Figure 4. EBV/HIV dual-infected humanized mice replicate HIV-1 in B cells.

(A) Representative IHC stainings of EBNA2, CD20, CD4, CD8, and co-IHC for PAX5 (brown, nuclear stain) and p24 (red, cytoplasmic stain) of formalin-fixed, paraffin-embedded spleen sections from EBV/HIV–infected and EBV/HIV–infected OKT8-treated mice (EBV/HIV OKT8). (B) Representative co-immunohistochemistry stainings for EBNA2 (red, nuclear staining) and p24 (brown, cytoplasmic staining). (A, B) Scale bar: 40 μm. Inserts are a 2× magnification of a section of the main image. (C) Quantification of PAX5⁺/p24⁺ (left) and EBNA2⁺/p24⁺ (mid) cells in the spleen from the indicated experimental groups and EBNA2⁺/p24⁺ as % of total p24⁺ (right) cells in non-tumorous spleen and tumor tissue within the EBV/HIV OKT8 group. N = respectively; Mock: 4 & 5, HIV: 6 & 3, HIV OKT8: 7 & 5, EBV/HIV: 14 & 12, EBV/HIV OKT8: 15 & 14, “Spleen”: 5, “Tumor”: 4. PAX5⁺/p24⁺ P = 0.112; EBNA2⁺/p24⁺: **P = 0.006, Spleen versus Tumor: *P = 0.032 (Mann–Whitney test). (D) Experimental setup for cell fraction transfer assay: Newborn NSG mice were irradiated and transplanted with fetal liver derived CD34⁺ hematopoietic progenitor cells. Donor mice were infected with EBV and 1 wk later with X4-tropic HIV-1. At week 2 and 4, donor mice either received the CD8-depleting antibody (OKT8) treatment or PBS. At week 5 or 6, the donors were sacrificed, and splenic lymphocytes were MACS separated into CD19⁺ and CD19-depleted fractions. Recipient mice received either CD19⁺ or CD19-depleted cells i.p. from either EBV/HIV or EBV/HIV OKT8-treated mice. Recipient mice were monitored up to week 12 for HIV-1 RNA in plasma and for EBV DNA in whole blood. (E) The highest number of HIV-1 RNA copies measured in plasma of individual-recipient mice and the frequency of recipients with detectable HIV RNA in the plasma and EBV DNA in the peripheral blood after receiving either CD19⁺ or CD19-depleted cells from either EBV/HIV or EBV/HIV OKT8-treated donor mice.