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Abstract Background Gene editing of immunomodulating molecules is a potential transplantation strategy to control immune rejection. As we noticed the successful transplantation of retinal pigment epithelium (RPE) derived from embryonic stem cells of a cynomolgus monkey that accidentally lacked MHC class II (MHC-II) molecules, we hypothesized immune rejection could be evaded by suppressing MHC-II. Methods Gene editing by the Crispr/Cas9 system was performed in induced pluripotent stem cells derived from a cynomolgus monkey (miPSCs) for targeted deletion of the gene coding class II MHC trans-activator (CIITA). Then the CIITA-knocked out miPSCs were differentiated into RPE cells to generate miPSC-derived MHC-II knockout RPE. The MHC-II knockout or wild-type RPEs were transplanted into the eyes of healthy cynomolgus monkeys. All monkeys used in this study were male. Results Here we show when MHC-II knockout RPE are transplanted into monkey eyes, they show suppressed immunogenicity with no infiltration of inflammatory cells, leading to successful engraftment. Conclusions Our results reasonably evidence the efficacy of MHC-II knockout iPSC-RPE transplants for clinical application.

Immune-related drug delivery systems (DDSs) in humanized mouse models are at the forefront of cancer research and serve as bridges between preclinical studies and clinical applications. These systems offer unique platforms for exploring new therapies and understanding their interactions with human cells and the immune system. Here, we focus on a DDS and a peripheral blood mononuclear cell (PBMC)-engrafted humanized mouse model that we recently developed, and consider some of the key components, challenges, and applications to advance these systems towards better cancer treatment on the basis of a better understanding of the immune response. Our DDS is unique and has a dual function, an anticancer effect and a capacity to fine-tune the immune reaction. The PBL-NOG-hIL-4-Tg mouse system is superior to other available humanized mouse systems for the development of such multifunctional DDSs because it supports the rapid reconstruction of an individual donor’s immunity and avoids the onset of graft-versus-host disease.

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.

Objectives: SARS-CoV-2 transmission and epidemic potential is related to the population's immunity levels. As such, assessing different regions’ preexisting immune responses to SARS-CoV-2 is important to understand the transmission potential of emerging SARS-CoV-2 variants. Design: In 975 serum samples from Vietnam (2014 to 2019), anti-SARS-CoV-2 Immunoglobulin G levels were determined by enzyme-linked immunosorbent assay. Plaque reduction neutralization test (PRNT) was performed using Wuhan strain and variants of concern (VOCs). Cross-reactivity was confirmed by analyzing B-cell receptor (BCR) repertoire sequences and identifying BCR repertoire sequences-derived T-cell epitopes. Results: Overall, 20.9% (n = 76/364) and 9.2% (n = 7) demonstrated SARS-CoV-2 neutralizing activity (PRNT50) against the Wuhan and Alpha strain, respectively. Neutralizing activity against Beta, Gamma, and Delta strains was absent (PRNT50

Pig skeletal muscle-derived stem cells (SK-MSCs) were transplanted onto the common peroneal nerve with a collagen tube as a preclinical large animal experiment designed to address long nerve gaps. In terms of therapeutic usefulness, a human family case was simulated by adjusting the major histocompatibility complex to 50% and 100% correspondences. Swine leukocyte antigen (SLA) class I haplotypes were analyzed and clarified, as well as cell transplantation. Skeletal muscle-derived CD34+/45− (Sk-34) cells were injected into bridged tubes in two groups (50% and 100%) and with non-cell groups. Therapeutic effects were evaluated using sedentary/general behavior-based functional recovery score, muscle atrophy ratio, and immunohistochemistry. The results indicated that a two-Sk-34-cell-transplantation group showed clearly and significantly favorable functional recovery compared to a non-cell bridging-only group. Supporting functional recovery, the morphological reconstitution of the axons, endoneurium, and perineurium was predominantly evident in the transplanted groups. Thus, Sk-34 cell transplantation is effective for the regeneration of peripheral nerve gap injury. Additionally, 50% and 100% SLA correspondences were therapeutically similar and not problematic, and no adverse reaction was found in the 50% group. Therefore, the immunological response to Sk-MSCs is considered relatively low. The possibility of the Sk-MSC transplantation therapy may extend to the family members beyond the autologous transplantation.

IntroductionParkinson’s disease (PD) is a neurodegenerative and polygenic disorder characterised by the progressive loss of neural dopamine and onset of movement disorders. We previously described eight SINE-VNTR-Alu (SVA) retrotransposon-insertion-polymorphisms (RIPs) located and expressed within the Human Leucocyte Antigen (HLA) genomic region of chromosome 6 that modulate the differential co-expression of 71 different genes including the HLA classical class I and class II genes in a Parkinson’s Progression Markers Initiative (PPMI) cohort.Aims and methodsIn the present study, we (1) reanalysed the PPMI genomic and transcriptomic sequencing data obtained from whole blood of 1521 individuals (867 cases and 654 controls) to infer the genotypes of the transcripts expressed by eight classical HLA class I and class II genes as well as DRA and the DRB3/4/5 haplotypes, and (2) examined the statistical differences between three different PD subgroups (cases) and healthy controls (HC) for the HLA and SVA transcribed genotypes and inferred haplotypes.ResultsSignificant differences for 57 expressed HLA alleles (21 HLA class I and 36 HLA class II alleles) up to the three-field resolution and four of eight expressed SVA were detected at p

Polymorphisms of HLA genes, which play a crucial role in presenting peptides with diverse sequences in their peptide-binding pockets, are also thought to affect HLA gene expression, as many studies have reported associations between HLA gene polymorphisms and their expression levels. In this study, we devised an ectopic expression assay for the HLA class I genes in the context of the entire gene, and used the assay to show that the HLA-C*03:03:01 and C*04:01:01 polymorphic differences observed in association studies indeed cause different levels of RNA expression. Subsequently, we investigated the C*03:23N null allele, which was previously noted for its reduced expression, attributed to an alternate exon 3 3’ splice site generated by G/A polymorphism at position 781 within the exon 3. We conducted a thorough analysis of the splicing patterns of C*03:23N, and revealed multiple aberrant splicing, including the exon 3 alternative splicing, which overshadowed its canonical counterpart. After confirming a significant reduction in RNA levels caused by the G781A alteration in our ectopic assay, we probed the function of the G-rich sequence preceding the canonical exon 3 3’ splice site. Substituting the G-rich sequence with a typical pyrimidine-rich 3’ splice site sequence on C*03:23N resulted in a marked elevation in RNA levels, likely due to the enhanced preference for the canonical exon 3 3’ splice site over the alternate site. However, the same substitution led to a reduction in RNA levels for C*03:03:01. These findings suggested the dual roles of the G-rich sequence in RNA expression, and furthermore, underscore the importance of studying polymorphism effects within the framework of the entire gene, extending beyond conventional mini-gene reporter assays.

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.

Immunity: Crucial cell surface proteins New genetic sequencing and bioinformatic analysis techniques are improving our understanding of the human major histocompatibility complex (MHC), a large and complicated group of genes that encode cell surface proteins crucial to the human immune system. In a review, Jerzy Kulski and co-workers at the Tokai University School of Medicine in Isehara, Japan report that new sequencing technologies have now allowed researchers to map fine-scale and large-scale differences between different subgroups of genes within the MHC that are inherited together, called haplotypes. Study of haplotypes has illuminated the relatedness and evolutionary history of various human ethnic groups. Large-scale bioinformatic studies have revealed associations between MHC genes and disease. These advances will potentially provide better diagnosis or treatment for many diseases, both infectious and autoimmune, and may help improve transplant donor selection.

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.

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.

Progesterone (P4) and glucocorticoid (GC) play crucial roles in the immunoregulation of a mother to accept and maintain a semi-allogenic fetus. P4 concentration increases during pregnancy and becomes much higher in the placenta than in the other peripheral tissues, wherein the concentration of cortisol (COR), the most abundant GC and a strong immunosuppressor, remains uniform throughout the rest of the body. Here, we evaluated the effect of a high-P4 environment on pregnant immunity by comparing it with COR. Naïve T cell proportion increased transiently in peripheral blood of pregnant women just after delivery and decreased after one month. T cells stimulated with superantigen toxic-shock-syndrome-1 (TSST-1) in the presence of P4 stayed in the naïve state and did not increase, irrespective of the presence of COR, and reactive T cells could not survive. Treatment of T cells with P4 without T cell receptor (TCR) stimulation transiently suppressed T cell activation and proliferation, whereas the levels remain unaltered if P4 was not given before stimulation. Comparison of the engraftment and response against specific antigens using hu-PBL-NOG-hIL-4-Tg mice showed that P4-pretreated lymphocytes preserved CD62L expression and engrafted effectively in the spleen. Moreover, they produced antigen-specific antibodies, whereas COR-pretreated lymphocytes did not. These results suggest that a high-P4 environment suppresses T cell activation and induces T cell migration into lymphoid tissues, where they maintain the ability to produce anti-pathogen antibodies, whereas COR does not preserve T cell function. The mechanism may be pivotal in maintaining non-fetus-specific T cell function in pregnancy.

Acute graft-versus-host disease (aGVHD) is defined as a syndrome of an immunological response of graft to the host that occurs early after allogeneic hematopoietic stem cell transplantation (HCT). This disease is frequently observed even in HCT matched for human leukocyte antigen (HLA) alleles at multiple gene loci. Although the HLA region represents complex and diverse genomic characteristics, detailed association analysis is required for the identification of uncharacterized variants that are strongly associated with aGVHD. We genotyped three loci, OR2H2, HLA-F-AS1, and HLA-G, that are located in the 460 kb of HLA telomeric region and statistically analyzed the genotypes including HLA-DPB1 with clinical and transplantation outcomes using 338 unrelated bone marrow transplantation (UR-BMT) patient–donor pairs who were matched for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 (HLA-10/10). Multivariate analyses demonstrated that HLA-F-AS1 and HLA-DPB1 mismatches were associated with grade II–IV aGVHD (hazard ratio (HR), 1.76; 95% CI, 1.07–2.88; p = 0.026; and HR, 1.59; CI, 1.02–2.49; p = 0.042, respectively). There was no confounding between HLA-F-AS1 and HLA-DPB1 (p = 0.512), suggesting that the HLA-F-AS1 mismatch has a strong effect on aGVHD independently of HLA-DPB1. Moreover, a stratified analysis suggested possible associations of HLA-F-AS1, HLA-DPB1, and/or HLA-G mismatches with grade II–IV aGVHD and the more severe grade III–IV aGVHD. These findings provide new insights into understanding the molecular mechanism of aGVHD caused by HLA-matched UR-BMT.

T-cell recognition of antigen epitopes is a crucial step for the induction of adaptive immune responses, and the identification of such T-cell epitopes is, therefore, important for understanding diverse immune responses and controlling T-cell immunity. A number of bioinformatic tools exist that predict T-cell epitopes; however, many of these methods highly rely on evaluating conventional peptide presentation by major histocompatibility complex (MHC) molecules, but they ignore epitope sequences recognized by T-cell receptor (TCR). Immunogenic determinant idiotopes are present on the variable regions of immunoglobulin molecules expressed on and secreted by B-cells. In idiotope-driven T-cell/B-cell collaboration, B-cells present the idiotopes on MHC molecules for recognition by idiotope-specific T-cells. According to the idiotype network theory formulated by Niels Jerne, such idiotopes found on anti-idiotypic antibodies exhibit molecular mimicry of antigens. Here, by combining these concepts and defining the patterns of TCR-recognized epitope motifs (TREMs), we developed a T-cell epitope prediction method that identifies T-cell epitopes derived from antigen proteins by analyzing B-cell receptor (BCR) sequences. This method allowed us to identify T-cell epitopes that contain the same TREM patterns between BCR and viral antigen sequences in two different infectious diseases caused by dengue virus and SARS-CoV-2 infection. The identified epitopes were among the T-cell epitopes detected in previous studies, and T-cell stimulatory immunogenicity was confirmed. Thus, our data support this method as a powerful tool for the discovery of T-cell epitopes from BCR sequences.

Polymorphisms of canine leukocyte antigen (DLA) class I (DLA-88 and DLA-12/88L) and class II (DLA-DRB1) genes are important for disease susceptibility studies, but information on the genetic diversity among dog breeds is still lacking. To better elucidate the polymorphism and genetic diversity between breeds, we genotyped DLA-88, DLA-12/88L, and DLA-DRB1 loci using 829 dogs of 59 breeds in Japan. Genotyping by Sanger sequencing identified 89, 43, and 61 alleles in DLA-88, DLA-12/88L, and DLA-DRB1 loci, respectively, and a total of 131 DLA-88–DLA-12/88L–DLA-DRB1 haplotypes (88-12/88L-DRB1) were detected more than once. Of the 829 dogs, 198 were homozygotes for one of the 52 different 88-12/88L-DRB1 haplotypes (homozygosity rate: 23.8%). Statistical modeling suggests that 90% of the DLA homozygotes or heterozygotes with one or other of the 52 different 88-12/88L-DRB1 haplotypes within somatic stem cell lines would benefit graft outcome after 88-12/88L-DRB1-matched transplantation. As previously reported for DLA class II haplotypes, the diversity of 88-12/88L-DRB1 haplotypes varied remarkably between breeds but was relatively conserved within most breeds. Therefore, the genetic characteristics of high DLA homozygosity rate and poor DLA diversity within a breed are useful for transplantation therapy, but they may affect biological fitness as homozygosity progresses.

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.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), which is one of the most serious fish diseases leading to mass mortality in a wide range of fish species worldwide. Although a few injectable inactivated vaccines are commercially available, there is a need for more labor-saving, cost-effective, and fish-friendly immunization methods. The use of transgenic plants expressing pathogen-derived recombinant antigens as edible vaccines is an ideal way to meet these requirements. In this study, chloroplast genetic engineering was successfully utilized to overexpress the red-spotted grouper NNV capsid protein (RGNNV-CP). The RGNNV-CP accumulated at high levels in all young, mature, and old senescent leaves of transplastomic tobacco plants (averaging approximately 3 mg/g leaf fresh weight). The RGNNV-CP efficiently self-assembled into virus-like particles (RGNNV-VLPs) in the chloroplast stroma of the transgenic lines, which could be readily observed by in situ transmission electron microscopy. Furthermore, intraperitoneal injection and oral administration of the crudely purified protein extract containing chloroplast-derived RGNNV-VLPs provided the sevenband grouper fish with sufficient protection against RGNNV challenge, and its immunogenicity was comparable to that of a commercial injectable vaccine. These findings indicate that chloroplast-derived VLP vaccines may play a promising role in the prevention of various diseases, not only in fish but also in other animals, including humans.

The major histocompatibility complex (MHC) on chromosome 6p21 is one of the most single-nucleotide polymorphism (SNP)-dense regions of the human genome and a prime model for the study and understanding of conserved sequence polymorphisms and structural diversity of ancestral haplotypes/conserved extended haplotypes. This study aimed to follow up on a previous analysis of the MHC class I region by using the same set of 95 MHC haplotype sequences downloaded from a publicly available BioProject database at the National Center for Biotechnology Information to identify and characterize the polymorphic human leukocyte antigen (HLA)-class II genes, the MTCO3P1 pseudogene alleles, the indels of transposable elements as haplotypic lineage markers, and SNP-density crossover (XO) loci at haplotype junctions in DNA sequence alignments of different haplotypes across the extended class II region (∼1 Mb) from the telomeric PRRT1 gene in class III to the COL11A2 gene at the centromeric end of class II. We identified 42 haplotypic indels (20 Alu, 7 SVA, 13 LTR or MERs, and 2 indels composed of a mosaic of different transposable elements) linked to particular HLA-class II alleles. Comparative sequence analyses of 136 haplotype pairs revealed 98 unique XO sites between SNP-poor and SNP-rich genomic segments with considerable haplotype shuffling located in the proximity of putative recombination hotspots. The majority of XO sites occurred across various regions including in the vicinity of MTCO3P1 between HLA-DQB1 and HLA-DQB3, between HLA-DQB2 and HLA-DOB, between DOB and TAP2, and between HLA-DOA and HLA-DPA1, where most XOs were within a HERVK22 sequence. We also determined the genomic positions of the PRDM9-recombination suppression sequence motif ATCCATG/CATGGAT and the PRDM9 recombination activation partial binding motif CCTCCCCT/AGGGGAG in the class II region of the human reference genome (NC_ 000006) relative to published meiotic recombination positions. Both the recombination and anti-recombination PRDM9 binding motifs were widely distributed throughout the class II genomic regions with 50% or more found within repeat elements; the anti-recombination motifs were found mostly in L1 fragmented repeats. This study shows substantial haplotype shuffling between different polymorphic blocks and confirms the presence of numerous putative ancestral recombination sites across the class II region between various HLA class II genes.

Tumorigenicity of induced pluripotent stem cells (iPSCs) is anticipated when cells derived from iPSCs are transplanted. It has been reported that iPSCs formed a teratoma in vivo in autologous transplantation in a nonhuman primate model without immunosuppression. However, there has been no study on tumorigenicity in major histocompatibility complex (MHC)-matched allogeneic iPSC transplantation with immune-competent hosts. To examine the tumorigenicity of allogeneic iPSCs, we generated four iPSC clones carrying a homozygous haplotype of the MHC. Two clones were derived from female fibroblasts by using a retrovirus and the other two clones were derived from male peripheral blood mononuclear cells by using Sendai virus (episomal approach). The iPSC clones were transplanted into allogenic MHC-matched immune-competent cynomolgus macaques. After transplantation of the iPSCs into subcutaneous tissue of an MHC-matched female macaque and into four testes of two MHC-matched male macaques, histological analysis showed no tumor, inflammation, or regenerative change in the excised tissues 3 months after transplantation, despite the results that iPSCs formed teratomas in immune-deficient mice and in autologous transplantation as previously reported. The results in the present study suggest that there is no tumorigenicity of iPSCs in MHC-matched allogeneic transplantation in clinical application.

The genomic region (~4 Mb) of the human major histocompatibility complex (MHC) on chromosome 6p21 is a prime model for the study and understanding of conserved polymorphic sequences (CPSs) and structural diversity of ancestral haplotypes (AHs)/conserved extended haplotypes (CEHs). The aim of this study was to use a set of 95 MHC genomic sequences downloaded from a publicly available BioProject database at NCBI to identify and characterise polymorphic human leukocyte antigen (HLA) class I genes and pseudogenes, MICA and MICB, and retroelement indels as haplotypic lineage markers, and single-nucleotide polymorphism (SNP) crossover loci in DNA sequence alignments of different haplotypes across the Olfactory Receptor (OR) gene region (~1.2 Mb) and the MHC class I region (~1.8 Mb) from the GPX5 to the MICB gene. Our comparative sequence analyses confirmed the identity of 12 haplotypic retroelement markers and revealed that they partitioned the HLA-A/B/C haplotypes into distinct evolutionary lineages. Crossovers between SNP-poor and SNP-rich regions defined the sequence range of haplotype blocks, and many of these crossover junctions occurred within particular transposable elements, lncRNA, OR12D2, MUC21, MUC22, PSORS1A3, HLA-C, HLA-B, and MICA. In a comparison of more than 250 paired sequence alignments, at least 38 SNP-density crossover sites were mapped across various regions from GPX5 to MICB. In a homology comparison of 16 different haplotypes, seven CEH/AH (7.1, 8.1, 18.2, 51.x, 57.1, 62.x, and 62.1) had no detectable SNP-density crossover junctions and were SNP poor across the entire ~2.8 Mb of sequence alignments. Of the analyses between different recombinant haplotypes, more than half of them had SNP crossovers within 10 kb of LTR16B/ERV3-16A3_I, MLT1, Charlie, and/or THE1 sequences and were in close vicinity to structurally polymorphic Alu and SVA insertion sites. These studies demonstrate that (1) SNP-density crossovers are associated with putative ancestral recombination sites that are widely spread across the MHC class I genomic region from at least the telomeric OR12D2 gene to the centromeric MICB gene and (2) the genomic sequences of MHC homozygous cell lines are useful for analysing haplotype blocks, ancestral haplotypic landscapes and markers, CPSs, and SNP-density crossover junctions.

The major histocompatibility complex (MHC) is a highly polymorphic and duplicated genomic region that encodes transplantation and immune regulatory molecules. Although it is well-known that particular MHC allelic polymorphisms and haplotypes are genetically relate to immune-mediated diseases detailed information of the cat MHC (Feline Leukocyte Antigen; FLA) genetic and haplotypic structure and diversity is limited in comparison to humans and many other species. In this study, to better understand the degree and types of allele and allelic haplotype diversity of FLA-class I (FLA-I) and FLA-DRB loci in domestic cats, we identified six expressible FLA-I loci in peripheral white blood cells by in silico estimation of the coding exons and NGS-based amplicon sequencing using five unrelated cats. We then used a newly developed NGS-based genotyping method to genotype and annotate 32 FLA-I and 16 FLA-DRB sequences in two families of 20 domestic cats. A total of 14 FLA-I and seven FLA-DRB were identified as novel polymorphic sequences. Phylogenetic analyses grouped the sequences into six FLA-I (FLA-E/H/K, FLA-A, FLA-J, FLA-L, FLA-O and a tentatively named FLA-E/H/K_Rec) and four FLA-DRB (FLA-DRB1, FLA-DRB3, FLA-DRB4, and FLA-DRB5) lineages. Pedigree analysis of two cat families revealed eight distinct FLA structural haplotypes (Class I – DRB) with five to eight FLA-I and two to three FLA-DRB transcribed loci per haplotype. It is evident that the eight FLA haplotypes were generated by gene duplications and deletions, and rearrangements by genetic recombination with the accumulation and/or inheritance of novel polymorphisms. These findings are useful for further genetic diversity analysis and disease association studies among cat breeds and in veterinary medicine.

The highly polymorphic human major histocompatibility complex (MHC) also known as the human leukocyte antigen (HLA) encodes class I and II genes that are the cornerstone of the adaptive immune system. Their unique diversity (>25,000 alleles) might affect the outcome of any transplant, infection, and susceptibility to autoimmune diseases. The recent rapid development of new next-generation sequencing (NGS) methods provides the opportunity to study the influence/correlation of this high level of HLA diversity on allele expression levels in health and disease. Here, we describe the NGS capture RNA-Seq method that we developed for genotyping all 12 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5) and assessing their allelic imbalance by quantifying their allele RNA levels. This is a target enrichment method where total RNA is converted to a sequencing-ready complementary DNA (cDNA) library and hybridized to a complex pool of RNA-specific HLA biotinylated oligonucleotide capture probes, prior to NGS. This method was applied to 161 peripheral blood mononuclear cells and 48 umbilical cord blood cells of healthy donors. The differential allelic expression of 10 HLA loci (except for HLA-DRA and HLA-DPA1) showed strong significant differences (P < 2.1 × 10−15). The results were corroborated by independent methods. This newly developed NGS method could be applied to a wide range of biological and medical questions including graft rejections and HLA-related diseases.

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.

IntroductionInclusion body myositis (IBM) is an idiopathic inflammatory myopathy, characterized by unique clinical features including finger flexor and quadriceps muscle weakness and a lack of any reliable treatment. The human leukocyte antigen (HLA)-DRB1 allele and autoantibody profiles in Japanese IBM patients have not been fully elucidated.MethodsWe studied 83 Japanese IBM patients with a mean age of 69 years (49 males and 34 females) who participated in the 'Integrated Diagnosis Project for Inflammatory Myopathies' from January 2011 to September 2016. IBM was diagnosed by histological diagnosis. Various autoantibodies were screened by RNA immunoprecipitation and enzyme-linked immunosorbent assays. HLA-DRB1 genotyping was performed using polymerase chain reaction-sequence based typing. A total of 460 unrelated healthy Japanese controls were also studied.ResultsThe allele frequencies of DRB1*01:01, DRB1*04:10, and DRB1*15:02 were significantly higher in the IBM group than in the healthy control group (Corrected P = 0.00078, 0.00038 and 0.0046). There was a weak association between the DRB1*01:01 allele and severe leg muscle weakness and muscle atrophy. While hepatitis type C virus infection and autoantibodies to cytosolic 5'-nucleotidase 1A were found in 18 and 28 patients, respectively, no significant association with HLA-DRB1 alleles was observed.ConclusionJapanese IBM patients had the specific HLA-DRB1 allele and autoantibody profiles.

Abstract Cynomolgus macaques are useful experimental animals that are physiologically and genetically close to humans. We have developed two kinds of experimental usage of cynomolgus macaque: transplantation and disease models. First, we identified certain major histocompatibility complex (MHC) haplotypes including homozygotes and heterozygotes in cynomolgus macaques native to the Philippines, because they have less polymorphism in the MHC than that in other origins such as Vietnam and Indonesia. As a preclinical model of the induced pluripotent stem cell (iPSC) stock project, we established iPSCs from various types of MHC homozygous macaques, which were transplanted into compatible MHC heterozygous macaques, the iPSC stock project was experimentally shown to be effective. Second, to obtain disease models of cynomolgus macaques for studies on regenerative medicine including cell therapies, we established two kinds of genetic technology to modify cynomolgus macaques: transgenic technology and gene editing technology using CRISPR-Cas9. We will establish disease models, such as Alzheimer’s disease and progeria (Werner syndrome). In future, we will distribute the MHC-identical cynomolgus monkeys and genetically modified macaques to researchers, especially those engaging in regenerative medicine.

Abstract In the Mauritian macaque experimentally inoculated with SIV, gene polymorphisms potentially associated with the plasma virus load at a set point, approximately 100 days post inoculation, were investigated. Among the 42 animals inoculated with 50 AID50 of the same strain of SIV, none of which received any preventive or curative treatment, nine individuals were selected: three with a plasma virus load (PVL) among the lowest, three with intermediate PVL values and three among the highest PVL values. The complete genomes of these nine animals were then analyzed. Initially, attention was focused on variants with a potential functional impact on protein encoding genes (non-synonymous SNPs (NS-SNPs) and splicing variants). Thus, 424 NS-SNPs possibly associated with PVL were detected. The 424 candidates SNPs were genotyped in these 42 SIV experimentally infected animals (including the nine animals subjected to whole genome sequencing). The genes containing variants most probably associated with PVL at a set time point are analyzed herein.

Objective Microminipigs are a novel animal model with extensive applications in laboratory studies owing, in part, to their extremely small body sizes. In this study, the relationship between swine leukocyte antigen (SLA) class II haplotype and body weight was evaluated in the Microminipig population. Methods A total of 1,900 haplotypes, covering SLA class II haplotypes Lr−0.7, Lr−0.23, Lr−0.17, Lr−0.37, Lr−0.16, Lr−0.11, Lr−0.13, and Lr−0.18, were analyzed in 950 piglets. Birth weights and weights on postnatal day 50 were examined in piglets with eight different SLA class II haplotypes. Results The mean birth weight of piglets with the Lr−0.23 haplotype (0.415 kg, n = 702) was significantly lower than that of piglets with Lr−0.17 (0.445 kg, n = 328) and Lr−0.37 (0.438 kg, n = 383) haplotypes. At postnatal day 50, the mean body weight of piglets with the Lr−0.23 haplotype (3.14 kg) was significantly lower than that of piglets with the Lr−0.13 haplotype (3.46 kg, p

Summary: Antibody-mediated rejection is characterized by donor-specific antibody produced by B cells. However, to our knowledge, B cell invasion and antibody in the inflamed retina after transplantation of retinal pigment epithelial (RPE) cells has not been reported. To determine if RPE transplantation could be performed using allografts, we established in vivo immune rejection models with induced pluripotent stem cell (iPSC)-RPE allografts and determined whether RPE-specific antibody could be detected in these models. We detected alloantibodies in the serum from recipient monkeys that had immune attacks in the retina in an immunofluorescent assay using the transplanted iPSC-RPE cells as the antigen. In addition to T cell and antigen-presenting cell immunity, peripheral blood cells and lymph nodes in animal models with allogeneic iPSC-RPE cells also had activated B cells, which were probably secreting alloantibodies. Using serum and transplanted cells, alloreactive antibody can be detected for the diagnosis of immune rejection after transplantation. : In this article, Sugita and colleagues show that B cells, as well as T cells, are associated with immune attack on iPSC-derived retinal pigment epithelial cells (RPE) after allogeneic transplantation. In addition, it shows that B cells can produce alloantibodies against iPSC-RPE cells. Keywords: induced pluripotent stem cells, retinal pigment epithelial cells, B cells, antibody, immune rejection

Major histocompatibility complex (MHC) matching improves graft survival rates after organ transplantation. Here the authors show that in macaques, MHC-matched iPSC-derived neurons provide better engraftment in the brain, with a lower immune response and higher survival of the transplanted neurons.

Abstract A number of autoimmune diseases are associated with the genotypes of human leukocyte antigen class II (HLA), some of which present peptides derived from self-proteins, resulting in clonal expansion of self-reactive T cells. Therefore, selective inhibition of self-peptide loading onto such disease-associated HLA could ameliorate the diseases. To effectively identify such compounds, in this study, we established, for the first time, a cell- and 96-well microplate-based high-throughput screening system for inhibitors of antigen presentation. A panel of DRB1 genes plus DRA*01:01 gene were expressed in HEK293T cells and in 3T3 cells, and their binding with biotinylated known self-antigen peptides was measured by flow cytometry. HLA-DR1 (DRB1*01:01) and DR15 (DRB1*15:01) showed a high affinity with myelin basic protein peptide (MBP83-98). Therefore, in 96-well plate wells, MBP83-99 was allowed to bind to DR1 or DR15 on 3T3 cells in competition with a test compound, and the HLA-bound peptide was detected by streptavidin-conjugated β-galactosidase, thereby identifying inhibitor compounds for rheumatoid arthritis or multiple sclerosis. Our assay system has a potential for broad applications, including designing peptide vaccines.

The enteric protozoa Entamoeba histolytica is the causative agent of amebiasis, which is one of the most common parasitic diseases in developed and developing countries. Entamoeba nuttalli is the genetically closest species to E. histolytica in current phylogenetic analyses of Entamoeba species, and is prevalent in wild macaques. Therefore, E. nuttalli may be a key organism in which to investigate molecules required for infection of human or non-human primates. To explore the molecular signatures of host-parasite interactions, we conducted de novo assembly of the E. nuttalli genome, utilizing self-correction of PacBio long reads and polishing corrected reads using Illumina short reads, followed by comparative genomic analysis with two other mammalian and a reptilian Entamoeba species. The final draft assembly of E. nuttalli included 395 contigs with a total length of approximately 23 Mb, and 9,647 predicted genes, of which 6,940 were conserved with E. histolytica. In addition, we found an E. histolytica-specific repeat known as ERE2 in the E. nuttalli genome. GO-term enrichment analysis of mammalian host-related molecules indicated diversification of transmembrane proteins, including AIG1 family and BspA-like proteins that may be involved in the host-parasite interaction. Furthermore, we identified an E. nuttalli-specific protein that contained 42 repeats of an octapeptide ([G,E]KPTDTPS). This protein was shown to be localized on the cell surface using immunofluorescence. Since many repeat-containing proteins in parasites play important roles in interactions with host cells, this unique octapeptide repeat-containing protein may be involved in colonization of E. nuttalli in the intestine of macaques. Overall, our draft assembly provides a valuable resource for studying Entamoeba evolution and host-parasite selection.

There is an ongoing controversy as to whether major histocompatibility complex (MHC) matching is a solution for allogeneic stem cell transplantation. In the present study, we established retinal pigment epithelial (RPE) cells from induced pluripotent stem cells (iPSCs) in MHC homozygote donors. We observed no rejection signs in iPSC-derived RPE allografts of MHC-matched animal models without immunosuppression, whereas there were immune attacks around the graft and retinal tissue damage in MHC-mismatched models. In an immunohistochemical examination of MHC-mismatched allografts, the transplanted RPE sheets/cells were located in the subretinal space, but the RPE exhibited inflammatory and hypertrophic changes, and many inflammatory cells, e.g., Iba1+ cells, MHC class II+ cells, and CD3+ T cells, invaded the graft area. Conversely, these inflammatory cells poorly infiltrated the area around the transplanted retina if MHC-matched allografts were used. Thus, cells derived from MHC homozygous donors could be used to treat retinal diseases in histocompatible recipients.

Induced pluripotent stem cells (iPSCs) can serve as a source of cardiomyocytes (CMs) to treat end-stage heart failure; however, transplantation of genetically dissimilar iPSCs even within species (allogeneic) can induce immune rejection. We hypothesized that this might be limited by matching the major histocompatibility complex (MHC) antigens between the donor and the recipient. We therefore transplanted fluorescence-labeled (GFP) iPSC-CMs donated from a macaque with homozygous MHC haplotypes into the subcutaneous tissue and hearts of macaques having heterozygous MHC haplotypes (MHC-matched; group I) or without identical MHC alleles (group II) in conjunction with immune suppression. Group I displayed a higher GFP intensity and less immune-cell infiltration in the graft than group II. However, MHC-matched transplantation with single or no immune-suppressive drugs still induced a substantial host immune response to the graft. Thus, the immunogenicity of allogeneic iPSC-CMs was reduced by MHC-matched transplantation although a requirement for appropriate immune suppression was retained for successful engraftment.

Lipid antigens have been added to the antigenic repertoire in recent years. Here, the authors have identified Mamu-B*098 as a lipopeptide antigen presenting molecule and using structural and biochemical analysis have shown that it has a different antigen binding pocket to previously analysed proteins.

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

The transcription of photosynthesis genes encoded by the plastid genome is mainly mediated by a prokaryotic-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP). Standard PEP-dependent promoters resemble bacterial sigma-70-type promoters containing the so-called -10 and -35 elements. On the other hand, an unusual light- and stress-responsive promoter (psbD LRP) that is regulated by a 19-bp AAG-box immediately upstream of the -35 element has been mapped upstream of the psbD-psbC operon in some angiosperms. However, the occurrence of the AAG-box containing psbD LRP in plant evolution remains elusive. We have mapped the psbD promoters in eleven embryophytes at different evolutionary stages from liverworts to angiosperms. The psbD promoters were mostly mapped around 500–900 bp upstream of the psbD translational start sites, indicating that the psbD mRNAs have unusually long 5′-UTR extensions in common. The -10 elements of the psbD promoter are well-conserved in all embryophytes, but not the -35 elements. We found that the AAG-box sequences are highly conserved in angiosperms and gymnosperms except for gnetaceae plants. Furthermore, partial AAG-box-like sequences have been identified in the psbD promoters of some basal embryophytes such as moss, hornwort, and lycophyte, whereas liverwort has the standard PEP promoter without the AAG-box. These results suggest that the AAG-box sequences of the psbD LRP may have evolved from a primitive type of AAG-box of basal embryophytes. On the other hand, monilophytes (ferns) use another type of psbD promoter composed of a distinct cis-element upstream of the potential -35 element. Furthermore, we found that psbD expression is not regulated by light in gymnosperms or basal angiosperms, although they have the well-conserved AAG-box sequences. Thus, it is unlikely that acquisition of the AAG-box containing psbD promoter is directly associated with light-induced transcription of the psbD-psbC operon. Light- and stress-induced transcription may have evolved independently and multiple times during terrestrial plant evolution.

The human Major Histocompatibility Complex (MHC) genes are part of the supra-locus on chromosome 6p21 known as the human leukocyte antigen (HLA) system [...]

Among the non-human primates used in experimental medicine, cynomolgus macaques (Macaca fascicularis hereafter referred to as Mafa) are increasingly selected for the ease with which they are maintained and bred in captivity. Macaques belong to Old World monkeys and are phylogenetically much closer to humans than rodents, which are still the most frequently used animal model. Our understanding of the Mafa genome has progressed rapidly in recent years and has greatly benefited from the latest technical advances in molecular genetics. Cynomolgus macaques are widespread in Southeast Asia and numerous studies have shown a distinct genetic differentiation of continental and island populations. The major histocompatibility complex of cynomolgus macaque (Mafa MHC) is organized in the same way as that of human, but it differs from the latter by its high degree of classical class I gene duplication. Human polymorphic MHC regions play a pivotal role in allograft transplantation and have been associated with more than 100 diseases and/or phenotypes. The Mafa MHC polymorphism similarly plays a crucial role in experimental allografts of organs and stem cells. Experimental results show that the Mafa MHC class I and II regions influence the ability to mount an immune response against infectious pathogens and vaccines. MHC also affects cynomolgus macaque reproduction and impacts on numerous biological parameters. This review describes the Mafa MHC polymorphism and the methods currently used to characterize it. We discuss some of the major areas of experimental medicine where an effect induced by MHC polymorphism has been demonstrated.

The effects of swine leukocyte antigen (SLA) molecules on numerous production and reproduction performance traits have been mainly reported as associations with specific SLA haplotypes that were assigned using serological typing methods. In this study, we intended to clarify the association between SLA class II genes and reproductive traits in a highly inbred population of 187 Microminipigs (MMP), that have eight different types of SLA class II haplotypes. In doing so, we compared the reproductive performances, such as fertility index, gestation period, litter size, and number of stillbirth among SLA class II low resolution haplotypes (Lrs) that were assigned by a polymerase chain reaction-sequence specific primers (PCR-SSP) typing method. Only low resolution haplotypes were used in this study because the eight SLA class II high-resolution haplotypes had been assigned to the 14 parents or the progenitors of the highly inbred MMP herd in a previous publication. The fertility index of dams with Lr-0.13 was significantly lower than that of dams with Lr-0.16, Lr-0.17, Lr-0.18, or Lr-0.37. Dams with Lr-0.23 had significantly smaller litter size at birth than those with Lr-0.17, Lr-0.18, or Lr-0.37. Furthermore, litter size at weaning of dams with Lr-0.23 was also significantly smaller than those dams with Lr-0.16, Lr-0.17, Lr-0.18, or Lr-0.37. The small litter size of dams with Lr-0.23 correlated with the smaller body sizes of these MMPs. These results suggest that SLA class II haplotypes are useful differential genetic markers for further haplotypic and epistatic studies of reproductive traits, selective breeding programs, and improvements in the production and reproduction performances of MMPs.

HLA molecules play an important role for immunoreactivity in allogeneic hematopoietic stem cell transplantation. To elucidate the effect of specific HLA alleles on acute graft-versus-host disease, we conducted a retrospective analysis using 6967 Japanese patients transplanted with T-cell-replete marrow from an unrelated donor. Using unbiased searches of patient and donor HLA alleles, patient and/or donor HLA-B*51:01 (patient: HR, 1.37, P

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.

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

The common marmoset (Callithrix jacchus) is considered a novel experimental animal model of non-human primates. However, due to antibody unavailability, immunological and pathological studies have not been adequately conducted in various disease models of common marmoset. Quantitative real-time PCR (qPCR) is a powerful tool to examine gene expression levels. Recent reports have shown that selection of internal reference housekeeping genes are required for accurate normalization of gene expression. To develop a reliable qPCR method in common marmoset, we used geNorm applets to evaluate the expression stability of eight candidate reference genes (GAPDH, ACTB, rRNA, B2M, UBC, HPRT, SDHA and TBP) in various tissues from laboratory common marmosets. geNorm analysis showed that GAPDH, ACTB, SDHA and TBP were generally ranked high in stability followed by UBC. In contrast, HPRT, rRNA and B2M exhibited lower expression stability than other genes in most tissues analyzed. Furthermore, by using the improved qPCR with selected reference genes, we analyzed the expression levels of CD antigens (CD3ε, CD4, CD8α and CD20) and cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12β, IL-13, IFN-γ and TNF-α) in peripheral blood leukocytes and compared them between common marmosets and humans. The expression levels of CD4 and IL-4 were lower in common marmosets than in humans whereas those of IL-10, IL-12β and IFN-γ were higher in the common marmoset. The ratio of Th1-related gene expression level to that of Th2-related genes was inverted in common marmosets. We confirmed the inverted ratio of CD4 to CD8 in common marmosets by flow cytometric analysis. Therefore, the difference in Th1/Th2 balance between common marmosets and humans may affect host defense and/or disease susceptibility, which should be carefully considered when using common marmoset as an experimental model for biomedical research.

We made an H1N1 vaccine candidate from a virus library consisting of 144 ( = 16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically naïve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.