Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors by Caroline A. Evans, Shahrul Ariffin, Andrew Pierce, and Anthony D. Whetton Blood Volume 100(9):3164-3174 November 1, 2002 ©2002 by American Society of Hematology

Sequence of transmembrane region of the hGM/βc chimera Sequence of transmembrane region of the hGM/βc chimera.The transmembrane domain sequence of the chimeric hGM/βcreceptor mutant is shown with the adjacent amino acids. Sequence of transmembrane region of the hGM/βc chimera.The transmembrane domain sequence of the chimeric hGM/βcreceptor mutant is shown with the adjacent amino acids. The sequences from wild-type hGM Rα and hβc subunits are shown in plain and bold text, respectively. The putative transmembrane region is underlined and is based on sequence data for the hGM Rα and h/βc.51 52 Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology

Sequence comparison of cytosolic domains of hGM Rα, IL –3 Rα, and IL-5 Rα.Aligned cytosolic sequences of hGM Rα, IL-3 Rα, and the related IL-5 Rα are shown. Sequence comparison of cytosolic domains of hGM Rα, IL –3 Rα, and IL-5 Rα.Aligned cytosolic sequences of hGM Rα, IL-3 Rα, and the related IL-5 Rα are shown. Amino acids present in all 3 Rα subunits or that are subject to conservative change are shown in bold. Those conserved between species are boxed and those present in 2 α subunits are marked with an asterisk (*). The PIG and KLN sequences of hIL-3 Rα and hGM Rα are indicated by underlining. Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology

Effect of expression of hGMβcchimera with or without hβc in FDCP-mix cells.(A) Analysis of receptor subunit expression. Effect of expression of hGMβcchimera with or without hβc in FDCP-mix cells.(A) Analysis of receptor subunit expression. Parental and hGM/βc transfected cells were analyzed for expression of the extracellular domain of the (i) hGM Rα and (ii) hβcsubunit using a 2-step labeling procedures and flow cytometry. The solid gray histogram represents the cell autofluorescence obtained in the absence of antibody staining. Representative histograms are shown for labeling obtained in the presence of secondary antibody only (gray) and in the presence of both primary and secondary antibody (black). (B) Effects on cellular proliferation. Cells expressing hGM/βc (○) and hGM/βc, hβc(●) were cultured in hGM-CSF (1 ng/mL) for 14 days. Cellular viability was assessed at intervals and the results are expressed as viable cell number (× 105/mL) and are mean ± SEM from 3 experiments. The results obtained with the wild-type hGM Rα,βc are shown for comparison (■). (C) Effect of activation of hGM/βc expressed alone or in combination with the hβc subunit on cell morphology. (i) hGM/βc cells, (ii) hGM/βc,hβc, and (iii) wild-type hGM Rα, hβc were cultured in hGM-CSF (1 ng/mL) for 7 days and photomicrographs were prepared following May-Grünwald-Giemsa staining of cytospin preparations. The morphology of cells cultured in the presence of murine cytokines (G/M Diff conditions), which promote myeloid development, are shown for comparison for (iv) hGM/βccells and (v) hGM/βc,βc and (vi) wild-type hGM Rα, hβc. Results are shown from an experiment representative of 3. Bar is 10 μm. Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology

Effects of hIL-3 (M) Rα activation. (A) Cell surface expression Effects of hIL-3 (M) Rα activation.(A) Cell surface expression. hIL-3 (M) Rα, hβc cell transfects were analyzed for expression of the extracellular domains of the (i) hIL-3 Rα and (ii) hβc by flow cytometry. Effects of hIL-3 (M) Rα activation.(A) Cell surface expression. hIL-3 (M) Rα, hβc cell transfects were analyzed for expression of the extracellular domains of the (i) hIL-3 Rα and (ii) hβc by flow cytometry. Cells were sequentially incubated with anti–hIL-3 Rα antibody and fluorescein isothiocyanate (FITC)–conjugated antimouse secondary antibody. The solid gray histogram represents the cell autofluorescence obtained in the absence of antibody staining. Representative histograms are shown for labeling obtained in the presence of secondary antibody only (gray) and in the presence of both primary and secondary antibody (black). (B) Cell survival proliferation. Cells expressing wild-type hIL-3 Rα, hβc (░) or hIL-3 (M) Rα, hβc (▪) were cultured in hIL-3 (0-100 ng/mL). (i) Cell viability was assessed at 48 hours using trypan blue. The results are expressed as cell viability (percent rmIL-3 response) and are mean values ± SEM from at least 3 experiments. (ii) [3H]-thymidine incorporation was assessed after 16 hours in culture. The results are expressed as percentage of the response obtained with rmIL-3 (10 ng/mL). (C) Cell morphology. Cells expressing (i) wild-type hIL-3 Rα, hβc or (ii) hIL-3 (M) Rα, hβc were cultured in hIL-3 (1 ng/mL) for 7 days. Photomicrographs were prepared from May-Grünwald-Giemsa–stained cytospin preparations. Results are representative of 3 experiments. Bar is 10 μm. Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology

Coexpression of hGM (M) Rα and hβc in FDCP-mix cells Coexpression of hGM (M) Rα and hβc in FDCP-mix cells.(A) Cell surface expression. Coexpression of hGM (M) Rα and hβc in FDCP-mix cells.(A) Cell surface expression. Cells transfected with hGM (M) Rα in combination with hβc cells were analyzed for expression of the extracellular domains of the hGM Rα and hβc by flow cytometry. Cells were sequentially incubated with (i) anti-hGM Rα antibody or (ii) anti-hβc antibody and FITC-conjugated antimouse secondary antibody. The solid gray histogram represents the cell autofluorescence obtained in the absence of antibody staining. Representative histograms are shown for labeling obtained in the presence of secondary antibody only (gray) and in the presence of both primary and secondary antibody (black). (B) Effects on cell survival. Cells coexpressing hGM (M) Rα, hβc (░) were cultured in the presence of hGM-CSF (0-100 ng/mL) for 48 hours prior to assessment of cell viability based on trypan blue exclusion. The data obtained for the chimeric hGM/βc, hβc (▪) are shown for comparison. Results are expressed as a percentage of the rmIL-3 (10 ng/mL) response and are mean values ± SEM of 3 experiments. (C) Long-term proliferation. Cells coexpressing either hGM/βc, hβc (●) or hGM (M) Rα, hβc (▴) were cultured for 60 days in the presence of hGM-CSF (1 ng/mL). The growth rate was determined from the initial and subsequent cultures of cells seeded at 1 × 105/mL and resuspended in fresh media when the cell number was more than 5 × 105/mL. The results are expressed as log viable cell number/mL. The growth rates of hGM/βc, hβc( ) and hGM (M) Rα, hβc( ) cells in response to rmIL-3 (10 ng/mL) are also shown. Results are mean ± SEM of 3 experiments. Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology

Effects of activation of hGM (M) Rα, hβcand hGM/βc, hβc on cell development.(A) Cell morphology. Effects of activation of hGM (M) Rα, hβcand hGM/βc, hβc on cell development.(A) Cell morphology. (i) hGM (M) Rα, hβc and (ii) hGM/βc, hβc cells were cultured in hGM-CSF (1 ng/mL) for 60 days and cytospin samples prepared at intervals during this time. Morphology was assessed following May-Grünwald-Giemsa staining. The morphology results are expressed as percentage of the total cells scored and pooled data from 3 experiments. The SEMs were less than 10%. (B) Ability to differentiate following long-term culture in hGM-CSF. Photomicrographs are shown of (i) hGM (M) Rα, hβc cells and (ii) hGM/βc, hβc cells cultured in hGM-CSF (1 ng/mL) for 60 days. After this time, (iii) hGM (M) Rα, hβc cells and (iv) hGM/βc, hβc cells were harvested, washed, and cultured in murine cytokines that promote myeloid development for a further 7 days. Cytospin preparations were May-Grünwald-Giemsa stained and are representative of 3 separate experiments. Bar is 10 μm. Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology

hGM-CSF mediated changes in expression of differentiation markers of cells expressing mutant hGM-CSF receptors.Cells expressing (A) hGM (M) Rα, hβc and (B) hGM/βc, hβc cells were cultured in 1 ng/mL hGM-CSF for 7 days. hGM-CSF mediated changes in expression of differentiation markers of cells expressing mutant hGM-CSF receptors.Cells expressing (A) hGM (M) Rα, hβc and (B) hGM/βc, hβc cells were cultured in 1 ng/mL hGM-CSF for 7 days. The levels of expression of the differentiation markers are shown as representative histograms from at least 3 experiments (____). Nonspecific labeling was determined using the corresponding isotype control (- - -). The results obtained for cells cultured in murine cytokines that promote G/M Diff for 7 days are also shown for comparison. Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology

hGM-CSF mediated changes in expression of differentiation markers of cells expressing mutant hGM-CSF receptors.Cells expressing (A) hGM (M) Rα, hβc and (B) hGM/βc, hβc cells were cultured in 1 ng/mL hGM-CSF for 60 days. hGM-CSF mediated changes in expression of differentiation markers of cells expressing mutant hGM-CSF receptors.Cells expressing (A) hGM (M) Rα, hβc and (B) hGM/βc, hβc cells were cultured in 1 ng/mL hGM-CSF for 60 days. The levels of expression of the differentiation markers are shown as representative histograms from at least 3 experiments (____). Nonspecific labeling was determined using the corresponding isotype control (- - -). The results obtained for cells cultured in murine cytokines that promote G/M Diff for 7 days after 60 days culture in hGM-CSF are also shown. Caroline A. Evans et al. Blood 2002;100:3164-3174 ©2002 by American Society of Hematology