Junior Recital: Leah McArthur Sexton, mezzo-soprano

This recital is presented in partial fulfillment of requirements for the degree Bachelor of Music in Performance. Ms. Sexton studies voice with Jana Young.https://digitalcommons.kennesaw.edu/musicprograms/1212/thumbnail.jp

Student Recital from the Studio of Jana Young

KSU School of Music presents Student Recital from the studio of Jana Young.https://digitalcommons.kennesaw.edu/musicprograms/1329/thumbnail.jp

Studio Recital: Students of Jana Young

Kennesaw State University School of Music presents Studio Recital: Students of Jana Young.https://digitalcommons.kennesaw.edu/musicprograms/1367/thumbnail.jp

Studio Recital: Jana Young\u27s Voice Studio

KSU School of Music presents Jana Young\u27s Voice Studio.https://digitalcommons.kennesaw.edu/musicprograms/1202/thumbnail.jp

Student Compostiton Recital

Kennesaw State University School of Music presents Student Composition Recital.https://digitalcommons.kennesaw.edu/musicprograms/1390/thumbnail.jp

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Super-resolution mapping of Rho IS surface labeling in mouse rods.

Z-projection SIM images of thin plastic sections of full (unpeeled) retinas from (A) WT, (B) Rho-GFP/+, and (C) RhoGFP-1D4 mice that were all labeled with WGA (cyan) and NbGFP-A647 (magenta) using normal, permeabilizing immunostaining conditions. Note that NbGFP-A647 only labels Rho-GFP/+ and Rho-GFP-1D4/+ rod OS tips as in Fig 1E and 1F. (D–F) Z-projection SIM images from full retinas labeled with WGA (cyan) and Rho-N-4D2 antibody (magenta) from WT (D), Rho-GFP/+ (E), or Rho-GFP-1D4/+ (F) mice using a surface labeling (no fixative, no detergent) protocol (see Materials and methods). Inverted WGA and Rho-N-4D2 single channel images are shown adjacent to the composite image. Red brackets indicate positive Rho-N-4D2 fluorescent signal in the IS. (G) Z-projection SIM image from a full WT retina labeled with WGA (cyan) and Rho-C-1D4 antibody (magenta). Lack of Rho-C-1D4 surface immunolabeling demonstrates Rho-N-4D2 extracellular labeling specificity and effectiveness. The OS, IS, and ONL layers, based on the WGA labeling pattern, are indicated for each SIM image. Scale bar values match adjacent panels when not labeled. (H–J) STORM reconstruction examples of rods from WT (H), Rho-GFP/+ (I), or Rho-GFP-1D4/+ (J) full retinas that were surface labeled with WGA (cyan) and Rho-N-4D2 antibody (magenta). The OS and IS regions are indicated. Adjacent to each STORM image is the Rho-N-4D2 molecular coordinates for the IS region. The number of Rho-N-4D2 molecules in (H) = 2,257, in (I) = 2,022, and in (J) = 2,764. Surface localization of Rho-N-4D2 molecules is indicated with orange arrows. Nearest distance-to-hull measurements for Rho-N-4D2 (magenta) and the corresponding set of random molecules (green) are plotted in a frequency graph above the molecule plots. For each rod analyzed with STORM, the distance to hull frequency within 0.2 μm was divided by the distance to hull frequency within 0.2 μm of the corresponding random coordinates to acquire “normalized frequency K). WT n value (for number of rods analyze) = 23, “Rho-GFP” for Rho-GFP/+ rods n = 19, “Rho-GFP-1D4” for Rho-GFP-1D4/+ rods n = 22. No significant difference in these data were based on a one-way ANOVA test (P value = 0.144). In the violin plot graph, circles = median values and dashed lines = mean values. Numerical values corresponding to all graphical data are provided in Table D in S1 Data. IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; ONL, outer nuclear layer; OS, outer segment; Rho, rhodopsin; SIM, structured illumination microscopy; STORM, stochastic optical reconstruction microscopy; WGA, wheat germ agglutinin; WT, wild-type.</p

STX3 coimmunoprecipitates with Rho in vivo.

Co-IP results from (A) Rho-GFP/+ IS-enriched retinal lysates incubated with GFP-Trap agarose beads, (B) Rho-GFP-1D4/+ IS-enriched retinal lysates incubated with GFP-Trap agarose beads, (C) Rho-GFP/+ IS-enriched retinal lysates incubated with anti-1D4 IgG agarose beads, and (D) WT IS-enriched retinal lysates incubated with anti-1D4 IgG agarose beads. In all western blots, input lanes correspond to 2% (% vol/vol) of the starting lysate volume, unbound lanes correspond to 2% (% vol/vol) of lysate volume post bead incubation, and bound lanes correspond to half the total eluate from each co-IP. Antibodies/nanobodies used for immunodetection are listed to the right of each corresponding western blot scan. Molecular weight marker sizes are indicated in kDa. Black arrows mark the correct size bands when other bands are present on the scan. Red asterisks indicate mouse IgG bands. In (B), the red arrow indicates the endogenous Rho band and the green arrow indicates the Rho-GFP-1D4 band. co-IP, coimmunoprecipitation; IgG, immunoglobulin G; Rho, rhodopsin; STX3, syntaxin 3; WT, wild-type.</p

S4 Fig -

(A, B) Violin plot graphs of STORM distance to hull normalized frequency values within (A) 0.1 μm and (B) 0.3 μm. N values are the same as in Fig 6F. Comparisons were tested for statistical significance using the Mann–Whitney U test. (A) PDC vs. Rho-GFP **P value = 0.0011; PDC vs. Rho-GFP-1D4 ***P value P = 0.0003; PDC vs. 4D2 *P = 0.0172. (B) PDC vs. Rho-GFP ***P value = 0.0003; PDC vs. Rho-GFP-1D4 ***P value P = 0.0033. (C, D) Immunogold localization of STX3 and Rho in mouse rods. (C) Single rod IS electron micrograph examples from a WT mouse retinas immunolabeled with STX3 antibody and nanogold secondary antibody. In a threshold image showing only the STX3+ immunogold particles, the approximate location of the IS plasma membrane is outlined in cyan. The outline is also continuous with the CC membrane. (D) Electron micrograph examples of rod ISs from IS-enriched WT mouse retinas immunolabeled with Rho-C-1D4. The IS plasma membrane is outlined in cyan in the threshold image. Some non-punctate staining from the BB and CC axoneme is present in the threshold images. mito = mitochondria. Numerical values corresponding to all graphical data are provided in Table H in S1 Data. BB, basal body; CC, connecting cilium; IS, inner segment; PDC, phosducin; Rho, rhodopsin; STORM, stochastic optical reconstruction microscopy; STX3, syntaxin 3; WT, wild-type. (PDF)</p

OS peeling generates IS-enriched mouse retina samples.

(A) Diagram of the mouse retina OS peeling method. Mouse retina slices are iteratively peeled from filter paper 8 times to remove rod OSs (yellow) from the IS layer (magenta). (B) Western blot analysis comparing control full retina samples vs. retinas after OS peeling/IS-enriched retinas; both from Rho-GFP/+ mice. Approximately 2% of total volume from lysates from 1 retina (either control or peeled) were used from each condition for SDS-PAGE. Molecular weight marker sizes are indicated in kDa. Immunolabeled bands from antibodies targeting OS proteins, including NbGFP-A647, Rho-C-1D4, and ROM1 (black arrow points to the ROM1 monomer band), were reduced in peeled lysates, whereas IS proteins STX3 and Rab11a were roughly equal demonstrating IS enrichment. Tubulin immunoblotting was performed as a loading control. (C) Control full retina slices and (D) IS-enriched retinal slices from Rho-GFP/+ mice were fixed and stained for resin embedding, ultrathin sectioning, and TEM ultrastructure analysis. TEM images were pseudocolored to point out key rod structures as follows: OS = yellow, IS = magenta, CCs/BBs = blue. Single rod examples are magnified from both conditions to emphasize intact CC ultrastructure and the preservation of the IS plasma membrane (magenta arrows). The locations of the DAPs and rootlet are annotated in (D). (E) Z-projection SIM images of Rho-GFP/+ IS-enriched retinas immunolabeled with NbGFP-A647 (magenta) and centrin antibody (green). Fluorescence in the single rod example corresponding to the OS, IS, and CC are annotated. (F) Control SIM image of a WT IS-enriched retina immunolabeled and imaged as in (E) to demonstrate NbGFP-A647 labeling specificity. Scale bar values match adjacent panels when not labeled. BB, basal body; CC, connecting cilium; DAP, distal appendage; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; OS, outer segment; Rho, rhodopsin; STX3, syntaxin 3; SIM, structured illumination microscopy; TEM, transmission electron microscopy; WT, wild-type.</p