Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies by Julie Gertner-Dardenne, Cecile Bonnafous, Christine Bezombes, Aude-Hélène Capietto, Virginie Scaglione, Sophie Ingoure, Delphine Cendron, Emilie Gross, Jean-François Lepage, Anne Quillet-Mary, Loîc Ysebaert, Guy Laurent, Hélène Sicard, and Jean-Jacques Fournié Blood Volume 113(20):4875-4884 May 14, 2009 ©2009 by American Society of Hematology

Freshly isolated TCRVγ9+ PBMCs are reactive to both PAg and RTX Freshly isolated TCRVγ9+ PBMCs are reactive to both PAg and RTX. (A) Representative phenotypes of unactivated and PAg-activated TCRVγ9+ T lymphocytes. Freshly isolated TCRVγ9+ PBMCs are reactive to both PAg and RTX. (A) Representative phenotypes of unactivated and PAg-activated TCRVγ9+ T lymphocytes. (B) Flow cytometry of intracellular phosphoZAP70 and phosphoERK1/2 in gated TCRVγ9+ γδ T cells after stimulation with PAg and/or cross-linked RTX (a representative experiment of 6 performed, GAH: goat anti–human IgG). (C) Depletion of B cells from PBMCs of healthy subjects (n = 8) upon 24 hours of treatment with PAg (400nM BrHPP) and/or RTX (10 μg/mL) in vitro; *P < .05 versus control and RTX versus RTX+PAg by 1-way paired Student t test. (D) Culture with PAg (400 nM BrHPP) for 7 and 13 days in medium containing 100 U/mL IL-2 amplifies the number of TCRVγ9+ γδ T lymphocytes in the PBMCs of healthy subjects (n = 10; bars: group means); *P < .05 versus day 0 by 1-way paired t tests. Julie Gertner-Dardenne et al. Blood 2009;113:4875-4884 ©2009 by American Society of Hematology

BrHPP plus mAbs optimize binding of γδ T lymphocytes to cancer cells in vitro. BrHPP plus mAbs optimize binding of γδ T lymphocytes to cancer cells in vitro. (A) Representative experiment showing γδ T-cell binding to CD20+HER2/Neu− RAJI cells in various conditions. BrHPP (400 nM), TTZ (20 μg/mL), and RTX (10 μg/mL) were added to cells as indicated. Cell binding (acquired MESF) was obtained by subtracting the value at 0 minutes from that at 60 minutes. (B) Binding to CD20+ B-cell lymphoma cell lines by TCRVγ9+ γδ cells from different subjects in the specified conditions. The data are means and 1 SD from n = 3-8 different donors; nt, not tested; *P < .05 for significant difference of the BrHPP+RTX group to the other groups by 1-way ANOVA on ranks and multiple comparison. (C) Representative TCRVγ9+ γδ cell binding to the CD20+CD52+ mantle cell lymphoma cell line GRANTA in the various conditions described in panel B, including anti-CD52 ALZ (10 μg/mL). Julie Gertner-Dardenne et al. Blood 2009;113:4875-4884 ©2009 by American Society of Hematology

BrHPP plus mAbs increase TCRVγ9+ γδ T-cell–mediated ADCC BrHPP plus mAbs increase TCRVγ9+ γδ T-cell–mediated ADCC. (A) The percentage of CD107a+ cells in total TCRVγ9 lymphocytes u pon 4-hour incubation with various CD20+ B-cell lymphomas in the specified conditions. BrHPP plus mAbs increase TCRVγ9+ γδ T-cell–mediated ADCC. (A) The percentage of CD107a+ cells in total TCRVγ9 lymphocytes u pon 4-hour incubation with various CD20+ B-cell lymphomas in the specified conditions. (B) ADCC of various CD20+ lymphoma cells by RTX and BrHPP-activated γδ T cells. Tumor cell lysis by RTX alone (100, 50, and 10 μg/mL): ; PAg-activated γδ T cells alone (E/T ratio 30:1, 10:1, and 1:1): ▨; RTX (10 μg/mL) plus PAg-activated γδ T cells (E/T ratio 30:1, 10:1, and 1:1): ; data show means ± SD from 3 to 8 experiments, each with different donor. (C) Specific lysis of CD20+ RL cells by RTX and PAg-activated γδ T lymphocytes results from ADCC (means ± 1 SD from 3 experiments each with a different donor. (D) ADCC of CD52+ RL cells with ALZ plus PAg-activated γδ T cells, as in panel C. (E) ADCC of HER2+ SKBR3 mammary carcinoma cells with TTZ plus PAg-activated γδ T cells, as in panel C. P < .05 by Mann-Whitney rank sum tests. Julie Gertner-Dardenne et al. Blood 2009;113:4875-4884 ©2009 by American Society of Hematology

Depletion of autologous B cells in vitro from PBMCs of CLL patients. Depletion of autologous B cells in vitro from PBMCs of CLL patients. (A) The percentage of B cells (□) and TCRVγ9+ γδ T cells () remaining in the PBMCs of CLL patients (mean ± SD, n = 8) after 1, 7, and 13 days culture in medium supplemented with PAg and therapeutic mAbs, as indicated. Data are means plus or minus SD; NS: no significant change as compared with day 0, Mann-Whitney tests with α = 5%. (B) Amplification of TCRVγ9+ γδ T cells from PBMCs of the same CLL patients as in panel A after 13 days culture in medium supplemented with IL2 and the specified reagent; *P < .05 versus “control IL2 medium” by 1-way paired t tests. (C) Cytotoxic degranulation by TCRVγ9+ γδ T cells (amplified from patient CLL017 as specified) in response to 4-hour incubation with autologous PBMCs and PAg, as indicated. (D) 51Cr-release assays for specific lysis of target cells (autologous PBMCs or allogeneic MEC2 CLL, as specified) by CLL patients' PBMCs that were either untreated or activated by 13 days' culture with BrHPP and IL2 as specified. Data are means of triplicates from 1 of 3 experiments. (E,F) Same experiments as in panels C and D with RTX-treated CLL target cells. Julie Gertner-Dardenne et al. Blood 2009;113:4875-4884 ©2009 by American Society of Hematology

B-cell depletion in macaques is enhanced by treatment with RTX, PAg, and IL2. B-cell depletion in macaques is enhanced by treatment with RTX, PAg, and IL2. Age- and sex-matched cynomolgus macaques received intravenous RTX alone (group 1; n = 4; empty circles and bars), RTX and IL2 (group 2; n = 4; gray circles and bars) or RTX, PAg, and IL2 (group 3; n = 6; black circles and bars) at the intervals specified on the graphs. Data shown are group means for blood (graphs, left) and group means ± SEM for bone marrow and lymph nodes (histograms, right) showing the percentage of cells that were TCRVγ9+ cells (A) and CD20+ B cells (B). (C) Group means for serum concentrations of RTX; *P < .05 by 1-tailed Mann-Whitney tests. Julie Gertner-Dardenne et al. Blood 2009;113:4875-4884 ©2009 by American Society of Hematology