Detection of spliced and unspliced forms of germline TCR-Vβ transcripts in extrathymic lymphoid sites

Abstract

Germline TCR-Vβ transcription is commonly considered an event coupled with rearrangement of TCR genes in T cells. The extent of germline Vβ transcription is studied here in a range of cell types and in several mouse strains. A sensitive semi-quantitative RT-PCR method was developed to specifically detect germline and not rearranged transcripts. Germline transcription of a range of different Vβ genes was detected along with rearranged transcripts in bone marrow, thymus, mesenteric lymph node and spleen. Some transcripts were also detected in low level in non-lymphoid tissues including heart, liver and brain. Expression was also studied in the C57BL/6J-β₂microglobulin^−/− (C57BL/6J-β₂M^−/−) mouse model that lacks NK1.1 T cells and predominantly utilises Vβ8.2 in the formation of a TCR. β₂M^−/− mice, which lack both CD1-dependent NK1.1 T cells and CD8⁺ T cells, showed germline TCR-Vβ8 transcription in most tissues indicating that germline transcription is not specifically related to CD1-dependent NK1.1 T cells. In many tissues, multiple transcripts were amplified representing both spliced and unspliced forms of germline Vβ. For most Vβ genes, the expression of spliced and unspliced forms was equivalent. Given an abundance of unspliced transcripts, the presence of alternative ORFs encoding a novel protein was investigated within the TCR-Vβ genes. Sequence analysis of ORFs showed only genes with a high level of similarity to TCR-β. All data reflect the prevalence of germline transcripts in vivo and raise questions about their functional role.

Introduction

Germline transcription is the production of mRNA transcripts from either the unrearranged TCR or Ig loci which are in ‘germline’ configuration. A common view has been that production of germline transcripts is tightly coupled to rearrangement events that take place at these loci to produce a functional T cell receptor (TCR) or B cell receptor (BCR). If this is the case, one would expect germline transcripts to be produced just prior to VDJ recombination when the chromatin is open and fully accessible to the transcriptional machinery. Contradictory evidence shows germline TCR-Vβ transcripts in mice well before the TCR-Vβ rearrangement event, when TCR rearrangement is blocked (Candeias et al., 1994, Chen et al., 2001, Jolly and O’Neill, 1995), and in tissues that contain cells expressing a fully rearranged TCR (Candeias et al., 1994, Jolly and O’Neill, 1997, O’Neill and Jolly, 1995). This evidence refutes a model for tight linkage between the TCR rearrangement process and the germline transcription event. In addition, germline transcripts were thought to be sterile and non-functional. However, there is evidence that these transcripts may encode and present a TCR-Vβ peptide on the cell surface in the absence of a Cβ region (Abbey et al., 2006, Jolly and O’Neill, 1995). This is a novel finding since the Vβ domain confers antigen specificity whilst the Cβ domain anchors the receptor in the membrane. This raises many questions about the functional role of germline (GL)-Vβ transcripts within the cell, whether they are translated, and whether they play a functional role in vivo.

Earlier studies on the prevalence of germline (GL) TCR-Vβ transcription identified only GL-Vβ8.2 transcripts in lymphoid tissues and in the lymphoid precursor cell line, C1-V13D (Abbey and O’Neill, 2004, Candeias et al., 1994, Jolly and O’Neill, 1995, O’Neill and Jolly, 1995). Expression of GL-Vβ genes other than Vβ8.2 was then demonstrated in the C1-V13D cell line (Abbey and O’Neill, 2004). An extensive analysis of Vβ gene expression in lymphoid tissues using germline-specific primers has not been attempted. A semi-quantitative study by Chen et al. (2001) examined GL-Vβ expression in thymus of RAG-2^−/− mouse. This experimental system relied entirely on RAG-2^−/− mice to avoid detection of rearranged transcripts and forward primers were located in the Leader (L) and Variable (V) exon of the particular Vβ gene. Primers would not distinguish between rearranged and germline transcripts if the RAG-2 mutation was leaky, and gave no indication of the relative levels of rearranged versus germline transcripts in any tissue or cell type. In addition, this semi-quantitative assay did not standardise gene expression in relation to a house-keeping gene, but to genomic DNA. Whilst the copy number of each gene may be known, this method does not account for differences between samples in efficiency of RNA extraction and cDNA synthesis.

Tissues studies so far have failed to clarify whether GL-Vβ8.2 transcripts are produced by a single cell type analogous to C1-V13D or whether they are produced by a range of cells. C1-V13D cell surface markers have been characterised, and cells found to bind anti-NK1.1 antibody (O’Neill, 1992). This raises the question of whether C1-V13D is an NK1.1 T cell line, and whether the presence of GL-Vβ8.2 expression in lymphoid tissues reflects the presence of NK1.1 T cells, consistent with the fact that some NK1.1 T cells express a mature T cell receptor (TCR) which preferentially utilises the Vβ8.2 gene (Arase et al., 1992). However no information exists on germline TCR-Vβ8.2 transcription in NK1.1 T cells.

A comparison of the expression levels of both germline and rearranged Vβ8.2 transcripts involved primers specific for both leader (L)5.1 and L8.2. Analysis was performed in several strains of mice including C57BL/6J, DBA/2j and β₂microglobulin^−/− (C56BL/6J-β₂M^−/−) mice which have limited numbers of NK1.1 T cells (Arase et al., 1992, Coles and Raulet, 1994). β₂M forms part of the MHC class I complex and also plays an indirect role in the development of NK1.1 T cells through association with CD1 (Ohteki and MacDonald, 1994). In β₂M^−/− mice, CD1 is not expressed and development of CD1-dependent NK1.1 T cells is impeded (Bendelac et al., 1997). Comparison of germline transcription in β₂M^−/− and parent C57BL/6J mice, should reveal any association between germline transcripts and NK1.1 T cells. A semi-quantitative RT-PCR method has been used here to detect a range of germline TCR-Vβ transcripts including GL-Vβ8.2.