isotype matched control antibody  (Proteintech)

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells do not induce an allogeneic immune response in NZB/W lupus mice (A) One million WT or HIP CAR T cells were intravenously injected into fully allogeneic NZB/W lupus mice and the host immune response was quantified after 6 days. CD8 T cells were recovered from the spleen of these mice, NK cells and macrophages were taken from C57BL/6 mice, and IgM and IgG donor CAR T cell-specific antibodies (DSA) were quantified. (B) Interferon-γ (IFN-γ) ELISpot assays were performed with CD8 lymphocytes isolated from the NZB/W recipients and the injected WT or HIP CAR T cells as stimulators. Spot frequencies were automatically enumerated (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). (C) Impedance cytotoxicity assays with WT and or HIP CAR T cells as targets and isolated CD8 lymphocytes from the lupus mice as effector cells (5 animals per group, mean ± SD per time point). The cell index is normalized at time point zero hours and a drop of the curve indicates target cell killing, while a stable signal indicates target cell survival. Fluctuations in the first few hours reflect cell culture perturbations from the addition of the effector cells. (D) IgM and IgG DSAs were quantified by flow cytometry (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). The background of this assay is shown in a dashed line. (E and F) Impedance cytotoxicity assays with WT or HIP CAR T cells or MHC class I and II-deficient double knockout (DKO) cells as targets and C57BL/6 NK cells (E) or macrophages (Mac; F) as effector cells (5 animals per group, mean ± SD per time point).

Article Snippet: For MHC class I, the PerCP-eFlour710-labeled anti-MHC class I antibody (clone AF6–88.5.5.3, eBioscience) with PerCP-eFlour710-labeled mouse IgG2a isotype-matched control antibody (clone eBM2a, eBioscience) were used.

Techniques: Injection, Enzyme-linked Immunospot, Isolation, MANN-WHITNEY, Cell Culture, Flow Cytometry, Double Knockout

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells deplete CD19 cells and suppress total IgG and donor-specific IgG antibodies in irradiated NZB/W mice (A) NZB/W mice with established proteinuria were irradiated with 5 Gy 2 days before receiving 7 million allogeneic WT or HIP CAR T cells. Animals were followed for 21 weeks for lupus-specific disease parameters and overall survival. The study was started with 10 untreated NZB/W mice and 8 WT and 10 HIP CAR T cell animals. Animals that died dropped out of subsequent analyses. (B–D) The percentages of blood CD3 cells (B), CAR T cells (C), and CD19 B cells (D) were longitudinally quantified over the study period (all single animals are shown for all time points, mean ± SD). (E and F) Total IgM (E) and IgG (F) antibodies were quantified using ELISA assays in untreated NZB/W mice and those that received WT or HIP CAR T cells. (G and H) Anti-DNA IgM (G) and IgG (H) were quantified using ELISA assays (all single animals are shown for all time points, mean ± SD).

Article Snippet: For MHC class I, the PerCP-eFlour710-labeled anti-MHC class I antibody (clone AF6–88.5.5.3, eBioscience) with PerCP-eFlour710-labeled mouse IgG2a isotype-matched control antibody (clone eBM2a, eBioscience) were used.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells deplete CD19 cells and suppress total IgG and donor-specific IgG antibodies in non-irradiated NZB/W mice (A) NZB/W mice with established proteinuria received 7 million allogeneic WT or HIP CAR T cells. Animals were followed for 21 weeks for lupus-specific disease parameters and overall survival. The study was started with 10 WT and 10 HIP CAR T cell animals. Animals that died dropped out of subsequent analyses. (B–D) The percentages of blood CD3 cells (B), CAR T cells (C), and CD19 B cells (D) were longitudinally quantified over the study period (all single animals are shown for all time points, mean ± SD). (E and F) Total IgM (E) and IgG (F) antibodies were quantified using ELISA assays in untreated NZB/W mice and those that received WT or HIP CAR T cells (all single animals are shown for all time points, mean ± SD). (G and H) Anti-DNA IgM (G) and IgG (H) were quantified using ELISA assays (all single animals are shown for all time points, mean ± SD).

Article Snippet: For MHC class I, the PerCP-eFlour710-labeled anti-MHC class I antibody (clone AF6–88.5.5.3, eBioscience) with PerCP-eFlour710-labeled mouse IgG2a isotype-matched control antibody (clone eBM2a, eBioscience) were used.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells do not induce an allogeneic immune response in NZB/W lupus mice (A) One million WT or HIP CAR T cells were intravenously injected into fully allogeneic NZB/W lupus mice and the host immune response was quantified after 6 days. CD8 T cells were recovered from the spleen of these mice, NK cells and macrophages were taken from C57BL/6 mice, and IgM and IgG donor CAR T cell-specific antibodies (DSA) were quantified. (B) Interferon-γ (IFN-γ) ELISpot assays were performed with CD8 lymphocytes isolated from the NZB/W recipients and the injected WT or HIP CAR T cells as stimulators. Spot frequencies were automatically enumerated (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). (C) Impedance cytotoxicity assays with WT and or HIP CAR T cells as targets and isolated CD8 lymphocytes from the lupus mice as effector cells (5 animals per group, mean ± SD per time point). The cell index is normalized at time point zero hours and a drop of the curve indicates target cell killing, while a stable signal indicates target cell survival. Fluctuations in the first few hours reflect cell culture perturbations from the addition of the effector cells. (D) IgM and IgG DSAs were quantified by flow cytometry (5 animals per group, all single animals are shown, mean ± SD, Mann-Whitney test). The background of this assay is shown in a dashed line. (E and F) Impedance cytotoxicity assays with WT or HIP CAR T cells or MHC class I and II-deficient double knockout (DKO) cells as targets and C57BL/6 NK cells (E) or macrophages (Mac; F) as effector cells (5 animals per group, mean ± SD per time point).

Article Snippet: PerCP-eFlour710-labeled mouse IgG2a isotype-matched control antibody , eBioscience , clone eBM2a cat.no. 46-4724-82.

Techniques: Injection, Enzyme-linked Immunospot, Isolation, MANN-WHITNEY, Cell Culture, Flow Cytometry, Double Knockout

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells deplete CD19 cells and suppress total IgG and donor-specific IgG antibodies in irradiated NZB/W mice (A) NZB/W mice with established proteinuria were irradiated with 5 Gy 2 days before receiving 7 million allogeneic WT or HIP CAR T cells. Animals were followed for 21 weeks for lupus-specific disease parameters and overall survival. The study was started with 10 untreated NZB/W mice and 8 WT and 10 HIP CAR T cell animals. Animals that died dropped out of subsequent analyses. (B–D) The percentages of blood CD3 cells (B), CAR T cells (C), and CD19 B cells (D) were longitudinally quantified over the study period (all single animals are shown for all time points, mean ± SD). (E and F) Total IgM (E) and IgG (F) antibodies were quantified using ELISA assays in untreated NZB/W mice and those that received WT or HIP CAR T cells. (G and H) Anti-DNA IgM (G) and IgG (H) were quantified using ELISA assays (all single animals are shown for all time points, mean ± SD).

Article Snippet: PerCP-eFlour710-labeled mouse IgG2a isotype-matched control antibody , eBioscience , clone eBM2a cat.no. 46-4724-82.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Hypoimmune CD19 CAR T cells treat allogeneic mice with features of spontaneous systemic lupus erythematosus

doi: 10.1016/j.isci.2025.112806

Figure Lengend Snippet: HIP CAR T cells deplete CD19 cells and suppress total IgG and donor-specific IgG antibodies in non-irradiated NZB/W mice (A) NZB/W mice with established proteinuria received 7 million allogeneic WT or HIP CAR T cells. Animals were followed for 21 weeks for lupus-specific disease parameters and overall survival. The study was started with 10 WT and 10 HIP CAR T cell animals. Animals that died dropped out of subsequent analyses. (B–D) The percentages of blood CD3 cells (B), CAR T cells (C), and CD19 B cells (D) were longitudinally quantified over the study period (all single animals are shown for all time points, mean ± SD). (E and F) Total IgM (E) and IgG (F) antibodies were quantified using ELISA assays in untreated NZB/W mice and those that received WT or HIP CAR T cells (all single animals are shown for all time points, mean ± SD). (G and H) Anti-DNA IgM (G) and IgG (H) were quantified using ELISA assays (all single animals are shown for all time points, mean ± SD).

Article Snippet: PerCP-eFlour710-labeled mouse IgG2a isotype-matched control antibody , eBioscience , clone eBM2a cat.no. 46-4724-82.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay