ( left ) or IgG ( right ) antibodies from TB patients ( TB ) compared to HD as indicated . Mean and SEM are indicated . Data are representative of three independent experiments with three technical replicates each . ( B ) Direct comparison of purified serum IgA and IgG antibodies on epithelial cell invasion as measured in ( A ) . P - value was determined using Wilcoxon - Mann - Whitney test ; * * * * p < 0 . 0001 .

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Figure 7(A) Absolute bacterial counts (CFUs) in human lung epithelial A549 cells 1 h after infection with MTB pre-incubated with purified serum IgA (left) or IgG (right) antibodies from TB patients (TB) compared to HD as indicated. Mean and SEM are indicated. Data are representative of three independent experiments with three technical replicates each.(B) Direct comparison of purified serum IgA and IgG antibodies on epithelial cell invasion as measured in (A). P-value was determined using Wilcoxon-Mann-Whitney test; ****p<0.0001.


Figure 1Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of tumor progression ( average ± SD ) Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) . Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of ( C ) tumor weight at sacrifice , ( D ) vessel number , Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) . Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of percentage of necrotic area by histology , Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) . Effects of DC and Beva treatment in 786 - O orthoxenograft . Evaluation of survival by Kaplan Meier plot in control and treated animals . Box plots indicate median , Q1 / Q3 and max / min value whiskers in Control ( black ) , DC101 ( red ) and Beva ( blue ) treatment groups . 5 - 8 tumors / group were analyzed by Mann - Whitney test , and Mantel - Cox test for survival where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 and DC * p < 0 . 05 and Beva # p < 0 . 05 vs Control ) . G ) Representative invasive front profile of 786 - O control and short - term DC and Beva treated tumors in vimentin stained sections ( 4X ) . Front ( red line ) and rear ( white line ) edges of tumor protrusion into kidney determine the depth of invasion ( green arrow ) perpendicularly connecting red and white lines . Box plot shows the quantification of invasion indicating median , Q1 / Q3 and max / min value whiskers of fold - invasion ( ∆ invasion ) in treated vs control tumors ( 7 samples / group ; * p < 0 . 05 by Mann - Whitney test ) . H ) Evaluation of lung micrometastases in survival experiments . Incidence ( pie chart , * p < 0 . 05 by Chi - square ) and proportion of mice with 0 , 1 , 2 or > 3 metastases ( mets ) per lungs ( * p < 0 . 05 by Mann - Whitney test ) are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of tumor progression (average±SD) Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of (C) tumor weight at sacrifice, (D) vessel number, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of percentage of necrotic area by histology, Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).Effects of DC and Beva treatment in 786-O orthoxenograft. Evaluation of survival by Kaplan Meier plot in control and treated animals. Box plots indicate median, Q1/Q3 and max/min value whiskers in Control (black), DC101 (red) and Beva (blue) treatment groups. 5-8 tumors/group were analyzed by Mann-Whitney test, and Mantel-Cox test for survival where *p < 0.05, **p < 0.01 and ***p < 0.001 and DC * p < 0.05 and Beva # p < 0.05 vs Control).G) Representative invasive front profile of 786-O control and short-term DC and Beva treated tumors in vimentin stained sections (4X). Front (red line) and rear (white line) edges of tumor protrusion into kidney determine the depth of invasion (green arrow) perpendicularly connecting red and white lines. Box plot shows the quantification of invasion indicating median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) in treated vs control tumors (7 samples/group; *p < 0.05 by Mann-Whitney test).H) Evaluation of lung micrometastases in survival experiments. Incidence (pie chart, *p < 0.05 by Chi-square) and proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs (*p < 0.05 by Mann-Whitney test) are shown.


Figure 2Overall mass architecture of paired human specimens and Ren - PDOX tumors observed by HE staining ( 20X ) . TNM staging and Fuhrman grade ( F ) of implanted tumors are reported . For Ren - PDOXs , lung micrometastases in HE and vimentin stained sections are shown ( 40X ) . Human to mouse stroma substitution . CD34 / CD31 staining for respectively human ( h ) and mouse ( m ) blood vessels in original human specimens and paired Ren13 and 28 - PDOX are shown ( 20X ) . Ren - PDOX OT referred to tumor generated from first implantation of human specimen in mice . Insets show human and mouse kidney as positive / negative controls of staining ( 20X ) . Frequency histogram of SNV and / or indels found in Ren - PDOX by high - throughput sequencing in Ren13 , Ren28 , Ren36 , Ren86 ( targeted - NGS analysis by Haloplex ) and Ren50 - PDOX ( WES ) . Only non - synonymous / stop gain SNVs and frameshift indels novel or with MAF < 0 . 01 are included .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Overall mass architecture of paired human specimens and Ren-PDOX tumors observed by HE staining (20X). TNM staging and Fuhrman grade (F) of implanted tumors are reported. For Ren-PDOXs, lung micrometastases in HE and vimentin stained sections are shown (40X).Human to mouse stroma substitution. CD34/CD31 staining for respectively human (h) and mouse (m) blood vessels in original human specimens and paired Ren13 and 28-PDOX are shown (20X). Ren-PDOX OT referred to tumor generated from first implantation of human specimen in mice. Insets show human and mouse kidney as positive/negative controls of staining (20X).Frequency histogram of SNV and/or indels found in Ren-PDOX by high-throughput sequencing in Ren13, Ren28, Ren36, Ren86 (targeted-NGS analysis by Haloplex) and Ren50-PDOX (WES). Only non-synonymous/stop gain SNVs and frameshift indels novel or with MAF<0.01 are included.


Figure 3Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Evaluation of tumor progression average and SD in control ( black ) compared to DC ( red , * p < 0 . 05 ) and Beva ( blue , # p < 0 . 05 ) treatments from 5 - 13 animals per tumor and treatment group by Mann Whitney test . Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Quantification of tumor weight in control and treated tumors from 4 - 5 samples / tumor / treatment group by Mann Whitney test . Box plots indicate median , Q1 / Q3 and max / min value whiskers . Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Quantification of vessel number in control and treated tumors from 4 - 5 samples / tumor / treatment group by Mann Whitney test . Box plots indicate median , Q1 / Q3 and max / min value whiskers . Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Quantification of tumor necrosis in control and treated tumors from 4 - 5 samples / tumor / treatment group by Mann Whitney test . Box plots indicate median , Q1 / Q3 and max / min value whiskers . Effects of DC and Beva treatments on Ren13 , Ren86 , Ren28 and Ren50 - PDOX . Evaluation of overall survival comparing Control ( black ) , DC101 ( red ) and Beva ( blue ) treated mice from 4 - 13 mice / tumor / treatment group by Mantel - Cox test where * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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Figure 3Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of tumor progression average and SD in control (black) compared to DC (red, *p < 0.05) and Beva (blue, # p < 0.05) treatments from 5-13 animals per tumor and treatment group by Mann Whitney test. Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor weight in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.  Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of vessel number in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.  Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Quantification of tumor necrosis in control and treated tumors from 4-5 samples/tumor/treatment group by Mann Whitney test. Box plots indicate median, Q1/Q3 and max/min value whiskers.  Effects of DC and Beva treatments on Ren13, Ren86, Ren28 and Ren50-PDOX. Evaluation of overall survival comparing Control (black), DC101 (red) and Beva (blue) treated mice from 4-13 mice/tumor/treatment group by Mantel-Cox test where *p < 0.05, **p < 0.01 and ***p < 0.001.


Figure 4Effects of DC and Beva treatment on Ren13 , Ren86 , Ren28 and Ren50 - PDOX on capsular invasion Incidence of Capsular invasion ( stripes ) in each model and treatment . 5 - 8 animals per model and treatment group were analyzed by Chi - square test where * p < 0 . 05 , * * p < 0 . 01 . C ) Effects of DC and Beva treatment on Ren13 , Ren86 , Ren28 and Ren50 - PDOX on tumor front of invasion Representative images of Vimentin staining ( 4X ) , as described in Figure 1 , and box plot quantification of fold - invasion ( ∆ invasion ) of treated vs control tumors indicating median , Q1 / Q3 and max / min value whiskers . 6 - 12 animals per model and treatment group were analyzed by Mann - Whitney test where * p < 0 . 05 , * * p < 0 . 01 . D ) Effects of sunitinib treatment on Ren13BM and Ren28 on capsular invasion and tumor front invasion . Bar graphs show incidence of capsular invasion ( stripes ) in control and sunitinib treated tumors . 3 - 9 animals per model and treatment group were analyzed by Chi - square test where * p < 0 . 05 , * * p < 0 . 01 . Box plots represent median , Q1 / Q3 and max / min value whiskers of fold - invasion ( ∆ invasion ) of sunitinib - treated vs control tumors . 5 - 8 animals per model and treatment group were analyzed by Mann - Whitney test . * p < 0 . 05 , * * p < 0 . 01 .

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Figure 4Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on capsular invasion Incidence of Capsular invasion (stripes) in each model and treatment. 5-8 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01. C) Effects of DC and Beva treatment on Ren13, Ren86, Ren28 and Ren50-PDOX on tumor front of invasion Representative images of Vimentin staining (4X), as described in Figure 1, and box plot quantification of fold-invasion (∆ invasion) of treated vs control tumors indicating median, Q1/Q3 and max/min value whiskers. 6-12 animals per model and treatment group were analyzed by Mann-Whitney test where *p < 0.05, **p < 0.01. D) Effects of sunitinib treatment on Ren13BM and Ren28 on capsular invasion and tumor front invasion. Bar graphs show incidence of capsular invasion (stripes) in control and sunitinib treated tumors. 3-9 animals per model and treatment group were analyzed by Chi-square test where *p < 0.05, **p < 0.01. Box plots represent median, Q1/Q3 and max/min value whiskers of fold-invasion (∆ invasion) of sunitinib-treated vs control tumors. 5-8 animals per model and treatment group were analyzed by Mann-Whitney test. *p < 0.05, **p < 0.01.


Figure 5Evaluation of lung micrometastases using HE and vimentin - stained lung sections upon antiangiogenic therapies in Ren13BM Representative images of lung metastasis in Ren13BM upon antiangiogenic therapies . Evaluation of lung micrometastases using HE and vimentin - stained lung sections upon antiangiogenic therapies in Ren13BM and Ren28 . proportion of mice with 0 , 1 , 2 or > 3 metastases ( mets ) per lungs ( * p < 0 . 05 by Mann - Whitney test ) are shown .

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Figure 5Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM Representative images of lung metastasis in Ren13BM upon antiangiogenic therapies. Evaluation of lung micrometastases using HE and vimentin-stained lung sections upon antiangiogenic therapies in Ren13BM and Ren28. proportion of mice with 0, 1, 2 or > 3 metastases (mets) per lungs (*p < 0.05 by Mann-Whitney test) are shown.


Figure 6Tumor specific ( human ) enriched gene sets in Ren13 and Ren50 Ren - PDOX tumors , after GSEA analysis from RNAseq data . Gene sets most significantly enriched in Ren13 or Ren50 are indicated in the table ( left ) , including their normalized enrichment score ( NES ) , p - value and false discovery rate ( FDR q - value ) . Heatmaps illustrate expression levels of the genes responsible for the enrichment of KRAS signaling up and down in Ren13 and Ren50 , respectively ( right ) . Validation of candidate gene expression in unique series of 39 RCC patients ( GSE29609 ) , where microvascular invasion ( MVI ) and renal vein involvement ( RV ) are specifically annotated . Progressive invasion relates to the three progressive degrees of invasion toward the vena cava in this series : 1st , no invasion ; 2nd , microvascular invasion ; and 3rd , renal vein involvement . The gene expression is shown for each variable and condition in box plots representing median , Q1 / Q3 and max / min value whiskers , * p < 0 . 05 * * p < 0 . 01 by Mann - Whitney test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Tumor specific (human) enriched gene sets in Ren13 and Ren50 Ren-PDOX tumors, after GSEA analysis from RNAseq data. Gene sets most significantly enriched in Ren13 or Ren50 are indicated in the table (left), including their normalized enrichment score (NES), p-value and false discovery rate (FDR q-value). Heatmaps illustrate expression levels of the genes responsible for the enrichment of KRAS signaling up and down in Ren13 and Ren50, respectively (right).Validation of candidate gene expression in unique series of 39 RCC patients (GSE29609), where microvascular invasion (MVI) and renal vein involvement (RV) are specifically annotated. Progressive invasion relates to the three progressive degrees of invasion toward the vena cava in this series: 1st, no invasion; 2nd, microvascular invasion; and 3rd, renal vein involvement. The gene expression is shown for each variable and condition in box plots representing median, Q1/Q3 and max/min value whiskers, *p < 0.05 **p < 0.01 by Mann-Whitney test.


Figure 7 Representative images of ALDH1A3 expression on pro - invasive ( Ren13 and Ren86 ) and non - pro - invasive ( Ren28 and Ren50 ) tumors . Confirmation of ALDH1A3 mRNA levels of gene expression in pro - invasive ( Ren13 and Ren86 ) and non - pro - invasive ( Ren28 and Ren50 ) tumors . Average ± SD of 4 - 6 independent replicates per group were analyzed by Mann - Whitney test * p < 0 . 05 * * p < 0 . 01 . Association of pre - treatment ALDH1A3 protein levels by IHC on a series of Ren - PDOX models treated with sunitinib . ( C ) Representation of ALDH1A3 expression on pro - invasive and non - pro - invasive Ren - PDOX after suntinib treatment ( * * p < 0 . 01 by Chi - Square test ) . D ) Association of pre - treatment ALDH1A3 protein levels by IHC on a series of Ren - PDOX models treated with sunitinib . ( D ) Correlation of pre - treatment ALDH1A3 protein levels by IHC with increased tumor invasion found after sunitinib treatment . n = 7 , Spearman ' s non - parametric correlation , p = 0 . 0095 . F ) Representation of ALDH1A3 score ( 0 = absent , 1 = low , 2 = high expression ) on pre - treatment samples of pro - aggressive and non - pro - aggressive ccRCC patients described above . n = 15 , Chi - square test for independence p = 0 . 015 , and Chi - square test for trend p = 0 . 030 ( * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 Representative images of ALDH1A3 expression on pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors. Confirmation of ALDH1A3 mRNA levels of gene expression in pro-invasive (Ren13 and Ren86) and non-pro-invasive (Ren28 and Ren50) tumors. Average ± SD of 4-6 independent replicates per group were analyzed by Mann-Whitney test *p < 0.05 **p < 0.01.Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (C) Representation of ALDH1A3 expression on pro-invasive and non-pro-invasive Ren-PDOX after suntinib treatment (**p < 0.01 by Chi-Square test). D) Association of pre-treatment ALDH1A3 protein levels by IHC on a series of Ren-PDOX models treated with sunitinib. (D) Correlation of pre-treatment ALDH1A3 protein levels by IHC with increased tumor invasion found after sunitinib treatment. n=7, Spearman's non-parametric correlation, p=0.0095. F) Representation of ALDH1A3 score (0=absent, 1=low, 2=high expression) on pre-treatment samples of pro-aggressive and non-pro-aggressive ccRCC patients described above. n=15, Chi-square test for independence p=0.015, and Chi-square test for trend p=0.030 (*).


Figure 1Representative fluorescence microscopy images of total , CD142 - and CD142 + ASPCs isolated with the use of the respective anti - CD142 antibodies : SinoBiological ( SB ) , SinoBiological - LL ( SB - LL ) , BiOrbyt ( BO ) and R & D Systems ( RnD ) ( Appendix Fig . S2B , Appendix Fig . S5A , Methods ) , after in vitro adipogenic differentiation ; Fraction of differentiated cells per ASPC type shown in E , as quantified by the " adiposcore " ; marker shapes correspond to different anti - CD142 antibodies used for isolation as indicated , n = 9 - 15 , 3 - 4 biological replicates , 3 - 5 independent wells for each ; Representative fluorescence microscopy images of CD142 - , 20 % CD142 + and ( 5 - 7 % ) CD142 + ASPCs isolated following the sorting strategy shown in Appendix Fig . S7A , after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails ( Methods ) ; Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J , as quantified by the " adipo - score " ; marker shapes correspond to different biological replicates , n = 8 - 17 , 3 - 5 biological replicates , 2 - 5 independent wells for each ; Representative fluorescence microscopy images of male - and female - derived total , CD142 - and CD142 + ASPCs , isolated following the sorting strategy shown in Appendix Fig . S8A after in vitro adipogenic differentiation ; Fraction of differentiated cells per ASPC type and per sex shown in L , as quantified by the " adiposcore " ; bar colour shading corresponds to male - and female - derived cells as indicated ; marker shapes correspond to different biological replicates , n = 9 - 10 , 4 biological replicates , 2 - 3 independent wells for each ;

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative fluorescence microscopy images of total, CD142- and CD142+ ASPCs isolated with the use of the respective anti-CD142 antibodies: SinoBiological (SB), SinoBiological-LL (SB-LL), BiOrbyt (BO) and R&D Systems (RnD) (Appendix Fig. S2B, Appendix Fig. S5A, Methods), after in vitro adipogenic differentiation;Fraction of differentiated cells per ASPC type shown in E, as quantified by the "adiposcore"; marker shapes correspond to different anti-CD142 antibodies used for isolation as indicated, n=9-15, 3-4 biological replicates, 3-5 independent wells for each;Representative fluorescence microscopy images of CD142-, 20% CD142+ and (5-7%) CD142+ ASPCs isolated following the sorting strategy shown in Appendix Fig. S7A, after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails (Methods);Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J, as quantified by the "adipo- score"; marker shapes correspond to different biological replicates, n=8-17, 3-5 biological replicates, 2-5 independent wells for each;Representative fluorescence microscopy images of male- and female-derived total, CD142- and CD142+ ASPCs, isolated following the sorting strategy shown in Appendix Fig. S8A after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type and per sex shown in L, as quantified by the "adiposcore"; bar colour shading corresponds to male- and female-derived cells as indicated; marker shapes correspond to different biological replicates, n=9-10, 4 biological replicates, 2-3 independent wells for each;


Figure 2Representative fluorescence microscopy images of P0 - , P12 - 17 - , 4wo - , 7wo - and 11wo - derived total , CD142 - and CD142 + APSCs after in vitro adipogenic differentiation ; Fraction of differentiated cells per ASPC type shown in D , as quantified by the " adiposcore " ; bar colour shading corresponds to pre - and post - weaning - derived cells as indicated ; marker shapes correspond to different biological replicates , n = 14 - 65 , 12 - 13 biological replicates , 2 - 6 independent wells for each

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Figure 2Representative fluorescence microscopy images of P0-, P12-17-, 4wo-, 7wo- and 11wo-derived total, CD142- and CD142+ APSCs after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type shown in D, as quantified by the "adiposcore"; bar colour shading corresponds to pre- and post-weaning-derived cells as indicated; marker shapes correspond to different biological replicates, n=14-65, 12-13 biological replicates, 2-6 independent wells for each


Figure 3Representative fluorescence microscopy images of CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type ( WT ) CD142 + ASPCs after in vitro adipogenic differentiation ; Fraction of differentiated CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type CD142 + ASPCs ( non - treated or treated with control scrambled siRNA ) after in vitro adipogenic differentiation and as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each Representative fluorescence microscopy imaged of human - derived stromal vascular fraction ( SVF ) , containing the ASPCs , after in vitro adipogenic differentiation when co - cultured with total , CD142 - or CD142 + mouse ASPCs ; Fraction of differentiated human - derived SVF , containing the ASPCs co - cultured with total , CD142 - or CD142 + mouse ASPCs after in vitro adipogenic differentiation ( Methods ) shown in E ; marker shapes correspond to different biological replicates , n = 9 , 3 biological replicates , 3 independent wells for each Fraction of differentiated adult - derived CD142 - ASPCs , as quantified by the " adiposcore " , treated with the indicated recombinant proteins shown in J ; marker shapes correspond to different biological replicates , n = 11 - 59 , 2 - 10 biological replicates , 3 - 9 independent wells for eachRepresentative fluorescence microscopy images of adult - derived CD142 - ASPCs after in vitro adipogenic differentiation ; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg - specific candidates : CD142 , MGP , GDF10 , CLEC11A , CPE and BGN at 100 ng /

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Representative fluorescence microscopy images of CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type (WT) CD142+ ASPCs after in vitro adipogenic differentiation;   Fraction of differentiated CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type CD142+ ASPCs (non-treated or treated with control scrambled siRNA) after in vitro adipogenic differentiation and as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each  Representative fluorescence microscopy imaged of human-derived stromal vascular fraction (SVF), containing the ASPCs, after in vitro adipogenic differentiation when co-cultured with total, CD142- or CD142+ mouse ASPCs;   Fraction of differentiated human-derived SVF, containing the ASPCs co-cultured with total, CD142- or CD142+ mouse ASPCs after in vitro adipogenic differentiation (Methods) shown in E; marker shapes correspond to different biological replicates, n=9, 3 biological replicates, 3 independent wells for each  Fraction of differentiated adult-derived CD142- ASPCs, as quantified by the "adiposcore", treated with the indicated recombinant proteins shown in J; marker shapes correspond to different biological replicates, n=11-59, 2-10 biological replicates, 3-9 independent wells for eachRepresentative fluorescence microscopy images of adult-derived CD142- ASPCs after in vitro adipogenic differentiation; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg-specific candidates: CD142, MGP, GDF10, CLEC11A, CPE and BGN at 100 ng/m