Journal: Cancer Immunology Research

Article Title: Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors

doi: 10.1158/2326-6066.CIR-22-0640

Figure Lengend Snippet: The dFab-CCR heterodimerization format activates a broad range of cytokine receptors families in primary human T cells. A, Schematic of the main cytokine receptors family members and the viral vector design selected for the generation of the dFab_CCR library. The illustration was created with BioRender.com (RRID:SCR_018361). B, Quantitated proliferation of T cells engineered with either IL2 or IL7 dFab_CCRs, expressed as CTV MFI dilution (left), and fold expansion (right) of RQR8 + CD3 + T cells ( n = 4, one-way ANOVA; ns P > 0.05, ****, P ≤ 0.0001). C, Immunoblot analysis of STAT5 phosphorylation, Y694. Nontransduced T cells were cultured in cytokine starvation for 24 hours. dFab_CCR-IL2 or -IL7 were cytokine starved for 96 hours. GAPDH was used as loading control. Data are representative of three independent experiments. D, Immunoblot analysis of in vitro STAT6 phosphorylation, Y641. Nontransduced and dFab_CCR-IL4 T cells were cultured in cytokine starvation for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. E, Immunoblot analysis of in vitro STAT3 phosphorylation, Y705. Nontransduced, IL9, and IL21 dFab_CCR T cells were cultured in cytokine starvation for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. F, IFNγ secreted by IL2, IL12, IL23, and IL27 dFab_CCR transduced T cells cultured for either 24 or 48 hours in cytokine starvation ( n = 3, one-way ANOVA; ns P > 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). G, Immunoblot analysis of in vitro STAT1 and STAT3 phosphorylation. Nontransduced, dFab_CCR-IL12, -IL23, and -IL27 T cells were cytokine starved for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. H, IFNγ secreted by IL2 and IL18 dFab_CCR T cells cultured for either 24 or 48 hours in cytokine starvation ( n = 3, one-way ANOVA; ns P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01). I, Immunoblot analysis of in vitro ERK1/2 phosphorylation, T202 and Y204. Nontransduced, IL1, IL18, and IL33 dFab_CCR T cells were cytokine starved for 24 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. J, Quantitated in vitro proliferation of T cells engineered with either -IL3, -IL5 and -GMCSF dFab_CCRs, expressed as CTV MFI dilution (left), and fold expansion (right) of RQR8 + /CD3 + T cells ( n = 4, one-way ANOVA; ns P > 0.05; *, P ≤ 0.05). K, Immunoblot analysis of in vitro STAT3 and STAT5 phosphorylation. Nontransduced T cells were cytokine starved for 24 hours. IL3, IL5, and GMCSF dFab_CCRs T cells were cytokine starved for 96 hours. GAPDH was used as loading control in the same membrane. Data are representative of three independent experiments. All data are presented as mean ± SEM.

Article Snippet: Isolated splenocytes were activated with 2 μg Concanavalin A (Sigma, C0412–5MG) and 1 ng murine IL7 (Miltenyi Biotec, 130–094–066) per 1.5 × 10 6 cells for 24 hours.

Techniques: Plasmid Preparation, Western Blot, Phospho-proteomics, Cell Culture, Control, In Vitro, Membrane

Journal: Cancer Immunology Research

Article Title: Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors

doi: 10.1158/2326-6066.CIR-22-0640

Figure Lengend Snippet: Coexpression of the dFab_CCRs with GD2 CAR in primary human T cells reveal differential effects on CAR T-cell functionality. A, Schematic of the of the tetra-cistronic γ-RV vectors coexpressing RQR8, 2 nd generation 41bbζ GD2-specific CAR and dFab_CCR. The illustration was created with BioRender.com (RRID:SCR_018361). B, Day 7 quantification of in vitro proliferation of GD2-specific CAR T cells engineered with the library of dFab_CCRs, expressed as fold expansion of RQR8 + CD3 + GD2 CAR T cells ( n = 11, GD2CAR + dFab_CCR-IL7 n = 4, one-way, ANOVA; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001). C, Killing of SupT1-NT (black) or SupT1-GD2 + (red) after 48-hour coculture with CAR-T cells coexpressing the library of dFab_CCRs at a 1:4 effector:target ratio. Data show mean percentage (± SD) of live cells compared with nontransduced (NT) control ( n = 11, GD2CAR + dFab_CCR-IL7 n = 4, two-way ANOVA). D, Extended in vitro persistence of IL2, IL7, IL3, IL5, or GMCSF dFab_CCR coexpressing GD2-specific CAR T cells cultured in cytokine-free complete cell culture media. Live cells were counted weekly using DAPI/acridine orange (AO)I staining. Data show mean 10 6 live cells (± SD; n = 6). E, Killing of SupT1-GD2 + after 48-hour coculture with GD2 CAR-T cells recovered after 7 days of cytokine starvation at 1:2 and 1:4 effector:target ratio. Data shows mean percentage (± SD) of live cells compared with NT control ( n = 4, two-way ANOVA; **, P ≤ 0.01; ****, P ≤ 0.0001). All data are presented as mean ± SEM.

Article Snippet: Isolated splenocytes were activated with 2 μg Concanavalin A (Sigma, C0412–5MG) and 1 ng murine IL7 (Miltenyi Biotec, 130–094–066) per 1.5 × 10 6 cells for 24 hours.

Techniques: In Vitro, Control, Cell Culture, Staining

Journal: Cancer Immunology Research

Article Title: Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors

doi: 10.1158/2326-6066.CIR-22-0640

Figure Lengend Snippet: dFab_CCR coexpression sustains GD2-specific human CAR T-cell function after chronic antigen exposure. A, Chronic antigen stimulation experimental design. The first coculture was initiated with 1 × 10 6 GD2-specific CAR T cells together with 0.1 × 10 6 SKOV3 GD2 + cells for 7 days, in the absence of exogenous cytokines. For the second and third cocultures, T cells were harvested from the previous coculture and then replated in new culture medium with fresh tumor cells at the same 10:1 E:T ratio. The illustration was created with BioRender.com (RRID:SCR_018361). B, Cumulative expansion of selected GD2-specific CAR T cells from A after three rounds of coculture ( n = 11, n = 4 GD2CAR + dFab_CCR-IL7, one-way ANOVA; ns P > 0.05; ***, P ≤ 0.001). C, Quantitated in vitro proliferation of GD2-specific CAR T cells recovered after three rounds of coculture ( B ) and cultured for 7 days in cytokine starvation condition. Proliferation expressed as fold expansion of RQR8 + CD3 + GD2-specific CAR T cells ( n = 11; GD2CAR + dFab_CCR-IL7 n = 4; one-way ANOVA; **, P ≤ 0.01; ****, P ≤ 0.0001). All data are presented as mean ± SEM. D, Killing of SupT1 nontransduced (NT; black) and GD2 + (red) after 48-hour coculture with GD2-specific CAR T cells recovered after three rounds of coculture ( B ) at 1:4 E:T ratio. Data show mean percentage (± SD) of live cells compared with NT control ( n = 11; GD2CAR + dFab_CCR-IL7 n = 4, GD2CAR + dFab_CCR-IL7; two-way ANOVA; **, P ≤ 0.01). E, Exhaustion phenotype of CAR T cells after three rounds of chronic antigen exposure. Exhaustion evaluated by the expression of TIM3, LAG3, PD-1, or KLRG1 in either CD4 + (left) or CD8 + (right) T cells. Stacked bars show percentage of cells expressing 0, 1, 2, 3, or 4 markers per individual donors ( n = 11; GD2CAR + dFab_CCR-IL7 n = 4). F, Memory phenotype of CAR T cells after three rounds of chronic antigen exposure. Memory phenotype evaluated by the expression of CD45RA and/or CCR7 in either CD4 + (left) or CD8 + (right) T cells. Memory phenotype defined as: naïve T cells (T N , CD45RA + , CCR7 + ), central memory (T CM ) T cells (CD45RA − , CCR7 + ), effector memory (T EM ) T cells (CD45RA − , CCR7), and terminally differentiated (T EMRA ) T cells (CD45RA + , CCR7 − ). Stacked bars show percentage of cells in each population markers per individual donors ( n = 11; GD2CAR + dFab_CCR-IL7 n = 4). All data are presented as mean ± SEM.

Article Snippet: Isolated splenocytes were activated with 2 μg Concanavalin A (Sigma, C0412–5MG) and 1 ng murine IL7 (Miltenyi Biotec, 130–094–066) per 1.5 × 10 6 cells for 24 hours.

Techniques: Cell Function Assay, In Vitro, Cell Culture, Control, Expressing

Journal: Cancer Immunology Research

Article Title: Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors

doi: 10.1158/2326-6066.CIR-22-0640

Figure Lengend Snippet: IL18 and GMCSF dFab_CCRs deliver functionally and transcriptomically different output to GD2-specific human CAR T cells. A, PCA analysis of each individual dFab_CCR transcriptome in activated CAR T cells (left) or nonactivated (resting) CAR T cells (right). B, Volcano plot representing the comparison of dFab_CCR-IL18 CAR T cells against either dFab_CCR-IL7, dFab_CCR-GMCSF, or CAR alone in activated CAR T cells. C, Volcano plot representing the comparison of dFab_CCR-IL18 CAR T cells against either dFab_CCR-IL7 or dFab_CCR-GMCSF or CAR alone in nonactivated CAR T cells. D and E, The oxygen consumption rates (OCR) and the individual metabolic parameter were analyzed either after 24 hours of antigen starvation (nonactivated CAR T cells, D ) or after 24 hours of activation (activated CAR T cells, F ; n = 5, one-way ANOVA; ***, P ≤ 0.001). All data are presented as mean ± SEM.

Article Snippet: Isolated splenocytes were activated with 2 μg Concanavalin A (Sigma, C0412–5MG) and 1 ng murine IL7 (Miltenyi Biotec, 130–094–066) per 1.5 × 10 6 cells for 24 hours.

Techniques: Comparison, Activation Assay

Journal: Cancer Immunology Research

Article Title: Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma

doi: 10.1158/2326-6066.cir-21-0778

Figure Lengend Snippet: Figure 1. Soluble factors mediate FRC activation and mitochondrial imbalance. A, Experimental scheme used to investigate tumor-draining factors in vivo. B, Draining lymph nodes were harvested from female C57BL/6 mice that had received 10 daily subcutaneous doses of PBS, RPMI medium, or B16.F10 TCM into the shoulder. Flow cytometric analysis allowed reporting of the total number of lymph node cells (left), flow cytometry gating scheme, FRCs (right), and FRC Pdpn histogram (furthest right). n ¼ 4 to 9 animals per group. C, qRT-PCR analysis of Pdpn (left), Thy1 (middle), and Il7 (right) in FRCs cultured in vitro and treated for 4 days with CCM, B16.F10 TCM, or 4T1 TCM. n ¼ 3 to 10 independent experiments. D, IL7 protein quantification in supernatants of FRCs cultured in vitro and treated with CCM or B16.F10 TCM, assessed by ELISA. n ¼ 4 independent experiments. Flow cytometric quantification of Pdpn protein (E) and Thy1 protein (F) expression for FRCs cultured in vitro and treated with CCM or 4T1 TCM for 4 days. n ¼ 3 independent experiments. G, Flow cytometric analysis of TMRM incorporation by FRCs treated for 4 days with CCM or B16 TCM. n ¼ 4 independent experiments performed in triplicate. H, OCR of FRCs treated with CCM or B16.F10 TCM at baseline and in response to oligomycin, FCCP, and rotenone plus antimycin A. One representative of three experiments with five replicates. I, Baseline OCR from CCM or B16.F10 TCM-treated FRCs. n ¼ 3 experiments with 5 replicates. J, Representative confocal imaging of live cells treated with CCM or B16.F10 TCM. Green, Mitochondria (Mitotracker green); blue, nuclei (Hoechst). Scale bar: 7 mm. Data are mean with SEM. Significance (, P < 0.05; , P < 0.01; , P < 0.001; and , P < 0.0001) was determined by unpaired two-tailed t test or one-way ANOVA with Tukey post hoc (B). s.c., subcutaneous; B16, B16.F10; max, maximum; NS, not significant.

Article Snippet: Protein quantification per ELISA was performed according to the R&D System Quantikine IL7 ELISA Kit (R&D Systems, #M70000,) product manual.

Techniques: Activation Assay, In Vivo, Cytometry, Quantitative RT-PCR, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Imaging, Two Tailed Test

Journal: Cancer Science

Article Title: Newcastle disease virus co‐expressing interleukin 7 and interleukin 15 modified tumor cells as a vaccine for cancer immunotherapy

doi: 10.1111/cas.13468

Figure Lengend Snippet: Construction of recombinant virus. A, Schematic representation of recombinant LX construct. The coding sequence interleukin 15 ( IL 15) and IL 7 was inserted into the sequence between P and M genes of LX genome. B, Multistep growth curve for LX and LX / IL (15+7). The virus titers were determined in triplicate by TCID 50 in DF ‐1 cells at 0, 8, 16, 24, 32 and 40 h post‐infection. The data shown are representative of the 3 experiments

Article Snippet: The expression s of IL7 and IL15 in the supernatants were measured using ELISA as described by mouse IL7 DuoSet ELISA and IL15 DuoSet ELISA (R&D Systems, Minneapolis, MN USA), respectively.

Techniques: Recombinant, Virus, Construct, Sequencing, Infection

Journal: Cancer Science

Article Title: Newcastle disease virus co‐expressing interleukin 7 and interleukin 15 modified tumor cells as a vaccine for cancer immunotherapy

doi: 10.1111/cas.13468

Figure Lengend Snippet: Expression of the interleukin 15 ( IL 15) and IL 7 in tumor cells infected with LX / IL (15+7). A, Irradiated B16 were incubated with LX / IL (15+7) or LX / RFP (100 HAU virus per 10 6 cells). After incubation for 24, 48, 72 and 96 h, the supernatants were collected and analyzed for the production of IL 15 and IL 7 (A) or IL 15‐2A‐ IL 7 (B) by ELISA

Article Snippet: The expression s of IL7 and IL15 in the supernatants were measured using ELISA as described by mouse IL7 DuoSet ELISA and IL15 DuoSet ELISA (R&D Systems, Minneapolis, MN USA), respectively.

Techniques: Expressing, Infection, Irradiation, Incubation, Virus, Enzyme-linked Immunosorbent Assay