Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens

Abstract

The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neo-antigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.

Keywords: HLA; TILs.

Figure 1. Tandem mass spectra of endogenous mutant peptides identified in 12T

a. KLFEDRVGTIK b. DANSFLQSV. Mutation sites are indicated with the amino acid underlined.

Figure 2. Response of the TIL to candidate neo-antigens identified from its autologous tumor

IFN-γ release measured after overnight co-culture of the TIL with autologous EBV transformed B cells that were pulsed with 1μM of the mutated or wild-type peptides. Error bars represent standard deviation of triplicates.

Figure 3. Response of the TILs to MED15 and TPD52L2 mutant and wild-type peptides in the context of HLA-B*51

A. IFN-γ release measured after overnight co-culture of the TILs with T2 cells that were pulsed with 1μM of the mutated or wild-type peptides or with the autologous and allogenic melanoma cells. B. IFN-γ release measured after overnight co-culture of 12T TILs with T2 cells that were pulsed with tittered concentrations of the MED15 mutated and wild-type peptides. Error bars represent standard deviation of triplicates.

Figure 4. Intracellular staining of IFN-γ after stimulation with the peptides and melanoma

Intracellular IFN-γ was stained after 6 hours co-culture of the TILs with T2 cells that were pulsed with 1μM of the mutated or wild-type peptides or with the autologous melanoma.