Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 as a pH-Sensing regulator in T Cells and generation of GPCR68 fl/fl CD4 Cre mice. (A) Schematic diagram of the effect of pH on T cell GPCR68 as well as tumor. (B) Naïve CD4 + T cells were isolated and activated using anti-CD3 and anti-CD28 using the culture media with varying pH. RT-qPCR was performed to determine the expression of GPCR68 at various pH. (C) Naïve CD4 + T cells were activated with anti-CD3 and anti-CD28 under different pH conditions, and GPCR68 protein expression was assessed by Western blot analysis. (D) To generate conditional knockout (CKO) of GPCR68 in T cells, GPCR68 fl/fl mice were crossed with CD4 Cre mice and generated GPCR68 fl/fl CD4 Cre (CKO). (E) Flow cytometry was used to determine the population of CD4 and CD8 cells in the lymph nodes (LN), thymus (THY), and spleen (SP) at the basal level in CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (F) Flow cytometry was used to determine the population of Foxp3+ Treg cells in the lymph nodes, thymus, and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (G-H) The population of F4/80+, CD11c+ (G), and B220+ (H) cells was determined in the lymph nodes and spleen at the basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. (I-J) Flow cytometry was used to evaluate the CD4 + or CD8 + T cells for the determination of intracellular cytokines IFN-γ+ (I), or TNF-α+ (J) from the spleen and lymph nodes at basal level in the CD4 Cre or GPCR68 fl/fl CD4 Cre mice. Student t-test was performed for comparison between the two groups. Data are mean ± SEM (n = 5), ∗ p < 0.05.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Western Blot, Knock-Out, Generated, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: GPCR68 fl/fl CD4 Cre mice exhibit improved anti-tumor mmune responses. (A-C) Naïve CD4 + T cells were isolated from CD4 Cre or GPCR68 fl/fl CD4 Cre mice and activated using anti-CD3 and anti-CD28 using the culture media under physiologic neutral pH (7.4) or varying pH 6.0, 6.5, or 7.8. Flow cytometry plots showing the expression of IFN-γ and IL-2 in CD4 + T cells from CD4 Cre and GPCR68 fl/fl CD4 Cre mice. Each panel represents the frequency of IFN-γ + and IL-2 + cells. (B) Bar graph summarizing the percentage of IFN-γ + CD4 + T cells at each pH level for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (C) Bar graph showing the percentage of IL-2 + CD4 + T cells at each pH for CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (D) Experimental timeline depicting tumor induction and treatment protocol in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (E) Tumor growth curves in CD4 Cre and GPCR68 fl/fl CD4 Cre mice. (F) Tumor weight in CD4 Cre versus GPCR68 fl/fl CD4 Cre mice at the time of harvesting on day 21. (G) Representative images of excised tumors at day 21. (H) Flow cytometric analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (I) Flow cytometric analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns = not significant.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Isolation, Flow Cytometry, Expressing, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Anti-tumor effects of borate bioactive glass (BOLT) in B16 tumor. (A) Schematic illustration depicting the induction of B16 melanoma tumors, followed by treatment with BOLT at various time points, and tumor harvesting for subsequent analysis. (B) Tumor growth curves showing tumor volume in Control and BOLT-treated B16 melanoma tumors in mice. (C) Tumor weight at the time of harvesting in the BOLT-treated group compared to the Control. (D) Representative images of excised tumors from Control and BOLT-treated mice. (E) In vivo imaging of tumor-bearing mice in both the Control and BOLT-treated groups. (F) Flow cytometry analysis showing IFN-γ production in CD4 + and CD8 + T cells following BOLT treatment compared to Control. (G) Flow cytometry analysis demonstrated TNF-α production in CD4 + and CD8 + T cells in the BOLT-treated group, with a significant increase observed in CD8 + T cells. Student t-test was performed for comparison between the two groups. Two-way ANOVA was used for multiple comparisons. Data represent the mean ± SEM (n = 5). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Control, In Vivo Imaging, Flow Cytometry, Comparison

Journal: Bioactive Materials

Article Title: pH-neutralization strategy to suppress GPCR68 spatiotemporally activates T cells and enhances anti-tumor immunity

doi: 10.1016/j.bioactmat.2026.02.039

Figure Lengend Snippet: Combinational treatment of BOLT and anti-CTLA-4 blockade enhances anti-tumor immune response in B16 melanoma. (A) C57BL/6 mice were subcutaneously injected with 1 × 10 5 B16 melanoma cells on day 0 to induce tumors. On day 7, mice were randomly divided into groups and treated with either BOLT alone (intratumoral injection administered on alternate days starting from day 7), anti-CTLA-4 (intraperitoneal injection administered on days 9, 11, 13, and 15), or a combination of both treatments. PBS was used as a vehicle Control, while IgG was used as anti-CTLA-4 Control. Tumor growth was monitored throughout the treatment period, and tumors were harvested for analysis on day 21. (B-C) Tumor growth curves and area under the curve (AUC) analysis for WT mice treated with BOLT, with or without anti-CTLA-4 antibody, following subcutaneous injection of B16 melanoma cells. Tumor growth was monitored, and analysis was conducted on day 21. (D) Representative images of excised tumors at day 21, showed reduced tumor size in combination-treated mice. (E, F) Flow cytometry analysis of IFN-γ production by tumor-infiltrating CD4 + and CD8 + T cells. (G, H) Flow cytometry analysis of TNF-α production by tumor-infiltrating CD4 + and CD8 + T cells. Two-way ANOVA was used for multiple comparisons. Data are mean ± SEM (n = 5), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Naïve T cells were purified from lymph nodes as well as spleens of C57/BL6, CD4 Cre , GPCR68 fl/fl CD4 Cre (CKO) mice by using the mouse naïve CD4 + T Cell Isolation Kit (#130-104-453; Miltenyi Biotec) or naïve CD8 + T Cell Isolation Kit (#130-096-543; Miltenyi Biotec) for negative selection.

Techniques: Injection, Control, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: CpG 1018 augments mRNA vaccine-induced anti-tumor immunity by potentiating CD8+ T cell responses

doi: 10.1016/j.omton.2026.201230

Figure Lengend Snippet: CpG 1018 enhances OVA mRNA-induced anti-tumor immunity in preventive B16F10-OVA models (A) Schematic illustration of experimental design in preventive B16F10-OVA tumor models. (B–E) Cellular immune responses were analyzed 1 week after boost. PBMCs were stimulated with OVA overnight followed by intracellular cytokine staining and flow cytometry analysis. Cells were first gated based on SSC and FSC and then CD4 and CD8 ( A). CD4 + and CD8 + T cells were then gated based on GrB and IFNγ ( A). Percentages of GrB + and IFNγ + cells in CD8 + T cells were shown in (B) and (D), respectively. MFI of GrB and IFNγ in GrB + CD8 + and IFNγ + CD8 + T cells was shown in (C) and (E), respectively. (F–G) Mice were challenged with 10 6 OVA-expressing B16F10-OVA melanoma cells 3 weeks after boost. Tumor growth rate was shown in (F). Mice with measurable tumor growth were euthanized 2 weeks after challenge. Tumors were dissected and weighed. Tumor weights of all groups were shown in (G). Tumor weights of mRNA vaccine groups were shown in (H). Tumor images were shown in (I). Percentages of tumor-free mice were shown in (J). One-way ANOVA with Fisher’s LSD test was used to compare differences between groups in (B–E), and (G–H). Two-way ANOVA with Dunnett’s multiple comparisons test was used to compare differences between groups in (F). Logrank (Mantel-Cox) test was used to compare tumor-free percentage between groups (vs. OVA mRNA) in (J). n = 5; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Experiments were repeated once with similar results.

Article Snippet: Cells were then subjected to magnetic beads-based negative purification of naive CD8 + T cells with a commercial kit (130–096–543, Miltenyi Biotec).

Techniques: Staining, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: CpG 1018 augments mRNA vaccine-induced anti-tumor immunity by potentiating CD8+ T cell responses

doi: 10.1016/j.omton.2026.201230

Figure Lengend Snippet: CpG 1018 enhances OVA mRNA-induced anti-tumor immunity in therapeutic B16F10-OVA models (A) Schematic illustration of experimental design in therapeutic B16F10-OVA melanoma models. (B–D) Cellular immune responses were analyzed 1 week after boosting. PBMCs were stimulated with OVA overnight followed by intracellular cytokine staining and flow cytometry analysis. Cells were first gated based on SSC and FSC and then CD4 and CD8 ( A). CD4 + and CD8 + T cells were then gated based on GrB, IFNγ, and TNFα ( A). Representative dot plots with gated GrB + cells in CD8 + T cells were shown in (B). Percentages of GrB + (left), IFNγ + (middle), and TNFα + cells (right) in CD8 + T cells were shown in (C). Percentages of GrB + (left), IFNγ + (middle), and TNFα + cells (right) in CD4 + T cells were shown in (D). (E) Overall tumor growth rate in different groups. (F) Tumor growth rate of individual mice in each group. (G) Percentages of tumor-infiltrating CD8 + T cells in CD3 + T cells. (H) Percentages of tumor-infiltrating CD4+ T cells in CD3+ T cells. Gating strategies (G and H) were shown in B. (I) Ratios of tumor-infiltrating CD8 + to CD4 + T cells. One-way ANOVA with Fisher’s LSD test was used to compare differences between groups in (C and D), and (G–I). Two-way ANOVA with Dunnett’s multiple comparisons test was used to compare differences between groups in (E). n = 5; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Experiments were repeated twice with similar results.

Article Snippet: Cells were then subjected to magnetic beads-based negative purification of naive CD8 + T cells with a commercial kit (130–096–543, Miltenyi Biotec).

Techniques: Staining, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: CpG 1018 augments mRNA vaccine-induced anti-tumor immunity by potentiating CD8+ T cell responses

doi: 10.1016/j.omton.2026.201230

Figure Lengend Snippet: Crucial roles of CD8+ T cells in CpG 1018-adjuvanted OVA mRNA vaccine in therapeutic B16F10-OVA models (A) Schematic illustration of experimental design. (B) Tumor growth was monitored and compared among groups. (C) Tumors were harvested at the endpoint and imaged. (D) Tumor weight was compared among groups. n = 5. One-way ANOVA with Fisher’s LSD test was used to compare differences between groups in (D). Two-way ANOVA with Dunnett’s multiple comparisons test was used to compare differences between groups in (B). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Experiments were repeated once with similar results.

Article Snippet: Cells were then subjected to magnetic beads-based negative purification of naive CD8 + T cells with a commercial kit (130–096–543, Miltenyi Biotec).

Techniques:

Journal: Molecular Therapy Oncology

Article Title: CpG 1018 augments mRNA vaccine-induced anti-tumor immunity by potentiating CD8+ T cell responses

doi: 10.1016/j.omton.2026.201230

Figure Lengend Snippet: CpG 1018 increases NeoAg mRNA-induced anti-tumor immunity in therapeutic B16F10 model (A) Illustration of NeoAg mRNA design. (B) Illustration of experimental design. (C–G) PBMCs were collected 6 days after boost and stimulated with M27 and M30 peptide mixture overnight. Cells were collected 5 h later after addition of Brefeldin A and subjected to immunostaining and flow cytometry analysis. Cells were first gated based on FSC and SSC and then CD4 and CD8 expression. Cells were then gated based on GrB expression. Representative dot plots showing GrB + cells in CD8 + T cells were shown in (C). Percentages of GrB + cells in CD8 + T cells were shown in (D). MFI of GrB in GrB + CD8 + T cells was shown in (E). Percentages of IFNγ + and perforin + cells in CD8 + T cells were shown in (F) and (G), respectively. Gating strategies (C–G) were shown in C. (H) Summary of tumor growth rate. (I) Tumor growth rate of individual mice in each group. One-way ANOVA with Fisher’s LSD test was used to compare differences between groups in (D–G). Two-way ANOVA with Fisher’s LSD test was used to compare differences between groups in (H). n = 5. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. Experiments were repeated once with similar results.

Article Snippet: Cells were then subjected to magnetic beads-based negative purification of naive CD8 + T cells with a commercial kit (130–096–543, Miltenyi Biotec).

Techniques: Immunostaining, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: CpG 1018 augments mRNA vaccine-induced anti-tumor immunity by potentiating CD8+ T cell responses

doi: 10.1016/j.omton.2026.201230

Figure Lengend Snippet: CpG 1018 increases cytokine-secreting OT-I T cells following adoptive transfer OT-I T cells were magnetically purified from OT-I mice, stained with CFSE, and adoptively transferred to naive C57BL/6 mice. After 24 h, recipient mice were i.m. immunized with OVA mRNA alone or OVA mRNA/CpG 1018. After 4 days, draining popliteal and inguinal LNs were collected and stimulated overnight with OVA followed by immunostaining and flow cytometry analysis. Cells were first gated based on FSC and SSC and then CD4 and CD8. CD8+ T cells were then gated based on CFSE. CFSE+ cells were further analyzed for IFNγ and TNFα expression. Gating strategies were shown in H. (A) Illustration of experimental procedures. (B) Representative dot plots showing IFNγ and TNFα single-positive or double-positive cells. (C) Percentages of IFNγ and TNFα single-positive or double-positive cells in CFSE+ cells in popliteal LNs. (D) Percentages of IFNγ and TNFα single-positive or double-positive cells in CFSE+ cells in inguinal LNs. One-tailed Student’s t test was used to compare differences between groups in (B) and (C). n = 5; ∗ p < 0.05; ∗∗ p < 0.01. Experiments were repeated once with similar results.

Article Snippet: Cells were then subjected to magnetic beads-based negative purification of naive CD8 + T cells with a commercial kit (130–096–543, Miltenyi Biotec).

Techniques: Adoptive Transfer Assay, Purification, Staining, Immunostaining, Flow Cytometry, Expressing, One-tailed Test

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A – C ) Gli1 KO mice have phenotypically normal peripheral T cells. Splenocytes were isolated from Gli1 WT, Gli1 HET, and Gli1 KO mice. ( A ) Splenocytes were stained for flow cytometry analysis of T cells (CD3 + , top panel), CD4 + and CD8 + T cells (middle panel), and naive (CD62L + , CD44 - ), central memory (CM, CD62L + , CD44 + ), and effector memory (EM, CD62L - , CD44 - ) subsets (bottom two panels). Representative flow cytometry plots for WT, HET, and KO mice are shown. ( B , C ) Quantitative analysis of percentages and cell numbers from stainings in ( A ). n = 12–19 biological replicates from 9 independent experiments. Every circle (WT), square (HET), and triangle (KO) represents a single mouse. Error bars indicate SD. Statistical significance was assessed using a one-way ANOVA with Dunnett’s multiple comparison test. ( D – F ) Tumour-infiltrating Gli1 WT and Gli1 KO CD8 + lymphocytes are phenotypically similar. Tumour-infiltrating CD8 + lymphocytes (CD8 + TILs) were isolated from tumours on day 27 (experimental setup shown in Fig. ). ( D ) Representative gating strategy. In brief, cells were gated on live, singlet CD45 + CD8 + cells for further analysis of CD44, CD62L, CD27, TIM3 and PD1 expression. Gating was performed based on FMO controls. ( E ) Absolute numbers of CD45 + and CD8 + cells within the tumour. ( F ) Mean fluorescence intensities (MFIs) of CD62L, CD44, TIM3, PD1 and CD27 on CD8 + cells 27 ( Gli1 WT n = 8 , Gli1 KO n = 6 biological replicates pooled from 2 independent experiments). Bars represent the mean; error bars indicate SD. Statistical significance was assessed using an unpaired two-tailed Student’s t test ( E —left panel), a Mann-Whitney two-tailed test ( E —right panel) or an unpaired two-tailed Student’s t test ( F ). ( G ) Viability of human CTLs was determined by flow cytometry after treatment with 5 mM GANT61 for 8–18 h on day 12–14 post stimulation. n = 6 biological replicates from 2 independent experiments. Symbols indicate individual human donors. Bars represent the mean; error bars shown are SD. Statistical significance was assessed using an paired two-tailed Student’s t test. ns = not significant.

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Isolation, Staining, Flow Cytometry, Comparison, Expressing, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A ) CTLs were generated from Gli1 WT, Gli1 HET and Gli1 KO mice and restimulated on day 10 with plate-bound anti-CD3ε antibodies for 24 h. Cells were lysed and lysates blotted for protein expression of Gli1 and Tubulin. n = 2 biological replicates from 2 independent experiments. Molecular masses are shown in kilodaltons. ( B , C ) On day 7 post stimulation CTLs from Gli1 WT and Gli1 KO OT-I mice were co-cultured with ovalbumin-pulsed EL4 target cells for 4 h at the indicated effector to target ratios and subjected to an LDH cytotoxicity assay. ( B ) Representative killing assay. Data points represent mean of three technical replicates; error bars indicate SD. ( C ) Quantification of n = 6 biological replicates from 6 independent experiments normalised to killing of Gli1 WT cells at a 10:1 Effector:Target ratio. Individual biological replicates are shown. p -values were calculated using two-way ANOVA (10:1 p = 0.0021, 5:1 p = 0.0071, 2.5:1 p = 0.0362). ( D ) Experimental setup for in vivo tumour model: Rag2 KO mice were injected 0.5 × 10 6 MC38-OvaT4 cells sc. On day 13 post tumour cell injection, mice received 4 × 10 6 OT-I CTLs, either wild type or knockout for Gli1 , via tail vein injection. ( E ) Tumour size of MC38-OvaT4 tumours as determined by caliper measurements ( Gli1 WT n = 8 , Gli1 KO n = 6 biological replicates pooled from 2 independent experiments). Arrow indicates the timepoint of Gli1 WT and Gli1 KO OT-I CTL injection. Data points represent the mean; error bars indicate SEM. P -values were calculated using two-way ANOVA with Tukey’s multiple comparisons test, pooled from two independent experiments (day 26 p = 0.0311, day 27 p = 0.0070, no significant differences were observed at other timepoints). ( F ) Tumour size in grams on day 27 ( Gli1 WT n = 8 , Gli1 KO n = 6 biological replicates pooled from 2 independent experiments). Bars represent the mean; error bars indicate SD. p -values were calculated using an unpaired two-tailed Student’s t test ( p = 0.0337). ( G ) Flow cytometric analysis of percentages of differentiation and exhaustion/activation markers on tumour-infiltrating CD8 + T cells ( Gli1 WT n = 8 , Gli1 KO n = 6 biological replicates pooled from 2 independent experiments ) . Bars represent the mean; error bars indicate SD. Statistical significance was assessed using multiple unpaired Welch’s tests with Holm-Sidak multiple comparisons test (left and middle panel) or an unpaired two-tailed Student’s t test (right panel). ( H ) Basic schematic overview of the Hedgehog pathway. GANT61 (red) is a small-molecule Gli inhibitor. ( I , J ) On day 12–21 post stimulation CTLs from healthy human donors were co-cultured with anti-CD3ε-pulsed P815 target cells for 3 h in the presence or absence of GANT61 at the indicated effector to target ratios and subjected to an LDH cytotoxicity assay. ( I ) Representative killing assay. Data points represent mean of three technical replicates; error bars indicate SD. ( J ) Quantification of n = 5 biological replicates from 3 independent experiments (except for 0.31:1 effector:target ratio, n = 3 biological replicates from 2 independent experiments) normalised to killing of carrier-treated cells at a 10:1 effector:target ratio. Individual biological replicates are shown. p -values were calculated using two-way ANOVA with Tukey’s multiple comparisons test (10:1 p = 0.0027, 5:1 p = 0.0284 & p = 0.0016, 2.5:1 p = 0.0129, 1.25:1 p = 0.0375, 0.625:1 p = 0.0404). * p < 0.05, ** p < 0.01, and *** p < 0.001. ns = not significant. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Generated, Expressing, Cell Culture, LDH Cytotoxicity Assay, In Vivo, Injection, Knock-Out, Two Tailed Test, Activation Assay

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A ) Schematic overview of signalling downstream of the TCR culminating in the activation of transcription factors NFAT, NF-κB and AP-1. Small molecule agonists (green) as well as antagonists and dominant negative constructs (red) of the branches of TCR signalling are shown. ( B – D ) CD8 + T cells were isolated from spleens and inguinal lymph nodes of Rag2 KO OT-I mice. ( B ) Naive CD8 + T cells or CTLs were treated with indicated doses of PMA and/or Ionomycin for 3 h (naive, top panel) (CTL, bottom panel) before being subjected to qRT-PCR analysis. For double treatment with PMA and Ionomycin, 50 ng/ml PMA and 1 μg/ml Ionomycin was used. n = 3 biological replicates from 3 independent experiments. Data is normalised to CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Bars represent the mean; error bars indicate SD. p values were calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparison test comparing each condition to PMA/Iono (Naive—PMA 1 p = 0.0092, PMA 5 p = 0.0706, PMA 10 p = 0.0108, PMA 50 p = 0.0055, Iono 10 p = 0.0024, Iono 50 p = 0.0039, Iono 100 p = 0.0022, Iono 1000 p = 0.0030, CTL—PMA 1 p = 0.0095, PMA 5 p = 0.0017 PMA 10 p = 0.0299, PMA 50 p = 0.2308, Iono 10 p = 0.0006, Iono 50 p = 0.0003, Iono 100 p = 0.0002, Iono 1000 p = 0.0102). ( C ) CTLs were nucleofected with GFP, GFP-VIVIT or Dominant Negative IκBα (DN IκBα) on day 6. Cells were restimulated with plate-bound anti-CD3ε antibodies on day 7 for 3 h for qRT-PCR analysis. n = 3 biological replicates from 3 independent experiments. Data is normalised to CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Bars represent the mean; error bars indicate SD. Statistical significance was assessed using a two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Naive CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 antibodies (left) and CTLs were restimulated with plate-bound anti-CD3ε (right) for 15 h in the presence of 10 μM U0126 or carrier control before RNA was extracted for qRT-PCR analysis. n = 4 (left panel) or n = 3 (right panel) biological replicates from 2 and 3 independent experiments, respectively. Data is normalised to Tbp as a reference gene. Similar results were obtained when CD3ε was used as a reference gene. Bars represent the mean; error bars indicate SD. p values were calculated using an unpaired two-tailed Student’s t test (Naive p = 0.1335, CTL p = 0.0389). Symbols indicate biological replicates. * p < 0.05, ** p < 0.01, and *** p < 0.001. ns = not significant. ( E ) Analysis of ChIP-Seq data from ( Kurachi et al) showing binding of AP-1 family members (c-Jun, JunB, JunD) at the Gli1 promoter. Binding at the Ifng promoter serves as a positive control and no binding at the Gli2 and Gli3 promoters serve as negative controls. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Activation Assay, Dominant Negative Mutation, Construct, Isolation, Quantitative RT-PCR, Comparison, Control, Two Tailed Test, ChIP-sequencing, Binding Assay, Positive Control

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A – C ) Validation of TCR signalling inhibitors. ( A ) CTLs were nucleofected with GFP or GFP-VIVIT on day 6. Cells were restimulated with PMA/Ionomycin on day 7 prior to intracellular flow cytometric staining for IL-2 and IFN-γ. FACS plots are gated on GFP + cells. ( B ) CTLs were nucleofected with GFP or Dominant Negative I κ Bα (DN I κ Bα) on day 6. Cells were restimulated with anti-CD3 on day 7 for 1 h prior to ImageStream analysis. ( C ) Naive CD8 + T cells were isolated from spleens and peripheral lymph nodes of Rag2 KO OT-I mice and stimulated with cross-linked soluble anti-CD3/CD28 in the presence of 10 µM U0126 or carrier control prior to analysis by flow cytometry of phosphorylated Erk1/2 (pErk1/2). ( A – C ) One representative experiment of n = 2–3 biological replicates from 2–3 independent experiments is shown. ( D , E ) Cell viability of mouse CD8 + T cells upon Ca 2+ blockade. ( D ) Murine CTLs were restimulated with plate-bound anti-CD3ε antibodies for 3 h in the presence of indicated doses of the cell permeable Ca 2+ chelator BAPTA-AM or carrier control before viability was assessed via the Beckman Coulter Vi-Cell XR. n = 4–5 biological replicates from 4–5 independent experiments. Symbols indicate biological replicates. Bars represent the mean; error bars shown are SD. Statistical significance was assessed using a one-way ANOVA with Dunnett’s multiple comparison test comparing each condition to DMSO (0.025%). ( E ) Murine CTLs were restimulated with plate-bound anti-CD3e in the presence of the indicated Ca 2+ channel inhibitors or carrier control for 15 h before flow cytometric analysis for cell viability. n = 3 biological replicates from 3 independent experiments. Symbols indicate biological replicates. Bars represent the mean; error bars indicate SD. Statistical significance was assessed using a one-way ANOVA with Dunnett’s multiple comparison test comparing each condition to carrier. ( F – I ) Characterization of dLckCre + Ihh KO, GzmBER T2 Cre + Smo KO mice and retroviral transduction constructs. ( F ) Splenocytes were isolated from dLckCre + Ihh +/fl ( Ihh HET) and dLckCre + Ihh fl/fl ( Ihh KO) mice and subjected to flow cytometric phenotypic analysis. Representative FACS plots are shown (left 2 panels). Quantification of relative percentages, cell numbers and steady-state phenotype: naive (CD62L + , CD44 - ), central memory (CM, CD62L + , CD44 + ), and effector memory (EM, CD62L - , CD44 - ) shown on the right two panels. Every square represents one HET, and every triangle one individual KO mouse. n = 5–6 biological replicates from 5–6 independent experiments. Error bars indicate SD. Statistical significance was assessed using multiple unpaired Welch’s tests with Holm-Sidak multiple comparisons test. ( G ) Splenocytes were isolated from GzmBER T2 Cre + Smo +/+ ( Smo WT) and GzmBER T2 Cre + Smo fl/fl ( Smo KO) mice and subjected to flow cytometric phenotypic analysis. Representative FACS plots are shown (left). Quantification of relative percentages and steady-state memory phenotype shown (middle). Every square represents one WT, and every triangle one individual KO mouse. CTLs were generated from these mice and restimulated at day 8/9 for qRT-PCR analysis of Smo (right). n = 4–7 biological replicates from 4 independent experiments. Error bars indicate SD. p values were calculated using multiple unpaired Welch’s tests with Holm-Sidak multiple comparisons (0 h p = 0.0296, 3 h p = 0.0222). (H) CD8 + T cells were retrovirally transduced with constructs encoding empty vector (EV), SmoM2 or Ihh, respectively. Representative flow cytometry plots of sorted, transduced (Thy1.1 + ) cell populations shown (left panel). Sorted, transduced CTLs were restimulated at day 8/9 for qRT-PCR analysis of Smo (middle panel) or Ihh (right panel), respectively. n = 3 biological replicates from 3 independent experiments. p values were calculated using a two-way ANOVA with Sidak’s multiple comparison test (Smo 0 h p = 0.0073, Smo 4 h p < 0.0001, Smo 15 h p < 0.0001, Ihh 0 h p = 0.084, Ihh 4 h p = 0.0098, Ihh 15 h p = 0.0007). ( I ) CD8 + T cells were retrovirally transduced with pMig constructs encoding empty vector (EV), SmoM2 or Ihh, respectively. Sorted, transduced CTLs were restimulated on day 8/9 for qRT-PCR analysis. n = 3 biological replicates from 3 independent experiments. Statistical significance was assessed using a two-way ANOVA with Tukey’s multiple comparison test. ( H , I ) Data is normalised to CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Symbols indicate biological replicates. Bars represent the mean; error bars indicate SD. * p < 0.05, ** p < 0.01, **** p < 0.0001. ns = not significant.

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Staining, Dominant Negative Mutation, Isolation, Control, Flow Cytometry, Comparison, Retroviral, Transduction, Construct, Generated, Quantitative RT-PCR, Plasmid Preparation

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A ) Naive CD8 + T cells (left panel) or CTLs (middle panel) from RAG1 KO OT-I mice were stimulated with plate-bound anti-CD3/CD28 or anti-CD3ε antibodies alone for 3 h in the presence of indicated doses of the cell permeable Ca 2+ chelator BAPTA-AM or carrier control. RNA was extracted for qRT-PCR analysis. Data is normalised using CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Immunoblot analysis and quantification of Gli1 and tubulin of CTLs left unstimulated or restimulated with plate-bound anti-CD3ε for 3 h in the presence of BAPTA-AM or carrier control (right panel). Molecular masses are shown in kilodaltons. n = 3 (left panel) or n = 4 (right panel) biological replicates from 3 and 4 independent experiments, respectively. Bars represent the mean; error bars indicate SD. p values were calculated using a one-way ANOVA with Dunnett’s multiple comparison test (naive 5 µM p = 0.003, naive 10 µM p < 0.0001, naive 25 µM p < 0.0001, A CTL 25 µM p = 0.0437) or a one-way ANOVA with Holm-Sidak multiple comparison test (10 µM p = 0.0147, 25 µM p = 0.0010). ( B ) CTLs were restimulated for 3 h with plate-bound anti-CD3ε in the presence of 1.25 mM BAPTA, 1.25 mM EGTA or carrier control before RNA was extracted for qRT-PCR analysis (right panel). n = 3 biological replicates from 3 independent experiments. Data is normalised using CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Bars represent the mean; error bars indicate SD. p values were calculated using a one-way ANOVA with Holm-Sidak multiple comparison test (EGTA p = 0.0003, BAPTA p = 0.0053). ( C ) Schematic overview of Ca 2+ channels with respective antagonists (red) used in this study. ( D ) CTLs were restimulated with plate-bound anti-CD3ε in the presence of the indicated inhibitors or carrier control for 15 h before RNA was extracted for qRT-PCR analysis. n = 3–4 biological replicates from 3–4 independent experiments. Data is normalised using CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Bars represent the mean; error bars indicate SD. p values were calculated using a one-way ANOVA with Holm-Sidak multiple comparison test (carrier vs Nif50µM p = 0.0202, carrier vs Nif100µM p = 0.0002, U0126 vs U0126+Nif50µM p = 0.0311, U0126 vs U0126+Nif50µM p = 0.0118). ( E , F ) On day 6–8 post stimulation murine CTLs were loaded with Calcium Sensor Dye eFluor TM 514 and Ca 2+ flux was analysed by flow cytometry in the presence or absence of 100 μM nifedipine. After 30 s, the TCR was activated. ( E ) Representative flow cytometry plot is shown. Arrow indicates time of TCR-cross-linking. ( F ) Quantification of Ca 2+ flux after TCR cross-linking (between 30s-480s). n = 4 biological replicates from 4 independent experiments. Data points represent individual biological replicates. p values were calculated using a paired two-tailed Student’s t test ( p = 0.0456). AUC = area under the curve. ( G ) CTLs were restimulated with 10 µg/ml cross-linked soluble anti-CD3ε in the presence of nifedipine, 10 µM U0126 or carrier control. Cells were subsequently prepared for intracellular flow cytometric analysis. n = 2 biological replicates from 2 independent experiments. ( H ) Naive CD8 + T cells were stimulated with plate-bound anti-CD3ε in the presence of the indicated doses of nifedipine or carrier control. Data is normalised using CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Bars represent the mean; error bars indicate SD. p values were calculated using a two-way ANOVA with Sidak’s multiple comparison test (3 h carrier vs Nif50µM p = 0.0185, 6 h carrier vs Nif50µM p = 0.0455, 6 h carrier vs Nif50µM p = 0.001). n = 3 biological replicates from 3 independent experiments. ( I ) Naive CD8 + T cells were stimulated with 50 ng/mL PMA in the presence of carrier control or 100 μM nifedipine for 3 h before RNA was extracted for qRT-PCR analysis. Tbp served as a reference gene. n = 5 biological replicates from 2 independent experiments. Bars represent the mean; error bars indicate SD. p values were calculated using a one-sample Student’s t-test ( p = 0.0008). Symbols indicate biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. ns = not significant. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Control, Quantitative RT-PCR, Western Blot, Comparison, Flow Cytometry, Two Tailed Test

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A ) CTLs were generated from Gli1 WT and Gli1 KO mice. On day 7, cells were co-cultured with either Ova-pulsed EL4 cells or anti-CD3ε antibody-coated P815 target cells at the indicated effector to target ratios and subjected to an LDH cytotoxicity assay in the presence of 100 μM nifedipine or carrier control. Representative killing assay (left, data points represent mean of three technical replicates; error bars indicate SD) and quantification of n = 4 biological replicates from 4 independent experiments normalised to killing of Gli1 WT carrier condition at a 10:1 effector:target ratio (right, bars represent the mean; error bars indicate SD) are shown. p values were calculated using a two-way ANOVA with Tukey’s multiple comparison test (10:1 p < 0.0001 & p < 0.0001, 5:1 p = 0.0051 & p = 0.0001, 2.5:1 p = 0.0118). ( B ) CTLs were generated from GzmBER T2 Cre + Smo +/+ ( Smo WT) or GzmBER T2 Cre + Smo fl/fl ( Smo KO) mice. Cells were treated with 4-OH-Tamoxifen for the duration of the in vitro experiments. CTLs were restimulated for 3 h on day 8/9 for qRT-PCR analysis. n = 5 biological replicates from 5 independent experiments per condition. Bars represent the mean; error bars indicate SD. Statistical significance was assessed using a one sample Student’s t test (left panel) or a paired Student’s t test (right panel). Fold change of Gli1 expression relative to 0 h (set to “1”) is shown for Smo WT (left bar) and Smo KO (right bar) using their respective 0 h timepoint. Symbols indicate biological replicates. ( C ) CD8 + T cells were isolated from dLckCre + Ihh fl/+ ( Ihh Het) or dLckCre + Ihh fl/fl ( Ihh KO) mice. CTLs were restimulated for 15 h on day 8/9 in the presence of 100 µM nifedipine and/or 10 µM U0126 or carrier control for qRT-PCR analysis. n = 5 (naive) and n = 3 (restimulation) biological replicates from 5 and 3 independent experiments, respectively. ( B , C ) Data is normalised to CD3ε as a reference gene. Similar results were obtained when Tbp was used as a reference gene. Bars represent the mean; error bars indicate SD. Statistical significance was assessed using a one sample Student’s t test (left panel) or multiple unpaired Welch’s tests with Holm-Sidak multiple comparisons test (right panel). ( D , E ) On day 6–8 of culture, Rag2 KO OT-I CTLs were pre-treated with either carrier or 2.5 μM cyclopamine for 18 h before being co-cultured with ovalbumin-pulsed EL4 target cells for 4 h in the presence or absence of 50 μM nifedipine at the indicated effector to target ratios. ( D ) representative LDH cytotoxicity assay ( E ) n = 3 biological replicates from 3 independent experiments. p values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test ( E , all p < 0.0001 except for 1.25:1 Cyclopamine vs Nifedipine+Cyclopamine p = 0.0193). Bars represent the mean; error bars indicate SD. * p < 0.05, *** p < 0.001 **** p < 0.0001. ns = not significant. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Generated, Cell Culture, LDH Cytotoxicity Assay, Control, Comparison, In Vitro, Quantitative RT-PCR, Expressing, Isolation

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A ) RNA was extracted from murine CTLs on day 8 post stimulation mRNA expression of Ca v 1 isoforms was determined by qRT-PCR. Data is normalised to Tbp as a reference gene. Similar results were obtained when B2m was used as a reference gene. n = 3 biological replicates from 1 independent experiment. Symbols indicate biological replicates. Bars represent the mean; error bars indicate SD. ( B – H ) OT-I CD8 + T cells were electroporated with RNP complexes on day 2/3 post stimulation to generate Ca v 1.3/1.4 -/- (double KO) or non-targeting control (NTC) CTLs. ( B ) PCR amplification of the edited loci was performed prior to a T7EI mismatch cleavage assay to quantify genome editing efficiency on day 8/9 post stimulation. n = 3 biological replicates from 3 independent experiments. p values were calculated using an unpaired two-tailed Student’s t test ( p < 0.0001). Symbols indicate biological replicates. Bars represent the mean; error bars indicate SD. ( C , D ) CTLs were restimulated on day 8/9 with 10 µg/ml cross-linked soluble anti-CD3ε for flow cytometric analysis of pErk (C) or restimulated with plate-bound anti-CD3ε for mRNA expression of Gli1 (D). p values were calculated using a two-way ANOVA ( p = 0.0019). n = 3 biological replicates from 3 independent experiments. Symbols indicate biological replicates. ( E ) On day 5-7 CTLs were loaded with Calcium Sensor Dye eFluor TM 514 and calcium (Ca 2+ ) flux was analysed by flow cytometry. After 30 s, the TCR was cross-linked. The Ca 2+ chelator BAPTA serves as a negative control. Representative flow cytometry plot is shown. ( F ) Quantification of the area under the curve (AUC) of Ca 2+ flux of the period after TCR cross-linking (between 30s-300s). p values were calculated using a paired two-tailed Student’s t test ( p = 0.0472). n = 5 biological replicates from 3 independent experiments. ( G ) On day 6 post stimulation, CTLs were subjected to an Incucyte cytotoxicity assay with MC38-OvaN4 target cells at an effector:target ratio of 0.5:1. n = 2 biological replicates from 2 independent experiments normalised to killing of NTC cells at 45 h time point. Error bars indicate SD. p values were calculated using a two-way ANOVA ( p = 0.0029). ( H ) Rag2 KO mice were injected with 1.6 × 10 6 Celltracker TM Violet BMQC-labelled, untransduced EL4 (EL4 UT) and 1.6 × 10 6 GFP-expressing EL4-OvaN4 cells intraperitoneally (ip). Two hours post tumour cell injection 0.5 × 10 6 NTC or Cav1.3/1.4 KO CTLs were adoptively transferred via i.p . injection. At endpoint, 20 h post CTL injection, cells were collected from the peritoneal cavity and analysed by flow cytometry. Bar chart showing the ratio between EL4 UT and EL4-OvaN4 normalised to the ratio of PBS (no effector) injected mice. n = 2 (no effector), n = 8 (NTC), n = 7 (Ca v 1.3/1.4 -/- ) biological replicates from 2 independent experiments. p values were calculated using an unpaired two-tailed Student’s t test ( p = 0.0414). Bars represent the mean; error bars indicate SD. * p < 0.05, ** p < 0.01, **** p < 0.0001. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Amplification, Cleavage Assay, Two Tailed Test, Flow Cytometry, Negative Control, Cytotoxicity Assay, Injection

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A , B ) Naive human CD8 + T cells, freshly isolated from peripheral blood of healthy donors ( A ), or human CTLs on day 13–26 post activation ( B ) were processed for qRT-PCR analysis of human Ca v 1.1 ( CACNA1S ), Ca v 1.2 ( CACNA1C ), Ca v 1.3 ( CACNA1D ) and Ca v 1.4 ( CACNA1F ) channel expression. Values were normalised to TBP as a reference gene. Similar results were obtained when ACTB was used as a reference gene. ND: not detected. Bars represent the mean; error bars indicate SEM. Each symbol indicates a different healthy donor. ( A ) n = 7 ( B ) n = 8 biological replicates from 2 independent experiments. ( C ) Naive human CD8 + T cells were isolated from PBMCs of healthy donors and stimulated with human T-activator CD3/CD28 beads for 3 days in the presence of nifedipine or carrier control before flow cytometric analysis for cell viability. Bars represent the mean; error bars indicate SD. Statistical significance was assessed by a paired two-tailed Student’s t test. n = 5 biological replicates. Each symbol indicates a different healthy donor. ( D , E ) Human CD8 + T cells were isolated from peripheral blood of healthy donors and CTLs were generated. On day 12–15 post stimulation CTLs were loaded with Calcium Sensor Dye eFluor TM 514 and Ca 2+ flux was assessed by flow cytometry in the presence or absence of 50 μM nifedipine. After 30 s, TCR cross-linking was induced by antibody cross-linking. After 8 min, ionomycin was added to measure maximal Ca 2+ flux. ( D ) Representative flow cytometry plot is shown. ( E ) Quantification of the AUC of Ca 2+ flux of the period after TCR cross-linking (between 30 s–480 s). n = 5 biological replicates from 4 independent experiments. p values were calculated using a paired two-tailed Student’s t test ( p = 0.01). ( F ) T cells were stimulated for 3 days and again restimulated on d10 for 3 days. On d14–d15, CD8 + CTLs were co-cultured with P815 target cells in the presence of the indicated concentrations of nifedipine or carrier control at the indicated effector to target ratios and subjected to an LDH cytotoxicity assay. Left panel: representative donor shown. Right panel: data points represent mean of three technical replicates; error bars indicate SD, n = 5 biological replicates from 3 independent experiments. p values were calculated using a two-way ANOVA with Tukey’s multiple comparisons test (10:1 p = 0.0079, 5:1 p = 0.0008 & p < 0.0001, 2.5:1 p = 0.007 & p = 0.0016, 1.25:1 p = 0.0029 & p = 0.0075, 0.625:1 p = 0.0153 & p = 0.0179, 0.3125:1 p = 0.0109 & p = 0.0165). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns = not significant. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Isolation, Activation Assay, Quantitative RT-PCR, Expressing, Control, Two Tailed Test, Generated, Flow Cytometry, Cell Culture, LDH Cytotoxicity Assay

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: Human CD8 + T cells were stimulated with soluble anti-CD3/CD28/CD2 antibodies for 72 h for CTL generation. Between day 9 and 11 of culture, CTLs were fixed and stained with ( A ) phalloidin and antibodies against Ca v 1.4, ( B ) antibodies against Ca v 1.4 and TCR, ( C ) phalloidin and antibodies against Ca v 1.4 and AP1G1, ( D ) phalloidin and antibodies against Ca v 1.4 and VPS35, or ( E ) phalloidin and antibodies against Ca v 1.4. Nuclei were stained with Hoechst 33342. For ( E ), CTLs were plated on anti-CD3ε-coated wells for 4 min prior to fixation to generate immune synapses. ( A ) n = 8 biological replicates, ( B ) n = 3 biological replicates, ( C , D ) n = 2 biological replicates, ( E ) n = 4 biological replicates. Single Z-stack is shown. Representative donor shown. Scale bar = 10 μm. White arrowheads indicate areas of colocalisation between Ca v 1.4 and phalloidin ( A ), Ca v 1.4 and TCR ( B ), Ca v 1.4 and AP1G1 ( C ). White box indicates the region shown at higher magnification in ( A – D ). Bar graphs show the Pearson’s correlation coefficient between Ca v 1.4 and the indicated markers. Bars represent the mean; error bars indicate SD. ( A ) n = 562 cells, from 8 images. ( B ) n = 568 cells, from 8 images. ( C ) n = 132 cells, from 6 images. ( D ) n = 74 cells, from 3 images. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Staining

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A ) Murine OT-I CTLs were restimulated on day 9/10 for 3 h with plate-bound anti-CD3ε in the presence of the indicated concentrations of FPL 64176 or carrier control before RNA was extracted for qRT-PCR analysis. n = 4 biological replicates from 2 independent experiments. Data is normalised to Tbp as a reference gene. Similar results were obtained when CD3ε was used as a reference gene. Bars represent the mean; error bars indicate SD. p values were calculated using a one-way ANOVA with Tukey’s multiple comparison test (Carrier vs 5 µM p = 0.0009, Carrier vs 10 µM p = 0.0201). ( B ) On day 7 post stimulation, murine CTLs were co-cultured with Ova-pulsed EL4 tumour target cells in the presence of 10 µM FPL 64176 or carrier control at the indicated effector to target ratios. LDH cytotoxicity assay was performed after 3 h. Representative assay (left, data points represent mean of three technical replicates; error bars indicate SD) and quantification of n = 4 biological replicates from 2 independent experiments normalised to killing of carrier-treated cells at a 10:1 effector:target ratio (right, bars represent the mean; error bars indicate SD) are shown. p values were calculated using a two-way ANOVA with Sidak’s multiple comparison test (10:1 p < 0.0001, 5:1 p < 0.0001, 2.5:1 p < 0.0001, 1.25:1 p = 0.0406). ( C , D ) Human CD8 + T cells were isolated from peripheral blood of healthy donors and CTLs were generated. ( C ) Human CTLs were restimulated on day 15–20 for 24 h with plate-bound anti-CD3ε in the presence of 10 µM of FPL 64176 or carrier control before RNA was extracted for qRT-PCR analysis. n = 3 biological replicates from 2 independent experiments. Data is normalised to TBP as a reference gene. Similar results were obtained when CD3ε was used as a reference gene. Bars represent the mean; error bars indicate SD. p values were calculated using a one-way ANOVA with Tukey’s multiple comparison test ( p = 0.0171). ( D ) On day 16–24, CD8 + T cells were co-cultured with P815 target cells at indicated effector to target ratios for 3 h and subjected to a flow-cytometry-based cytotoxicity assay in the presence of 10 µM FPL64176 or carrier control. Representative killing assay (left) and quantification of n = 4 biological replicates from 2 independent experiments normalised to killing of carrier-treated cells at a 10:1 effector:target ratio (right) are shown. Bars represent the mean; error bars indicate SD. p values were calculated using a two-way ANOVA with Sidak’s multiple comparison test (10:1 p < 0.0001, 5:1 p < 0.0001, 2.5:1 p < 0.0004, 1.25:1 p = 0.0003, 0.625:1 p = 0.0092). ( E – H ) On day 6–8 poststimulation murine CTLs were loaded with Calcium Sensor Dye eFluor TM 514 and Ca 2+ flux was analysed by flow cytometry in the presence or absence of 10 µM FPL 64176. After 30 s, TCR cross-linking was induced with IgG antibodies ( E , F ) or store-operated Ca 2+ entry was induced by 1 µM thapsigargin ( G , H ). After 8 min, ionomycin was added to measure maximal Ca 2+ flux. Conditions with the Ca 2+ chelator BAPTA serve as negative controls. Representative flow cytometry plots shown ( E , G ). ( F ) Quantification of the area under the curve (AUC) of Ca 2+ flux of the period after TCR cross-linking (between 30 s and 480 s). n = 4 biological replicates from 4 independent experiments. p values were calculated using a paired two-tailed Student’s t test ( p = 0.0295). ( H ) Quantification of the AUC of Ca 2+ flux of the period after thapsigargin addition (between 30 s and 480 s). n = 4 biological replicates from 4 independent experiments. p values were calculated using a paired two-tailed Student’s t test ( p = 0.1506). ( I ) Murine naive CD8 + T cells were stimulated for 3 h and 24 h with plate-bound anti-CD3ε and anti-CD28 antibodies in the presence of 10 mM FPL 64176 or carrier control before RNA was extracted for RNA-Seq analysis. Heatmap depicting a selection of biologically relevant, statistically significant differentially downregulated and upregulated genes, respectively. n = 6 biological replicates. * p < 0.05, ** p < 0.01, **** p < 0.0001. Symbols indicate biological replicates or individual human donors. ns = not significant. Boxes indicate genes with one of 11 Gli consensus sequences as shown in Appendix Table (Winklmayr et al, ). .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Control, Quantitative RT-PCR, Comparison, Cell Culture, LDH Cytotoxicity Assay, Isolation, Generated, Flow Cytometry, Cytotoxicity Assay, Two Tailed Test, RNA Sequencing, Selection

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A – I ) FPL 64176 does not lead to a Ca 2+ response in the absence of TCR stimulation, enhances CTL killing via extracellular Ca 2+ influx, and does not affect CD8 + T cell viability, differentiation, proliferation or cytokine production. ( A ) Murine CTLs on day 6–8 post stimulation were loaded with Calcium Sensor Dye eFluor TM 514, stained with anti-CD8 and anti-CD3e and calcium (Ca 2+ ) flux was analysed by flow cytometry. After 30 s, FPL 64176 or carrier solution was added to a final concentration of 10 µM. After 8 min, ionomycin was added to measure maximal Ca 2+ flux. Data shown is representative of n = 4 biological replicates from 4 independent experiments. ( B ) On day 7–8 post stimulation, murine CTLs were co-cultured with ovalbumin-pulsed EL-4 target cells at the indicated effector to target ratios in the presence of 10 µM FPL64176 and/or 1.25 mM BAPTA and their respective carrier controls and subjected to an LDH cytotoxicity assay for 3 h. n = 4 biological replicates from 4 independent experiments normalised to killing of carrier-treated cells at a 10:1 effector:target cell ratio. Error bars indicate SD. p values were calculated using a two-way ANOVA with E:T ratio and treatment as factors with Dunnett’s multiple comparisons test comparing each treatment condition ( p = 0.0195). ( C – E ) Naive CD8 + T cells were stimulated with plate-bound anti-CD3/CD28 antibodies for 24 h in the presence of 10 µM FPL64176 or carrier control before flow cytometric analysis. Bars represent the mean; error bars indicate SD. ( C ) Quantitative analysis of live cell numbers normalised to carrier n = 4 biological replicates from 2 independent experiments. Statistical significance was assessed using a two-tailed one sample t test. ( D ) Percentage of apoptotic cells as detected by Apotracker™ Green staining n = 4 biological replicates from 2 independent experiments. Statistical significance was assessed using a two-tailed paired Student’s t test. ( E ) Analysis of T cell subsets based on the expression of CD44 and CD62L: CD44 - CD62L + (naive), CD44 + CD62L + (CM = central memory), and CD44 + CD62L - (EM = effector memory). n = 4 biological replicates from 2 independent experiments. Statistical significance was assessed using a two-way ANOVA with Sidak’s multiple comparison test. ( F , G ) CTLs were restimulated on day 10 with plate-bound anti-CD3ε antibody for 24 h in the presence of 10 µM FPL64176 or carrier control before flow cytometric analysis. Quantitative analysis of live cell numbers normalised to carrier condition ( F ) and percentage of apoptotic cells as detected by Apotracker™ Green staining ( G ) are shown. n = 4 biological replicates from 2 independent experiments. Bars represent the mean; error bars indicate SD. Statistical significance was assessed using a two-tailed one sample t test ( F ) or two-tailed paired Student’s t test ( G ). ( H , I ) Between day 11 and 13 post stimulation, human CTLs were left unstimulated or were re-stimulated with 1 µg/mL plate-bound anti-CD3ε antibodies for 4.5 h in the presence of 10 µM FPL64176 or carrier control. IFN-γ and TNF-α production was assessed by intracellular cytokine staining. Representative flow cytometry plots (top) and quantification of percentages and median fluorescence intensity (MFI) (bottom) are shown. n = 4 individual healthy donors from 2 independent experiments. Symbols indicate individual healthy donors. Bars represent the mean and error bars indicate SD. Statistical significance was assessed using a two-tailed paired Student’s t test. * p < 0.05. ns = not significant.

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Staining, Flow Cytometry, Concentration Assay, Cell Culture, LDH Cytotoxicity Assay, Control, Two Tailed Test, Expressing, Comparison, Fluorescence

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A – C ) L-type voltage-gated Ca 2+ channel agonist FPL 64176 enhances the killing capability of Vδ1 + T lymphocytes. ( A ) Human Vγ9Vδ2 + cells were expanded from αβ T cell-depleted PBMCs using 1 µM zoledronate before checking purity by flow cytometry on day 14. ( B ) Human Vδ1 + cells were expanded from αβ T cell-depleted PBMCs using anti-CD3ε antibodies and cytokines before checking purity by flow cytometry on day 18. ( C ) On day 22, human Vδ1 + T cells were co-cultured with P815 target cells at indicated effector to target ratios for 3 h and subjected to a flow cytometry-based cytotoxicity assay in the presence of 10 µM FPL 64176 or carrier control. Left panel shows a representative killing assay. Data points represent mean of two technical replicates; error bars indicate SD. Right panel shows quantification of n = 2 biological replicates from 1 independent experiment normalised to killing of carrier-treated cells at a 10:1 effector:target ratio. Bars represent the mean. ( D , E ) Sorting of human CD8 + T cells expressing Ca v 1.4 constructs and characterization of activation and differentiation markers. ( D ) Human CTLs were electroporated with PiggyBac vectors encoding GFP only (EV), human Ca v 1.4 (WT), or human Ca v 1.4 harbouring the gain-of-function mutation I745T (I745T) 48 h post stimulation and sorted between d17–28 of CTL culture based on GFP expression levels. Representative flow cytometry plots pre- and post-sorting are shown. n = 7 biological replicates from 5 independent experiments. ( E ) Sorted human CTLs expressing EV, WT, or I745T were restimulated for 72 h on day 19–20 of culture using plate-bound anti-CD3ε before being analysed by flow cytometry for activation and differentiation markers on day 28. Top Expression of CD25 and CD69 (top panel), CCR7 and CD45RA (middle panel) and CD27 (bottom panel) are shown. FMO control is shown in grey. Data shown is from n = 2 healthy donors.

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Cell Culture, Cytotoxicity Assay, Control, Expressing, Construct, Activation Assay, Mutagenesis

Journal: EMBO Reports

Article Title: L-type voltage-gated Ca 2+ channels control T cell killing via non-canonical Hedgehog signalling

doi: 10.1038/s44319-026-00810-8

Figure Lengend Snippet: ( A ) Schematic overview of Ca v 1.4 construct overexpression in human CD8 + T cells based on the gain-of-function mutation I745T identified in Hope et al, . Ca v 1.4 constructs were introduced into human CTLs via electroporation using the PiggyBac transposon system. Created with BioRender.com. ( B ) On day 26–34, human CTLs cells expressing Ca v 1.4 constructs were co-cultured with P815 target cells for 3 h at 5:1 effector to target ratio and subjected to a flow cytometry-based cytotoxicity assay. n = 7 biological replicates from 5 independent experiments normalised to killing of empty vector (EV) construct expressing cells. Bars represent the mean; error bars shown are SD. p values were calculated using a Friedman test with Dunn’s multiple comparisons test ( p = 0.0099). ( C ) Human CTLs expressing Cav1.4 constructs were sorted based on GFP expression and restimulated with plate-bound anti-CD3ε antibodies for 48–72 h and counted again on day 8–12 after sorting. n = 7 biological replicates from 5 independent experiments normalised to the number of cells obtained on the day of sorting. Bars represent the mean; error bars shown are SD. Statistical significance was assessed using a one-way ANOVA with Tukey’s multiple comparisons test. ( D, E ) Sorted Human CTLs expressing Cav1.4 constructs were analysed by flow cytometry for activation and differentiation markers 8–9 days post restimulation with plate-bound anti-CD3ε for 72 h. ( D ) Quantitative analysis of percentage of cells expressing activation markers CD25 and CD69. n = 2 biological replicates. Symbols indicate individual human donors. Bars represent the mean. ** ( E ) Flow cytometric analysis of the percentage of cells expressing differentiation markers CCR7 and CD45RA. Naive = CCR7 + CD45RA + , CM = CCR7 + CD45RA - , EM = CCR7 - CD45RA - , TEMRA = CCR7 - CD45RA + . n = 2 biological replicates. Symbols indicate individual human donors. Bars represent the mean. ** p < 0.01. ns = not significant. .

Article Snippet: Naive murine CD8 + T cells were isolated using negative selection (Naive CD8 + T Cell Isolation Kit, Cat no. 130-096-543, Miltenyi Biotec) from 6 Rag1 KO OT-I mice according to the manufacturer’s instructions.

Techniques: Construct, Over Expression, Mutagenesis, Electroporation, Expressing, Cell Culture, Flow Cytometry, Cytotoxicity Assay, Plasmid Preparation, Activation Assay

Journal: bioRxiv

Article Title: Immunodominance Hierarchy of Endogenous BBN963 Bladder Cancer Antigens Remains Stable Under Anti-PD1 and Anti-CTLA4 Immunotherapy

doi: 10.64898/2026.05.20.726664

Figure Lengend Snippet: Three BBN963 tumors were analyzed by whole exome and transcriptome sequencing. Data were analyzed using the Landscape of Effective Tumor antigen Software (LENS) to predict tumor antigens derived from a set of genomic sources (SNVs, indels, fusions, splice variants, viruses, endogenous retroviruses, and cancer/testis antigens). The set of predicted tumor antigen peptide epitopes was evaluated using ACE Configurator for ELISpot (ACE) to construct peptide matrices for large scale ELISpot testing. IFN-γ production measured by ELISpot was used to determine relative immunodominance of MHC Class I restricted epitopes recognized by CD8+ T cells.

Article Snippet: A kit was used for CD8+ T cell enrichment (Miltenyi Biotec, Cat No 130-104-075) via magnetic column, with calculations and procedures done according to the manufacturer instructions.

Techniques: Sequencing, Software, Derivative Assay, Enzyme-linked Immunospot, Construct

Journal: bioRxiv

Article Title: Immunodominance Hierarchy of Endogenous BBN963 Bladder Cancer Antigens Remains Stable Under Anti-PD1 and Anti-CTLA4 Immunotherapy

doi: 10.64898/2026.05.20.726664

Figure Lengend Snippet: 6-week-old C57BL/6 female mice were inoculated with 3 x 10 6 BBN963 cells, sacrificed at 10 days post tumor inoculation, and assessed for tumor antigen-specific CD8+ T cell responses via IFN-g ELISpot in pooled animal studies n=15 and individual animal studies (biological replicates n=9). (A) IFN-g spot count of matrixed peptide pools in pooled animal studies. Peptide pools containing positive antigens are shown in magenta and pools that are negative shown in light blue. (B) Single peptide validation of tumor antigens via ELISpot. Tumor antigens are mapped to genomic source and predicted MHC restriction (H2-Kb and H2-Db). (C) Immunodominance hierarchy of Tumor Antigens across immunogenic antigens with peptide biophysical similarity cladogram displaying ELISpot response to peptides with same core sequences. (D) Validation of immunodominance hierarchy of immunoprevalent antigens in single animal studies.

Article Snippet: A kit was used for CD8+ T cell enrichment (Miltenyi Biotec, Cat No 130-104-075) via magnetic column, with calculations and procedures done according to the manufacturer instructions.

Techniques: Enzyme-linked Immunospot, Biomarker Discovery

Journal: bioRxiv

Article Title: Immunodominance Hierarchy of Endogenous BBN963 Bladder Cancer Antigens Remains Stable Under Anti-PD1 and Anti-CTLA4 Immunotherapy

doi: 10.64898/2026.05.20.726664

Figure Lengend Snippet: (A) Experimental design: 6-week-old C57BL/6 female mice were inoculated with 3 x 10 6 BBN963 cells. Assessment of antigen-specific CD8 + T cell responses was done as outlined in (n=10 per treatment arm). (B) 145 peptide matrixed IFN-γ ELISpot results of anti-PD1 treated tumor-bearing animals as deconvoluted by ACE. (C) Single peptide validation of anti-PD1 treated animals showing the genomic source of each antigen. (D) 145 peptide matrixed IFN-γ ELISpot results of anti-CTLA4 treated tumor bearing animals as deconvoluted by ACE. (E) Single peptide validation of anti-CTLA4 treated animals showing the genomic source of each antigen.

Article Snippet: A kit was used for CD8+ T cell enrichment (Miltenyi Biotec, Cat No 130-104-075) via magnetic column, with calculations and procedures done according to the manufacturer instructions.

Techniques: Enzyme-linked Immunospot, Biomarker Discovery