Figure 1. Phenotype of CD8⁺ T cells and CD4⁺ T cells in human immunodeficiency virus (HIV)–seropositive women who are seropositive or seronegative for herpes simplex virus type 2 (HSV-2). Percentage of CD8⁺ (A) and CD4⁺ (C) T cells among PBMC and, within each of these populations, the frequency of cells expressing the indicated cell-surface markers by cross-sectional analysis comparing group effects among HIV⁺/HSV-2⁻ (n = 15) and HIV⁺/HSV-2⁺ (n = 49) women. The data represent the percentage of cells (+SEM). There was a significant difference in the frequency of cells expressing indicated markers among the CD4⁺ (P = .002) but not CD8⁺ (P = .65) cells. The asterisks indicate significant differences in CXCR4⁺ (P = .005) and CCR6⁺ (P = .04) cells from the model. B, The frequency of HIV Gag or cytomegalovirus (CMV)–specific CD8⁺ T cells by class I major histocompatibility complex (MHC) tetramer staining among human leukocyte antigen (HLA)-A2⁺subjects (n = 8 HIV⁺/HSV-2⁻ and n = 29 HIV⁺/HSV-2⁺). Each symbol represents an individual subject. From: Role of Interleukin 32 in Human Immunodeficiency Virus Reactivation and Its Link to Human Immunodeficiency Virus–Herpes Simplex Virus Coinfection J Infect Dis. 2016;215(4):614-622. doi:10.1093/infdis/jiw612 J Infect Dis | © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Figure 2. Differences in CD4⁺ T cells in herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus (HIV)–infected and –uninfected women. The percentage of live CD4⁺ T cells expressing the indicated markers in 4 cohorts of women were compared by 2-way analysis of variance (HIV⁻/HSV-2⁻: n = 10; HIV⁻/HSV-2⁺: n = 18; HIV⁺/HSV-2⁻: n = 15; and HIV⁺/HSV-2⁺: n = 49). There was a significant effect of HSV-2 infection on the frequency of CD69⁺ (P = .02) (A), CCR10⁺ (P = .008) (B), and naive CD45RA⁺/CCR7⁺ (P = .04) (C) T cells. The P value comparing PD-1 expression among HSV-2⁺ versus HSV-2⁻ women was .06 (D). From: Role of Interleukin 32 in Human Immunodeficiency Virus Reactivation and Its Link to Human Immunodeficiency Virus–Herpes Simplex Virus Coinfection J Infect Dis. 2016;215(4):614-622. doi:10.1093/infdis/jiw612 J Infect Dis | © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Figure 3. Human immunodeficiency virus (HIV) cell-associated DNA in HIV-infected women. Total cell-associated HIV DNA in CD4⁺ cells of study participants (HIV⁺/HSV-2⁻: n = 14; HIV⁺/HSV-2⁺: n = 21) was determined by real-time quantitative polymerase chain reaction. Each sample was assessed in duplicate, and results were normalized to cellular input. Data are presented for each subject, and the line indicates the median. Samples below the limit of quantification were assigned 1.67 copies/million cells. (P = .05, Fisher’s exact test comparing detectable viral loads in the 2 groups). From: Role of Interleukin 32 in Human Immunodeficiency Virus Reactivation and Its Link to Human Immunodeficiency Virus–Herpes Simplex Virus Coinfection J Infect Dis. 2016;215(4):614-622. doi:10.1093/infdis/jiw612 J Infect Dis | © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Figure 4. Herpes simplex virus type 2 (HSV-2) infection was associated with a decrease in interleukin 32 (IL-32) expression. A, Interleukin 32 expression was assessed by intracellular cytokine staining in CD4⁺ and CD8⁺ T cells isolated from women in all 4 groups (HIV⁻/HSV-2⁻: n = 9; HIV⁻/HSV-2⁺: n = 8; HIV⁺/HSV-2⁻: n = 9; HIV⁺/HSV-2⁺: n = 14) and compared using a 2-way analysis of variance. Results are presented as mean fluorescence intensity (MFI) + SEM; ** P = .007 comparing HSV-2⁺ versus HSV-2⁻ subjects. B–D, RNA was extracted from CD4⁺ T cells, converted to complementary DNA, and analyzed by real-time quantitative polymerase chain reaction for IL-32, PD-1, and CD69 expression. Each symbol represents the gene expression of an individual subject relative to the HIV⁺/HSV-2⁻ mean, and the line indicates the mean for the group; *P < .05, by unpaired t test, comparing HIV⁺/HSV-2⁺ versus HIV⁺/HSV-2⁻. From: Role of Interleukin 32 in Human Immunodeficiency Virus Reactivation and Its Link to Human Immunodeficiency Virus–Herpes Simplex Virus Coinfection J Infect Dis. 2016;215(4):614-622. doi:10.1093/infdis/jiw612 J Infect Dis | © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Figure 5. Interleukin 32 (IL-32) expression in CD4⁺ T-cell subsets. Interleukin 32 (IL-32) expression was assessed by intracellular cytokine staining in PD-1⁺ (A), CXCR5⁺ (B), and T follicular helper cells (Tfh; identified as CD4⁺PD-1⁺CXCR5⁺) (C) T cells in women in the 4 study groups. (HIV⁻/HSV-2⁻: n = 9; HIV⁻/HSV-2⁺: n = 8; HIV⁺/HSV-2⁻: n = 9; HIV⁺/HSV-2⁺: n = 14). The mean fluorescence intensity (MFI) in HSV-2⁺ versus HSV-2⁻ women was compared by 2-way analysis of variance. (*PD-1: P = .046; **CXCR5: P = .009; *Tfh: P = .03). From: Role of Interleukin 32 in Human Immunodeficiency Virus Reactivation and Its Link to Human Immunodeficiency Virus–Herpes Simplex Virus Coinfection J Infect Dis. 2016;215(4):614-622. doi:10.1093/infdis/jiw612 J Infect Dis | © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Figure 6. Effects of interleukin 32 (IL-32) on human immunodeficiency virus (HIV) reactivation, RUNX1 gene expression, and blockade of HIV reactivation by addition of RUNX1 inhibitor. A, CD4⁺ T cells from a representative HIV⁺/herpes simplex virus type 2–positive (HSV-2⁺) subject were exposed to medium or medium supplemented with tumor necrosis factor (TNF; 100 U/mL), phytohemagglutinin (PHA) (5 µg/mL), or phorbol 12-myristate 13-acetate (PMA) (2nM) for 24 hours in the absence or presence of recombinant human IL-32γ (100 ng/mL). Cells were washed, and fresh medium with or without IL-32γ was added. Seven days after stimulation, HIV cell-associated RNA was quantified; results are presented as copies per microgram RNA and are means + SEM of duplicate wells (**P = .002, t test). B, Average reduction in HIV RNA transcription following PHA stimulation and IL-32 treatment (n = 6; P = .03, paired t test). C, CD4⁺ T cells were cultured with PHA or PHA plus recombinant IL-32γ. After 24 hours, RNA was prepared from the cells, and gene expression was analyzed by nanostring. The individual symbols show the 238 genes with detectable signals after filtering as the log₂ fold change (PHA+rIL-32γγ/PHA) versus P value (by paired t test). The dotted lines represent 1.5-fold change in gene expression and a P value of .05. The red symbols indicate top genes that were differentially expressed. D, CD4⁺ T cells from 2 additional women were treated with medium, medium supplemented with PHA, PHA plus recombinant IL-32γ, or PHA plus recombinant IL-32γ, and (after removal of PHA) recombinant IL-32γ plus Ro5-3335 (5 μM). Results are presented relative to HIV RNA detected in PHA-treated cells 7 days after stimulation (*P < .05 and **P < .01, analysis of variance). From: Role of Interleukin 32 in Human Immunodeficiency Virus Reactivation and Its Link to Human Immunodeficiency Virus–Herpes Simplex Virus Coinfection J Infect Dis. 2016;215(4):614-622. doi:10.1093/infdis/jiw612 J Infect Dis | © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.