Prediction and identification of HLA-A*0201-restricted epitopes from cancer testis antigen CT23

Figures & data

Table 1. Websites for epitope prediction.

Name	Website
BIMAS	http://www.bimas.cit.nih.gov/molbio/hlabind
SYFPEITHI	http://www.syfpeithi.de
NetCTL 1.2	http://www.cbs.dtu.dk/services/NetCTL
NetMHC 4.0	http://www.cbs.dtu.dk/services/NetMHC
NetChop 4.0	http://www.cbs.dtu.dk/services/NetChop
IEDB	http://tools.immuneepitope.org/analyze/html/mhc_processig.html
NetMHCpan-4.1	http://www.cbs.dtu.dk/services/NetMHCpan/

Table 2. In silico analysis of CT23 epitopes restricted to HLA-A*0201.

		BIMAS	SYFTEPITHI	NetCTL	NetChop	IEDB	NetMHC	NetMHCpan
Sequence（name）	Pos	Scores				Rank%
ALLTPTWKA（P1）	47	137.8	23	1.0365	0.03142	0.13	0.6	0.136
RLSNNVEEL（P2）	170	24	26	1.0827	0.66106	0.16	1.10	0.150
VLCSQPVSI（P3）	120	17.7	23	0.7542	0.66247	0.55	2.0	0.532
QLPHTEALL（P4）	317	21.3	22	0.6383	0.12721	2.5	5	2.5
MIMENIQEL（P5）	279	174.2	27	1.3720	0.68066	0.03	0.04	0.024
LLASQSLSI（P6）	407	17.7	25	1.1862	0.97493	0.32	0.5	0.363
ALLVLCYSI（P7）	323	38	26	1.1268	0.88896	1.9	0.4	1.9

Pos: amino acid start position in the protein sequence.

Figure 1. Affinity and stability of CT23 candidate peptides binding to HLA-A2 analyzed by flow cytometry.

(a) HLA-A2 status of T2 cells with CT23 candidate peptides (P1-P7), positive (P⁺)/negative (P⁻)control peptide, and without peptide (NP)

(b) Binding stability of CT23 candidate peptides (P1-P7) to HLA-A2 molecule on the surface of T2 cells .

P⁺: positive control peptide; P⁻: negative control peptide; NP: none peptide loading. MFI: mean fluorescence intensity.

Table 3. The HLA-A2 binding affinity and stability of CT23 candidate peptides and control peptides.

Epitope	MFI^#	FI^##	DC50*
ALLTPTWKA（P1）	15223.5	1.27	6-8 h
RLSNNVEEL（P2）	13851.5	1.06	4-6 h
VLCSQPVSI（P3）	12470	0.86	6-8 h
QLPHTEALL（P4）	14925	1.22	6-8 h
MIMENIQEL（P5）	13690.5	1.04	6-8 h
LLASQSLSI（P6）	14103	1.10	4-6 h
ALLVLCYSI（P7）	21424.5	2.19	6-8 h
KTPFVSPLL（P^−）	11116	0.66	4-6 h
GILGFVFTL（P^+）	15432	1.30	6-8 h

^#: MFI: mean fluorescence intensity.

^##: FI = (mean FITC fluorescence with the given peptide – mean FITC fluorescence without peptide)/(mean FITC fluorescence without peptide).

*: DC50 was defined as the time (h) required for 50% dissociation of the HLA-A2/peptide complex stabilized at t = 0 h.

Figure 2. Expression of CT23 protein in tumor cells detected by immunoblot.

Figure 3. Morphological features of DCs in different stages.

(a) DCs on the 3th day culture;（b）DCs on the 5th day culture; (c) DCs on the 8th day culture.

Figure 4. The surface molecules of DCs detected by flow cytometry.

(a) DCs cultured for 5 days; (b) DCs cultured for 8 days.

Figure 5. Proliferation assay of D8⁺T cell stimulated by peptide-loaded DCs.

P1 ~ 7-DC+T group: CD8⁺T cells co-cultured with P1-P7 peptide-loaded DCs;

CT23-DC+T group: CD8⁺T cells co-cultured with CT23 recombinant protein-loaded DCs;

P⁺-DC+T group:CD8⁺T cells co-cultured with positive control peptide -loaded DCs;

P⁻-DC+T group:CD8⁺T cells co-cultured with negative control peptide-loaded DCs;

NP-DC+T group:CD8⁺T cells co-cultured with No peptide -loaded DCs;

SI: stimulation index of CD8+T cells in each group (Peptides-DC+T group vs. NP-DC+T group;   **p < .01).

Figure 6. IFN-γ production ability of CT23 peptide-induced CTLs.

(a) ELISA analysis of IFN-γ amount in the 12 day co-culture medium of DCs and CD8⁺T cells.

(b, c) ELISPOT analysis of the number of IFN-γ secreting CTLs.

P1-7-CTL: CTL induced by CT23 peptides P1~P7;

CT23-CTL: CTL induced by CT23 recombinant protein.

P⁻-CTL: CTL induced by negative control peptide.

P⁺-CTL: CTL induced by positive control peptide.

NP-CTL: CTL without using any peptide.

Peptides-CTL vs. NP-CTL, *p < .05, **p < .01.

Figure 7. Cytotoxic activity of CT23 peptide-induced CTLs to target cells detected by LDH release.

(a) lysis rate of P7-CTLs to HLA-A*0201⁺ tumor cells. P7-CTLs vs. NP-CTL, **p < .01.

(b) lysis rate of P7-CTLs to T2 cells without or with loading the peptide P7（P7-T2）. **p < .01.