NSG mice adoptively transferred with LTR-Tax CD4⁺ T cells exhibit CD4⁺ T-cell proliferation. CD4⁺ T cells from LTR-Tax or littermate control mice were activated with anti-CD3 and anti-CD28 for 2 weeks in vitro and transferred into NSG mice. Recipient mice were euthanized at 2-4 months. (A) Geimsa staining of blood smear from a representative LTR-Tax CD4⁺ T cell–inoculated NSG mouse showing the presence of cells exhibiting lymphoblastoid phenotype adjacent to a neutrophil. Micrographs were obtained by visualization with a 40×/0.6 objective lens, using a Nikon EclipseTE2000-S microscope, Roper Scientific Photometrics imaging system, and Image Pro plus image acquisition software Version 4.5.0.29. (B) Flow cytometric analysis of blood leukocytes from NSG mice. 32.6% of cells in LTR-Tax CD4⁺ cell–inoculated NSG mouse (same as panel A) were CD4^+;, compared with 10% in control cell inoculated mice. (C) Total CD4 ⁺ T-cell counts in the blood and spleen of control or LTR-Tax cell–inoculated NSG mice, calculated as the percentage of gated CD4⁺ cells multiplied by the total number of blood leukocytes or splenocytes. (D) Histologic analysis (H&E staining) of a representative lymph node from the LTR-Tax-injected NSG mice, demonstrating diffuse infiltration of lymphoid cells (using 10×/0.2, 40×/0.65, and 100×/1.25 objectives, on a Leica DME microscope, Nikon Coldpix4500 camera and Nikon View 5.0 software). No lymph nodes were detected in littermate control inoculated NSG mice. (E) Percentage of lymph node cells expressing CD4, as determined by flow cytometry (representative specimen). (F) Tax expression in adoptively transferred NSG mice. NSG mice treated with LTR-Tax CD4⁺ T cells are positive for Tax. PCR for Tax expression (∼ 1kb) was performed from DNA (∼ 1 ug) that was extracted from various tissue taken from NSG mice adoptively transferred with LTR-Tax or littermate control CD4⁺ T cells that were previously activated ex vivo and cultured for 2 weeks prior to transfer. As shown NSG mice treated with LTR-Tax CD4⁺ T cells are positive for Tax in spleen and lymph node derived DNA.