Table S1. Viral load and CD4 count of HIV-infected patient population
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1 Table S1. Viral load and CD4 count of HIV-infected patient population Subject ID Viral load (No. of copies per ml of plasma) CD4 count (No. of cells/µl of blood) 28 7, , , , , , , , , , , , , , ,36, , , , , , , , , , 142
2 Table S2. Monoclonal antibodies used in the study Antibody Clone Source Anti-human CD2 2D7 ebiosciences, CA Anti-human CD123 6H6 ebiosciences, CA Anti-human CD123 7G3 BD biosciences, CA Anti-human CD11c S-HCL-3 BD biosciences, CA Anti-human HLA-DR G46-6 BD biosciences, CA Anti-human CD14 RMO2 Beckman Coulter, CA Anti-human CD8 L37.4 BD biosciences, CA Anti-human CD4 SK3 BD biosciences, CA Anti-human CD8 SK7 BD biosciences, CA Anti-human β7-integrin FIB4 Biolegend, CA/ BD bioscience, CA Anti-human CD49d 9F1 BD biosciences, CA Anti-human CD123 (IHC) SC-681 Santa Cruz biotechnology, CA Anti-human CCR7 13 R&D Systems, MN Anti- IDO ZMD.42 Invitrogen, CA Anti- Ki-67 B6 BD bioscience, CA Anti-human IFNα 22.C Chromaprobe Inc Anti-IFN-γ B27 BD bioscience, CA Anti-TNF-α MAb11 ebiosciences, CA
3 Figure S1. (A) FACS plots depict gating for pdc (CD123+ HLA-DR+ LIN-) and mdc (CD11c+ HLA-DR+ LIN-). (B, C) Frequency of pdc and mdc as a percentage of HLA-DR+ LIN- cells and absolute numbers per ml of blood following SIV infection. (D) Proportion of pdc expressing CCR7 in the blood. Data represents 7 rhesus macaques at each time-point over the course of infection. Figure S2. Expression of α4β7 and staining of β7-integrin. (A) FACS plots depict the coexpression of β7-integrin with CD49d (α4) on pdc from the blood of one animal. (B) Longitundinal data on the proportion of β7-integrin + pdc in the blood of uninfected RM (n=16). (C) β7-integrin stain intensity of SM, RM and human PBMC. FACS plots show staining of β7-integrin on total cells (includes lymphocytes, dendritic cells, monocytes) from the blood of SM, RM and human. (D) FACS plots show pdc from RM (black) overlayed on the HLA-DR+ LIN- population (grey). Stains were done using the β7-integrin antibody, florescent minus one (FMO) or isotype control antibody. Figure S3. Sort purity of DC or T cells (A) FACS plots show the purity of CD3+ CD4RA+ cells obtained from one experiment. (B) FACS plots show presorted cells and the purity of sorted pdc obtained from one experiment. Figure S4. pdc from SIV- RM are efficient stimulators of CD8 Tc1 differentiation. Number of IFN-γ+ CD8 or CD4 T cells stimulated with anti-cd3 in the presence of pdc or mdc from healthy RM. DC were stimulated with AT-2 SIVmac21 and cocultured at a DC:T cell ratio of 1: for 3. days. Following this, cells were restimulated with PMA-ionomycin for 4 hr prior to intracellular
4 staining for flow cytometry. Cell counts represent the difference between DC and no DC controls. Each number represents an individual RM. Figure S. Frequency of pdc in (A) blood of animals from the untreated control group and the antiα4β7-integrin antibody-treated group. (B) Frequency of Ki-67+ CD8 T cells in the blood post SIV infection. Charts show the geomean and individual data from the treated and untreated groups. An average of three preinfection timepoints was used for week. The increase in Ki-67+ CD8+ T cells was calculated by subtracting the proportion of these cells at week from the proportion of Ki-67+ CD8 T cells at a specific post infection time point. (C) Frequency of CD8 T cells in the colorectum post SIV infection. Frequency is expressed as a percentage of the total live cells gated within the lymphocyte region. (D) Association between viral DNA copies and MX1 or OAS2 expression levels in the colorectum at week 3 postinfection. Each dot represents an individual RM either from antiα4β7-integrin antibody treated or untreated group. Non-parametric correlation was analyzed using the Spearman two-tailed test.
5 Figure S1 A SSC-A FSC-A FSC-H FSC-A SSC-W SSC-H CD2 CD3 CD2 CD2-CD3- CD14 CD2-CD3-CD14- (LIN-) HLA-DR SSC-A HLA-DR pdc CD123 mdc HLA-DR CD11c B 3. pdc C 6 mdc % HLA-DR+ LIN- No. of pdc per µl of blood No. of mdc per µl of blood % % of HLA-DR+ LIN D
6 Figure S2 A B 1 SIV- RM β7-integrin α4 (CD49d)- integrin 2 day day day 24 CD123 time after 1st sampling C SM RM Human D β7-integrin β7-integrin FMO CD3 isotype CD123
7 Figure S3 A B Presort Postsort CD3 HLA-DR CD4RA CD123
8 Figure S pdc mdc
9 Figure S Untreated!4"7 antibody-treated A pre-infection RAI12 RSJ12 RDS7 RKJ pre-infection RQJ12 RQU11 RSQ7 RYI B 2 2 Geomean Untreated anti-!4"7 antibody-treated 2 2 Untreated 2 2!4"7 antibody-treated C Untreated!4"7-antibody treated D MX1: Relative fold gene expression in the colorectum r=.33 P= viral DNA copies per ng DNA OAS2: Relative fold gene expression in the colorectum r=.9 P= viral DNA copies per ng DNA
Supplementary Figure 1
Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition
Spleen. mlns. E Spleen 4.1. mlns. Spleen. mlns. Mock 17. Mock CD8 HIV-1 CD38 HLA-DR. Ki67. Spleen. Spleen. mlns. Cheng et al. Fig.
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