Journal: Molecular Therapy

Article Title: Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection

doi: 10.1016/j.ymthe.2025.01.009

Figure Lengend Snippet: CORA_sh-Ts mediate effective and target-specific T cell signaling, activation and cytotoxicity CORA receptors with either a short (CORA_sh) or long (CORA_lo) spacer domain were transduced into (A) Jurkat-based reporter cells or (B and C) primary CD8 + T cells. Respective untransduced cells served as controls. (A) After cultivation of transduced reporter cells without (w/o) target cells or with HB-82 cells (anti-HLA-A∗02) in an E:T ratio of 1:1 for 24 h, transcription factor activity was determined by evaluation of NF-κB-induced enhanced cyan fluorescent protein (eCFP) reporter expression by flow cytometry ( n = 4). (B) After co-cultivation of transduced CD8 + T cells with HB-82 cells in the indicated E:T ratios for 48 h, expression of CD137 as activation marker was assessed by flow cytometry ( n = 6–8). (C) Cytotoxicity by CORA-Ts was assessed by LDH assay. Data are shown as mean ± SD ( n = 5). Green and red asterisks indicate comparisons of CORA_sh- and CORA_lo-Ts, respectively, with untransduced T cells. (A–C) Data are shown as scattered dot plot with mean ± SD, whereby each symbol represents an independent donor. Statistical analysis was performed by using Mann-Whitney test. ns: not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Article Snippet: CORA-Ts for the HB-82 mouse model were generated by using a small-scale protocol similar to the manufacturing of CAR-Ts at the CliniMACS Prodigy : CD4 + and CD8 + T cells were isolated by combination of positive selection kits (Miltenyi Biotec), activated with T cell TransAct (Miltenyi Biotec), and then cultured and transduced with CORA_sh_mod receptors as described above.

Techniques: Activation Assay, Activity Assay, Expressing, Flow Cytometry, Marker, Lactate Dehydrogenase Assay, MANN-WHITNEY

Journal: Molecular Therapy

Article Title: Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection

doi: 10.1016/j.ymthe.2025.01.009

Figure Lengend Snippet: Modification of the CORA_sh receptor prevents activation and proliferation of CD8 + T cells CORA_sh receptors comprising either a truncated wild-type or a modified (D227K, T228A; CORA_sh_mod) HLA-A∗02 molecule were transduced into SPI-801 cells, whereby respective HLA-A∗02 domains are expected to dimerize with β 2 m and to be loaded with SPI-801-derived peptides. (A–K) CD8 + T cells were isolated from HLA-A∗02 + donors and co-cultured with untransduced or transduced SPI-801 cells in an E:T ratio of 1:1 for 7 days. (A–C) Expression of activation markers and (D and E) proliferation of CD8+ T cells as (D) representative histograms or (E) mean ± SD was assessed by flow cytometry. (F–I) Release of soluble mediators into the supernatant was assessed by LEGENDplex. (J–L) Before co-culture with CD8 + T cells, transduced SPI-801 cells were exogenously loaded with the HLA-A∗02-restricted, and CMVpp65-derived peptide NLVPMVATV. (F and G) After co-culture, the frequency of expanded HLA-A∗02/pp65 NLV -specific T cells was assessed by multimer staining using flow cytometry and is shown as (F) representative dot plots or (G) relative values, whereby respective frequencies of HLA-A∗02/pp65 NLV -specific T cells present in co-cultures with untransduced SPI-801 cells were subtracted from all values. (L) For evaluation of cytotoxicity toward transduced and pp65 NLV -loaded SPI-801 cells, they were transduced to express mCherry before and co-cultured with HLA-A∗02/pp65 NLV -specific T cells enriched from CD8 + of HLA-A∗02-positive donors using multimer. Live-cell imaging using the Incucyte Live-Cell Analysis System (Sartorius) was performed to evaluate elimination of mCherry-labeled cells. Total red object areas were analyzed by using the Incucyte software (Sartorius) and normalized to the time point of T cell addition, respectively. Data are shown as mean + SEM ( n = 3). (A–C, E–I, K) Data are shown as scattered dot plot with mean ± SD, whereby each symbol represents an independent donor ( n = 6). Statistical analysis was performed by using (A–C and E) Mann-Whitney test and (K) Wilcoxon matched-pairs signed rank test. ns: not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01.

Article Snippet: CORA-Ts for the HB-82 mouse model were generated by using a small-scale protocol similar to the manufacturing of CAR-Ts at the CliniMACS Prodigy : CD4 + and CD8 + T cells were isolated by combination of positive selection kits (Miltenyi Biotec), activated with T cell TransAct (Miltenyi Biotec), and then cultured and transduced with CORA_sh_mod receptors as described above.

Techniques: Modification, Activation Assay, Derivative Assay, Isolation, Cell Culture, Expressing, Flow Cytometry, Co-Culture Assay, Staining, Live Cell Imaging, Cell Analysis, Labeling, Software, MANN-WHITNEY

Journal: Molecular Therapy

Article Title: Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection

doi: 10.1016/j.ymthe.2025.01.009

Figure Lengend Snippet: CORA_sh- and CORA_sh_mod-Ts exhibit effective and target-specific T cell signaling, activation, and cytokine release CORA_sh and CORA_sh_mod receptors were transduced into (A, B, F, G) Jurkat-based reporter cells or (C–E and H–J) primary CD8 + T cells. Respective untransduced cells served as controls. (A, B, F, and G) After cultivation of transduced reporter cells without (w/o) target cells or with the indicated target cells in an E:T ratio of 1:1 for 24h (A and F) NF-κB-induced eCFP or (B and G) NFAT-induced eGFP reporter expression was evaluated by flow cytometry ( n = 4). (C and H) Transduced and untransduced (ø) T cells were co-cultured with the indicated target cells in an E:T ratio of 1:1 for 48 h. Release of soluble mediators into the supernatant was assessed by LEGENDplex. Fold increase to respective T cell cultures w/o target is shown as mean ( n = 4–8). (D, E, I, and J) CORA-Ts were co-cultured with target cells in the indicated E:T ratios for 48 h. Expression of activation markers was evaluated by flow cytometry. Data are shown as scattered dot plot with mean ± SD, whereby each symbol represents an independent donor ( n = 4–10). (A–J) Statistical analysis was performed by using two-way ANOVA with Tukey’s multiple comparisons test. ns: not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: CORA-Ts for the HB-82 mouse model were generated by using a small-scale protocol similar to the manufacturing of CAR-Ts at the CliniMACS Prodigy : CD4 + and CD8 + T cells were isolated by combination of positive selection kits (Miltenyi Biotec), activated with T cell TransAct (Miltenyi Biotec), and then cultured and transduced with CORA_sh_mod receptors as described above.

Techniques: Activation Assay, Expressing, Flow Cytometry, Cell Culture

Journal: Molecular Therapy

Article Title: Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection

doi: 10.1016/j.ymthe.2025.01.009

Figure Lengend Snippet: CORA_sh- and CORA_sh_mod-Ts mediate target-specific cytotoxicity resulting in effective reduction of anti-HLA antibody release (A–H) CORA_sh- and CORA_sh_mod-Ts were co-cultured with indicated target cells in the indicated E:T ratios for 48 h. Respective co-cultures with untransduced T cells served as controls. (A–C) Prior to the co-culture, target cells were labeled with CTV to determine the frequency of living target cells (CTV + 7-AAD − ) after co-culture ( n = 6–7). Frequencies of living target cells in co-cultures with indicated T cells were then normalized to corresponding frequencies of viable target cells in cultures without (w/o) T cells. (D–H) Co-culture supernatants were evaluated for LDH levels as indicator for T cell-mediated cytotoxicity ( n = 4–7). (I) Generated CORA-Ts were co-cultured with HB-82 cells in an E:T ratio of 5:1. At the indicated time points, supernatants were analyzed for released anti-HLA-A∗02 antibody by Luminex. After 24 h, co-culture media were replaced by fresh medium, after which further measurements were performed at indicated time points (“+”). Data are shown as mean + SEM ( n = 5). Significances are shown in comparison with HB-82 cultured without (w/o) T cells and evaluated at the respective same time points. (A–H) Data are shown as mean ± SEM. Statistical analysis was performed by using two-way ANOVA with Dunnett’s multiple comparisons test. Significances are shown in comparison with respective untransduced T cells cultured in the same E:T ratio. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: CORA-Ts for the HB-82 mouse model were generated by using a small-scale protocol similar to the manufacturing of CAR-Ts at the CliniMACS Prodigy : CD4 + and CD8 + T cells were isolated by combination of positive selection kits (Miltenyi Biotec), activated with T cell TransAct (Miltenyi Biotec), and then cultured and transduced with CORA_sh_mod receptors as described above.

Techniques: Cell Culture, Co-Culture Assay, Labeling, Generated, Luminex, Comparison

Journal: Molecular Therapy

Article Title: Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection

doi: 10.1016/j.ymthe.2025.01.009

Figure Lengend Snippet: CORA_sh_mod-Ts significantly reduce anti-HLA B-cell growth in vivo (A) Female NSG mice were injected 5 × 10 5 ffluc + HB-82 cells followed by injection of 5 × 10 5 CORA_sh_mod-Ts or untransduced T cells 2 days later. Respective mice injected with ffluc + HB-82 cells but not treated with T cells (untreated) served as controls. CORA_sh_mod-Ts were generated by transduction of CD4 + and CD8 + T cells and expansion using a protocol similar to manufacturing of CAR-Ts on the CliniMACS Prodigy. (B and C) Bioluminescence imaging was performed every week (w) to assess growth of HB-82 cells. Images in w0 were taken 15–20 min after injection of ffluc + HB-82 cells. (C) Quantification of bioluminescence was performed by setting a defined square area set on each animal to obtain average (Avg) radiance values. Data are shown as mean + SEM ( n = 5). Statistical analysis was performed by using Mann-Whitney test. Significances are shown in comparison with respective mice treated with untransduced T cells at the same time points. ∗ p ≤ 0.05.

Article Snippet: CORA-Ts for the HB-82 mouse model were generated by using a small-scale protocol similar to the manufacturing of CAR-Ts at the CliniMACS Prodigy : CD4 + and CD8 + T cells were isolated by combination of positive selection kits (Miltenyi Biotec), activated with T cell TransAct (Miltenyi Biotec), and then cultured and transduced with CORA_sh_mod receptors as described above.

Techniques: In Vivo, Injection, Generated, Transduction, Imaging, MANN-WHITNEY, Comparison

Journal: Molecular Therapy

Article Title: Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection

doi: 10.1016/j.ymthe.2025.01.009

Figure Lengend Snippet: CORA-Ts mediate specific elimination of HLA-specific B cells in target-cell mixtures and can be modified to resist tacrolimus treatment (A) CORA_sh- and CORA_sh_mod-Ts were co-cultured in a 1:1 ratio with target-cell mixtures composed of mCherry + HB-82 with CFSE + TÜ165 cells in the indicated ratios. Respective co-cultures with untransduced T cells served as controls. Live-cell imaging using the Incucyte Live-Cell Analysis System (Sartorius) was performed to evaluate specific target-cell elimination. (B) Representative pictures of selected time points of co-cultures with target-cell mixtures in a 1:5 ratio. (C) Total orange or green object areas were analyzed by using the Incucyte software (Sartorius) and normalized to respective values of target-cell mixtures cultured without T cells (target cells only) for every time point. Data are shown as mean ± SD of two independent experiments with two donors each. In one experiment, HFF cells were seeded on all wells as scaffold 1 day before setting the triple co-cultures. (D–K) CORA_sh receptors were transduced into CD8 + T cells, followed by transfection with FKBP12-targeting RNP complex (FKBP ko ). Respective untransfected CORA_sh-Ts served as controls (untransf.). (D) CRISPR efficiency was assessed by analysis of sequencing results by TIDE. (E–G) CORA-Ts were co-cultured with HB-82 cells for in an E:T ratio of 1:1 for 48 h in presence or absence (w/o) of 5 ng/mL tacrolimus, after which expression of activation markers was evaluated by flow cytometry ( n = 4). (E and H–K) Prior to the co-culture, CORA-Ts were labeled with CTV. After 2 days of co-culture, CORA-Ts were re-stimulated with the same number of HB-82 cells ( n = 3). After 5–7 days of co-culture, (H) the frequency of living target cells (CTV − CD3 − 7-AAD − ) was assessed by flow cytometry. HB-82 cells cultured alone (only) for 5–7 or 3–5 days, respectively, are shown as first and second bar. (I and J) Proliferation of CORA-Ts was assessed via CTV dilution assay by flow cytometry and is shown as (I) representative histograms or (J) mean. (K) Release of soluble mediators into the supernatant was assessed by LEGENDplex. Fold increase to respective co-cultures in absence of tacrolimus is shown as mean. Statistical analysis was performed by using two-way ANOVA with Šídák’s multiple comparisons test. (D, F–H, and J) Data are shown as scattered dot plot with mean ± SD, whereby each symbol represents an independent donor. Statistical analysis was performed by using two-way ANOVA with Tukey’s multiple comparisons test. ns: not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: CORA-Ts for the HB-82 mouse model were generated by using a small-scale protocol similar to the manufacturing of CAR-Ts at the CliniMACS Prodigy : CD4 + and CD8 + T cells were isolated by combination of positive selection kits (Miltenyi Biotec), activated with T cell TransAct (Miltenyi Biotec), and then cultured and transduced with CORA_sh_mod receptors as described above.

Techniques: Modification, Cell Culture, Live Cell Imaging, Cell Analysis, Software, Transfection, CRISPR, Sequencing, Expressing, Activation Assay, Flow Cytometry, Co-Culture Assay, Labeling, Dilution Assay