1. Modular Three-component Delivery System Facilitates HLA Class I Antigen Presentation and CD8+ T-cell Activation Against Tumors 
Benjamin J Umlauf, Chin-Ying Chung, Kathlynn C Brown 
Molecular Therapy 
Volume 23, Issue 6, Pages (June 2015)
DOI: /mt
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
The minimal antigen delivery system consists of three components. (a) PEGylated stealth liposomes are loaded with an immunogenic human leukocyte antigen (HLA) class 1 restricted peptide derived from measles virus, named H250. The surface of the liposome is modified with a cancer-specific targeting peptide, referred to as H1299.3, which mediates binding and internalization of the liposome into the target cell. (b) The structure of the H peptide dimer. A unique thiol, indicated by the box, is incorporated into the lysine core for downstream conjugation to the liposome. Selection and characterization of the H peptide is discussed in the Introduction Section and reported in ref. 10.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Characterization of human and murine immune and antigen presenting cells. (a) Results of PCR based HLA typing to identify H1993 as a non-small cell lung cancer human cell line that is HLA A*02 positive to serve as antigen-presenting cells. (b) Results of polymerase chain reaction-based HLA typing to identify human peripheral blood mononuclear cells (PBMCs) that are HLA A*02 positive to use in coculture assays. (c) IFNγ secretion of D4 and D9 PBMCs when cultured with 1 μmol/l of H250 peptide or without addition of H250 (blank). (d) C57BL/6 mice were vaccinated with 50 μg of PSMYRVFEVGVIRNP (termed major histocompatibility complex (MHC) class II peptide) in Complete Freund's Adjuvant followed by boast at 2 weeks of 50 μg peptide in Incomplete Freund's Adjuvant. Lymphocytes harvested from vaccinated mice at 4 and 5 weeks postvaccination generated robust IFNγ response from lymphocytes stimulated with HLA Class 1 restricted H250 peptide compared to lymphocytes not cultured with H250 peptide (blank).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
H targeting peptide facilitates presentation of encapsulated H250 immunogenic peptide in MHC and HLA class I molecules. (a) H1993 cells were treated with liposomes containing H250 targeted by previously established cancer specific targeting peptides that internalize into H1993 (H1299.2, H2009.1, and H1299.3) and one non-internalizing ligand (H460.1) as indicated on the x-axis. Treated H1993 cells were then cocultured with D4 PBMCs (described in Figure 2b,c) and culture supernatants were analyzed for IFNγ secretion. Free H250 peptide (5 μmol/l) is used as positive control (H250 labeled group). The inset table contains amino acid sequence of the targeting peptides used in this figure. (b) H1993 cells treated with Blank liposomes (lacking both H250 and H1299.3), Quenched liposomes (containing H250 but lacking H targeting peptide), or H targeted liposomes containing H250. Post treatment, H1993 cells were then cocultured with D4 PBMCs and culture supernatants were analyzed for IFNγ secretion. Free H250 peptide (5 μmol/l) is used as positive control. CD8− group indicate coculture with CD8+ depleted PBMCs. (c) Repeat of assay presented in (b) using second human donor PBMCs, termed D9. (d) LLC1 cells treated with same groups as presented in b, then cocultured with lymphocytes from H250 vaccinated C57BL/6 mice and analyzed for IFNγ secretion. CD8− group indicates coculture with CD8 depleted peripheral blood leukocytes (PBLs). Single * indicates a P value < 0.05, and ** indicates P value < 0.01.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
H colocalizes with autophagic vesicles. (a) LLC1 cells expressing GFP-LC3B (green) are incubated with 100 nmol/l of H AF546 (red). Cells are imaged using live cell confocal microscopy. Blue represents nuclei stained with Hoechst White arrows indicate colocalization of the peptide and LC3B. (b) LLC1 cells expressing GFP-LC3B treated with 100 nmol/l scrambled H AF546. (c) H1993 cells expressing GFP-LC3B treated with 100 nmol/l of H AF546; white arrows indicate colocalization. (d) H1993 cells expressing GFP-LC3B treated with 100 nmol/l of scrambled H AF546. (e) LLC1 cells expressing Lamp1-GFP (green) via Bacmam 2.0 system. Cells are then treated with 100 nmol/l of H AF546 (red) then imaged using live cell confocal microscopy. Blue indicates nuclei stained with Hoechst White arrows indicate colocalization. (f) LLC1 cells expressing Lamp1-GFP treated with 100 nmol/l scrambled H AF546. (g) H1993 cells expressing Lamp1-GFP using Bacmam 2.0 system, treated with 100 nmol/l H AF546. White arrows indicate colocalization. (h) H199.3 cells expressing Lamp1-GFP treated with 100 nmol/l scrambled H AF546. Scale bars represent 20 μm.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Pharmacological and genetic perturbation of autophagy results in loss H facilitated presentation. (a) LLC1 cells are treated with H targeted liposomes containing H250 in the presences of pharmacological inhibitors, fixed, washed and subjected to coculture with PBLs. Coculture supernatants were analyzed for IFNγ secretion. Fixed LLC1 cells incubated with free H250 peptide (5 μmol/l) served as positive control (labeled H250). (b) ATG7 or control siRNA treated LLC1 are subjected to H targeted liposomes containing H250 then cocultured with lymphocytes and analyzed for IFNγ secretion. Free H250 peptide (5 μmol/l) is used as positive control. (c) Western blot quantifying ATG7 levels in LLC1 cells treated with siRNA. β-actin levels are presented as loading control. (d) IFNγ secretion from H1993 treated with siRNA against ATG7 then treated with H targeted liposomes. D9 PBMCs were used in the coculture assay and culture supernatant is analyzed for IFNγ secretion. Free H250 peptide (5 μmol/l) is used as positive control. (e) Western blot quantifying ATG7 protein levels in H1993 cells treated with siRNA. A western blot demonstrating α-tubulin levels is presented as loading control. A single * indicates P value < 0.05.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Treatment with H targeted liposomes containing H250 results in significant reduction in LLC1 tumor growth in vaccinated C57BL/6 mice. (a) Subcutaneous LLC1 tumor growth rate in C56BL/6 mice vaccinated against H250 that are treated every other day I.V. with H targeted liposomes containing H250 as treatment group or lacking H250 as vehicle control (n = 5). (b) Ex vivo IFNγ secretion from tumor and liver cells derived from nonvaccinated C57BL/6 mice treated three times I.V. with either H targeted liposomes containing H250 or vehicle control lacking H250. Isolated, in vivo treated cells, are cultured with PBLs from vaccinated C57BL/6 mice and IFNγ levels are quantified from coculture supernatants. Free H250 peptide (5 μmol/l) is used as positive control (labeled H250). (c) Mean weights of explanted tumors separated by group. (d) Quantification of CD8+ cells present in tumor sections from either treated or control groups. Five fields were counted from each group. (e) Picture of explanted LLC1 tumors with treated group on the left and vehicle group on the right. A single * indicates P value < 0.05 while ** indicates P value < 0.01.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions