Immunization with truncated sequence of Telomerase Reverse Transcriptase induces a specific antitumor response in vivo

Figures & data

Table

Rank	Start position	Subsequence residue listing	Score (Estimate of half time of disassociation of a molecule containing this subsequence)
H2-Dd binding epitope
1	760	LQPYMGQFL	20.000
2	822	QGIPQGSSL	20.000
3	899	NFPVEPGTL	20.000
4	826	QGSSLSTLL	20.000
5	920	LFPWCGLLL	20.000
6	907	LGGAAPYQL	20.000
H2-Kd binding epitope
1	627	SYSMGTRAL	2400.000
2	612	RFIPKPNGL	2304.000
3	862	DFLLVTPHL	1920.000
4	682	AFVLRVRAL	1920.000
5	838	CFGDMENKL	1382.400
6	749	SFRRQVTTL	960.000
7	643	HFTQRLKTL	800.000
8	899	NFPVEPGTL	800.000
9	920	LFPWCGLLL	800.000
10	798	LFDFFLHFL	800.000
11	858	RFVDDFLLV	691.200
12	676	IYRTWRAFV	500.000
H2-Db binding epitope
1	777	SALRNSVVI	5588.352
2	668	SSVLGMNDI	86.619
3	652	FSMLNYERT	81.331
4	790	SMNESSSSL	52.557
5	752	RQVTTLSDL	21.470
H2-Kb binding epitope
1	882	HGVPEYGCM	24.000
2	834	LCSLCFGDM	11.000
3	937	CDYSGYAQT	10.000

Figure 1.  (A) The expression of TERT^t in B16 and CT26 cells. 1: DNA marker I; 2: RT-PCR product of -actin in B16 cells; 3:RT-PCR product of TERT^t in B16 cells; 4: RT-PCR product of -actin in CT26 cells; 5: RT-PCR result of CT26 cells using TERT^t primers. (B) Linearization of pBluescriptIIKS(+)-TERT^t : 1. DNA marker I 2. Linearized pBluescriptIIKS(+)-TERT^t template 3.Double enzyme digestion of pBluescriptIIKS(+)-TERT^t. (C) In vitro transcription of TERT^t mRNA: 1. RNA marker 2.Capped TERT^t mRNA 3. Capped TERT^t mRNA with poly-A tail.

Figure 2.  DCs were eletroporated with EGFP mRNA to evaluate the transfection efficiency. Post EGFP mRNA electroporation 24h, EGFP expression can be detected in CD11c⁺ DCs using FCM analysis. (A) The DCs of 8th day culture system showed no EGFP expression; (B) After EGFP mRNA electroporated for 24h, The DCs of 7th day culture system showed EGFP expression.

Figure 3.  TERT^t mRNA transfected DCs immunization can induce B16 specific CTLs in C57B/L mice. (A) TERT^t mRNA transfected DC immunization can induce IFN-γ secreting specific Splenocytes responding to Mitomycin C treated B16 cells. 1: Splenocytes of Blank DC immunized mice; 2: Splenocytes of TERT^t-DC immunized mice; 3: Splenocytes of hMAGEn-DC immunized mice; 4: Negtive control (Splenocytes of Blank DC immunized mice, no target). 5: Positive control (Splenocytes of Blank DC immunized mice, no target, 1mg/L PHA stimulated). (B) Splenocytes obtained from TERT^t mRNA transfected DCs immunized mice showed escalated cytotoxic activity against Mitomycin C treated B16 cells at E:T ratios indicated. ▴: Splenocytes of TERT^t-DC immunized mice; •: Splenocytes of Mn-DC immunized mice; ▪: Splenocytes of Blank-DC immunized mice. (Values are the mean±SD of triplicate wells)

Figure 4.  (A) Primary CT26 cells showed undetectable TERT activity. After stimulated with target cells for 24 hours, splenocytes derived from TERT^t-DC immunized mice only menifested IFN-γ secreting response against CT26-TERT^t cells but not CT26 or CT26-hMAGEn cells. 1: Splenocytes of Blank DC immunized mice; 2: Splenocytes of TERT^t-DC immunized mice; (Values are the mean±SD of triplicate wells) (B) TERT^t mRNA transfected DC immunization can generate TERT^t specific CTLs in BALB/c mice in vivo. Splenocytes of TERT^t-DC immunized mice displayed strong cytolytic activity against CT26-TERT^t at E:T ratio of 10:1, 20:1 and 40:1. Left: Splenocytes of Blank DC immunized mice; Middle: Splenocytes of TERT^t-DC immunized mice. (Values are the mean±SD of triplicate wells)

Figure 5.  The Survival curve of different mRNA transfected DCs immunized/ B16-challenged mice. Log-rank test showed that the median survival time was different in these three groups of mice (p = 0.029). The mice vaccinated with TERT^t-DC showed longer survival time than those vaccinated with Blank DC or hMAGEn-DC. 1: Blank DC immunized mice with B16 challenged; 2: TERT^t-DC immunized mice with B16 challenged; 3: hMAGEn-DC immunized mice with B16 challenged.

Image1.  The TERT^t expression can be detected in Dendritic cells by using immunofluorescence assay 24 hours post TERT^t mRNA electroporation. (First antibody: rabbit anti mouse TERT; Second antibody: FITC-goat anti rabbit IgG)