JM Dijkstra

CD4 and LAG-3 are related molecules that are receptors for MHC class II molecules. Their major functional differences are situated in their cytoplasmic tails, in which CD4 has an activation motif and LAG-3 an inhibitory motif. Here, we identify shark LAG-3 and show that a previously identified shark CD4-like gene has a genomic location, expression pattern, and motifs similar to CD4 in other vertebrates. In nurse shark (Ginglymostoma cirratum) and cloudy catshark (Scyliorhinus torazame), the highest CD4 expression was consistently found in the thymus whereas such was not the case for LAG-3. Throughout jawed vertebrates, the CD4 cytoplasmic tail possesses a Cx(C/H) motif for binding kinase LCK, and the LAG-3 cytoplasmic tail possesses (F/Y)xxL(D/E) including the previously determined FxxL inhibitory motif resembling an immunoreceptor tyrosine-based inhibition motif (ITIM). On the other hand, the acidic end of the mammalian LAG-3 cytoplasmic tail, which is believed to have an inhibitory function as well, was acquired later in evolution. The present study also identified CD4-1, CD4-2, and LAG-3 in the primitive ray-finned fishes bichirs, sturgeons, and gars, and experimentally determined these sequences for sterlet sturgeon (Acipenser ruthenus). Therefore, with CD4-1 and CD4-2 already known in teleosts (modern ray-finned fish), these two CD4 lineages have now been found within all major clades of ray-finned fish. Although different from each other, the cytoplasmic tails of ray-finned fish CD4-1 and chondrichthyan CD4 not only contain the Cx(C/H) motif but also an additional highly conserved motif which we expect to confer a function. Thus, although restricted to some species and gene copies, in evolution both CD4 and LAG-3 molecules appear to have acquired functional motifs besides their canonical Cx(C/H) and ITIM-like motifs, respectively. The presence of CD4 and LAG-3 molecules with seemingly opposing functions from the level of sharks, the oldest living vertebrates with a human-like adaptive immune system, underlines their importance for the jawed vertebrate immune system. It also emphasizes the general need of the immune system to always find a balance, leading to trade-offs, between activating and inhibiting processes.

Superficial discolored spots on Atlantic salmon (Salmo salar) fillets are a serious quality problem for commercial seafood farming. Previous reports have proposed that the black spots (called melanized focal changes (MFCs)) may be melanin, but no convincing evidence has been reported. In this study, we performed chemical characterization of MFCs and of red pigment (called red focal changes (RFCs)) from salmon fillets using alkaline hydrogen peroxide oxidation and hydroiodic acid hydrolysis. This revealed that the MFCs contain 3,4-dihydroxyphenylalanine (DOPA)-derived eumelanin, whereas the RFCs contain only trace amounts of eumelanin. Therefore, it is probable that the black color of the MFCs can be explained by the presence of eumelanin from accumulated melanomacrophages. For the red pigment, we could not find a significant signature of either eumelanin or pheomelanin; the red color is probably predominantly hemorrhagic in nature. However, we found that the level of pigmentation in RFCs increased together with some melanogenic metabolites. Comparison with a "mimicking experiment", in which a mixture of a salmon homogenate + DOPA was oxidized with tyrosinase, suggested that the RFCs include conjugations of DOPAquinone and/or DOPAchrome with salmon muscle tissue proteins. In short, the results suggest that melanogenic metabolites in MFCs and RFCs derive from different chemical pathways, which would agree with the two different colorations deriving from distinct cellular origins, namely melanomacrophages and red blood cells, respectively.

Group 1 innate lymphoid cells (G1-ILCs), including circulating natural killer (NK) cells and tissue-resident type 1 ILCs (ILC1s), are innate immune sentinels critical for responses against infection and cancer. In contrast to relatively uniform NK cells through the body, diverse ILC1 subsets have been characterized across and within tissues in mice, but their developmental and functional heterogeneity remain unsolved. Here, using multimodal in vivo approaches including fate-mapping and targeting of the interleukin 15 (IL-15)-producing microenvironment, we demonstrate that liver parenchymal niches support the development of a cytotoxic ILC1 subset lacking IL-7 receptor (7 R- ILC1s). During ontogeny, fetal liver (FL) G1-ILCs arise perivascularly and then differentiate into 7 R- ILC1s within sinusoids. Hepatocyte-derived IL-15 supports parenchymal development of FL G1-ILCs to maintain adult pool of 7 R- ILC1s. IL-7R+ (7R+) ILC1s in the liver, candidate precursors for 7 R- ILC1s, are not essential for 7 R- ILC1 development in physiological conditions. Functionally, 7 R- ILC1s exhibit killing activity at steady state through granzyme B expression, which is underpinned by constitutive mTOR activity, unlike NK cells with exogenous stimulation-dependent cytotoxicity. Our study reveals the unique ontogeny and functions of liver-specific ILC1s, providing a detailed interpretation of ILC1 heterogeneity.

Since the description of some peculiar symptoms by James Parkinson in 1817, attempts have been made to define its cause or at least to enlighten the pathology of "Parkinson's disease (PD)." The vast majority of PD subtypes and most cases of sporadic PD share Lewy bodies (LBs) as a characteristic pathological hallmark. However, the processes underlying LBs generation and its causal triggers are still unknown. ɑ-Synuclein (ɑ-syn, encoded by the SNCA gene) is a major component of LBs, and SNCA missense mutations or duplications/triplications are causal for rare hereditary forms of PD. Thus, it is imperative to study ɑ-syn protein and its pathology, including oligomerization, fibril formation, aggregation, and spreading mechanisms. Furthermore, there are synergistic effects in the underlying pathogenic mechanisms of PD, and multiple factors-contributing with different ratios-appear to be causal pathological triggers and progression factors. For example, oxidative stress, reduced antioxidative capacity, mitochondrial dysfunction, and proteasomal disturbances have each been suggested to be causal for ɑ-syn fibril formation and aggregation and to contribute to neuroinflammation and neural cell death. Aging is also a major risk factor for PD. Iron, as well as neuromelanin (NM), show age-dependent increases, and iron is significantly increased in the Parkinsonian substantia nigra (SN). Iron-induced pathological mechanisms include changes of the molecular structure of ɑ-syn. However, more recent PD research demonstrates that (i) LBs are detected not only in dopaminergic neurons and glia but in various neurotransmitter systems, (ii) sympathetic nerve fibres degenerate first, and (iii) at least in "brain-first" cases dopaminergic deficiency is evident before pathology induced by iron and NM. These recent findings support that the ɑ-syn/LBs pathology as well as iron- and NM-induced pathology in "brain-first" cases are important facts of PD pathology and via their interaction potentiate the disease process in the SN. As such, multifactorial toxic processes posted on a personal genetic risk are assumed to be causal for the neurodegenerative processes underlying PD. Differences in ratios of multiple factors and their spatiotemporal development, and the fact that common triggers of PD are hard to identify, imply the existence of several phenotypical subtypes, which is supported by arguments from both the "bottom-up/dual-hit" and "brain-first" models. Therapeutic strategies are necessary to avoid single initiation triggers leading to PD.

DLA-88 is a classical major histocompatibility complex (MHC) class I gene in dogs, and allelic DLA-88 molecules have been divided into two categories named "DLA-88*0" and "DLA-88*5." The defining difference between the two categories concerns an LQW motif in the α2 domain helical region of the DLA-88*5 molecules that includes the insertion of an extra amino acid compared to MHC class I consensus length. We here show that this motif has been exchanged by recombination between different DLA-88 evolutionary lineages. Previously, with pDLA-88*508:01, the structure of a molecule of the DLA-88*5 category was elucidated. The present study is the first to elucidate a structure, using X-ray crystallography, of the DLA-88*0 category, namely DLA-88*001:04 complexed with β2m and a nonamer peptide derived from canine distemper virus (CDV). The LQW motif that distinguishes DLA-88*5 from DLA-88*0 causes a shallower peptide binding groove (PBG) and a leucine exposed at the top of the α2 domain helix expected to affect T cell selection. Peptide ligand amino acid substitution and pMHC-I complex formation and stability analyses revealed that P2 and P3 are the major anchor residue positions for binding to DLA-88*001:04. We speculate that the distribution pattern of the LQW motif among canine classical MHC class I alleles represents a strategy to enhance allogeneic rejection by T cells of transmissible cancers such as canine transmissible venereal tumor (CTVT).

Abstract The dark pigment neuromelanin (NM) is abundant in cell bodies of dopamine (DA) neurons in the substantia nigra (SN) and norepinephrine (NE) neurons in the locus coeruleus (LC) in the human brain. During the progression of Parkinson’s disease (PD), together with the degeneration of the respective catecholamine (CA) neurons, the NM levels in the SN and LC markedly decrease. However, questions remain among others on how NM is associated with PD and how it is synthesized. The biosynthesis pathway of NM in the human brain has been controversial because the presence of tyrosinase in CA neurons in the SN and LC has been elusive. We propose the following NM synthesis pathway in these CA neurons: (1) Tyrosine is converted by tyrosine hydroxylase (TH) to L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted by aromatic L-amino acid decarboxylase to DA, which in LC neurons is converted by dopamine β-hydroxylase to NE; (2) DA or NE is autoxidized to dopamine quinone (DAQ) or norepinephrine quinone (NEQ); and (3) DAQ or NEQ is converted to eumelanic NM (euNM) and pheomelanic NM (pheoNM) in the absence and presence of cysteine, respectively. This process involves proteins as cysteine source and iron. We also discuss whether the NM amounts per neuromelanin-positive (NM⁺) CA neuron are higher in PD brain, whether NM quantitatively correlates with neurodegeneration, and whether an active lifestyle may reduce NM formation.

Parvalbumins are small molecules with important functions in Ca2+ signaling, but their sequence comparisons to date, especially in fish, have been relatively poor. We here, characterize sequence motifs that distinguish parvalbumin subfamilies across vertebrate species, as well as those that distinguish individual parvalbumins (orthologues) in fish, and map them to known parvalbumin structures. As already observed by others, all classes of jawed vertebrates possess parvalbumins of both the α-parvalbumin and oncomodulin subfamilies. However, we could not find convincing phylogenetic support for the common habit of classifying all non-α-parvalbumins together as "β-parvalbumins." In teleost (modern bony) fish, we here distinguish parvalbumins 1-to-10, of which the gene copy number can differ between species. The genes for α-parvalbumins (pvalb6 and pvalb7) and oncomodulins (pvalb8 and pvalb9) are well conserved between teleost species, but considerable variation is observed in their copy numbers of the non-α/non-oncomodulin genes pvalb1-to-5 and pvalb10. Teleost parvalbumins 1-to-4 are hardly distinguishable from each other and are highly expressed in muscle, and described allergens belong to this subfamily. However, in some fish species α-parvalbumin expression is also high in muscle. Pvalb5 and pvalb10 molecules form distinct lineages, the latter even predating the origin of teleosts, but have been lost in some teleost species. The present study aspires to be a frame of reference for future studies trying to compare different parvalbumins.

Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244-CXCR6-), C1 (CD244-CXCR6+), or C2 (CD244+CXCR6+) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell-like features, whereas C1 iNKT cells showed more T cell-like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244+CXCR6+ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell-like properties distinct from conventional tissue-resident iNKT cells.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and-at least bitiscetin-1-induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein-bitiscetin-3-were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit-which differs from that of both bitiscetin-3 subunits-the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Two classes of major histocompatibility complex (MHC) molecules, MHC class I and class II, play important roles in our immune system, presenting antigens to functionally distinct T lymphocyte populations. However, the origin of this essential MHC class divergence is poorly understood. Here, we discovered a category of MHC molecules (W-category) in the most primitive jawed vertebrates, cartilaginous fish, and also in bony fish and tetrapods. W-category, surprisingly, possesses class II-type α- and β-chain organization together with class I-specific sequence motifs for interdomain binding, and the W-category α2 domain shows unprecedented, phylogenetic similarity with β2-microglobulin of class I. Based on the results, we propose a model in which the ancestral MHC class I molecule evolved from class II-type W-category. The discovery of the ancient MHC group, W-category, sheds a light on the long-standing critical question of the MHC class divergence and suggests that class II type came first.

Psychotherapies aim to relieve patients from mental distress by guiding them toward healthier attitudes and behaviors. Psychotherapies can differ substantially in concepts and approaches. In this review article, we compare the methods and science of three established psychotherapies: Morita Therapy (MT), which is a 100-year-old method established in Japan; Cognitive Behavioral Therapy (CBT), which-worldwide-has become the major psychotherapy; and Acceptance and Commitment Therapy (ACT), which is a relatively young psychotherapy that shares some characteristics with MT. The neuroscience of psychotherapy as a system is only beginning to be understood, but relatively solid scientific information is available about some of its important aspects such as learning, physical health, and social interactions. On average, psychotherapies work best if combined with pharmacotherapies. This synergy may rely on the drugs helping to "kickstart" the use of neural pathways (behaviors) to which a patient otherwise has poor access. Improved behavior, guided by psychotherapy, can then consolidate these pathways by their continued usage throughout a patient's life.

Cartilaginous fish are the most primitive extant species with MHC molecules. Using the nurse shark, the current study is, to the best of our knowledge, the first to present a peptide-loaded MHC class I (pMHC-I) structure for this class of animals. The overall structure was found to be similar between cartilaginous fish and bony animals, showing remarkable conservation of interactions between the three pMHC-I components H chain, β2-microglobulin (β2-m), and peptide ligand. In most previous studies, relatively little attention was given to the details of binding between the H chain and β2-m, and our study provides important new insights. A pronounced conserved feature involves the insertion of a large β2-m F56+W60 hydrophobic knob into a pleat of the β-sheet floor of the H chain α1α2 domain, with the knob being surrounded by conserved residues. Another conserved feature is a hydrogen bond between β2-m Y10 and a proline in the α3 domain of the H chain. By alanine substitution analysis, we found that the conserved β2-m residues Y10, D53, F56, and W60-each binding the H chain-are required for stable pMHC-I complex formation. For the β2-m residues Y10 and F56, such observations have not been reported before. The combined data indicate that for stable pMHC-I complex formation β2-m should not only bind the α1α2 domain but also the α3 domain. Knowing the conserved structural features of pMHC-I should be helpful for future elucidations of the mechanisms of pMHC-I complex formation and peptide editing.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

In the spring of 2020, we and others hypothesized that T cells in COVID-19 patients may recognize identical protein fragments shared between the coronaviruses of the common cold and COVID-19 and thereby confer cross-virus immune memory. Here, we look at this issue by screening studies that, since that time, have experimentally addressed COVID-19 associated T cell specificities. Currently, the identical T cell epitope shared between COVID-19 and common cold coronaviruses most convincingly identified as immunogenic is the CD8 + T cell epitope VYIGDPAQL if presented by the MHC class I allele HLA-A*24:02. The HLA-A*24:02 allele is found in the majority of Japanese individuals and several indigenous populations in Asia, Oceania, and the Americas. In combination with histories of common cold infections, HLA-A*24:02 may affect their protection from COVID-19.

Structures of peptide-loaded major histocompatibility complex class I (pMHC-I) and class II (pMHC-II) complexes are similar. However, whereas pMHC-II complexes include similar-sized IIα and IIβ chains, pMHC-I complexes include a heavy chain (HC) and a single domain molecule β2-microglobulin (β2-m). Recently, we elucidated several pMHC-I and pMHC-II structures of primitive vertebrate species. In the present study, a comprehensive comparison of pMHC-I and pMHC-II structures helps to understand pMHC structural evolution and supports the earlier proposed-though debated-direction of MHC evolution from class II-type to class I. Extant pMHC-II structures share major functional characteristics with a deduced MHC-II-type homodimer ancestor. Evolutionary establishment of pMHC-I presumably involved important new functions such as (i) increased peptide selectivity by binding the peptides in a closed groove (ii), structural amplification of peptide ligand sequence differences by binding in a non-relaxed fashion, and (iii) increased peptide selectivity by syngeneic heterotrimer complex formation between peptide, HC, and β2-m. These new functions were associated with structures that since their establishment in early pMHC-I have been very well conserved, including a shifted and reorganized P1 pocket (aka A pocket), and insertion of a β2-m hydrophobic knob into the peptide binding domain β-sheet floor. A comparison between divergent species indicates better sequence conservation of peptide binding domains among MHC-I than among MHC-II, agreeing with more demanding interactions within pMHC-I complexes. In lungfishes, genes encoding fusions of all MHC-IIα and MHC-IIβ extracellular domains were identified, and although these lungfish genes presumably derived from classical MHC-II, they provide an alternative mechanistic hypothesis for how evolution from class II-type to class I may have occurred.

<ns4:p>SARS-CoV-2 is the coronavirus agent of the COVID-19 pandemic causing high mortalities. In contrast, the widely spread human coronaviruses OC43, HKU1, 229E, and NL63 tend to cause only mild symptoms. The present study shows, by <ns4:italic>in silico</ns4:italic> analysis, that these common human viruses are expected to induce immune memory against SARS-CoV-2 by sharing protein fragments (antigen epitopes) for presentation to the immune system by MHC class I. A list of such epitopes is provided. The number of these epitopes and the prevalence of the common coronaviruses suggest that a large part of the world population has some degree of specific immunity against SARS-CoV-2 already, even without having been infected by that virus. For inducing protection, booster vaccinations enhancing existing immunity are less demanding than primary vaccinations against new antigens. Therefore, for the discussion on vaccination strategies against COVID-19, the available immune memory against related viruses should be part of the consideration.</ns4:p>

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are related cytokines that signal through receptors possessing the β common (βc) chain. As a family, these cytokines combine rather non-specific hematopoietic growth factor properties with a special importance for eosinophils, basophils, and mast cells. In fish the cytokines of this family are called IL-5fam, and the present study, using carp, constitutes their first functional analysis. Carp il-5fam expression was enhanced by stimulation with phytohemagglutinin and killed bacteria. Reminiscent of mammalian IL-3/IL-5/GM-CSF family members, recombinant carp IL-5fam (rcIL-5fam) induced activation of transcription factor STAT5 and efficiently promoted proliferation and colony-formation of eosinophil/basophil/mast-cell type (EBM) granulocytes. Upon addition of recombinant carp βc the growth effect of rcIL-5fam was reduced, suggesting βc participation in the signaling route. In summary, despite differences in individual cytokines and cell populations, fish and mammalian IL-3/IL-5/GM-CSF family members share growth factor functions for non-neutrophil granulocytes.

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

Humans have a number of nonclassical major histocompatibility complex (MHC) class I molecules that are quite divergent from the classical ones, and that may have separated from the classical lineage in pre-mammalian times. To estimate when in evolution the respective nonclassical lineages separated from the classical lineage, we first identified “phylogenetic marker motifs” within the evolution of classical MHC class I the selected motifs are rather specific for and rather stably inherited within clades of species. Distribution of these motifs in nonclassical MHC class I molecules indicates that the lineage including the nonclassical MHC class I molecules CD1 and PROCR separated from the classical lineage before the emergence of tetrapod species, and that the human nonclassical MHC class I molecules FCGRT, MIC/ULBP/RAET, HFE, MR1, and ZAG show similarity with classical MHC class I at the avian/reptilian level. An MR1-like α1 exon sequence was identified in turtle. Our system furthermore indicates that the lineage UT, hitherto only found in non-eutherian mammals, predates tetrapod existence, and we identified UT genes in reptiles. If only accepting wide distribution of a lineage among extant species as true evidence for ancientness, the oldest identified nonclassical MHC class I lineage remains the fish-specific lineage Z, which was corroborated in the present study by finding both Z and classical-type MHC class I sequences in a primitive fish, the bichir. In short, we gained important new insights into the evolution of classical MHC class I motifs and the probable time of origin of nonclassical MHC class I lineages.

MHC molecules evolved with the descent of jawed fishes some 350-400 million years ago. However, very little is known about the structural features of primitive MHC molecules. To gain insight into these features, we focused on the MHC class I Ctid-UAA of the evolutionarily distant grass carp (Ctenopharyngodon idella). The Ctid-UAA H chain and beta 2-microglobulin (Ctid-beta 2m) were refolded in vitro in the presence of peptides from viruses that infect carp. The resulting peptide-Ctid-UAA (p/Ctid-UAA) structures revealed the classical MHC class I topology with structural variations. In comparison with known mammalian and chicken peptide-MHC class I (p/MHC I) complexes, p/Ctid-UAA structure revealed several distinct features. Notably, 1) although the peptide ligand conventionally occupied all six pockets (A-F) of the Ag-binding site, the binding mode of the P3 side chain to pocket D was not observed in other p/MHC I structures; 2) the AB loop between beta strands of the alpha 1 domain of p/Ctid-UAA complex comes into contact with Ctid-beta 2m, an interaction observed only in chicken p/BF2*2101-beta 2m complex; and 3) the CD loop of the alpha 3 domain, which in mammals forms a contact with CD8, has a unique position in p/Ctid-UAA that does not superimpose with the structures of any known p/MHC I complexes, suggesting that the p/Ctid-UAA to Ctid-CD8 binding mode may be distinct. This demonstration of the structure of a bony fish MHC class I molecule provides a foundation for understanding the evolution of primitive class I molecules, how they present peptide Ags, and how they might control T cell responses.

The related cytokine genes IL-3, 1L-5 and GM-CSF map to the (extended) T(H)2 cytokine locus of the mammalian genome. For chicken an additional related cytokine gene, KK34, was reported downstream of the IL-3 plus GM-CSF cluster, but hitherto it was believed that mammalian genomes lack this gene. However, the present study identifies an intact orthologue of chicken KK34 gene in many mammals like cattle and pig, while remnants of KK34 can be found in human and mouse. Bovine KK34 was found to be transcribed, and its recombinant protein could induce STAT5 phosphorylation and proliferation of lymphocytes upon incubation with bovine PBMCs. This concludes that KK34 is a fourth functional cytokine of the IL-3/IL-5/GM-CSF/KK34-family (alias IL-5 family) in mammals. While analyzing KK34, the present study also made new identifications of cytokine genes in the extended T(H)2 cytokine loci for reptiles, birds and marsupials. This includes a hitherto unknown cytokine gene in birds and reptiles which we designated "IL-5famE". Other newly identified genes are KK34, GM-CSF(-like), IL-5, and IL-13 in reptiles, and IL-3 in marsupials. (C) 2016 Elsevier Ltd. All rights reserved.

A phenomenon already discovered more than 25 years ago is the possibility of naïve helper T cells to polarize into TH1 or TH2 populations. In a simplified model, these polarizations occur at opposite ends of an “immune 1-2 axis” (i1-i2 axis) of possible conditions. Additional polarizations of helper/regulatory T cells were discovered later, such as for example TH17 and Treg phenotypes although these polarizations are not selected by the axis-end conditions, they are affected by i1-i2 axis factors, and may retain more potential for change than the relatively stable TH1 and TH2 phenotypes. I1-i2 axis conditions are also relevant for polarizations of other types of leukocytes, such as for example macrophages. Tissue milieus with “type 1 immunity” (“i1”) are biased towards cell-mediated cytotoxicity, while the term “type 2 immunity” (“i2”) is used for a variety of conditions which have in common that they inhibit type 1 immunity. The immune milieus of some tissues, like the gills in fish and the uterus in pregnant mammals, probably are skewed towards type 2 immunity. An i2-skewed milieu is also created by many tumors, which allows them to escape eradication by type 1 immunity. In this review we compare a number of i1-i2 axis factors between fish and mammals, and conclude that several principles of the i1-i2 axis system seem to be ancient and shared between all classes of jawed vertebrates. Furthermore, the present study is the first to identify a canonical TH2 cytokine locus in a bony fish, namely spotted gar, in the sense that it includes RAD50 and bona fide genes of both IL-4/13 and IL-3/ IL-5/GM-CSF families.

Background MHC class I (MHCI) molecules are the key presenters of peptides generated through the intracellular pathway to CD8-positive T-cells. In fish, MHCI genes were first identified in the early 1990's, but we still know little about their functional relevance. The expansion and presumed sub-functionalization of cod MHCI and access to many published fish genome sequences provide us with the incentive to undertake a comprehensive study of deduced teleost fish MHCI molecules. Results We expand the known MHCI lineages in teleosts to five with identification of a new lineage defined as P. The two lineages U and Z, which both include presumed peptide binding classical/typical molecules besides more derived molecules, are present in all teleosts analyzed. The U lineage displays two modes of evolution, most pronouncedly observed in classical-type alpha 1 domains; cod and stickleback have expanded on one of at least eight ancient alpha 1 domain lineages as opposed to many other teleosts that preserved a number of these ancient lineages. The Z lineage comes in a typical format present in all analyzed ray-finned fish species as well as lungfish. The typical Z format displays an unprecedented conservation of almost all 37 residues predicted to make up the peptide binding groove. However, also co-existing atypical Z sub-lineage molecules, which lost the presumed peptide binding motif, are found in some fish like carps and cavefish. The remaining three lineages, L, S and P, are not predicted to bind peptides and are lost in some species. Conclusions Much like tetrapods, teleosts have polymorphic classical peptide binding MHCI molecules, a number of classical-similar non-classical MHCI molecules, and some members of more diverged MHCI lineages. Different from tetrapods, however, is that in some teleosts the classical MHCI polymorphism incorporates multiple ancient MHCI domain lineages. Also different from tetrapods is that teleosts have typical Z molecules, in which the residues that presumably form the peptide binding groove have been almost completely conserved for over 400 million years. The reasons for the uniquely teleost evolution modes of peptide binding MHCI molecules remain an enigma.

In a recent publication in Science, Wang et al. found a long noncoding RNA (lncRNA) expressed in human dendritic cells (DC), which they designated lnc-DC. Based on lentivirus-mediated RNA interference (RNAi) experiments in human and murine systems, they concluded that lnc-DC is important in differentiation of monocytes into DC. However, Wang et al. did not mention that their so-called "mouse lnc-DC ortholog? gene was already designated " Wdnm1-like? and is known to encode a small secreted protein. We found that incapacitation of the Wdnm1-like open reading frame (ORF) is very rare among mammals, with all investigated primates except for hominids having an intact ORF. The null-hypothesis by Wang et al. therefore should have been that the human lnc-DC transcript might only represent a non-functional relatively young evolutionary remnant of a protein coding locus. Whether this null-hypothesis can be rejected by the experimental data presented by Wang et al. depends in part on the possible off-target (immunogenic or otherwise) effects of their RNAi procedures, which were not exhaustive in regard to the number of analyzed RNAi sequences and control sequences. If, however, the conclusions by Wang et al. on their human model are correct, and they may be, current knowledge regarding the Wdnm1-like locus suggests an intriguing combination of different functions mediated by transcript and protein in the maturation of several cell types at some point in evolution. We feel that the article by Wang et al. tends to be misleading without the discussion presented here.

Eomesodermin (Eomes), a T-box transcription factor, is a key molecule associated with function and differentiation of CD8(+) T cells and NK cells. Previously, two teleost Eomes genes (Eomes-a and -b), which are located on different chromosomes, were identified and shown to be expressed in zebrafish lymphocytes. For the present study, we identified these genes in rainbow trout and ginbuna crucian carp. Deduced Eomes-a and -b amino acid sequences in both fish species contain a highly conserved T-box DNA binding domain. In RT-PCR, both Eomes transcripts were readily detectable in a variety of tissues in rainbow trout and ginbuna. The high expression of Eomes-a and -b in brain and ovary suggests involvement in neurogenesis and oogenesis, respectively, while their expression in lymphoid tissues presumably is associated with immune functions. Investigation of separated lymphocyte populations from pronephros indicated that both Eomes-a and -b transcripts were few or absent in IgM(+) lymphocytes, while relatively abundant in IgM(-)/CD8 alpha(+) and IgM(-)/CD8 alpha(-) populations. Moreover, we sorted trout CD8 alpha(+) lymphocytes from mucosal and non-mucosal lymphoid tissues and compared the expression profiles of Eomes-a and -b with those of other T cell-related transcription factor genes (GATA-3, T-bet and Runx3), a Th1 cytokine gene (IFN-gamma) and a Th2 cytokine gene (IL-4/13A). Interestingly, the tissue distribution of Eomes-a/b, T-bet, and Runx3 versus IFN-gamma transcripts did not reveal simple correlations, suggesting tissue-specific properties of CD8 alpha(+) lymphocytes and/or multiple modes that drive IFN-gamma expressions. (C) 2013 Elsevier Ltd. All rights reserved.

Background: Classical major histocompatibility complex (MHC) class II molecules play an essential role in presenting peptide antigens to CD4§ssup§+§esup§ T lymphocytes in the acquired immune system. The non-classical class II DM molecule, HLA-DM in the case of humans, possesses critical function in assisting the classical MHC class II molecules for proper peptide loading and is highly conserved in tetrapod species. Although the absence of DM-like genes in teleost fish has been speculated based on the results of homology searches, it has not been definitively clear whether the DM system is truly specific for tetrapods or not. To obtain a clear answer, we comprehensively searched class II genes in representative teleost fish genomes and analyzed those genes regarding the critical functional features required for the DM system. Results: We discovered a novel ancient class II group (DE) in teleost fish and classified teleost fish class II genes into three major groups (DA, DB and DE). Based on several criteria, we investigated the classical/non-classical nature of various class II genes and showed that only one of three groups (DA) exhibits classical-type characteristics. Analyses of predicted class II molecules revealed that the critical tryptophan residue required for a classical class II molecule in the DM system could be found only in some non-classical but not in classical-type class II molecules of teleost fish. Conclusions: Teleost fish, a major group of vertebrates, do not possess the DM system for the classical class II peptide-loading and this sophisticated system has specially evolved in the tetrapod lineage. © 2013Dijkstra et al. licensee BioMed Central Ltd.

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in proliferation of polyclonal CD4(+) alpha beta T cells as described previously. These bulk-cultured T cells expressed transcripts for both T helper 1 cells (Th1) master regulator (T-bet) and T helper 2 cells (Th2) master regulator (GATA-3). To identify the Th subsets in bulk-cultured T cells, single cells were picked up from the bulk culture, proliferated, and characterized. The majority of the clones displayed characteristics consistent with CD4(+) alpha beta T cell identity. These clones expressed both TCR alpha and TCR beta, but could not produce a TCR gamma delta heterodimer since they typically only expressed either TCR gamma or TCR delta. These clones also expressed the TCR co-receptor genes CD4-1 or CD4-2, whereas they did not express CD8 alpha or CD8 beta. In addition, GATA-3 was expressed whereas T-bet was not. Among these clones, one clone (KoThL5) continued to proliferate on the supporting cells and was successively transferred for more than 10 months and 90-100 passages. To characterize the KoThL5 cells by their cytokine production profile, they were stimulated with PHA and investigated by real-time RT-PCR. mRNA expression of Th2-related cytokine (IL-4/13B) was only enhanced in KoThL5 cells whereas both Th1 -related cytokine (IFN gamma) and Th2-related cytokines (IL-4/13A and IL-4/13B) were significantly enhanced in bulk-cultured T cells. Taken together, KoThL5 cells share some features with mammalian Th2 cells. This is the first study to describe in vitro cultures of teleost cell with Th2-like features. The KoThL5 cell line has considerable potential for addressing questions concerning the properties of teleost Th2 cells. (C) 2012 Elsevier Ltd. All rights reserved.

Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor gamma-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLC gamma 2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution.

Rainbow trout and Atlantic salmon interleukin-4/13A (IL-4/13A) genes were identified. They were found expressed at high level in thymus, gill, and skin, in concert with the transcription factor gene GATA-3. High expression levels of IL-4, IL-13, and GATA-3 were also detected in murine thymus, suggesting similar importance of the fish and mammalian homologues for early T cell development. In mammals, combined high expression of IL-4/13 and GATA-3 in tissues other than thymus is mostly indicative of Th2 responses. Th2-skewage may protect fish skin and gill from parasites and from damage by inflammatory Th1 and Th17 responses. The immune milieus of fish gill and skin are relevant to aquaculture, because these tissues are preferred sites for vaccine administration. The similarities between the immune milieus of fish gill and thymus may reflect an evolutionary relationship, since these tissues map close together lining the gill cavity. Expression patterns of IL-4/13A and interferon gamma (IFN-gamma) in isolated trout gill cells and pronephrocytes were consistent with Th2 identity of IL-4/13A. (C) 2011 Elsevier Ltd. All rights reserved.

In modern bony fishes, or teleost fish, the general lack of leucocyte markers has greatly hampered investigations of the anatomy of the immune system and its reactions involved in inflammatory responses. We have previously reported the cloning and sequencing of the salmon CD3 complex, molecules that are specifically expressed in T cells. Here, we generate and validate sera recognizing a peptide sequence of the CD3 epsilon chain. Flow cytometry analysis revealed high numbers of CD3 epsilon+ or T cells in the thymus, gill and intestine, whereas lower numbers were detected in the head kidney, spleen and peripheral blood leucocytes. Subsequent morphological analysis showed accumulations of T cells in the thymus and spleen and in the newly discovered gill-located interbranchial lymphoid tissue. In the latter, the T cells are embedded in a meshwork of epithelial cells and in the spleen, they cluster in the white pulp surrounding ellipsoids. The anatomical organization of the salmonid thymic cortex and medulla seems to be composed of three layers consisting of a sub-epithelial medulla-like zone, an intermediate cortex-like zone and finally another cortex-like basal zone. Our study in the salmonid thymus reports a previously non-described tissue organization. In the intestinal tract, abundant T cells were found embedded in the epithelium. In non-lymphoid organs, the presence of T cells was limited. The results show that the interbranchial lymphoid tissue is quantitatively a very important site of T cell aggregation, strategically located to facilitate antigen encounter. The interbranchial lymphoid tissue has no resemblance to previously described lymphoid tissues.

In teleost fish, a novel gene G6F-like was identified, encoding a type I transmembrane molecule with four extracellular Ig-like domains and a cytoplasmic tail with putative tyrosine phosphorylation motifs including YxN and an immunoreceptor tyrosine-based activation motif (ITAM). G6F-like maps to a teleost genomic region where stretches corresponding to human chromosomes 6p (with the MHC), 12p (with CD4 and LAG-3), and 19q are tightly linked. This genomic organization resembles the ancestral "Ur-MHC" proposed for the jawed vertebrate ancestor. The deduced G6F-like molecule shows sequence similarity with members of the CD4/LAG-3 family and with the human major histocompatibility complex-encoded thrombocyte marker G6F. Despite some differences in molecular organization, teleost G6F-like and tetrapod G6F seem orthologous as they map to similar genomic location, share typical motifs in transmembrane and cytoplasmic regions, and are both expressed by thrombocytes/platelets. In the crucian carps goldfish (Carassius auratus auratus) and ginbuna (Carassius auratus langsdorfii), G6F-like was found expressed not only by thrombocytes but also by erythrocytes, supporting that erythroid and thromboid cells in teleost fish form a hematopoietic lineage like they do in mammals. The ITAM-bearing of G6F-like suggests that the molecule plays an important role in cell activation, and G6F-like expression by erythrocytes suggests that these cells have functional overlap potential with thrombocytes.

The cytoplasmic tail of mammalian CD8 alpha binds the kinase LCK in a zinc-dependent manner. In analogy with a previous study for humans (Kim et al., 2003) peptides were synthesized from rainbow trout CD8a and LCK. Surface plasmon resonance (SPR) analysis indicated that also in fish these molecules bind to each other in a zinc-dependent manner. (C) 2009 Elsevier Ltd. All rights reserved.

In mammals CD4 is a membrane glycoprotein on Th cells with four extracellular immunoglobulin-like (Ig-like) domains (D1-D4). It functions as a co-receptor during immune recognition between the TCR and the MHC II/peptide complex. The cytoplasmic domain binds p561ck, a protein kinase responsible for phosphorylating CD3 which is the first interaction in a cascade leading to T cell activation. We have previously reported a CD4-2 gene in rainbow trout (Oncorhynchus mykiss) which was found adjacent to the CD4-1 gene by synteny analysis. There are two subtypes (a and b) of CD4-2 in rainbow trout, with two Ig-like extracellular domains. Here we present the homologues of mammalian CD4 in Atlantic salmon (Salmo salar): CD4-1 with four extracellular domains and CD4-2a and CD4-2b with two extracellular domains. A Southern blot analysis shows two copies of the CD4-1 gene in the genomic DNA of the closely related rainbow trout. The genes for CD4-1 and CD4-2 have been sequenced and show typical traits for CD4 genes, such as the code for the first domain (D1) being divided between two exons and the other domains being largely coded for by single exons. The corresponding translated cDNAs show little (13-17%) identity to higher vertebrates and are approximately 37% similar to other translated, teleost sequences but are 89% identical to the closely related rainbow trout. However they exhibit conserved features such as the Lck binding motif in their cytoplasmic domains and the order of variable and constant type Ig-like domains. qRT-PCR data are presented describing the differential tissue expression of these genes together with other T cell markers (TCR and CD3) in several individuals. (C) 2008 Elsevier Ltd. All rights reserved.

Interleukins 4 and 13 (IL-4 and IL-13) are related cytokines important for Th2 immune responses and encoded by adjacent genes on human chromosome 5. Efforts were made previously to detect these genes in fish, but research was hampered by a lack of sequence conservation. A Tetraodon nigrovirides (green spotted pufferfish) gene was annotated as IL-4 by Li et al. (Mol Immunol, 44:2078-2086, 2007), but this annotation was not well substantiated. However, the present study concludes that the reported pufferfish gene belongs to the IL-4/13 lineage indeed, while also describing an additional IL-4/13 copy in a paralogous genomic region. Our analyses of IL-4/13 loci in fish describe (1) genomic region history, (2) characteristic intron-exon organization, (3) deduced IL-4/13 molecules for several teleost fish species, (4) IL-4/13 lineage-specific protein motifs including a cysteine pair (pair 1), and (5) computer software predictions of a type I cytokine fold. Teleost IL-4/13 molecules have an additional cysteine pair (pair 2) or remnants thereof, which is absent in mammalian IL-4 and IL-13. We were unable to determine if the teleost IL-4/13 genes are orthologous to either IL-4 or IL-13, or if these mammalian genes separated later in evolution.

We have cloned cDNAs encoding the alpha and beta chains of CD8 from the tiger pufferfish (fugu), Takifugu rubripes. The cDNA sequences encode a putative leader peptide, extracellular immunoglobulin variable region-like domain, stalk region, transmembrane region, and cytoplasmic tail. A protein tyrosine kinase p56lck binding motif was not found in the putative fugu CD8alpha cytoplasmic tail. O-linked glycosylation sites were found in the stalk of both CD8 chains, suggesting possible stalk formation. Phylogenetic analysis showed that fugu CD8alpha and CD8beta chains cluster with other vertebrate CD8alpha and CD8beta chains, respectively. The fugu CD8 genes comprise six exons separated by five introns. The genes are tandemly aligned 3.6 kb apart and are in the same transcription orientation. Quantitative RT-PCR analysis demonstrated that fugu CD8 is expressed predominantly in lymphoid tissues. In situ hybridization showed that fugu CD8 genes are expressed in thymocytes and lymphocytes within lymphoid organs. Molecular characterization of CD8 in fish provides the basis for development of specific antibodies to identify T-cell subsets, as well as potentially important insights into the evolution of CD8 and the adaptive immunity.

Most of the previously studied teleost MHC class I molecules can be classified into two broad lineages: "U" and "Z/ZE." However, database reports on genes in cyprinid and salmonid fishes show that there is a third major lineage, which lacks detailed analysis so far. We designated this lineage "L" because of an intriguing linkage characteristic. Namely, one zebrafish L locus is closely linked with MHC class II loci, despite the extensively documented nonlinkage of teleost class I with class II. The L lineage consists of highly variable, nonclassical MHC class I genes, and has no apparent orthologues outside teleost fishes. Characteristics that distinguish the L lineage from most other MHC class I are (1) absence of two otherwise highly conserved tryptophan residues W51 and W60 in the alpha1 domain, (2) a low GC content of the alpha1 and alpha2 exons, and (3) an HINLTL motif including a possible glycosylation site in the alpha3 domain. In rainbow trout (Oncorhynchus mykiss) we analyzed several intact L genes in detail, including their genomic organization and transcription pattern. The gene Onmy-LAA is quite different from the genes Onmy-LBA, Onmy-LCA, Onmy-LDA, and Onmy-LEA, while the latter four are similar and categorized as "Onmy-LBA-like." Whereas the Onmy-LAA gene is organized like a canonical MHC class I gene, the Onmy-LBA-like genes are processed and lack all introns except intron 1. Onmy-LAA is predominantly expressed in the intestine, while the Onmy-LBA-like transcripts display a rather homogeneous tissue distribution. To our knowledge, this is the first description of an MHC class I lineage with multiple copies of processed genes, which are intact and transcribed. The present study significantly improves the knowledge of MHC class I variation in teleosts.

Previous typing of microsatellite markers in a BC1F1 backcross rainbow trout Oncorhynchus mykiss revealed two quantitative trait loci (QTL) with a significant impact on susceptibility to infectious pancreatic necrosis. In the present study, additional genetic markers were mapped using the same strain in order to increase QTL resolution. A total of 199 individuals were screened at 226 marker loci (106 microsatellites, 116 amplified fragment length polymorphism markers, classical major histocompatibility complex molecules [MHC class I, nonclassical MHC class I, MHC class II] and tyrosinase) and the QTL were determined by single-point and interval mappings. Seven QTL were distinguished in the linkage groups RT-11, 12,17, 23, 26, 29 and 31, in addition to the two already known in RT-3 and 22. The major QTL were those in RT-3, 12 and 22; whereas the classical MHC class I locus Onmy-UBA and the MHC class II locus Onmy-DAB were not associated with any of the QTL, a nonclassical MHC class I region with Onmy-UCA, Onmy-UDA, and Onmy-UEA mapped to the major QTL on RT-3.