Figure 3 ( A ) Quantification of gene expression in HEK293 cells after transduction with 0 . 5x106 , 1x106 , and 2x106 TU / cell of RGD4C . TPA , RGD4C . AAVP or non - targeted ( NT ) particles carrying Lucia . Luciferase activity was measured at days 1 to 6 post - transduction . Data shown are representative of three independent experiments ( n = 3 ) , and are expressed as mean + SEM . One - way ANOVA with Tukey ' s HSD test was used for data analysis . ( B ) Representative images of FITC - labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post - inoculation ( n = 3 ) . Scale bar , 100 μm . ( C ) Particle diffusion using a vertical trans - well system partitioned by Matrigel . Samples were collected from the wells below the Matrigel at days 1 to 7 , n = 3 , technical replicates . Data are expressed as mean + SEM , independent t - test was used for data analysis . ( D ) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins , then quantified by FACS at 2 and 4 hours post - transduction , 0 . 1X106TU / cell , and normalised to particle length . Data are expressed as mean + SEM of n = 3 independent experiments , independent t - test was used for data analysis . ( E ) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post - transduction with 0 . 5x106 TU / cell of RGD4C . TPA and RGD4C . AAVP , using qPCR with primers specific to AAV - 2 ITRs . Data are representative of n = 3 independent experiments and expressed as mean + SEM , independent t - test was used for data analysis . ( F ) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma - derived neural stem cells with 1x106 TU / cell . Data are expressed as mean + SEM of n = 3 independent experiments . One - way ANOVA with Tukey ' s HSD test was used for data analysis . ( G ) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia . Experiments were repeated three times and performed in triplicates . Data are shown as mean + SEM and are representative of one experiment . Independent t - test was used for data analysis .

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Figure 3(A) Quantification of gene expression in HEK293 cells after transduction with 0.5x106, 1x106, and 2x106 TU/cell of RGD4C.TPA, RGD4C.AAVP or non-targeted (NT) particles carrying Lucia. Luciferase activity was measured at days 1 to 6 post-transduction. Data shown are representative of three independent experiments (n = 3), and are expressed as mean + SEM. One-way ANOVA with Tukey's HSD test was used for data analysis.(B) Representative images of FITC-labelled TPA and AAVP particle diffusion in Matrigel at 5 and 30 minutes post-inoculation (n = 3). Scale bar, 100 μm. (C) Particle diffusion using a vertical trans-well system partitioned by Matrigel. Samples were collected from the wells below the Matrigel at days 1 to 7, n = 3, technical replicates. Data are expressed as mean + SEM, independent t-test was used for data analysis.(D) Comparison of AAVP and TPA internalisation efficiency in HEK293 cells as evaluated by staining of the phage capsid proteins, then quantified by FACS at 2 and 4 hours post-transduction, 0.1X106TU/cell, and normalised to particle length. Data are expressed as mean + SEM of n = 3 independent experiments, independent t-test was used for data analysis.(E) Quantification of ITRs in the cytosol and nucleus of HEK293 cells 24 hours post-transduction with 0.5x106 TU/cell of RGD4C.TPA and RGD4C.AAVP, using qPCR with primers specific to AAV-2 ITRs. Data are representative of n = 3 independent experiments and expressed as mean + SEM, independent t-test was used for data analysis.(F) Quantification of Lucia reporter gene expression following transduction of human primary SEBTA003 glioblastoma and G26 glioblastoma-derived neural stem cells with 1x106 TU/cell. Data are expressed as mean + SEM of n = 3 independent experiments. One-way ANOVA with Tukey's HSD test was used for data analysis.(G) 3D primary GBM001 tumor spheres subjected to transduction by increasing doses of TPA and AAVP encoding Lucia. Experiments were repeated three times and performed in triplicates. Data are shown as mean + SEM and are representative of one experiment. Independent t-test was used for data analysis.


Figure 4 ( B ) ELISA quantification of TNFα production in media collected on days 3 to 5 post - transduction of human primary GBM001 tumor spheres at 1x106 TU / cell of targeted ( RGD4C ) or non - targeted ( NT ) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data analysis . ( C ) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C . TPA . TNFαIL2 to NOD / SCID mice bearing subcutaneous human GBM001 . Tumors and healthy organs were harvested at day 18 post - transduction and analysed using RT - qPCR to quantify the TNFαIL2 transcripts . Data shown are expressed as mean + SEM , from n = 3 mice . Two - way ANOVA was used for data analysis . ( D ) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene , at day 18 post targeted TNFαIL2 cytokine treatment . ( E ) Analysis of tumor growth in representative mice , from D , using the tumor bioluminescence data ( total flux ) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0 .

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 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 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

Figure 4(B) ELISA quantification of TNFα production in media collected on days 3 to 5 post-transduction of human primary GBM001 tumor spheres at 1x106 TU/cell of targeted (RGD4C) or non-targeted (NT) TPA particles carrying native TNFα or recombinant TNFαIL2 sequences. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis.(C) Biodistribution of gene delivery upon intravenous administration of 5x1010 TU of NT or RGD4C.TPA.TNFαIL2 to NOD/SCID mice bearing subcutaneous human GBM001. Tumors and healthy organs were harvested at day 18 post-transduction and analysed using RT-qPCR to quantify the TNFαIL2 transcripts. Data shown are expressed as mean + SEM, from n = 3 mice. Two-way ANOVA was used for data analysis.(D) BLI of Luc showing representative mice with subcutaneous GBM001 labelled with Luc gene, at day 18 post targeted TNFαIL2 cytokine treatment. (E) Analysis of tumor growth in representative mice, from D, using the tumor bioluminescence data (total flux) and expressed as fold changes of tumor luminescence flux at different time points compared to day 0.


Figure 5 ( B ) Investigation of the TPA . IL15IgK in comparison to the native TPA . IL15 in B16 . F1 and CT26 . CL25 tumor cells . The TPA DNA constructs were transfected into tumor cells , then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL - 2 . Data are representative of one experiment and expressed as mean + SEM . Experiments were repeated twice and performed in triplicates , one - way ANOVA with Tukey ' s HSD test was used for data analysis ( C ) Targeted ( RGD4C ) and non - targeted ( NT ) TPA particles carrying IL15IgK were produced and used to transduce B16 . F1 or CT26 . CL25 cells at 0 . 5x106 or 1x106 TU / cell . Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post - transduction . RGD4C . TPA without IL15IgK ( mock ) was also included as control . Experiments were repeated twice and shown are the results from a representative experiment . Data are expressed as mean + SEM , one - way ANOVA with Tukey ' s HSD test was used for data analysis . ( D ) Evaluation of CTLL - 2 proliferation in the presence of media collected from B16 . F1 or CT26 . CL25 tumor cells after transduction with TPA particles carrying IL15IgK . Cells treated with a recombinant IL2 cytokine were included as a positive control . Data are representative of one experiment , expressed as mean + SEM and shown as change in CTLL - 2 number relative to treatment initiation day . Experiments were repeated twice and in triplicates , one - way ANOVA with Tukey ' s HSD test was used to analyse the data .

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 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 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

Figure 5(B) Investigation of the TPA.IL15IgK in comparison to the native TPA.IL15 in B16.F1 and CT26.CL25 tumor cells. The TPA DNA constructs were transfected into tumor cells, then the bioactivity of secreted IL15 was assessed by evaluating the effects of media from transfected cells on the proliferation of mouse CTLL-2. Data are representative of one experiment and expressed as mean + SEM. Experiments were repeated twice and performed in triplicates, one-way ANOVA with Tukey's HSD test was used for data analysis(C) Targeted (RGD4C) and non-targeted (NT) TPA particles carrying IL15IgK were produced and used to transduce B16.F1 or CT26.CL25 cells at 0.5x106 or 1x106 TU/cell. Then secreted IL15 was quantified by ELISA in tumor cell media collected on day 5 post-transduction. RGD4C.TPA without IL15IgK (mock) was also included as control. Experiments were repeated twice and shown are the results from a representative experiment. Data are expressed as mean + SEM, one-way ANOVA with Tukey's HSD test was used for data analysis.(D) Evaluation of CTLL-2 proliferation in the presence of media collected from B16.F1 or CT26.CL25 tumor cells after transduction with TPA particles carrying IL15IgK. Cells treated with a recombinant IL2 cytokine were included as a positive control. Data are representative of one experiment, expressed as mean + SEM and shown as change in CTLL-2 number relative to treatment initiation day. Experiments were repeated twice and in triplicates, one-way ANOVA with Tukey's HSD test was used to analyse the data.


Figure 6 ( A ) Tumor - bearing BALB / c mice with established subcutaneous CT26 . CL25 tumors were intravenously injected with a single dose , 5x1010 TU / mouse , of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration . Data are representative of one experiment , n = 3 , and expressed as mean + SEM . Two - way ANOVA test was used for data analysis . ( B ) Safety of dose escalation regimens assessed at day 5 following TPA delivery , by evaluating the serum levels of LDH from n = 2 mice / TPA dose of targeted ( RGD4C ) or non - targeted ( NT ) TPA . IL15IgK . Serums from untreated mice were also analysed . ( C ) Representative tumor - bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C . TPA . IL15IgK ( RGD4C ) or non - targeted ( NT ) particles . The CT26 . CL25 tumor cells stably express a luciferase Luc reporter gene . Untreated mice were also included as controls ( CTRL ) . ( D ) Changes in tumor viability in mice ( n = 4 ) from day 0 to day 5 post TPA administration . Tumor viability was measured by bioluminescence activity . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Kruskal - Wallis test was used for data analysis . ( E ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from all experimental groups . ( F ) Animal weights ( n = 4 ) were monitored during therapy experiments ad expressed as mean + SEM .

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Figure 6(A) Tumor-bearing BALB/c mice with established subcutaneous CT26.CL25 tumors were intravenously injected with a single dose, 5x1010 TU/mouse, of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Gene delivery was evaluated by quantifying the IL15IgK mRNA expression in tumors and healthy tissues at day 5 post TPA administration. Data are representative of one experiment, n = 3, and expressed as mean + SEM. Two-way ANOVA test was used for data analysis.(B) Safety of dose escalation regimens assessed at day 5 following TPA delivery, by evaluating the serum levels of LDH from n = 2 mice/TPA dose of targeted (RGD4C) or non-targeted (NT) TPA.IL15IgK. Serums from untreated mice were also analysed.(C) Representative tumor-bearing mice showing bioluminescence imaging of luciferase on days 0 and 5 post administration with 5x1010 TU of RGD4C.TPA.IL15IgK (RGD4C) or non-targeted (NT) particles. The CT26.CL25 tumor cells stably express a luciferase Luc reporter gene. Untreated mice were also included as controls (CTRL).(D) Changes in tumor viability in mice (n = 4) from day 0 to day 5 post TPA administration. Tumor viability was measured by bioluminescence activity. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Kruskal-Wallis test was used for data analysis.(E) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from all experimental groups.(F) Animal weights (n = 4) were monitored during therapy experiments ad expressed as mean + SEM.


Figure 7 ( A ) The immune profile of CT26 . CL25 tumors was investigated using RT - qPCR at day 5 after TPA . IL15IgK administration . A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8 + T cell population ; NKp46 for NK cell population . Markers for cytotoxic cell - killing by CD8 + T cells ( GZMa , GZMb ) and by NK cells ( Prf1 ) were also analysed . Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma ( IFN - γ ) , and FoxP3 for Treg cells . Data shown are representative of n = 5 mice and expressed as mean + SEM . Non - parametric Mann - Whitney test was used for GZMb and TBX2 data analysis , and independent t - test to analyse data of the other markers . ( B ) TPA particles encoding IL12 were used to treat immunocompetent BALB / c mice bearing subcutaneous CT26 . CL25 tumors labelled with a Luc reporter gene . Representative tumor - bearing mice at day 5 post - treatment with targeted RGD4C . TPA . IL12 ( RGD4C ) or non - targeted TPA . IL12 ( NT ) . ( C ) Analysis of total flux change in tumors , n = 4 mice , after IL12 immunotherapy . The central band represents the median of the data . The boxes upper and lower lines represent quartile 3 and quartile 1 of the data , respectively . The whiskers represent the maximum and minimum outliers of the data . Non - parametric Mann - Whitney test was used for data analysis . ( D ) Kaplan Meier curves showing survival benefit for tumor - bearing mice ( n = 4 ) from two experimental groups . ( E ) Serial quantification of IL12 in media of B16 . F10 cells treated with RGD4C . TPA . IL12 or controls . Data are representative of one experiment , n = 3 biological repeats , and shown as mean + SD Experiments were repeated three times . One - way ANOVA with Tukey ' s HSD test was used for data analysis . ( F ) RGD4C . TPA . IL12 immunotherapy of subcutaneous B16 . F10 tumors in C57BL / 6 mice ( n = 4 ) . Data are shown as mean + SEM independent t - test was used for data analysis . Tumor volume changes are shown over time .

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 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 O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 7(A) The immune profile of CT26.CL25 tumors was investigated using RT-qPCR at day 5 after TPA.IL15IgK administration. A panel of immunological mRNA transcripts were selected for analysis and included CD8a and CD8b as markers for CD8+ T cell population; NKp46 for NK cell population. Markers for cytotoxic cell-killing by CD8+ T cells (GZMa, GZMb) and by NK cells (Prf1) were also analysed. Expression of TBX21 was evaluated as a marker for the Th1 response mediated by interferon gamma (IFN-γ), and FoxP3 for Treg cells. Data shown are representative of n = 5 mice and expressed as mean + SEM. Non-parametric Mann-Whitney test was used for GZMb and TBX2 data analysis, and independent t-test to analyse data of the other markers.(B) TPA particles encoding IL12 were used to treat immunocompetent BALB/c mice bearing subcutaneous CT26.CL25 tumors labelled with a Luc reporter gene. Representative tumor-bearing mice at day 5 post-treatment with targeted RGD4C.TPA.IL12 (RGD4C) or non-targeted TPA.IL12 (NT).(C) Analysis of total flux change in tumors, n = 4 mice, after IL12 immunotherapy. The central band represents the median of the data. The boxes upper and lower lines represent quartile 3 and quartile 1 of the data, respectively. The whiskers represent the maximum and minimum outliers of the data. Non-parametric Mann-Whitney test was used for data analysis.(D) Kaplan Meier curves showing survival benefit for tumor-bearing mice (n = 4) from two experimental groups.(E) Serial quantification of IL12 in media of B16.F10 cells treated with RGD4C.TPA.IL12 or controls. Data are representative of one experiment, n = 3 biological repeats, and shown as mean + SD Experiments were repeated three times. One-way ANOVA with Tukey's HSD test was used for data analysis.(F) RGD4C.TPA.IL12 immunotherapy of subcutaneous B16.F10 tumors in C57BL/6 mice (n = 4). Data are shown as mean + SEM independent t-test was used for data analysis. Tumor volume changes are shown over time.


Figure 1Kaplan - Meier survival curves stratified by the patient MICA - 129 genotype for all patients ( n = 446 ) . The survival curve is displayed for the first 66 months ; 7 . 6 % of the patients were followed longer . Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2 . The HR indicates the risk per MICA - 129Met allele carried by the patients ( additive risk model ) . The numbers of patients carrying the three genotypes and the number of events ( in brackets ) are indicated . Kaplan - Meier survival curves for patients receiving a graft matched for the MICA - 129 genotype ( n = 404 ) . Kaplan - Meier survival curves for patients who experienced aGVDH ( any grade , n = 244 ) . Kaplan - Meier survival curves for patients not experiencing aGVHD ( n = 189 ) . The HR indicates the risk of patients carrying two MICA - 129Met alleles ( recessive risk model ) . Kaplan - Meier survival curves for patients who did not receive a T - cell - depleting treatment with ATG ( n = 193 ) . Kaplan - Meier survival curves for patients treated with ATG ( n = 250 ) . Kaplan - Meier survival curves for patients with the MICA - 129Val / Val genotype ( n = 229 ) stratified by treatment with ATG . Kaplan - Meier survival curves for patients carrying one or two MICA - 129Met alleles ( n = 214 ) stratified by treatment with ATG .

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Figure 1Kaplan-Meier survival curves stratified by the patient MICA-129 genotype for all patients (n = 446). The survival curve is displayed for the first 66 months; 7.6% of the patients were followed longer. Effects on overall survival were determined by Cox regression with covariate adjustment as indicated in Table2. The HR indicates the risk per MICA-129Met allele carried by the patients (additive risk model). The numbers of patients carrying the three genotypes and the number of events (in brackets) are indicated.Kaplan-Meier survival curves for patients receiving a graft matched for the MICA-129 genotype (n = 404).Kaplan-Meier survival curves for patients who experienced aGVDH (any grade, n = 244).Kaplan-Meier survival curves for patients not experiencing aGVHD (n = 189). The HR indicates the risk of patients carrying two MICA-129Met alleles (recessive risk model).Kaplan-Meier survival curves for patients who did not receive a T-cell-depleting treatment with ATG (n = 193).Kaplan-Meier survival curves for patients treated with ATG (n = 250).Kaplan-Meier survival curves for patients with the MICA-129Val/Val genotype (n = 229) stratified by treatment with ATG.Kaplan-Meier survival curves for patients carrying one or two MICA-129Met alleles (n = 214) stratified by treatment with ATG.


Figure 2The linear regression of MICA expression intensity and binding of a recombinant NKG2D - Fc fusion protein both determined as MFI by flow cytometry is displayed for L - MICA - 129Met ( n = 79 , left panel ) and L - MICA - 129Val clones ( n = 81 , right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated . Purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells ( 106 ) were stimulated with immobilized MICA - 129Met - mIgG2a - Fc or MICA - 129Val - mIgG2a - Fc or OVA - mIgG2a - Fc fusion proteins ( 10 μg / ml ) for 3 , 10 , or 30 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb , an anti - phospho - SRC family ( Tyr419 ) kinases Ab , and an anti - β - actin mAb as a loading control . The arrow points toward phosphorylated SRC family kinases . Blots obtained from three independent experiments were analyzed by densitometry , and the means plus SD of the ratio between phospho - SRC family kinase and β - actin signals is displayed . The difference between NK cells stimulated for 10 min by MICA - 129Met - Fc or MICA - 129Val - Fc proteins was assessed by t - test . Purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days , 106 ) were incubated with the SRC kinase inhibitor PP2 ( 25 μM ) , the vehicle DMSO , or medium only ( Ø ) for 30 min before being added to immobilized MICA - 129Met - Fc , MICA - 129Val - Fc , or OVA - Fc fusion proteins ( 10 μg / ml ) for 10 min . The protein lysates of these cells were separated by SDS - PAGE , and the blot was probed subsequently with an anti - phospho - Tyr mAb and an anti - β - actin mAb as a loading control . The blot is representative for two independent experiments . In parallel , degranulation of the NK cells was measured by anti - CD107a staining in flow cytometry . The difference between DMSO - and PP2 - treated cells with respect to CD107a + cells and the MFI of CD107a is indicated in the histograms . The results are representative for two independent experiments .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 2The linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met (n = 79, left panel) and L-MICA-129Val clones (n = 81, right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (106) were stimulated with immobilized MICA-129Met-mIgG2a-Fc or MICA-129Val-mIgG2a-Fc or OVA-mIgG2a-Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases.Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t-test.Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS-PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.


Figure 3The degranulation ( CD107a expression ) of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells in response to immobilized MICA - 129Met - Fc and MICA - 129Val - Fc , and as negative control , OVA - Fc fusion proteins was determined by flow cytometry after 1 h ( n = 3 ) . Displayed are means and SD of the MFI ( left panel ) and the percentage of CD107a + cells ( right panel ) . Differences between MICA - 129Met - Fc - and MICA - 129Val - Fc - induced NK - cell degranulation were analyzed by two - way ANCOVA adjusted for MICA protein concentration , and the respective P - values are indicated . The degranulation of IL - 2 - stimulated LAK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 27 ) or L - MICA - 129Val clones ( n = 27 ) for 1 h was determined by flow cytometry . CD107a cell surface expression was analyzed after gating on CD56 + NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of CD107a on NK cells ( upper panels ) or the proportion of CD107a + NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated . A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L - MICA - 129Met and an L - MICA - 129Val clone . L - con cells served as a negative and K562 cells as a positive control . The means of specific lysis of triplicates plus SD at different E : T ratios ( 200 : 1 to 3 : 1 ) were measured in an 51chromium - release assay . The MICA expression intensity and the binding of a recombinant NKG2D - Fc fusion protein to the target cells were determined in parallel by flow cytometry , and the MFIs are indicated .

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 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 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 3The degranulation (CD107a expression) of purified IL-2-stimulated (100 U/ml for 4 days) NK cells in response to immobilized MICA-129Met-Fc and MICA-129Val-Fc, and as negative control, OVA-Fc fusion proteins was determined by flow cytometry after 1 h (n = 3). Displayed are means and SD of the MFI (left panel) and the percentage of CD107a+ cells (right panel). Differences between MICA-129Met-Fc- and MICA-129Val-Fc-induced NK-cell degranulation were analyzed by two-way ANCOVA adjusted for MICA protein concentration, and the respective P-values are indicated.The degranulation of IL-2-stimulated LAK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 27) or L-MICA-129Val clones (n = 27) for 1 h was determined by flow cytometry. CD107a cell surface expression was analyzed after gating on CD56+ NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of CD107a on NK cells (upper panels) or the proportion of CD107a+ NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.A representative of 21 experiments is shown demonstrating the specific cytotoxic activity of LAK cells against an L-MICA-129Met and an L-MICA-129Val clone. L-con cells served as a negative and K562 cells as a positive control. The means of specific lysis of triplicates plus SD at different E:T ratios (200:1 to 3:1) were measured in an 51chromium-release assay. The MICA expression intensity and the binding of a recombinant NKG2D-Fc fusion protein to the target cells were determined in parallel by flow cytometry, and the MFIs are indicated.


Figure 4The IFNγ expression of purified IL - 2 - stimulated ( 100 U / ml for 4 days ) NK cells exposed for 4 h to L - MICA - 129Met ( n = 23 ) or L - MICA - 129Val clones ( n = 23 ) was determined by flow cytometry . The IFNγ expression was analyzed after gating on CD56brightCD16 − NK cells . In parallel , the MICA expression on target cells was determined . Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16 − NK cells ( upper panels ) or the proportion of IFNγ + CD56brightCD16 − NK cells ( lower panels ) and the MICA expression intensity on target cells ( MFI ) for the L - MICA - 129Met ( left panels ) and L - MICA - 129Val clones ( right panels ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated . The IFNγ release of purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days ) co - cultured with L - MICA - 129Met ( n = 34 ) or L - MICA - 129Val clones ( n = 32 ) for 24 h was measured in the supernatant by ELISA . In parallel , the MICA expression intensity on target cells was determined by flow cytometry . The linear regressions of IFNγ release ( pg / ml ) by NK cells and MICA expression on targets ( MFI ) are displayed for the L - MICA - 129Met clones ( left panel ) and the L - MICA - 129Val clones ( right panel ) . The coefficients of determination ( R2 ) , the regression coefficients ( reg . coeff . ) , and the P - values for Pearson correlation are indicated .

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 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

Figure 4The IFNγ expression of purified IL-2-stimulated (100 U/ml for 4 days) NK cells exposed for 4 h to L-MICA-129Met (n = 23) or L-MICA-129Val clones (n = 23) was determined by flow cytometry. The IFNγ expression was analyzed after gating on CD56brightCD16−NK cells. In parallel, the MICA expression on target cells was determined. Displayed are the linear regressions of the MFI of IFNγ in CD56brightCD16−NK cells (upper panels) or the proportion of IFNγ+CD56brightCD16−NK cells (lower panels) and the MICA expression intensity on target cells (MFI) for the L-MICA-129Met (left panels) and L-MICA-129Val clones (right panels). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.The IFNγ release of purified IL-2-stimulated NK cells (100 U/ml for 4 days) co-cultured with L-MICA-129Met (n = 34) or L-MICA-129Val clones (n = 32) for 24 h was measured in the supernatant by ELISA. In parallel, the MICA expression intensity on target cells was determined by flow cytometry. The linear regressions of IFNγ release (pg/ml) by NK cells and MICA expression on targets (MFI) are displayed for the L-MICA-129Met clones (left panel) and the L-MICA-129Val clones (right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.


Figure 5NKG2D expression on purified IL - 2 - stimulated NK cells ( 100 U / ml for 4 days ) exposed to L - MICA - 129Met ( n = 25 ) or L - MICA - 129Val clones ( n = 25 ) for 0 , 4 , and 24 h was analyzed by flow cytometry . NK cells ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating of CD3 − CD56 + cells . The means and SD of the percentage of NKG2D + NK cells ( left panel ) and of the MFI of NKG2D ( right panel ) are shown . Differences between the groups were analyzed by repeated measures ANOVA , and P - values for pairwise comparisons are indicated .

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

Figure 5NKG2D expression on purified IL-2-stimulated NK cells (100 U/ml for 4 days) exposed to L-MICA-129Met (n = 25) or L-MICA-129Val clones (n = 25) for 0, 4, and 24 h was analyzed by flow cytometry. NK cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating of CD3−CD56+ cells. The means and SD of the percentage of NKG2D+ NK cells (left panel) and of the MFI of NKG2D (right panel) are shown. Differences between the groups were analyzed by repeated measures ANOVA, and P-values for pairwise comparisons are indicated.


Figure 6MACS - separated CD8 + T cells were cultured in triplicate on an immobilized anti - CD3 mAb ( 0 . 005 μg / ml [ upper panel ] or 0 . 01 μg / ml [ lower panel ] ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , and OVA - Fc proteins at various concentrations ( 1 . 0 , 0 . 5 , 0 . 1 , 0 . 0 μg / ml ) . After 72 h , 25 % of the supernatant was harvested and IL - 2 concentrations were measured by ELISA . The harvested medium was replaced by the same volume containing 1 μCi 3H - labeled thymidine . After 12 h , the plates were completely harvested and the DNA - bound radioactivity was determined . The means and SD of the stimulation index ( SI ) are displayed ( n = 4 ) . Significant differences between MICA - 129Met / Val - Fc and OVA - Fc proteins were found when the antigen - specific signal ( anti - CD3 ) was limited ( * P < 0 . 05 , t - test ; upper left panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0372 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0366 ; upper right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0499 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0192 ; 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0164 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0357 ; lower left panel : 0 . 5 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0287 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0232 ; lower right panel : 1 . 0 μg / ml : MICA - 129Met - Fc versus OVA - Fc P = 0 . 0171 and MICA - 129Val - Fc versus OVA - Fc P = 0 . 0484 ) . Purified CFSE - stained CD8 + T cells were stimulated by immobilized anti - CD3 ( 0 . 005 μg / ml ) in combination with recombinant MICA - 129Met - Fc , MICA - 129Val - Fc , OVA - Fc proteins , or co - stimulatory mAb ( anti - CD28 , anti - NKG2D ) or an isotype control ( mIgG1 ) . The proliferation of CD3 + CD8 + T cells was assessed at 60 h by flow cytometry . Results of a representative out of 6 experiments are displayed . Untreated CFSE - stained CD8 + T cells are included for comparison . The percentage of proliferating cells and MFI for CFSE are indicated . The MFI of CFSE in unstimulated CD8 + T cells ( control ) was set to 100 % in individual experiments ( n = 6 ) , and the relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( * P = 0 . 0277 , Wilcoxon test ) between MICA - 129Met - Fc versus MICA - 129Val - Fc and OVA - Fc proteins were found at slightly higher anti - CD3 concentrations ( 0 . 1 and 0 . 05 μg / ml ) than at later time points ( see A ) . Anti - CD28 and anti - NKG2D mAb were used in parallel as a positive control , mean + SD are shown , and significant differences ( * P = 0 . 0277 , Wilcoxon test ) to the isotype control ( mIgG1 ) are indicated ( n = 6 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6MACS-separated CD8+ T cells were cultured in triplicate on an immobilized anti-CD3 mAb (0.005 μg/ml [upper panel] or 0.01 μg/ml [lower panel]) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, and OVA-Fc proteins at various concentrations (1.0, 0.5, 0.1, 0.0 μg/ml). After 72 h, 25% of the supernatant was harvested and IL-2 concentrations were measured by ELISA. The harvested medium was replaced by the same volume containing 1 μCi 3H-labeled thymidine. After 12 h, the plates were completely harvested and the DNA-bound radioactivity was determined. The means and SD of the stimulation index (SI) are displayed (n = 4). Significant differences between MICA-129Met/Val-Fc and OVA-Fc proteins were found when the antigen-specific signal (anti-CD3) was limited (*P < 0.05, t-test; upper left panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0372 and MICA-129Val-Fc versus OVA-Fc P = 0.0366; upper right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0499 and MICA-129Val-Fc versus OVA-Fc P = 0.0192; 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0164 and MICA-129Val-Fc versus OVA-Fc P = 0.0357; lower left panel: 0.5 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0287 and MICA-129Val-Fc versus OVA-Fc P = 0.0232; lower right panel: 1.0 μg/ml: MICA-129Met-Fc versus OVA-Fc P = 0.0171 and MICA-129Val-Fc versus OVA-Fc P = 0.0484).Purified CFSE-stained CD8+T cells were stimulated by immobilized anti-CD3 (0.005 μg/ml) in combination with recombinant MICA-129Met-Fc, MICA-129Val-Fc, OVA-Fc proteins, or co-stimulatory mAb (anti-CD28, anti-NKG2D) or an isotype control (mIgG1). The proliferation of CD3+CD8+T cells was assessed at 60 h by flow cytometry. Results of a representative out of 6 experiments are displayed. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (*P = 0.0277, Wilcoxon test) between MICA-129Met-Fc versus MICA-129Val-Fc and OVA-Fc proteins were found at slightly higher anti-CD3 concentrations (0.1 and 0.05 μg/ml) than at later time points (see A).Anti-CD28 and anti-NKG2D mAb were used in parallel as a positive control, mean + SD are shown, and significant differences (*P = 0.0277, Wilcoxon test) to the isotype control (mIgG1) are indicated (n = 6).


Figure 7NKG2D expression on purified CD8 + T cells exposed to L - MICA - 129Met ( n = 19 ) or L - MICA - 129Val clones ( n = 19 ) for 0 , 4 , and 24 h was analyzed by flow cytometry . CD8 + T cells ( 2 . 5 × 105 ) were co - cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3 + CD8 + T cells . The means and SD of the MFI of NKG2D ( left panel ) and of the percentage of NKG2D + CD8 + T cells ( right panel ) are displayed . Differences between the groups were analyzed by repeated measures ANOVA , and the P - values are indicated . Purified CD8 + T cells were cultured on plate - bound anti - NKG2D ( 1 μg / ml ) or isotype control ( mIgG1 ) for 24 h before the NKG2D expression was measured by flow cytometry . Means and SD of MFI ( upper panel ) and percentage of NKG2D + cells ( lower panel ) are shown ( n = 6 ) . Differences between the groups were analyzed by t - tests , and the P - values are indicated . These CD8 + T cells were subsequently CFSE - stained and cultured on plates coated with anti - CD3 ( 0 . 005 μg / ml ) in combination with anti - CD28 ( 0 . 5 μg / ml ) as a positive control or anti - NKG2D ( 0 . 5 μg / ml ) . Proliferation was measured after 60 h by flow cytometry . Untreated CFSE - stained CD8 + T cells are included for comparison . The percentage of proliferating cells and the MFI for CFSE are indicated . The MFI of CFSE in unstimulated CD8 + T cells ( control ) was set to 100 % in individual experiments ( n = 6 ) , and the relative decrease due to proliferation was calculated . Means + SD are shown . Significant differences ( Wilcoxon test ) between CD8 + T cells pre - exposed to anti - NKG2D and isotype control ( mIgG1 ) were found in these experiments at anti - CD3 concentrations of 0 . 01 and 0 . 005 μg / ml .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7NKG2D expression on purified CD8+T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.These CD8+T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.


Figure 1A - D Appearance of autophagic structures ( marked with GFP - Atg8a ) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1 , Atg5 , Atg6 , Atg7 or Atg12 . All constructs are expressed in the epidermis under the control of the A58 - Gal4 driver . ( A ) Time points from movies of wounded epidermis . The wounds have closed by 2 h in all cases . ( B ) Higher magnification of the areas marked by magenta boxes at t = 120 min . ( C ) Quantification of the appearance of GFP - Atg8a puncta in the imaged area ( 10000 µm2 ) 3 control larvae , shown for each individual larva . ( D ) Quantification of number of GFP - Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure ; n = 4 - 10 larvae each genotype , for the detail see Data analysis .

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Figure 1A-D Appearance of autophagic structures (marked with GFP-Atg8a) during wound closure in the epidermis of control third instar larvae and after epidermal knockdown of the autophagy pathway components Atg1, Atg5, Atg6, Atg7 or Atg12. All constructs are expressed in the epidermis under the control of the A58-Gal4 driver. (A) Time points from movies of wounded epidermis. The wounds have closed by 2 h in all cases. (B) Higher magnification of the areas marked by magenta boxes at t=120 min. (C) Quantification of the appearance of GFP-Atg8a puncta in the imaged area (10000 µm2) 3 control larvae, shown for each individual larva. (D) Quantification of number of GFP-Atg8a puncta in different genetic conditions measured in an area of 10000 µm2 at the time of wound closure; n=4-10 larvae each genotype, for the detail see Data analysis.


Figure 3Control of epidermal autophagy by TOR signalling . ( A ) Epidermis of third instar larvae expressing the autophagosome marker GFP - Atg8a together with constructs for up - or down - regulating the autophagy pathway in the epidermis . Healthy epidermis contains few autophagosomes , but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes . ( B ) Higher magnification of the areas marked by magenta boxes in ( A ) . Control of epidermal autophagy by TOR signalling . ( C ) Quantification of Atg8a puncta in an area of 10000 µm2 , n = 6 - 8 larvae each genotype . We assumed unequal sample size and unequal variances and calculations were performed . Values are presented as box plots . Box plot elements are : center line , median ; box limits , upper and lower quartiles ; whiskers , 1 . 5x interquartile range ; points , outliers .

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Figure 3Control of epidermal autophagy by TOR signalling. (A) Epidermis of third instar larvae expressing the autophagosome marker GFP-Atg8a together with constructs for up- or down-regulating the autophagy pathway in the epidermis. Healthy epidermis contains few autophagosomes, but artificially activating autophagy through overexpression of Atg1 or blocking TOR signalling leads to accumulation of autophagosomes. (B) Higher magnification of the areas marked by magenta boxes in (A).Control of epidermal autophagy by TOR signalling. (C) Quantification of Atg8a puncta in an area of 10000 µm2, n=6-8 larvae each genotype. We assumed unequal sample size and unequal variances and calculations were performed. Values are presented as box plots. Box plot elements are: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.


Figure 5A - D Fluorescence loss in photobleaching ( FLIP ) to test free cytoplasmic GFP motility within the epidermis . A small area ( magenta circle ; 179 or 1098 µm2 ) was laser - illuminated for the indicated times ( 3 or 20 min ) in control or Atg1 - expressing epidermis also expressing free GFP . ( A ) Snapshots before bleaching and at 3 points of recovery . ( B ) Kymographs along the broken line in ( A ) during recovery . ( C , D ) Quantification of fluorescence recovery after bleaching , shown separately for 3 and 20 min bleaching protocols .

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Figure 5A-D Fluorescence loss in photobleaching (FLIP) to test free cytoplasmic GFP motility within the epidermis. A small area (magenta circle; 179 or 1098 µm2) was laser-illuminated for the indicated times (3 or 20 min) in control or Atg1-expressing epidermis also expressing free GFP. (A) Snapshots before bleaching and at 3 points of recovery. (B) Kymographs along the broken line in (A) during recovery. (C, D) Quantification of fluorescence recovery after bleaching, shown separately for 3 and 20 min bleaching protocols.


Figure 8B - D Atg1 - induced syncytium formation is abolished when Atg5 function is downregulated ( compare to rows 2 and 3 in Fig . 5A , B ) ; same representation as shown in Fig . 5A - D .

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Figure 8B-D Atg1-induced syncytium formation is abolished when Atg5 function is downregulated (compare to rows 2 and 3 in Fig. 5A, B); same representation as shown in Fig. 5A-D.


Figure 9C , D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy . ( C ) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493 / 503 to mark lipid accumulations . ( D ) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions . A - D , n = 7 - 20 larvae each genotype .

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Figure 9C, D Accumulation of neutral lipid in the cytoplasm under conditions of uncontrolled autophagy. (C) Fixed epidermis from larvae expressing the indicated constructs was stained with BodiPy 493/503 to mark lipid accumulations. (D) Quantification of the number of lipid puncta in an epidermal area of 10000 µm2 in the indicated conditions. A-D, n=7-20 larvae each genotype.


figf1 ( a , b ) Bacteria in the spleens of Slamf1 − / − Rag1 − / − and Rag1 − / − BALB / c mice ( a ) or Slamf1 − / − and Slamf1 + / + BALB / c mice ( b ) 48 h after intraperitoneal injection of virulent S . typhimurium 14028s or attenuated S . typhimurium SseB − . CFU , colony - forming units ; ND , not detectable . Data are representative of four independent experiments ( mean and s . d . ) . ( c - e ) Killing of bacteria by peritoneal macrophages from Slamf1 + / + and Slamf1 − / − BALB / c mice exposed to S . typhimurium SseB − ( c ) , E . coli F18 ( d ) or S . aureus ( e ) , assessed by gentamycin assay . Data are representative of five independent experiments ( mean and s . d . ) . ( f ) Uptake of bacteria by Slamf1 + / + ( red solid lines ) or Slamf1 − / − ( black solid lines ) BALB / c peritoneal macrophages incubated at 37 ° C with E . coli - eGFP or S . typhimurium - eGFP or by Slamf1 + / + macrophages incubated for 60 min at 4 ° C with the bacteria ( dotted lines ) . Right , mean fluorescence intensity ( MFI ) . Data are representative of three independent experiments ( mean and s . d . ) .

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figf1(a,b) Bacteria in the spleens of Slamf1−/−Rag1−/− and Rag1−/− BALB/c mice (a) or Slamf1−/− and Slamf1+/+ BALB/c mice (b) 48 h after intraperitoneal injection of virulent S. typhimurium 14028s or attenuated S. typhimurium SseB−. CFU, colony-forming units; ND, not detectable. Data are representative of four independent experiments (mean and s.d.).(c-e) Killing of bacteria by peritoneal macrophages from Slamf1+/+ and Slamf1−/− BALB/c mice exposed to S. typhimurium SseB− (c), E. coli F18 (d) or S. aureus (e), assessed by gentamycin assay. Data are representative of five independent experiments (mean and s.d.).(f) Uptake of bacteria by Slamf1+/+ (red solid lines) or Slamf1−/− (black solid lines) BALB/c peritoneal macrophages incubated at 37 °C with E. coli-eGFP or S. typhimurium-eGFP or by Slamf1+/+ macrophages incubated for 60 min at 4 °C with the bacteria (dotted lines). Right, mean fluorescence intensity (MFI). Data are representative of three independent experiments (mean and s.d.).


figf2 ( a ) NOX2 activity in Slamf1 + / + and Slamf1 - / - BALB / c peritoneal macrophages stimulated for 0 - 100 min with E . coli F18 , S . aureus or PMA , assessed with lucigenin . Data are representative of five independent experiments ( mean ± s . d . ) . ( b ) Phagosomal pH of Slamf1 + / + , Slamf1 - / - and gp91phox - / - B6 primary macrophages loaded for 0 - 200 min with pHrodo - coated E . coli or S . aureus , analyzed by flow cytometry . Data are representative of three independent experiments ( mean ± s . d . ) . ( c ) NOX2 activity in primary macrophages in response to LPS , purified OmpC , peptidoglycan ( PGN ) or PMA , assessed with lucigenin . TLR4 - KO , TLR4 - deficient ( strain del / Jtht ; C3H ) . Data are representative of five independent experiments ( mean ± s . d . ) .

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figf2(a) NOX2 activity in Slamf1+/+ and Slamf1-/- BALB/c peritoneal macrophages stimulated for 0-100 min with E. coli F18, S. aureus or PMA, assessed with lucigenin. Data are representative of five independent experiments (mean ± s.d.).(b) Phagosomal pH of Slamf1+/+, Slamf1-/- and gp91phox-/- B6 primary macrophages loaded for 0-200 min with pHrodo-coated E. coli or S. aureus, analyzed by flow cytometry. Data are representative of three independent experiments (mean ± s.d.).(c) NOX2 activity in primary macrophages in response to LPS, purified OmpC, peptidoglycan (PGN) or PMA, assessed with lucigenin. TLR4-KO, TLR4-deficient (strain del/Jtht; C3H). Data are representative of five independent experiments (mean ± s.d.).


figf3 ( a ) Fluorescence microscopy of E . coli - containing phagosomes in primary macrophages transfected with RFP - conjugated LAMP - 1 , showing colocalization with E . coli - eGFP or S . aureus - eGFP . Right , quantification of LAMP - 1 + phagosomes in the microscopy at left . ( b ) Immunoblot analysis of LAMP - 1 in phagosomes isolated by sucrose - gradient flotation from RAW264 . 7 macrophages after phagocytosis by Slamf1 - or mock - transfected RAW264 . 7 macrophages of beads coated with E . coli outer membrane extract . WCL , whole - cell lysate . Right , quantification of LAMP - 1 in the immunoblot at left . ( c ) Localization of MHC class II - eGFP ( MHCII GFP ) in phagosomes of primary macrophages from Slamf1 + / + or Slamf1 − / − MHC class II - eGFP ( B6 ) mice and 3 - μm beads coated with E . coli outer membrane extract . ( d ) Entry of 3 - μm beads coated with E . coli outer membrane extract into lysosomes loaded with Texas red - dextran . Right ( c , d ) , quantification of fluorescence in the microscopy at left . Numbers in bottom right corners ( a , c , d ) indicate time ( in min ) . Original magnification ( a - c ) , × 60 . DIC , differential interference contrast . Data are representative of three combined experiments ( a ) or at least three independent experiments ( b - d ) with at least 100 beads or 80 bacteria in each ( error bars , s . d . ) .

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figf3(a) Fluorescence microscopy of E. coli-containing phagosomes in primary macrophages transfected with RFP-conjugated LAMP-1, showing colocalization with E. coli-eGFP or S. aureus-eGFP. Right, quantification of LAMP-1+ phagosomes in the microscopy at left.(b) Immunoblot analysis of LAMP-1 in phagosomes isolated by sucrose-gradient flotation from RAW264.7 macrophages after phagocytosis by Slamf1- or mock-transfected RAW264.7 macrophages of beads coated with E. coli outer membrane extract. WCL, whole-cell lysate. Right, quantification of LAMP-1 in the immunoblot at left.(c) Localization of MHC class II-eGFP (MHCII GFP) in phagosomes of primary macrophages from Slamf1+/+ or Slamf1−/− MHC class II-eGFP (B6) mice and 3-μm beads coated with E. coli outer membrane extract. (d) Entry of 3-μm beads coated with E. coli outer membrane extract into lysosomes loaded with Texas red-dextran. Right (c,d), quantification of fluorescence in the microscopy at left. Numbers in bottom right corners (a,c,d) indicate time (in min). Original magnification (a-c), ×60. DIC, differential interference contrast. Data are representative of three combined experiments (a) or at least three independent experiments (b-d) with at least 100 beads or 80 bacteria in each (error bars, s.d.).


figf4 ( a , b ) Association of EEA - 1 ( a ) or Rab5 ( b ) with phagosomes generated in primary macrophages by 3 - μm beads coated with E . coli outer membrane extract ; numbers in bottom corners indicate time ( in min ) . α - , anti - . Original magnification , × 60 . Right ( a , b ) , quantification of fluorescence . Data are representative of at least three independent experiments ( error bars , s . d . ) .

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figf4(a,b) Association of EEA-1 (a) or Rab5 (b) with phagosomes generated in primary macrophages by 3-μm beads coated with E. coli outer membrane extract; numbers in bottom corners indicate time (in min). α-, anti-. Original magnification, ×60. Right (a,b), quantification of fluorescence. Data are representative of at least three independent experiments (error bars, s.d.).


figf5 ( a ) Colocalization of SLAM and bacteria in RAW264 . 7 macrophages transfected with SLAM - mCherry and allowed to phagocytose E . coli - eGFP or S . aureus - eGFP for 0 - 120 min ( time ( in min ) , bottom right corners at left ) . Original magnification , × 60 . Right , quantification of fluorescence at left . Data are representative of two independent experiments with a minimum of 40 bacteria per time point ( error bars , s . d . ) . ( b ) Immunoblot analysis of phagosome isolates from RAW264 . 7 macrophages transiently transfected with cDNA encoding Myc - tagged SLAM ( Slamf1 - Myc ) or mock transfected and allowed to phagocytose beads coated with E . coli outer membrane extract for 60 or 120 min . kDa , kilodaltons . Data are representative of at least three independent experiments .

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figf5(a) Colocalization of SLAM and bacteria in RAW264.7 macrophages transfected with SLAM-mCherry and allowed to phagocytose E. coli-eGFP or S. aureus-eGFP for 0-120 min (time (in min), bottom right corners at left). Original magnification, ×60. Right, quantification of fluorescence at left. Data are representative of two independent experiments with a minimum of 40 bacteria per time point (error bars, s.d.).(b) Immunoblot analysis of phagosome isolates from RAW264.7 macrophages transiently transfected with cDNA encoding Myc-tagged SLAM (Slamf1-Myc) or mock transfected and allowed to phagocytose beads coated with E. coli outer membrane extract for 60 or 120 min. kDa, kilodaltons. Data are representative of at least three independent experiments.


figf6 ( a ) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter ( as described in Results ) , plus a renilla luciferase reporter , then exposed to heat - killed bacteria ( top ) . ( b ) Luciferase activity in Jurkat cells transfected with the fusion in a ( Slamf1 - CD3ζ ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ ( ΔIgV - Slamf1 - CD3ζ ) , and luciferase reporters as in a , then exposed to heat - killed E . coli F18 . ( c ) Luciferase activity in Jurkat cells transfected as in a , then left uninoculated ( − ) or inoculated with 10 × 108 E . coli F18 ( + ) , followed by the addition of monoclonal antibody ( mAb ; amount , under graph ) 9D1 to SLAM ( α - SLAM ) or rat immunoglobulin G2b isotype - matched control antibody ( Rat IgG2b ) . ( d ) Luciferase activity in Jurkat cells transfected as in a , then exposed to medium alone ( Med ) , wild - type E . coli ( JM101 ) , or JM101 E . coli mutants lacking both Omp C and Omp F ( HN705 ) or lacking either OmpC ( ΔOmpC ) or OmpF ( ΔOmpF ) . Data are representative of at least three independent experiments ( error bars ( a ) , s . d . ) .

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figf6(a) Luciferase activity in Jurkat cells transfected with a fusion of SLAM and CD3ζ and a luciferase reporter (as described in Results), plus a renilla luciferase reporter, then exposed to heat-killed bacteria (top).(b) Luciferase activity in Jurkat cells transfected with the fusion in a (Slamf1-CD3ζ) or a fusion of SLAM ectodomain construct lacking the immunoglobulin V domain and CD3ζ (ΔIgV-Slamf1-CD3ζ), and luciferase reporters as in a, then exposed to heat-killed E. coli F18.(c) Luciferase activity in Jurkat cells transfected as in a, then left uninoculated (−) or inoculated with 10 × 108 E. coli F18 (+), followed by the addition of monoclonal antibody (mAb; amount, under graph) 9D1 to SLAM (α-SLAM) or rat immunoglobulin G2b isotype-matched control antibody (Rat IgG2b).(d) Luciferase activity in Jurkat cells transfected as in a, then exposed to medium alone (Med), wild-type E. coli (JM101), or JM101 E. coli mutants lacking both Omp C and Omp F (HN705) or lacking either OmpC (ΔOmpC) or OmpF (ΔOmpF). Data are representative of at least three independent experiments (error bars (a), s.d.).


figf7 ( a , b ) Production of phagosomal PtdIns ( 3 ) P in E . coli - containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP - tagged PX domain of p40phox ( p40 PX - eGFP ) and treated with DsRed - expressing E . coli ( E . coli - DsRed ; a ) or beads coated with E . coli outer membrane extract ( b ) . Numbers in bottom right corners indicate time ( in min ) . Original magnification , × 60 . Right , quantification of fluorescence in microscopy at left . ( c ) HPLC analysis of the production of PtdIns ( 3 ) P in primary macrophages labeled with 3H - tagged myoinositol and treated with beads coated with E . coli outer membrane extract . ( d ) PtdIns ( 3 ) P production in RAW264 . 7 cells stably expressing SLAM or a mock construct , transfected with eGFP - tagged p40 PX and treated with beads coated with E . coli outer membrane extract . Data are from three combined experiments ( a ) or are representative of at least three independent experiments ( b , d ) or two independent experiments ( c ) with at least 100 beads or bacteria per experiment ( error bars , s . d . ) .

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figf7(a,b) Production of phagosomal PtdIns(3)P in E. coli-containing phagosomes of primary peritoneal macrophages transfected with reporter cDNA encoding an eGFP-tagged PX domain of p40phox (p40 PX-eGFP) and treated with DsRed-expressing E. coli (E. coli-DsRed; a) or beads coated with E. coli outer membrane extract (b). Numbers in bottom right corners indicate time (in min). Original magnification, ×60. Right, quantification of fluorescence in microscopy at left.(c) HPLC analysis of the production of PtdIns(3)P in primary macrophages labeled with 3H-tagged myoinositol and treated with beads coated with E. coli outer membrane extract.(d) PtdIns(3)P production in RAW264.7 cells stably expressing SLAM or a mock construct, transfected with eGFP-tagged p40 PX and treated with beads coated with E. coli outer membrane extract. Data are from three combined experiments (a) or are representative of at least three independent experiments (b,d) or two independent experiments (c) with at least 100 beads or bacteria per experiment (error bars, s.d.).


figf8 ( a , b ) Immunoassay of 293 cells transfected with various combinations of full - length SLAM ( a ) or hemagglutinin - tagged ( - HA ) tail - less SLAM ( b ) , EAT - 2A , V5 - tagged ( - V5 ) Vps34 - Vps15 , and beclin - 1 ; proteins immunoprecipitated ( IP ) from lysates with monoclonal antibody to SLAM , as well as whole - cell lysates ( WCL ) , were analyzed by immunoblot ( IB ) with anti - beclin - 1 , anti - Vps34 , anti - V5 , anti - hemagglutinin , anti - SLAM or anti - EAT2 . ( c ) Microscopy of RAW264 . 7 cells transiently transfected with cDNA encoding GFP - tagged beclin - 1 and SLAM - mCherry , then treated with beads coated with E . coli outer membrane extract ( at a ratio of 10 : 1 , beads / cells ) and fixed after 60 min . Data are representative of six ( a ) or two ( b , c ) independent experiments .

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figf8(a,b) Immunoassay of 293 cells transfected with various combinations of full-length SLAM (a) or hemagglutinin-tagged (-HA) tail-less SLAM (b), EAT-2A, V5-tagged (-V5) Vps34-Vps15, and beclin-1; proteins immunoprecipitated (IP) from lysates with monoclonal antibody to SLAM, as well as whole-cell lysates (WCL), were analyzed by immunoblot (IB) with anti-beclin-1, anti-Vps34, anti-V5, anti-hemagglutinin, anti-SLAM or anti-EAT2.(c) Microscopy of RAW264.7 cells transiently transfected with cDNA encoding GFP-tagged beclin-1 and SLAM-mCherry, then treated with beads coated with E. coli outer membrane extract (at a ratio of 10:1, beads/cells) and fixed after 60 min. Data are representative of six (a) or two (b,c) independent experiments.


Figure 1B . Two - hybrid interaction between GBD - AC40 and WT or mutant GAD - IN1578 - 635 . Alanine substitutions in IN1578 - 635 are indicated . Cells were plated in two - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were detected ( Figs EV1A - B ) . + , interaction ; - , no interaction . C . Two - hybrid interaction between GBD - AC40 and different IN1 regions fused to GAD , as indicated . Cells were plated in ten - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were detected ( Figs EV1C - D ) . + , interaction ; - , no interaction . D . In vitro interaction between AC40 and IN1 proteins co - expressed in E . coli . Co - precipitation of WT or mutated ( K617A , S621A , or L622A ) IN1 - C - tag using AC40 - Twin - Strep - tag as bait from bacterial protein extracts co - expressing ( + ) or not ( - ) these proteins . Expected sizes are 41 kDa for AC40 - Twin - Strep - tag and 100 kDa for IN1 - C - tag ( WT and mutants ) .

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 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

Figure 1B. Two-hybrid interaction between GBD-AC40 and WT or mutant GAD-IN1578-635. Alanine substitutions in IN1578-635 are indicated. Cells were plated in two-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1A-B). +, interaction; -, no interaction.C. Two-hybrid interaction between GBD-AC40 and different IN1 regions fused to GAD, as indicated. Cells were plated in ten-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detected (Figs EV1C-D). +, interaction; -, no interaction.D. In vitro interaction between AC40 and IN1 proteins co-expressed in E. coli. Co-precipitation of WT or mutated (K617A, S621A, or L622A) IN1-C-tag using AC40-Twin-Strep-tag as bait from bacterial protein extracts co-expressing (+) or not (-) these proteins. Expected sizes are 41 kDa for AC40-Twin-Strep-tag and 100 kDa for IN1-C-tag (WT and mutants).


Figure 2A . Localization of GFP2 - IN1 bNLS variants in yeast cells by direct fluorescence microscopy . Nup49 - mCherry signal was used to visualize the location of the nuclear envelope . Corresponding DIC images are shown . Scale bar is 5 μm . B . Retrotransposition frequency ( log scale ) of pGAL1 - Ty1his3AI bearing the indicated substitutions of conserved residues in a spt3 - 101 rad52 ∆ strain . IN1 catalytic core domain mutant D154A is defective for integration and NLS mutant ( KKR628 - 630GGT ) for nuclear import . Values are mean ± SD , n = 4 experiments , each performed with four independent colonies . * * * p ≤ 0 . 001 , otherwise not significant ( Student t - test ) .

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Figure 2A. Localization of GFP2-IN1 bNLS variants in yeast cells by direct fluorescence microscopy. Nup49-mCherry signal was used to visualize the location of the nuclear envelope. Corresponding DIC images are shown. Scale bar is 5 μm.B. Retrotransposition frequency (log scale) of pGAL1-Ty1his3AI bearing the indicated substitutions of conserved residues in a spt3-101 rad52∆ strain. IN1 catalytic core domain mutant D154A is defective for integration and NLS mutant (KKR628-630GGT) for nuclear import. Values are mean±SD, n=4 experiments, each performed with four independent colonies. ***p ≤ 0.001, otherwise not significant (Student t-test).


Figure 3A . Co - immunoprecipitation of ectopic IN1 using C160 - HA as bait , from yeast protein extracts expressing WT or mutated IN1 - Strep ( K617A , S621A , or L622A ) from the GAL1 promoter in the presence of galactose . Expected sizes are 160 kDa for C160 - HA and 100 kDa for IN1 - Strep ( WT and mutants ) . B . Quantitative ChIP analysis of HA - IN1 enrichment at Pol III - transcribed genes . Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input . Pol III transcribed - genes : tDNA - Ile and tDNA - Leu families ( 16 and 22 genes , respectively ) and the unique SCR1 gene . GAL1 ORF serves as a control . Data represent means ± SD ( n ≥ 3 ) . C . Fold enrichment of WT HA - IN1 over input at all the genes where HA - IN1 has been detected by ChIP - seq analysis . Each dot represents a gene . Grey , tDNAs ; Red , other Pol III - transcribed genes ; Blue , Pol II - transcribed genes . The dashed line indicates the separation between Pol II and Pol III - transcribed genes . D . WT and mutant IN1 association with all Pol III - transcribed genes . Values obtained from ChIP seq analysis have been normalized in log2 RPKM ( reads per kilobase per million mapped reads ) . Control is anti - HA immunoprecipitation of chromatin extracts in cells expressing IN1 - Strep . Immunopurified DNA from 3 - 4 independent experiments was combined in one replicate . Data are presented as boxplots showing the median , the first quartile , and the third quartile . Error bars show minimum and maximum values . E . Genome - wide occupancy profiles ( top ) and heatmaps ( bottom ) of WT and HA - IN1 mutants over a 1kb window upstream and downstream of all tDNAs . Pearson correlation to WT values corresponding to Fig EV2B are indicated . F . WT and mutant IN1 association to all Pol - II transcribed genes respectively . Control , replicates and data presentation , as described for panel D . Regions used for calculation are gene bodies . G . Recruitment of IN1 along the SCR1 gene locus . Top . ChIP ‐ qPCR analysis as described for panel Fig 3B . Data represent means ± SD ( n = 3 ) . Bottom . Schematic representation of the SCR1 locus with DNA amplicon positions . TATA , A and B boxes and terminator of transcription ( T ) are indicated .

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Figure 3A. Co-immunoprecipitation of ectopic IN1 using C160-HA as bait, from yeast protein extracts expressing WT or mutated IN1-Strep (K617A, S621A, or L622A) from the GAL1 promoter in the presence of galactose. Expected sizes are 160 kDa for C160-HA and 100 kDa for IN1-Strep (WT and mutants).B. Quantitative ChIP analysis of HA-IN1 enrichment at Pol III-transcribed genes. Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input. Pol III transcribed-genes: tDNA-Ile and tDNA-Leu families (16 and 22 genes, respectively) and the unique SCR1 gene. GAL1 ORF serves as a control. Data represent means ± SD (n ≥ 3).C. Fold enrichment of WT HA-IN1 over input at all the genes where HA-IN1 has been detected by ChIP-seq analysis. Each dot represents a gene. Grey, tDNAs ; Red, other Pol III-transcribed genes ; Blue, Pol II-transcribed genes. The dashed line indicates the separation between Pol II and Pol III-transcribed genes.D. WT and mutant IN1 association with all Pol III-transcribed genes. Values obtained from ChIP seq analysis have been normalized in log2 RPKM (reads per kilobase per million mapped reads). Control is anti-HA immunoprecipitation of chromatin extracts in cells expressing IN1-Strep. Immunopurified DNA from 3-4 independent experiments was combined in one replicate. Data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values.E. Genome-wide occupancy profiles (top) and heatmaps (bottom) of WT and HA-IN1 mutants over a 1kb window upstream and downstream of all tDNAs. Pearson correlation to WT values corresponding to Fig EV2B are indicated.F. WT and mutant IN1 association to all Pol-II transcribed genes respectively. Control, replicates and data presentation, as described for panel D. Regions used for calculation are gene bodies.G. Recruitment of IN1 along the SCR1 gene locus. Top. ChIP‐qPCR analysis as described for panel Fig 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the SCR1 locus with DNA amplicon positions. TATA, A and B boxes and terminator of transcription (T) are indicated.


Figure 4A . Co - immunoprecipitation of ectopic IN1 using TAP - tagged - A190 as bait from yeast protein extracts expressing WT or the indicated IN1 - HBH mutants from a pTet - Off promoter in the absence of doxycycline . Expected sizes are 204 kDa for A190 - HA and 82 kDa for IN1 - Strep ( WT and mutants ) . B . Genome browser visualization of HA - IN1 occupancy at the RDN1 locus . Occupancy of WT HA - IN1 and K617A , S621A and L622A HA - IN1 mutants is represented in each panel . Control , as described for panel 3D . C . Top . IN1 is recruited at Pol I - transcribed genes . ChIP ‐ qPCR analysis as described for panel 3B . Data represent means ± SD ( n = 3 ) . Bottom . Schematic representation of the RDN1 locus with DNA amplicon positions . D . Recruitment of WT and mutant HA - IN1 ( K617A , S621A , or L622A ) at the 18S rDNA locus transcribed by Pol I . ChIP ‐ qPCR analysis as described for panel 3B . Data represent means ± SD ( n = 3 ) .

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 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 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 O O O O O O O O O O O O O O O O O O

Figure 4A. Co-immunoprecipitation of ectopic IN1 using TAP-tagged-A190 as bait from yeast protein extracts expressing WT or the indicated IN1-HBH mutants from a pTet-Off promoter in the absence of doxycycline. Expected sizes are 204 kDa for A190-HA and 82 kDa for IN1-Strep (WT and mutants).B. Genome browser visualization of HA-IN1 occupancy at the RDN1 locus. Occupancy of WT HA-IN1 and K617A, S621A and L622A HA-IN1 mutants is represented in each panel. Control, as described for panel 3D.C. Top. IN1 is recruited at Pol I-transcribed genes. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the RDN1 locus with DNA amplicon positions.D. Recruitment of WT and mutant HA-IN1 (K617A, S621A, or L622A) at the 18S rDNA locus transcribed by Pol I. ChIP‐qPCR analysis as described for panel 3B. Data represent means ± SD (n = 3).


Figure 5A . Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 ( red triangle ) and a primer in the locus of interest ( blue triangle ) . Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant ( IN1 K617A , S621A and L622A ) Ty1his3AI from the GAL1 promoter . Total genomic DNA was extracted from His + cells obtained from independent cultures . B . Genome - Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap . Score is computed in column Z - score . ORFs , all Pol II - transcribed genes except gene at subtelomeres ; Pol I , one RDN37 copy ; up 1kb Pol III , 1kb upstream of all Pol III transcribed genes ; Subtelomeres , genomic coordinates corresponding to chromatin covered by Sir2 and Sir3 , when they are co - overexpressed ( Hocher et al , 2018 ) ; Random , 100 000 random Ty1 computed insertions in the genome . C . Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A . Four total genomic DNA preparation from panel A were randomly tested by PCR . D . Ty1 insertion profile upstream of tDNAs . Total genomic DNA extracted in ( B ) was prepared for Ty1 de novo integration event sequencing . Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs ( position 0 in the graph ) . Each position is divided by the number of insertions at this position ( weight ) . The Smoothing curves indicate the general trend . Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from ( Brogaard et al , 2012 ) . E . Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap . Score is computed in row Z - score . Random , as described for panel B . High level of WT Ty1 - HIS3 integration in II - L is due to the presence of the tRNA gene tF ( GAA ) B .

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 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 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 O O O O O O O O O

Figure 5A. Detection of de novo Ty1 insertions upstream of the SUF16 and SEO1 genes by PCR using a primer in HIS3 (red triangle) and a primer in the locus of interest (blue triangle). Ty1 retrotransposition was induced in cells transformed with plasmids expressing WT or mutant (IN1 K617A, S621A and L622A) Ty1his3AI from the GAL1 promoter. Total genomic DNA was extracted from His+ cells obtained from independent cultures.B. Genome-Wide Ty1 insertion frequencies at each genomic feature are clustered in a heatmap. Score is computed in column Z-score. ORFs, all Pol II-transcribed genes except gene at subtelomeres; Pol I, one RDN37 copy; up 1kb Pol III, 1kb upstream of all Pol III transcribed genes; Subtelomeres, genomic coordinates corresponding to chromatin covered by Sir2 and Sir3, when they are co-overexpressed (Hocher et al, 2018); Random, 100 000 random Ty1 computed insertions in the genome.C. Detection of de novo Ty1 insertions upstream of RDN5 and in RDN37 genes by PCR as described for panel 5A. Four total genomic DNA preparation from panel A were randomly tested by PCR.D. Ty1 insertion profile upstream of tDNAs. Total genomic DNA extracted in (B) was prepared for Ty1 de novo integration event sequencing. Ty1 insertions are computed in a 1kb window upstream of all the 275 nuclear tDNAs (position 0 in the graph). Each position is divided by the number of insertions at this position (weight). The Smoothing curves indicate the general trend. Nucleosome center positions for the three first nucleosomes upstream of all tDNAs are from (Brogaard et al, 2012).E. Ty1 insertion frequencies for each left and right subtelomere of chromosomes are clustered in a heatmap. Score is computed in row Z-score. Random, as described for panel B. High level of WT Ty1-HIS3 integration in II-L is due to the presence of the tRNA gene tF(GAA)B.


Figure 6A . Two - hybrid interaction between GBD - AC40 ( left ) or GBD - Sir4 ( right ) and different GAD - IN5 or GAD - IN1 constructions . Cells were plated in five - fold serial dilutions on DO - Leu - Trp - His plates . No growth or protein expression defects were detectedB . Genome - wide occupancy profiles ( top ) and heatmaps ( bottom ) of IN5 , WT , and indicated mutants , + / - 1kb upstream and downstream of all tDNAs . C . Genome browser visualization of different HA - IN occupancy for chromosome V ( chrV : 431129 . . 443275 ) . Occupancy of the indicated integrase is represented in each panel . Control , as panel 3D . The region contains tH ( GUG ) E2 , tK ( CUU ) E2 , tV ( ACC ) E1 , tI ( AAU ) E1 , SNR52 and SCR1 , all transcribed by Pol III . D . Detection of Ty5 , Ty5ΔTD5 + bNLS or Ty1 de novo integration events at HMR and HML loci , SCR1 and upstream of all glycine tDNAs , by PCR using a primer in HIS3 and a primer in the locus of interest . Retrotransposition was induced in cells transformed with WT or mutated pGAL1 - Ty5his3AI ( Ty5 or Ty5ΔTD5 + bNLS ) and pGAL1 - Ty1his3AI ( Ty1 ) . Total genomic DNA was extracted from His + cells obtained from independent cultures . Nucleosome position ( green circles ) is indicated on the right of the panels ( Brogaard et al , 2012 ) .

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Figure 6A. Two-hybrid interaction between GBD-AC40 (left) or GBD-Sir4 (right) and different GAD-IN5 or GAD-IN1 constructions. Cells were plated in five-fold serial dilutions on DO-Leu-Trp-His plates. No growth or protein expression defects were detectedB. Genome-wide occupancy profiles (top) and heatmaps (bottom) of IN5, WT, and indicated mutants, +/- 1kb upstream and downstream of all tDNAs.C. Genome browser visualization of different HA-IN occupancy for chromosome V (chrV:431129..443275). Occupancy of the indicated integrase is represented in each panel. Control, as panel 3D. The region contains tH(GUG)E2, tK(CUU)E2, tV(ACC)E1, tI(AAU)E1, SNR52 and SCR1, all transcribed by Pol III.D. Detection of Ty5, Ty5ΔTD5+bNLS or Ty1 de novo integration events at HMR and HML loci, SCR1 and upstream of all glycine tDNAs, by PCR using a primer in HIS3 and a primer in the locus of interest. Retrotransposition was induced in cells transformed with WT or mutated pGAL1-Ty5his3AI (Ty5 or Ty5ΔTD5+bNLS) and pGAL1-Ty1his3AI (Ty1). Total genomic DNA was extracted from His+ cells obtained from independent cultures. Nucleosome position (green circles) is indicated on the right of the panels (Brogaard et al, 2012).


Figure 1 ( B ) Violin plots displaying RNA - seq readings by FPKM ( normalized Fragments Per Kilobase Million ) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls ( n = 49 , 42 ; unpaired Kolmogorov - Smirnov test ; continuous lines : median ; dashed lines : quartiles ) . ( D ) Mean and SEM of mRNA levels of Xbp1s , Bip / Hspa5 and Chop / Ddit3 were determined in dissected hippocampus of i . p . treated mice by quantitative RT - PCR . ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups ) . ( E ) Animals received bilateral intra - hippocampal injections of 2 μl tunicamcyin ( 5 mg / mL ) or vehicle for 24 h . Graphs indicate mean and s . e . m . of Xbp1s mRNA levels in the hippocampus ( n = 3 - 4 animals / group . One - way ANOVA followed by Tukey ' s post - test compared tunicamycin treated groups . ( F - G ) Mean and SEM of relative levels of S - nitrosylated IRE1 ( f ) and total IRE1 ( g ) in brains from young and aged mice . Histograms show relative levels of SNO - IRE1 / input IRE1α measured by biotin - switch assay and total IRE1 / GAPDH quantified by densitometry on standard immunoblots for young ( 3 - month - old ) versus aged ( 24 month - old ) mouse brains ( n = 3 , unpaired Student ' s t test ) . Data information :

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Figure 1(B) Violin plots displaying RNA-seq readings by FPKM (normalized Fragments Per Kilobase Million) of chaperones HSPA1A and HSPA1B of human hippocampus samples of demented patients and controls (n = 49,42; unpaired Kolmogorov-Smirnov test; continuous lines: median; dashed lines: quartiles).(D) Mean and SEM of mRNA levels of Xbp1s, Bip/Hspa5 and Chop/Ddit3 were determined in dissected hippocampus of i.p. treated mice by quantitative RT-PCR. (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups).(E) Animals received bilateral intra-hippocampal injections of 2 μl tunicamcyin (5 mg/mL) or vehicle for 24 h. Graphs indicate mean and s.e.m. of Xbp1s mRNA levels in the hippocampus (n = 3-4 animals/group. One-way ANOVA followed by Tukey's post-test compared tunicamycin treated groups.(F-G) Mean and SEM of relative levels of S-nitrosylated IRE1 (f) and total IRE1 (g) in brains from young and aged mice. Histograms show relative levels of SNO-IRE1/input IRE1α measured by biotin-switch assay and total IRE1/GAPDH quantified by densitometry on standard immunoblots for young (3-month-old) versus aged (24 month-old) mouse brains (n = 3, unpaired Student's t test). Data information: