The common HLA class I-restricted tumor-infiltrating T cell response in HPV16-induced cancer

Abstract

Immunotherapies targeting truly tumor-specific targets focus on the expansion and activation of T cells against neoantigens or oncogenic viruses. One target is the human papilloma virus type 16 (HPV16), responsible for several anogenital cancers and oropharyngeal carcinomas. Spontaneous and vaccine-induced HPV-specific T cells have been associated with better clinical outcome. However, the epitopes and restriction elements to which these T cells respond remained elusive. To identify CD8⁺ T cell epitopes in cultures of tumor infiltrating lymphocytes, we here used multimers and/or a functional screening platform exploiting single HLA class I allele-engineered antigen presenting cells. This resulted in the detection of 20 CD8⁺ T cell responses to 11 different endogenously processed HLA-peptide combinations within 12 HPV16-induced tumors. Specific HLA-peptide combinations dominated the response in patients expressing these HLA alleles. T cell receptors (TCRs) reactive to seven different HLA class I-restricted peptides could be isolated and analysis revealed tumor reactivity for five of the six TCRs analyzed. The tumor reactive TCRs to these dominant HLA class I peptide combinations can potentially be used to engineer tumor-specific T cells for adoptive cell transfer approaches to treat HPV16-induced cancers.

Keywords: HPV16; TCR gene transfer; TIL.

Fig. 1

Experimental set-up for the detection and isolation of HPV16-specific CD8⁺ T cells. Pipelines for the detection and isolation of CD8⁺ T cells from tumor infiltrated lymphocytes (TILs) through pMHC class I multimer staining and the functional screening platform. TIL batches were obtained from HPV16 positive tumors by culturing tumor fragments with 1000 U/ml IL-2. HPV16 E6 and E7 epitopes presented in common and relevant HLA class I were predicted using the Net-MHC platform. The obtained predicted epitopes were curated from published validated ones and used to generate pMHC class I multimers for the detection of HPV16 E6/E7-specific CD8⁺ T cells. In parallel, K562 engineered to express single defined HLA class I and target antigen (shuffled E6 (E6sh) or E7 (E7sh)) were generated and used to screen TIL batches for HPV-specific CD8⁺ T cells. Detection and single cell sorting of HPV-specific CD8⁺ T cells was done following co-culture by making use of the flow cytometry-based IFNγ-secretion assay. Single cell sorted HPV-specific CD8⁺ T cells were used to generate T cell clones and single cell PCR was applied to identify the TCR

Fig. 2

HPV16 E6 and E7-specific CD8⁺ T cells that were restricted to various HLA class I alleles could be detected with our pMHC class I multimer and/or functional screening platform. HPV16 E6- and E7-specific CD8⁺ T cells were detected in TIL batches of HPV16 positive tumors following co-culture with K562 expressing a single defined HLA class I allele and shuffled E6 (E6sh) or E7 (E7sh). a Graphs depicting the amount of IFNγ (pg/ml) produced in response to E6sh (grey) and E7sh (black) expressing K562 cells for eight TIL batches. Each used K562 cell batch was transfected with the depicted HLA class I allele. The asterisk depicts the detection of a positive response, which is defined as IFNγ production that is at least two times that of the T cells alone (dotted line). To confirm the presence of HPV16-specific CD8⁺ T cells in the TIL batches, pMHC class I multimer staining b or CD8/IFNγ staining c of the TIL batches was performed. b pMHC class I multimer double staining of the HLA-A*02:01-restricted epitopes E6_29–38, E7_11–19 and E6_15–23 are depicted for three TIL batches. c CD8/IFNγ staining of unstimulated and K562-HLA-B*15:01/E7sh, K562-HLA-C*07:02/E6sh or K562-HLA-B*40:01/E7sh-stimulated T cells are depicted for four TIL batches

Fig. 3

TCR transduced CD8⁺ T cells are capable of recognizing endogenously processed and presented epitopes on HLA class I allele and E6 or E7 engineered K562 cells. In vitro reactivity of TCR transduced (Td) CD8⁺ T cells against HLA class I and shuffled E6 (E6sh; gray), E7 (E7sh; black) or non-transduced (empty; white) engineered K562 cells was assessed by intracellular IFNγ staining and is depicted as percentage of IFNγ⁺ of TCR Td CD8⁺ T cells. Reactivity of TCR Td T cells alone is depicted by the dotted line. n.t. means not tested

Fig. 4

TCR transduced CD8⁺ T cells are capable of recognizing endogenously processed and presented epitopes on tumor cells. In vitro reactivity of TCR transduced (Td) CD8⁺ T cells against HPV16-expressing tumor cell lines that express the HLA class I allele of interest either naturally or after retroviral engineering (indicated underneath the cell line’s name) as assessed by intracellular IFNγ staining and depicted as percentage IFNγ⁺ of TCR Td CD8⁺ T cells. The percentage IFNγ⁺ of TCR Td T cells alone (TCR alone) is given as negative control and that in response to PMA and ionomycin (PMA/ion) or K562 engineered to express the HLA class I allele and E6sh or E7sh is given as positive control