Antigen-specific human T-cell responses and T cell-dependent production of human antibodies in a humanized mouse model

Abstract

Humanized mice with a functional human immune system would be very useful for in vivo studies of human immunobiology. We have previously shown that cotransplantation of human fetal thymus/liver tissues and CD34(+) fetal liver cells into immunodeficient nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice leads to the development of multiple lineages of human lymphohematopoietic cells and formation of secondary lymphoid organs with normal architecture. Here, we evaluated the ability of these humanized mice to develop antigen-specific, T cell-dependent antibody responses after in vivo immunization with T-dependent antigen, 2,4-dinitrophenyl hapten-keyhole limpet hemocyanin (DNP(23)-KLH). Human T cells from DNP(23)-KLH-immunized mice showed strong proliferation in response to KLH in vitro. Furthermore, T cell-dependent production of DNP-specific human antibodies (mainly IgG1 and IgG2) was detected in all immunized mice. These results confirm that a functional human immune system can be established in immunodeficient mice through cotransplantation of human fetal thymus/liver tissues and CD34(+) hematopoietic stem/progenitor cells.

Figure 1

Human lymphohematopoietic cell reconstitution in human Thy/Liv/CD34⁺ FLC-grafted NOD/SCID mice. (A) Levels of total human lymphohematopoietic (CD45⁺) cells, CD3⁺ T cells, and CD19⁺ B cells in PBMCs were analyzed by flow cytometry at week 9 after human tissue/cell transplantation. ■ and □ represent hu-mice that were subsequently used for immunization with DNP₂₃-KLH (n = 7) or adjuvant alone (ie, PBS controls; n = 5), respectively. (B,C) Levels of human CD3⁺ cells (B) and human CD19⁺ cells (C) in PBMCs, spleen, and LNs of DNP₂₃-KLH–immunized (n = 7) and PBS-injected control (n = 5) hu-mice were analyzed by flow cytometry at time of death (ie, week 2 or week 4 after booster immunization). Error bars represent SEM. (D) White pulp formation in hu-mouse spleen. Shown are sections prepared from a representative hu-mouse spleen stained with hematoxyin and eosin, anti-human CD3, CD20, and CD68.

Figure 2

Antigen-specific T-cell and antibody responses in immunized hu-mice. (A) Proliferation of human CD3⁺ T cells in response to KLH (left) and Con A (right). Stimulation index of each individual hu-mouse in DNP₂₃-KLH–immunized (●) and control (○) groups are shown. (B) Representative histograms showing CFSE levels in gated human CD3⁺ T cells from DNP₂₃-KLH–immunized (top) and control (bottom) groups. Cells stimulated with KLH, Con A, and medium are shown in left, middle, and right panels, respectively. (C) Serum levels of DNP-specific IgG in DNP₂₃-KLH immunized (●) and PBS control (○) mice at week 1 after booster immunization (left) and at time of death (ie, 2 or 4 weeks after booster immunization; right). Each symbol represents an individual hu-mouse. (D) Serum levels of DNP-specific human IgG1, IgG2, IgG3, and IgG4 in DNP₂₃-KLH–immunized (●) and PBS control (○) hu-mice at time of death (ie, 2 or 4 weeks after booster immunization). Each symbol represents an individual mouse. Horizontal lines in Figure 2A,D represent mean values.