A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia

Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy

The Role of Antigen Cross-presentation From Leukemia Blasts on Immunity to the Leukemia-associated Antigen PR1

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Since neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B-cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination and proteasome processing for cross-presentation. Using anti-PR1/HLA-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, while DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classical MHC class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia

Delta-24-RGD oncolytic adenovirus elicits anti-glioma immunity in an immunocompetent mouse model

Background: Emerging evidence suggests anti-cancer immunity is involved in the therapeutic effect induced by oncolytic viruses. Here we investigate the effect of Delta-24-RGD oncolytic adenovirus on innate and adaptive anti-glioma immunity. Design: Mouse GL261-glioma model was set up in immunocompetent C57BL/6 mouse for Delta-24-RGD treatment. The changes of the immune cell populations were analyzed by immunohistochemistry and flow cytometry. The anti-glioma immunity was evaluated with functional study of the splenocytes isolated from the mice. The efficacy of the virotherapy was assessed with animal survival analysis. The direct effect of the virus on the tumor-associated antigen presentation to CD8+ T cells was analyzed with an in vitro ovalbumin (OVA) modeling system. Results: Delta-24-RGD induced cytotoxic effect in mouse glioma cells. Viral treatment in GL261-glioma bearing mice caused infiltration of innate and adaptive immune cells, instigating a Th1 immunity at the tumor site which resulted in specific anti-glioma immunity, shrunken tumor and prolonged animal survival. Importantly, viral infection and IFNγ increased the presentation of OVA antigen in OVA-expressing cells to CD8+ T-cell hybridoma B3Z cells, which is blocked by brefeldin A and proteasome inhibitors, indicating the activity is through the biosynthesis and proteasome pathway. Conclusions: Our results demonstrate that Delta-24-RGD induces anti-glioma immunity and offers the first evidence that viral in

The Role of Antigen Cross-presentation From Leukemia Blasts on Immunity to the Leukemia-associated Antigen PR1

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Since neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B-cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination and proteasome processing for cross-presentation. Using anti-PR1/HLA-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, while DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classical MHC class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia

Delta-24-RGD infection and IFNγ increased the presentation of TAA to CD8+ T cells.

<p>A. OVA expression in GL261-OVA and B16-OVA cells. Whole cell lysates were analyzed by immunoblotting for the expression of OVA protein. Actin protein levels are shown as a loading control. P: parental cells; O: OVA-expressing cells. B. Presentation of OVA antigen by OVA-expressing tumor cells to OVA-specific CD8+ T cells. Cells were infected with Delta-24-RGD (GL261 and GL261-OVA at 100 pfu/cell, B16 and B16-OVA at 20 PFU/cell) in the presence or absence of IFNγ (200 u/ml, added 4hrs post infection) for 48 h. After fixed with PFA, cells were incubated with OVA-specific CD8+ T cells for 18 h and then the activation of the T cells (upregulation of IL-2 promoter activity) were assessed with β-Glo assay. The relative β-gal activity refers to the folds of activity compared to the activity from the group with mock-treated parental cells (assigned as 1). Data are represented as mean ± S.E.; <i>n</i> = 3. <i>P</i><0.05 (Student's t-test, double sided). C. MHC I expression on the cell surface as assessed by flow cytometry analysis. Cells were infected with Delta-24-RGD as in B. The cells were then immunostained with mouse anti-mouse MHC I (H-2Kd) APC and mouse IgG2a APC and analyzed with flowcytometry for cell surface MHC I expression. Top panels in B and C: GL261-OVA; bottom panels in B and C: B16-OVA.</p

Delta-24-RGD triggered anti-tumor immunity and prolonged the survival of glioma-bearing mice.

<p>A. Delta-24-RGD was injected into the intracranial tumor on days 4, 7, 9 after tumor implantation. On day 15, the spleens of 4 mice from each group were harvested, grouped together, and the splenocytes were isolated and incubated with the indicated target cells (wt-MEFs, Gl261, or Delta-24-RGD-infected Gl261). Then, IFNγ secreted by the splenocytes was quantified by ELISA. No tumor: naïve mice; Tumor: mice with GL261-glioma; Tumor + virus: mice with GL261-glioma treated with Delta-24-RGD. Data are represented as mean ± S.E.; <i>n</i> = 3. <i>P</i><0.05 (Student's t-test, double sided). B. Kaplan–Meier curves of overall survival of Delta-24-RGD–treated (<i>n</i> = 17) and PBS–treated (<i>n</i> = 16) glioma-bearing mice. <i>P</i><0.0001 (Log-rank test). C. Histological examination of the brains from the long-term surviving Delta-24-RGD-treated mice. Shown is a close-up of a brain section from a representative mouse that was sacrificed 91 days after tumor implantation. The brain was fixed and stained with hematoxylin-and-eosin. Note the needle track (arrow) and tumor sequela (TS) in the implantation site.</p

Delta-24-RGD-mediated presentation of TAA to CD8+ T cells depended on biosynthetic and proteasome activity.

<p>Cells were infected with Delta-24-RGD as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097407#pone-0097407-g005" target="_blank">Figure 5</a>. Brefeldin A (BFA, 6 µg/ml), MG132 (1 µM) or Lactacystin (Lac, 10 µM) was added during the last 24 h of the experiment. After fixed with PFA, the cells were incubated with CD8+ cells for 18 h and then the T-cell activation was assessed by β-Glo assay. Relative data are represented as mean ± S.E.; <i>n</i> = 3. <i>P</i><0.05 (Student's t-test, double sided).</p