NK-like Cells Research Articles

Regulation of Development of CD56brightCD11c+ NK-like Cells with Helper Function by IL-18

Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56brightCD11c+ cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56brightCD11c+ cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56brightCD11c+ cells. CD14+ monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56intCD11c+ cells to promote their differentiation to CD56brightCD11c+ cells with helper function. The development of CD56brightCD11c+ cells was suppressed in an IFN-α dependent manner. These results indicate that CD14+ monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56brightCD11c+ cells, which in turn modulate the expansion of γδ T cells. CD56brightCD11c+ NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation.

Swiprosin-1 is a novel actin bundling protein that regulates cell spreading and migration.

Protein functions are often revealed by their localization to specialized cellular sites. Recent reports demonstrated that swiprosin-1 is found together with actin and actin-binding proteins in the cytoskeleton fraction of human mast cells and NK-like cells. However, direct evidence of whether swiprosin-1 regulates actin dynamics is currently lacking. We found that swiprosin-1 localizes to microvilli-like membrane protrusions and lamellipodia and exhibits actin-binding activity. Overexpression of swiprosin-1 enhanced lamellipodia formation and cell spreading. In contrast, swiprosin-1 knockdown showed reduced cell spreading and migration. Swiprosin-1 induced actin bundling in the presence of Ca2+, and deletion of the EF-hand motifs partially reduced bundling activity. Swiprosin-1 dimerized in the presence of Ca2+ via its coiled-coil domain, and a lysine (Lys)-rich region in the coiled-coil domain was essential for regulation of actin bundling. Consistent with these observations, mutations of the EF-hand motifs and coiled-coil region significantly reduced cell spreading and lamellipodia formation. We provide new evidence of how swiprosin-1 influences cytoskeleton reorganization and cell spreading.

IL-22: An Evolutionary Missing-Link Authenticating the Role of the Immune System in Tissue Regeneration

Tissue regeneration is a critical component of organ maintenance. The ability of lymphocytes to kill pathogen-infected cells has been well-studied. However, the necessity for lymphocytes to participate in reconstruction of destroyed tissues has not been explored until recently. Interleukin (IL)-22, a newly defined cytokine exclusively produced by subsets of lymphocytes, provides the strongest proof yet for the tissue regenerative potentials of the immune system. IL-22 plays an obligatory role in epithelial homeostasis in the gut, liver and lung. The receptor for IL-22 (IL-22R1 and IL-10R2) is predominantly expressed by epithelial cells. While the pro-inflammatory effect is questioned, the pro-constructive potential of IL-22 is well established. It is evident from the response to IL-22, that epithelial cells not only produce anti-microbial peptides but also actively proliferate. Aryl hydrocarbon receptor (AhR) and retinoic acid-related orphan receptor (RORγt) transcription factor are required for IL-22 generation from Lymphoid Tissue inducer cells LTi, Th22 and NK-like cells. However, IL-22 production from conventional NK cells is independent of AhR and RORγt. In this review, we present a case for a paradigm shift in how we define the function of the immune system. This would include tissue regeneration as a legitimate immune function.

Abstract 2722: Active recruitment of immune effector cells mediates in vivo tumor growth inhibition by enavatuzumab, an antibody to human TWEAK receptor

Abstract Enavatuzumab (PDL192) is a humanized anti-TWEAK receptor antibody currently under clinical evaluation for the treatment of solid malignancies. Enavatuzumab is postulated to exert its potent anti-tumor activity in xenograft models through two distinct mechanisms of action: direct inhibition of tumor cell growth and Fc-mediated function. The goal of this study was to elucidate the role of immune effector cells in Fc-mediated function. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models which responded to enavatuzumab but did not respond to a version of enavatuzumab containing a mutation in the FcαRIII binding region. Subsequent analysis of circulating effector cells showed increased expression of activation markers DX5 and CD27 on monocyte-like cells (CD45+CD11bHigh) and DX5 on NK-like cells (CD45+CD11bLow) after enavatuzumab treatment in xenograft models dependent on Fc-FcR interactions for enavatuzumab efficacy. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in non-responders. Consistent with the in vivo xenograft studies, human effector cells migrated towards enavatuzumab-treated tumor cells in vitro. Effector cells preferentially migrated toward in vivo responsive tumor cells, and the majority of migratory cells were monocytes and NK cells. Conditioned media from enavatuzumab-treated tumor cells also induced migration, and contained elevated levels of cytokines/chemokines, including IL8 and MCP-1. The presence of an anti-MCP-1 neutralizing antibody in enavatuzumab-treated tumor cell conditioned medium significantly blocked effector cell migration, suggesting that MCP-1 might be responsible for effector cell migration triggered by enavatuzumab. We also observed elevated levels of human MCP-1 in the serum of both enavatuzumab-treated xenograft mouse models and in a subset of patients from the enavatuzumab Phase I clinical study. In summary, in vivo and in vitro studies suggest that enavatuzumab exerts its potent anti-tumor activity, in part, by actively recruiting effector cells and activating effectors to kill tumor cells. In addition, enavatuzumab-induced chemokines may be potential pharmacodynamic biomarkers and warrant further evaluation in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2722. doi:1538-7445.AM2012-2722

Development of a new antibody for detecting natural killer enhancing factor (NKEF)‐like protein in infected salmonids

The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up-regulation in infected fish, suggesting a trigger effect upon NK-like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF-like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF-like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.

In vitro effects of PCDDs/Fs on NK-like cell activity of Eisenia andrei earthworms

In this study, we assessed<em> in vitro</em> the effects of PCDD/Fs on the NK-like cell activity in<em> Eisenia andrei</em> earthworms using flow cytometry for analysis. NK-like coelomocytes isolated from <em>E. andrei</em> and used as effectors were exposed to various concentrations of PCDDs/Fs mixture, C1 (6.25x10-3 ng 2378- TCDD/mL), C2 (12.5x10-3 ng 2378-TCDD/mL) and C3 (25x10-3 ng 2378-TCDD/mL), before adding them to human tumoral cells (K562) used as targets. We evaluated the percentage of targets lysed by Nk-like cells. The results showed a significant stimulation of the NKlike activity at C3 when PCDD/Fs were not removed from effectors before contact with targets, while no effects were noted when the effectors were washed (PCDD/Fs removed) or fixed. Assessment of the viability of the targets (K562), exposed alone and separately from effectors, to the three concentrations of PCDD/Fs, C1, C2 and C3, showed that all these concentrations were cytotoxic for K562. Results suggest that PCDD/Fs concentrations tested in this assay may be considered too low to induce suppressive effects on the immune function such as the NK-like activity in <em>E. andrei</em> earthworms.

Molecular evidence for natural killer-like cells in equine endometrial cups

ObjectivesTo identify equine orthologs of major NK cell marker genes and utilize them to determine whether NK cells are present among the dense infiltration of lymphocytes that surround the endometrial cup structures of the horse placenta during early pregnancy. Study designPCR primers were developed to detect the equine orthologs of NKP46, CD16, CD56, and CD94; gene expression was detected in RNA isolated from lymphocytes using standard 2-step reverse transcriptase (RT) PCR and products were cloned and sequenced. Absolute real-time RT-PCR was used to quantitate gene expression in total, CD3+, and CD3- peripheral lymphocytes, and invasive trophoblast. Lymphocytes surrounding the endometrial cups (ECL) of five mares in early pregnancy were isolated and NK marker gene expression levels were assayed by quantitative RT-PCR. Main outcome measuresAbsolute mRNA transcript numbers were determined by performing quantitative RT-PCR and comparing values to plasmid standards of known quantities. ResultsNKP46 gene expression in peripheral CD3- lymphocytes was higher than in CD3+ lymphocytes, CD16 levels were higher in the CD3+ population, and no significant differences were detected for CD56 and CD94 between the two groups. Expression of all four NK cell markers was significantly higher in lymphocytes isolated from the endometrial cups of pregnant mares compared to PBMC isolated from the same animal on the same day (NKP46, 14-fold higher; CD94, 8-fold higher; CD16, 20-fold higher; CD56, 44-fold higher). ConclusionsThese data provide the first evidence for the expression of major NK cell markers by horse cells and an enrichment of NK-like cells in the equine endometrium during pregnancy.

Natural killer (NK) and NK-like cells at mucosal epithelia: Mediators of anti-microbial defense and maintenance of tissue integrity

Natural killer (NK) cells are innate lymphocytes that play important roles in the defense against microbial pathogens through secretion of IFN-γ and recognition and lysis of virally or bacterially infected host cells. A recently identified population of NK-like cells that shares characteristics of both NK cells and lymphoid tissue inducer (LTi) cells promotes innate immune responses in epithelial tissue through the secretion of IL-22. In contrast to classical NK cells, NK-like cells are localized preferentially at mucosal sites, such as the intestinal mucosa. In this review, we consider the function of NK and NK-like cells in anti-microbial defense as well as the maintenance of tissue integrity in the mucosal epithelium of the intestine, lung, and female reproductive tract. Current experimental evidence supports an important protective role for IL-22-producing NK-like cells during intestinal infections, whereas classical NK cells are crucial in the early defense against many pathogens in the respiratory tract. NK cells isolated from the pregnant uterus differ significantly in phenotype and function from those at other tissue locations. Uterine NK cells clearly contribute to the tissue remodeling that takes place during placentation, but their role in anti-microbial defense remains largely undefined.

Graft T Cells Retard Stem Cell Derived Lymphoid Organ Reconstitution and Immune Function After Allogeneic Transplantation,

Abstract 4030Impaired immune function is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Adoptively transferred donor T cells (TC) mediate graft-versus-host disease (GVHD), and make the use of pharmacological immunosuppression necessary, yet are believed to protect against infections post-HCT (pTX). In an MHC-matched, minor antigen mismatched mouse model, we studied the impact of allogeneic doTC on GVH-related damage of lymphoid organs, their recovery and immune function pTX. Lethally irradiated BALB.B mice received purified hematopoietic stem cells (HSC; cKit+Sca1+Thy1.1loLin−) +/− TC from C57BL/6 (B6) donors. Recipient lymphoid organs, marrow and livers were analyzed at multiple time points. Mice given HSC+TC promptly became full donor chimeras in all lineages and developed moderate GVHD. HSC recipients retained some residual host cells, and had no signs of GVHD. By tetramer analysis and ELISPOT assay, we showed that in pure HSC recipients early pTX residual host TC protected against murine cytomegalovirus infection. Once a nascent donor HSC-derived TC pool was established, these TC reacted robustly against the virus. In contrast, transferred mature allogeneic TC proliferated and expanded in HSC+TC recipients, but did not respond or protect against the virus. To better understand why MHC-matched mature donor TC did not react against this pathogen, even when derived from pre-immunized donors, we studied the lymphatic microenvironment, hypothesizing that only highly organized interactions between cellular and humoral immunity, antigen-presenting and innate immune cells provide full immune function. Comparing tissues of mice given pure HSC +/− TC we found that: (i) lethal radiation caused marked reduction in lymphoid organ cellularity at 2 wks pTX in all transplanted groups. While the cellularity increased rapidly in HSC recipients, severe hypocellularity persisted in HSC+TC recipients, was associated with significantly decreased organ size (lymph nodes [LN] and thymuses) and marked disruption of the histological architecture; (ii) TC phenotypes present in reconstituted mice comprised a mixture of naïve (CD62L+CD44−), central memory (CM; CD62L+CD44+) and effector memory (EM; CD62L−CD44+) cells in HSC recipients, but were largely EM and rarely naïve TC in HSC+TC recipients; (iii) at ∼3 wks pTX donor B cells (BC) dominated lymphoid tissues in HSC recipients, but were absent in HSC+ToTC recipients; (iv) NK cells regenerated promptly and steadily in HSC recipients, constituting up to 20% of all lymphoid cells in the liver at 3 wks pTX, while in HSC+TC recipients they incessantly decreased to near-complete absence; (v) in mesenteric (m) LN of HSC-, but not HSC+TC- recipients, CD4+CD8+CD3− TC were observed, derived from donor HSC. This latter population resembled immature thymic TC, suggesting that peripheral lymphoid organs participate in lymphoid reconstitution in adult recipients; (vi) at 2 wks pTX we noted only in HSC, but not HSC+ToTC recipients persisting and regenerating innate lymphoid cells (ILC). No ‘classical’ lymphoid tissue inducer cells, as described in embryogenesis of lymphoid organs, but cells with features of ILC were identified. Donor HSC-derived NKp46+RORgt+CD3− NK-like cells and IL-17A-secreting residual host CD4 TC were present, especially in livers and mLN. Cells that secreted IL-17A only and IL-17A+IFNγ were identified. IL-17A was not detected in recipients of HSC+ToTC, rather their donor TC were characterized by high IFNγ levels. Our studies show that, paradoxically, pure HSC grafts have advantages over TC-replete grafts with regard to immune function pTX. We hypothesize this superiority may be due to the presence of ILC that participate in the repair of lymphoid organs, and the whole spectrum of immune cells, including TC, BC, innate and immature cells emerging in HSC recipients. An intact microenvironment appears to be critical for optimal activation of TC to respond to pathogens. Moreover, our studies imply that donor TC cannot exert their protective immune function, when rapidly expanding alloreactive TC prevent the development of other lymphoid and innate immune cell, and disrupt the lymphoid tissue architecture. Controlling the negative effects of mature donor TC remains a challenge, and requires better understanding of the complexity of functional immune reconstitution pTX. Disclosures:No relevant conflicts of interest to declare.

Salivary Gland NK Cells Are Phenotypically and Functionally Unique

Natural killer (NK) cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV). However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV) infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.

Lineage Relationship Analysis of RORγt + Innate Lymphoid Cells

Lymphoid tissue-inducer (LTi) cells initiate the development of lymphoid tissues through the activation of local stromal cells in a process similar to inflammation. LTi cells express the nuclear hormone receptor RORγt, which also directs the expression of the proinflammatory cytokine interleukin-17 in T cells. We show here that LTi cells are part of a larger family of proinflammatory RORγt(+) innate lymphoid cells (ILCs) that differentiate from distinct fetal liver RORγt(+) precursors. The fate of RORγt(+) ILCs is determined by mouse age, and after birth, favors the generation of cells involved in intestinal homeostasis and defense. Contrary to RORγt(+) T cells, however, RORγt(+) ILCs develop in the absence of microbiota. Our study indicates that RORγt(+) ILCs evolve to preempt intestinal colonization by microbial symbionts.

An Early T Cell Lineage Commitment Checkpoint Dependent on the Transcription Factor Bcl11b

The identities of the regulators that mediate commitment of hematopoietic precursors to the T lymphocyte lineage have been unknown. The last stage of T lineage commitment in vivo involves mechanisms to suppress natural killer cell potential, to suppress myeloid and dendritic cell potential, and to silence the stem cell or progenitor cell regulatory functions that initially provide T cell receptor-independent self-renewal capability. The zinc finger transcription factor Bcl11b is T cell-specific in expression among hematopoietic cell types and is first expressed in precursors immediately before T lineage commitment. We found that Bcl11b is necessary for T lineage commitment in mice and is specifically required both to repress natural killer cell-associated genes and to down-regulate a battery of stem cell or progenitor cell genes at the pivotal stage of commitment.

An Essential Developmental Checkpoint for Production of the T Cell Lineage

In early T cell development, progenitors retaining the potential to generate myeloid and natural killer lineages are eventually determined to a specific T cell lineage. The molecular mechanisms that drive this determination step remain unclarified. We show that, when murine hematopoietic progenitors were cultured on immobilized Notch ligand DLL4 protein in the presence of a cocktail of cytokines including interleukin-7, progenitors developing toward T cells were arrested and the arrested cells entered a self-renewal cycle, maintaining non-T lineage potentials. Reduced concentrations of interleukin-7 promoted T cell lineage determination. A similar arrest and self-renewal of progenitors were observed in thymocytes of mice deficient in the transcription factor Bcl11b. Our study thus identifies the earliest checkpoint during T cell development and shows that it is Bcl11b-dependent.

Reprogramming T cells into NK-like cells for use in cancer immunotherapy via B-cell CLL/lymphoma 11B (BCL11B) deletion

Reprogramming T cells into NK-like cells for use in cancer immunotherapy via B-cell CLL/lymphoma 11B (BCL11B) deletion

Identification of an IL-7 Responsive CD4+ Lymphoid Tissue Inducer Cell from Adult Human Blood (36.24)

Abstract In mice, the lymphoid tissue inducer (LTi) cell forms a distinct lineage of hematopoietic cells required for the development of lymphoid tissues, and in the adult may be important for regeneration of lymphoid tissues following infection. LTi cells are rare and constitute a phenotypically mixed population of cells that may differentiate into NK-like cells, which has hindered their identification and characterization. Here, we isolated cells from adult human blood with phenotype and functional responses conserved with mouse CD4+ LTi cells. Human LTi-like cells expressed CD4 and IL-7 receptor-α, Rorc and Tnfsf8, signature markers for mouse CD4+ LTi cells, but lacked lineage markers for T and B lymphocytes, NK and myeloid cells. A culture system that allowed survival of human and mouse LTi cells was independent of IL-7, yet human LTi cells retained responsiveness to IL-7 by induction of lymphotoxin (LT)-α and LTβ, the key signaling system for lymphoid organogenesis. Human CD4+ LTi cells, like T cells, expressed the lymphoid tissue homing chemokine receptor, Ccr7, and the coreceptor for HIV, CXCR4. The identification of adult human CD4+ LTi cells should expand the investigation of these cells in human immunobiology and pathogenesis.

Salivary gland NK cells are phenotypically and functionally unique (89.20)

Abstract NK cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus. However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Notably, naïve salivary gland NK cells have a reduced expression of CD27, are nearly absent of the inhibitory receptor KLRG1, yet are mostly CD11b positive. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer (LTi) NK-like cells and are not thymic derived. During the course of MCMV infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited effector functions in vivo. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that latency in SMG may result from both the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3 −CD2 + and NKp46 + largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3 −CD2 +NKp46 +. The remainder of the NK-like cells comprised two minor populations, CD3 −CD2 +NKp46 − and CD3 −CD2 −NKp46 +; the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3 −CD2 + and NKp46 + NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8αα and CD8αβ and did not express CD21, WC1, CD14 or γδ TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3 −CD2 −NKp46 + population expressed CD8α compared to CD3 −CD2 +NKp46 + cells. Furthermore, a significantly greater proportion of the CD3 −CD2 +NKp46 − population expressed CD8 compared to total CD3 −CD2 + cells. Adult cattle had a significantly higher proportion of perforin + cells compared to calves aged ≤6 weeks. In this age group, the majority of perforin + cells expressed NKp46, while in adults the majority of perforin + cells were NKp46 −. However, the proportion of NKp46 + and CD3 −CD2 + cells that expressed perforin was not significantly different in any age group tested.

Cellular expression and antimicrobial function of a phylogenetically conserved novel histone 1x-like protein on mouse cells: a potential new class of pattern recognition receptor

A H1x-like protein (i.e., NCAMP-1) is expressed on the membrane and in GEs from fish NK-like cells. In the present study, we identify the imprinting control region mouse NCAMP-1 ortholog using NCAMP-1 polyclonal antibodies and mAb. Polychromatic flow cytometry revealed NCAMP-1 expression on PBLs (Gr-1(+) PMNs were 21.1% NCAMP-1(+); DX-5(+) NK cells were 12.2% NCAMP-1(+)), mesenteric LN cells (CD11c(+) DCs were 23.2% NCAMP-1(+); Gr-1(+) PMNs were 24.8% NCAMP-1(+); CD21(+) B cells were 17.8% NCAMP-1(+)), and splenocytes (CD11c(+) were 39.6% NCAMP-1(+); Gr-1(+) PMNs were 40.9% NCAMP-1(+); DX-5(+) NK cells were 24.3% NCAMP-1(+); CD21(+) B cells were 28.5% NCAMP-1(+)). Western blot analysis using pNCAMP-1 and GEs from RAW 264.7 cells produced a 32-kDa signal. GEs from RAW 264.7 cells produced a significant reduction in Escherichia coli CFU. This antimicrobial killing activity was inhibited by pretreatment of the extract with (polyclonal) anti-NCAMP-1. Treatment with preimmune serum did not reduce bacterial cell killing. Confocal microscopy using NCAMP-1 and LAMP-1 mAb demonstrated that NCAMP-1 was located on the membrane and in cytosolic vesicles of RAW 264.7 cells and did not appear to colocalize with LAMP-1. NCAMP-1 may participate as a bifunctional protein on cells. It is expressed on the membranes of phagocytic cells, NK cells, and APCs in mice as well as in the granules of macrophages. In phagocytic cells, NCAMP-1 may participate in a nonregulated exocytosis pathway of cellular secretion.

Alloantigen-specific killing is mediated by CD8-positive T cells in fish

CD8-positive (CD8 +) cytotoxic T lymphocytes (CTL) have antigen-specific cytotoxic activity. In fish, however, CTL expressing CD8 on their cell surface have not been identified. In order to characterize the cells involved in specific cell-mediated cytotoxicity in teleosts, we separated and sorted ginbuna kidney leucocytes into CD8α +, CD4 + and surface IgM (sIgM) + cells by magnetic activated cell sorting using monoclonal antibodies and examined their cytotoxic activities. Effector donor ginbuna (OB1 clone) were sensitized by allografting scales from S3N clone fish followed by injection of an allogeneic cell line (CFS) derived from S3N fish. In cytotoxic assays, target cells were labeled with CFSE and cytotoxicity was calculated based on the number of viable target cells using flow cytometry. CD8α + cells from sensitized OB1 fish showed relatively high cytotoxicity against CFS cells (immunogen) but not against allogeneic CFK cells (third party) nor isogeneic CFO cells. Pre-sensitized sIgM + cells exhibited cytotoxicity against not only CFS cells but also CFK cells. However, CD4 + or CD8α − CD4 −sIgM − cells as well as cells from non-sensitized fish did not show any significant cytotoxic activity. These results suggest that CD8α + cells in fish have characteristics similar to those of CTL in mammals, and that the sIgM + cells include NK-like cells which non-specifically killed the target cells.

Cloning and In Vitro Antiapoptotic Effects of Pig FLIPs

Introduction Cellular FLICE-like protein (cFLIP) inhibits death receptor-mediated apoptosis signal transduction, such as that induced by Fas and TNFR. The present study examined the role of antiapoptotic molecules to protect pig cells from human natural killer (NK) cells in vitro, as a model of delayed-type xenograft rejection. Methods Pig FLIPs were cloned using the TBLASTIN program to search for cDNA fragments of pig FLIPs. The sequence was identified using the dideoxy chain termination method and an ABI PRISM3100 genetic analyzer. The cDNA of pig FLIPs was inserted into the cloning site of the chicken beta-actin promoter (pCXN2). The cDNA was then transfected into pig endothelial cells (PEC), to establish several stable PEC clones containing the cDNA. Expression of the pig FLIP gene was evaluated by reverse-transcriptase polymerase chain reaction, and NK cell-mediated cytolysis assessed, using YT cells (an NK-like cell line). Results The full-length pig FLIP encoding sequence, total 5′-region to 3′-region, was defined for the first time. PEC transfectants with the FLIP showed moderate expression of FLIPs. Transfection of PEC with plasmids encoding FLIPs inhibited NK cell-mediated PEC lysis. While approximately half of parental PEC were injured by the human NK-like YT cells, the injury rate was relatively lower in the transfectants. Conclusion Overexpression of the antiapoptotic molecules, pig FLIPs, has the potential for use in protecting graft cells from human NK cells.