Spleen BALB/c BALB/c are shown 4 human from tissue slides and 4 eosin stain. Representative tissue sections slides were stained with hematoxylin and formalin BALB/c mouse lymphoid tissues tissues mouse and lymphoid analysis of Comparative Supplementary 1. Figure Human - mice. Scale Scale 200 bars: mice. fixed and paraffin embedded, and LymphoidOrgans the microanatomyhuman of Mouse Mouse μm . Human Human and were . Supplementary Figure 2. Processing of human lymphoid organoids and hematopoietic cells for constructing BLTS-humanized mice. (A) Human fetal organs (spleen, and thymus) are processed into 1-1.5 mm^2 pieces; representative pieces from a portion of respective organs are shown. (B) Human hematopoietic stem cells along with leukocytes (Before Selection) were isolated from the fetal liver and the fraction (Positive Fraction) was enriched using enriched via immuno-magnetic selection based on human CD34 expression; the leukocyte fraction (Negative Fraction) also confirms enrichment of the hematopoietic stem cell fraction. A representative flow cytometry analysis of human CD34+ hematopoietic stem cells enrichment is shown. Data presented are representative of 4 human fetal organs and human CD34+ hematopoietic stem cells isolation. A analysis of points post Representative histological (hematoxylin and eosin stain hCD68+) analysis of group) at 16 weeks post grossand histological (hematoxylin and eosin stain immune Human cells Supplementary 3. developmentin humanized Figure

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Supplementary Figure 4. Human immune cells development in lymphoid tissues in the BLT-humanized mouse model. (A) Representative gross and histological (hematoxylin and eosin stain – H&E) analysis of human thymus organoid (Thymus), humanized murine spleen (h-mSP), humanized murine lymph node (h-mLN) in BLT- humanized mice (n=3) at 16 weeks post-transplantation; black circles identifies tissues of interest. Representative human-specific immunohistochemical (IHC; Brown stain - T cells-hCD3+, B Cells-hCD20+, or macrophages-hCD68+) analysis of human thymus organoid (Thymus), humanized murine spleen (h-mSP), humanized murine lymph nodes (h-mLN) in BLT-humanized mice (n=3) at 16 weeks post-transplantation. Scale bars: 200 μm. (B) A representative clinical manifestation (excessive hair loss and wasting syndrome) of GVHD at 24 weeks post-transplantation in the BLT-humanized mouse model (n=4). Supplementary Figure 5. Human immune cells development in the peripheral blood in BLT-humanized mice. (A) Representative flow cytometry analysis of human immune cell (hCD45+) reconstitution, along with lymphocytes subsets, including T cells (CD3+) and CD4+ and CD8+ T cells subsets and B cells (CD19+) in peripheral blood mononuclear cells (PBMCs) of BLT-humanized mice at 10 weeks post-transplantation. (B) Quantification of human immune cells reconstitution (n=18; 2 independent experiments) and lymphocyte subsets (n=8) in peripheral blood mononuclear cells (PBMCs) of BLT-humanized mice at 10 weeks post-transplantation. Data is presented as mean +/- standard deviation. P values were determined using paired, 2-tailed Student’s t test between 2 groups. L o g 1 0 H IV G E C o p ie s p e r m L standard error error standard mean. of group. Data is presented as mean values analyzed using qPCR, with mock the as control 1x10^5IU per mouse in BLT the blood (n=4 per group) following inoculation at genome copies per mL The kinetics HIV of BLTkinetics replication in Supplementary 6. Figure HIV Analysisof

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I H H 0 M o c k H IV + A R T Supplementary Figure 7. Analysis of tissue fibrosis in humanized murine lymphoid organs in HIV-infected BLTS-humanized mice. (A) Kinetics of lymphoid tissue fibrosis (Sirius red-fast green stain; red-collagen positive and green-collagen negative) in humanized murine spleen following inoculation at 1x10^5 IU per mouse in BLTS- humanized mice (n=4 per group); mock inoculated mice, aged-matched to the 24 weeks post-infection (24 WPI) - mice served as controls; (B) lymphoid tissue fibrosis analysis was confirmed independently via Masson’s trichrome stain (MT; analine blue-collagen positive, dark red/purple-cell nuclei, and red/pink-cell cytoplasm) and human collagen 1 (hCOL1-brown stain) immunohistochemistry in HIV-infected BLTS-humanized mice at 10 weeks post-infection (10 WPI). (C) Analysis of lymphoid tissue fibrosis in humanized murine lymph nodes of ART-treated HIV-infected BLTS-humanized mice. Chronic HIV infection in BLTS-humanized mice was allowed to develop over a period of 8 weeks, and mice were subsequently treated with ART for 4 weeks, at which point humanized murine lymph nodes (n=3 per group) were analyzed for collagen deposition using Sirius red- fast green stain (red-collagen positive and green-collagen negative), aged-matched mice served as controls. Data is presented as mean values ± standard error of mean. P values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) were determined using one-way analysis of variance (ANOVA) between more than 2 groups or unpaired, 2-tailed Student’s t test between 2 groups for each lymphoid tissue, with mock as the control group. Scale bars: 200 μm.

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Supplementary Figure 8. Analysis of lymphoid tissue fibrosis in HIV-infected BLT-humanized mice. Chronic HIV infection in BLT-humanized mice was allowed to develop over a period of 12 weeks, at which point human thymus organoid, humanized murine spleen, and humanized murine lymph nodes were analyzed for collagen deposition using Sirius red-fast green stain (red-collagen positive and green-collagen negative), aged-matched mice served as controls. Shown are representative data (n = 4 per group). Scale bars: 200 μm.