Altered decamer and nonamer from an HLA-A0201-restricted epitope of Survivin differentially stimulate T-cell responses in different individuals

Abstract

Survivin is a universal tumor antigen that is being currently targeted in vaccine approaches against cancer. Our study here examined the immunogenicity of a novel variant of an HLA-A0201-binding decamer peptide from region 95 to 104 of Survivin (ELMLGEFLKL) with a T→M modification at position 3 in the peptide. We found that this new modified 10-mer peptide had enhanced HLA-A0201 binding and induced a stronger T-cell response over its wild type counterpart peptide (ELTLGEFLKL) in select HLA-A0201(+) normal donors. In addition, when compared to the previously characterized altered 96-104 peptide (LMLGEFLKL) from the same region of Survivin currently used in vaccine trials, we found that both peptides had similar immunogenicity, but donor T cells preferentially reacted strongly to either one or the other, but not strongly to both. These results suggest that these two closely related Survivin peptides yield distinct T-cell responses and that most individuals dominantly respond to one or the other altered peptide. We also found a novel association between positive reactivity to the new altered decamer Survivin peptide in some individuals and their expression of the HLA-C0701 allele along with HLA-A0201. Thus, vaccinating with both the 10-mer and 9-mer peptides would be required to immunize a maximum number of individuals in the HLA-A0201(+) population and could lead to more consistent T-cell responses against this region of Survivin.

Fig. 1

A. Binding of Survivin-derived peptides from the 95-104 amino acid sequence to the HLA-A0201 molecule. Wild type (wt) or modified (m) versions of the peptides were compared. 95-104wt (ELTLGEFLKL), 95-104m (ELMLGEFLKL), 96-104wt (LTLGEFLKL) or 96-104m (LMLGEFLKL) have been tested in a classical T2 binding assay for their capacity to stabilize HLA-A0201 expression on the surface of T2 cells (see methods). Peptide concentrations of 0.01-100uM have been tested. Results of one out two experiments with similar results are shown. B. Altered 95-104 and 96-104 peptides had increased immunogenicity in comparison to their wild type counterpart. Enumeration of cells secreting IFN-γ by ELISPOT after 3 rounds of stimulation using 95-104wt or 95-104m (left panel), 96-104wt or 96-104m (right panel). Data from left and right panel are derived from one donor. Similar data has been obtained with two additional donors.

Fig. 2

Response to 95-104 and 96-104 altered peptides in normal donors. A. Thirteen HLA-A0201 normal donor PBMCs were used to raise T cell lines against 95-104m or 96-104m Survivin peptides and reactivity to the cognate peptide was assessed after 3 rounds of stimulation by IFN-γ ELISPOT. Results are expressed in number of spots per 200,000 cells. HIV REV peptide (ILKEPVHGV) was used to control for non-specific IFN-γ secretion. For each donor, the two first columns on the left are testing the ability of the 95-104m raised T cell line to recognize 95-104m peptide (or irrelevant REV peptide). The two last columns on the right side of the graph represent the ability of the 96-104m raised T cell line to recognize 96-104m peptide (or irrelevant REV peptide). The donors have been classified according to the preference of the mounted response as 10-mer responders (top row), 9-mer responders (middle row) or no preference/very weak response (bottom row). B. Pulsing APCs with the two altered peptides simultaneously to raise T cell lines abrogates the response to any of the single peptide. Cell lines were raised against individual altered peptide or a mixture of both for 3 rounds of stimulation after which response to individual peptide or to the mixture of the two was assessed by IFN-γ ELISPOT. Left panel represents results from a 95-104m responder, and the right panel represents the results from a 96-104m responder. These experiments represent the results of two 95-104m responders and two 96-104m responders tested. C. Binding of either individual or mixed altered peptides to HLA-A0201 is equivalent. T2 binding assay using 20μm of either peptide or a mixture of both (20μM of each) was performed and % saturation of HLA-A0201 molecule was calculated relative to gp100(209-217)210M mean fluorescence intensity (MFI) at saturation.

Fig. 3

Recognition of the wild type Survivin peptides by T cell lines raised with altered Survivin peptides and plasticity of recognition between the 9-mer and 10-mer Survivin peptides. Results from one normal donor where 4 T cell lines were raised (one for each peptide) and after the third round of stimulation the cell lines were tested in IFN-γ ELISPOT for recognition of the four test peptides including the one they have been raised with. Results are representative of 8 normal donors assessed in separate experiments.

Fig. 4

T cell lines raised with Survivin altered peptides can kill T2 targets pulsed with the 4 test peptides. T cell lines raised against 95-104m (A) or 96-104m (B) from normal donors were tested in a ⁵¹Cr-release CTL assay to evaluate their ability to kill T2 cells pulsed with the 4 test peptides (or HIV REV negative control); the 10-mer 95-104 wild type or modified, the 9-mer 96-104 wild type or modified at the different effector-to-target (E:T) ratios shown on the right. Cytotoxicity assay was performed after the fourth round of stimulation. The two cell lines showed were derived from different donors since we could not have a donor who reacted strongly to both altered peptides. This is representative of 4 separate experiments in 4 different donors.

Fig. 5

T cells raised against the new 95-104m peptide can kill unpulsed targets. A. Survivin expression in MDA 453 breast cancer cell line transduced with HLA-A0201 molecule (MDA 453/A2). Cells were fixed, permeabilized, stained with anti-Survivin-FITC or isotype control and assessed by flow cytometry analysis (isotype control in broken line, Survivin expression in black line). B. T cell lines from the same HLA-A0201⁺ individual raised against the Sur 95-104m or Sur 96-104m peptide by 4 rounds of stimulation with peptide-pulsed APC preferentially kill MDA 453 breast cancer cells expressing HLA-A0201, as measured using a Caspase 3-cleavage CTL assay performed using wild-type MDA 453 (HLA-A0201-negative) or MBA 453/A2 cells as targets. C. INF-γ ELISPOT showing reactivity of T cell lines used in B to Survivin peptides after 3 rounds of stimulation. D-E. 95-104m raised T cell line can kill a target naturally expressing HLA-A0201. D. Killing of a primary HLA-A0201⁺ melanoma cell line freshly established from a patient enrolled in an adoptive cell therapy trial at MD Anderson was assessed in a standard ⁵¹Cr assay. Targets and T cells were incubated with 40 μg/ml of an anti- HLA-Class I antibody (clone W6/32), or with the same concentration of an isotype control antibody at 40:1 or 20:1 effector:target ratio for 5 h at 37°C. E. 95-104m raised T cell line can kill pancreatic cancer cell line CF-Pac-1 (HLA-A0201⁺). Targets and T cells were incubated with 20 μg/ml of an anti- HLA-Class I antibody (clone W6/32), or with the same concentration of an isotype control antibody at 30:1 effector:target ratio for 5h at 37°C.

Fig. 6

The predicted structure of Survivin peptides (green) into HLA-A0201 binding groove (red) was generated by adapting available crystallographic data from HIV gag peptide bound to HLA-A2*0201 (pdb2C7U). The HIV peptide was removed and replaced with Survivin peptides. From left to right are presented the peptide/MHC structure of the 96-104 in its wild type form (LTLGEFLKL), 96-104 modified (LMLGEFLKL) and finally our novel 95-104 modified (ELMLGEFLKL). The area circled in white highlights the difference in structure imposed by the mutation T→M at position 97, affecting both 96-104m and 95-104m peptides. The dotted line symbolizes the distance between the ‘floor’ of the HLA molecule and the highest point reached by the peptide.