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. 2022 Feb 11;23(1):5.
doi: 10.1186/s12865-022-00478-4.

Determining if T cell antigens are naturally processed and presented on HLA class I molecules

Jay Friedman  1 , Sreenivasulu Gunti  1 , Maxwell Lee  1 , Ke Bai  1 , Christian Hinrichs  2 , Clint T Allen  3
Affiliations

Determining if T cell antigens are naturally processed and presented on HLA class I molecules

Jay Friedman et al. BMC Immunol. .

Abstract

Background: Determining T cell responses to naturally processed and presented antigens is a critical immune correlate to determine efficacy of an investigational immunotherapeutic in clinical trials. In most cases, minimal epitopes and HLA restriction elements are unknown.

Results: Here, we detail the experimental use of ex vivo expanded autologous B cells as antigen presenting cells to overcome the limitation of unknown HLA restriction, and the use of electroporated full length mRNA encoding full length parental proteins to ensure that any observed T cell responses are specific for antigens that are naturally processed and presented.

Conclusions: This technique can serve as useful experimental approach to determine the induction or enhancement of specific responses to naturally processed and presented antigens on HLA class I molecules in peripheral blood or tumor infiltrating T cells.

Keywords: Antigen presenting cells; Electroporation; HLA restriction; Minimal epitope; T cell responses.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Fig. 1
Fig. 1
Schematic overview of determining antigen-specific T cell responses using electroporated B cells electroporated with full-length mRNA. a pcRNA2SL vector engineered to express a gene of interest is linearized through restriction digestion; b linearized expression plasmid is transcribed into full length 5′ capped and 3′ polyA tailed mRNA through in vitro transcription with a commercially available kit; c peripheral blood B cells are isolated from patient material through magnetic selection and full-length mRNA is introduced via electroporation; d peripheral blood or tumor infiltrating T cells are co-incubated with autologous B cells loaded with full-length mRNA and antigen-specific responses are detected. R.E. restriction element, TIL tumor infiltrating lymphocytes
Fig. 2
Fig. 2
B cells are readily expanded in culture and electroporated with full-length mRNA. a Cell counts of magnetically isolated B cells co-cultured with NIH3T3-CD40L feeder cells and IL-4, error bars represent standard deviation of three independent experiments; b phase contrast and immunofluorescent photomicrographs of ex vivo expanded B cells electroporated without or with mRNA encoding GFP. Images acquired 2 h after electroporation; c flow cytometric analysis of ex vivo expanded B cells electroporated with mRNA encoding GFP for fluorescent signal; d viability of B cells electroporated without mRNA (control), with GFP mRNA or with HPV16 mRNA was assessed via sytox staining on flow cytometry or via acridine orange/propidium iodide staining on the automated cell counter. e Flow cytometric analysis of cell surface CD80, CD86 and HLA-DR expression on B cells before and after in vitro expansion, and with or without electroporation. Hashed bars indicate B cells electroporated with no mRNA, GFP mRNA or HPV16 E7 mRNA. ***, p < 0.001
Fig. 3
Fig. 3
B cells naturally process and present minimal epitope from electroporated full-length mRNA. a Representative photographs of ELISpot wells resulting from co-culture of expanded autologous B cells loaded with E711–19 minimal peptide or 15mer overlapping peptide spanning the entire E7 protein, electroporated with mRNA encoding full-length E7, or controls for each condition; b quantification of IFNγ spots from all experimental controls and conditions. Spot counts greater than or equal to 1000 are depicted as 1000. Representative results from one of three independent assays with similar results. ***, p < 0.001
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