Fig 1.
Generation of NOG-hIL-4-Tg mice.
A, Genotyping of NOG-hIL-4-Tg mice. Human IL-4-specific bands (561 bp) were detected along with the internal control (151 bp). PC: positive control. These results confirmed the gene transfer. NC: negative control. Conventional NOG mice were used. B, ELISA of NOG-IL-4-Tg plasma corresponding to the mice submitted for genotyping. The mice producing more than 100 pg/ml hIL-4 (higher than the broken line) were selected and used for the assays. A representative assay is shown. C, Comparison of hIL-4 protein levels in HD plasma (n = 12) and NOG-hIL-4-Tg mice (n = 57). The broken line shows the 100 pg/ml level. The high hIL-4 (n = 47) and low hIL-4 groups (n = 10) are shown. D, Tissue-specific expression of hIL-4 mRNA. Representative data of 1 NOG and 2 NOG-hIL-4-Tg mice are shown. Human IL-4 specific bands (449 bp) were detected along with β-actin (569 bp).
Fig 2.
Decreased GVHD symptoms in PBMC-NOG-hIL-4-Tg mice.
A, Experimental protocol for hPBMC transfer to NOG-hIL-4-Tg and conventional NOG mice. Doses of 2.5x106 PBMCs were intravenously transferred into NOG-hIL-4-Tg and NOG mice 1 day after 2.5 Gy irradiation. After cell transfer, human lymphocytes were analyzed by FCM. B, Typical flow cytometric pattern of PBMC-transplanted mice. The CD45+ fraction was further analyzed for CD33+CD19- myeloid cells, CD19+ B cells, CD56+CD3- NK cells, CD3+CD4+ T cells and CD3+CD8+ T cells. The data presented in these panels show the percentages of these cell fractions. C, Suppression of GVHD in NOG-hIL-4-Tg mice. After HD PBMC transplantation, the body weights of NOG-hIL-4 Tg and NOG mice were measured biweekly for 20 weeks. Open circles and dashed lines: individual NOG-hIL-4-Tg mice; closed circles and solid lines: individual NOG mice. D-E, From 1 to 10 weeks after HD PBMC transplantation, the engraftment rates of human PBMCs were analyzed by FCM. D, Human CD45+ cells (%) in mononuclear cells (MNCs). E, Left panel; human CD3+ cells (%) in MNCs, right panel; human CD19+ cells (%) in MNCs. F, From 1 to 8 weeks after HD PBMC transplantation, the engraftment rates of human PBMCs were analyzed. Left panel; CD4+ T cells and CD8+ T cells in human CD3+ cells (%) in hCD45+ cells (%) of the mice, right panel; human CD4+ T cells and CD8+ T cells in human CD3+ cells (%) in hCD45+ cells (%) of the mice. Dotted lines: NOG-hIL-4-Tg mice; solid lines: NOG mice.
Fig 3.
Comparison of engrafted human lymphocytes in NOG-hIL-4-Tg and conventional NOG mice.
A, Cellularity in HD PBMC-transferred NOG or NOG-hIL-4-Tg mice. NOG-hIL-4-Tg and NOG mice were transplanted with PBMCs (5x106) from the same HD. After 4 weeks, the ratios of each T or B cell subset in BM or spleen obtained from PBMC-NOG (n = 6) and PBMC-NOG-hIL-4-Tg mice (n = 3) were analyzed by FCM. HD PBMCs (n = 20) are shown as a control. CD4-N; naïve CD4+ T cells, CD8-N; naïve CD8+ T cells, CD4-M; memory CD4+ T cells, CD8-M; memory CD8+ T cells, B-T; transitional B cells, B-N; naïve B cells, B-M; memory B cells, B-P; plasmablast/plasma cells. The mean ± SD is shown with the percentage score above each bar. B. Human naïve CD4+ T cells were purified and transferred to both NOG mice (n = 5) and NOG-hIL-4-Tg mice (n = 8). The left 4 panels show the typical expression profiles of CD45RA, CD45RO, CD3 and CD4 among purified naïve CD4+ T cells. MNCs were obtained from PBMC-NOG or PBMC-NOG-hIL-4-Tg mice, and the human CD4+ T cells were analyzed by FCM as shown in the 4 right panels. The lower panels were all gated on the upper gates surrounded by the lines. C, Isolated human CD4+ T cells were stimulated with PMA and ionomycin, and the expression levels of human IL-4 and IFN-γ were analyzed by FCM. Typical patterns are shown.
Fig 4.
The humoral immunity of transplanted NOG-hIL-4-Tg mice induced by antigen immunization.
A, Experimental protocol for immunization of PBMC-NOG-hIL-4-Tg mice. After transplantation with HD PBMCs, mice were immunized with CH401MAP or KLH emulsified in complete Freund’s adjuvant (CFA). As a negative control, PBS was emulsified with the adjuvant. After 14 days, CH401MAP or KLH was boosted with incomplete Freund’s adjuvant (IFA). Finally, after 28 days, the mice were sacrificed and analyzed. B, HE staining of NOG-IL-4-Tg spleens. Upper panels; the spleen sections of control PBS and CH401MAP-immunized PBMC-NOG-hIL-4-Tg mice. Lower panels; enlarged view of each spleen section. C, Cellularities of human lymphocytes in the spleen and BM of immunized PBMC-NOG-hIL-4-Tg mice. The ratio of each lymphocyte subset among CD45+ cells (%) is shown in the panels. Upper left: PBS and adjuvant-injected spleen; upper right: PBS and adjuvant-injected BM; middle left: CH401MAP and adjuvant-injected spleen; middle right: CH401MAP and adjuvant-injected BM; lower left: KLH and adjuvant-injected spleen; lower right: KLH and adjuvant-injected BM. The mean ± SD is shown with the percentage score above each bar. D, Typical B cell profiles of the lymphocytes in HD PBMCs and CH401MAP-immunized PBMC-NOG-hIL-4-Tg mouse spleen and BM cells. Upper panels; HD patterns. Middle panels: PBMC-NOG-hIL-4-Tg spleen cells; Lower panels: PBMC-NOG-hIL-4-Tg BM cells. All density plots were gated on CD45+CD19+ cells. E, Morphology of plasmablast/plasma cells in the BM or spleen of PBMC-NOG-hIL-4-Tg mice. After depletion of CD3+ T cells, Giemsa staining was performed. Upper panels: spleen; lower panels: BM. The plasmablast/plasma cells with typical morphology are shown with arrows. F, Plasma samples from immunized NOG-hIL-4-Tg mice were collected, and antigen-specific IgG was measured by ELISA. Upper panel: CH401MAP-immunized mouse plasma (n = 10); lower panel: KLH-immunized mouse plasma (n = 12). The PBMCs of HD-A, HD-B, HD-K and HD-L were transplanted into 3 mice, and PBS/CFA, CH401MAP/CFA and KLH/CFA were injected into each mouse. Therefore, the same control PBS/CFA data were used for titer comparisons between anti-CH401MAP IgG and anti-KLH IgG treatment with PBS controls. Thus, the total PBS control mouse number was 18. Open bars: mouse treated with PBS/CFA (PBS). Closed bars: CH401MAP/KLH-immunized mouse. The figure above each bar shows the accurate absorbance at 450 nm. Asterisks; indicate CH401MAP-immunized serum for which the reactivity was two-fold higher than the control sera in mice transplanted with the same donor PBMCs.
Table 1.
The list of classical HLA types of HDs.