Antigen/Adjuvant-Displaying Enveloped Viral Replica as a Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidate.

We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled ß-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to ∼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.

Coccomyxa sp.KJ extract affects the fate of T cells stimulated by toxic shock syndrome toxin-1, a superantigen secreted by Staphylococcus aureus.

T cell stimulation by bacterial superantigens induces a cytokine storm. After T cell activation and inflammatory cytokine secretion, regulatory T cells (Treg) are produced to suppress the immune response. Coccomyxa sp.KJ (IPOD FERM BP-22254), a green alga, is reported to regulate immune reactions. Therefore, we examined the effects of Coccomyxa sp.KJ extract (CE) on the superantigen-induced immune response. When human peripheral blood mononuclear cells (PBMCs) were stimulated with toxic shock syndrome-1 (TSST-1) in the presence of CE, the number of activated T cells decreased moderately. Purified T cells stimulated in the presence of CE comprised more non-proliferating cells than those stimulated in the absence of CE, whereas some T cells proliferated more quickly. The levels of activation markers on the stimulated T cells increased in the presence of CE. Most of the inflammatory cytokines did not change but IL-1ß, IL-17, IL-4, and IL-13 secretion increased, whereas that of IL-2, TNF-α, and IL-18 decreased. IL-10 secretion was also decreased by CE treatment, suggesting that the immune response was not suppressed by Treg cells. CE enhanced the expression of stem cell-like memory cell markers in T cells. These results suggest that CE can regulate the fate of T cells and can help to ameliorate superantigen-induced T cell hyperactivation and immune suppression.

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates.

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam3 CSK4 , α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.

Humanized Mice as an Effective Evaluation System for Peptide Vaccines and Immune Checkpoint Inhibitors.

Peptide vaccination was developed for the prevention and therapy of acute and chronic infectious diseases and cancer. However, vaccine development is challenging, because the patient immune system requires the appropriate human leukocyte antigen (HLA) recognition with the peptide. Moreover, antigens sometimes induce a low response, even if the peptide is presented by antigen-presenting cells and T cells recognize it. This is because the patient immunity is dampened or restricted by environmental factors. Even if the immune system responds appropriately, newly-developed immune checkpoint inhibitors (ICIs), which are used to increase the immune response against cancer, make the immune environment more complex. The ICIs may activate T cells, although the ratio of responsive patients is not high. However, the vaccine may induce some immune adverse effects in the presence of ICIs. Therefore, a system is needed to predict such risks. Humanized mouse systems possessing human immune cells have been developed to examine human immunity in vivo. One of the systems which uses transplanted human peripheral blood mononuclear cells (PBMCs) may become a new diagnosis strategy. Various humanized mouse systems are being developed and will become good tools for the prediction of antibody response and immune adverse effects.

Syntheses and Immunological Evaluation of Self-Adjuvanting Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with a T-helper Cell Epitope as Antitumor Vaccine Candidates.

Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen (TACA) rarely observed on healthy tissues. We synthesized two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn) vaccines possessing a T-helper epitope and a TLR2 agonist, since the clustered STn antigens are highly expressed on many cancer cells. Immunization of both vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine were less specific. These results illustrated that N-propionyl triSTn is a valuable unnatural TACA for anticancer vaccines.

Liposome-encapsulated progesterone efficiently suppresses B-lineage cell proliferation.

Progesterone suppresses several ancient pathways in a concentration-dependent manner. Based on these characteristics, progesterone is considered a candidate anticancer drug. However, the concentration of progesterone used for therapy should be higher than the physiological concentration, which makes it difficult to develop progesterone-based anticancer drugs. We previously developed liposome-encapsulated progesterone (Lipo-P4) with enhanced anticancer effects, which strongly suppressed triple-negative breast cancer cell proliferation in humanized mice. In this study, we aimed to clarify whether Lipo-P4 effectively suppresses the proliferation of B-lineage cancer cells. We selected six B-cell lymphoma and two myeloma cell lines, and analyzed their surface markers using flow cytometry. Next, we prepared liposome-encapsulated progesterone and examined its effect on cell proliferation in these B-lineage cancer cells, three ovarian clear cell carcinoma cell lines, two prostate carcinoma cell lines, and one triple-negative breast cancer adenocarcinoma cell line. Lipo-P4 suppressed the proliferation of all cancer cell lines. All B-lineage cell lines, except for the HT line, were more susceptible than the other cell types, regardless of the expression of differentiation markers. Empty liposomes did not suppress cell proliferation. These results suggest that progesterone encapsulated in liposomes efficiently inhibits the proliferation of B-lineage cells and may become an anticancer drug candidate for B-lineage cancers.

Coccomyxa subellipsoidea KJ Components Enhance the Expression of Metallothioneins and Th17 Cytokines during Human T Cell Activation.

Coccomyxa subellipsoidea KJ (C-KJ) is a green alga with unique immunoregulatory characteristics. Here, we investigated the mechanism underlying the modification of T cell function by C-KJ components. The water-soluble extract of C-KJ was fractionated into protein (P) and sugar (S) fractions acidic (AS), basic (BS), and neutral (NS). These fractions were used for the treatment of peripheral blood mononuclear cells stimulated with toxic shock syndrome toxin-1. Transcriptome analysis revealed that both P and AS enhanced the expression of the genes encoding metallothionein (MT) family proteins, inflammatory factors, and T helper (Th) 17 cytokine and suppressed that of those encoding Th2 cytokines in stimulated T cells. The kinetics of MT1 and MT2A gene expression showed a transient increase in MT1 and maintenance of MT2A mRNA after T cell stimulation in the presence of AS. The kinetics of Th17-related cytokine secretion in the early period were comparable to those of MT2A mRNA. Furthermore, our findings revealed that static, a STAT-3 inhibitor, significantly suppressed MT2A gene expression. These findings suggest that the expression of MTs is involved in the immune regulatory function of C-KJ components, which is partially regulated by Th17 responses, and may help develop innovative immunoregulatory drugs or functional foods.

Construction of the systemic anticancer immune environment in tumour-bearing humanized mouse by using liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated progesterone.

Immune checkpoint inhibitors highlight the importance of anticancer immunity. However, their clinical utility and safety are limited by the low response rates and adverse effects. We focused on progesterone (P4), a hormone produced by the placenta during pregnancy, because it has multiple biological activities related to anticancer and immune regulation effects. P4 has a reversible immune regulatory function distinct from that of the stress hormone cortisol, which may drive irreversible immune suppression that promotes T cell exhaustion and apoptosis in patients with cancer. Because the anticancer effect of P4 is induced at higher than physiological concentrations, we aimed to develop a new anticancer drug by encapsulating P4 in liposomes. In this study, we prepared liposome-encapsulated anti-programmed death ligand 1 (PD-L1) antibody-conjugated P4 (Lipo-anti-PD-L1-P4) and evaluated the effects on the growth of MDA-MB-231 cells, a PD-L1-expressing triple-negative breast cancer cell line, in vitro and in NOG-hIL-4-Tg mice transplanted with human peripheral blood mononuclear cells (humanized mice). Lipo-anti-PD-L1-P4 at physiological concentrations reduced T cell exhaustion and proliferation of MDA-MB-231 in vitro. Humanized mice bearing MDA-MB-231 cells expressing PD-L1 showed suppressed tumor growth and peripheral tissue inflammation. The proportion of B cells and CD4+ T cells decreased, whereas the proportion of CD8+ T cells increased in Lipo-anti-PD-L1-P4-administrated mice spleens and tumor-infiltrated lymphocytes. Our results suggested that Lipo-anti-PD-L1-P4 establishes a systemic anticancer immune environment with minimal toxicity. Thus, the use of P4 as an anticancer drug may represent a new strategy for cancer treatment.

Precise immunological evaluation rationalizes the design of a self-adjuvanting vaccine composed of glycan antigen, TLR1/2 ligand, and T-helper cell epitope.

Sialyl-Tn (STn), overexpressed on various tumors, has been investigated for its application in anti-cancer vaccine therapy. However, Theratope, an STn-based vaccine, failed in the phase III clinical trial due to poor immunogenicity and epitope suppression by the foreign carrier protein. We therefore developed a self-adjuvanting STn based-vaccine, a conjugate of clustered STn (triSTn) antigen, TLR1/2 ligand (Pam3CSK4), and T-helper (Th) cell epitope, and found that this three-component self-adjuvanting vaccine effectively resulted in the production of anti-triSTn IgG antibodies. We herein analyzed immune responses induced by this self-adjuvanting vaccine in detail. We newly synthesized two-component vaccines, i.e., Pam3CSK4- or Th epitope-conjugated triSTn, as references to evaluate the immune-stimulating functions of Pam3CSK4 and Th epitope. Immunological evaluation of the synthesized vaccine candidates revealed that Pam3CSK4 was essential for antibody production, indicating that the uptake of triSTn antigen by antigen-presenting cells (APCs) was promoted by the recognition of Pam3CSK4 by TLR1/2. The function of the Th epitope was also confirmed. Th cell activation was important for boosting antibody production and IgG subclass switching. Furthermore, flow cytometric analyses of immune cells, including T cells, B cells, dendritic cells, and other monocytes, were first employed in the evaluation of self-adjuvanting vaccines and revealed that the three-component vaccine was able to induce antigen-specific immune responses for efficient antibody production without excessive inflammatory responses. Importantly, the co-administration of Freund's adjuvants was suggested to cause excessive myeloid cell accumulation and decreased plasma cell differentiation. These results demonstrate that vaccines can be designed to achieve the desired immune responses via the bottom-up construction of each immune element.

Genetic Association between Farrowing Rates and Swine Leukocyte Antigen Alleles or Haplotypes in Microminipigs.

We have previously reported specific swine leukocyte antigen (SLA) haplotype associations with significant effects on several reproduction performance traits in a highly inbred miniature pig population of Microminipigs (MMPs). In this study, to clarify the effects on farrowing rates of SLA similarity between mating partners in the MMP population, we compared the farrowing rates as a measure of reproductive success after 1063-cumulative matings among the following three groups of mating partners: (1) completely sharing SLA class I or class II haplotypes or alleles between partners (CS), (2) only one sharing the haplotypes or alleles (OS), and (3) non-sharing the haplotypes or alleles (NS). Average farrowing rates in CS groups consisting of completely sharing SLA class II haplotypes or DRBI and DQB1 alleles were lowest in the three groups. Moreover, lower farrowing rates were indicated in mating pairs with smaller amino acid pairwise genetic distances of SLA-1, SLA-3, DRB1 and DQB1 alleles between the pairs. These results suggested that the dissimilarity of SLA class I and class II alleles between mating partners markedly improved reproductive performance; therefore, SLA alleles or haplotypes are potentially useful genetic markers for the selection of mating pairs in breeding programs and epistatic studies of reproductive traits of MMPs.

Stillbirth rates and their association with swine leucocyte antigen class II haplotypes in Microminipigs.

OBJECTIVE: Microminipig (MMP) is a miniature pig with an extra small body size for experimental use. In the present study, the occurrence of stillbirths and their genetic association with swine leukocyte antigen (SLA) class II haplotypes were evaluated in a population of MMPs. METHODS: The occurrences of stillbirth and genetic association with SLA class II haplotypes using 483 stillborn and 2,246 live piglets, and their parents were compared among the three groups of newborn piglet litters; an all stillborn (AS) group consisting of only stillborn piglet litters, a partial stillborn (PS) group consisting of stillborn and live piglet litters, and an all alive (AA) group consisting of only live piglet litters. RESULTS: The incidence of stillborn piglets was 483/2,729 (17.7%). Distributions of litter sizes, numbers of stillborn piglets in a litter, parities, and gestation periods were distinct among the three groups. The frequencies of low resolution haplotype (Lr)-0.7 or Lr-0.23 were higher in the AS group than in the PS or AA groups. In sires, the frequency of Lr-0.7 associated with the AS group was significantly higher in the AS group than with the AA group. In dams, the frequency of Lr-0.23 was significantly higher in the AS group than in the PS or AA groups, whereas the frequency of Lr-0.7 was not significantly different. CONCLUSION: The incidence of stillborn piglets in MMPs appears to be higher than those in other pig breeds. Several traits related with stillbirths such as the number of stillborn piglets and parities of the AS group were different from those of the PS and AA groups. Specific SLA class II haplotypes were associated significantly with a high incidence of stillbirths and could be used as genetic markers to adopt breeding strategies to lower the rate of stillbirth in MMPs.

HER2-antigen-specific humoral immune response in breast cancer lymphocytes transplanted in hu-PBL hIL-4 NOG mice.

The status of humoral immunity of cancer patients is not clear compared to cellular immunity because the ability of specific antibody production is difficult to analyze in vitro. We previously developed a humanized mouse model to evaluate antigen-specific antibody production by transplanting human peripheral blood mononuclear cells (PBMCs) into NOG-hIL-4-Tg mice (hu-PBL hIL-4 NOG). In this study, these mice were transplanted with PBMCs derived from breast cancer patients (BC) and immunized with a human epidermal growth factor receptor 2 (HER2) peptide, CH401MAP, to analyze humoral immunity of BCs. The hu-PBL hIL-4 NOG mice recapitulated immune environment of BCs as the ratio of CD8+/CD4+T cells was lower and that of PD-1 + T cells was higher compared to healthy donors (HDs). Diverse clusters were detected in BC-mouse (BC-M) plasma components involving immunoglobulins and complements unlike HD-M, and there was a significant diversity in CH401MAP-specific IgG titers in BC-M. The number of B cell clones producing high CH401MAP-specific IgG was not increased by immunization in BC-M unlike HD-M. These results demonstrated that the humoral immunity of BCs appeared as diverse phenotypes different from HDs in hu-PBL hIL-4 NOG mice, which may provide important information for the study of personalized medicine.

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs.

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

Syntheses and Functional Studies of Self-Adjuvanting Anti-HER2 Cancer Vaccines.

The 9-mer peptide MFCH401 (N: 165-173: DTILWKDIF), which is located in the extracellular domain of HER2, has been predicted to be a novel epitope. Self-adjuvanting anti-HER2 vaccine constructs were designed and synthesized via covalently attaching MFCH401 or its linear tandem repeats (2×MFCH401, 3×MFCH401) to a lipopeptide Pam3 CSK4 via iterative condensation reaction. The in vivo results showed the Pam3 CSK4 -MFCH401 vaccine construct can induce higher antibody titers of IgG and IgM than those of other conjugates, and the analysis of changes in plasma cytokines level indicate the activation of Th1 cells and NK cells. In addition, the Pam3 CSK4 -MFCH401 vaccine conjugate induced a specific immune response to HER2-overexpressing human BT474 cells. Our data clearly indicated that MFCH401 is a promising epitope; moreover, its linear tandem repeats were unsuitable for anticancer vaccine design when conjugating with Pam3 CSK4 , which provided useful evidence for developing further anti-HER2 cancer vaccines.

Expression of glucocorticoid receptor shows negative correlation with human B-cell engraftment in PBMC-transplanted NOGhIL-4-Tg mice.

The humanized mouse system is a promising tool for analyzing human immune responses in vivo. Recently, we developed a new humanized mouse system using the severely immunodeficient NOD/Shi-scid-IL2rγnull (NOG)-hIL-4-Tg mouse, which enabled us to evaluate the human humoral immune response after peripheral blood mononuclear cell (PBMC) transplantation. However, the mechanism by which hIL-4 enhances antigen-specific IgG production in these mice is not clear. In this study, we analyzed the relationship between human lymphocyte subsets and the expression level of the glucocorticoid receptor (GR) to clarify the humoral immune condition in human PBMC-transplanted NOG-hIL-4 mice. The results showed that the human GR mRNA level was significantly lower in NOG-hIL-4-Tg splenocytes than in conventional NOG splenocytes after immunization. Whereas no obvious difference of the proportion of T helper-cell subsets was observed between the NOG and NOG-hIL-4-Tg mouse strains, the B-cell proportion and antigen-specific IgG concentration in plasma showed strong negative correlations with the GR mRNA level. These results suggest that the GR expression level was changed in PBMCs in the humanized NOG-hIL-4-Tg mice, which may support B-cell survival and function in the mouse system.

Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

Differentiation ability of multipotent hematopoietic stem/progenitor cells detected by a porcine specific anti-CD117 monoclonal antibody.

CD117 is a cytokine receptor expressed on the surface of hematopoietic stem cells with a likely role in cell survival, proliferation and differentiation. In order to study the differentiation activity of porcine CD117 hematopoietic cells in vitro and in vivo we prepared an anti-swine CD117 Mab (2A1) with high specificity for flow-cytometrical analysis. The 2A1 Mab did not recognize mouse or human mast cells suggesting that 2A1 is species-specific. Swine bone marrow (BM) CD117+ cells differentiated in vitro mainly into erythroid and monocyte lineages in the methylcellulose-based colony assay. When the swine BM CD117+ cells were transplanted in vivo into immunodeficient NOG (NOD/SCID/IL-2gc-null) mice, a significant amount of swine CD45+ leukocytes, including CD3 positive T cells, were developed in the mice. These results revealed that the swine BM CD117+ cells possess hematopoietic stem/progenitor activity and when monitored in immunodeficient mice or in vitro they can develop into lymphoid, erythroid, and myeloid cells efficiently with the new monoclonal antibody.

Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells.

The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72 h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4(+)CD25(+) cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48 h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72 h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar kinetics to those of the three classical SLA class I genes. The expression profiles detected by flow cytometry of the SLA molecules on the cell surface of PBMCs were maintained at a consistently high level during cell stimulation with either TSST-1 or IFN-γ, which was distinct from the kinetics of mRNA expression. These results showed that miniature swine SLA class I mRNA expression was effectively and equally up-regulated among the three loci and coordinately with IRF-1 gene expression after stimulation of T cell activation by sAG or IFN-γ.