Cloning and large-scale expansion of epitope-specific equine cytotoxic T lymphocytes using an anti-equine CD3 monoclonal antibody and human recombinant IL-2

Abstract

Cytotoxic T lymphocytes are involved in controlling intracellular pathogens in many species, including horses. Particularly, CTL are critical for the control of equine infectious anemia virus (EIAV), a lentivirus that infects horses world-wide. In humans and animal models, CTL clones are valuable for evaluating the fine specificity of epitope recognition, and for adoptive immunotherapy against infectious and neoplastic diseases. Cloned CTL would be equally useful for similar studies in the horse. Here we present the first analysis of a method to generate equine CTL clones. Peripheral blood mononuclear cells were obtained from an EIAV-infected horse and stimulated with the EIAV Rev-QW11 peptide. Sorted CD8+ T cells were cloned by limiting dilution, and expanded without further antigen addition using irradiated PBMC, anti-equine CD3, and human recombinant IL-2. Clones could be frozen and thawed without detrimental effects, and could be subsequently expanded to numbers exceeding 2 x 10(9)cells. Flow cytometry of expanded clones confirmed the CD3+/CD8+ phenotype, and chromium release assays confirmed CTL activity. Finally, sequencing TCR beta chain genes confirmed clonality. Our results provide a reliable means to generate large numbers of epitope-specific equine CTL clones that are suitable for use in downstream applications, including functional assays and adoptive transfer studies.

Fig. 1

Time-line and general strategy for producing Rev-QW11-specific CTL clones. In the middle of each week starting with week 3, rapidly growing clones were often split 1:2 and fed with fresh media and rhuIL-2.

Fig. 2

Flow cytometric analysis of A2150 PBMC after one week of stimulation with the Rev-QW11 peptide. a. Stimulated PBMC immediately prior to sorting. b. Phenotype of cells sorted by FACS using anti-equine CD3 and CD8 monoclonal antibodies. Sorted cells were subsequently cloned by limiting dilution.

Fig. 3

Functional and phenotypic characteristics of representative Rev-QW11-specific CTL clones. a. Rev-QW11-specific CTL activity of five different CTL clones was determined on A2150 EK targets pulsed with increasing concentrations of Rev-QW11 peptide. E:T cell ratio was 20:1. Error bars are standard error. b. CD3+/CD8+ phenotype of CTL clones R8-0603 and R5-0603 following cryopreservation and four weeks of additional expansion to large numbers. c. Rev-QW11-specific CTL activity of CTL clones R8-0603 and R5-0603 assayed at the same time as b. Percent specific lysis was determined on A2150 EK targets pulsed with 10⁴ nM Rev-QW11 peptide. E:T cell ratio was 20:1. Error bars are standard error.

Fig. 4

TCRβ chain amino acid sequences encoded by cDNA clones derived from a. Rev-QW11-specific CTL clone R17-1005, b. CTL clones R3.3-1006 and R7.2-1006 (with the R17-1005 sequence shown for comparison), and c. bulk PBMC (with the R17-1005 sequence shown for comparison). For each, total RNA was extracted and the TCRβ variable segment genes amplified by RT-PCR using the 5' RACE procedure with a 3' primer in the constant region. RT-PCR products were TA cloned, sequenced, and aligned. The sequences shown for R3.3-1006 and R7.2-1006 represent 16 and 15 TA cloned sequences, respectively. Leader sequence, variable, diversity, joining, and constant domains were assigned based on published equine and human TCRβ sequences.