Fig 2: Comparison of the function of AnnV-IL2 and IL-2 in vitro. (A) Schematic of AnnV-IL2 construct. (B) Antigen experienced OT-1 cells were stimulated with anti-CD3 and anti-CD28 antibodies or left unstimulated for 5 hours. Cells were then pre-treated with or without excess unlabeled AnnV prepared in annexin V binding buffer for 30 minutes. Cells were washed and then stained with Alexa-647 labeled AnnV-IL2 or IL-2 protein. Histograms represent one sample from each group. (C) CTLL2 cells were IL-2 starved for 5 hours and then stimulated with the indicated concentration of AnnV-IL2 or IL-2 for 10 minutes and then immediately fixed. Cells were permeabilized and stained intracellularly with anti-pSTAT5 or isotype antibodies. (D) Antigen inexperienced or (G) antigen experienced CD8+ cells were cultured with anti-CD3 and anti-CD28 antibodies for 24 hours. After 24 hours, media was removed and replaced with media containing anti-CD28 with or without equimolar amounts of AnnV-IL2 or IL-2 and incubated for 5-6 days. Absolute cell counts were determined using a flow cytometer. (E) Antigen inexperienced or (H) antigen experienced CD8+ T cells were cultured with anti-CD3 and anti-CD28 in the presence of equimolar amounts of AnnV-IL2 or IL-2 for 3 days. Brefeldin A was added to culture media on day 3 and IFN? production was quantified by intracellular flow cytometry. ELISA for IFN? in supernatant of (F) antigen inexperienced or (I) antigen experienced cell cultures. (D–I) Control refers to cells stimulated with only anti-CD3 and anti-CD28. Data is represented by mean ± SEM. P values were calculated by ordinary one-way ANOVA with the Tukey-Kramer multiple comparison test, and P < 0.05 is considered statistically significant. ** = < 0.01,*** = < 0.001, **** = < 0.0001. AnnV, Annexin V; MFI, Median fluorescent intensity. NS, not significant.

Fig 3: Human Fc?R Polymorphisms Impact Response to Ipilimumab in Patients with Advanced Melanoma(A) Anti-CTLA-4 response rate analysis in two separate cohorts of advanced melanoma patients, as published by Van Allen et al. (2015) (top) and Snyder et al. (2014) (bottom). For each analysis patients are split into four groups: (1) high load of somatic mutations and presence of germline high-affinity CD16a-V158F polymorphism (SNP+), (2) high load of somatic mutations and absence of germline CD16a-V158F polymorphism (SNP-), (3) low load of somatic mutations and SNP+, and (4) low load of somatic mutations and SNP-. Both homozygous and heterozygous patients were included in the SNP+ groups. Two different measures of mutational load were tested (McGranahan et al., 2016, Turajlic et al., 2017): (left) the number of frameshift indel mutations and (right) the number of non-synonymous single-nucleotide variant (nsSNV) neoantigens. In all cases, high and low are defined as above or below the median value, respectively. In each analysis, patient group (1) is tested for a difference in response rate compared with groups 2–4 using Fisher's exact test. Meta-analysis for each measure (pmeta), across the two patient cohorts was conducted using the Fisher's method of combining p values from independent tests.(B) Survival analysis of patients with advanced melanoma treated with anti-CTLA-4 with low (=median) or high (>median) predicted neoantigen burden with or without the germline polymorphism CD16a-V158F. Log rank p values are displayed with hazard ratio (HR) and confidence interval (CI). In (A) and (B), patients from the Snyder et al. (2014) cohort treated with tremelimumab (n = 3) were excluded.(C) Boxplot showing the expression level of key immune markers from patients with available RNA-seq data from the Van Allen et al. (2015) cohort (n = 30): CD8A, ratio of CD8A divided by FOXP3 and cytolytic activity (defined as the log-average of GZMA and PRF expression). Patients are grouped into responders with high mutational load (based on either measure) and SNP+, compared with all other patients. Boxes show the middle quartile (25%–75%); horizontal bars represent the median; whiskers show either the maximum and minimum values in the dataset or ±1.5 times the interquartile range if the maximum and minimum values exceed these limits. TPM, transcripts per million.(D) Extension of the response rate analysis from (A), top left, with the following additional two groups: high mutational load (for both measures) plus high CD8A expression (>median) plus SNP+ and high mutational load (for both measures) plus SNP+ (top bar graph). In addition, high CD8A expression plus SNP+ and high CD8A expression plus SNP- were compared (bottom bar graph). Due to the small RNA-seq sample size (n = 30), differences were not tested for statistical significance in (C) and (D).See also Figure S5.

Fig 4: T cells infiltration in iExoSTINGa-treated tumors.A–D, quantitative analyses of flow cytometry measurement of CD45+ (A), CD3+ (B), CD4+, and CD8+ (C) cells in the tumors of the indicated groups. A, Kruskall–Wallis with Dunn's multiple comparison test performed; B and C, one-way ANOVA with Bonferroni's multiple comparison test performed. D and E, quantitative analyses of flow cytometry measurement of CD4+Ki67+ (D) and CD8+Ki67+ (E) cells in the tumors of the indicated groups. D and E, one-way ANOVA with Bonferroni's multiple comparison test performed. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Fig 5: Characterization of the tumor microenvironment in spontaneous HPV16-E6E7-expressing oral tumor model in HLA-A2 (AAD) transgenic mice treated with CRT/E7(N53S) DNA vaccine. HPV16-E6E7-expressing oral tumors were induced in HLA-A2 (AAD) mice (4 per group) and treated as described in Fig. 5. One week following the final vaccination, the tumor was surgically extracted for analysis. A Determination of E7(aa11-20) peptide-specific CD8 + T cells using E7 (aa11-20) peptide loaded HLA-A2 tetramer staining followed by flow cytometry analysis. Upper panel is an example of flow cytometry. Bottom panel is vertical scatter plot summarizing the number of E7(aa11-20) peptide-specific CD8 + T cells in the tumor of mice vaccinated with CRT/E7(N53S) or empty vector. B Determination of CD8 + T TILs by flow cytometry analysis. Upper panel is an example of flow cytometry. Bottom panel is the vertical scatter plot summarizing the percent of CD8 + TILs. C Determination of CD4 + TILs followed by flow cytometry analysis. Upper panel is an example of flow cytometry. Bottom panel is the vertical scatter plot summarizing the percent of CD4 + TILs. D Determination of MDSCs by flow cytometry analysis. Upper panel is example of flow cytometry. Bottom panel is the vertical scatter plot summarizing the percent of MDSCs