Figure 1. CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo myeloid-derived suppressor cell expansion by human immunodeficiency virus type 1 does not require viral replication. Peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with heat-inactivated HIV_BaL (multiplicity of infection, 0.01) or stimulated with phytohemagglutinin (PHA; 10 µg/mL; Sigma–Aldrich, St. Louis, MO) for 48 hours and infected with HIV_BaL (multiplicity of infection, 0.01) in the presence of recombinant interleukin 2 (10 units/mL; Roche Diagnostics, Mannheim, Germany). After 5 days, the percentages of CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo cells (A) and the relative numbers of CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo cells (B) were determined and compared to those for controls. Mean values and standard errors of the mean are shown for 4 healthy donors. From: HIV Type 1 gp120–Induced Expansion of Myeloid Derived Suppressor Cells Is Dependent on Interleukin 6 and Suppresses Immunity J Infect Dis. 2013;209(3):441-451. doi:10.1093/infdis/jit469 J Infect Dis | © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figure 2. Effect of envelope protein gp120 on expansion of CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo myeloid-derived suppressor cells. Peripheral blood mononuclear cells from healthy donors were treated with gp120 (1 µg/mL) or gp41 (1 µg/mL) and compared to untreated controls. After 5 days, cells were analyzed by flow cytometry using logical gating. A, Representative dot plot is shown. B and C, Percentages (B) and relative numbers (C) of CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo cells were determined. Mean values and standard errors of the mean are shown for 9 healthy donors. Abbreviation: PE, phycoerythrin. From: HIV Type 1 gp120–Induced Expansion of Myeloid Derived Suppressor Cells Is Dependent on Interleukin 6 and Suppresses Immunity J Infect Dis. 2013;209(3):441-451. doi:10.1093/infdis/jit469 J Infect Dis | © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figure 3. Soluble factors from gp120-treated peripheral blood mononuclear cells (PBMCs) mediate CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo myeloid-derived suppressor cell (MDSC) expansion. A, PBMCs were cultured in the absence or presence of gp120 (1 µg/mL), and untreated (control medium), and gp120-treated (gp120 medium) supernatants were used to culture PBMCs from different healthy donors. As additional controls, PBMCs from the same donors were left untreated or were treated with gp120 (1 µg/mL). After 5 days, the percentages (i) and relative numbers (ii) of CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo cells were determined by flow cytometry. Mean values and standard errors of the mean (SEM) from 3 donors are shown. B, PBMCs from healthy donors were left untreated or were treated with gp120. After 5 days, interleukin 6 (IL-6) concentrations were determined in culture supernatants by enzyme-linked immunosorbent assay. C, IL-6–neutralizing antibody was added to cultures to determine the effect of blocking IL-6 on MDSC expansion. PBMCs from healthy donors were cultured in medium alone or with gp120 (1 µg/mL) and either anti–IL-6 (10 µg/mL) or isotype control antibody (10 µg/mL) was added to some gp120 treated wells. After 5 days, the percentages (i) and relative numbers (ii) of CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo cells were determined. Mean values and SEM are shown for 4 healthy donors. D and E, PBMCs were treated with gp120 in presence of IL-6–neutralizing antibody for 5 days. Cells were surface stained with anti-CD11b and anti-CD33, followed by intracellular staining with anti-pSTAT3 or isotype control antibody. Phosphorylated STAT3 (pSTAT3) was analyzed in CD11b⁺CD33⁺ cells by flow cytometry. A representative histogram is shown. Abbreviation: MFI, mean fluorescence intensity, and mean values and SEM are shown for 4 healthy donors. From: HIV Type 1 gp120–Induced Expansion of Myeloid Derived Suppressor Cells Is Dependent on Interleukin 6 and Suppresses Immunity J Infect Dis. 2013;209(3):441-451. doi:10.1093/infdis/jit469 J Infect Dis | © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figure 4. gp120-expanded CD33⁺ cells inhibit T-cell–associated interferon γ (IFN-γ) production and require cell-to-cell contact. Peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with gp120 or medium for 5 days, and CD33⁺ cells were subsequently isolated by positive selection, using magnetic beads. CD33⁺ cells from gp120-treated or control PBMCs were either cocultured with freshly isolated autologous T cells or cultured in transwell and T cells in wells of a 24-well plate in the presence of anti-CD3/CD28 for 3 days. IFN-γ concentrations were then determined by enzyme-linked immunosorbent assay. gp120-expanded CD33⁺ cells were cultured with CD4⁺ cells (A), and gp120-expanded CD33⁺ cells were cultured with CD8⁺ T cells (B). Mean values and standard errors of the mean are shown for 7 healthy donors. From: HIV Type 1 gp120–Induced Expansion of Myeloid Derived Suppressor Cells Is Dependent on Interleukin 6 and Suppresses Immunity J Infect Dis. 2013;209(3):441-451. doi:10.1093/infdis/jit469 J Infect Dis | © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figure 5. gp120-expanded CD33⁺ cell mediated suppression is induced by reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS). Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in the presence or absence of gp120 (1 µg/mL) for 5 days, and CD33⁺ cells were isolated. A, Expression of p47^phox, arginase 1, and iNOS was assessed by quantitative polymerase chain reaction, relative to hypoxanthine–guanine phosphoribosyltransferase. Gene induction was compared to CD33⁺ cells isolated from untreated PBMCs. Isolated CD33⁺ cells were cocultured with autologous CD4⁺ (B) and CD8⁺ (C) T cells as described previously in presence of ROS inhibitor (catalase; 100 U/mL), arginase inhibitor (nor-NOHA; 0.5 mM), or iNOS inhibitor (L-NMMA; 0.5 mM) for 3 days. Supernatants were collected, and interferon γ (IFN-γ) concentrations were measured by enzyme-linked immunosorbent assay. Mean values and standard errors of the mean are for 5 separate donors. *P < .05. From: HIV Type 1 gp120–Induced Expansion of Myeloid Derived Suppressor Cells Is Dependent on Interleukin 6 and Suppresses Immunity J Infect Dis. 2013;209(3):441-451. doi:10.1093/infdis/jit469 J Infect Dis | © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figure 6. gp120-expanded CD33⁺ cells increase interleukin 10 (IL-10) expression in CD4 cocultures and induce T-regulatory cell expansion. A, Peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with gp120 or medium for 5 days, and CD33⁺ cells were subsequently isolated by positive selection, using magnetic beads. CD33⁺ cells from untreated and gp120-treated PBMCs were cocultured with autologous CD4⁺ T cells in the presence of anti-CD3/CD28 for 3 days. IL-10 concentrations were then determined by enzyme-linked immunosorbent assay. Mean values and standard errors of the mean (SEM) shown are for 7 separate donors. B, Autologous CD4⁺ T cells were cultured with control or gp120-expanded CD33⁺ cells in the presence of anti-CD3/CD28 for 3 days, and IL-10 secretion was analyzed by intracellular fluorescence-activated cell sorter analysis, as shown in the representative plot. C, IL-10 secretion was analyzed by gating on CD4⁺ T cells and CD33⁺ cells, and the percentage of IL-10–secreting cells was quantified. Mean values and SEM from 3 independent experiments are shown. D, Control and gp120-expanded CD33⁺ cells were cultured with CD4⁺ T cells either together or in transwells in the presence of anti-CD3/CD28 for 3 days. Staining for surface CD4 and CD25 and intracellular FoxP3 was performed using respective antibodies and percentages of CD4⁺CD25⁺FoxP3⁺ cells was analyzed by flow cytometry. Mean values and SEM are for 3 separate donors. From: HIV Type 1 gp120–Induced Expansion of Myeloid Derived Suppressor Cells Is Dependent on Interleukin 6 and Suppresses Immunity J Infect Dis. 2013;209(3):441-451. doi:10.1093/infdis/jit469 J Infect Dis | © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figure 7. CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo cell counts are increased in human immunodeficiency virus type 1–infected patients. Whole blood specimens from 5 HIV–infected persons and 5 healthy controls were stained with antibody to CD11b, CD33, CD14, and HLA-DR. Flow cytometry was used to determine percentages (A) and absolute counts (B) of CD11b⁺CD33⁺CD14⁺HLA-DR^−/lo cells. The upper and lower limits of the boxes denote the interquartile ranges. The horizontal lines within the boxes denote median values. The upper and lower lines outside the boxes denote the highest and lowest values, respectively. From: HIV Type 1 gp120–Induced Expansion of Myeloid Derived Suppressor Cells Is Dependent on Interleukin 6 and Suppresses Immunity J Infect Dis. 2013;209(3):441-451. doi:10.1093/infdis/jit469 J Infect Dis | © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.