Journal: Frontiers in Immunology

Article Title: Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo

doi: 10.3389/fimmu.2023.1072810

Figure Lengend Snippet: Design, pharmacokinetic and pharmacodynamic profiles of novel ACC TA99-HL2-KOA1. (A) Model depiction of engineered cytokine mimic HL2-KOA1 binding to IL-2Rα. IL-2 shown in blue, IL-2Rα shown in grey. Positions in contact with F42 shown as grey spheres. F42 and F42V shown as pink and red spheres respectively. Clash scores provided in total Rosetta energy units (REU). (B) Reducing SDS-PAGE analysis comparing the migration patterns of TA99-WT, TA99-Neo2/15, TA99-HL2-KOA1, pVAX backbone control transfection supernatants. (C) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human Tyrp1 compared to a murine isotype IgG2a control. (D) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rα compared to human IL2-Fc control. (E) Binding of recombinant TA99-WT, TA99-Neo2/15, and TA99-HL2-KOA1 to recombinant human IL2-Rβ compared to human IL2-Fc control. (F) Geometric mean fluorescence intensity of phosphorylated STAT5 in CD25+ CD4+ CD19- T cells from naïve mice (n=6) treated with indicated ACC. (G) Pharmacokinetic profile of recombinant TA99-WT and TA99-Neo-2/15 antibodies; C57BL/6 mice were injected intraperitoneally with 100µg of ACC constructs (n=5 mice per group). Serum at the indicated timepoints was assessed for TYRP1 binding by ELISA. (H) Total serum cytokine level over 14-day period in terms of area under the curve (AUC) in mice treated with ACC. (I) Timecourse of serum TNFα levels in sera of mice administered 100µg ACC. Two sera pools were used for (H, I) . (J) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-Neo2/15 (50µg) + TriVax (10µg each of Trp2, Gp100 and Tyrp1), or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax. 1x10 5 B16F10 cells subcutaneously into the right flank of C57BL/6 mice on Day 0. Green arrows indicate administration of treatment. (K) Survival curves of mice (n=10 mice per group) post tumor challenge following treatment with anti-PD1 alone, or anti-PD1 (200µg) + TA99-HL2-KOA1 (50µg) + TriVax, or anti-PD1 + TA99 (50µg) + Human IL2 (5µg) + TriVax. Challenge and treatment scheme was performed as in (J) Error bars represent standard deviation; non-parametric Mann Whitney T test compared with TA99-WT used in (F) ; non-parametric Kruskal-Wallis ANOVA was used in (I) ; log-rank test was used to compare between differences in all survival curves; *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: In addition, serially diluted recombinant protein variants of antibody-cytokine chimera or recombinant mouse Fc-tagged IL2 (IL2-Fc) (Molecular Innovations, Cat# MIL2-FC-0.05MG) were used in place of serially diluted mouse sera.

Techniques: Binding Assay, SDS Page, Migration, Control, Transfection, Recombinant, Fluorescence, Injection, Construct, Enzyme-linked Immunosorbent Assay, Standard Deviation, MANN-WHITNEY

Journal: Cells

Article Title: ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting

doi: 10.3390/cells4010056

Figure Lengend Snippet: Log Normal size distributions for cytokine ELISPOTs produced by CD8 and CD4 cells. ( A ) For CD8 cells, IFN-γ ELISPOT size distributions were studied for a total of 334 positive recall responses induced by the 32 individual CEF peptides. Each peptide is represented by an individual symbol type. ( B – F ) For CD4 cells, 80 individual IFN-γ, IL-2, IL- 4, IL-5, and IL-17 spot size distributions elicited by inactivated CMV and EBV were studied; each one specified by a different symbol. The Kolmogorov–Smirnov goodness of fit test was used to determine the normality of spot size distribution for each individual positive recall response. Experimental IDs of the positive donors are shown on the x-axis, and the y-axis represents the p-values for the donor/peptide combinations. The red line indicates the cut-off significance level of 5%.

Article Snippet: Human Interferon-γ ImmunoSpot® kits (CTL-HIFNG-1/5M), human IL-2- (CTL-HIL21M/5), IL-4- (CTL-HIL4-1M/5), IL-5- (CTL-HIL5-1M/5), and IL-17 kits (CTL-HIL17-1M/5) were obtained from CTL.

Techniques: Produced, Enzyme-linked Immunospot

Journal: Cells

Article Title: ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting

doi: 10.3390/cells4010056

Figure Lengend Snippet: ( A ) IFN-γ, ( B ) IL-2, ( C ) IL-4, ( D ) IL-5, and ( E ) IL-17 ELISPOTs produced by CD4 cells show a maximal three-fold size variation between donors and antigens. Inactivated EBV or CMV virions (indicated by the different symbols) were used to induce CD4 cells of different donors to produce the cytokines specified in the panels. The mean spot size (mm 2 ) and SD for each positive response (y-axis) are plotted in the logarithmic scale vs. the individual donors’ ID specified on the x-axis. For each data point 1000 spots were analyzed.

Article Snippet: Human Interferon-γ ImmunoSpot® kits (CTL-HIFNG-1/5M), human IL-2- (CTL-HIL21M/5), IL-4- (CTL-HIL4-1M/5), IL-5- (CTL-HIL5-1M/5), and IL-17 kits (CTL-HIL17-1M/5) were obtained from CTL.

Techniques: Produced

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Alpaca ( Vicugna pacos ), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset

doi: 10.1073/pnas.1909474117

Figure Lengend Snippet: BTN3-dependent PAg reactivity of transduced alpaca Vγ9Vδ2 TCRs. (A) Murine responder cells (53/4) expressing vpTCR(Jδ2), vpTCR(Jδ4), or a human Vγ9Vδ2 TCR (huTCR) were cocultured with 293T cell lines (WT), 293T BTN3KO, or BTN3KO vpBTN3 (phNGFR mCherry) in the presence of titrated HMBPP doses. Supernatants were tested for mIL-2 after 22 h. (B) In addition, WTH-5 (1 µg/mL) or mIgG2b,κ isotype were added to cultures of TCR transductants with wild-type or BTN3KO vpBTN3 in the presence of 10 µM HMBPP. Duplicates were measured in three experiments and mean + SD are shown. Statistical analysis is shown in SI Appendix, Table S6 for A and a two-way ANOVA (B) was carried out with a Bonferroni post hoc test (ns, not significant: P > 0.05, ****P < 0.0001).

Article Snippet: The activation of TCR transductants was measured via mIL-2 ELISA (BD).

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Alpaca ( Vicugna pacos ), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset

doi: 10.1073/pnas.1909474117

Figure Lengend Snippet: The alpaca B30.2 domain can sense PAgs. (A) A number of 1 × 104 293T BTN3KO or BTN3A1KO cells transduced with huBTN3A1, vpBTN3, hu/vpBTN3, or vp/huBTN3 (all in phNGFR mCherry) were plated overnight. Next, 5 × 104 Murine responder cells [53/4 huTCR or vpTCR(Jδ2)] were added as well as a dilution of HMBPP. Medium was used as a control, and the experiment was carried out in duplicates. Supernatants were tested for mIL-2 after 22 h with mIL-2 ELISA. Means of three independent experiments are shown with SDs. Statistical analysis is summarized in SI Appendix, Table S7. (B) Graphical summary of A with schematic representation of wild-type human (hu) and alpaca BTN3 (vp) and constructed chimeric molecules with an exchange of intracellular domains (hu/vp and vp/hu). The mIL-2 response of responder cells is indicated as high (+++), intermediate (++), low (+), or negative (−).

Article Snippet: The activation of TCR transductants was measured via mIL-2 ELISA (BD).

Techniques: Transduction, Enzyme-linked Immunosorbent Assay, Construct