Immune-related, lectin-like receptors are differentially expressed in the myeloid and lymphoid lineages of zebrafish

The identification of C-type lectin (Group V) natural killer (NK) cell receptors in bony fish has remained elusive. Analyses of the Fugu rubripes genome database failed to identify Group V C-type lectin domains (Zelensky and Gready, BMC Genomics 5:51, 2004) suggesting that bony fish, in general, may lack such receptors. Numerous Group II C-type lectin receptors, which are structurally similar to Group V (NK) receptors, have been characterized in bony fish. By searching the zebrafish genome database we have identified a multi-gene family of Group II immune-related, lectin-like receptors (illrs) whose members possess inhibiting and/or activating signaling motifs typical of Group V NK receptors. Illr genes are differentially expressed in the myeloid and lymphoid lineages, suggesting that they may play important roles in the immune functions of multiple hematopoietic cell lineages.     

The zebrafish activating immune receptor Nitr9 signals via Dap12

Both inhibitory and activating forms of natural killer (NK) cell receptors are found in mammals. The activating receptors play a direct role in the recognition of virally infected or transformed cells and transduce activating signals into the cell by partnering with an adaptor protein, which contains a cytoplasmic activation motif. Activating NK receptors encoded by the mammalian leukocyte receptor complex (e.g., killer cell immunoglobulin-like receptors) and the natural killer complex (e.g., Ly49s) partner with the adaptor protein DAP12, whereas NK receptors encoded in the CD94/NKG2 complex partner with the adaptor protein DAP10. Novel immune-type receptors (NITRs) found in bony fish share several common features with immunoglobulin-type NK receptors. Nitr9 is a putative activating receptor in zebrafish that induces cytotoxicity within the context of human NK cells. One isoform of Nitr9, Nitr9L, is shown here to preferentially partner with a zebrafish ortholog of Dap12. Cross-linking the Nitr9L–Dap12 complex results in activation of the phosphytidylinositol 3-kinase→AKT→extracellular signal-regulated kinase pathway suggesting that the DAP12-based activating pathway is conserved between bony fish and mammals. Sheng Wei and Jun-min Zhou contributed equally to this work.     

Identification of the non-specific cytotoxic cell receptor protein 1 (NCCRP1) in regenerating axolotl limbs

The teleost non-specific cytotoxic cells (NCC) are evolutionary precursors of the mammalian natural killer (NK) cells and an important element of innate immunity. The non-specific cytotoxic cell receptor protein (NCCRP1) is a characteristic cell surface protein with main functions in target cell recognition and cytotoxicity with sequence information available for many species of fish. We have isolated a cDNA encoding the Axolotl homologue of fish NCCRP1 out of limb regeneration blastema and analysed its expression by RT-PCR. Sequence analysis revealed a high degree of homology with teleost NCCRP1 on nucleotide and deduced amino acid levels. NCCRP1 contains a conserved C-terminal F-box-associated domain (FBA) and proline-rich motifs (PRM) characteristic for this protein family. NCCRP1 is expressed in multiple tissues with high levels in limb regeneration blastema. The present work describes for the first time the cloning of the NCCRP1 gene in a tetrapod vertebrate providing a valuable link between fish and higher vertebrates. Our findings suggest the existence of NCC in axolotl and a role of the innate immune system in the processes of limb regeneration.     

自然杀伤细胞参与肝脏恶性肿瘤免疫逃逸研究进展

自然杀伤细胞是机体固有免疫系统重要组成部分,在肝脏等免疫器官中含量丰富,而且免疫表型、功能等表现出器官特异性。在正常情况下,靶细胞表面的配体与自然杀伤细胞表面的活化性受体直接结合并释放细胞毒性物质,诱导活化靶细胞凋亡程序,从而发挥抗感染、抗肿瘤作用。然而肿瘤细胞仍能够通过多种途径逃逸机体的免疫监视功能,研究认为肿瘤细胞抗原异常表达、肿瘤微环境中细胞因子及其他免疫细胞相互作用等因素所引起的自然杀伤细胞活性降低对于诱导肿瘤免疫逃逸起重要作用。本文综述了自然杀伤细胞在肝脏恶性肿瘤发生过程中参与免疫逃逸的机制及研究进展,以期为临床抗肿瘤免疫治疗的研究提供参考。     

Phosphorylation-induced activation of tilapia nonspecific cytotoxic cells by serum cytokines.

Cytokines as soluble mediators of immunity are important in understanding immunological mechanisms against infectious organisms and during stress conditions. In the present study, the role of protein tyrosine phosphorylation is assessed in the activation of nonspecific cytotoxic cells (NCC) from tilapia Oreochromis niloticus by cytokine-like serum factors. NCC are the teleost equivalent of mammalian natural killer (NK) cells. In teleost fish, NCC are important mediators of innate immunity against bacterial and parasite insult and tumor growth. We have previously shown that exposure of tilapia (a tropical fish) to cold water temperatures (3 to 5 min at 5 to 10 degrees C) produces physiological stress responses characterized by immediate phenotypic and immunological changes. The serum obtained from stressed tilapia contains a 'stress activating serum factor' (SASF) which passively increases in vitro naive NCC cytotoxicity 2- to 4-fold over control levels. In an effort to identify the mechanisms of activation of cytotoxicity by SASF, the phosphorylation status of tyrosine residues in proteins from treated NCC was determined. NCC were incubated with heat-inactivated or untreated stress serum and Western blots of the cell lysates were probed with anti-phosphotyrosine monoclonal antibodies (mabs). The levels of tyrosine phosphorylation in several proteins of the SASF-activated NCC were higher than in control cells. Increased tyrosine phosphorylation was also induced by incubation of NCC in the presence of the tyrosine phosphatase inhibitor Na orthovanadate (vanadate). In every case, an increase in phosphorylation status shown by Western blotting was correlated with increases in cytotoxic activity of NCC against HL-60 target cells. The enzyme inhibitor Herbimycin A (HA) has been previously used to inhibit the activity of the src-family of tyrosine kinases. In the present study, a 4 h pretreatment of NCC with HA (2 microM), followed by treatment with SASF blocked the activation of cytotoxicity produced by SASF. These results suggested that activation of NCC by cytokine-like factors is mediated through activation of the src family of protein tyrosine kinases. Activation was associated with increased phosphorylation and higher cytotoxic effector functions.     

Tolerant and diverse natural killer cell repertoires in the absence of selection

Natural killer (NK) cells are innate lymphocytes that participate in the early control of viruses and tumors. The function of NK cells is under tight regulation by two complementary inhibitory receptor families that bind to classical and non-classical HLA class I molecules: the CD94/NKG2A receptors and the killer cell immunoglobulin-like receptors (KIRs). In this mini-review, recent data on the structure of human NK cell receptor repertoires and its relation to functional responses and tolerance to self are discussed. We propose that no active selection is required to generate diverse NK cell repertoires characterized by a dominant expression of receptors with specificity for self-HLA class I. Instead, the primary consequence of interactions with HLA class I molecules is a functional tuning of randomly generated NK cell repertoires.     

Human natural killer cells and cytotoxic T lymphocytes require cell surface carbohydrate determinants for lytic function

Cloned and uncloned populations of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) were treated with tunicamycin, an antibiotic that inhibits N-linked glycosylation, in order to study the potential role of cell surface carbohydrate determinants in lytic function. It is shown that tunicamycin-treated NK and CTL effector cells lose killer function in a dose-dependent manner. This effect is reversible; cells washed free of tunicamycin begin to recover their killer activity within 2 to 3 days after initial treatment. Conjugate experiments indicate that killer-target cell binding is not affected by tunicamycin treatment of the NK cells. It is also shown that tunicamycin treatment of target cells does not significantly affect their ability to be lysed by NK or CTL effector cells. These studies provide evidence that carbohydrate determinants are important in the lytic mechanism of both CTL and NK cells, rather than in specific effector-target cell binding.     

The phylogenetic origins of natural killer receptors and recognition: relationships,possibilities, and realities

Natural killer (NK) cells affect a form of innate immunity that recognizes and eliminates cells that are infected with certain viruses or have undergone malignant transformation. In mammals, this recognition can be mediated through immunoglobulin- (Ig) and/or lectin-type NK receptors (NKRs). NKR genes in mammals range from minimally polymorphic single-copy genes to complex multigene families that exhibit high levels of haplotypic complexity and exhibit significant interspecific variation. Certain single-copy NKR genes that are present in one mammal are present as expanded multigene families in other mammals. These observations highlight NKRs as one of the most rapidly evolving eukaryotic gene families and likely reflect the influence of pathogens, especially viruses, on their evolution. Although well characterized in human and mice, cytotoxic cells that are functionally similar to NK cells have been identified in species ranging from birds to reptiles, amphibians and fish. Although numerous receptors have been identified in non-mammalian vertebrates that share structural relationships with mammalian NKRs, functionally defining these lower vertebrate molecules as NKRs is confounded by methodological and interpretive complexities. Nevertheless, several lines of evidence suggest that NK-type function or its equivalent has sustained a long evolutionary history throughout vertebrate species.     

The proinflammatory cytokines IL-2, IL-15 and IL-21 modulate the repertoire of mature human natural killer cell receptors

Natural killer (NK) cells play a crucial role in the immune response to micro-organisms and tumours. Recent evidence suggests that NK cells also regulate the adaptive T-cell response and that it might be possible to exploit this ability to eliminate autoreactive T cells in autoimmune disease and alloreactive T cells in transplantation. Mature NK cells consist of a highly diverse population of cells that expresses different receptors to facilitate recognition of diseased cells and possibly pathogens themselves. Ex vivo culture of NK cells with cytokines such as IL-2 and IL-15 is an approach that permits significant expansion of the NK cell subpopulations, which are likely to have potent antitumour, antiviral, or immunomodulatory effects in autoimmunity. Our data indicate that the addition of IL-21 has a synergistic effect by increasing the numbers of NK cells on a large scale. IL-2 and IL-15 may induce the expression of killer cell immunoglobulin-like receptors (KIRs) in KIR-negative populations, the c-lectin receptor NKG2D and the natural cytotoxic receptor NKp44. The addition of IL-21 to IL-15 or IL-2 can modify the pattern of the KIR receptors and inhibit NKp44 expression by reducing the expression of the adaptor DAP-12. IL-21 also preserved the production of interferon-γ and enhanced the cytotoxic properties of NK cells. Our findings indicate that the proinflammatory cytokines IL-2, IL-15 and IL-21 can modify the peripheral repertoire of NK cells. These properties may be used to endow subpopulations of NK cells with specific phenotypes, which may be used in ex vivo cellular immunotherapy strategies.     

Spontaneous focal activation of invariant natural killer T (iNKT) cells in mouse liver and kidney

Background  

Invariant natural killer T (iNKT) cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity in vivo has so far been reported.     

Cytotoxic activities of fish leucocytes

Like mammalian leucocytes, white blood cells of fish are able to kill altered (e.g. virus-infected) and foreign (allogeneic or xenogeneic) cells. The existence of natural killer (NK)-like and specific cytotoxic cells in fish was first shown using allogeneic and xenogeneic effector/target cell systems. In addition to in vivo and ex vivo studies, very important contributions were made by in vitro analysis using a number of different long-term cytotoxic cell lines established from channel catfish. In mammals, specific cell-mediated cytotoxicity (CMC) as part of the adaptive immune response requires a number of key molecules expressed on effector leucocytes and target cells. CD8+ T lymphocytes kill infected cells only, if their antigen receptor (TCR) matches the MHC class I with bound peptide of the target cell. Expression patterns of the fish gene homologues for TCR, CD8 and MHC class I, as well as related genes, are in agreement with similar function. Convenient systems for the analysis of specific CMC have only recently become available for fish with the combination of clonal fish with syngeneic or allogeneic but MHC class I matching cell lines. It was demonstrated that both, NK- and cytotoxic T (Tc) cells are involved in the killing of virus infected MHC class I matching and mismatching target cells. Analysis of these lymphocyte subsets is only starting for fish. There is also evidence that the different viral proteins trigger different subsets of killer cells. This review further discusses findings on fish CMC with regard to temperature/seasons and ontogeny.     

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies.  There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.     

The expanded cattle <Emphasis Type="Italic">KIR</Emphasis> genes are orthologous to the conserved single-copy <Emphasis Type="Italic">KIR3DX1</Emphasis> gene of primates

Cattle are the only non-primate species for which expansion of the killer cell immunoglobulin-like receptor (KIR) genes has been reported. We analyzed cattle KIR sequences to determine their relationship to the two divergent lineages of primate KIR: one comprising the KIR3DX1 gene of unknown function, the second comprising all other primate KIR genes, which encode variable major histocompatibility complex class I receptors. Phylogenetics and analysis of repetitive elements shows that cattle KIR subdivide into the same two lineages as primate KIR. Unlike the primates, the lineage of variable and likely functional cattle KIR corresponds to the KIR3DX1 lineage of primate KIR, whereas the variable lineage of primate KIR is represented in cattle by one KIR gene and a related gene fragment.     

The gp49B1 inhibitory receptor regulates the IFN-gamma responses of T cells and NK cells

The magnitude and diversity of Ag-specific T cell effector activity have been proposed to be controlled by an integration of positive signals transduced by the TCR and negative signals originating from inhibitory cell surface molecules. Although the lectin family of NK cell-associated inhibitory receptors has been reported to regulate the function of murine CTLs, gp49B1, the Ig superfamily member is not known to be expressed on T cells. Moreover, the consequences of the lack of an endogenously expressed NK cell-associated inhibitory receptor on T cell functions are not known. We report that gp49B1 is expressed by nearly all activated CD8 and CD4 T cells in addition to NK cells during an immune response to viral, bacterial, or tumor challenge. Kinetics of gp49B1 expression parallel functional capability and subside in the memory phase. Following vaccinia viral infection, IFN-gamma production by both subsets of T cells and NK cells is enhanced in gp49B1-deficient mice compared with gp49B1(+/+) mice. The stimulation threshold for IFN-gamma production is also lower in gp49B1-deficient T cells. In contrast, no significant differences were observed in the cytotoxic responses. We conclude that gp49B1 is a unique inhibitory receptor that is induced in multiple lineages of innate and adaptive immune cells during an infection and controls their IFN-gamma, but not cytotoxic responses.     

A population of early fetal thymocytes expressing Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells.

We have identified a dominant fetal thymocyte population at day 14.5 of gestation in the mouse that lacks CD4 and CD8 but expresses Fc gamma RII/III several days prior to acquisition of the T cell receptor (TCR) in vivo. If maintained in a thymic microenvironment, this population of CD4-CD8-TCR-Fc gamma RII/III+ thymocytes differentiates first into CD4+CD8+TCRlowFc gamma RII/III- thymocytes and subsequently CD4+CD8-TCRhighFc gamma RII/III- and CD4-CD8+TCRhighFc gamma RII/III- mature Ti alpha-beta lineage T cells. However, if removed from the thymus, the CD4-CD8-TCR-Fc gamma RII/III+ thymocyte population selectively generates functional natural killer (NK) cells in vivo as well as in vitro. These findings show that a cellular pool of Fc gamma RII/III+ precursors gives rise to T and NK lineages in a microenvironment-dependent manner. Moreover, they suggest a hitherto unrecognized role for Fc receptors on primitive T cells.