Development of a Novel Platform for in-Vitro Immune-Monitoring of Antigen-Specific T Cells and Discovery of Novel Antigen-Specific TCRs Using Immortalized B Cells Transfected with mRNA of Antigen Fused to Mitd (IBMAM)

Introduction Immunomonitoring of antigen (Ag)-specific T cells is critical in evaluating cellular immune function. While peripheral mononuclear blood cells (PBMCs) are the most widely used study materials for Ag-specific T cells, low-frequency Ag-specific T cells in PBMCs remain as one unsolved technical limitation. While increasing the epitope expression level serves as an effective method in expanding low-frequency Ag-specific T cells, we propose to establish a novel platform where immortalized B cells processing the mRNA of antigens linked to MHC class-I trafficking domain (MITD) [IBMAM] are used as the means for increasing epitope expression levels. We optimized the B cell immortalization, tested epitope minigene arrangement, and compared wild-type (WT)-MITD to MITD-phosphorylational variants in antigen presentation. To determine if IBMAM platform can successfully detect and expand Ag-specific T cells, we applied this platform to HCMV-specific T cell detection and expansion using HCMV-seropositive donors’ PBMC samples. In addition, we hypothesized that the IBMAM platform would be an extremely effective tool in facilitating novel TCR discovery and validation, given the powerful high-throughput TCR sequencing nowadays.

Materials and Methods To establish immortalized B cells in vitro, B cells were isolated from the HCMV-seropositive normal healthy donors (NHDs), stimulated, and transduced with a retrovirus containing BCL-6 and BCL-XL. In vitro transcription constructs for mRNA synthesis were based on the pMRNA-120bp-vector. HCMV-MITD vectors were generated containing the HCMV pp65-full length gene with either MITD-WT, various MITD mutations mimicking phosphorylation, or no MITD. HCMV specific CD8+ T cells (asteria comp) and TCRαβ-KO Jurkat cells (Promega Corporation) were used for T cell activation. Purified CD4+ T lymphocytes obtained from the NHDs PBMC were co-cultured with autologous immortalized B cells transfected with HCMV-MITD-WT mRNA and then measured for activation using IFNγ ELISA. We developed two approaches for IBMAM mediated Ag-specific T cell activation and detection based on whether IFNγ is detectable from the PBMC. For the first approach, if IFNγ is not detected, NHDs PBMC is stimulated twice using IBMAM and subsequently Ag-specific T cells are detected using IBMAM again. For the second approach, if IFNγ is detected, T cells are isolated again from NHDs PBMC and expanded using rapid expansion procedures (REP). After the first REP, Ag-specific T cells are detected using IBMAM, sorted, and a second REP is performed for further study.

Results The three key components of the IBMAM system are the mRNA of antigen, MITD conjugation, and antigen presenting cells (APCs). Our data shows that B cells serving as potent APCs can be successfully immortalized by transfecting at least 5 x 10⁵ cells with retrovirus (1MOI) and co-culturing with CD40L-IL21 expressing human fibroblasts. After coincubation with anti-HCMV T cells, B cells transfected with HCMV-MITD-WT mRNA led to a significantly higher IFNγ secretion in comparison to using HCMV mRNA containing no MITD. While previous studies indicate phosphorylation on serine/tyrosine in MITD can potentially increase MITD's efficacy in antigen presentation, our results suggest a relatively comparable antigen-presentation ability of MITD-WT with the other MITD phosphorylational variants. Our results support a mRNA concentration of 7.5-30ug per 10⁶ B cell dose for B cell transfection, and T:B cell ratios of 1:1 or 1:2 for efficient induction of Ag-specific T cell responses. For approaches 1 and 2, successful detection, activation and expansion of HCMV-specific T cells were observed from low frequency NHDs samples using the IBMAM platform. A panel of TCRs theoretically complexed to NY-ESO-1-_60-72-HLA-B*07:02 were knocked in the TCRαβ-KO Jurkat cells for expression, followed by coincubation with IBMAM platform involving B cells processing the mRNA of NY-ESO-1-MITD. Via measuring Jurkat activation, we have shown that the IBMAM platform is effective in detecting the reactive TCRs.

Conclusions We have established a highly sensitive method of presenting naturally processed epitopes for immuno-monitoring that we will use for the detection of Ag-specific T cells that can be applied to patient samples with low frequency of Ag-specific T cells. This platform can also serve as an effective tool in novel antigen specific TCR discovery.

Neelapu:Bluebird Bio: Consultancy, Honoraria; Adicet Bio: Consultancy, Honoraria, Other: Personal fees, Research Funding; Medscape: Consultancy, Honoraria; Calibr: Consultancy, Honoraria, Other: Personal fees; Unum Therapeutics: Consultancy, Honoraria, Other: Personal fees, Research Funding; Legend Biotech: Consultancy, Honoraria, Other: Personal fees; Precision Biosciences: Consultancy, Honoraria, Other: Personal fees, Research Funding; Incyte: Consultancy, Honoraria, Other: Personal fees; Cell Medica/Kuur: Consultancy, Honoraria, Other: Personal fees; Allogene Therapeutics: Consultancy, Honoraria, Other: Personal fees, Research Funding; Pfizer: Consultancy, Honoraria, Other: Personal fees; Celgene: Consultancy, Honoraria, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; Bristol Myers Squibb: Consultancy, Honoraria, Other: Personal fees, Research Funding; Merck: Consultancy, Honoraria, Other: Personal fees, Research Funding; Kite: Consultancy, Honoraria, Other: Personal fees, Research Funding; Aptitude Health: Consultancy, Research Funding; Bio Ascend: Consultancy, Honoraria; Poseida: Research Funding; Cellectis: Research Funding; Karus Therapeutics: Research Funding; Acerta: Research Funding; Takeda Pharmaceuticals: Patents & Royalties: related to cell therapy..