Bovine gamma delta T cells and the function of gamma delta T cell specific WC1 co-receptors

Cellular Immunology 296 (2015) 76–86

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Bovine gamma delta T cells and the function of gamma delta T cell specific WC1 co-receptors Janice C. Telfer ⇑, Cynthia L. Baldwin Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA

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Article history: Received 7 January 2015 Revised 11 May 2015 Accepted 11 May 2015 Available online 16 May 2015 Keywords: Cattle Human Gamma delta T cell Mycobacteria Leptospira WC1 CD163 SRCR

a b s t r a c t The study of cd T cells in ruminants dates to the discovery of the cd TCR in humans and mice. It is important since cattle offer an alternative model to the mouse for evaluating the role of cd T cells in zoonotic disease research and for control of disease reservoirs in non-human animals. In addition, maintaining the health of cattle and other members of the order Artiodactyla is critical to meet the global human need for animal-source protein. In this review, we examine the bovine cd T cell responses to Mycobacteria, which infects a third of the human population, and bovine c and d TCR diversity and the relationship to the TCR of human mycobacteria-responsive cd T cells. We review the utilization of the cd T cell specific scavenger receptor cysteine-rich (SRCR) glycoproteins known as WC1, and that are part of the CD163 family, which function as both cd T cell activating co-receptors and pattern recognition receptors (PRR) for bovine cd T cells and highlight the presence and evolution of this multigenic array, with potential for the same function, in birds, reptiles, jawless and bony fishes, and prototherian and eutherian mammals. Ó 2015 Elsevier Inc. All rights reserved.

1. Importance of research in bovine immunology Cattle may provide a model for human infectious diseases. However, understanding the immune system and resistance to infectious diseases in cattle also provides benefits to humans in a number of additional ways. It is estimated that zoonotic infections are responsible for 60% of infectious diseases in humans (www. cdc.gov/onehealth/zoonotic-diseases). Examples of zoonotic bacterial diseases spread from cattle to humans include leptospirosis, tuberculosis and brucellosis. Under the One Health rubric, which is now prominent in the infectious disease world, it is proposed that we can attain optimal health for people, animals, and the environment by forming an integrated approach. Thus, the infected animals are not merely ‘models’ manipulated to recapitulate the disease in humans but in fact are the primary targets of the infectious agents, having co-evolved with the pathogens, and serve as a reservoir. Understanding cd T cells in cattle will shed light on the immune responses important for controlling infectious diseases. Cattle [1], sheep [2], swine [3] and chicken [4] are all known to have a high percentage of cd T cells in their peripheral blood and distributed throughout their tissues. They are referred to as ‘‘cd T cell high’’

⇑ Corresponding author. Tel.: +1 413 545 5564; fax: +1 413 545 6326. E-mail address: [email protected] (J.C. Telfer). http://dx.doi.org/10.1016/j.cellimm.2015.05.003 0008-8749/Ó 2015 Elsevier Inc. All rights reserved.

species while mice and humans have lower numbers of cd T cells and are referred to as ‘‘cd T cell low’’ species [5]. It is not yet known whether members of other animal groups such as the monotremes, amphibians, reptiles or marsupials are cd T cell high or cd T cell low species, and thus what is the norm, but clearly being selected for high levels of cd T cells would be expected to have a survival benefit. Control of infectious diseases in livestock has an ancillary benefit for human nutrition. Cattle and related species such as goats and sheep provide an important component of animal-source food in the human diet throughout much of the world, including the developing world where food security is an issue. Inclusion of animal source food in the diets of children has been shown to alleviate under-nutrition that results in physical stunting and diminished cognitive development in studies in sub-Saharan Africa [6–9]. Preventing or controlling infectious diseases in our food animals is necessary to reduce the cost and increase the availability of animal source food and also has an ethical component of alleviating suffering and enhancing the welfare of animals. Moreover, animals provide fiber and hides for clothing and housing, fertilizer in the form of animal waste, traction and even act as a de facto banking system. While some infectious diseases can be controlled with good management, such as mastitis, there are many intractable ones for which our best hope for control is development of vaccines. cd T cells may play an important role as targets of next generation vaccines for control of such diseases.

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2. Discovery of cd T cells in ruminants Highlights of the discovery of cd T cells are shown in Fig. 1 with key discoveries in ruminants juxtaposed with those in humans and mice. For example the discovery of a TCRc gene in 1984 [10] and the TCRc gene locus in 1985 [11] in humans and mice was closely followed by the identification in 1986-7 of a major third lymphocyte population in sheep that was not CD4+ or CD8+ [12,13]. Following discovery of the TCRd locus [14], several groups showed the expression of the new cd TCR by human cells [15–19]. The CD4/CD8 third population of cells identified in sheep was further defined using monoclonal antibodies reactive with a unique cell surface molecule designated T19 in sheep [20] and WC1 in cattle [21,22]. By 1989, ruminant cells expressing T19/WC1 had been shown to express the c and d TCRs [1,20,23]. The mAbs to T19 or WC1 provided an important tool for initially identifying and studying cd T cells in ruminants. However, WC1 cd T cells also exist in these species, being the majority of cd T cells in the spleen [24], the only cd T cells in the uterus [25] while in the blood 1% to 50% of cd T cells are WC1 [26]. Broadly speaking, WC1 cd T cells have myeloid characteristics while WC1+ cd T cells are pro-inflammatory, producing IFN-c [27,28] and promoting the production of IgG2 [29]. However, a subpopulation of the WC1+ cd T cells known as WC1.2+ also has a regulatory function producing IL-10 and TGFb [30,31].

3. Responses of WC1+ and WC1 bovine cd T cells to mycobacterial infections As for primates, the response of cd T cells to mycobacterial infections has been extensively studied in cattle since they are natural hosts for both tuberculous and non-tuberculous mycobacterial infections. Bovine tuberculosis [29,32–37] is typically associated with M. bovis rather than M. tuberculosis because cattle are relatively resistant to the later [38,39] while humans may be infected by either [40]. The lungs and associated lymphoid tissues are the principal sites of infection with M. bovis as occurs in humans. In contrast, infection of cattle with M. avium paratuberculosis (MAP) results in a chronic granulomatous disease in the gut tissues known as Johne’s disease [41] with diarrhea and wasting and eventually death once it progresses to the symptomatic stage after years of controlled infection. It has been hypothesized that MAP may be a causative agent of human inflammatory bowel disease. Both of these mycobacterial diseases are of economic importance in cattle and pose zoonotic disease reservoirs.

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M. bovis in cattle results in a type 1 or Th1 response with well-organized granulomas while Johne’s disease induces a lepromatous or type 2 (Th2) response and often unorganized granulomas [41]. cd T cells are associated with granulomas in both types of mycobacterial diseases [41–43] and the WC1+ cd T cells are postulated to have a role in protective immunity while other cd T cells may be involved in suppressing protective responses depending upon the timing of their arrival and progression of disease. That is, WC1+ cd T cells arrive at the M. bovis infection site first [44] and at sites of skin test antigen injection (i.e. purified protein derivative, PPD) before neutrophils [32]. The cd T cells also increase in representation in blood and this correlates with increased IFN-c, preceding the CD4 T cell response [45,46]. In lung granulomas in the bovine tuberculosis model [42] the cd T cells are WC1+ [43]. A particular subpopulation of the WC1+ cd T cells known as WC1.1+ cd T cells also increase in number in lungs and upper respiratory tract of cattle vaccinated with the M. bovis BCG intranasally [47] while during infections with the virulent strains of M. bovis, both major WC1+ subpopulations (WC1.1+ and WC1.2+) accumulate, along with WC1 cells [47,48]. Because of the propensity of the WC1+ cd T cells to produce IFN-c it may not be surprising that depleting the WC1+ cd T cells from M. bovis-infected cattle shifts the immune response away from a type 1 or Th1 response [29]. The WC1 cd T cells predominate in the gut tissues of cattle, the principal site of MAP infection. However, in vivo experiments show that both WC1 and WC1+ cd T cells accumulate in granulomas in M. paratuberculosis-infected cattle [41]. Using MAP in matrix biopolymers (e.g. Matrigel), Plattner et al. have shown that both subpopulations of cd T cells respond more vigorously at 7 days following infection than at later time points and that they are responsive before the CD4 T cells [49]. However, if WC1+ cd T cells respond initially and are found first in the granulomas, followed by the WC1 cd T cells [41], then the granuloma is well-organized. In contrast, if the WC1 cells respond first, the granuloma does not progress to an organized state. In well-organized granulomas, eventually the WC1+ cd T cells are found in the outer margins near the fibrous border while the WC1 cd T cells are in the central regions [43]. Plattner et al. hypothesize that the WC1+ cd T cells may play a decreasing role as the granuloma organizes. This difference in granuloma organization is interesting since both WC1+ and WC1 cd T cells made IFN-c in response to IL-2 with cells from MAP vaccinated animals and the WC1 cd T cells actually produced more [49]. Intriguingly, as much IFN-c is made by WC1 cd T cells in response to IL-2 alone (antigen-independent) when the cells are from naïve

Fig. 1. Timeline of cd T cell discovery in humans, mice and ruminants. Adapted from that presented by Willi Born at the 10th cd T cell conference in Chicago, Illinois May 2014 [1,10–20,117,118].

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animals as from vaccinated animals, while the WC1+ cd T cells require pre-priming by MAP vaccination for optimal IFN-c responses [49] and thus may have other qualities that facilitate granuloma organization. It has been shown that as Johne’s disease progresses to the symptomatic stage that there is an increase in the cd T cells in the lamina propria and a decrease in the CD4 T cells [50]. The majority of the cd T cells in the intraepithelial lymphoid population are WC1 cd T cells [50] and it has been hypothesized that these cells are suppressors of the CD4 cell-mediated immune response through their cytotoxic activities [51]. Subsets of bovine WC1+ cd T cells also have been shown to have regulatory activity through cytokine secretion [30,31] although the contribution of this subpopulation to Johne’s disease progression has not been investigated.

4. Mycobacterial antigens that stimulate bovine cd T cells WC1+ cd T cells from M. bovis-primed cattle respond to various mycobacterial components, including the protein antigen ESAT-6, in recall responses in vitro by producing IFN-c [34,37]. Recent studies have shown cd T cells derived from both M. bovis infected or BCG-vaccinated cattle are also stimulated by peptides and non-protein ligands in in vitro recall responses and these include both WC1+ and WC1 cd T cells [37,48]. For example, cd T cells from M. bovis infected cattle proliferate and produce IFNc in response to non-protein mycobacterial antigens lipoarabinomannan (LAM) and mycolyl-arabinogalactan peptidoglycan (mAGP) [48]. Anti-TCR mAbs block the response to both protein and nonprotein antigen demonstrating the involvement of the TCR and suggesting specificity [48]. WC1+ cd T cells are also responsive to M. bovis-infected dendritic cells (DCs) requiring DC:cd T cell direct interaction although the ligands involved have not been identified [52]. The human and non-human primate cd T cell response to prenyl pyrophosphates or phosphoantigens (pAg) such as IPP or HMBPP produced by bacteria including mycobacteria is well characterized [37]. WC1+ bovine cd T cells have also been reported to respond to IPP but this has not been reproduced in all labs [35]. Since the human Vc9Vd2 TCR (also known as Vc2Vd2 TCR) is expressed by cd T cells activated in response to pAg [53], it is of considerable interest to determine whether this TCR has a counterpart in a cd T cell high species like cattle (Fig. 2) to further our understanding of bovine cd T cell responses to mycobacterium. Unlike the human TRG locus, the two bovine TRG loci are organized into six V–J–C cassettes (Fig. 2C), each with a unique TRGC and V– J–C rearrangements occur within cassettes [54–56]. The bovine TRD locus is similar that of humans and mice except that the TRDV1 gene has expanded to more than 50 genes in cattle [57]. Bayesian analysis of the evolutionary history shows that of the six bovine TRGC genes, bovine TRGC5 is the closest to the two human TRGC genes (Fig. 2A). The cassette containing bovine TRG5 also contains the functional V region genes TRGV 3-1, 3-2, 4 and 7 [55]. Human TRGV9 gene is closest to bovine TRGV4. Bovine TRGV 3-1, 3-2, and 7 are also in the same clade as human TRGV9 but are more closely related to human TRGV10 and TRGV11 (Fig. 2B). It has been reported that the bovine equivalent of human TRGV9 is a pseudogene because of a stop codon interrupting translation [58]; however, the reported sequence does not correspond to that of the bovine TRGV4 open reading frame but may correspond to the ovine pseudogene TRGV11-1, mapped to the 50 end of the ovine TRGC5 cassette [56]. Bovine TRGV4-TRGC5 is transcribed in ex vivo peripheral blood cd cells, albeit at lower levels compared to its genomic neighbors TRGV3-TRGC5 and TRGV7-TRGC5 [55,59], but could be the

population that responds to pAg. The low and perhaps variable frequency of TRGV4-TRGC5-expressing cells among cattle may account for why some but not all labs have reported responses to pAg. It is not yet known whether bovine cd T cells expressing TRGV4-TRGC5 show a TRDV restriction equivalent to human Vd2. Theoretically, it is possible that most of the contacts with antigen made by a bovine TCR using TRGV4 would be through the c chain of the TCR, as is the case with the human Vc9Vd2 TCR and prenyl pyrophosphates [60] and thus the involvement of the TCRd may be secondary. The mystery of how the human Vc9Vd2 TCR can be activated by small soluble pAg, given that they should not be able to crosslink the TCR/CD3 complex, has been solved by the discovery that the ligand involves the transmembrane receptor butyrophilin 3A1, modified by interaction with pAg. Of the three human BTN3A gene products, only BTN3A1 effectively activates VcVd2 T cells in trans when cells expressing it, and an unknown cofactor not present in murine cells, are treated with pAg [61–64]. Prenyl pyrophosphates have been reported to bind to the BTN3A1 extracellular domain [65] or to a positively charged surface pocket in the intracellular B30.2 domain of BTN3A1 [66]. Mutations reversing the positive charge in the intracellular B30.2 domain abrogated pAg binding and VcVd2 T cell activation; conversely, gain of function mutations introduced in the non-functional BTN3A gene product conveyed both pAg binding and VcVd2 T cell activation [66]. While cattle possess one BTN3A gene, it is not known whether its expressed product participates in the reported activation of WC1+ cd T cells by pAg. In addition, the cDNA predicted from the genomic sequence contains in-frame stop codons leading to premature truncation; it is not known whether this is accurate or due to sequencing error or whether cattle are polymorphic for BTN3A with some animals expressing a functional gene. Other placental mammals that have been reported to have translatable human Vc9Vd2 TCR and BTN3A1 homologs include the sloth, armadillo, the primates gray mouse lemur and aye aye, as well as alpaca, bottlenose dolphin and killer whale [58], suggesting there may be a conserved ability to respond to pAg.

5. WC1 molecules function as co-receptors for the cd TCR While acting as a useful marker to identify the majority of peripheral blood cd T cells, WC1 transmembrane proteins, members of the ancient and conserved scavenger receptor cysteine rich (SRCR) receptor superfamily and closely related to CD163 molecules [67], functionally act as coreceptors for cd T cells [68,69]. WC1/CD163 molecules are characterized by the presence of multiple group B SRCR domains, as are CD5, CD6, Spa, and DMBT1 molecules [70]. Each group B SRCR domain is approximately 100 amino acids long with conserved spacing of 6 to 8 cysteines, 3–4 disulfide bonds and a hydrophobic core. Alternative splicing (WC1) or shedding of the protein (CD163A) can lead to a secreted form [71,72]. We have shown that subpopulations of the WC1+ cd T cells are the first T cells to respond to vaccines against the important zoonotic pathogen Leptospira and have used this system to define the role of WC1 molecules as detailed below. Coreceptors are known to potentiate activation of T cells. For example, the ab T cell CD8 coreceptor and the ab TCR both bind to MHC class I, with coreceptor dependence for T cell activation inversely correlated with TCR affinity for peptide plus MHC [73]. While not as much is known about cd TCR in species other than humans and mice, it is probable that they follow the same rules of binding to antigen directly rather than in the context of MHC class I or II, with membrane constraint required to activate signaling through the cd TCR/CD3 complex. What is known about how the cd TCR contacts its ligand is limited to four crystal structures

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Fig. 2. Comparison of human and bovine TRG genes. Human (Hs) and bovine (Bt) TCRGC (A) and TCRGV (B) amino acid sequences, with nomenclature determined by the International Immunogenetics Information System (imgt.org) [119], were aligned using ClustalX 2.0.8. Pairwise and multiple alignment penalties for gaps were 10.0, 0.10 for gap extension and Gonnet 250 for protein weight matrix. The evolutionary history of 8 TCRGC and 26 TCRGV taxa was inferred using MrBayes3.2, with 2 runs with 3 cold chains and 1 heated chain, temperature setting of 0.2, for 30,000 or 40,000 generations, respectively [120]. An amino acid mixed model was used to approximate the posterior probabilities of trees, shown at branch nodes. Trees were sampled every 100 generations and the burnin fraction was 0.25. The convergence diagnostic used was the average standard deviation of split frequencies, which were 0.01. The average standard deviation of split frequencies was 0.01 and the average potential scale reduction factor was 1.003 and 1.010. Trees were visualized using FigTree1.4.2 (http://tree.bio.ed.ac.uk). (C) Organization of human and bovine TRG loci (imgt.org; [54]). Human TRGV pseudogenes are indicated in gray, TRGC sequences are indicated by white boxes.

[74–77] and a mutational analysis of activation via human Vc9Vd2 TCR [60]. Common among these five cd TCRs is the observation that amino acids that contact ligand are often in the less variable germline-encoded CDR1 and CDR2 regions rather than the recombination junction-encoded CDR3 regions. When CDR3 regions contact ligand, only the CDR3c [60,77] or the CDR3d [74] is used, rather than the CDR3 regions of c and d chains in tandem contacting variable ligands as occurs with the ab TCR. It is also notable that the TCR d can incorporate multiple d diversity genes with P and N untemplated addition of nucleotides [78] (including in bovine [57,79]) and thus the CDR3d can be particularly long, resembling an immunoglobulin heavy chain, which may add flexibility [80]. The use of a coreceptor may determine whether the avidity of a broadly reactive, low affinity TCR for its ligand is sufficient for activation. Evidence that WC1 acts as a coreceptor comes from experiments showing that antibody-mediated crosslinking of bovine WC1 with the TCR-CD3 complex potentiates T cell activation, while ligation of WC1 alone has no effect or is inhibitory [69,81]. Following activation, WC1 colocalizes with the cd TCR-CD3 complex in lipid rafts [82]. Other studies showed that the WC1-mediated potentiation of TCR signaling requires the phosphorylation of a tyrosine in the cytoplasmic tails of WC1 and that if the WC1 is knocked down that primed WC1+ cd T cells can no longer respond to Leptospira in recall responses [83]. Among the 13 bovine WC1 proteins, there are three types of WC1 cytoplasmic tails with increasing lengths because of the insertion or substitution of additional exons: type I is the shortest, type II is an intermediate length with an insertion of 14–16 amino

acids and type III is the longest [68]. In WC1 proteins with type I or II cytoplasmic domains, phosphorylation of the membrane proximal tyrosine motif Y24EEL is required for WC1 coreceptor activity [68,69] (Fig. 3). In contrast, in WC1 proteins with a type III cytoplasmic domain, the membrane proximal tyrosine motif has mutated to Y24QEI. It is no longer phosphorylated, even though src family tyrosine kinases are capable of phosphorylating this motif [84]. Instead, phosphorylation of the C-terminal tyrosine motif Y199DDV in the type III cytoplasmic domain is required for WC1 coreceptor activity [68]. The C-terminal Y138DDV (type I) or YDDI/V (type II) motifs are neither phosphorylated or required for WC1 coreceptor activity [68,69], suggesting that the longer type III cytoplasmic domain adopts a different conformation, such that the C-terminal YDDV domain is accessible to the src family kinase and can substitute for the phosphorylated membrane proximal Y24EEL tyrosine motif in downstream signaling events. Differential tyrosine phosphorylation may lead to the recruitment of different signaling molecules, leading to the activation of different signaling cascades. Phosphorylated YEEL in type I or II WC1 cytoplasmic domains would be most likely to bind src family tyrosine kinases or the adaptor protein Shc [85]. Phosphorylated YDDV in type III WC1 cytoplasmic domains would be more likely to bind the adaptor proteins SLP-76 or Nck [86,87]. Mutation of two membrane proximal serines in the type I WC1 cytoplasmic domain, one of which is in a consensus protein kinase C (PKC) phosphorylation motif, also significantly decreased WC1 coreceptor activity [88]. WC1 coreceptor activity is modulated by a membrane proximal endocytic dileucine motif ([DE]XXXL[LIM]), which regulates endocytosis. Mutation of the endocytic dileucine motif in a type I

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Fig. 3. Model of cd TCR and WC1 signaling via co-ligation. Phosphorylation of serine by PKC and a membrane proximal or distal tyrosine by fyn is required for WC1 potentiation of TCR-mediated signaling. WC1 cross-linking in the absence of TCR signaling does not activate T cells. Different SH2-containing molecules are predicted to bind to the phosphorylated membrane proximal tyrosine versus the membrane distal tyrosine. Endocytosis via a dileucine motif decreases WC1/TCR signaling.

cytoplasmic domain significantly decreases PMA-induced endocytosis. This inhibition of endocytosis and sustained presence of WC1 at the cell surface is correlated with an increase in WC1 coreceptor activity at high concentrations of crosslinking antibody. Interestingly, coligation of WC1 cytoplasmic domains with TCR/CD3 results in differential levels of IL-2 secretion: type I < type II < type III. This can be correlated with differences in the nucleation of the signaling complex downstream of WC1 [68] but it may also be due to differences in the two endocytic dileucine motifs in type II and III cytoplasmic domains that would be predicted to decrease clathrin-mediated endocytosis and prolong signaling [88]. 6. WC1 molecules function as pattern recognition receptors Bovine WC1+ cd T cells are divided into subpopulations known as WC1.1+ and WC1.2+ based on the WC1 molecules they express. This is determined by staining with monoclonal antibodies recognizing SRCR a1 domains from particular expressed products of the 13 bovine WC1 genes [68]. Multiple WC1 genes are transcribed in one cd T cell (e.g. some WC1.1+ cells express WC1 genes WC1–3 and WC1–8 while WC1.2+ cells express WC1–4, WC1–7 and WC1– 9) and this expression divides the WC1+ cd T cell population into subsets [68]. Expression of particular WC1 molecules correlates with responses to different bacteria suggesting the WC1 molecules contribute to the specificity of the response. For example, only WC1.1+

cd T cells respond to Leptospira following vaccination of cattle [89] while only WC1.2+ cells were isolated as Anaplasma-responsive [90] in recall responses following infection of cattle. Moreover, WC1.1+ or WC1.2+ cd T cells share the same restriction of TCR gene usage (TRGV3/TRGV4/TRGV7, TRGJ5–1, TRGC5 and TRDJ1/TRDJ3) although a variety of TRDV genes may be used [59,90]. Knockdown of some WC1 gene products significantly decreases cd T cell responses to Leptospira [83], as indicated above, which may suggest that individual WC1 molecules act as pattern recognition receptors (PRR) and then transduce signals. The hypothesis is that WC1 binding has the capacity to determine whether a restricted set of c or d TCR-expressing cells can be activated by bacterial pathogen associated molecular patterns (PAMPs). The TCR is involved in antigen recognition by WC1+ cd T cells in both cases since anti-TCR antibodies block the responses [59,90]. Our model is that the variable WC1 proteins coligate specific bacterial ligands with the relatively low affinity restricted cd TCR, thereby increasing the avidity for those ligands above the threshold required for activation of the cd cells. The 13 diverse bovine WC1 open reading frames conserved among cattle could also add diversity to the cd TCR repertoire since collectively the 13 WC1 molecules have 137 SRCR domains [71,91]. There is precedent for other SRCR group B molecules closely related to WC1 to bind pathogens: CD6 [92], Spa [93], CD163A [94] and DMBT1 (aka gp340/salivary agglutinin/surfactant pulmonary-associated D-binding protein) [95] bind Gram-positive and Gram-negative bacteria and CD5 binds yeast [96]. Identified ligands include lipoteichoic acid, lipopolysaccharide, poly-phosphorylated and -sulfated compounds, leucine rich repeat proteins and fungal mannose [92–94,96–98]. Moreover, a linear binding motif (VEVLXXXXW) in an external loop of SRCR domains has been identified in CD163A and DMBT1 [94,99,100]. Comparing a representative of WC1 proteins expressed by WC1.1+ Leptospira-responsive cells (i.e. WC1-3) to a representative of WC1.2+ Leptospira-nonresponsive cells (i.e. WC1-4), we found that five out of eleven WC1-3 SRCR domains and none of the eleven WC1-4 SRCR domains bound to multiple serovars of two Leptospira species, L. borgpetersenii and L. interrogans. WC1 SRCR a1 domains from 5 WC1.1-type WC1 proteins bound Leptospira spp. (Fig. 4A). Mutational analysis of WC1-3 SRCR domains demonstrates that the active site for bacterial binding is different than the continuous VEVLXXXXW motif previously reported for other SRCR proteins. In the case of the WC1-3 b2 domain, the few critical amino acids lie at the edge of a hydrophobic pocket (Fig. 4B). In addition, soluble WC1 SRCR domain inhibits Leptospira growth in a binding-dependent manner [101]. Taken together with the knockdown experiments in which reduction of WC1.1-type receptors reduce cd T cell activation by Leptospira [83], the correlation of Leptospira-binding with expression of the WC1-3 gene product on Leptospira-responsive cd T cells suggests that expression of individual WC1 receptors encodes antigen specificity through direct co-ligation of bacteria products with cd TCR. The conservation of multiple large open reading frames encoding WC1 for millions of years of evolution implies that the each WC1 gene was selected for their ligation of bovine pathogens in conjunction with a cd T cell response. In support of this hypothesis, we have found that WC1 SRCR domains bind to Borrelia burgdorferi [101] and Mycobacteria spp. (H. Hsu and J.C. Telfer, unpublished results). A complete description of WC1 SRCR domain binding specificity remains to be determined. It is important to note that WC1-3 vs. WC1-4 transcription, and thus cd T cell function, remains stable in mature cd T cells, in a manner reminiscent of the CD4 and CD8 ab T cell subset identities established during thymic development. It is possible that a similar epigenetic modification of the WC1 locus occurs during bovine cd T cell development. However, WC1 mRNA is extensively

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Fig. 4. (A) Alignment of WC1 SRCR a1 domains, grouped into WC1.1-type Leptospira binding domains (top), WC1.1-type non-Leptospira binding domains (middle), and WC1.2-type non- Leptospira binding domains (bottom). The locations of six conserved cysteines (the bovine WC1 SRCR a1 domain lacks cysteine 2 and 7, which in other SRCR group B domains forms a disulfide bond) are numbered. Amino acid differences from the WC1-3a1 SRCR domain are underlined and bolded. The VEVLXXXXW-like motif is boxed; the locations of amino acids mutated in the WC1-3b2 SRCR domain are indicated by asterisks [101]. (B) Location of WC1 SRCR mutations mapped onto a predicted WC1 SRCR domain structure [83]. The sequence of the wild-type (WT) WC1-3 b2 SRCR domain is aligned with the sequences of the mutated WC1-3b2 SRCR domains (mut1, mut2, and mut3), with the variable amino acids underlined. The sequence of the wild-type (WT) WC1-13a1 SRCR domain is aligned with that of the sequence of the mutant WC1-13a1 SRCR domain, with the variable amino acids underlined. Adapted from Hsu et al. [101], with permission from Journal of Immunology. ‘Copyright 2015. The American Association of Immunologists, Inc.’

alternatively spliced [71] which has implications for ligand binding avidity, projection from the cell surface and localization in the immune synapse, WC1 signaling and whether WC1 is a secreted anti-microbial protein or a transmembrane receptor. 7. WC1/CD163 multigene arrays in evolution Unlike the single gene copies of ab TCR coreceptors CD8 and CD4, WC1 and CD163 genes are multigenic arrays (Fig. 5). In cattle, WC1 is expressed exclusively on cd T cells while CD163c-a and CD163A are expressed on cd T cells but on other cell types as well [67]. Although WC1 genes are not present in the murine or human genome, the CD163c-a homologs murine SCART1 and SCART2 and human CD163c-a are expressed on cd T cells [102–104]. Human CD163A is highly expressed on monocytes and macrophages [105], but whether it is expressed on cd T cells has not been documented. The SRCR domains in WC1 and CD163 proteins can be typed by phylogenetic analysis, with domains assigned an alphabet designation based on their clustering together in a clade. SRCR domains b, c, d, e, and d’ are shared by WC1 and CD163, with membrane

proximal d’ being the most conserved (Fig. 5). WC1 is distinguished by the presence of SRCR domain a, found always at the N-terminal of the eutherian mammalian WC1 proteins and thus also referred to as a1. CD163c-a is characterized by SRCR domains m, l, and n and CD163A by SRCR domains h, i, j and k. The SRCR domains of WC1 and CD163 are encoded by single exons which has led to exon divergence, duplication and shuffling over evolutionary time. It is notable that four species of the order Rodentia appear to possess WC1 genes (Fig. 6), even though the mouse does not. Evidence also exits that WC1 genes are present in the genomes of the jawless vertebrate sea lamprey, the sauropsids birds, turtle, lizards, alligator, and snakes, the bony lobe-finned fish coelacanth, the bony ray-finned fishes cichlid, Mexican tetra and spotted gar, the prototherian mammal duck-billed platypus, marsupials opossum and Tasmanian devil, order Carnivora bear, panda, cat, dog, ferret, walrus and seal, order Soricomorpha star-nosed mole and European shrew, order Chiroptera bat, order Perissodactyla white rhinoceros and horse, order Rodentia mole rat, chinchilla, guinea pig, thirteen-lined ground squirrel, order Cingulata armadillo, and order Artiodactyla cattle, yak, Tibetan antelope, water buffalo, goat, sheep, pig, alpaca, and camel (Fig. 6). The size of the WC1

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Fig. 5. Schematic of CD163 and WC1, with SRCR domain letter designations and cytoplasmic domain motifs indicated (Bo, bovine; Hu, human; Mu, mouse). WC1 gene products are divided into molecules having type I, type II or type III cytoplasmic domains. Serines (SS or SD), a functional dileucine motif (LL) or mutated motifs (LI or II), and tyrosine motifs are indicated. Phosphorylated tyrosines are indicated by a circled P [67–69,88].

multigenic array can differ among species since in the order Artiodactyla there 13 bovine [71,91], 26 ovine, 18 caprine and 2 swine WC1 genes (S. Kim, C.L. Baldwin, J.C. Telfer, unpublished results) while there are 18 predicted WC1/CD163 genes in the sauropsid chicken and 10 predicted for the prototherian mammal duck-billed platypus [67]. It is likely that our search of predicted proteins from species-specific genome projects under represents the number of WC1 genes in each genome, since genomic assemblies appear to count multiple nearly identical SRCR domains as belonging to one WC1 gene. For example, a BLAST search with the sequence of three bovine WC1 SRCR a1 domains picked up only one predicted WC1 protein from goat, while PCR amplification from one goat has resulted in over twenty distinct WC1 SRCR domain a sequences (J.C. Telfer, unpublished data). 8. WC1 predates the cd TCR The presence of WC1 genes in the genomes of less-derived species such as the sea lamprey suggests that WC1 evolved and presumably played a role in the immune response of a common vertebrate ancestor. In addition, there is precedent for a multigenic array of SRCR domain-containing proteins lending diversity to the immune response in the purple sea urchin Strongylocentrotus purpuratus, whose genome contains approximately 200 group A SRCR genes whose expression changes upon challenge with bacteria [106–108]. Thus, the use of multiple SRCR domain-containing proteins in the immune response predates the evolution of the cd TCR. Based on phylogenetic analysis of the TCR chains c, d, a, and b and immunoglobin, cd T cells are thought to be the least derived cells of the adaptive immune system [109]. Lymphocytes in jawed animals (gnathostomes), ranging from mammals to birds to cartilaginous fishes such as the shark, recombine and express c and d TCR [110]. In contrast, the jawless fishes (cyclostomes) sea lamprey

and hagfish, whose ancester diverged from the gnathostomes 500 million years ago, do not possess TCR. However, they do have lymphocytes expressing variable lymphoid receptors (VLRs), which acquire their diversity through the rearrangement of leucine-rich (LRR) repeat modules [111–114]. Most importantly, these VLRs can be classified as TCR ab-like (VLRA+), immunoglobin-like (VLRB+), and TCR cd-like (VLRC+) [112,115]. This is not dependent on sequence homology of the VLRs to TCR chains, but is correlated with the gene program in the cells in which they are expressed, the location of these cells, and their function [112]. VLRC+ lymphocytes originate from bi-potent precursors in the ‘thymoid’ region of the gills that can develop into either VLRA+ or VLRC+ cells. They proliferate when the host is immunized with Bacillus anthracis exosporium, express the cd T cell fate-determining transcription factor SOX13, and are found in high numbers with a restricted VLRC in the lamprey epidermis, similar to dendritic epithelial T cells in mice with restricted cd TCR [116]. This discovery suggests that the emergence of a functional analog of a cd T cell in evolution predated the emergence of the cd TCR. We have found seven predicted WC1 genes with multiple SRCR domains in the genome of the sea lamprey and obtained sequence from one of those domains (S. Paquette, C. Porth,R. D’Orazio, R. Grabar, S. Alpert, H. Benkiran, S. Brown, J.C. Telfer; UMass Amherst 2014 ANIMLSCI H01 Honors Colloquium, unpublished results), which is shown in the phylogenetic tree of WC1 and CD163 SRCR domains (Fig. 6). Although it is annotated in the sea lamprey genome as a DMBT1 gene, another multiple group B SRCR domain-containing protein, it clusters in the same clade as a WC1 homolog from the ray-finned fish cichlid and is closer to bovine WC1 than to the outliers human DMBT1 SRCR domains 1 and 2 (Fig. 6). It remains to be determined whether the sea lamprey WC1 genes are expressed in the cd T cell-like VLRC+ cells, but it is possible that WC1, like the cd T cell fate-determining transcription factor SOX13, was integral to cd T cell identity before the cd TCR arose.

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Fig. 6. WC1 SRCR domains in multiple vertebrate species were identified by BLAST search of the non-redundant protein database with the amino acid sequences of bovine WC1-3, WC1-4, and WC1-11 SRCR a1 domains. SRCR domains were aligned using ClustalX 2.0.8. Pairwise and multiple alignment penalties for gaps were 10.0, 0.10 for gap extension and Gonnet 250 for protein weight matrix. The evolutionary history of 97 taxa was inferred using MrBayes3.2, with 2 runs with 3 cold chains and 1 heated chain, temperature setting of 0.2, for 1 million generations [120]. Trees were sampled every 100 generations and the burnin fraction was 0.25. The convergence diagnostic used was the average standard deviation of split frequencies, at less than 0.03. The average standard deviation of split frequencies was 0.024 and the average potential scale reduction factor was 1.012. The resulting tree was visualized using FigTree1.4.2 (http://tree.bio.ed.ac.uk) and rooted on human (Hs) DMBT1 SRCR domain 1. The bovine (Bt) WC1, CD163A, and CD163c-a domains are designated by gene number (WC1 only), alphabetical class, and numbered according to their position relative to the N-terminus (e.g. BtWC1_3_5e is the WC1-3 e domain fifth from the N-terminus; Fig. 5).

9. Conclusions

Acknowledgments

In this age of global emerging zoonotic pathogens, it is important to understand differences and similarities between the immune response of humans and non-human animals, since a vaccine program may be most effective carried out in the primary pathogen reservoir, rather than in humans. As shown above, WC1 SRCR domains, appear to have evolved even before the cd TCR and are broadly distributed, appearing in the jawless vertebrate sea lamprey, the sauropsids birds and reptiles, bony fishes, prototherian mammals, and many orders of eutherian mammals. An understanding of bovine cd T cells and WC1/CD163 molecules offers a promise of intentional vaccine design to recruit the cd T cell response against important zoonotic pathogens such as Leptospira, Borrelia and Mycobacteria spp. We have shown that bovine cd T cells interact with infectious agents in a unique way via the cd TCR and WC1 acting as coreceptors and PRRs. Thus, cattle are a valuable alternative model for understanding the variety of ways that cd T cells may be engaged to respond to important pathogens of cattle and humans.

Thank you to Willi Born for allowing us to adapt his cd T cell discovery timeline to include milestones in ruminant biology. Studies in the Telfer and Baldwin laboratories are supported by AFRI competitive grant no. 2011-67015-30736 & NIH R01 HD070056-01 from the NIFA USDA-NIH program titled Dual Purpose with Dual Benefit: Research in Biomedicine and Agriculture Using Agriculturally Important Domestic Species. References [1] C.R. Mackay, W.R. Hein, A large proportion of bovine T cells express the gamma delta T cell receptor and show a distinct tissue distribution and surface phenotype, Int. Immunol. 1 (1989) 540–545. [2] W.R. Hein, C.R. Mackay, Prominence of gamma delta T cells in the ruminant immune system, Immunol. Today 12 (1991) 30–34. [3] M. Sinkora, J.E. Butler, The ontogeny of the porcine immune system, Dev. Comp. Immunol. 33 (2009) 273–283. [4] J.T. Sowder, C.L. Chen, L.L. Ager, M.M. Chan, M.D. Cooper, A large subpopulation of avian T cells express a homologue of the mammalian T gamma/delta receptor, J. Exp. Med. 167 (1988) 315–322.

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