High Prevalence of Functional Laa Specific Cytotoxic T Lymphocytes in Healthy Individuals-Implications for Strategies in Adoptive T Cell Therapies of Relapsed Leukemia

While adoptive transfer of virus antigen specific T cells has shown to be effective in therapy of resistant recurrent viremia which is frequently associated with the lack of protective immunity following hematopoietic stem cell transplantation, the transfer of leukemia associated antigen specific (LAA) T cells is less implemented and appears to depend on factors that hamper a successful translation into the clinic. Among them are low frequencies and low antigen affinity of LAA specific T cells which currently mandate laborious in vitro expansion protocols. Moreover, screening of healthy individuals with regard to the presence of LAA specific T cells revealed contradictory results. Since we failed to detect LAA specific T cells in healthy donors using single peptide specificities to known LAA epitopes coupled to MHC Streptamers, here we asked if the use of peptide mixes comprising 15mers overlapping by 11 amino acids and spanning the entire LAA protein could elicit in vitro T cell responses in healthy donors, otherwise undetectable by single peptide staining.

A cohort of 48 HLA A*0201 healthy individuals was screened using intracellular cytokine staining (ICS) after stimulation with tumor specific peptide mixes representing well known LAAs (WT1, PRAME, NY-ESO, Survivin and p53). While distinct T-helper cell responses were not observed in either of the specimen tested, cytotoxic T lymphocytes could be elicited and measured after incubation with peptide mixes for 5 hours and subsequent CD8⁺ IFNγ⁺ staining in 12 out of 48 healthy subjects. Only one individual displayed specifies against multiple antigens (WT1:0,1%; PRAME:0,5%; NY-ESO:0,1%; p53:0.06%), while the remaining responses were directed to one single antigen per individual. Most prevalent and highest T cell frequencies were found against PRAME in 5 out of all screened subjects (mean 0.4±0.3%; max. 0.8%), followed by WT1 in 4 (mean 0.07±0.03%; max. 0.1%) and NY-ESO in 3 individuals (mean 0,07±0,04%; max. 0,1%); one showed CD8 T cells specific against Survivin (0,03%) and 2 individuals had CD8 frequencies specific against p53 (0,05±0,01; max. 0,06%), respectively. The calculated limit of detection (LOD) for the enumeration of LAA specific T cells was 0,02%. In contrary, testing LAA positive individuals with according MHC Streptamers presenting single peptides of previously described epitopes showed no frequencies exceeding LOD. Further analysis showed LAA specific CD8⁺ IFNγ⁺ T cells exhibit mainly a less differentiated phenotype (CD45RA⁺, CCR7^+/-, TNFα⁺, IL-2^+/-) and could be immune-magnetically isolated to purities of 94.5±0.7% using a PRAME-specific IFN-γ capture assay yielding 1*10⁴ antigen specific T cells out of 4*10⁷ PBMCs. Simultaneous enrichment of helper T cells to a purity of 73.0±7.6% proofed their existence, despite no CD4⁺ response could be detected via ICS in the first place. The cytotoxic potential of the cell product was confirmed in an Europium assay using T2 cells loaded with PRAME peptide mix. The specific lysis accounted to 19.3% at an E:T ratio of 1:1 after 90 minutes of co-incubation.

In conclusion, using LAA specific peptide mixes in combination with ICS we were able to show a relatively high prevalence of LAA specific T cells, especially for PRAME, in healthy donors. These LAA specific T cells can be enriched without the need of in vitro expansion culturing ex vivo using the IFN-γ capture assay with regard to achieving a functional LAA specific T cell product for adoptive T cell transfer. Furthermore, a less differentiated phenotype exhibited by a large proportion of LAA specific T cells might contribute to their long term survival in a patient after transplantation.