Journal: Nagoya Journal of Medical Science

Article Title: T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation

doi: 10.18999/nagjms.85.4.779

Figure Lengend Snippet: Recognition of various tumor cell lines using clone #17 Fig. 2A: Clone #17 was co-incubated with various tumor cell lines with/without HLA-DPB1*09:01-P2A-HLA-DPA1*02:01 for 6 h. TNF-α and IFN-γ production was examined via intracellular staining. Fig. 2B: A series of myeloid and monocytic cell lines was further tested as shown above. Fig. 2C: Cytotoxicity of clone #17 was evaluated by the decrease of fluorescence-labeled target at graded E/T ratio. Percent lysis is indicated as the mean ±SD and a representative result of at least two independent experiments is shown. Statistical significance was determined using two-tailed Student’s t -test. (** P <0.01, *** P <0.001). Study acronyms are explained in Abbreviations to main body.

Article Snippet: Aliquots of T cell clones or TCR-T cells (1 × 10 5 ) were co-incubated with various tumor cells (1 × 10 5 ) forcibly expressing the HLA-DP restriction molecules for 6 h. For blocking studies, the target cells were pretreated with either control mouse IgG (Wako), anti-HLA-DR (BioLegend, USA), -DP (Cosmo Bio), or -DQ (Novus Bio, USA) antibody at a final concentration of 10 μg/mL for 1 h at room temperature, and subsequently cocultured with effector TCR-T cells for 6 h. TNF-α and IFN-γ production in the supernatant was evaluated using conventional sandwich enzyme-linked immunosorbent assays with a pair of relevant antibodies against TNF-α and IFN-γ (all from R&D Systems, USA) and 3,3′,5,5′-tetramethylbenzidine colorimetric reagents (Sigma-Aldrich, USA).

Techniques: Incubation, Staining, Fluorescence, Labeling, Lysis, Two Tailed Test

Journal: Nagoya Journal of Medical Science

Article Title: T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation

doi: 10.18999/nagjms.85.4.779

Figure Lengend Snippet: Cytokine production of clone #17 against primary leukemia cells Fig. 3A: Expression of HLA-DR and HLA-DP on three primary myeloid leukemia cells with or without TNF-α and IFN-γ pre-treatment for 48 h. Fig. 3B: Production of TNF-α and IFN-γ from clone #17 following cocultivation of leukemia cells and positive/negative control cells assessed via intracellular cytokine staining. Study acronyms are explained in Abbreviations to main body.

Article Snippet: Aliquots of T cell clones or TCR-T cells (1 × 10 5 ) were co-incubated with various tumor cells (1 × 10 5 ) forcibly expressing the HLA-DP restriction molecules for 6 h. For blocking studies, the target cells were pretreated with either control mouse IgG (Wako), anti-HLA-DR (BioLegend, USA), -DP (Cosmo Bio), or -DQ (Novus Bio, USA) antibody at a final concentration of 10 μg/mL for 1 h at room temperature, and subsequently cocultured with effector TCR-T cells for 6 h. TNF-α and IFN-γ production in the supernatant was evaluated using conventional sandwich enzyme-linked immunosorbent assays with a pair of relevant antibodies against TNF-α and IFN-γ (all from R&D Systems, USA) and 3,3′,5,5′-tetramethylbenzidine colorimetric reagents (Sigma-Aldrich, USA).

Techniques: Expressing, Negative Control, Staining

Journal: Nagoya Journal of Medical Science

Article Title: T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation

doi: 10.18999/nagjms.85.4.779

Figure Lengend Snippet: Specificity of TCR-T cells with clone #17 TCR Fig. 4A: Structure of retroviral vector. siRNA, shRNA specific to endogenous TCR mRNA. Fig. 4B: Expression of transduced TCR on cultured CD4 + T cells assessed with anti-TCR-Vβ18 antibody. Fig. 4C: TCR-T cells were co-cultured with K86 cells expressing HLA-DPB1*09:01 with the indicated blocking antibodies for 6 h. TNF-α and IFN-γ production was examined via intracellular staining. Fig. 4D: K86 cells were individually transduced with the retroviral vector encoding various HLA-DPA1 and -DPB1 combinations, which are frequently observed in the Japanese population. They were cocultured with TCR-T cells and cytokine production was evaluated as above. Data were processed as in Fig. 2. Study acronyms are explained in Abbreviations to main body.

Article Snippet: Aliquots of T cell clones or TCR-T cells (1 × 10 5 ) were co-incubated with various tumor cells (1 × 10 5 ) forcibly expressing the HLA-DP restriction molecules for 6 h. For blocking studies, the target cells were pretreated with either control mouse IgG (Wako), anti-HLA-DR (BioLegend, USA), -DP (Cosmo Bio), or -DQ (Novus Bio, USA) antibody at a final concentration of 10 μg/mL for 1 h at room temperature, and subsequently cocultured with effector TCR-T cells for 6 h. TNF-α and IFN-γ production in the supernatant was evaluated using conventional sandwich enzyme-linked immunosorbent assays with a pair of relevant antibodies against TNF-α and IFN-γ (all from R&D Systems, USA) and 3,3′,5,5′-tetramethylbenzidine colorimetric reagents (Sigma-Aldrich, USA).

Techniques: Retroviral, Plasmid Preparation, shRNA, Expressing, Cell Culture, Blocking Assay, Staining, Transduction

Journal: Nagoya Journal of Medical Science

Article Title: T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation

doi: 10.18999/nagjms.85.4.779

Figure Lengend Snippet: TCR-T cells can recognize target expressing HLA-DP, even at a low level. K86 cells transduced with the tetracycline-inducible vector encoding HLA-DPA1*02:01-P2A-DPB1*09:01 was manageable with the DOX concentration using the tetracycline-inducible system Fig. 6A: After 48 h of induction with a graded concentration of DOX, some K86 cells were stained for HLA-DP expression. Fig. 6B and 6C: The remaining cells were cocultured with TCR-T cells overnight. Thereafter, TNF-α and IFN-γ production was evaluated using intracellular cytokine staining. Study acronyms are explained in Abbreviations to main body.

Article Snippet: Aliquots of T cell clones or TCR-T cells (1 × 10 5 ) were co-incubated with various tumor cells (1 × 10 5 ) forcibly expressing the HLA-DP restriction molecules for 6 h. For blocking studies, the target cells were pretreated with either control mouse IgG (Wako), anti-HLA-DR (BioLegend, USA), -DP (Cosmo Bio), or -DQ (Novus Bio, USA) antibody at a final concentration of 10 μg/mL for 1 h at room temperature, and subsequently cocultured with effector TCR-T cells for 6 h. TNF-α and IFN-γ production in the supernatant was evaluated using conventional sandwich enzyme-linked immunosorbent assays with a pair of relevant antibodies against TNF-α and IFN-γ (all from R&D Systems, USA) and 3,3′,5,5′-tetramethylbenzidine colorimetric reagents (Sigma-Aldrich, USA).

Techniques: Expressing, Transduction, Plasmid Preparation, Concentration Assay, Staining

Journal: Nagoya Journal of Medical Science

Article Title: T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation

doi: 10.18999/nagjms.85.4.779

Figure Lengend Snippet: Cytokine production of TCR-T cells against primary leukemia cells Fig. 7A: Expression of HLA-DR and HLA-DP on three primary myeloid leukemia cells with or without TNF-α and IFN-γ pre-treatment for 48 h. Fig. 7B: Production of TNF-α and IFN-γ from TCR-T cells following cocultivation of leukemia cells and positive/negative control cells assessed via intracellular cytokine staining. Fig. 7C: Expression of granzyme B and CD107a by TCR-T cells tested as shown above. Study acronyms are explained in Abbreviations to main body.

Article Snippet: Aliquots of T cell clones or TCR-T cells (1 × 10 5 ) were co-incubated with various tumor cells (1 × 10 5 ) forcibly expressing the HLA-DP restriction molecules for 6 h. For blocking studies, the target cells were pretreated with either control mouse IgG (Wako), anti-HLA-DR (BioLegend, USA), -DP (Cosmo Bio), or -DQ (Novus Bio, USA) antibody at a final concentration of 10 μg/mL for 1 h at room temperature, and subsequently cocultured with effector TCR-T cells for 6 h. TNF-α and IFN-γ production in the supernatant was evaluated using conventional sandwich enzyme-linked immunosorbent assays with a pair of relevant antibodies against TNF-α and IFN-γ (all from R&D Systems, USA) and 3,3′,5,5′-tetramethylbenzidine colorimetric reagents (Sigma-Aldrich, USA).

Techniques: Expressing, Negative Control, Staining

Journal: GeroScience

Article Title: Resistance to mild cold stress is greater in both wild-type and long-lived GHR-KO female mice

doi: 10.1007/s11357-022-00706-0

Figure Lengend Snippet: Female mice chronically housed at 16 °C eT had increased levels of FGF21 and energy metabolism. Abbreviations: mWT, male WT mice; fWT, female WT mice; mKO, male GHR-KO mice; fKO, female GHR-KO mice. a mRNA levels of FGF21 in the liver. b Concentration of FGF21 in plasma. c mRNA levels of PGC-1α in interscapular BAT. Oxygen consumption (VO2) in WT mice (d and e) or in GHR-KO mice (f and g). Energy expenditure (EE) in WT mice (h and i) or in GHR-KO mice (j and k). Respiratory Quotient (RQ) in WT mice (l and m) or in GHR-KO mice (n and o). p mRNA levels of UCP1 in interscapular BAT. Data are means ± SEM (n = 8–20). A two-way ANOVA was used to determine p-values for the categorial variables (G = genotype and T = eT) and their interaction (G × T), which are shown for each bar graph. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 based on a two-tailed Student’s t-test

Article Snippet: As per the manufacturer’s protocol, FGF21 was measured with a Mouse FGF21 ELISA Kit (BioVendor R&D, Cat# RD291108200R), triglycerides (TG) with Pointe Scientific Triglycerides Liquid Reagents (Pointe Scientific, Cat# 23–666-410), a non-esterified fatty acids (NEFA) test (Roche, Ref# 11 383 175 001), testosterone with a Mouse Testosterone ELISA Kit (Crystal Chem, Cat# 80,552), corticosterone with a Corticosterone ELISA Kit (Cayman chemical, Cat# 501,320), and adrenocorticotropic hormone (ACTH) with a Adrenocorticotropic hormone Enzyme Immunoassay Kit (RayBiotech, Cat# EIA-ACTH).

Techniques: Concentration Assay, Two Tailed Test