Fig. 1.
Fig. 1. Development of CD4ζ/γ-expressing T cells, but not CD4ζ/γ-expressing myeloid, NK, and B cells is blocked following BMT. / (A) Bone marrow cells isolated from C3H mice were exposed to retroviral supernatant encoding CD4ζ, CD4γ, or CD4del (CD4Δ) in the presence of polybrene for 4 hours. Twenty-four hours later, cells were stained with antihuman CD4-phycoerythrin (CD4-PE; solid line) or isotype-matched control monoclonal antibody (dotted line) and analyzed by fluorescence-activated cell sorting (FACS) to determine the efficiency of transduction. Histograms for control cells stained with antihuman CD4-PE or isotype control were indistinguishable (data not shown). The percentage of human CD4-expressing cells over background is indicated. (B) The transduced bone marrow cells shown in panel A were infused into sublethally irradiated C3H mice. Six weeks after transplantation, peripheral blood was isolated and analyzed by 2-color flow cytometry using PE-conjugated antihuman CD4 and FITC-conjugated anti–Gr-1, anti-B220, anti-5E6, or anti-CD3. Forward and side-scatter properties were used to determine gates for myeloid-specific (Gr-1) and lymphoid-specific (B220, 5E6, and CD3) markers. The percentage of lineage-positive cells expressing human CD4 is shown in the top left hand corner of each dot plot. Control cells stained with antihuman CD4-PE or isotype control yielded indistinguishable results (data not shown). Results are representative of at least 15 additional mice. (C) Thymocytes were isolated from mice that received transplants of CD4ζ or CD4del (CD4−) 6 weeks after transplantation, stained with allophycocyanin (APC)–conjugated anti-CD3/anti-CD4/anti-CD8, PE-conjugated anti-CD25, fluorescein isothiocyanate (FITC)–conjugated anti-CD44, and energy-coupled dye (ECD)–conjugated antihuman CD4, and analyzed by 4-color flow cytometry. APC− (ie, TN cells) were subsequently analyzed for CD44 and CD25 expression to define TN subsets. The level of human CD4 expression (solid line) for CD25+CD44+(TN2), CD25+CD44− (TN3), and CD25−CD44− (TN4) cells is shown. APC+ TP (ie, CD3+CD4+CD8+) thymocytes were also analyzed for human CD4 expression (solid line). The percentage of peripheral blood–derived B cells (B220) expressing human CD4 in the same animals is also shown for comparison. Shaded histograms represent cells from control animals that received transplants of unmodified bone marrow in each case. The percentage of TN2, TN3, TN4, TP, or B cells (B220) expressing human CD4 is indicated in each histogram.

Development of CD4ζ/γ-expressing T cells, but not CD4ζ/γ-expressing myeloid, NK, and B cells is blocked following BMT.

(A) Bone marrow cells isolated from C3H mice were exposed to retroviral supernatant encoding CD4ζ, CD4γ, or CD4del (CD4Δ) in the presence of polybrene for 4 hours. Twenty-four hours later, cells were stained with antihuman CD4-phycoerythrin (CD4-PE; solid line) or isotype-matched control monoclonal antibody (dotted line) and analyzed by fluorescence-activated cell sorting (FACS) to determine the efficiency of transduction. Histograms for control cells stained with antihuman CD4-PE or isotype control were indistinguishable (data not shown). The percentage of human CD4-expressing cells over background is indicated. (B) The transduced bone marrow cells shown in panel A were infused into sublethally irradiated C3H mice. Six weeks after transplantation, peripheral blood was isolated and analyzed by 2-color flow cytometry using PE-conjugated antihuman CD4 and FITC-conjugated anti–Gr-1, anti-B220, anti-5E6, or anti-CD3. Forward and side-scatter properties were used to determine gates for myeloid-specific (Gr-1) and lymphoid-specific (B220, 5E6, and CD3) markers. The percentage of lineage-positive cells expressing human CD4 is shown in the top left hand corner of each dot plot. Control cells stained with antihuman CD4-PE or isotype control yielded indistinguishable results (data not shown). Results are representative of at least 15 additional mice. (C) Thymocytes were isolated from mice that received transplants of CD4ζ or CD4del (CD4−) 6 weeks after transplantation, stained with allophycocyanin (APC)–conjugated anti-CD3/anti-CD4/anti-CD8, PE-conjugated anti-CD25, fluorescein isothiocyanate (FITC)–conjugated anti-CD44, and energy-coupled dye (ECD)–conjugated antihuman CD4, and analyzed by 4-color flow cytometry. APC− (ie, TN cells) were subsequently analyzed for CD44 and CD25 expression to define TN subsets. The level of human CD4 expression (solid line) for CD25+CD44+(TN2), CD25+CD44− (TN3), and CD25−CD44− (TN4) cells is shown. APC+ TP (ie, CD3+CD4+CD8+) thymocytes were also analyzed for human CD4 expression (solid line). The percentage of peripheral blood–derived B cells (B220) expressing human CD4 in the same animals is also shown for comparison. Shaded histograms represent cells from control animals that received transplants of unmodified bone marrow in each case. The percentage of TN2, TN3, TN4, TP, or B cells (B220) expressing human CD4 is indicated in each histogram.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal