Journal: Oncoimmunology

Article Title: T cell-mediated elimination of cancer cells by blocking CEACAM6–CEACAM1 interaction

doi: 10.1080/2162402X.2021.2008110

Figure Lengend Snippet: Immunomodulatory characteristics of BAY 1834942. A . Analysis of CEACAM1, -5, -6, and -8 receptor expression on survivin T cells and virus-specific T cells by flow cytometry using anti-CEACAM1 (TPP-3006) and anti-CEACAM6 (BAY 1834942). All other antibodies as listed in Suppl. Table S1. B . Effects of combined antibody-mediated blockade of CEACAM6 and CEACAM1 on IFN-γ cytokine secretion. Co-culture of 10,000 FluM1 peptide-loaded HCC2935 cancer cells with 20,000 FluM1-specific T cells for 20 h. BAY 1834942 and CEACAM1 antibody (TPP-3006) were used at 30 µg/mL. In combination experiments, BAY 1834942 and isotype mAbs were used at 1 µg/mL. IFN-γ levels were measured by ELISA. The isotype control antibodies (30 µg/mL) used were TPP-1238 (huIgG2 isotype control) and TPP-754 (huIgG1 isotype) control. The percentages indicate the fold differences in cytokine secretion. C . The effect of BAY 1834942 on IL-2 secretion in 100,000 CEACAM1 knockout Jurkat E6.1 or wild-type Jurkat E6.1 cells (both CD3-preactivated) after a 20-hour co-culture with 50,000 HCC2935 lung cancer cells in the presence of T cell engaging antibody construct EpCAM/CD3 (10 ng/mL) and either BAY 1834942 (40 µg/mL in X–vivo 20 medium) or TPP-1238 isotype control antibody. IL-2 levels in supernatants were analyzed by ELISA. The percentages indicate the fold differences in cytokine secretion. D . Recombinant CEACAM6 and CEACAM1 loaded on beads suppressed T cell signaling as determined from phosphorylated zeta-chain-associated protein kinase 70 (pZAP70, Tyr319) levels in CD3-activated Jurkat T cell lysates by immunoprecipitation and Western blot. HuIgG1 mAb Eureka was used as the Fc-isotype control. E . CEACAM6-suppressed cytokine secretion was reconstituted by BAY 1834942 in survivin T cells (100,000 cells) co-incubated for 20 h with 1 × 10 6 CEACAM6-loaded beads as determined by IFN-γ levels measured with ELISA. The CD3/CEACAM6 beads were pre-incubated with BAY 1834942 or an isotype control antibody at room temperature for 20 min. The colored closed circles denote reactions as follows: Black circles: beads coated with huIgG1; Yellow circles: beads coated with anti-CD3 and huIgG1; Orange circles: beads coated with anti-CD3, huIgG1, and CEACAM6-Fc; Green circles: beads coated with anti-CD3, huIgG1, and CEACAM6-Fc + increasing concentrations of isotype control TPP-1238 in solution; Blue circles: beads coated with anti-CD3, huIgG1, and CEACAM6-Fc + increasing concentrations of BAY 1834942 in solution. F . Reversal of CEACAM6-induced suppression of cytokine secretion as described in (e) using an anti-CEACAM1 (TPP-9145) antibody and an isotype control. The colored closed circles denote reactions as follows: Black circles: beads coated with huIgG1; Yellow circles: beads coated with anti-CD3 and huIgG1; Orange circles: beads coated with anti-CD3, huIgG1, and CEACAM6-Fc; Green circles: beads coated with anti-CD3, huIgG1, and CEACAM6-Fc + increasing concentrations of isotype control TPP-754 in solution; Blue circles: beads coated with anti-CD3, huIgG1, and CEACAM6-Fc + increasing concentrations of the anti-CEACAM1 antibody (TPP-9145) in solution. The anti-CEACAM1 antibody was pre-incubated with Jurkat cells; Pink circles: beads coated with anti-CD3 and huIgG1 + anti-CEACAM1 antibody (TPP-9145) in solution; Dark purple circles: beads coated with huIgG1 + anti-CEACAM1 antibody (TPP-9145) in solution. Statistical analysis for (b-c) was performed using a linear model and corrected for family-wise error rate using Sidak’s method. ns, non-significant; *, p < .05; ***, p < .001.

Article Snippet: Jurkat E6.1 cells (ATCC) and Jurkat T-REx (Bayer AG) cells were used to generate CEACAM1 knockout T cells for experiments.

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Knock-Out, Construct, Recombinant, Immunoprecipitation, Western Blot, Incubation

Journal:

Article Title: Rapid Detection of Clonal T-Cell Receptor-? Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis

doi:

Figure Lengend Snippet: Optimization of PCR cycles. DNA samples from 100% Jurkat T-cell line (J, top curve), a mixture of 50% Jurkat and 50% tonsil (0.5 J + 0.5 T, middle curve), 100% tonsil (T, bottom curve), and a negative control including all reagents without DNA (H2O) were amplified for 20 (A), 30 (B), 40 (C), 50 (D), and 60 (E) cycles. B: Jurkat started to produce a sharp −dF/dT peak after 30 PCR cycles. D: The −dF/dT peak height ratio of 100% Jurkat/100% tonsil reached 4.1 at 50 cycles (Table 1). Additional cycles did not significantly improve this ratio. Therefore, the diagnosis of a clonal T-cell gene rearrangement sample was based on DNA melting after 50 PCR cycles. x axis = temperature, ° C; y axis = −dF/dT, where F = fluorescence and T = temperature.

Article Snippet: The polyclonal tonsil background had essentially no influence on the Tm values of the clonally rearranged Jurkat T-cell line DNA when amplified with either primer set A or C (compare lines 1 and 2 in ). table ft1 table-wrap mode="anchored" t5 caption a7 Cell lines Primer set A Primer set C n Tm, mean CV n Tm, mean CV 50% Jurkat 7 86.99 0.45 7 90.18 0.26 100% Jurkat 10 87.25 0.44 5 90.11 0.27 100% CEM 7 91.96 0.32 7 92.14 0.14 Open in a separate window Tm = DNA melting temperature, °C.

Techniques: Negative Control, Amplification, Biomarker Discovery, Fluorescence

Journal:

Article Title: Rapid Detection of Clonal T-Cell Receptor-? Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis

doi:

Figure Lengend Snippet: Optimization of LightCycler PCR Cycle Number

Article Snippet: The polyclonal tonsil background had essentially no influence on the Tm values of the clonally rearranged Jurkat T-cell line DNA when amplified with either primer set A or C (compare lines 1 and 2 in ). table ft1 table-wrap mode="anchored" t5 caption a7 Cell lines Primer set A Primer set C n Tm, mean CV n Tm, mean CV 50% Jurkat 7 86.99 0.45 7 90.18 0.26 100% Jurkat 10 87.25 0.44 5 90.11 0.27 100% CEM 7 91.96 0.32 7 92.14 0.14 Open in a separate window Tm = DNA melting temperature, °C.

Techniques:

Journal:

Article Title: Rapid Detection of Clonal T-Cell Receptor-? Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis

doi:

Figure Lengend Snippet: Coefficient of Variation (CV) of LightCycler System Melting Curve Analysis

Article Snippet: The polyclonal tonsil background had essentially no influence on the Tm values of the clonally rearranged Jurkat T-cell line DNA when amplified with either primer set A or C (compare lines 1 and 2 in ). table ft1 table-wrap mode="anchored" t5 caption a7 Cell lines Primer set A Primer set C n Tm, mean CV n Tm, mean CV 50% Jurkat 7 86.99 0.45 7 90.18 0.26 100% Jurkat 10 87.25 0.44 5 90.11 0.27 100% CEM 7 91.96 0.32 7 92.14 0.14 Open in a separate window Tm = DNA melting temperature, °C.

Techniques:

Journal:

Article Title: Rapid Detection of Clonal T-Cell Receptor-? Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis

doi:

Figure Lengend Snippet: Minimal detection of percent clonal T cell. Jurkat T-cell line DNA (100%, 50%, 25%, 12.5%, 6.25%, and 3.125%) was serially diluted into tonsil DNA. After 50 cycles of amplification in the LightCycler system, we could still detect a distinct peak with the expected Tm of Jurkat by DNA melting curve analysis at the 12.5% level with primer set A (A) and at 6.25% level with primer set C (B).

Article Snippet: The polyclonal tonsil background had essentially no influence on the Tm values of the clonally rearranged Jurkat T-cell line DNA when amplified with either primer set A or C (compare lines 1 and 2 in ). table ft1 table-wrap mode="anchored" t5 caption a7 Cell lines Primer set A Primer set C n Tm, mean CV n Tm, mean CV 50% Jurkat 7 86.99 0.45 7 90.18 0.26 100% Jurkat 10 87.25 0.44 5 90.11 0.27 100% CEM 7 91.96 0.32 7 92.14 0.14 Open in a separate window Tm = DNA melting temperature, °C.

Techniques: Amplification