Journal: The Journal of investigative dermatology

Article Title: Increased Activation of Innate Immunity and Pro-Apoptotic CXCR3B in Normal-Appearing Skin on the Lesional Site of Patients with Segmental Vitiligo.

doi: 10.1016/j.jid.2021.07.157

Figure Lengend Snippet: Figure 2. CXCR3B in the skin of patients with SV. (a) CXCR3B mRNA level and (b) the number of CXCR3Bþ melanocytes (gp100þ) in patients with SV taken from their NL (n ¼ 5) and L (n ¼ 5) site and compared with those of the healthy control skin (n ¼ 5). CXCR3B mRNA results are given as RQ of the target gene expressed compared with that of housekeeping gene RPLPO. The number of double-positive gp100/CXCR3B-positive cells (depicted in c as yellow colocalization) were counted over 10 nonoverlapping fields along the dermis, and results were given as the mean number of positive cells per field. Differences between groups were tested using Kruskal‒Wallis test followed by Dunn’s posthoc analysis. Significance was set at P < 0.05. Bar ¼ 30 mm (10 mm in close-up pictures). L, lesional; NL, nonlesional; RQ, relative quantity; SV, segmental vitiligo.

Article Snippet: Afterward, tissue was incubated overnight at 4 C with primary antibody directed against human HSP70 (HSPA1A/B, 1:50, Enzo Life Sciences, Farmingdale, New York), mtDNA (1:10, Progen, Heidelberg, Germany), or CXCR3B (1:50, ProteinTech, Rosemont, IL) and later in presence or absence of gp100 (1:200, PMEL, Abcam, Cambridge, United Kingdom).

Techniques: Control

Journal: bioRxiv

Article Title: Experimental and stochastic models of melanoma T-cell therapy define impact of subclone fitness on selection of antigen loss variants

doi: 10.1101/860023

Figure Lengend Snippet: (A) Experimental outline of HCmel12 WT melanomas treated with ACT METi . (B) Representative Pmel expression by immunohistochemistry in untreated and ACT METi -recurrent HCmel12 WT melanomas. (C) Quantification of the experiments from B. Two-sided Mann-Whitney-U test. (D) Pmel indel distribution in HCmel12 cells gene-edited with the indicated sgRNAs. Portions of frameshift and in-frame indels are indicated. (E) Experimental outline of HCmel12 pcMix melanomas treated with ACT METi . (F) Individual tumor growth curves of HCmel12 pcMix melanomas left untreated or treated with ACT METi . (G) Amplicon NGS based quantification of Pmel KO allele percentages in the tumor cell fraction of untreated or recurrent ACT METi -treated HCmel12 pcMix melanomas. (H) As G, but total Pmel indel percentages and the respective portions of frameshift and small in-frame indels. (I) Examples of small in-frame indels detected in G, H.

Article Snippet: Tissues were embedded in paraffin and sections stained with rabbit anti-mouse Pmel pAb (NBP1-69571; Novus Biologicals) followed by enzyme-conjugated secondary antibodies and the LSAB-2 color development system (DAKO).

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY, Amplification

Journal: bioRxiv

Article Title: Experimental and stochastic models of melanoma T-cell therapy define impact of subclone fitness on selection of antigen loss variants

doi: 10.1101/860023

Figure Lengend Snippet: (A) Interaction diagram displaying cells/molecules (filled circles) and mechanisms that are incorporated in the mathematical model. Arrows represent possible changes to the state of the system, e.g., cell division, cytokine secretion, or cell death. Formulae describe the frequencies at which these population changes occur. (B) Tumor growth curves from experimental data published in Glodde et al. (C) Tumor growth curves generated by simulations for different initial tumor sizes, shown as tumor diameter [mm]. Vertical lines mark beginning of METi injections, injection of Pmel-1 T cells, and end of METi injections. Dashed lines indicate tumors undergoing eradication.

Article Snippet: Tissues were embedded in paraffin and sections stained with rabbit anti-mouse Pmel pAb (NBP1-69571; Novus Biologicals) followed by enzyme-conjugated secondary antibodies and the LSAB-2 color development system (DAKO).

Techniques: Generated, Injection

Journal: bioRxiv

Article Title: Experimental and stochastic models of melanoma T-cell therapy define impact of subclone fitness on selection of antigen loss variants

doi: 10.1101/860023

Figure Lengend Snippet: Simulations of the evolution of different cell/molecule types for the untreated case (A) and under ACT METi ( B, left and right zoom-in panel), shown as number of cells in 10e7. (C) Simulations of the evolution of tumor size, shown as diameter [mm], and percentage of Pmel KO cells. Initial tumor of medium size and α=4. For A-C vertical lines mark beginning of METi injections (‘), injection of Pmel-1 T-cells (#), and end of METi injections (“) and the time that 10 mm tumor diameter is reached (&). (D) Comparison of experimental data (Exp.) from and simulation results (Sim.) for the frequency of Pmel KO alleles in ACT METi -recurrent HCmel12 pcMix melanomas. Percentage determined at a random time +-0.5 mm of the measured diameter in experiments. (E) Predictions for enrichment of Pmel KO cells under varying subclone fitness rKO=bKO-dKO and different tumor sizes at time point of harvesting. Predicted variability of enrichment by different tumor sizes at time point of harvesting (§) verus different sublcone fitness (¶). (F) Pmel indel distribution in untreated (upper row) and ACT METi -recurrent (lower row) HCmel12 pcMix melanomas determined by amplicon NGS. Orange dots indicate respective indel frequency at the time of tumor cell injection ( , right panel)

Article Snippet: Tissues were embedded in paraffin and sections stained with rabbit anti-mouse Pmel pAb (NBP1-69571; Novus Biologicals) followed by enzyme-conjugated secondary antibodies and the LSAB-2 color development system (DAKO).

Techniques: Injection, Comparison, Amplification

Journal: bioRxiv

Article Title: Experimental and stochastic models of melanoma T-cell therapy define impact of subclone fitness on selection of antigen loss variants

doi: 10.1101/860023

Figure Lengend Snippet: (A-B) Experimental outline of HCmel12 sscMix melanomas treated with ACT METi . Mixing ratios of HCmel12 WT and Pmel KO single cell clones are indicated. (C-F) Individual tumor growth curves of HCmel12 sscMix melanomas described in A-B left untreated or treated with ACT METi . (G) Percentages of individual Pmel KO single cell clones (subclones) in ACT METi -recurrent tumor from D and F determined by amplicon NGS. (H) Quantification of in vitro cell growth of individual Pmel KO single cell clones compared to HCmel12 WT cells. Two-sided t test. ***p<0.001. (I) Experimental outline of color-coding approach comparing early outgrowth of HCmel12 Pmel KO single cell clones versus WT cells in untreated mice. (J) Representative whole-mount fluorescent images corresponding to experimental setup described in I. eGFP signal represented in false color blue. (K) Corresponding quantification of J. One sample t-test with hypothetical mean of 50%. *p<0.05, **p<0.01.

Article Snippet: Tissues were embedded in paraffin and sections stained with rabbit anti-mouse Pmel pAb (NBP1-69571; Novus Biologicals) followed by enzyme-conjugated secondary antibodies and the LSAB-2 color development system (DAKO).

Techniques: Clone Assay, Amplification, In Vitro

Journal: bioRxiv

Article Title: Experimental and stochastic models of melanoma T-cell therapy define impact of subclone fitness on selection of antigen loss variants

doi: 10.1101/860023

Figure Lengend Snippet: (A) Right and left zoom-in panel: Simulation of the evolution of different cell/molecule types under ACT METi , shown as number of cells in 10e7. Initial pure WT tumor size below critical threshold for therapy success. Natural mutation to Pmel KO cells at rate of m=10e-7. (B) Simulations of tumor growth curves, shown as diameter [mm] on a log-scale. Red curve shows no successful mutation while first successful mutations of blue and green curves are marked with crosses. For A-B vertical lines mark beginning of METi injections (‘), injection of Pmel-1 T-cells (#), and end of METi injections (“) and the time that 10 mm tumor diameter is reached (&). (C) Number of tumors going into relapse (reaching diameter of 10 mm) within one year after tumor inoculation, out of 100 runs under varying subclone fitness rKO=bKO-dKO.

Article Snippet: Tissues were embedded in paraffin and sections stained with rabbit anti-mouse Pmel pAb (NBP1-69571; Novus Biologicals) followed by enzyme-conjugated secondary antibodies and the LSAB-2 color development system (DAKO).

Techniques: Mutagenesis, Injection