Lentiviral delivered aflibercept OXB-203 for treatment of neovascular AMD.

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness in the aging population, with vascular endothelial growth factor (VEGF) playing a key role. Treatment with recombinant anti-VEGFs is the current standard of care; however, it is only effective for 1-2 months at a time and requires re-administration. Gene therapy could pave the way for stable, long-term expression of therapeutic anti-VEGF with a single dose, reducing the frequency of treatment and potentially improving clinical outcomes. As such, we have developed OXB-203, a lentiviral-based gene therapy encoding the anti-VEGF protein aflibercept. Aflibercept derived from OXB-203 exhibited comparable in vitro binding characteristics to VEGF as recombinant aflibercept. Furthermore, its biological potency was demonstrated by the equivalent inhibition of VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and tubule formation as recombinant aflibercept. In a rat choroidal neovascularization (CNV) model of nAMD, a single subretinal administration of OXB-203 reduced laser-induced CNV lesion areas analogous to an intravitreal bolus of recombinant aflibercept. Finally, in a head-to-head comparative study, aflibercept derived from OXB-203 was shown to be expressed at significantly higher levels in ocular tissues than from an AAV8-aflibercept vector following a single subretinal delivery to rats. These findings support the therapeutic potential of OXB-203 for the management of nAMD.

Heterogeneous populations from in vitro cultures of antigen presenting cells in pigs.

Dendritic cells (DCs) are the most potent antigen presenting cells (APCs). Because of the difficulty in obtaining these cells directly from tissues, different sources of DCs are frequently used for in vitro experimentation and many of their biological and functional characteristics were studied using these systems. Until recently, it was assumed that specific culture conditions polarized the differentiation of either DCs or macrophages (Macs); however, it was shown that some DC culture systems in other species generate heterogeneous cell populations that can be identified according to their CD11c and MHC class II (MHC-II) expression. Following this approach, porcine DCs were directly isolated from peripheral blood or differentiated in vitro by culturing bone marrow (BM) progenitor cells or blood monocytes treated with growth factors. Mostly homogeneous monocyte-derived DCs (MoDCs) were obtained with similar phenotype and phagocytic characteristics to that of blood DCs. On the contrary, BM-derived DC (BMDC) cultures generated two distinct heterogeneous populations identified as MHC-II+ and MHC-II++ cells. BMDCs MHC-II+ had similar phenotypic and phagocytic characteristics to those of MoDCs and blood DCs. However, BMDCs MHC-II++ population expressed a higher amount of surface markers and transcribed genes associated with Macs-lineage exhibiting a higher phagocytic capacity than all the other cells. Noteworthy, every cell system expressed different genetic signatures. These results will help interpreting and re-interpreting data obtained using in vitro systems.

Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling.

Viruses routinely employ strategies to prevent the activation of innate immune signaling in infected cells. Respiratory syncytial virus (RSV) is no exception, as it encodes two accessory proteins (NS1 and NS2) which are well established to block interferon signaling. However, RSV-encoded mechanisms for inhibiting NF-κB signaling are less well characterized. In this study, we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV-infected cells, we demonstrated that the p65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured p65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following tumor necrosis factor alpha (TNF-α) stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalize the RSV N protein and p65 within bovine RSV (bRSV) IBs, which are granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterization of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signaling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate-a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally, they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells.IMPORTANCE Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalize their life cycle to provide favorable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterized. In this manuscript, we show that bovine respiratory syncytial virus (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signaling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease.

A Single Shot Pre-fusion-Stabilized Bovine RSV F Vaccine is Safe and Effective in Newborn Calves with Maternally Derived Antibodies.

Achieving safe and protective vaccination against respiratory syncytial virus (RSV) in infants and in calves has proven a challenging task. The design of recombinant antigens with a conformation close to their native form in virus particles is a major breakthrough. We compared two subunit vaccines, the bovine RSV (BRSV) pre-fusion F (preF) alone or with nanorings formed by the RSV nucleoprotein (preF+N). PreF and N proteins are potent antigenic targets for neutralizing antibodies and T cell responses, respectively. To tackle the challenges of neonatal immunization, three groups of six one-month-old calves with maternally derived serum antibodies (MDA) to BRSV received a single intramuscular injection of PreF, preF+N with MontanideTM ISA61 VG (ISA61) as adjuvant or only ISA61 (control). One month later, all calves were challenged with BRSV and monitored for virus replication in the upper respiratory tract and for clinical signs of disease over one week, and then post-mortem examinations of their lungs were performed. Both preF and preF+N vaccines afforded safe, clinical, and virological protection against BRSV, with little difference between the two subunit vaccines. Analysis of immune parameters pointed to neutralizing antibodies and antibodies to preF as being significant correlates of protection. Thus, a single shot vaccination with preF appears sufficient to reduce the burden of BRSV disease in calves with MDA.

Familial 3-Way Balanced Translocation Causes 1q43→qter Loss and 10q25.2→qter Gain in a Severely Affected Male Toddler.

Constitutional complex chromosomal rearrangements (CCRs) are rare events that typically involve 2 or more chromosomes with at least 3 breakpoints and can result in normal or abnormal phenotypes depending on whether they disturb the euchromatic neighborhood. Here, we report an unusual balanced CCR involving chromosomes 1, 9, and 10 that causes an unbalanced karyotype in a severely affected toddler. The CCR was initially reported as a maternal 2-way translocation but was reclassified as a 3-way translocation after a microarray analysis of the propositus revealed the involvement of another chromosome not identified by G-banding in his phenotypically normal mother. FISH assays on maternal metaphase cells confirmed that the 1qter region of der(1) was translocated to der(10), whereas the 10qter segment was translocated to der(9), which in turn donated a segment to der(1). Subsequently, this CCR was also identified in her phenotypically normal father (the patient's grandfather). Thus, the patient inherited the previously unreported pathogenic combination of der(1) with a loss of 1q43→qter (including AKT3, ZBTB18, HNRNPU, and SMYD3) and der(9) with a gain of 10q25.2→qter (including FGFR2), leading to a compound phenotype with key features of the 1q43→qter deletion and distal 10q trisomy syndromes. Our observations suggest that the loss of SMYD3 accounts for cardiac defects in a subset of patients. Moreover, due to recurrent miscarriages in this family, our findings allowed improved genetic counseling.

Bovine Derived in vitro Cultures Generate Heterogeneous Populations of Antigen Presenting Cells.

Antigen presenting cells (APC) of the mononuclear phagocytic system include dendritic cells (DCs) and macrophages (Macs) which are essential mediators of innate and adaptive immune responses. Many of the biological functions attributed to these cell subsets have been elucidated using models that utilize in vitro-matured cells derived from common progenitors. However, it has recently been shown that monocyte culture systems generate heterogeneous populations of cells, DCs, and Macs. In light of these findings, we analyzed the most commonly used bovine in vitro-derived APC models and compared them to bona fide DCs. Here, we show that bovine monocyte-derived DCs and Macs can be differentiated on the basis of CD11c and MHC class II (MHCII) expression and that in vitro conditions generate a heterologous group of both DCs and Macs with defined and specific biological activities. In addition, skin-migrating macrophages present in the bovine afferent lymph were identified and phenotyped for the first time. RNA sequencing analyses showed that these monophagocytic cells have distinct transcriptomic profiles similar to those described in other species. These results have important implications for the interpretation of data obtained using in vitro systems.

Contributions of Farm Animals to Immunology.

By their very nature, great advances in immunology are usually underpinned by experiments carried out in animal models and inbred lines of mice. Also, their corresponding knock-out or knock-in derivatives have been the most commonly used animal systems in immunological studies. With much credit to their usefulness, laboratory mice will never provide all the answers to fully understand immunological processes. Large animal models offer unique biological and experimental advantages that have been and continue to be of great value to the understanding of biological and immunological processes. From the identification of B cells to the realization that γδ T cells can function as professional antigen presenting cells, farm animals have contributed significantly to a better understanding of immunity.

Ag85A-specific CD4+ T cell lines derived after boosting BCG-vaccinated cattle with Ad5-85A possess both mycobacterial growth inhibition and anti-inflammatory properties.

There is a need to improve the efficacy of the BCG vaccine against human and bovine tuberculosis. Previous data showed that boosting bacilli Calmette-Guerin (BCG)-vaccinated cattle with a recombinant attenuated human type 5 adenovirally vectored subunit vaccine (Ad5-85A) increased BCG protection and was associated with increased frequency of Ag85A-specific CD4+ T cells post-boosting. Here, the capacity of Ag85A-specific CD4+ T cell lines - derived before and after viral boosting - to interact with BCG-infected macrophages was evaluated. No difference before and after boosting was found in the capacity of these Ag85A-specific CD4+ T cell lines to restrict mycobacterial growth, but the secretion of IL-10 in vitro post-boost increased significantly. Furthermore, cell lines derived post-boost had no statistically significant difference in the secretion of pro-inflammatory cytokines (IL-1ß, IL-12, IFNγ or TNFα) compared to pre-boost lines. In conclusion, the protection associated with the increased number of Ag85A-specific CD4+ T cells restricting mycobacterial growth may be associated with anti-inflammatory properties to limit immune-pathology.

Protection of calves by a prefusion-stabilized bovine RSV F vaccine.

Bovine respiratory syncytial virus, a major cause of respiratory disease in calves, is closely related to human RSV, a leading cause of respiratory disease in infants. Recently, promising human RSV-vaccine candidates have been engineered that stabilize the metastable fusion (F) glycoprotein in its prefusion state; however, the absence of a relevant animal model for human RSV has complicated assessment of these vaccine candidates. Here, we use a combination of structure-based design, antigenic characterization, and X-ray crystallography to translate human RSV F stabilization into the bovine context. A "DS2" version of bovine respiratory syncytial virus F with subunits covalently fused, fusion peptide removed, and pre-fusion conformation stabilized by cavity-filling mutations and intra- and inter-protomer disulfides was recognized by pre-fusion-specific antibodies, AM14, D25, and MPE8, and elicited bovine respiratory syncytial virus-neutralizing titers in calves >100-fold higher than those elicited by post-fusion F. When challenged with a heterologous bovine respiratory syncytial virus, virus was not detected in nasal secretions nor in respiratory tract samples of DS2-immunized calves; by contrast bovine respiratory syncytial virus was detected in all post-fusion- and placebo-immunized calves. Our results demonstrate proof-of-concept that DS2-stabilized RSV F immunogens can induce highly protective immunity from RSV in a native host with implications for the efficacy of prefusion-stabilized F vaccines in humans and for the prevention of bovine respiratory syncytial virus in calves.

Modulation of the transcription factor NF-κB in antigen-presenting cells by bovine respiratory syncytial virus small hydrophobic protein.

Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease in young cattle and is closely related to human RSV (HRSV), which causes severe respiratory disease in infants and the elderly. The RSV genome encodes a small hydrophobic (SH) protein with viroporin activity. Previous studies have shown that recombinant BRSV lacking the SH gene (rBRSVΔSH) is attenuated in the lungs, but not in the upper respiratory tract, of calves and mucosal vaccination with rBRSVΔSH induced long-lasting protective immunity. Attenuation of rBRSVΔSH may be due to the ability of this virus to induce an early innate response as rBRSVΔSH induces higher levels of pro-inflammatory cytokines than wild-type (wt) rBRSV. In this study, we investigated the effects of the BRSV SH protein on NF-κB p65 phosphorylation, a master step in the regulation of pro-inflammatory cytokines. Expression of SH resulted in the inhibition of NF-κB p65 phosphorylation in response to BRSV infection and extracellular lipopolysaccharide, and a reduction in the production of pro-inflammatory cytokines. In contrast, rBRSVΔSH does not inhibit NF-κB p65 phosphorylation in bovine antigen-presenting cells, including monocytes, macrophages and dendritic cells, resulting in increased expression of pro-inflammatory cytokines and increased activation of T cells compared to cells infected with wt BRSV. These findings highlight an important role for the BRSV SH protein in immune modulation.

Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway.

Dendritic cells (DC) are central to the initiation of immune responses, and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication-deficient AdV-based vaccine vectors. We found that, despite lack of expression of Coxsackie B-Adenovirus Receptor and other known adenovirus receptors, AdV readily enters skin-draining DC via an actin-dependent endocytosis. Virus exit from endosomes was pH independent, and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that, in the absence of Coxsackie B-Adenovirus Receptor and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC, and its relevance to vaccination strategies and vaccine vector targeting is discussed.

Efficacy of a virus-vectored vaccine against human and bovine respiratory syncytial virus infections.

Human respiratory syncytial virus (HRSV) is a major cause of lower respiratory tract disease in children and the elderly for which there is still no effective vaccine. We have previously shown that PanAd3-RSV, which is a chimpanzee adenovirus-vectored vaccine candidate that expresses a secreted form of the HRSV F protein together with the N and M2-1 proteins of HRSV, is immunogenic in rodents and nonhuman primates, and protects mice and cotton rats from HRSV challenge. Because the extent to which protection demonstrated in rodent models will translate to humans is unclear, we have exploited the calf model of bovine RSV (BRSV) infection, which mimics HRSV disease in children more closely than do experimental models of unnatural laboratory hosts, to evaluate the safety and efficacy of the PanAd3-RSV vaccine. We show that PanAd3-RSV alone and in combination with a modified vaccinia Ankara expressing the same HRSV antigens (MVA-RSV) induced neutralizing antibodies and cellular immunity in young seronegative calves and protected against upper and lower respiratory tract infection and pulmonary disease induced by heterologous BRSV challenge. There was no evidence either of enhanced pulmonary pathology or of enhanced respiratory disease in vaccinated calves after BRSV challenge. These findings support the continued evaluation of the vectored RSV vaccines in man.

Immunology of bovine respiratory syncytial virus in calves.

Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease in young calves. The virus is genetically and antigenically closely related to human (H)RSV, which is a major cause of respiratory disease in young infants. As a natural pathogen of calves, BRSV infection recapitulates the pathogenesis of respiratory disease in man more faithfully than semi-permissive, animal models of HRSV infection. With the increasing availability of immunological reagents, the calf can be used to dissect the pathogenesis of and mechanisms of immunity to RSV infection, to analyse the ways in which the virus proteins interact with components of the innate response, and to evaluate RSV vaccine strategies. Passively transferred, neutralising bovine monoclonal antibodies, which recognise the same epitopes in the HRSV and BRSV fusion (F) protein, can protect calves against BRSV infection, and depletion of different T cells subsets in calves has highlighted the importance of CD8(+) T cells in viral clearance. Calves can be used to model maternal-antibody mediated suppression of RSV vaccine efficacy, and to increase understanding of the mechanisms responsible for RSV vaccine-enhanced respiratory disease.

The relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species.

Adenovirus vaccine vectors generated from new viral serotypes are routinely screened in pre-clinical laboratory animal models to identify the most immunogenic and efficacious candidates for further evaluation in clinical human and veterinary settings. Here, we show that studies in a laboratory species do not necessarily predict the hierarchy of vector performance in other mammals. In mice, after intramuscular immunization, HAdV-5 (Human adenovirus C) based vectors elicited cellular and humoral adaptive responses of higher magnitudes compared to the chimpanzee adenovirus vectors ChAdOx1 and AdC68 from species Human adenovirus E. After HAdV-5 vaccination, transgene specific IFN-γ(+) CD8(+) T cell responses reached peak magnitude later than after ChAdOx1 and AdC68 vaccination, and exhibited a slower contraction to a memory phenotype. In cattle, cellular and humoral immune responses were at least equivalent, if not higher, in magnitude after ChAdOx1 vaccination compared to HAdV-5. Though we have not tested protective efficacy in a disease model, these findings have important implications for the selection of candidate vectors for further evaluation. We propose that vaccines based on ChAdOx1 or other Human adenovirus E serotypes could be at least as immunogenic as current licensed bovine vaccines based on HAdV-5.

Bovine γδ T cells are a major regulatory T cell subset.

In humans and mice, γδ T cells represent <5% of the total circulating lymphocytes. In contrast, the γδ T cell compartment in ruminants accounts for 15-60% of the total circulating mononuclear lymphocytes. Despite the existence of CD4(+)CD25(high) Foxp3(+) T cells in the bovine system, these are neither anergic nor suppressive. We present evidence showing that bovine γδ T cells are the major regulatory T cell subset in peripheral blood. These γδ T cells spontaneously secrete IL-10 and proliferate in response to IL-10, TGF-ß, and contact with APCs. IL-10-expressing γδ T cells inhibit Ag-specific and nonspecific proliferation of CD4(+) and CD8(+) T cells in vitro. APC subsets expressing IL-10 and TFG-ß regulate proliferation of γδ T cells producing IL-10. We propose that γδ T cells are a major regulatory T cell population in the bovine system.

Recombinant bovine respiratory syncytial virus with deletion of the SH gene induces increased apoptosis and pro-inflammatory cytokines in vitro, and is attenuated and induces protective immunity in calves.

Bovine respiratory syncytial virus (BRSV) causes inflammation and obstruction of the small airways, leading to severe respiratory disease in young calves. The virus is closely related to human (H)RSV, a major cause of bronchiolitis and pneumonia in young children. The ability to manipulate the genome of RSV has provided opportunities for the development of stable, live attenuated RSV vaccines. The role of the SH protein in the pathogenesis of BRSV was evaluated in vitro and in vivo using a recombinant (r)BRSV in which the SH gene had been deleted. Infection of bovine epithelial cells and monocytes with rBRSVΔSH, in vitro, resulted in an increase in apoptosis, and higher levels of TNF-α and IL-1ß compared with cells infected with parental, wild-type (WT) rBRSV. Although replication of rBRSVΔSH and WT rBRSV, in vitro, were similar, the replication of rBRSVΔSH was moderately reduced in the lower, but not the upper, respiratory tract of experimentally infected calves. Despite the greater ability of rBRSVΔSH to induce pro-inflammatory cytokines, in vitro, the pulmonary inflammatory response in rBRSVΔSH-infected calves was significantly reduced compared with that in calves inoculated with WT rBRSV, 6 days previously. Virus lacking SH appeared to be as immunogenic and effective in inducing resistance to virulent virus challenge, 6 months later, as the parental rBRSV. These findings suggest that rBRSVΔSH may be an ideal live attenuated virus vaccine candidate, combining safety with a high level of immunogenicity.

Bovine γδ T cells: cells with multiple functions and important roles in immunity.

The γδ T-cell receptor (TCR)-positive lymphocytes are a major circulating lymphocyte population in cattle, especially in young calves. In contrast, human and mice have low levels of circulating γδ TCR(+) T cells (γδ T cells). The majority of the circulating γδ T cells in ruminants express the workshop cluster 1 (WC1) molecule and are of the phenotype WC1(+) CD2(-) CD4(-) CD8(-). WC1 is a 220000 molecular weight glycoprotein with homology to the scavenger receptor cysteine-rich (SRCR) family, closely related to CD163. The existence of 13 members in the bovine WC1 gene family has recently been demonstrated and although murine and human orthologues to WC1 genes exist, functional gene products have not been identified in species other than ruminants and pigs. Highly diverse TCRδ usage has been reported, with expanded variable genes in cattle compared to humans and mice. Differential γ chain usage is evident between populations of bovine γδ T cells, this may have implications for functionality. There is a growing body of evidence that WC1(+) γδ T cells are important in immune responses to mycobacteria and may have important roles in T cell regulation and antigen presentation. In this review, we will summarize recent observations in γδ T cell biology and the importance of γδ T cells in immune responses to mycobacterial infections in cattle.

Induction of a cross-reactive CD8(+) T cell response following foot-and-mouth disease virus vaccination.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. Current inactivated FMDV vaccines generate short-term, serotype-specific protection, mainly through neutralizing antibody. An improved understanding of the mechanisms of protective immunity would aid design of more effective vaccines. We have previously reported the presence of virus-specific CD8(+) T cells in FMDV-vaccinated and -infected cattle. In the current study, we aimed to identify CD8(+) T cell epitopes in FMDV recognized by cattle vaccinated with inactivated FMDV serotype O. Analysis of gamma interferon (IFN-γ)-producing CD8(+) T cells responding to stimulation with FMDV-derived peptides revealed one putative CD8(+) T cell epitope present within the structural protein P1D, comprising residues 795 to 803 of FMDV serotype O UKG/2001. The restricting major histocompatibility complex (MHC) class I allele was N*02201, expressed by the A31 haplotype. This epitope induced IFN-γ release, proliferation, and target cell killing by αß CD8(+) T cells, but not CD4(+) T cells. A protein alignment of representative samples from each of the 7 FMDV serotypes showed that the putative epitope is highly conserved. CD8(+) T cells from FMDV serotype O-vaccinated A31(+) cattle recognized antigen-presenting cells (APCs) loaded with peptides derived from all 7 FMDV serotypes, suggesting that CD8(+) T cells recognizing the defined epitope are cross-reactive to equivalent peptides derived from all of the other FMDV serotypes.

Cattle MIC is a ligand for the activating NK cell receptor NKG2D.

Major histocompatibility complex (MHC) class I chain-related (MIC) genes encode molecules that are expressed in response to stress, signalling immune system cells primarily via the activating receptor NKG2D. We investigated the expression of receptors for MIC in lymphocyte subsets found in peripheral blood, lymph node and gut in cattle and demonstrated their presence on natural killer (NK) cells, gammadelta T cells and CD8(+) T cells. Recognition of MIC by NKG2D was formally demonstrated using recombinant protein and an ELISA. Staining with a cross-reactive monoclonal antibody recognising both human and cattle MIC showed that MIC is constitutively expressed within cattle intestinal epithelium. A functional response to soluble MIC was observed in receptor-bearing cells in blood, lymph node and gut, the latter requiring relatively high levels of MIC to trigger a response. Results suggest that NKG2D is a functionally important activating receptor in cattle.

Genomic location and characterisation of MIC genes in cattle.

Major histocompatibility complex (MHC) class I chain-related (MIC) genes have been previously identified and characterised in human. They encode polymorphic class I-like molecules that are stress-inducible, and constitute one of the ligands of the activating natural killer cell receptor NKG2D. We have identified three MIC genes within the cattle genome, located close to three non-classical MHC class I genes. The genomic position relative to other genes is very similar to the arrangement reported in the pig MHC region. Analysis of MIC cDNA sequences derived from a range of cattle cell lines suggest there may be four MIC genes in total. We have investigated the presence of the genes in distinct and well-defined MHC haplotypes, and show that one gene is consistently present, while configuration of the other three genes appears variable.