Human lymphocyte engraftment and functional human T and B cell development in huPBMC-transplanted Rag2^−/−γc^−/− mice. (A) The percentage of human CD45⁺ cells and CD3⁺ T cells in peripheral blood nucleated cells in mice (n = 5) at indicated time after transplantation. (B) The percentage of human lymphocyte subsets in peripheral blood lymphocytes in mice (n = 5) at day 28 after transplantation. The data are representative of four independent experiments. (C) A booster of TT vaccine (↓) was given at day 14 after priming in humanized mice 4 wk after huPBMC transplantation. The percentages of CD4⁺ T, CD8⁺ T, CD19⁺ B cells and IFN-γ–producing cells in CD4⁺ and CD8⁺ T cells from peripheral blood nucleated cells, and the levels of serum TT-specific antibodies at indicated times after the first dose of TT vaccination, are shown (n = 6). The data shown as mean ± SEM are representative of two independent experiments.

PAM selectively expands human Vγ9Vδ2 T cells in humanized mice. (A) The percentage (mean ± SEM) of Vγ9Vδ2 T cells in peripheral blood lymphocytes in humanized mice (n = 4) after 1 dose of PAM (10 mg/kg body weight) via i.p injection. (B) Accumulation of Vγ9Vδ2 T cells (brown) in different tissues at day 2 after 1 dose of PAM or PBS. Representative immunohistochemical sections from spleen, liver, lung, and intestine in uninfected humanized mice stained with an anti–human Vδ2 mAb (7B6). Bar, 100 µm. (C) Representative data for the percentages of Vγ9Vδ2 T cells, CD3⁺ T, CD4⁺ T, CD8⁺ T, CD19⁺ B, and CD56⁺ NK cells in the peripheral blood lymphocytes at day 2 after 1 dose of PAM or PBS in uninfected humanized mice (n = 4 per group). (D) Body weight changes of humanized (n = 5 per group) or Rag2^−/−γc^−/− mice (n = 5 per group) after PAM or PBS treatment on day 0, 2, 4, and 6. The data are representative of two independent experiments.

PAM inhibits influenza virus replication in lung. Humanized mice were infected with human H1N1 (n = 5 per group), avian H5N1 (n = 5 per group), or PR/8 (n = 4 per group) viruses, and treated with PAM or PBS as indicated in the legend of Fig. 5. The virus titers in the supernatants of homogenized lung tissue were determined at the indicated times after infection. *, P < 0.05. The data shown as mean ± SEM are representative of two to three independent experiments.

PAM cannot control influenza diseases in humanized mice without Vγ9Vδ2 T cells. Vγ9Vδ2 T cell–depleted huPBMCs were obtained after double depletions of Vδ2 T cells by positive selection with magnetic microbeads. Rag2^−/−γc^−/− mice were transplanted with 30 × 10⁶ of huPBMCs or Vγ9Vδ2 T cell-depleted huPBMCs i.p. from the same healthy human donor. In contrast with the mice reconstituted with whole huPBMCs, there were no or very few human Vγ9Vδ2 T cells in the mice reconstituted with Vγ9Vδ2 T cell–depleted huPBMCs (Fig. S5). Humanized mice reconstituted with whole huPBMCs (PBMC) or Vγ9Vδ2 T cell–depleted huPBMCs (PBMC-γδ-T) were infected with PR/8 virus, and treated with PAM or PBS on day 3, 5, 7 and 9 after infection. The body weight change and survival in virus-infected humanized mice (n = 4 per group) are shown. The data are representative of two independent experiments.

Cytotoxicity of PAM-expanded Vγ9Vδ2 T cells against virus-infected MDMs. (A) MDMs were infected with PR/8 virus at an MOI of 2, and then cultured alone (vMDMs alone) or with purified PAM-expanded Vγ9Vδ2 T cells at a ratio of 1:10 for 6 h. The percentages of dead MDMs among target cells (CD3⁻ population), identified as CD3⁻EthD2⁺ for four different experiments are shown. To determine the viral copy, PR/8 virus–infected MDMs were cultured alone or with purified PAM-expanded Vγ9Vδ2 T cells for 48 h. Total RNA was extracted from both cells and supernatant, and viral matrix gene copies were quantified, followed by reverse transcription. The results were shown as virus gene copies per 10⁵ MDMs for four different experiments. *, P < 0.05. (B) PAM-expanded Vγ9Vδ2 T cells were co-cultured with PR/8 virus–infected MDMs at a ratio of 10:1 for 6 h. The perforin inhibitor CMA, granzyme B inactivator Bcl-2, anti-NKG2D (aNKG2D), anti-TRAIL (aTRAIL), anti-FasL (aFasL) blocking antibodies, or their relevant isotype controls, were used. The cytotoxicity was analyzed by flow cytometry as the percentage of EthD-2⁺ cells in the CD3⁻ population and calculated as the percentage of inhibition relative to those without any treatment. The data shown as mean ± SEM are representative of four independent experiments. *, P < 0.05 as compared with their relevant isotype control. mIgG1, mouse IgG1; mIgG2b, mouse IgG2b; gIgG, goat IgG.

Antiviral activity of human Vγ9Vδ2 T cells against influenza in vivo. (A) The body weight changes in wild-type and Rag2^−/−γc^−/− mice after infection with human H1N1 virus or mock (PBS) i.n. (n = 6 per group). (B) The body weight changes in humanized mice and Vγ9Vδ2 T cell–depleted humanized mice (n = 6 per group) after infection with human H1N1 virus i.n. As the control, humanized mice (n = 6) were treated with PBS (Mock). (C–E) After adoptive transfer of in vitro PAM-expanded Vγ9Vδ2 T cells or PBS i.v. at day 2, 4, and 6 after infection, the body weight changes (n = 8 per group; C), lung viral titers (n = 4 per group; D) and survival (n = 8 per group; E) in PR/8 virus–infected humanized mice were measured at indicated time after infection. (F–H) After adoptive transfer of in vitro PAM-expanded Vγ9Vδ2 T cells or PBS i.v. at day 2, 4, and 6 after infection, the body weight changes (n = 8 per group; F), lung viral titers (n = 4 per group; G), and survival (n = 8 per group; H) in PR/8 virus–infected Rag2^−/−γc^−/− mice were measured at indicated time of after infection. The data are representative of two independent experiments. *, P < 0.05.

PAM reduces influenza disease severity and mortality. (A and B) Mice were infected with PR/8 virus and treated with PAM or PBS on day 3, 5, 7, and 9 after infection. The body weight change and survival in virus-infected humanized mice (n = 6 per group; A), and Rag2^−/−γc^−/− mice (n = 6 per group; B) are shown. (C and D) Humanized mice were infected with human H1N1 virus and treated with PAM or PBS on day 3, 5, 7, and 9 after infection. The body weight changes and survival (C; n = 6 per group), and representative immunohistochemical staining for Vγ9Vδ2 T cells (brown) in the lungs (D) at the indicated time after PAM or PBS treatment are shown. (E–G) Mice were infected with avian H5N1 virus and treated with PAM or PBS on day 1, 3, 5, 7, and 9 after infection. The body weight changes and survival (E; n = 12 per group) and representative immunohistochemical staining for Vγ9Vδ2 T cells (brown) in the lungs (F) at indicated time after PAM or PBS treatment are shown. The body weight changes and survival in avian H5N1 virus–infected Rag2^−/−γc^−/− mice (n = 5 per group; G) are also shown. The data are representative of two to three independent experiments. Bars, 100 µm.

PAM prevents inflammation in infected lung. Humanized mice were infected with human H1N1 (n = 4 per group; A), or avian H5N1 (n = 4 per group; B) viruses, and treated with PAM or PBS as indicated in the legend of Fig. 5. The levels of human proinflammatory cytokines and chemokines in the supernatants of homogenized lung tissue were determined at the indicated times after infection (A and B). The data are shown as mean ± SEM. *, P < 0.05. (C and D) Histopathologic changes in mouse lung tissues collected at indicated times after infection are shown. Representative histological sections of the lung tissues from human H1N1 (C) and avian H5N1 (D) virus–infected humanized mice after PAM or PBS treatment as indicated in the legend of Fig. 5 were stained with hematoxylin and eosin. Bars, 100 µm. The data are representative of three independent experiments.