Intratumoral and Peripheral Blood T Cells Recognize Mutated Neo-Antigens in Follicular Lymphoma

Introduction: The tumor microenvironment of follicular lymphoma is heavily infiltrated with CD4⁺ and CD8⁺ T cells that are tumor-reactive and produce cytokines such as IFN-g, TNF-a, and GM-CSF that may play a role in tumor control or regression. However, the nature of the tumor antigen(s) recognized by these T cells is unknown. Here, we investigated the possibility that intratumoral T cells recognize mutated tumor antigens, which may be recognized as neo-antigens by the host immune system as opposed to non-mutated proteins that are recognized as self-antigens.

Methods: Whole exome sequencing was performed on three follicular lymphoma tumors and their corresponding germline (peripheral blood mononuclear cells, PBMC) samples obtained at initial diagnosis. Nonsynonymous mutations were identified. The binding affinities of the peptides spanning each mutation to their respective MHC class I and class II molecules were determined in silico by MHC binding algorithms. Peptides predicted to have high binding affinity (IC50<500 nM) were selected for synthesis. For MHC Class I binding peptides, nonamer peptides predicted to bind to HLA-A*0201 (all three follicular lymphoma patients expressed HLA-A*0201) were synthesized and for MHC Class II binding peptides, 40-mer polypeptides were synthesized with the mutated amino acid in the middle. Corresponding wild type peptides were synthesized where necessary. Autologous T cells isolated from PBMC or tumors obtained at initial diagnosis were used to test reactivity to mutated and/or wild type peptides. Autologous PBMCs depleted of T cells were used as antigen-presenting cells in co-culture assays with peripheral blood or intratumoral T cells. IFN-g was assessed in culture supernatants by ELISA.

Results: In patient 1, of 61 nonsynonymous mutations, 9 nonamer peptides and 16 pentadecamer peptides were predicted to bind with high affinity to HLA-A*0201 and HLA Class II alleles expressed in the patient, respectively. Of the 9 nonamer HLA-A*0201-binding peptides, two were recognized by autologous peripheral blood and intratumoral T cells. Of the 16 HLA Class II-binding peptides, autologous peripheral blood and intratumoral T cells recognized four. HLA Class II and HLA-DR blocking antibodies but not HLA Class I and HLA-DP or DQ blocking antibodies inhibited production of IFN-g in response to the HLA Class II-binding peptides suggesting that they were recognized by CD4⁺ T cells. More importantly, the T cells were not reactive against the wild type peptides tested suggesting that the mutations converted the non-immunogenic wild type proteins into immunogenic neo-antigens. Testing of mutated peptides from additional patients is ongoing.

Conclusions: Our results suggest that peripheral blood and intratumoral T cells recognize mutated neo-antigens in follicular lymphoma. These results are consistent with recent results from solid tumors that showed intratumoral CD4⁺ and CD8⁺ T cells primarily recognize mutated neo-antigens. More importantly, administration of such neo-antigen-reactive CD4⁺ and CD8⁺ T cells as adoptive T-cell therapy resulted in long-term tumor regressions in humans. Thus, identification of immunogenic neo-antigens may facilitate development of novel personalized forms of cancer immunotherapy including therapeutic vaccines and adoptive T-cell therapy strategies. Moreover, characterization of such cancer regression antigens will be valuable to understand the effects of immune checkpoint antagonists and other novel immunotherapies.