Journal: Scientific Reports

Article Title: Cryopreserved leukapheresis enables scalable and distributed CAR-T manufacturing: a multi-platform comparative study

doi: 10.1038/s41598-025-14865-5

Figure Lengend Snippet: Comparison of quality between fresh leukapheresis, cryopreserved leukapheresis, and cryopreserved PBMCs. ( A ) Flow chart of direct cryopreservation of leukapheresis versus cryopreservation of leukapheresis after isolation of PBMCs. ( B ) WBC Classification by Sysmex XS-1000i. NEUT = Neutrophil; LYMPH = Lymphocyte; MONO = Monocyte, Cryo = Cryopreserved. ( C ) T-, B-, and NK- cell subsets in Lymphocytes were analyzed by flow cytometry. ( D ) A flow cytometer analyzed CD4 and CD8 subsets in T-cells. ( E ) Different phenotypes of differentiated subpopulations were analyzed by flow cytometry. Tn = Naïve T cell; TCM = Central Memory T cell; TEM = Effector Memory T cell; Teff = Effector T cell. ( F ) Cell viability in different stages of the process. Pre-separation, Before T cell separation; Post-separation, After T cell separation; Pre-transduction, Before CAR transduction. The p -values for panels B-F were derived from two-way ANOVA (Fresh-LEUK n = 5, Cryo-LEUK n = 8, Cryo-PBMC n = 3). ( G ) Cell recovery from cryopreserved leukapheresis and cryopreserved PBMCs. Cell recovery = concentration of thawed cells/ concentration of frozen cells ( n = 3). ( H ) Cell cryopreservation recovery from leukapheresis and PBMCs, Cell cryopreservation recovery = cell number of frozen cells/ cell number of leukapheresis. ( n = 5). ( I ) Cell sorting yield from cryopreserved leukapheresis and cryopreserved PBMCs. ( n = 5). Scatter dot plots with bars show the mean and SEM. The p -values for panels G-I were calculated using t-tests. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Specifically, 2 μL of CliniMACS CD4 (Miltenyi, 200-070-213, DE) and CD8 (Miltenyi, 200-070-215, DE) microbeads were added per 1 × 10 7 cells for positive selection, with purity validated by flow cytometry.

Techniques: Comparison, Isolation, Flow Cytometry, Transduction, Derivative Assay, Cell Recovery, Concentration Assay, FACS

Journal: Scientific Reports

Article Title: Cryopreserved leukapheresis enables scalable and distributed CAR-T manufacturing: a multi-platform comparative study

doi: 10.1038/s41598-025-14865-5

Figure Lengend Snippet: Generation of CAR-T cells by non-viral vector and lentiviral transduction. ( A ) Cell viability during the preparation of non-viral CAR-T cells from day 0 to day 9. ( n = 3). ( B ) Fold expansion during the generation of non-viral CAR-T cells from day 0 to day 9. ( n = 3). ( C ) Different phenotypes of differentiated subpopulations and CAR + within T cells from fresh and cryopreserved leukapheresis were analyzed by flow cytometry. ( n = 3). ( D ) Exhaustion markers PD-1, TIM-3, and LAG-3 in non-viral CAR-T cells were analyzed by Flow cytometry. ( n = 3). ( E ) Cytotoxicity of non-viral CAR T cells co-cultured with tumor cells in a 2:1 and 4:1 E: T ratio for 24 h. ( n = 3). ( F ) Cell viability in the process of LV CAR-T preparation through lentiviral technology. MOI = 1.5. ( n = 3). ( G ) Fold expansion during the generation of LV CAR-T cell cultures. ( n = 3). ( H ) Different phenotypes of differentiated subpopulations and CAR + within T cells from fresh and cryopreserved leukapheresis were analyzed by flow cytometry. ( n = 3). ( I ) Exhaustion markers PD-1, TIM-3, and LAG-3 in LV CAR-T cells were analyzed by Flow cytometry. ( n = 3). ( J ) Cell viability of the Fast CAR-T cell process. ( n = 2). ( K ) Recovery of Fast CAR-T cells from thawing. ( n = 2). ( L ) CD4 + and CD8 + T cell subsets of fast CAR-T cells 3 days post-thaw from fresh and cryopreserved leukapheresis. ( n = 2). ( M ) Different phenotypes of differentiated subpopulations and CAR + within T cells on 3 days post-thaw from fresh and cryopreserved leukapheresis. ( n = 2). ( N ) Cytotoxicity of fast CAR T cells co-cultured with tumor cells in a 1:2, 1:1, 2:1, and 4:1 E: T ratio for 24 h. ( n = 2). Scatter dot plots with bars show the mean and SEM. P -values were from t-tests.

Article Snippet: Specifically, 2 μL of CliniMACS CD4 (Miltenyi, 200-070-213, DE) and CD8 (Miltenyi, 200-070-215, DE) microbeads were added per 1 × 10 7 cells for positive selection, with purity validated by flow cytometry.

Techniques: Plasmid Preparation, Transduction, Flow Cytometry, Cell Culture

Journal: Scientific Reports

Article Title: Cryopreserved leukapheresis enables scalable and distributed CAR-T manufacturing: a multi-platform comparative study

doi: 10.1038/s41598-025-14865-5

Figure Lengend Snippet: Comparability study of immune cell culture in cryopreserved leukapheresis with cryopreserved PBMCs. ( A ) CD4 and CD8 subsets in Lymphocytes were analyzed by flow cytometry. ( n = 3). ( B ) Different phenotypes of differentiated subpopulations within T cells were analyzed by flow cytometry. ( n = 3). ( C ) Cell viability during the T cells from day 0 to day 9. ( n = 3). ( D ) Fold expansion of the T cells from day 0 to day 9. ( n = 3). ( E ) Cell viability during the NK and NKT cells from day 0 to day 15. ( n = 3). ( F ) Total fold expansion during the NK and NKT cells from day 0 to day 15. ( n = 3). ( G ) NK and NKT cell subsets in Lymphocytes were analyzed by flow cytometry on day 11 of the cell cultures. ( n = 3). ( H ) Specific fold expansion of NK cells and NKT cells at day 11 of cell cultures. ( n = 3). Scatter dot plots with bars show the mean and SEM. P -values were from t-tests.

Article Snippet: Specifically, 2 μL of CliniMACS CD4 (Miltenyi, 200-070-213, DE) and CD8 (Miltenyi, 200-070-215, DE) microbeads were added per 1 × 10 7 cells for positive selection, with purity validated by flow cytometry.

Techniques: Cell Culture, Flow Cytometry

Journal: Cancer immunology research

Article Title: Multiple Immune-Suppressive Mechanisms in Fibrolamellar Carcinoma

doi: 10.1158/2326-6066.CIR-18-0499

Figure Lengend Snippet: CD8+ T cells correlate with B7H3+, PD-L1+, and IDO+ tumor cells and TILs/TAMs. A, Median CD8+ T-cell density between B7-H3+ tumors, TILs, TAMs (orange bar), and B7-H3− tumors, TILs, TAMs (blue) are compared in the left side of the panel. Mean CD8+ cell density values are indicated above each bar. Median CD8+ T-cell density between PD-L1+ (orange) and PD-L1− (blue) specimens is shown on the right. A total of 32 tumor regions per specimen were used in the analysis. Quintile regression of medians was used with a P value of ≤ 0.05 to define statistical significance. B, Correlation analysis between the cell densities for IDO+ and CD8+ T cells was performed using linear regression and correlation coefficient. TIL/TAM: tumor-infiltrating lymphocyte/tumor-associated macrophage.

Article Snippet: IHC staining for PD-L1, CD8, Foxp3, PD-1, IDO, LAG3, CD11c, CD68, and B7-H3 was performed by the Tumor Microenvironment Center at Johns Hopkins School of Medicine and the IHC laboratory in Johns Hopkins Hospital, using the reagents listed in Supplementary Table S1 .

Techniques:

Journal: Cancer immunology research

Article Title: Multiple Immune-Suppressive Mechanisms in Fibrolamellar Carcinoma

doi: 10.1158/2326-6066.CIR-18-0499

Figure Lengend Snippet: Coexpression of different immune-checkpoint markers. A, PD-1 (red) and IDO (gray) median cell densities in tumors with and without PD-L1 expression and B, in tumors with and without B7H3 expression. 32 tumor regions were analyzed. Mean cell densities are indicated above each bar. Quantile regression was used with a P value of ≤ 0.05 to define statistical significance. C, Percentage of B7-H3 expression in tumors and TILs/TAMs with PD-L1 expression was compared with specimens without PD-L1 expression. Percentage is indicated above each bar. Pearson χ2 test was used with a P value of ≤ 0.05 for significance, as noted by **.

Article Snippet: IHC staining for PD-L1, CD8, Foxp3, PD-1, IDO, LAG3, CD11c, CD68, and B7-H3 was performed by the Tumor Microenvironment Center at Johns Hopkins School of Medicine and the IHC laboratory in Johns Hopkins Hospital, using the reagents listed in Supplementary Table S1 .

Techniques: Expressing