Briefly, single cells were generated by gently mashing the spleen using the rubber stopper end of a 1 mL syringe followed by passing through a 0.45m strainer. of mesenteric lymph nodes from (A-C) three HIV infected human and (D-F) seven HIV infected hDRAGA mice stained for (A, D) CD20 (magenta) and DAPI (blue), (B, E) CD4 (green) and CD20 (magenta), and (C, F) CD8 (green) and CD20 (magenta). Bars equal 200 m. Graphs depict percentages of (G) CD20+, (H) CD4+, and (I) CD8+ pixel area as determined by visual inspection and quantitative image analysis in CD20hi and CD20lo areas in seven infected hDRAGA mice mesenteric lymph nodes. Statistical analyses were performed using Wilcoxon tests (*p<0.05). Image_3.tif (3.5M) GUID:?8993D1F7-AEC0-451C-AD96-58F653E20E93 Supplementary Figure?4: Lymphocyte population measurements in spleen from HIV infected (n=11) and uninfected hDRAGA mice (n=7) euthanized more than 250 days post stem cell infusion. Percentages of positive pixels for (A) CD4, (B) CD8, and (C) CD20 were determined by staining spleen sections with immunofluorescent antibodies and quantitative image analysis. Statistical analyses were performed using Mann Whitney tests (*p<0.05, **p<0.01, ns=not significant). Image_4.tif (81K) GUID:?7691D4E1-CD41-4AAF-A064-DC7FDE0D6BFC Supplementary Figure?5: hDRAGA mice mesenteric lymph nodes do not form traditional SPN germinal centers and lack canonical FDC distribution. Representative images of HIV infected human (n=3) and hDRAGA mice lymph nodes (n=7) stained for (A, B) Ki67 (red) and IgD (green). Cisapride Bars equal 200 m. (C) HIV infected human inguinal lymph nodes (n=3) were stained for human FDC (yellow) and (D) HIV infected hDRAGA mice mesenteric lymph nodes (n=7) were stained for mouse FDC (green) and CD20 (not shown) (n=7). CD20hi areas are demarcated by white lines. Bars equal 100 m. Image_5.tif (2.1M) GUID:?38386F0C-C821-4226-991F-66A95672921F Supplementary Figure?6: Spleens from hDRAGA mice contain mouse FDC with aberrant distribution. (A) Representative image of normal distribution of mouse FDC in C57/BL6 mouse spleen (n=1). (B) Representative image of HIV infected hDRAGA mouse spleen stained with human FDC antibody (n=32). (C-G) Representative images of aberrant distribution of mouse FDC in hDRAGA mice spleen (n=32). Spleens were stained with antibodies to CD20 (not shown) and mouse FDC (green). CD20hi areas are demarcated by blue line. No obvious difference in Cisapride FDC distribution was observed between (C-E) HIV infected and (F, G) uninfected hDRAGA mice. Image_6.tif (1.4M) GUID:?BA0BF783-C5A9-4617-9FF1-56D58F4F8B9B Supplementary Figure?7: HIV vRNA+ cells and vRNA particles are detected Cisapride in mesenteric lymph nodes of infected hDRAGA mice. Representative image of hybridization for HIV RNA (red) in a (A) mesenteric lymph node section from a chronically infected hDRAGA mouse (n=7; shown, mouse 53). Mouse FDC are shown in green. CD20 staining is not shown but CD20hi area is demarcated Cisapride by green line. vRNA+ cells are marked by arrows and representative individual HIV particles are indicated by arrowheads. Bars equal 50 m and 30 m on the left and right image, respectively. (C) Frequencies of vRNA+ cells were quantified in CD20hi and CD20lo areas by visual inspection and quantitative image analysis and (D) adjusted for CD4 positive pixel area (n=7). Statistical analyses were performed using a Wilcoxon test (*p 0.05; ns=not significant). Image_7.tif (1.2M) GUID:?65E947A2-7C7C-4327-9E47-560D2C1EBA11 Table_1.pdf (8.6K) GUID:?D7D056CF-63F2-4260-AB3F-FCFD1F683655 Data Availability StatementThe original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author. Abstract A major barrier in the use of humanized mice as models of HIV-1 (HIV) infection is the inadequate generation of virus-specific antibody responses. Humanized DRAGA (hDRAGA) mice generate antigen-specific class switched antibodies to several pathogens, but whether they do so in HIV infection and the extent to which their secondary lymphoid tissues (sLT) support germinal center responses is unknown. hDRAGA mice were evaluated for their ability to support HIV replication, generate virus-specific antibody responses, develop splenocyte subsets, and organize sLT architecture. hDRAGA mice supported persistent HIV replication and developed modest levels of gp41-specific human IgM and IgG. Spleens from uninfected and HIV infected hDRAGA mice contained differentiated B and CD4+ T cell subsets including germinal center (GC) B cells and T follicular helper cells (TFH); relative expansions of TFH and CD8+ T cells, but not GC B cells, occurred in HIV-infected hDRAGA mice compared to uninfected animals. Immunofluorescent staining of spleen and mesenteric lymph node sections demonstrated atypical morphology. Most CD4+.