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Abstract Background WC1 co-receptors belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1+ γδ T cells. We have previously identified partial or complete genomic sequences for thirteen different WC1 genes through annotation of the bovine genome Btau_3.1 build. We also identified two WC1 cDNA sequences from other cattle that did not correspond to sequences in the Btau_3.1 build. Their absence in the Btau_3.1 build may have reflected gaps in the genome assembly or polymorphisms among animals. Since the response of γδ T cells to bacterial challenge is determined by WC1 gene expression, it was critical to understand whether individual cattle or breeds differ in the number of WC1 genes or display polymorphisms. Results Real-time quantitative PCR using DNA from the animal whose genome was sequenced (“Dominette”) and sixteen other animals representing ten breeds of cattle, showed that the number of genes coding for WC1 co-receptors is thirteen. The complete coding sequences of those thirteen WC1 genes is presented, including the correction of an error in the WC1-2 gene due to mis-assembly in the Btau_3.1 build. All other cDNA sequences were found to agree with the previous annotation of complete or partial WC1 genes. PCR amplification and sequencing of the most variable N-terminal SRCR domain (domain 1 which has the SRCR “a” pattern) of each of the thirteen WC1 genes showed that the sequences are highly conserved among individuals and breeds. Of 160 sequences of domain 1 from three breeds of cattle, no additional sequences beyond the thirteen described WC1 genes were found. Analysis of the complete WC1 cDNA sequences indicated that the thirteen WC1 genes code for three distinct WC1 molecular forms. Conclusion The bovine WC1 multi-gene family is composed of thirteen genes coding for three structural forms whose sequences are highly conserved among individual cattle and breeds. The sequence diversity necessary for WC1 genes to function as a multi-genic pattern recognition receptor array is encoded in the genome, rather than generated by recombinatorial diversity or hypermutation.

Abstract Background The scavenger receptor cysteine rich (SRCR) domain is an ancient and conserved protein domain. CD163 and WC1 molecules are classed together as group B SRCR superfamily members, along with Spα, CD5 and CD6, all of which are expressed by immune system cells. There are three known types of CD163 molecules in mammals, CD163A (M130, coded for by CD163), CD163b (M160, coded for by CD163L1) and CD163c-α (CD163L1 or SCART), while their nearest relative, WC1, is encoded by a multigene family so far identified in the artiodactyl species of cattle, sheep, and pigs. Results We annotated the bovine genome and identified genes coding for bovine CD163A and CD163c-α but found no evidence for CD163b. Bovine CD163A is widely expressed in immune cells, whereas CD163c-α transcripts are enriched in the WC1+ γδ T cell population. Phylogenetic analyses of the CD163 family genes and WC1 showed that CD163c-α is most closely related to WC1 and that chicken and platypus have WC1 orthologous genes, previously classified as among their CD163 genes. Conclusion Since it has been shown that WC1 plays an important role in the regulation of γδ T cell responses in cattle, which, like chickens, have a high percentage of γδ T cells in their peripheral blood, CD163c-α may play a similar role, especially in species lacking WC1 genes. Our results suggest that gene duplications resulted in the expansion of CD163c-α-like and WC1-like molecules. This expanded repertoire was retained by species known as "γδ T cell high", but homologous SRCR molecules were maintained by all mammals.

Abstract Background γδ T cells differ from αβ T cells with regard to the types of antigen with which their T cell receptors interact; γδ T cell antigens are not necessarily peptides nor are they presented on MHC. Cattle are considered a "γδ T cell high" species indicating they have an increased proportion of γδ T cells in circulation relative to that in "γδ T cell low" species such as humans and mice. Prior to the onset of the studies described here, there was limited information regarding the genes that code for the T cell receptor delta chains of this γδ T cell high species. Results By annotating the bovine (Bos taurus) genome Btau_3.1 assembly the presence of 56 distinct T cell receptor delta (TRD) variable (V) genes were found, 52 of which belong to the TRDV1 subgroup and were co-mingled with the T cell receptor alpha variable (TRAV) genes. In addition, two genes belonging to the TRDV2 subgroup and single TRDV3 and TRDV4 genes were found. We confirmed the presence of five diversity (D) genes, three junctional (J) genes and a single constant (C) gene and describe the organization of the TRD locus. The TRDV4 gene is found downstream of the C gene and in an inverted orientation of transcription, consistent with its orthologs in humans and mice. cDNA evidence was assessed to validate expression of the variable genes and showed that one to five D genes could be incorporated into a single transcript. Finally, we grouped the bovine and ovine TRDV1 genes into sets based on their relatedness. Conclusions The bovine genome contains a large and diverse repertoire of TRD genes when compared to the genomes of "γδ T cell low" species. This suggests that in cattle γδ T cells play a more important role in immune function since they would be predicted to bind a greater variety of antigens.

Abstract Background WC1 co-receptors are group B scavenger receptor cysteine-rich molecules that are found exclusively on γδT cells and are thought to be encoded by a multi-gene family. Previous studies have shown γδT cells that respond to a particular stimulus have unique WC1 molecules expressed. Prior to the onset of the studies described here only one full-length WC1 nucleotide sequence was publicly available, though three WC1 molecules had been distinguished based on monoclonal antibody reactivity. Furthermore, the number of WC1 genes found in the bovine genome and their sequences had not yet been resolved. Results By annotating the bovine genome Btau_3.1 assembly, here we show the existence of 13 members in the WC1 gene family and their organization within two loci on chromosome 5 including three distinct exon-intron gene structures one of which coded for a potentially more primitive and smaller WC1 molecule that is similar to the swine WC1 gene. We also provide cDNA evidence as verification for many of the annotated sequences and show transcripts for isoforms derived by alternative splicing. Conclusion It is possible that WC1 diversity contributes to functional differences that have been observed between γδT cell populations. The studies described here demonstrate that WC1 molecules are encoded by a large, multi-gene family whose transcripts undergo extensive alternative splicing. Similar to other non-rearranging immunoreceptors, it is likely that the WC1 gene repertoire underwent expansion in order to keep pace with rapidly changing ligands.

The WC1 cell surface family of molecules function as hybrid gamma delta (γδ) TCR co-receptors, augmenting cellular responses when cross-linked with the TCR, and as pattern recognition receptors, binding pathogens. It is known that following activation, key tyrosines are phosphorylated in the intracytoplasmic domains of WC1 molecules and that the cells fail to respond when WC1 is knocked down or, as shown here, when physically separated from the TCR. Based on these results we hypothesized that the colocalization of WC1 and TCR will occur following cellular activation thereby allowing signaling to ensue. We evaluated the spatio-temporal dynamics of their interaction using imaging flow cytometry and stochastic optical reconstruction microscopy. We found that in quiescent γδ T cells both WC1 and TCR existed in separate and spatially stable protein domains (protein islands) but after activation using Leptospira , our model system, that they concatenated. The association between WC1 and TCR was close enough for fluorescence resonance energy transfer. Prior to concatenating with the WC1 co-receptor, γδ T cells had clustering of TCR-CD3 complexes and exclusion of CD45. γδ T cells may individually express more than one variant of the WC1 family of molecules and we found that individual WC1 variants are clustered in separate protein islands in quiescent cells. However, the islands containing different variants merged following cell activation and before merging with the TCR islands. While WC1 was previously shown to bind Leptospira in solution, here we showed that Leptospira bound WC1 proteins on the surface of γδ T cells and that this could be blocked by anti-WC1 antibodies. In conclusion, γδ TCR, WC1 and Leptospira interact directly on the γδ T cell surface, further supporting the role of WC1 in γδ T cell pathogen recognition and cellular activation. [ABSTRACT FROM AUTHOR]

The immediate objective of our research is to understand the molecular mechanisms underlying activation and potentiation of the protective functional response of WC1+ γδ T cells to pathogens afflicting livestock species. The long‐term goal is to incorporate stimulation of these cells into the next generation of vaccine constructs. γδ T cells have roles in the immune response to many infectious diseases including viral, bacterial, protozoan and worm infections, and their functional responses overlap with those of canonical αβ T cells, for example they produce cytokines including interferon‐γ and IL‐17. Stimulation of non‐conventional lymphocytes including γδ T cells and αβ natural killer T (NKT) cells has been shown to contribute to protective immunity in mammals, bridging the gap between the innate and adaptive immune responses. Because of their innate‐like early response, understanding how to engage γδ T‐cell responses has the potential to optimize strategies of those that aim to induce pro‐inflammatory responses as discussed here. [ABSTRACT FROM AUTHOR]

Assessed the efficacy of four group evaluation methods used to examine change in high school students' attitudes after participation in a drug abuse prevention group counseling treatment, including pre- and posttreatment measurement with a Likert scale or an open-ended questionnaire, and a case study approach. Discusses the relative merits of various outcome research and data collection strategies. (KS)

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. [ABSTRACT FROM AUTHOR]

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by theUnited StatesNational Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings. [ABSTRACT FROM AUTHOR]

WCl proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WCl cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WCl is endocytosed in response to PMA. Because WCl endocytosis may play a role in the activation of γδ T cells, we examined WCl endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WCl. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WCl cytoplasmic domain significantly reduced PMAinduced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WCl caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WCl endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WCl inhibited IL-2 production stimulated by cocrosslinking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WCl gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens. [ABSTRACT FROM AUTHOR]

The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI)-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI)-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform. [ABSTRACT FROM AUTHOR]