T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

Abstract

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer.

Keywords: Ebola; Library; Peptide-specific; T-cell clone; Tumour; Type 1 diabetes.

Fig. 1

Overview of T-cell library methodology. (A–C) T-cells were enriched from fresh or frozen peripheral blood mononuclear cells (PBMC) via magnetic separation using anti-CD8 ⁺ or -CD4 ⁺ microbeads, prior to seeding into multiple 96U-well plates (range tested from 300–1500 cells per well) with CD3/CD28 beads at a 1:2 cell:bead ratio. (D) Approximately 2 weeks after initial T-cell activation with the beads, libraries were screened ± peptide(s) by IFNγ enzyme-linked immunospot assay (ELISpot). (E & F) Peptide-reactive wells identified from the screen were then enriched for peptide-specific T-cells using either an IFNγ or dual IFNγ/TNFα capture method. (G) T-cells were then cloned to the single-cell level * or expanded as a line. (H) Clone validation was performed by peptide titration (dose–response), phenotyping, pMHC multimer staining, clonotyping and cytotoxicity assays.

Fig. 2

Generation of clones that recognise EBV, influenza A, and insulin β chain peptides from a type 1 diabetes (T1D) CD8⁺ T-cell library. (A) A CD8⁺ T-cell library (96 wells at 1000 cells per well) from an HLA-A2⁺ donor with T1D was screened by IFNγ enzyme-linked immunospot assay (ELISpot), using T2 as antigen presenting cells. Library cells were tested alone (top), against HLA-A2-restricted peptides from Epstein Barr virus (EBV) (BMFL1_280–288) and influenza A (flu) virus (matrix protein (MP)_58–66) (middle), and against a pool of four HLA-A2-restricted T1D-relevant peptides (preproinsulin (PPI)_15–24, insulin β chain (InsB)_10–18, glutamic acid decarboxylase (GAD65)_114–123 and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)_265–273) (bottom). The number of spot forming cells (SFC) per 3.3 × 10⁴ cells, as determined by the ELISpot reader, is shown for each well. Those used for single-cell cloning following IFNγ enrichment are colour coded (EBV: red, flu: black, T1D: blue). (B) T-cells cloned from the EBV (A, red) and flu (A, black) peptide-reactive wells were stained with cognate pMHC tetramer. (C) A T-cell clone (GD.InsB.4) from the positive well for the T1D peptide pool (A, blue) was tested against individual peptides (data not shown), titrated against the InsB peptide in a MIP-1β ELISA (top), and stained with pMHC dextramer assembled with the same epitope (bottom).

Fig. 3

CD8⁺ T-cell clone generation from T-cell libraries screened with tumour peptides. A CD8⁺ T-cell library (576 wells at 1000 cells per well) from an HLA-A2⁺ donor was screened ± an HLA-A2-restricted tumour peptide pool by IFNγ enzyme-linked immunospot assay (ELISpot), using T2 as antigen presenting cells. (A) Spot forming cells (SFC) per 2.5 × 10⁴ cells is shown for the 10 peptide-reactive wells. (B) Positive wells were then pooled, and tested for individual peptide-specificity by IFNγ ELISpot. SFC per 2.5 × 10⁴ cells for each well is shown (SFC for duplicate wells has been shown in brackets). The pooled cells were then enriched for reactive T-cells based on IFNγ production, and subjected to single-cell cloning. (C & D) One of the clones (THEAK.gp100) was specific for the gp100-derived peptide by MIP-1β ELISA, and also successfully killed multiple HLA-A2⁺ melanoma cell lines (Mel 624, Mel 526, and MM909.24) in a ⁵¹Cr-release assay after 18 h, at a T-cell:tumour cell ratio of 10:1. A library from a second HLA-A2⁺ donor (288 wells at 500 cells per well) was screened as in (A), but with two pools of HLA-A2-restricted tumour peptides. (E) SFC per 5 × 10⁴ cells for the 14 peptide-reactive wells. (F) Cloned T-cells were screened against individual peptides by IFNγ ELISA, and were found to recognise a peptide from either cadherin-3 (CDH3) or Engrailed-2 (EN2). (G) Both the CDH3-specific clone (GD.FIL.6/30) and EN2-3-specific clone (GD.RPA.6/2) were tested for sensitivity to cognate peptide by MIP-1β ELISA. (H) The GD.FIL.6/30 clone was also tested for cytotoxicity towards an HLA-A2⁺ breast cancer cell line (MCF-7), and an HLA-A2⁺ metastatic melanoma cell line (MM909.24) in a ⁵¹Cr-release assay after 4 h.

Fig. 4

Isolation of peptide-specific CD4⁺ T-cells, and Zaire Ebola virus (EBOV-Z) specific CD8⁺ T-cells from T-cell libraries. T-cell libraries (192 wells per library at 1000 cells per well) were established from a healthy HLA-DR1⁺ donor (A–C), and a healthy HLA-A2⁺ donor who had previously participated in an EBOV DNA vaccine trial (D–F). (A) The healthy HLA-DR1⁺ library was screened by IFNγ enzyme-linked immunospot assay (ELISpot) against two pools of peptides, using T2-DR1s as antigen presenting cells (APC). Spot forming cells (SFC) per 5 × 10⁴ cells are shown for the peptide-reactive wells. 3 wells were positive for the influenza A (flu) pool (three putative peptides from haemagglutinin: Flu-1, -2 and -3), and 9 wells were positive for the 5T4 oncofetal protein pool (five putative peptides: 5T4-2, -12, -20, -38 and -PMS). 3 of the positive library wells (C11, D8 and D10), shown to respond to different 5T4 peptides, have been colour coded to illustrate their progression to validated 5T4-specific clones (B). Peptide dose–responses for the T-cell clones grown from these wells (GD.C112.DC, GD.D821.DC and GD.D104.DC) have been illustrated in (C). (D) A second library established from a healthy HLA-A2⁺ EBOV vaccinated individual was screened by IFNγ ELISpot, using T2 cells as APC. SFC per 3 × 10⁴ cells has been shown for the peptide-reactive wells. 2 wells showed a positive response to the pool of three HLA-A2-restricted epitopes (EBOV-Z-NP_150–158, EBOV-Z-NP_202–210, and EBOV-Z-NP_404–412) from EBOV-Z nucleoprotein (NP). These wells were pooled, subjected to IFNγ/TNFα dual enrichment, and then cloned to the single-cell level. (E) All six clones generated a response to EBOV-Z-NP_150–158 peptide. Dose–response curves (MIP-1β ELISA) for three of these clones (ST3.ebola.FLS, ST13.ebola.FLS and ST17.ebola.FLS) are depicted in (F).