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Publication Non-Canonical Functions of a Disintegrin and Metalloproteases in Development and Cancer(2024-09) Pandey, AnkitCranial neural crest (CNC) cells are transient stem cells induced at the neural plate's border and migrate ventrally to produce most of the facial structures. They are induced by a combination of FGF, Wnt, and BMP4 signaling that are subtly modulated at the right position to define the neural crest territory. A Disintegrin and Metalloproteases (ADAMs) are transmembrane metalloproteases that have been shown to regulate signaling, adhesion, and cell migration in the CNC by proteolytically cleaving their many substrates. While the role of these proteases is well understood, a significant number of ADAMs do not have a catalytic active site and are, therefore, not proteases at all. Mechanistic studies concerning these non-proteolytic ADAM are lacking almost entirely. The cytoplasmic domain of ADAM10 has been shown to translocate into the nucleus following γ-secretase cleavage. Considering this, we can divide ADAM function into two categories, one dependent directly on proteolytic activity (Canonical ADAM function) and functions not directly dependent on proteolytic activity (Non-canonical function). In this thesis, we highlight the non-canonical function of ADAMs, by studying two ADAMs, Adam11 and Adam13. vii Adam11 is not a protease and has a very short cytoplasmic domain (11 amino acids), and Adam13 is a protease but has been previously shown to translocate to the nucleus and regulate gene expression. This study design lets us investigate two non-canonical aspects of ADAM function separately. In this thesis, we show that Adam11 is expressed within the CNC and binds to the BMP receptor BMR1A and Wnt receptor Frizzled7. We show that Adam11 positively regulates BMP4 signaling while negatively regulating β-catenin activity. Since the Adam11 cytoplasmic domain is unlikely to contribute to these functions, we next wanted to investigate the mechanism of ADAM cytoplasmic domain function. We show that Adam13 interacts with histone-modification enzymes and regulates histone modification in CNC. We also show that Adam13, along with Arid3a, regulates histone modifications at the promoter region of transcription factor Tfap2α. We show that Adam13 selectively promotes transcription of one variant of Tfap2α (Tfap2α-S1) while inhibiting another (Tfap2α-S3).Publication Hormonal Control in Mammary Development and Uterine Health: Insights from Cell Lines and Animal Models(2024-09) Goral, CerenMammary gland development is a complex physiological process regulated by a delicate balance of hormonal signals, among which estrogens play a pivotal role. Estrogens, primarily estradiol, mediate their effects through binding to estrogen receptors (ERs), notably ERα (ESR1) and ERβ (ESR2). These receptors are transcription factors that, upon activation, regulate the expression of genes involved in cell proliferation, differentiation, and apoptosis. The differential roles of ERα and ERβ in mammary tissue are critical for normal development and are implicated in the pathogenesis of breast cancer. The objectives of this research are to evaluate the endogenous and exogenous activation of ERα and ERβ receptors in various cellular contexts. This involves a detailed examination of the transactivation capacities of ESR1 and ESR2 in different cell lines by assessing the responses of ERα and ERβ to estrogenic compounds. Furthermore, the study characterizes the estrogen-induced responses in Esr2 knock-out mice, backcrossed onto a BALB/c background. This analysis sheds light on the role of ERβ in mammary gland development and tumorigenesis, as well as its influence on metabolism, providing a comprehensive understanding of its systemic effects. Additionally, the research explores the control of the estrus cycle and uterine disease treatment in equids. It examines the systemic effects of intrauterine device (IUD) placement in mares and investigates the efficacy of copper-banded IUDs in promoting bacterial clearance and preventing uterine diseases. This aspect aims to enhance reproductive health management in equids, addressing the control of the estrus cycle and the treatment and prevention of uterine conditions. Overall, these objectives are designed to provide a holistic view of estrogen receptor functions in both mammary gland and reproductive health, contributing to advancements in human medicine and animal husbandry.Publication Investigating the regulation of divalent cation homeostasis during fertilization and preimplantation embryo development in mice(2024-05) Gupta, NehaThe role of calcium ions (Ca2+) as the universal signal for egg activation has been acknowledged for nearly a century, with subsequent research unveiling the necessity of Ca2+ oscillations for egg activation in mammals. However, understanding the precise triggering pathway(s) behind the prolonged and protracted Ca2+ responses during mammalian fertilization posed a challenge due to limited methods to investigate these events and knowledge of the mechanisms involved. Pivotal breakthroughs came with the discovery of fluorescent dyes to monitor ion changes and that injecting sperm extracts into eggs replicated fertilization-induced oscillations and egg activation. The culminating demonstration of this process was the birth of offspring through Intra Cytoplasmic Sperm Injection (ICSI) without previous interaction of the gametes. This milestone was molecularly underpinned by the identification of sperm-specific PLCζ, whose injection in the form of mRNA, induced Ca2+ oscillations identical to those observed during fertilization. Interestingly, infertile patients with repeated ICSI failure exhibited the absence of PLCζ expression or point mutations on the sequence of PLCζ, suggesting that PLCζ represented the main, and possibly sole Ca2+-active factor in mammalian sperm. However, this notion was challenged by the observation that Plcζ-null males initiated subdued Ca2+ oscillations and displayed subfertility, indicating the existence of an additional factor(s) in mouse sperm triggering sufficient Ca2+ responses to support embryo development. Yet, its identity and involvement during fertilization with wild-type sperm remain unknown. Our contribution here is to identify the remaining Ca2+ active component(s) in sperm that could function as a “backup mechanism” of egg activation. This contribution is significant because it is a necessary step in our goal to identify all the sperm molecules that induce the Ca2+ signal required for embryo development. TRPM7 is expressed ubiquitously across cell types and tissues, and exhibits diverse subcellular localization, including the plasma membrane, cytoplasm, and nucleus, suggesting multifunctionality. Numerous studies have highlighted the essential role of TRPM7 in numerous cell types and organs, including a vital role in pre-implantation embryo development, where its absence causes embryonic death. Significantly, its molecular mechanism in embryogenesis remains unknown. Additionally, despite its fundamental role in fertility, the expression and localization of TRPM7 in gametes and embryos have not been fully elucidated. Our contribution seeks to precisely identify the expression, localization, and molecular mechanism of TRPM7 in embryo development, a crucial step in understanding the function of this essential channel regulating divalent cation homeostasis during embryogenesis. Overall, this contribution holds significant implications for perfecting parthenogenetic methods to address egg activation failure, enhancing embryo development, identifying new indicators of male and female fertility, and exploring potential contraception targets. This study helped in understanding the molecules and regulatory mechanisms initiating embryo development in these species and advances our knowledge substantially.Publication ANALYSIS OF LETHAL GENES DURING MAMMALIAN OOGENESIS AND EARLY EMBRYOGENESIS(2024-02) Miao, XiaosuMammalian oogenesis begins with the proliferation of primordial germ cells which undergo meiosis and arrest at prophase of the MI. Oocyte development is crucial for female fertility and early embryogenesis, marked by highly regulated gene expression across different stages and cell populations. Identifying and characterizing embryonic lethal phenotypes plays an important role in understanding the functions and mechanisms of essential genes during oogenesis and early embryogenesis. Chapters 2, 3, and 5 of my research unveil Polr1d, Zc3h4, and Rbbp4 as lethal genes influencing early embryonic development, particularly during preimplantation and implantation stages. Furthermore, we generated oocyte-specific conditional knockout (OcKO) mice of Rbbp4 and discovered that absence of maternal RBBP4 leads to subfertility in female mice and disrupts transcriptional activity during oocyte growth due to altered histone modification. The maternal-to-zygotic transition is also impeded without maternal RBBP4, showing lower two-cell formation and higher transcription activity in two-cell embryos. In Chapter 4 of my study, we found that the gene of Rpa1 is essential for mouse oogenesis. Absence of RPA1 significantly impairs folliculogenesis, leading to a substantial reduction in oocyte number and female infertility. GV oocytes lacking RPA1 display severe DNA damage, triggering the canonical DNA damage response pathways, including the activation of ATM, ATR, DNA-PK as well as p53. Apart from lethal genes, small molecules from the environment also affect early embryo development. Chapter 6 explores the impact of Berberine, a natural compound from Berberis, which can alleviate LPS-induced apoptosis, oxidation, and skewed lineage specification during preimplantation development. As a part of this work in Chapter 7, we found surface functionalization of poly (dimethylsiloxane) (PDMS) substrates, one of the most ubiquitous polymers used in biological applications, facilitates the culture of embryos in vitro by blocking nonselective adsorption. The goal of this dissertation is to report the roles and mechanisms of lethal genes, including Polr1d, Zc3h4, Rbbp4, and Rpa1 during mouse oogenesis and early embryogenesis. This knowledge contributes to enhancing reproductive efficiency in animals and holds promise to develop targeted therapies or interventions associated with human genetic abnormalities.Publication The role of LKB1 spliceoforms in iTreg-Th17 plasticity and their interactions with PKCθ and SIRT1 downstream of IL-6 signaling(2023-05) Mohan, DeekshaFollowing activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that iTreg exposure to IL-6 culminates in Stk11 splicing downstream of PKCθ. Altogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL, to regulate Stk11 splice variants and induce Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be induced in iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity. Sirtuin 1 (Sirt1) plays a crucial role in metabolism and inflammatory responses. Sirt1 is a deacetylase that can regulate different transcription factors important for modulating immune responses. Sirt1 has been shown to increase iTreg conversion to Th17 cells by deacetylating the Foxp3 transcription factor in iTregs. LKB1, encoded by Stk11, has been identified as a mediator of Treg induction and function through its effects on TSDR methylation and metabolism. Sirt1 has been shown to activate LKB1, but the molecular mechanisms of these signaling pathways are not known. Our data suggest Stk11 splicing may lie at the heart of iTreg responses to IL-6 exposure and may represent a unique target for developing stable, cell-based iTreg therapies. Examining the contribution of Sirt1 to this process may provide additional insight as to how LKB1 isoforms regulate iTreg-Th17 cell fate decisions.Publication HUMAN MILK: FROM COVID-19 IMMUNITY TO BREAST CANCER RISK ASSESSMENT(2023-02) Narayanaswamy, VigneshBreastmilk is a complex biological fluid containing macromolecules including lipids, oligosaccharides, proteins as well as several types of cells. Several studies have reported in detail of these components. My focus is studying the protein component of breastmilk, specifically antibodies, cytokines, and other secreted factors in the setting of different pathogenicity in women. Studies have demonstrated that the levels of numerous cytokines as well as the levels of pathogen-specific antibodies are altered in milk upon either maternal or infant infections. Additionally, there are reports that the levels of certain inflammatory markers are altered in milk among women with breast cancer or at increased risk of breast cancer. In my thesis, I sought to elucidate the inflammatory profile of breastmilk obtained from women who: (1) were infected and/or recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (2) were vaccinated against the disease caused by SARS-CoV-2, coronavirus disease 2019 (COVID-19), and (3) were at high risk of breast cancer because of germline BRCA mutations. In my first study, I evaluated the immune response to SARS-CoV-2 in colostrum (the first secretion from the mammary gland after birth) from women who tested positive for the virus at delivery or during pregnancy. My focus was on trying to better understand the antibody and cytokine responses among the 15 enrolled women. The first study presents a proof-of-concept on how colostrum collected on paper spot cards can serve as a reliable surrogate for liquid samples. Because women express very small volumes of colostrum, in the first study, I found that colostrum from more than 70% of the women contained detectable levels of anti-SARS-CoV-2 antibodies. One of my main findings was that comparable levels of antibodies against SARS-CoV-2 were present in colostrum expressed by women testing positive for the virus over four months ago and women testing positive at delivery. I concluded that a strong humoral response is present in the colostrum of women with an active infection and women who recovered from it. I also provide evidence of how future large-scale studies can be conducted with milk easily collected on paper spot cards. However, the durability of antibody responses and the presence of other immune cells needed to be determined. In my next COVID-19 study, I analyzed serial milk obtained from 30 women collected over a period of 35 days, with an additional sample collected at around 4 months after a positive COVID-19 test. Milk from nearly 90% of women contained detectable levels of anti-SARS-CoV-2 antibodies. Importantly, I found that the levels of a specific subtype of T-cells, called effector-memory T cells, are upregulated in milk expressed when women recovered from COVID-19. Additionally, antibodies in milk were able to neutralize the virus and select variants of concern. Both the studies informed that SARS-CoV-2 infection elicits a robust humoral and cellular response, with the humoral response predominantly IgA-driven. In my next study, I determined whether these responses differed among women who received an mRNA-based COVID-19 vaccine. In this longitudinal study of 30 enrolled lactating women, I found that the humoral response is IgG-driven. In fact, there was no significant difference in the levels of anti-receptor binding domain (RBD)-IgA between milk expressed prior to vaccination and milk expressed three weeks after the second dose. To determine whether RBD-specific antibodies were vertically transmitted via breastmilk to infants, I measured anti-RBD IgG and IgA in stool samples from infants of vaccinated mothers. A third of these infant samples contained low, but detectable levels of antibodies, providing compelling evidence that breastmilk-derived antibodies are indeed being transmitted to breastfed infants. In my pursuit to profile immune markers in breastmilk, I assessed the levels of a panel of immune biomarkers in the milk of women with a pathogenic germline BRCA mutation. A total of 202 milk samples from BRCA carriers and 78 milk samples from non-carrier controls were analyzed. I hypothesized that the levels of these biomarkers would be significantly different in the milk of BRCA carriers compared to non-carrier controls. I found that the levels of certain inflammatory cytokines are elevated whereas levels of the member of the cytokine superfamily, osteoprotegerin (OPG) was lower in BRCA carriers compared to controls. Intriguingly, the alteration of these biomarkers was significant among BRCA1 carriers and not BRCA2 carriers, who more resembled the non-carrier controls for the panel assayed. Assessment of these biomarkers in women at high risk of breast cancer may be useful in assessing individual risk and in developing effective prevention measures.Publication FUNCTIONAL NANOPARTICLES FOR INFLAMMASOME-TARGETED APPLICATIONS: FROM DIAGNOSIS TO THERAPY.(2022-09) Nandi, DipikaChronic inflammatory disorders such as colitis, Alzheimer’s, arthritis, cardiovascular diseases contribute to about 60% of deaths worldwide. Such lifelong debilitating illness degrades the quality of life for patients and their caregivers while contributing to enormous social and economic burdens often associated with high costs for therapy and care. According to CDC, about 90% of the nation’s total health care costs contribute to chronic illnesses. This suggests that there’s an unmet need for advanced tools and techniques for the improvement of current diagnostic and therapeutic procedures. Inflammasomes have been extensively shown to be associated with the pathogenesis of various inflammatory diseases. It’s a form of innate immune response which is triggered when any form of pathogenic or sterile insult is recognized by the cytosolic threat sensors. This further trigger the assembling of three different proteins including sensor, adaptor and zymogen into the multimeric inflammasome protein complex which immediately leads to proximity-induced cleavage of caspase-1 zymogen into its active form. Active caspase-1 enzyme cleaves the downstream modulators, pore-forming protein gasdermin D, and procytokines into their active forms which subsequently leads to a specialized form of cell death, called pyroptosis, causing enhanced inflammation. Inflammasome is a host defense mechanism that, upon dysregulation, leads to several disorders. Thus, inflammasome signaling components could act as a common target for devising efficient diagnostic and therapeutic approaches for various inflammatory disorders. This thesis is focused on developing nanoplatforms, offering inflammasome-specific diagnostics and therapeutic opportunities, with proof of principal applications demonstrated in inflammatory disease models like gouty arthritis, colitis, and sepsis. We first aimed at understanding the interactions of nanomaterial and immune cells to identify the inflammasome activating nanoparticle-associated-molecular-patterns. This study identified nanoparticle core hydrophobicity as a core and tunable determinant of inflammasome activation. We further demonstrated that nanoparticle core hydrophobicity can be tuned to obtain different degrees of inflammasome assembly via distinct signaling inclinations and kinetics. This knowledge could be utilized for a myriad of applications, viz, the particles that don’t activate inflammasome could be utilized for immunotherapy as well as diagnostics, and the particles activating inflammasome can be employed as vaccine adjuvants or for prophylactic responses where the controlled inflammasome activation is desired. In the two follow-up studies, we developed reporter and theranostic nanoplatforms that can monitor inflammasome activation in real-time and offer simultaneous therapeutic benefits. We engineered two pair of nanoparticles termed as MATIN I and MATIN II, where MATIN corresponds to Monitoring and Therapeutics of Inflammasome via Nanoparticles. Each pair included a reporter and a theranostic platform encapsulating either just the caspase-1 responsive probe or an additional inflammasome inhibiting drug. Both the MATIN I and II platforms offer robust monitoring as well as therapeutic potential in-vitro in nigericin-treated immortalized macrophages and in-vivo in inflammatory disease models of gouty arthritis and DSS-induced colitis. The final study aimed at developing an advanced synergistic therapeutic platform by codelivering rational combinations of inflammasome-inhibiting drugs via nanoparticles. Here we synthesized and characterized a nanoparticle co-encapsulating two potent inflammasome inhibitors, MCC-950 and disulfiram. These dual-drug nanoparticles exhibited a synergistic response and significantly higher efficiency in-vitro when compared to single-drug nanosystems as well as free drugs. Additionally, they offered complete protection against LPS-mediated sepsis model, proving their heightened in-vivo efficacy. Overall, this thesis aims to serve as a guide to developing platforms that can offer inflammasome-specific diagnostics and therapeutics as well as leverage them for prophylactic responses to obtain the desired level of inflammation for host defense.Publication The Paternal Role Before, During and After Fertilization(2022-09) Tourzani, DaryaMale reproduction is dynamic cascade of events in which a stem cell undergoes several steps of differentiation and morphogenesis to produce a viable sperm cell during spermatogenesis. However, the newly formed sperm is still unable to fertilize and is immotile. Uniquely, sperm are both transcriptionally and translationally silent, therefore, in order to undergo molecular changes rendering the sperm capable of fertilization, they rely on post-translational modifications. The complex cellular dynamics of the epididymis as well as the length of time required for sperm to undergo sperm maturation has made investigating changes occurring during maturation challenging. Researchers have focused on recovering sperm from specific epididymal regions to compare molecular events. However, while it provides momentary differences, we may still be missing key regulators of the acquisition motility and fertilization capabilities. Nevertheless, post-maturation events during sperm capacitation have been very well documented, and due to our advancements in understanding the generation of assisted reproductive technologies (ART) has improved our ability to overcome infertility. However, while advances have been made in improving ART protocols, like enhancements towards in vitro fertilization (IVF), major limitations still occur reducing the overall efficiency. Some groups have made efforts to reduce the limiting factors by improving sperm selection techniques, which suggests a more significant role the sperm contribution aside from the paternal genome and activation of the oocyte by the sperm-specific factor, PLCz. Recent studies have revealed specific populations of small RNA contributions delivered to sperm during sperm maturation are contribute to embryo development. Specifically, when immature sperm were used with an isolated population of small RNAs from the mature sperm region, embryo development was improved. This data suggested that having a more complete understanding of both sperm maturation and the contribution of sperm on embryo development can improve our ability to overcome limiting factors observed in ART. The goal of this dissertation is to characterize an undescribed post-translational modification as it pertains to the acquisition of sperm motility and sperm maturation. Additionally, to investigate the role of mature sperm after fertilization and how changes to preincubation conditions can influence the success of pre-implantation development. Hence, the understanding the contributions of sperm after fertilization as occurred will provide pivotal insights into the mechanisms that regulate early pre-implantation development. Ultimately the goal is to use this knowledge to aid in the treatment of male infertility in human, in addition to enhancing the reproduction of high-value animals like livestock species, endangered animal preservation and transgenic animal production.Publication WC1 AND TCR INTERACTIONS FOR γδ T CELL ACTIVATION(2022-02) Gillespie, AlexandriaMajor subpopulations of gamma delta T cells within ruminant and pigs are defined by expression of WC1, a hybrid pattern recognition receptor/co-receptor to the T cell receptor (TCR). It is known that when WC1 is knocked down cells fail to respond. Showing that WC1 plays an active role in the stimulation of bovine gamma delta T cells. Here we explored the spatio-temporal dynamics of WC1 and TCR interaction using imaging flow cytometry and stochastic optical reconstruction microscopy. We found that in quiescent gamma delta T cells both WC1 and TCR existed in separate protein domains (protein islands) but after activation using Leptospira, our model system, that they concatenated. In cattle, WC1+ gamma delta T cells have been shown to use TCR gamma genes from only one of the six available cassettes (TRGC5). We postulated that this structure may be necessary to interact with WC1 for signal transduction. If correct, other species should have the same restriction of their T cell receptor (TCR) gene usage by their WC1+ cells. When evaluated by RT-PCR and PacBio sequencing we found that caprine WC1+ gamma delta T cells exhibited the same restriction as found in cattle while porcine WC1+ gamma delta T cells used all TCR gamma C genes, although they preferentially expressed TCR chains from their TRGC5 homologue cassette. Next, we addressed WC1 and TCR roles of determining antigen specificity. One model is that co-ligation of WC1 and the gamma delta TCR by antigen increases a low affinity gamma delta TCR-antigen interaction. In this paradigm, WC1 is the main determining element regarding pathogen recognition. If correct, we predict the TCR CDR3 sequences in the responding gamma delta T cells to be relatively unrestricted. The alternative model is that gamma delta T cells that respond to a pathogen have a TCR with higher affinity to antigen and thus would be restricted while TCR expressed on cell that do not respond would be polyclonal. To test these models, we performed next generation sequencing of sorted antigen-responding and non-responding WC1+ gamma delta T cells. As baseline data, populations of responding and nonresponding WC1+ gamma delta T cells were also evaluated for their overall transcriptome differences using RNA-Seq.Publication GENOMIC ORGANIZATION AND EXPRESSION OF THE WC1 HYBRID CORECEPTOR AND PATTERN RECOGNITION RECEPTOR ON PORCINE GAMMA DELTA T CELLS(2022-02) Le Page, Laurengd T cells are a crucial component of the immune response to a number of increasingly relevant and largely zoonotic pathogens to which efficacious vaccination is lacking. In ruminants and swine, gd T cells represent a major population of peripheral blood and epithelial tissue-resident lymphocytes. gdT cells respond to both protein and non-protein antigens independently of MHC presentation and possess immunological memory. Upon activation, gamma delta T cells illicit a variety of effector functions and play an indispensable role of orchestrating the downstream immune response. These characteristics make gamma delta T cells a promising candidate for recruitment by vaccination, however, methods for effectively priming these cells remain to be elucidated. The type I transmembrane receptor Workshop Cluster One (WC1) is expressed as a multigenic array on gd T cells in swine and ruminants. In cattle there are 13 unique WC1 genes (WC1-1 to WC1-13) each comprised of 6-11 SRCR domains that selectively bind unprocessed antigen in a manner that resembles a pattern recognition receptor (PRRs). WC1 functions as a hybrid PRR and co-receptor for the gamma delta TCR as it potentiates activation signals from the TCR and dictates antigen specificity of expressing gd T cells. cDNA evidence suggests that porcine WC1 is expressed as a multigenic array consisting of 9 genes (WC1-1 to WC1-9) each encoding 6 SRCR domains with unique pathogen binding potential. The objective of this study is to characterize the multigenic array of porcine WC1, investigate its propensity for pathogen binding, and evaluate its expression on gd T cells. Using the MAKER annotation pipeline, we annotated Sscrofa11.1 for sequence derived from full-length cDNA transcripts representing the 9 porcine WC1 genes. We were able to map 8 of the 9 genes, leaving one (WC1-8) unplaced in the current assembly. We defined three subpopulations of porcine gd T cells based on expression of WC1 and CD2. Finally, we confirmed that porcine WC1 SRCR domains are capable of directly binding whole fixed bacteria including Leptospira spp and Mycobacterium bovis.