MOESM1 of Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2–CH3 IgG spacer domains

Additional file 1: Figure S1. Replication kinetics of HCMV in HFF. Shown is the number of infectious virus particles obtained from 1x106 HFF at different time points after infection with HCMV (AD169; MOI as indicated). (A) Infectious particles released into the supernatant. (B) Cell associated infectious particles obtained from centrifuged supernatant after ultrasonic homogenization of the HFF

GTPase-specific binding to their effectors.

<p>A) Coomassie-stained SDS-gel of precipitates of pull down experiments from HEp2 cell lysate. Arrows indicate GST-Rac1/−Rac1 S71E (48 kDa) used as bait and a coprecipitated 190 kDa protein that was identified as IQGAP1 by MALDI-TOF/TOF analysis. B) The interaction of active, GTP-bound Rac1/Cdc42 and their active forms (Q61L) with specific effectors was analyzed by immunoblot analyses of precipitates from pull down assays. Non-specific binding was tested by GST-loaded glutathione beads as control. C) Representative input control of pull down analyses using constitutively active (Q61L) mutants of Rac1 and Cdc42 and their S71E mutants. D) Representative immunoblots of pull down precipitates showing the interaction of constitutively active Rac1 and Cdc42 and constitutively active S71E mutants with their effector proteins. Rac1 Q61L/S71E and Cdc42 Q61L/S71E did hardly bind to their specific effectors Sra-1 and N-WASP, respectively and to their common effector protein PAK1. Both phosphomimetic GTPases, however bound to their common effectors IQGAP and MRCK alpha, although to a lesser extent. The bars show the arithmetic mean value ± SD of densitometrical evaluation of 3 independent experiments.</p

Activation of PAK by Rac1 and Rac1 S71E.

<p>The effect of Rac1 and Rac1 S71E on PAK phosphorylation was shown in HEp2 cells stably expressing either GTPase. A) A brief characterization of these stable transfected cell lines was done by scanning electron microscopy showing surface topology of the cells. B) Immunoblot analysis revealed comparable ectopic expression of HA-tagged Rac1 and Rac1 S71E and concomitant Ser-144 phosphorylation of PAK1 and Thr-402 phosphorylation of PAK2. C) densitometrical evaluation of three separate Immunoblots showing phosphorylation of PAK 1/2. Shown are mean values ± SD. D) Propidium iodide staining of stable cell lines indicates populations of cells with 2n (G1 phase) or 4n (G2/M phase) set of chromosomes. Shown are percentages of cells within different cell cycle phases (mean values of five separate experiments).</p

Activity status of Rac1 S71E and Cdc42 S71E.

<p>A) Cell lysates of <i>Rac1^fl/fl</i> and <i>rac1</i>^−/− mouse fibroblasts were analyzed for Ser-71 phosphorylation of Rac1 and Cdc42 after treatment with 100 ng/ml EGF for 2 h by immunoblot using anti-pRac1/pCdc42 (Ser71) antibody. Lack of specific signal in <i>rac1</i>^−/− cells strongly suggested specific phosphorylation of Rac1 and no detectable phosphorylation of Cdc42. Immunoblot is representative for three separate experiments B) GTP-binding of phosphomimetic (▴) Rac1 S71E and Cdc42 S71E in comparison with wild-type (○) Rac1 and Cdc42 was analyzed by a [γ-³²P]-GTP-binding assay. The diagrams show mean values ± SD of three separate experiments. C) Intracellular localization of pRac1/pCdc42 (Ser71). Phosphorylated Rac1/Cdc42 is exclusively present in the membrane fraction of cells. D) Pull down assay of pRac1/pCdc42 and Rac1 using PAK-PBD after overexpression of Rho-GDI. Total Rac, total pRac1/pCdc42, and expression of Rho GDI were checked by immunoblot of whole cell lysate (lower panel). E) Pull down assay in a recombinant system showed nucleotide dependent binding of wild-type Rac1, Rac1 S71E and Rac1 S71A to the PAK-p21 binding domain. E) The nucleotide-dependent binding of Rac1, Rac1 S71E and Rac1 S71A to full length PAK1 as determined by pull down experiments with HEp2 cell lysates using GTPases as bait.</p

Phosphomimetic Rac1 S71E induces filopodia formation.

<p>A) Treatment with 100 ng/ml EGF for 2 h induces pronounced formation of filopodia. Cells were stained for nuclei (DAPI, blue), actin cytoskeleton (rhodamin-phalloidin, red), and VASP (Alexa-488, green). B) HEp2 cells transfected with HA-tagged Rac1, Rac1 S71E, Cdc42, and Cdc42 S71E. Expression of GTPases was visualized by HA-staining, the actin cytoskeleton was stained with rhodamin-phalloidin. Only Rac1 S71E induced morphotype that is comparable with EGF-induced alterations. C) Phenotypes of HEp2 cells transfected with HA-tagged constitutive active mutants of Rac1 and Cdc42 as well as their phosphomimetic mutants S71E. Constitutively active (Q61L) Rac1 induced membrane ruffling whereas Rac1 S71E induced formation of filopodia. Filopodia formation is less pronounced in Cdc42 Q61L and Cdc42 Q61L/S71E transfected cells. Stained are nuclei (blue) and HA-tag (green); bar represents 10 µm. D) Active, GTP-bound form of Cdc42 was determined by G-LISA 24 h post transfection with constructs as indicated. Cdc42 Q61L was used for transfection experiments as positive control for experimental setup. Additionally, <i>C. difficile</i> toxin A (TcdA) was used as negative control for inactivation of Cdc42. The bar chart shows mean values ± SD of three (for TcdA) or four separate experiments.</p

Construction and characterization of the chNKG2D.

<p><i>A,</i> The figure schematically illustrates the domain architecture of wildtype NKG2D and chNKG2D (N = N-terminus, C = C-terminus, CM = cytoplasma membrane). The N-terminal truncation of the extracellular NKG2D domain at aa81 is indicated by a bold bar. The native C-terminal end of NKG2D was fused to the N-terminus of the IgG1-Fc/CD28/CD3ζ transmembrane signaling platform creating a transmembrane receptor of type I orientation in which the relative orientation and positioning of the NKG2D ectodomain has not been changed. <i>B,</i> efficient transfection and strong surface expression of chNKG2D by electroporation of mRNA into primary T cells. The histograms show the surface expression of chNKG2D 24 hours after electroporation of quiescent or anti-CD3-activated human T cells (thick line, open histogram). Analysis was performed by flow cytometry using an anti-CD314 antibody. The expression of endogenous wildtype NKG2D in T transfected with a CMV-gH-specific CAR is shown for comparison (thin line, open histograms; filled histograms = isotype controls). <i>C,</i> chNKG2D triggers cytotoxicity of activated T cells. Quiescent or anti-CD3-activated CD8^pos or CD4^pos T cells were transfected with RNA encoding either the chNKG2D or a CMV-gH-specific control receptor. One day after transfection, the T cells were co-cultured with dye-labeled murine B cells (Ba/F3-MICA) stably expressing the human NKG2D-L MICA. The diagram shows the percentage of Annexin V^pos murine target cells after 5 hours of co-culture at the indicated E∶T ratios (data pooled from three independent experiments using different cell donors; mean ± S.D.). Statistical significance was calculated for the effects of activated T cells expressing the chNKG2D versus T cells expressing the control receptor.</p

Expression of NKG2D-Ls in ESFT.

<p>The histograms show the surface expression of the respective NKG2D-Ls recognized by specific monoclonal antibodies and analyzed by flow cytometry (filled histograms = specific antibodies; open histograms = isotype controls). The two stably transfected murine B cell lines (Ba/F3) expressing either human MICA or ULBP2 are displayed for comparison.</p

Stability of receptor surface expression upon target-cell encounter.

<p>The histograms show the flow cytometric analysis of chNKG2D expression (detected in CD4^pos T cells by an anti-CD314 antibody or in CD8^pos T cells by an anti-human-IgG1-Fc antibody; log scale in all histograms; note: amplification voltage in flow cytometric analysis was lowered for lentivirally transduced T cells due to the strong expression of the receptor). <i>A,</i> comparison of lentivirally transduced and mRNA transfected T cells. 75,000 chNKG2D^pos CD4^pos T cells were cultured 24 hours after electroporation for further 24 hours in presence or absence of 200,000 STA-ET-3 cells (filled grey histograms = presence of STA-ET-3, open histograms bold line = absence of STA-ET-3, open histograms dotted line = T cells not transfected). Shown is one representative experiment out of three independent repetitions. <i>B,</i> chNKG2D down-regulation in response to different ESFT cell lines. 100,000 chNKG2D^pos CD8^pos T cells were co-cultured 50 hours after mRNA electroporation for further 24 hours with 100,000 cells of different ESFT cell lines as indicated (unmodified T cells = “without chNKG2D”; without co-culture = “without target cells”). <i>C,</i> kinetics of chNKG2D down-regulation upon target-cell encounter at different times after transfection. 20,000 chNKG2D^pos CD4^pos T cells were co-cultured with 60,000 STA-ET-3 target cells for different times (1, 4, or 48 hours) before chNKG2D expression was determined (6, 30, or 74 hours after transfection) (one experiment). <i>D,</i> comparison of differentially stabilized mRNA species. ChNKG2D^pos CD4^pos T cells were co-cultured with STA-ET-3 target cells at different effector∶target ratios and at different times after transfection. Target cells were added at the indicated ratios either two or 26 hours after transfection and chNKG2D expression was determined by flow cytometry 6, 30, 50, or 74 hours after transfection. Shown is one out of three experiments. <i>E,</i> effect of patient serum containing soluble NKG2D-Ls. 15,000 chNKG2D^pos CD4^pos T cells were cultured 24 hours after electroporation for further 24 hours with or without serum of two different neuroblastoma patients (final serum concentration 33%).</p

Lentiviral transduction for transfer of chNKG2D.

<p><i>A,</i> antigen-specific enrichment of lentivirally transduced chNKG2D^pos MNCs. On day 13 after transduction the cells were co-cultured either with Ba/F3, ULBP2^pos Ba/F3 or without target cells. FACS analysis was performed 6 days later (open histogram, bold line: co-culture with the ULBP2^pos Ba/F3, 86.7% chNKG2D^pos T cells; filled histogram, dotted line: co-culture with Ba/F3, 60.7% chNKG2D^pos T cells; thin line: cells cultured without target cells, 54.1% chNKG2D^pos T cells; open histogram, dotted line = secondary antibody only). <i>B,</i> cytokine production. The diagram shows TNF secretion of purified CD4^pos T cells lentivirally transduced with either chNKG2D (enriched to 83–92% positive cells) or a similar chimeric CMV-specific control receptor (enriched to 69–81%) in response to the indicated cell lines or only medium as control (measured by ELISA; mean of duplicates). Ba/F3 or ULBP2^pos Ba/F3 cells were used for negative and positive control. Statistical significance was calculated for the effects of T cells expressing the chNKG2D versus T cells expressing the control receptor. <i>C,</i> Comparison of the cytotoxic response triggered by chNKG2D either stably or transiently transferred. CD8^pos T cells either unmodified (“without chNKG2D”) or expressing chNKG2D by lentiviral transduction (“LV”; enriched for chNKG2D^pos >90%) or mRNA transfection (“mRNA”; 20 hours after electroporation) were co-cultured with dye-labeled murine Ba/F3 cells expressing human ULBP2 (ULBP2-Ba/F3) or not (CTRL-Ba/F3). The diagram shows the percentage of Annexin V^pos Ba/F3 target cells after 4 hours of co-culture at the indicated E∶T ratios (mean ± S.D. of triplicates).</p