Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony-stimulating factor therapy during a chronic Staphylococcus aureus infection in lactating Holsteins.

Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (gCSF). Pegylated gCSF (PEG-gCSF; Imrestor, Elanco Animal Health, Greenfield, IN) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. Pegylated gCSF stimulates neutrophil expansion but also induces changes in monocyte and lymphocyte circulating numbers, surface protein expression changes, or both. We hypothesized that PEG-gCSF modulates surface expression of monocytes and neutrophils and facilitates their migration to the mammary gland. We challenged 8 mid-lactation Holsteins with approximately 150 cfu of Staphylococcus aureus (Newbould 305) in a single quarter via intramammary infusion. All animals developed chronic infections as assessed by bacteria counts and somatic cell counts (SCC). Ten to 16 wk postchallenge, 4 of the animals were treated with 2 subcutaneous injections of PEG-gCSF 7 d apart. Complete blood counts, SCC, bacterial counts, milk yield, feed intake, neutrophils extracellular trap analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 d after the first PEG-gCSF injection. The PEG-gCSF-treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared with control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF-treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophages sourced from milk from treated animals compared with cells isolated from controls. The PEG-gCSF-treated cows did not clear the S. aureus infection, nor did they significantly differ in SCC from controls. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, although surface MPO+ cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggests an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and possibly chronic mammary infections in general.

Symposium review: Omics in dairy and animal science-Promise, potential, and pitfalls.

Sequencing the first genome took 15 yr and $3 billion to complete. Currently, a genome can be sequenced in a day for a few thousand dollars. Comparing the relative abundance of nearly every mRNA transcript and small RNAs from cells and tissues from different experimental conditions has become so easy that it can take longer to transfer the data between computers than to perform the experiment. Nucleotide sequencing techniques have become so sensitive that the greatest concern is not detecting a gene or transcript but rather, falsely identifying one. Better genome sequencing has led to more complete transcriptomic and proteomic databases and, combined with more sensitive instrumentation and separation techniques, is bringing us closer to detecting complete transcriptomes and proteomes. The promise of these powerful omics techniques is to lead us to new and unexpected connections between molecular processes in the context of animal health. This promise cannot be achieved without hypothesis-driven research that connects omics data with animal health experiments. Any researcher who wishes to invest the time and resources in omics experiments should be aware of the common pitfalls and limitations of these techniques so they can avoid these issues and maximize the use of these research tools. Several important questions must be asked: What is the quality of the databases and how they are annotated? Are the annotations based on experimental results or computational predictions? What assumptions are made by the analysis algorithms, and how will this affect the result? Finally, how can the research community use the vast amount of data being generated by omics experiments in ways to achieve the goals of better animal health and production (which is the promise of omics technologies)? Until the observations shown in omics data sets are used to achieve the goals of better animal health and production, the potential of omics technology will not be fully realized.

The effect of pegylated granulocyte colony-stimulating factor treatment prior to experimental mastitis in lactating Holsteins.

Neutrophils are the first-acting and most prominent cellular defense against mastitis-causing pathogens. This makes neutrophil activation and expansion obvious candidates for targeted therapeutics. The granulocyte colony-stimulating factor (G-CSF) cytokine stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream, which results in neutrophilia as well as increasing the presence of other progenitor cells in the bloodstream. A pegylated form of G-CSF (PEG-gCSF) has been shown to significantly decrease naturally occurring mastitis rates in cows postpartum. The use of PEG-gCSF had not been evaluated in response to an experimental mastitis challenge. In an effort to examine the effect and mechanism of PEG-gCSF treatment, we challenged 11 mid-lactation Holsteins with ∼400 cfu Escherichia coli P4 by intramammary infusion. Five cows received 2 PEG-gCSF injections, one at 14 d and the other at 7 d before disease challenge, and 6 cows remained untreated. To evaluate the response of cows to the PEG-gCSF treatment, we measured complete blood counts, somatic cell counts, bacterial counts, milk yield, and feed intake data. The PEG-gCSF-treated cows had significantly increased circulating levels of neutrophils and lymphocytes after each PEG-gCSF injection, as well as following mastitis challenge. The PEG-gCSF-treated cows had significantly lower bacterial counts and lower milk BSA levels at the peak of infection. In addition, control cows had significant decreases in milk yield postinfection and significantly reduced feed intake postinfection compared with PEG-gCSF-treated cows. Collectively, PEG-gCSF treatment resulted in reduced disease severity when administered before a bacterial challenge. Mechanistically, we show that G-CSF treatment increases cell surface expression of an E-selectin ligand before infection on neutrophils and monocytes found in the blood. These cells quickly disappear from the blood shortly after infection, suggesting a mechanism for the reduced mastitis severity by priming immune cells for quick targeting to the site of infection.

Quantification of bovine oxylipids during intramammary Streptococcus uberis infection.

Streptococcus uberis mastitis results in severe mammary tissue damage in dairy cows due to uncontrolled inflammation. Oxylipids are potent lipid mediators that orchestrate pathogen-induced inflammatory responses, however, changes in oxylipid biosynthesis during S. uberis mastitis are unknown. Thus, the current objective was to determine how oxylipid concentrations change in milk and mammary tissues during different stages of S. uberis mastitis. Increased arachidonic acid and linoleic acid-derived oxylipids were significantly increased in S. uberis-infected bovine mammary tissue. Linoleic acid metabolites, hydroxyoctadecadienoic acid (HODE) and oxooctadecadienoic acid, predominated in tissue and milk. Furthermore, in vitro exposure of bovine mammary endothelial cells to 13-hydroperoxyoctadecadienoic acid, upstream metabolite of HODE, significantly increased cyclooxygenase-2 expression, but 13-HODE exposure had no effect. The findings in the current study indicate lipidomic profiling may explain some of the dynamics of inflammation during bacterial challenge, however continued research is necessary to determine sample compartments which best reflect disease pathogenesis.

Seasonal variation in vitamin D status of beef cattle reared in the central United States.

The objective was to retrospectively measure seasonal sunlight-associated variation in serum concentrations of 25-hydroxyvitamin D (25OHD) in beef cattle. The concentration of 25OHD was measured in crossbred animals born from March to May in 2011 and 2012. Vitamin D status 2 to 3 mo after birth (period 1) was only available for 2012 calves and was measured in June 2012. Period 1 animals had serum 25OHD concentrations of 26.3 ± 1.5 ng/mL. The 25OHD concentrations for late summer (period 2) were 46.6 ± 1.4 and 51.0 ± 1.5 ng/mL for 2011 and 2012, respectively. Serum concentration of 25OHD in early fall (period 3) were 63.8 ± 1.4 and 55.2 ± 1.5 ng/mL for calves in 2011 and 2012, respectively. Values observed for both late summer and early fall indicated vitamin D sufficiency (P < 0.001) compared with period 1. With diminishing exposure to ultraviolet B and consuming ∼800 IU or 1800 IU (2011 and 2012, respectively) of supplemental vitamin D, the calves' midwinter (period 4) 25OHD concentrations fell to 15.2 ± 1.6 and 16.7 ± 1.5 ng/mL for 2011 and 2012, respectively, after 4 to 5 mo on a finishing diet (P < 0.0001). This is considered vitamin D insufficiency in most species. Results indicate that calves are marginally sufficient to insufficient for vitamin D based on serum 25OHD concentrations soon after birth and during winter. Some individual animals would be classified vitamin D deficient. In the absence of sufficient UVB exposure, the dietary vitamin D requirements for rapidly growing beef cattle may need to be increased.

Acute phase response elicited by experimental bovine diarrhea virus (BVDV) infection is associated with decreased vitamin D and E status of vitamin-replete preruminant calves.

Studies in young animals have shown an association between vitamin deficiencies and increased risk of infectious disease; however, there is a paucity of information regarding the effect of acute infection on the vitamin status of the vitamin-replete neonate. To characterize the effects of acute infection on vitamin D and E status of the neonate, 6 vitamin-replete preruminant Holstein bull calves were experimentally infected with bovine viral diarrhea virus (BVDV; strain BVDV2-1373). Six mock-inoculated calves served as controls. Sustained pyrexia, leukopenia, and asynchronous increases in serum haptoglobin and serum amyloid A characterized the response of calves to infection with BVDV. Infection was also associated with increased serum IFN-γ, IL-2, and IL-6 concentrations. During the last 8 d of the 14-d postinoculation period, serum 25-hydroxyvitamin D and α-tocopherol concentrations in infected calves decreased by 51 and 82%, respectively. The observed inverse association between vitamin D and E status and serum amyloid A in infected calves suggests that the infection-induced acute phase response contributed to the reduced vitamin status of these animals. Additional studies are necessary to determine if the negative effect of infection on status are unique to this specific infection model or is representative of preruminant calf's response to acute infection. Studies are also needed to characterize mechanisms underlying infection-related changes in vitamin D and E status and to determine whether additional vitamin D or E supplementation during an acute infection diminishes disease severity and duration in the young animal.

A genomewide association study identified CYP2J2 as a gene controlling serum vitamin D status in beef cattle.

Vitamin D is an important modulator of calcium homeostasis and has several effects on the immune system. The objective of the study was to estimate its heritability and to identify genomic regions associated with concentration of circulating 25-hydroxyvitamin D (25OHD) in beef cattle. Status of vitamin D was measured in crossbred animals from Cycle VII of the United States Meat Animal Research Center (USMARC) Germplasm Evaluation Project. Progeny were born from March through May in 2008 and in 2010. Heritability was estimated and a genomewide association study was conducted on the concentration of 25OHD measured in 1,432 animals at preconditioning and 1,333 animals at weaning. Genotyping of the population was done by imputing from the parental generation genotyped with a high density array (777,000 SNP) to a target population genotyped with a medium density SNP array (50,000 SNP). After imputation, 675,018 SNP were used in the genomewide association study. Heritability of concentration of circulating 25OHD in cattle at preconditioning and at weaning was 0.41 ± 0.08 and 0.32 ± 0.11, respectively. A region on chromosome 3 was associated with circulating 25OHD. The region on BTA3 had 7 SNP significantly (P < 7.4 × 10(-8)) associated at the genomewide level with serum concentrations of serum 25OHD. Genomewide significant SNP spanned the region between 84.93 and 86.65 megabases (Mb); however, 6 SNP reside between 86.64 and 86.65 Mb. The gene CYP2J2 was identified as a candidate gene associated with concentrations of serum 25OHD in cattle. This is 1 of 6 enzymes involved in metabolizing vitamin D to 25OHD. Results from the present study suggest that CYP2J2 is a gene controlling serum 25OHD concentrations in cattle. CYP2J2 should be considered a prime candidate for understanding both genetic and physiological factors affecting serum 25OHD concentrations in cattle and, therefore, vitamin D status.

Modulation of the bovine innate immune response by production of 1alpha,25-dihydroxyvitamin D(3) in bovine monocytes.

In cattle, the kidney has been the only known site for production of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] from 25-hydroxyvitamin D(3) [25(OH)D(3)] by 1alpha-hydroxylase (1alpha-OHase). Based on human studies, it was hypothesized that bovine monocytes could produce 1,25(OH)(2)D(3) upon activation and 1,25(OH)(2)D(3) would regulate expression of vitamin D-responsive genes in monocytes. First, the effects of 1,25(OH)(2)D(3) on bovine monocytes isolated from peripheral blood were tested. Treatment of nonstimulated monocytes with 1,25(OH)(2)D(3) increased expression of the gene for the vitamin D 24-hydroxylase (24-OHase) enzyme by 51+/-13 fold, but 1,25(OH)(2)D(3) induction of 24-OHase expression was blocked by lipopolysaccharide (LPS) stimulation. In addition, 1,25(OH)(2)D(3) increased the gene expression of inducible nitric oxide synthase and the chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) in LPS-stimulated monocytes 69+/-13 and 40+/-12 fold, respectively. Next, the ability of bovine monocytes to express 1alpha-OHase and produce 1,25(OH)(2)D(3) was tested. Activation of monocytes with LPS, tripalmitoylated lipopeptide (Pam3CSK4), or peptidoglycan caused 43+/-9, 17+/-3, and 19+/-3 fold increases in 1alpha-OHase gene expression, respectively. Addition of 25(OH)D(3) to LPS-stimulated monocytes enhanced expression of inducible nitric oxide synthase and RANTES and nitric oxide production in a dose-dependent manner, giving evidence that activated monocytes convert 25(OH)D(3) to 1,25(OH)(2)D(3). In conclusion, bovine monocytes produce 1,25(OH)(2)D(3) in response to toll-like receptor signaling, and 1,25(OH)(2)D(3) production in monocytes increased the expression of genes involved in the innate immune system. Vitamin D status of cattle might be important for optimal innate immune function because 1,25(OH)(2)D(3) production in activated monocytes and subsequent upregulation of inducible nitric oxide synthase and RANTES expression was dependent on 25(OH)D(3) availability.

Centennial paper: Proteomics in animal science.

Proteomics holds significant promise as a method for advancing animal science research. The use of this technology in animal science is still in its infancy. The ability of proteomics to simultaneously identify and quantify potentially thousands of proteins is unparalleled. In this review, we will discuss basic principles of doing a proteomic experiment. In addition, challenges and limitations of proteomics will be considered, stressing those that are unique to animal sciences. The current proteomic research in animal sciences will be discussed, and the potential uses for this technology will be highlighted.

Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk.

Shotgun proteomics, using amine-reactive isobaric tags (iTRAQ), was used to quantify protein changes in milk fat globule membranes (MFGM) that were isolated from d 1 colostrum and compared with MFGM from d 7 milk. Eight Holstein cows were randomly assigned to 2 groups of 4 cow sample pools for a simple replication of this proteomic analysis using iTRAQ. The iTRAQ labeled peptides from the experiment sample pools were fractionated by strong cation exchange chromatography followed by further fractionation on a microcapillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Data analysis identified 138 bovine proteins in the MFGM with 26 proteins upregulated and 19 proteins downregulated in d 7 MFGM compared with colostrum MFGM. Mucin 1 and 15 were upregulated greater than 7-fold in MFGM from d 7 milk compared with colostrum MFGM. The tripartite complex of proteins of adipophilin, butyrophilin, and xanthine dehydrogenase were individually upregulated in d 7 MFGM 3.4-, 3.2-, and 2.6-fold, respectively, compared with colostrum MFGM. Additional proteins associated with various aspects of lipid transport synthesis and secretion such as acyl-CoA synthetase, lanosterol synthase, lysophosphatidic acid acyltransferase, and fatty acid binding protein were upregulated 2.6- to 5.1-fold in d 7 MFGM compared with colostrum MFGM. In contrast, apolipoproteins A1, C-III, E, and A-IV were downregulated 2.6- to 4.3-fold in d 7 MFGM compared with colostrum MFGM. These data demonstrate that quantitative shotgun proteomics has great potential to provide new insights into mammary development.

Immunological signaling networks: integrating the body's immune response.

The role of the immune system is to protect against infection and to eliminate disease from the host. Nonimmune cells can not only act as physical barriers, but also respond to microbial stimulation to release antimicrobial molecules, whereas immune cells are primarily responsible for eliminating pathogens or cancerous cells. In addition, immune cells regulate the immune response affecting the types of cells that are activated or suppressed. The following discussion is an overview of the immune system and its interconnection with the host. How nonimmune cells and innate and adaptive immune cells work separately and together to respond to a pathogenic challenge is discussed. In addition, how the immune system can be affected by factors such as nutrition and stress, and how the immune system can affect factors such as fertility demonstrates the integration of the immune system in processes other than elimination of pathogens.

Cumulative physiological events influence the inflammatory response of the bovine udder to Escherichia coli infections during the transition period.

A high proportion of intramammary coliform infections present at parturition develop disease characterized by severe inflammatory signs and sepsis during the first 60 to 70 d of lactation. In the lactating bovine mammary gland, the innate immune system plays a critical role in determining the outcome of these infections. Since the beginning of the 1990s, research has increased significantly on bovine mammary innate defense mechanisms in connection with the pathogenesis of coliform mastitis. Neutrophils are key effector cells of the innate immune response to intramammary infection, and their function is influenced by many physiological events that occur during the transition period. Opportunistic infections occur when the integrity of the host immune system is compromised by physical and physiological conditions that make the host more susceptible. The innate immune system of many periparturient cows is immunocompromised. It is unlikely that periparturient immunosuppression is the result of a single physiological factor; more likely, several entities act in concert, with profound effects on the function of many organ systems of the periparturient dairy cow. Their defense system is unable to modulate the complex network of innate immune responses, leading to incomplete resolution of the pathogen and the inflammatory reaction. During the last 30 yr, most efforts have been focused on neutrophil diapedesis, phagocytosis, and bacterial killing. How these functions modulate the clinical outcome of coliform mastitis, and how they can be influenced by hormones and metabolism has been the subject of intensive research and is the focus of this review. The afferent (sensing) arm of innate immunity, which enables host recognition of a diverse array of pathogens, is the subject of intense research interest and may contribute to the variable inflammatory response to intramammary infections during different stages of lactation. The development of novel interventions that modulate the inflammatory response or contribute to the elimination of the pathogen or both may offer therapeutic promise in the treatment of mastitis in periparturient cows.

Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors.

We have studied the contributions of proteasome inhibitor-sensitive and -insensitive proteases to the generation of class I MHC-associated peptides. The cell surface expression of 13 different human class I MHC alleles was inhibited by as much as 90% or as little as 40% when cells were incubated with saturating concentrations of three different proteasome inhibitors. Inhibitor-resistant class I MHC expression was not due to TAP-independent expression or preexisting internal stores of peptides. Furthermore, it did not correlate with the amount or specificity of residual proteasome activity as determined in in vitro proteolysis assays and was not augmented by simultaneous incubation with multiple inhibitors. Mass spectrometry was used to directly characterize the peptides expressed in the presence and absence of proteasome inhibitors. The number of peptide species detected correlated with the levels of class I detected by flow cytometry. Thus, for many alleles, a significant proportion of associated peptide species continue to be generated in the presence of saturating levels of proteasome inhibitors. Comparison of the peptide-binding motifs of inhibitor-sensitive and -resistant class I alleles further suggested that inhibitor-resistant proteolytic activities display a wide diversity of cleavage specificities, including a trypsin-like activity. Sequence analysis demonstrated that inhibitor-resistant peptides contain diverse carboxyl termini and are derived from protein substrates dispersed throughout the cell. The possible contributions of inhibitor-resistant proteasome activities and nonproteasomal proteases residing in the cytosol to the peptide profiles associated with many class I MHC alleles are discussed.

Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65).

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4(+) T cell response in IDDM.

Cytotoxic T lymphocytes from HLA-A2.1 transgenic mice define a potential human epitope from simian virus 40 large T antigen.

Recent reports have documented the presence of SV40 large T antigen (T ag) sequences in a number of human tumors and raised the question of whether cellular immunity to T ag is elicited in such individuals. We used HLA-A2.1 transgenic C57BL/6 mice to identify an epitope from T ag recognized by CD8+ CTLs when presented by this human MHC class I molecule. Immunization of HLA-A2.1 transgenic mice with syngeneic T ag-transformed cells resulted in the induction of HLA-A2.1-restricted, T ag-specific CTLs. The target epitope, residues 281-289 (KCDDVLLLL) of T ag, was identified using both cell lines expressing T ag variants and synthetic T ag peptides. Peptide 281-289 bound stably to HLA-A2.1 molecules, effectively sensitized target cells for CTL lysis, and was efficiently processed from endogenous T ag in cells of both mouse and human origin. CTLs were not cross-reactive on the human BK or JC virus T ags. Thus, SV40 T ag 281-289 represents a potential specific CTL recognition epitope for humans.

Role of CTL host responses and their implication for tumorigenicity testing and the use of tumour cells as vaccine substrate.

Viral oncogenes, mutated cellular oncogenes, or other adventitious agents that might contaminate vaccine preparations on inoculation of the host will encounter a T cell-mediated immune response which will play a determining role in the progression of neoplastic events or replication of contaminating viral agents. Using SV40 T antigen tumour systems as a model we discuss the regions of the oncoprotein that have an impact on tumourigenicity and the role of CD8 T lymphocyte immune responses in eliminating potential tumour cells. In addition, we discuss measures that counteract T cell immune responses to abrogate T cell-mediated immunosurveillance.

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways.

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.

Hierarchy among multiple H-2b-restricted cytotoxic T-lymphocyte epitopes within simian virus 40 T antigen.

Simian virus 40 large tumor (T) antigen contains three H-2Db-restricted (I, II/III, and V) and one H-2Kb-restricted (IV) cytotoxic T lymphocyte (CTL) epitopes. We demonstrate that a hierarchy exists among these CTL epitopes, since vigorous CTL responses against epitopes I, II/III, and IV are detected following immunization of H-2b mice with syngeneic, T-antigen-expressing cells. By contrast, a weak CTL response against the H-2Db-restricted epitope V was detected only following immunization of H-2b mice with epitope loss variant B6/K-3,1,4 cells, which have lost expression of CTL epitopes I, II/III, and IV. Limiting-dilution analysis confirmed that the lack of epitope V-specific CTL activity in bulk culture splenocytes correlated with inefficient expansion and priming of epitope V-specific CTL precursors in vivo. We examined whether defined genetic alterations of T antigen might improve processing and presentation of epitope V to the epitope V-specific CTL clone Y-5 in vitro and/or overcome the recessive nature of epitope V in vivo. Deletion of the H-2Db-restricted epitopes I and II/III from T antigen did not increase target cell lysis by epitope V-specific CTL clones in vitro. The amino acid sequence SMIKNLEYM, which species an optimized H-2Db binding motif and was found to induce CTL in H-2b mice, did not further reduce epitope V presentation in vitro when inserted within T antigen. Epitope V-containing T-antigen derivatives which retained epitopes I and II/III or epitope IV did not induce epitope V-specific CTL in vivo: T-antigen derivatives in which epitope V replaced epitope I failed to induce epitope V-specific CTL. Recognition of epitope V-H-2Db complexes by multiple independently derived epitope V-specific CTL clones was rapidly and dramatically reduced by incubation of target cells in the presence of brefeldin A compared with the recognition of the other T-antigen CTL epitopes by epitope specific CTL, suggesting that the epitope V-H-2Db complexes either are labile or are present at the cell surface at reduced levels. Our results suggest that processing and presentation of epitope V is not dramatically altered (reduced) by the presence of immunodominant CTL epitopes in T antigen and that the immunorecessive nature of epitope V is not determined by amino acids which flank its native location within simian virus 40 T antigen.

Functional analysis of amino acid residues encompassing and surrounding two neighboring H-2Db-restricted cytotoxic T-lymphocyte epitopes in simian virus 40 tumor antigen.

Simian virus 40 tumor (T) antigen contains three H-2Db-and one H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes (sites). Two of the H-2Db-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this study, we determine if the amino acids separating these two H-2Db-restricted CTL epitopes are dispensable for efficient processing and presentation. In addition, the importance of amino acid residues lying within and flanking the H-2Db-restricted epitopes I and II/III for efficient processing, presentation, and recognition by site-specific CTL clones was determined by using T-antigen mutants containing single-amino-acid substitutions between residues 200 and 239. Using synthetic peptides in CTL lysis and major histocompatibility complex class I stabilization assays, CTL recognition site I has been redefined to include residues 206 to 215. Substitutions in amino acids flanking either site I or site II/III did not affect recognition by any of the T-antigen-specific CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstrating that these two epitopes when arranged in tandem in the native T antigen can be efficiently processed and presented to CTL clones. Differences in fine specificities of two CTL clones which recognize the same epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) have been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR recognition and/or peptide-major histocompatibility complex association.

Comparative analysis of core amino acid residues of H-2D(b)-restricted cytotoxic T-lymphocyte recognition epitopes in simian virus 40 T antigen.

Simian virus 40 (SV40) tumor (T) antigen expressed in H-2b SV40-transformed cells induces the generation of Lyt-2+ (CD8+) cytotoxic T lymphocytes (CTL), which are involved in tumor rejection, in syngeneic mice. Five CTL recognition sites on T antigen have been described by using mutant T antigens. Four of the sites (I, II, III, and V) are H-2Db restricted and have been broadly mapped with synthetic peptides of 15 amino acids in length overlapping by 5 residues at the amino and carboxy termini. The goal of this study was to define the minimal and optimal amino acid sequences of T antigen which would serve as recognition elements for the H-2Db-restricted CTL clones Y-1, Y-2, Y-3, and Y-5, which recognizes sites I, II, III, and V, respectively. The minimal and optimal residues of T antigen recognized by the four CTL clones were determined by using synthetic peptides truncated at the amino or carboxy terminus and an H-2Db peptide-binding motif. The minimal site recognized by CTL clone Y-1 was defined as amino acids 207 to 215 of SV40 T antigen. However, the optimal sequence recognized by CTL clone Y-1 spanned T-antigen amino acids 205 to 215. The T-antigen peptide sequence LT223-231 was the optimal and minimal sequence recognized by both CTL clones Y-2 and Y-3. Site V was determined to be contained within amino acids 489 to 497 of T antigen. The lytic activities of CTL clones Y-2 and Y-3, which recognize a single nonamer peptide, LT223-231, were affected differently by anti-Lyt-2 antibody, suggesting that the T-cell receptors of these two CTL clones differ in their avidities. As the minimal and optimal H-2Db-restricted CTL recognition sites have been defined by nonamer synthetic peptides, it is now possible to search for naturally processed H-2Db-restricted epitopes of T antigen and identify critical residues involved in processing, presentation, and recognition by SV40-specific CTL.