Variegated Transcription of the WC1 Hybrid PRR/Co-Receptor Genes by Individual γδ T Cells and Correlation With Pathogen Responsiveness

Abstract

γδ T cells have broad reactivity and actively participate in protective immunity against tumors and infectious disease-causing organisms. In γδ-high species such as ruminants and other artiodactyls many γδ T cells bear the lineage-specific markers known as WC1. WC1 molecules are scavenger receptors coded for by a multigenic array and are closely related to SCART found on murine γδ T cells and CD163 found on a variety of cells. We have previously shown that WC1 molecules are hybrid pattern recognition receptors thereby binding pathogens as well as signaling co-receptors for the γδ T cell receptor. WC1⁺ γδ T cells can be divided into two major subpopulations differentiated by the WC1 genes they express and the pathogens to which they respond. Therefore, we hypothesize that optimal γδ T cell responses are contingent on pathogen binding to WC1 molecules, especially since we have shown that silencing WC1 results in an inability of γδ T cells from primed animals to respond to the pathogen Leptospira, a model system we have employed extensively. Despite this knowledge about the crucial role WC1 plays in γδ T cell biology, the pattern of WC1 gene expression by individual γδ T cells was not known but is critical to devise methods to engage γδ T cells for responses to specific pathogens. To address this gap, we generated 78 γδ T cell clones. qRT-PCR evaluation showed that approximately 75% of the clones had one to three WC1 genes transcribed but up to six per cell occurred. The co-transcription of WC1 genes by clones showed many combinations and some WC1 genes were transcribed by both subpopulations although there were differences in the overall pattern of WC1 genes transcription. Despite this overlap, Leptospira-responsive WC1⁺ memory γδ T cell clones were shown to have a significantly higher propensity to express WC1 molecules that are known to bind to the pathogen.

Keywords: T cell receptor; WC1; co-receptors; pattern recognition receptors; γδ T cells.

Figure 1

Efficiency of Taqman assays for WC1 transcripts. Standard curves for individual WC1 genes’ scavenger receptor cysteine rich a1 domains were amplified and detected with the Taqman primer/probe assays using serially diluted pST2a-WC1 templates. In each case, WC1-12 efficiency is shown as a benchmark.

Figure 2

Memory markers of Leptospira stimulated cultures. Cultures of peripheral blood mononuclear cells from a vaccinated animal subjected to the T_CM and T_EM protocol and cells stained for T cell receptor (TCR)δ with monoclonal antibody (mAb) GB21A (FITC) at the times indicated. The TCRδ⁺ cells were flow cytometrically gated on mAb GB21A⁺ and evaluated for their surface marker expression of either CD44 or CD62L (PE) by two-color immunofluorescence shown as histograms. The mean fluorescence intensity (MFI) is shown in the bar graphs.

Figure 3

Percentage of WC1 transcripts expressed per γδ T cell clone. The percentage of clones with a particular number of different WC1 gene transcripts is shown.

Figure 4

Time-course evaluation of WC1 gene transcription. Evaluation of WC1 gene transcription by selected clones at two different time-points 4 weeks apart are shown. Filled bars show earlier time point and open bars the later. Transcripts are represented as moles of the mean ± SE of replicates. This was done three times with triplicate samples.

Figure 5

WC1 gene transcripts associated with individual WC1.1⁺ or WC1.2⁺ clones. The various combinations of WC1 gene transcripts are shown for clones derived by flow cytometrically sorted cell subpopulations in a heat map. * = WC1 gene products that bind Leptospira.

Figure 6

Diversity index of individual WC1 gene transcription by γδ T cell clones in each WC1 cohort. (A) For each clone the predominately expressed WC1 gene, based on highest mean transcript level, was noted and the total number of times a WC1 gene was the predominant is plotted. (B) Diversity index shows the percentage of times transcripts for the WC1 genes occurred in a cohort of clones (y-axis). WC1 genes most frequently transcribed within the repertoire of WC1 transcripts is indicated as a number x (e.g., WC1-x) in appropriate boxes to add emphasis.

Figure 7

Model for WC1 interaction with pathogens. γδ T cells may express variable numbers of different WC1 molecules thus potentially enabling them to interact with more than one ligand on the same or different pathogens. However, since the number of WC1 co-receptors is necessarily limited on a cell an increase in receptor diversity could affect the avidity of the interaction with any particular ligand.