EMBO/soda-nel huggingface dataset:
Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions (2020)
Figure 1

Figure 1. Farnesylated hGBP1 binds directly to Shigella flexneri in a nucleotide-dependent manner.

(A) Structure of full-length, nucleotide-free, farnesylated hGBP1 (PDB entry 6k1z) and GDP·AlFX-bound LG-domain dimer (PDB entry 2B92). Insert (a) shows the farnesyl moiety and the triple arginine stretch (3R = R584-586). Insert (b) highlights residues required for nucleotide binding and hydrolysis.

(B) Experimental design: fluorescently labeled recombinant hGBP1 variants and nucleotides were mixed with broth-cultured live or fixed bacteria; bacterial binding was monitored by confocal microscopy.

(C) Confocal images of formaldehyde fixed GFP+ S. flexneri following 20 min incubation with 2 mM GTP and 10 µM Alexa-Fluor647-labeled protein. Bacteria associated with hGBP1 mutants after 20 min were quantified. Mean frequencies ± SEM of combined data from two independent experiments are shown. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ***, P ≤ 0.001. Scale bars equal 5 µm. Flow diagram depicts effects of K51A, R48A, and H74A hGBP1 mutations on nucleotide binding and hydrolysis.
Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions (2020)
Figure 2

Figure 2. hGBP1 polymerization is required for bacterial binding.

(A) Translocation of ectopically expressed mCherry-hGBP1 to cytosolic GFP+ S. flexneri ΔipaH9.8 in HeLa hGBP1-KO cells was monitored by time-lapse microscopy. Individual time frames of Movie EV1 starting at 55 min post infection (mpi) are shown.

(B) Confocal time-lapse microscopy was used to image 10 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP in the presence or absence of formaldehyde fixed GFP+ S. flexneri. Individual time frames of Movie EV2 depict hGBP1F fluorescence intensity. Merged images of hGBP1F and S. flexneri fluorescence are shown for the 60 min time points.

(C) Images were taken at 45 min after addition of 10 µM Alexa-Fluor647-hGBP1F to formaldehyde fixed GFP+ S. flexneri in the presence of indicated nucleotides (GTP, natural substrate; GppNHp, non-hydrolysable GTP analog; GTPγS, slowly hydrolysable GTP analog; GDP·AlFX, GTP transition state analog). hGBP1-associated bacteria after 45 min were quantified. Combined data from two independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.

(D) Model: hGBP1 polymers bind to S. flexneri directly and transition into a bacterium-encapsulating protein coat.

Data information: All scale bars equal 5 µm.
Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions (2020)
Figure 3

Figure 3. A C-terminal hGBP1 polybasic motif is required for sustained binding to S. flexneri.

(A) Polymerization of 10 µM hGBP1F and hGBP1FR584-586A in the presence of 2 mM GTP was monitored over time by absorption spectroscopy at 350 nm. Absorbance signals were superimposed with nucleotide composition of the same solution analyzed at defined time points, revealing the characteristic first phase of slow polymer nucleation and the second phase of fast polymer growth and cooperative hydrolysis.

(B) Scanning-electron micrographs of live S. flexneri incubated with no protein or with 5 µM of either hGBP1F, hGBP1FR584-586A, or hGBP1FR48A (non-polymerizing mutant) in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces.

(C) Confocal time-lapse microscopy frames of formaldehyde fixed GFP+ S. flexneri following admixture of 10 µM Alexa-Fluor647-labeled hGBP1F or hGBP1FR584-586A and 2 mM GTP. Binding of hGBP1F polymers to bacteria at 5 min and enclosure of bacteria with hGBP1F protein coats after 60 min were quantified. Mean frequencies ± SEM of combined data from at least three independent experiments are shown. Significance was determined by by unpaired t-tests, two-tailed. ***, P ≤ 0.001; ****, P ≤ 0.0001.

Data information: All scale bars equal 5 µm.
Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions (2020)
Figure 4

Figure 4. Direct binding of hGBP1F to LPS mediates its association with gram-negative bacteria

(A) Representative confocal images taken at 60 min after admixture of 2 mM GTP and 10 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F to formaldehyde fixed gram-negative and gram-positive bacteria expressing GFP (S. enterica Typhimurium [ST], L. monocytogenes [Lm]), or RFP (S. flexneri [Sf], uropathogenic E. coli [UPEC]), or dsRed (L. pneumophila [Lp], S. aureus [Sa]).

(B) (Left panel) Confocal time-lapse microscopy frames of Alexa-Fluor488-LPS-O55:B5 after addition of 5 µM Alexa-Fluor647-hGBP1F supplemented with 2 mM GTP. (Right panel) Confocal images taken of 5 µM Alexa-Fluor488-LPS-O55:B5 20 min after addition of 5 µM Alexa-Fluor647-hGBP1F or -hGBP1FR48A supplemented with 2 mM GTP. Graphs depict average aggregate area of Alexa-Fluor488-LPS-O55:B5 supplemented with 2 mM GTP and 5 µM of the indicated hGBP1 variant. Mean area ± SEM of combined data from three independent experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test.. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.

(C) Polymerization of 5 µM hGBP1F induced by addition of 2 mM GTP in the presence and absence of 5 µM LPS-O55:B5 was monitored by absorption spectroscopy at 350 nm. The absorbance signal was superimposed with nucleotide composition of the same solution analyzed at defined time points. Maximal hydrolysis rates (turnover numbers) were determined for hGBP1F variants in the presence and absence of 5 µM LPS-O55:B5. Graphs show mean turnover numbers ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ***, P ≤ 0.001; ****, P ≤ 0.0001.

(D) Formaldehyde fixed GFP+ S. flexneri supplemented with varying LPS-O55:B5 concentrations were mixed with 5 µM hGBP1F and 2 mM GTP. After 60 min hGBP1F-enclosed S. flexneri were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.

(E) Confocal images of formaldehyde fixed GFP-, RFP-, or dsRed-expressing gram-negative bacteria 60 min after addition of 5 µM Alexa-Fluor647- or Alexa-Fluor488-hGBP1F and 2 mM GTP in the presence and absence of 100 µM LPS-O55:B5. After 60 min hGBP1F-enclosed gram-negative bacteria were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.

Data information: All scale bars equal 5 µm.
Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions (2020)
Figure 5

Figure 5. Bacterial O-antigen drives the transition from bacteria-bound hGBP1F polymers into bacteria-encasing hGBP1F protein coats.

(A) Graphic depiction of the bacterial outer membrane composition and LPS structure of S. flexneri. Arrow indicates the LPS truncation site in the O-antigen deficient rfaL mutant. O-antigen oligosaccharide subunit composition of S. flexneri serotype 2a and E. coli serotypes O8 and O25 are shown.

(B) Time-lapse microscopy of fixed GFP+ co-isogenic S. flexneri wild type and rfaL mutant after adding 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP. Bacteria bound by hGBP1F at 5 min and hGBP1F-enclosed bacteria after 60 min were quantified. Combined data from three independent experiments are shown as mean ± SEM. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; ****, P ≤ 0.0001.

(C) Scanning-electron micrographs of live wild type and rfaL mutant strains incubated with no protein or with 5 µM of hGBP1F in the presence of 2 mM GTP for 4 min. Arrowheads point to unattached hGBP1 polymers, arrows point to hGBP1 polymers attached to bacteria, and asterisks mark polymeric structures that appear to fuse with bacterial surfaces

(D) Model: O-antigen drives the transition of surface-docked hGBP1F polymers into bacteria-enveloping hGBP1F protein sheets.

(E) Confocal images of formaldehyde fixed, co-isogenic GFP+ wild type and mutant S. flexneri strains harboring rfb regions of E. coli serotypes O8 and O25 after 60 min of incubation time in the presence of 10 µM Alexa-Fluor647-hGBP1F and 2 mM GTP.

Data information: All scale bars equal 5 µm.
Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions (2020)
Figure 6

Figure 6. Binding of hGBP1F to bacteria disrupts the O-antigen barrier function.

(A) Following incubation for 1.5 h with buffer in the absence or presence of 10 µM hGBP1F and 5 mM GTP, live bacteria were treated with antibiotics (2.5 µg/ml PMB or 5 mg/ml lysozyme) for 30 min or left untreated. The number of viable bacteria was subsequently determined by colony forming unit (CFU) counts. Graphs show mean CFUs ± SEM of combined data from at least three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.

(B) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A supplemented with 2 mM GTP. Following 60 min of incubation hGBP1F-enclosed UPEC were quantified. Graphs depict mean ± SEM of combined data from three independent experiments. Significance was determined by unpaired t-test, two-tailed. ****, P ≤ 0.0001.

(C) Formaldehyde fixed RFP+ UPEC were mixed with 10 µM Alexa-Fluor488-hGBP1F or -hGBP1FR584-586A and 2 mM GTP. Samples were fixed after 60 min incubation and stained with an antibody against the inner core/ lipid A fragment of E. coli LPS (anti-LPS). The frequency of anti-LPS stained UPEC was quantified. Graphs show mean ± SEM of combined data from three independent experiments. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. n.s., not significant; ****, P ≤ 0.0001.

(D) IFNγ-primed wild type and hGBP1-KO HeLa cells stably expressing inactive mutant YFP-Caspase-4C258S were infected with either cytosol-entering S. enterica Typhimurium mutant ΔsifA (MOI = 25) or with S. flexneri ΔospC3ΔipaH9.8 (MOI = 6), a mutant strain lacking Caspase-4 antagonist OspC3 as well as hGBP1 antagonist IpaH9.8. Cells were fixed at 4 hpi (ST ΔsifA) or at 2 hpi (Sf ΔospC3ΔipaH9.8) and stained for endogenous hGBP1. YFP-Caspase-4C258S associated bacteria were quantified and mean ± SEM of combined data from three independent experiments are shown. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. ****, P ≤ 0.0001.

Data information: All scale bars equal 5 µm.
Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions (2020)
Figure 7

Figure 7. hGBP1 disrupts polar localization of Shigella IcsA and blocks the recruitment of the host actin polymerization machinery.

(A) Schematic depicts the molecular mechanism by which S. flexneri co-opts the host actin polymerization machinery: the bacterial autotransporter IcsA localizes to one bacterial pole where it recruits and activates host actin nucleation promoting factor N-WASP. N-WASP then recruits and activates the host actin nucleator Arp2/3 complex to initiate actin polymerization.

(B - E). Cells were infected with poly-D-lysine treated GFP+ S. flexneri strains at an MOI of 6. Cells were stained for indicated proteins and Z-stacks were recorded using confocal fluorescence microscopy. Actin tails were classified as tails when ≥ 2.5 µm. All graphs show mean values ± SEM of combined data from three independent experiments. All scale bars are 5 µm. Arrows point to bacteria associated with indicated proteins, arrowheads point to bacteria lacking indicated proteins, and asterisks mark bacteria lacking unipolar IcsA localization. (B) Unprimed HeLa cells were infected with wild type or rfaL S. flexneri. Total and unipolar localization of IcsA as well as the actin tail formation were quantified at 1 hpi. Significance was determined by unpaired two-tailed t-tests. n.s., not significant; *, P ≤ 0.05; **, P ≤ 0.01. (C- D) mCherry-hGBP1 was expressed in HeLa hGBP1-KO cells infected with S. flexneri ΔipaH9.8 and stained for IcsA, N-WASP, Arp2, and F-actin at 2.5 hpi. Significance was determined by unpaired t-tests, two-tailed. n.s., not significant; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) IFNγ-primed and unprimed wild type and hGBP1-KO HeLa cells were infected with S. flexneri ΔipaH9.8 and subcellular localization of IcsA, N-WASP, Arp2, and F-actin was assessed and quantified at 2.5 hpi. Significance was determined by two-way ANOVA with Tukey's multiple comparison test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

EXPANDED VIEW FIGURE LEGENDS
Integrative multi-omics analysis of intestinal organoid differentiation (2018)
Figure 4

Figure 4: Hnf4g drives differentiation of enterocytes.

(A) Gene Set Enrichment Analysis (GSEA) of Hnf4g KO organoids compared to WT organoids. Significant changing intestinal cell type gene sets from Haber et al (2017) are shown (FDR < 0.05).

(B) Hematoxylin and (Periodic acid-Shiff) PAS staining of WT (left) and Hnf4g KO (right) intestine. Nuclei are visualized using Hematoxylin and goblet cells stain positive for PAS.

(C) Alcian Blue and Nuclear Fast Red staining of WT (left) and Hnf4g KO (right) small intestinal organoids. Cells are visualized using Nuclear Fast Red. Intra- and extracellular mucus that is produced in goblet cells stain positive for Alcian Blue.

(D) Strand specific RNA-seq data, Hnf4g ChIP-seq in EN, and promoter specific histone modification H3K4me3 over the Hnf4a locus is shown. Data from different organoid cultures are color coded. RNA-seq reads mapping to the positive strand (sense strand) are plotted in the top window of every sample, while RNA-seq reads that map to the negative strand are mirrored and shown in the bottom window of every sample. The two isoforms of Hnf4a that are expressed are shown in the bottom, together with the antisense transcript Hnf4aos. The "phyloP30way" track from the UCSC genome browser is plotted at the bottom to show sequence conservation.

(E) Quantification of the amount of goblet cells per villus for WT and Hnf4g KO intestine. Dots represent the amount of goblet cells counted per villus. (n = 7 to 25 villi from 11 WT mice and 3 to 17 villi from 14 Hnf4g KO mice). P value is from two-tailed Mann-Whitney U test.

Expanded View Figure Legends
Novel Alzheimer risk genes determine the microglia response to amyloid-β but not to TAU pathology (2019)
Figure 1

Fig.1: Enrichment of AD risk genes in APPtg and not in TAUtg mice.

A) Visualisation of the pathology load at 9M of age of TAUwt, TAUtg and APPtg mice at 9M of age. Immunofluorescent staining for X34 (a fluorescent derivative of Congo Red), Iba1 (microglia) and TO-PRO-3 (nuclei) has been pseudocolored. Scale bar = 100μm.

B) Experimental design for mRNA sequencing using n=12 per experimental group.

C) Explanation of the 2x2 linear model, where those cells labeled with 1 are compared to the cells labeled with 0. In the age comparison, mRNA expression in all 10-month-old (10M) mice is compared to all 4-month-old (4M) mice. In the genotype comparison, mRNA expression in all transgenic (TG) mice is compared to all wild-type (WT) mice. In the age*genotype comparison, we assess which transcripts are differentially expressed in the 10M TG mice compared to all other groups.

D) Based on Marioni et al. (Marioni et al, 2018) various sets of AD GWAS risk genes were created using different cut-off p-values indicated on the x-axis (number of genes within each set is written in grey). Enrichment for AD risk genes was assessed among the different statistical comparisons for APPtg and TAUtg mice (Int: age*genotype, Gen: genotype), using gene set enrichment analysis (GSEA (Subramanian et al, 2005)). Colors represent -log10 (Benjamini-Yuketieli adjusted p-value) for the enrichment; blank means no significant enrichment.
Novel Alzheimer risk genes determine the microglia response to amyloid-β but not to TAU pathology (2019)
Figure 6

Fig.6. Single microglia sequencing demonstrates a strong ARM response in APPtg in comparison to TAUtg mice.

A) UMAP clustering of sorted Cd11b+/Cd45+ myeloid cells from mouse hippocampus. ARM: activated response microglia; CAM: CNS-associated macrophages; CPM: cycling and proliferating microglia; HM.1: homeostatic microglia cluster 1; HM.2: homeostatic microglia cluster 2; IRM: interferon-response microglia; MHC.high: high MHC-expressing microglia; Mnc: monocytes; TRM: transitioning microglia.

B) Distribution of cells across the different clusters per experimental group (n=2 mice pooled for all experimental groups, except for TAUtg-11M and TAUwt-11M with n=3 mice pooled).

C) Distribution of cells per experimental group over the different clusters, expressed in percentages as stated on top of each bar. E.g. of all cells within APPtg-11M, 56.7% of cells are ARM, while 12.5% are HM.1 cells.

D) Scorpius-based ranking of all cells on a pseudotime from homeostatic to activated microglia. Whereas all cells within the WT groups are clustered within the homeostatic part of the pseudotime trajectory, APPtg-4M and APPtg-11M mice demonstrate a shift towards more activated microglia.

E) Differential expression of ARM vs HM.1 microglia for APPtg and TAUtg mice separately. Genes with a positive log(fold change) (LFC) are higher expressed in ARMs compared to HM.1 microglia. As can be seen which genes are more highly expressed in ARMs vs HM.1 cells are highly similar among APPtg and TAUtg mice (Spearman correlation r=0.91, p=2.2e-16). LFC > |0.2| with padj<0.05 are considered significant.

F) Predicted contribution of microglial expression of each of the top18 genes to the observed bulk RNA-seq expression data, based on linear regression (see Materials & Methods). For 16/18 genes the microglial expression contributes significantly and 67% or more to the observed expression from the bulk RNA-seq. ***= Benjamini-Yuketieli-adjusted p-value (padj) <0.001; **= padj<0.01; *= padj<0.05.

Expanded View Figure Legends
Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel (2021)
Figure 1

Figure 1. Establishment of a cell-based reporter assay measuring FcγR activation in response to sICs.

A) BW5147 reporter cells stably expressing human FcγR-ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD.

B) FCS coating of an ELISA microtiter plate allows for suspension of subsequently added IgG. Plate bound IgG was quantified via ELISA. Bar graph shows means of one experiment performed in technical triplicates. Error bars = SD.

C) Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X-Axis shows sample concentration determined by antibody molarity. Y-Axis shows FcγR activation determined by reporter cell mouse IL-2 production (OD 450nm). Two independent experiments performed in technical duplicates. Error bars = SD.

D) Schematic of used assay setups. BW5147 reporter cells expressing chimeric human FcγR receptors express endogenous CD69 or secrete mouse IL-2 in response to FcγR activation by clustered IgG. sICs are generated using mAbs and multivalent antigens. sIC suspension requires pre-blocking of an ELISA plate using PBS supplemented with 10% FCS (FCS coat, grey-dashed).
Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel (2021)
Figure 2

Figure 2. FcγRs are activated by sICs formed from multivalent antigens.

Ultra-pure antigen (Ag, TNF-α) mixed with therapy-grade mAb (infliximab, Ifx) was used to generate sICs. X-Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR-specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL-2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL-2 was measured via anti-IL-2 ELISA (A450nm) and IL-2 concentrations were calculated from an IL-2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. MFI were normalized to untreated cells (ctrl.) and are presented as fold-change increase.
Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel (2021)
Figure 3

Figure 3. The FcγR-dependent activation pattern of primary NK cells or primary neutrophils depends on IC solubility.

A) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry. NK cells were incubated with immobilized IgG (mepolizumab, Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 106 cells). Incubation with PMA and Ionomycin (Iono) served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one donor measured in technical duplicates. Error bars = SD. One-way ANOVA (Tukey); *p<0.05, ****p<0.0001.

B) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry. Neutrophils were incubated with immobilized IgG (Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 2*105 cells). Incubation with PMA or immobilized rituximab served as positive controls. Incubation with medium served as a negative control. Immobilized FcγRII and FcγRIII specific mAbs served as functional controls. Mean florescence intensity (MFI) values at t=30 minutes of incubation are presented as increase over t=0 min. Each dot represents one donor measured in technical duplicates. Error bars = SD. Two-way ANOVA compared to medium (Dunnett); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel (2021)
Figure 4

Figure 4. FcγRIIB/C and FcγR-IIIA respond to sIC size reproducing a Heidelberger-Kendall like precipitation curve.

A) infliximab (mAb) and rhTNF-α (Ag) were mixed at different ratios (17 µg total protein, calculated from monomer molarity) and analysed via AF4. sIC size is maximal at a 1:3 ratio of mAb:Ag and reduced when either mAb or Ag are given in excess. <M>w = mass-weighted mean of the molar mass distribution. Three independent experiments. Error bars = SD. Data taken from Table 1.

B) sICs of different size were generated by cross-titration according to the AF4 measurement. Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNF-α, right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either antigen or antibody, respectively (TNF-α calculated as monomer). Two independent experiments performed in technical duplicates. Error bars = SD.

C) Purified primary NK cells from three different donors were incubated with cross-titrated sICs as in B. NK cells were measured for MIP-1β expression (% positivity). Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one experimental replicate, the dashed line represents the mean values.