Case Study:
Pro5® Pentamers identify allo-restricted T cells in study to advance research into cancer immunotherapy

Stronen, E. et al. (2009). Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumor cells. Scand J Immunol. 69(4): 319-328. [PubMed ID: 19284496]

Many tumor-associated antigens (TAA) are derived from self proteins that are expressed at low levels in normal tissues. Cancer patients therefore do not produce a cytotoxic T lymphocyte (CTL) response to tumors over-expressing these TAA as they are recognized as self proteins. If CTL can be generated for TAA that are presented by foreign MHC molecules and transferred to the cancer patient, self-tolerance could be avoided. These allo-restricted CD8+ T cells could be used as an immunotherapy to kill the patient cancer.

Stronen et al investigated the use of dendritic cells (DC) as antigen-presenting cells to generate highly specific, functional allo-restricted T cells. Monocyte derived dendritic cells from HLA-A*02:01 negative individuals were transfected with A*02:01 and loaded with MART-1 (ELAGIGILTV) peptide. The transfected DC were co-cultured with monocyte-depleted PBMCs from the A*02:01 negative donor and reactive T cells identified using a Pro5® A*02:01/MART-1 Pentamer. Pentamer-positive cells were sorted by FACS or magnetic bead isolation and then expanded in vitro (figure 1). These MART-1 Pentamer positive T cells effectively killed A*02:01 melanoma tumor cell lines indicating that the sorted and expanded cells remain peptide specific.

Data showing anti-CD8 and A2/MART-1 Pentamer staining Figure 1: 

Induction of MART-1-positive cytotoxic T lymphocytes (CTL) from human leucocyte antigen (HLA)-A2-negative donors. Data plots are representative of PBMC from HLA-A*02:01 donors showing anti-CD8 and A2/MART-1 Pentamer immediately after isolation (left) or after 12 days of co-culture with MART-1 peptide-pulsed A2-monocyte-derived dendritic cells (right).

The validity of this novel methodology was confirmed by generating cell lines for peptides from 2 other leukemia associated self-antigens for CD33 and CD19. Peptides were selected using binding algorithms to predict potential epitopes. Custom Pro5® Pentamers were synthesized for both complexes (A*02:01/CD33 (9-17) LLWAGALAM and A*02:01/CD19 (279-287) VLWHWLLRT). Pentamer-positive cells could be detected ex vivo after 19 days in culture and these cells were successfully expanded whilst retaining peptide specificity against peptide pulsed A2 transfected EBV-LCLs.

Data showing anti-CD8 and A2/CD19 or A2/CD33 Pentamer staining Figure 2: 

Allo-restricted T cells specific for CD19 (279-287) and CD33 (9-17). Data plots are representative of PBMC from HLA-A*02:01 donors showing anti-CD8 and A2/CD19 or A2/CD33 Pentamer immediately after isolation (left), after 19 days (centre) or 38 days (right) of co-culture with specific peptide-pulsed A2-monocyte-derived dendritic cells.

These data show that it is possible to isolate allo-restricted, antigen-specific T cells and expand these cells to high numbers whilst still retaining functional specificity. The use of both catalogue and custom Pro5® Pentamers to identify and confirm specificity of the allo-restricted T cells was essential in this study.