human ctla 4  (R&D Systems)

Journal: Cancers

Article Title: Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation

doi: 10.3390/cancers14235738

Figure Lengend Snippet: iFRET quantifies high and low PD-1/PD-L1 interaction states in lung metastases, detecting inter- and intratumoral heterogeneity. Representative FLIM images demonstrating high and low CTLA-4/CD80 interaction states. Top panel: greyscale images indicate CTLA-4 or CD80 expression in donor-only or donor-acceptor tissue slices for one patient. No difference is observed between CTLA-4 expression between the donor-only and donor-acceptor slides. The lifetime map indicates the mean lifetime per pixel of an image. The donor only lifetime (2.27 ± 0.24 ns) is represented by blue/green in the pseudo-color scale. In the donor-acceptor slide, the lifetime of the donor is reduced to 1.53 ± 0.32 ns, yielding a FRET efficiency of 32.77%. This is indicative of a high CTLA-4/CD80 interaction state. Bottom panel: here, a good expression of CTLA-4 and CD80 are observed and no significant differences in expression profiles are seen between the top and bottom panels. The donor-only lifetime here is 2.32 ± 0.29 ns, which is reduced to 2.28 ± 0.27 ns in the presence of the acceptor, giving a FRET efficiency of 1.55%. This indicates that CTLA-4/CD80 are undergoing little to no interaction in this sample, despite the presence of both receptor and ligand. Critically, in both examples, high tissue level coincidence is observed between the donor-only and donor-acceptor slices, meaning that donor lifetime changes are due to the presence of the acceptor chromophore and are not reporting on intratumoral heterogeneity.

Article Snippet: Monoclonal antibodies mouse anti-PD-1, rabbit anti-PD-L1, and mouse anti-CTLA-4 were purchased from Abcam (catalogue numbers ab52587, ab205921, and ab19792, respectively).

Techniques: Expressing

Journal: Cancers

Article Title: Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation

doi: 10.3390/cancers14235738

Figure Lengend Snippet: iFRET detects intra- and intertumoral heterogeneity in both CTLA-4/CD80 and PD-1/PD-L1 interactions in metastases from lung one (pre-RFA). Box and whisker plots show the statistical distribution of all interaction states recorded for CTLA-4/CD80 (top panel) and PD-1/PD-L1 (bottom panel) in metastases taken from lung one, before RFA was carried out. Each point represents one region of interest for an analyzed patient sample. Note, no data were analyzed for CTLA-4/CD80 interactions in patient 17 due to poor a poor signal-to-noise ratio. Interaction states are generally higher in CTLA-4/CD80 than PD-1/PD-L1 with two patients having no PD-1/PD-L1 interaction across the sample. Some patients have differential interaction profiles for the two pathways. Patient 05 has a low CTLA-4/CD80 interaction and high PD-1/PD-L1 interaction. A contrasting trend is seen in patient 20, who has a significantly higher CTLA-4/CD80 interaction state than PD-1/PD-L1. Some patients, such as patient 13, showed no significant differences between CTLA-4/CD80 and PD-1/PD-L1 interaction states.

Article Snippet: Monoclonal antibodies mouse anti-PD-1, rabbit anti-PD-L1, and mouse anti-CTLA-4 were purchased from Abcam (catalogue numbers ab52587, ab205921, and ab19792, respectively).

Techniques: Whisker Assay

Journal: Cancers

Article Title: Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation

doi: 10.3390/cancers14235738

Figure Lengend Snippet: iFRET detects intra- and intertumoral heterogeneity in both CTLA-4/CD80 and PD-1/PD-L1 interactions in metastases from lung two (post-RFA). Box and whisker plots show the statistical distribution of all interaction states recorded for CTLA-4/CD80 (left-hand side) and PD-1/PD-L1 (right-hand side) in metastases taken from lung two, after RFA was carried out in lung one. Each point represents one region of interest for an analyzed patient sample. Interaction states are generally higher in CTLA-4/CD80 than PD-1/PD-L1. Some patients have differential interaction profiles for the two pathways. Patient 03 has a low CTLA-4/CD80 interaction state and regions of high PD-1/PD-L1 interaction. A contrasting trend is seen in patient 01, who has a significantly higher CTLA-4/CD80 interaction state than PD-1/PD-L1. Some patients, such as patient 06, showed no significant differences between CTLA-4/CD80 and PD-1/PD-L1 interaction states.

Article Snippet: Monoclonal antibodies mouse anti-PD-1, rabbit anti-PD-L1, and mouse anti-CTLA-4 were purchased from Abcam (catalogue numbers ab52587, ab205921, and ab19792, respectively).

Techniques: Whisker Assay

Journal: Cancers

Article Title: Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation

doi: 10.3390/cancers14235738

Figure Lengend Snippet: Checkpoint interaction does not correlate with ligand expression in pre- or post-RFA-treated lung metastases. The expression of each checkpoint ligand (CD80 or PD-L1) does not correlate with checkpoint interaction. ( A ) In lung metastases from lung 1, prior to RFA treatment, median CTLA-4/CD80 interaction states did not correlate with CD80 expression (r s = −0.134, p = 0.632). ( B ) From the same lung, pre-RFA, PD-1/PD-L1 interaction states did not correlate with PD-L1 expression (r s = −0.171, p = 0.541). ( C ) In patient samples taken from lung 2, post-RFA treatment of lung 1, checkpoint ligand expression failed to correlate with median CTLA-4/CD80 interaction (r s = 0.234, p = 0.439). ( D ) PD-1/PD-L1 interaction states failed to correlate with PD-L1 expression (r s = −0.265, p = 0.378, respectively).

Article Snippet: Monoclonal antibodies mouse anti-PD-1, rabbit anti-PD-L1, and mouse anti-CTLA-4 were purchased from Abcam (catalogue numbers ab52587, ab205921, and ab19792, respectively).

Techniques: Expressing

Journal: Cancers

Article Title: Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation

doi: 10.3390/cancers14235738

Figure Lengend Snippet: PD-1/PD-L1 interaction state negatively correlates with intratumoral CD3 and CD8 density in lung 1. (A) Scatter plots demonstrate a lack of correlation between intratumoral CD3 density (cells/mm 2 ) and median CTLA-4/CD80 interaction (r s = −0.154, p = 0.613) in metastases analyzed from lung one (pre-RFA). ( B ) A moderate negative correlation exists between intratumoral CD3 density and median PD-1/PD-L1 interaction state (r= −0.654, p = 0.014). This indicates, with significance, that higher median PD-1/PD-L1 interaction states are detected in areas with lower intratumoral CD3 density. ( C ) Scatter plots demonstrate a lack of correlation between intratumoral CD8 density (cells/mm 2 ) and median CTLA-4/CD80 interaction (r s = −0.017, p = 0.960) in metastases analyzed from lung one (pre-RFA). ( D ) A moderate negative correlation exists between intratumoral CD8 density and median PD-1/PD-L1 interaction state (r s = −0–537, p = 0.051). Whilst not statistically significant, this is a moderate trend that may achieve significance with higher sample numbers.

Article Snippet: Monoclonal antibodies mouse anti-PD-1, rabbit anti-PD-L1, and mouse anti-CTLA-4 were purchased from Abcam (catalogue numbers ab52587, ab205921, and ab19792, respectively).

Techniques: