Carolyn T.A. Herzig

WC1+ γδ T cell memory population is induced by killed bacterial vaccine

Limited studies have addressed the ability of γδ T cells to become memory populations. We previously demonstrated that WC1.1+ γδ T cells from ruminants vaccinated with killed Leptospira borgpetersenii proliferate and produce IFN‐γ in recall responses. Here we show that this response is dependent upon antigen‐responsive CD4 T cells, at least across transwell membranes; this requirement cannot be replaced by IL‐2. The response was also dependent upon in vivo priming, since γδ T cells from leptospira vaccine‐naive animals did not respond to antigen even when co‐cultured across membranes from antigen‐responsive PBMC. γδ T cells were the major antigen‐responding T cell population for the first 4 wks following vaccination and replicated more rapidly than CD4 T cells. Primed WC1+ γδ T cells circulated as CD62Lhi/CD45ROint/CD44lo, characteristics of TCM cells. When stimulated with antigen, they decreased CD62L, increased CD44 and CD25, and had no change in CD45RO expression. These changes paralleled those of the leptospira antigen‐responsive CD4 T cells but differed from those of γδ T cells proliferating to mitogen stimulation. This system for in vivo γδ T cell priming is unique, since it relies on a killed antigen to induce memory and may be pertinent to designing vaccines that require type 1 pro‐inflammatory cytokines.

Genomic organization and classification of the bovine WC1 genes and expression by peripheral blood gamma delta T cells.

BackgroundWC1 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.ResultsBy 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.ConclusionIt 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.

Bovine T cell receptor gamma variable and constant genes: combinatorial usage by circulating γδ T cells

Studies here describe expression and sequence of several new bovine T cell receptor gamma (TRG) genes to yield a total of 11 TRG variable (TRGV) genes (in eight subgroups) and six TRG constant (TRGC) genes. Publicly available genomic sequences were annotated to show their placement. Homologous TRG genes in cattle and sheep were assigned, using four accepted criteria. New genes described here include the bovine TRGC6, TRGV2, and TRGV4, homologues of ovine TRGC4, TRGV2, and TRGV4, respectively. The bovine Vγ7 and BTGV1 clones (previously TRGV4 and TRGV2, respectively) were reassigned to new subgroups TRGV7 and TRGV8, respectively, with approval by the IMGT Nomenclature Committee. Three TRGV subgroups (TRGV5, TRGV6, and TRGV8) were further designated as TRGV5-1 and TRGV5-2, TRGV6-1 and TRGV6-2, and TRGV8-1 and TRGV8-2 because each subgroup is comprised of two mapped genes. The complete sequence of bovine TRGC5 is also reported, for which a limited number of nucleotides was previously available, and shown to be most closely related to ovine TRGC5. Analysis of circulating γδ T cells revealed that rearrangement of TRGV genes with TRGC genes is largely dictated by their proximity within one of the six genomic V-J-C cassettes, with all TRG genes expressed by bovine peripheral blood γδ T cells. Cattle are useful models for γδ T cell biology because they have γδ T cells that respond to isopentenylpyrophosphate (IPP) antigens, while mice do not, and some bovine TRGV genes cluster closely with human genes.

Tyrosine phosphorylation of scavenger receptor cysteine-rich WC1 is required for the WC1-mediated potentiation of TCR-induced T-cell proliferation

Workshop cluster 1 (WC1) molecules are transmembrane glycoproteins uniquely expressed by γδ T cells. They belong to the scavenger receptor cysteine‐rich superfamily and are encoded by a multi‐gene family, which is divided on the basis of antibody reactivity, into three groups, WC1.1, WC1.2, and WC1.3. The potential role of WC1 as a co‐stimulatory molecule for the γδ TCR is suggested by the presence of several tyrosine‐based motifs in their intracellular domains. In this study, we found that WC1 was constitutively phosphorylated in ex vivo bovine γδ T cells and associated with src family tyrosine kinases. Crosslinking of WC1 molecules resulted in an increase in WC1 phosphorylation and co‐crosslinking of WC1 and γδ TCR together prolonged WC1 phosphorylation. We identified the second tyrosine residue as the primary phosphorylation target in WC1.1 and WC1.2 intracellular sequences in both in vitro and in vivo assays. The cytoplasmic tails of WC1.1 and WC1.2 were phosphorylated on serine and PKC activity was required for PMA‐induced endocytosis of WC1.1 or WC1.2. We found that phosphorylation of the second tyrosine in the WC1 cytoplasmic domain was required for the WC1‐mediated potentiation of TCR‐induced T‐cell proliferation, suggesting that WC1 acts as a co‐stimulatory molecule for γδ TCR.

Scavenger receptor WC1 contributes to the γδ T cell response to Leptospira.

WC1 molecules are exclusively expressed on the surface of γδ T cells. They belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. WC1 molecules have been grouped on the basis of antibody reactivity. The expression of WC1 molecules from these serologically defined groups is correlated with differences in γδ T cell responses. The expression of receptors within the WC1.1 group correlates with the capacity of γδ T cells to respond to Leptospira antigen. In this study, we used RNA interference to directly investigate the role of WC1 expression in the response to Leptospira borgpetersenii. We found that when three out of thirteen WC1 gene products were downregulated by RNA interference, γδ T cell proliferation and IFN-γ production in response to Leptospira antigen was significantly reduced. Our data demonstrate that specific receptors in the WC1 family directly participate in Leptospira recognition and/or activation of γδ T cells.

Antigenic basis of diversity in the γδ T cell co-receptor WC1 family

WC1 co-receptors are transmembrane glycoproteins with 11 extracellular scavenger receptor cysteine rich (SRCR) domains. They are related to the CD163 family but are uniquely expressed by gammadelta T cells. We recently showed that at least 13 members comprise the WC1 gene family in cattle, a model animal species for studies of gammadelta T cell biology. Since WC1 co-receptors participate in directing functional responses by gammadelta T cells either through the ligands they bind or the signals they transduce, availability of reagents to identify the expression of individual WC1 molecules of this diverse family would be valuable in further elucidating mechanisms of gammadelta T cell responsiveness. Although monoclonal antibodies (mAbs) have been widely used to identify WC1 co-receptors on gammadelta T cells, the locations of the antigenic epitopes recognized are unknown. Here, we mapped the epitopes to particular SRCR domains and evaluated their distribution among WC1 molecules. To do this, cDNA representing the extracellular domains of seven different WC1 genes was expressed in mammalian cells and analyzed for reactivity with anti-WC1 mAbs using ELISA and Western blotting. The study included mAbs that are broadly reactive with WC1(+) gammadelta T cells and those that divide WC1(+) gammadelta T cells into functionally distinct subpopulations. We found that mAb CC15 is a pan-reactive anti-WC1 mAb recognizing an epitope in the closely related SRCR domains 2 and 7 and that this epitope is present in at least domain 2 or 7 of all seven WC1 molecules evaluated here. Five other anti-WC1 mAbs, typified by mAb IL-A29, were found to be broadly reactive, recognizing epitopes in the related SRCR domains 4 and 9 but each having a unique pattern of reactivity with the seven WC1 molecules. Finally, the subpopulation-specific anti-WC1 mAbs, including those that recognize either the archetypal WC1.1 or WC1.2 molecule, were found to react with epitopes in the most variable WC1 domain, i.e. domain 1, of a restricted number of WC1 co-receptors.

Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes.

BackgroundWC1 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.ResultsReal-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.ConclusionThe 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.

Identification of three new bovine T-cell receptor delta variable gene subgroups expressed by peripheral blood T cells

To understand the biology of γδ T cells in ruminants, it is necessary to have a comprehensive picture of γδ T-cell receptor gene diversity and expression. In this study, three new subgroups of bovine T-cell receptor δ (TRD) variable genes were identified by RT-PCR and sequencing and homology with TRDV genes from other mammals determined. Previously unidentified TRDV subgroup genes described in this study include the bovine homologues of ovine TRDV2, TRDV3, and TRDV4 which were named accordingly. TRDV2 subgroup has two genes (TRDV2-1 and TRDV2-2) while we found the previously identified TRDV1 has at least eight genes corresponding to separate genomic sequences. Nucleotide and amino acid sequences for particular gene subgroups between cattle and sheep were more than 87% identical but identities among TRDV subgroups within a species were much less, with bovine TRDV4 having <45% identity to the other three bovine TRDV gene subgroups. Analysis of circulating bovine γδ T cells revealed that genes from all four TRDV subgroups were expressed in combination with TRDJ1, TRDJ3, and TRDC, although TRDV4 was the least represented, and all displayed a variety of CDR3 junctional lengths. Finally, some genes within the TRDV1, TRDV2, and TRDV3 subgroups recombined with TRAV incorporating TRAJs, suggesting dual use.

Central Line-Associated Bloodstream Infection Reduction and Bundle Compliance in Intensive Care Units: A National Study.

OBJECTIVES To describe compliance with the central line (CL) insertion bundle overall and with individual bundle elements in US adult intensive care units (ICUs) and to determine the relationship between bundle compliance and central line-associated bloodstream infection (CLABSI) rates. DESIGN Cross-sectional study. PARTICIPANTS National sample of adult ICUs participating in National Healthcare Safety Network (NHSN) surveillance. METHODS Hospitals were surveyed to determine compliance with CL insertion bundle elements in ICUs. Corresponding NHSN ICU CLABSI rates were obtained. Multivariate Poisson regression models were used to assess associations between CL bundle compliance and CLABSI rates, controlling for hospital and ICU characteristics. RESULTS A total of 984 adult ICUs in 632 hospitals were included. Most ICUs had CL bundle policies, but only 69% reported excellent compliance (≥95%) with at least 1 element. Lower CLABSI rates were associated with compliance with just 1 element (incidence rate ratio [IRR] 0.77; 95% confidence interval [CI], 0.64-0.92); however, ≥95% compliance with all 5 elements was associated with the greatest reduction (IRR, 0.67; 95% CI, 0.59-0.77). There was no association between CLABSI rates and simply having a written CL bundle policy nor with bundle compliance <75%. Additionally, better-resourced infection prevention departments were associated with lower CLABSI rates. CONCLUSIONS Our findings demonstrate the impact of transferring infection prevention interventions to the real-world setting. Compliance with the entire bundle was most effective, although excellent compliance with even 1 bundle element was associated with lower CLABSI rates. The variability in compliance across ICUs suggests that, at the national level, there is still room for improvement in CLABSI reduction. Infect Control Hosp Epidemiol 2016;37:805-810.

Expressed gene sequence of bovine IL23A and IL23R

The cloning and characterization of bovine IL23A and IL23 receptor cDNA from total RNA of PBMC and the genomic organization of the coding sequences are reported. The IL23A partial coding region was found to be 578 nucleotides coded for in 4 exons and shared 84% and 76% identity with human and mouse sequences, respectively. The IL23R complete coding region had 1890 nucleotides coded for in 10 exons and shared 87% and 73% homology with the human and mouse sequences, respectively. Both bovine sequences were more closely related to the human sequences than were mouse sequence. This work was done as part of the U.S. Veterinary Immune Reagent Network whose goal is to develop reagents for investigating diseases in livestock species, poultry and fish.