A pipeline for identification and validation of tumor-specific antigens in a mouse model of metastatic breast cancer

Figures & data

Table 1. NetH2pan PyMT peptide predictions. The PyMT protein amino acid sequence was entered into NetH2pan with the following search parameters: Peptide Length: 8-11mer peptides, Species/loci: Mouse (H-2), Allele: H-2D^q, and sorted by predicted affinity. Bold: eluted PyMT peptides from soluble MHC/tumors

Rank	Peptide	% Rank
1	VPQLIPPPI	0.0148
2	YPRTPPELL	0.0420
3	LPRQLWGDF	0.0472
4	LPSLLSNPTY	0.0557
5	MPMEDLYLDI	0.1372
6	VPQLIPPPII	0.2031
7	IPRAGLSPW	0.2755
8	TPTRDVLNL	0.3715
9	RLPSLLSNPTY	0.4070
10	LPSLLSNPT	0.4477
11	YPRTPPEL	0.4783
12	QVPQLIPPPI	0.4825
13	HPHILLEEDEI	0.5053
14	LPSLLSNPTYS	0.5601
15	LGRTLLLVTF	0.6029
16	MPLTCLVNV	0.6511
17	LAALLGICL	0.6614
18	YMPMEDLYLDI	0.6718
19	FIASMPIDW	0.7104
20	YPRTPPELLY	0.7917
21	HPDKGGSHAL	0.7939
22	LKLPRQLWGDF	0.8135
23	MPMEDLYLDIL	0.8690
24	LPGEQVPQLI	0.8777
25	IPPPIIPRAGL	0.8827

Figure 1. Soluble MHC elution matches H-2D^q profile and identifies H-2D^q PyMT-peptides . a) ELISA results of sMHC production from HeLa cells plotted against a standard curve of purified VLDL-tagged sMHC. b) Evaluation of PyMT expression using flow cytometry in HeLa-H-2D^q cells. An N-terminal flag-tag was engineered onto PyMT and an anti-FLAG antibody was used for staining. c) Peptide length distribution, d) Binding motif generated with Seq2Logo, e) Source protein distribution of eluted peptides, c-e) represent data from 8424 peptides eluted from soluble H-2D^q and PyMT co-transfected HeLa cells, f,g) MS2 fragment spectra from DDA acquisition containing b/y ions from synthetic MT_241-250 and MT_288-296 and sMHC-eluted peptides

Figure 2. MMTV-PyMT tumors present PyMT-specific class I MHC peptides. a) Experimental approach to identify peptides directly from tumors. Peptides were extracted and identified using two types of data acquisition. DDA = Data Dependent Acquisition, DIA/SWATH = Data Independent Acquisition. b) MS2 spectra from DDA acquisition: b/y ions of PyMT peptides eluted from tumors vs. high-purity synthetic peptides. c) SWATH acquisition data matched to PyMT synthetics using Skyline Software. Peaks were matched by retention times and peak area. Top panel: precursor, [M + 1], [M + 2] compared to theoretical isotopic distribution, bottom panel: b/y ions compared to library. Analysis of idotp/dotp show close agreement to expected/library values. RT = Retention Time

Table 2. Summary of PyMT peptides: ELISPOT responses and complementary approaches used for each peptide identification; NetH2pan rank corresponds to “Rank%” from NetH2pan H-2D^q output

Figure 3. DNA vaccination confirms H-2D^q PyMT epitope immunogenicity: a) PyMT and GM-CSF western blot following transient transfection of 293T cells with DNA plasmid vaccine: (-) untransfected 293T, (+) transfected 293T. GM-CSF transfected cells were treated with cellular transport inhibitor for 4 hours prior to analysis, b) DNA vaccine timeline, c) Overlapping 15mer design. MT_19–27: pool 1 (#1-10), MT_107–115: pool 4 (#31-40), MT_241–250: pool 8 (#71-80) and 9 (#81-90), MT_288–296: pool 10 (#91-100), MT_326–335: pool 11(#101-110), d) Anti-IFNγ ELISPOT following 24 hour overlapping peptide restimulation of splenocytes from vaccinated mice, e) Anti-IFNγ responses with representative ELISPOT picture to minimal epitopes of PyMT H-2D^q peptides following 24 hour peptide stimulation of splenocytes from vaccinated mice. Each row represents one mouse; columns represent duplicates of peptide or DMSO-only restimulation

Figure 4. PyMT DNA vaccination prevented MMTV-PyMT tumor growth

a) Prime/boost and tumor implantation timeline: mice were immunized with PyMT+GM-CSF (or control plasmid + GM-CSF) and boosted every 2 weeks, 250,000 MMTV-PyMT tumor cells were implanted at 6 weeks, and animals euthanized at 10 weeks or when tumors reached 2cm³, b) Tumor size measurements (0 = time of implantation) reveal PyMT+GM-CSF vaccine prevents tumor growth (**p < .005, linear regression analysis), c) Survival curve shows vaccine promotes survival up to 15 weeks (*p < .05, log-rank test), d) ELISA for PyMT-specific antibodies in the plasma of vaccinated mice one day after the 1^st booster (n = 5).

Figure 5. PyMT-D^q tetramers label antigen-specific CD8 + T cells after vaccination. a) Top Row) Naïve FVB mice were vaccinated and boosted twice with PyMT + mGM-CSF DNA plasmids. Two weeks after the final booster, splenocytes were harvested and analyzed with flow cytometry. Bottom Row) Naïve FVB mice received mammary fat pad transplants of 250,000 MMTV-PyMT tumor cells. Once tumors reached 2cm³, mice were euthanized and splenocytes were stained with tetramers for PyMT-specific T cells. Left-to-Right) H-2D^q tetramers in separate staining panels: MT_107-115, MT_241-250, and MT_288-296. These data have previously been gated on viability/CD3+/CD8α+. b) Quantification of tetramer staining, c) Phenotype of CD8 + T cells in naïve, tumor-bearing, vaccinated, or antigen-specific (vaccinated) settings. Each of the panels were gated on viability/CD3+/CD8 α +, with an additional gate for tetramer+ staining. d) Quantification of CD62L^lowCD44^hi populations. Panels B&D groups (n = 2/group): Naïve Spleen (clear), Unvaccinated Tumor-bearing (dark gray), PyMT Vaccinated (black), PyMT Vaccinated>Tetramer+ (light gray)