Cultured NK Cells Research Articles

Polymorphic alleles of murine CD244 exhibit divergent function in NK cells (89.1)

Abstract CD244 (2B4) is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors and is expressed on all murine NK cells and a subset of CD8+ T cells. The SLAM family of receptors exists as one of two stable haplotypes (b and z haplotype) in common laboratory strains of mice. The b haplotype is associated with C57-derived mouse strains while the z haplotype is found in most other inbred strains. Previous studies have shown that CD244 exhibits inhibitory function in NK cells from C57BL/6 (B6) mice. Here we employ a congenic mouse strain (B6.Sle1b) that differs from the parental solely at a region of chromosome 1 containing the SLAM family of genes to characterize the function of the z haplotype of CD244. We show that the more common haplotype of CD244 exhibits activating function in an SH2D1a dependent manner as evidenced by enhanced lytic activity towards targets expressing the ligand for CD244, CD48. The activating function of the z version of CD244 is due to polymorphic differences that appear to be clustered in the ligand-binding region of the receptor. In addition, we observed differential baseline phosphorylation and adaptor association for each of the CD244 haplotypes in cultured NK cells accounting for their functional differences.

A Soluble Form of the MHC Class I-Specific CD160 Receptor Is Released from Human Activated NK Lymphocytes and Inhibits Cell-Mediated Cytotoxicity

CD160 is a GPI-anchored lymphocyte surface receptor in which expression is mostly restricted to the highly cytotoxic CD56(dim)CD16(+) peripheral blood NK subset. We previously reported that MHC class I (MHC-I) molecules bind to CD160 receptors on circulating NK lymphocytes and that this interaction triggers their cytotoxic activity and cytokine production. We also observed that CD160 surface expression on NK cells is down-modulated upon activation with PMA or IL-2. In this study, we further report that short-time incubation of NK lymphocytes with IL-15 converts the membrane-bound CD160 to a soluble form through a proteolytic cleavage involving a metalloprotease. Thus, CD160 is no longer detected at the cell surface, but can be immunoprecipitated from the NK cell culture medium. Interestingly, CD160 transcript remains highly expressed during the process of protein shedding. In addition, we demonstrate that CD160 mRNA synthesis can be induced in CD56(bright) separated lymphocytes following exposure to IL-15. By producing a Flag-tagged soluble CD160 protein, we establish that its binding to MHC-I molecules results in the inhibition of the cytotoxic CD8(+) T lymphocyte activity and of the CD160-mediated NK cell cytotoxicity. Thus, we show that activated NK lymphocytes release a soluble form of CD160 that functionally impairs the MHC-I-specific cytotoxic CD8(+) T lymphocyte responsiveness.

Three common alleles of KIR2DL4 (CD158d) encode constitutively expressed, inducible and secreted receptors in NK cells

Genetic polymorphism of KIR2DL4 results in alleles with either 9 or 10 consecutive adenines in exon 6, which encodes the transmembrane domain. "10A" alleles encode a membrane-expressed receptor that is constitutively expressed on resting CD56bright NK cells and on CD56dim cells after culture. However, in some individuals with the 10A allele, KIR2DL4 cannot be detected on their resting CD56bright NK cells. "9A" alleles have been predicted to encode a secreted receptor due to the splicing out of the transmembrane region. In this publication, we show that those individuals with a 10A allele who lack detectable KIR2DL4 on CD56bright NK cells express a KIR2DL4 receptor in which the D0-domain is excised. This Delta-D0 receptor cannot be detected by the available anti-KIR2DL4 monoclonal antibodies. In such individuals, KIR2DL4 becomes detectable on cultured NK cells due to up-regulation of the full-length KIR2DL4 transcript. In all individuals with 10A alleles, KIR2DL4 ceases to be expressed at the cell surface 16 days after activation, despite the maintenance of maximal levels of KIR2DL4 mRNA transcription, suggesting the existence of a negative regulator of cell surface expression. Finally, we show that the 9A allele can produce a secreted KIR2DL4 receptor.

Ly49B Is Expressed on Multiple Subpopulations of Myeloid Cells

Using a novel mAb specific for mouse Ly49B, we report here that Ly49B, the last remaining member of the C57 Ly49 family to be characterized, is expressed at low levels on approximately 1.5% of spleen cells, none which are NK cells or T cells but which instead belong to several distinct subpopulations of myeloid cells defined by expression of CD11b and different levels of Gr1. Much larger proportions of bone marrow and peritoneal cells expressed Ly49B, all being CD11b+ and comprising multiple subpopulations defined by light scatter, F4/80, and Gr1 expression. Costaining for Ly49Q, also expressed on myeloid cells, revealed that Ly49B and Ly49Q were most strongly expressed on nonoverlapping subpopulations, Ly49Q(high) cells being mostly B220+CD4+ and/or CD8+, Ly49B+ cells lacking these markers. Myeloid populations that developed from bone marrow progenitors in vitro frequently coexpressed both Ly49B and Ly49Q, and Ly49B expression could be up-regulated by LPS, alpha-IFN, and gamma-IFN, often independently of Ly49Q. PCR analysis revealed that cultured NK cells and T cells contained Ly49B transcripts, and Ly49B expression could be detected on NK cells cultured in IL-12 plus IL-18, and on an immature NK cell line. Immunohistochemical studies showed that Ly49B expression in tissues overlapped with but was distinct from that of all other myeloid molecules examined, being particularly prominent in the lamina propria and dome of Peyer's patches, implicating an important role of Ly49B in gut immunobiology. In transfected cells, Ly49B was found to associate with SHP-1, SHP-2, and SHIP in a manner strongly regulated by intracellular phosphorylation events.

The ProtozoanNeospora caninumDirectly Triggers Bovine NK Cells To Produce Gamma Interferon and To Kill Infected Fibroblasts

Natural killer (NK) cells are considered to be key players in the early innate responses to protozoan infections, primarily indirectly by producing gamma interferon (IFN-gamma) in response to cytokines, like interleukin 12 (IL-12). We demonstrate that live, as well as heat-inactivated, tachyzoites of Neospora caninum, a Toxoplasma-like protozoan, directly trigger production of IFN-gamma from purified, IL-2-activated bovine NK cells. This response occurred independently of IL-12 but was increased by the addition of the cytokine. A similar IFN-gamma response was measured in cocultures of NK cells and N. caninum-infected autologous fibroblasts. However, no NK cell-derived IFN-gamma response was detected when cells were cultured with soluble antigens from the organism, indicating that intact tachyzoites or nonsoluble components are necessary for NK cell triggering. Furthermore, N. caninum-infected autologous fibroblasts had increased susceptibility to NK cell cytotoxicity compared to uninfected fibroblasts. This cytotoxicity was largely mediated by a perforin-mediated mechanism. The activating receptor NKp46 was involved in cytotoxicity against fibroblasts but could not explain the increased cytotoxicity against infected targets. Interestingly, N. caninum tachyzoites were able to infect cultured NK cells, in which tachyzoites proliferated inside parasitophorous vacuoles. Together, these findings underscore the role of NK cells as primary responders during a protozoan infection, describe intracellular protozoan infection of NK cells in vitro for the first time, and represent the first functional study of purified bovine NK cells in response to infection.

Single-cell analysis of the human NK cell response to missing self and its inhibition by HLA class I

AbstractNatural killer (NK) cells activate quickly in response to pathogens, tumors, and allogeneic hematopoietic cell transplants. Modulating the NK cell response are clonally distributed NK cell receptors that survey cells for change in the expression of major histocompatibility complex (MHC) class I and structurally related ligands. Here the enzyme-linked immunospot (ELISPOT) assay, intracellular cytokine staining (ICS), and short-term culture were used to quantify the response of bulk NK cell populations from human donors to HLA class I–deficient 221 cells and to 221 cells transfected with single HLA class I allotypes. NK cells in cultures containing interleukin-2 (IL-2) or IL-12 exhibited specificities of HLA class I–mediated inhibition that correlated well with those previously defined using NK cell clones in long-term culture and with the frequencies of cells expressing particular inhibitory HLA class I receptors. Culture with IL-12, but not IL-2, gave an increased frequency of cells expressing CD94: NKG2A but no change in killer immunoglobulin-like receptor (KIR) expression. For some heterozygote combinations of KIR3DL1 alleles, ICS can be used to compare the functional properties of the 2 allotypes. Thus, both the low-expressing KIR3DL1*005 and the high-expressing KIR3DL1*002 gave similar inhibitory response on challenge with an HLA-B*5801 ligand. The single-cell assays developed here should facilitate future population study and clinical analysis of human NK cell regulation by MHC class I.

The corticosteroid-induced inhibitory effect on NK cell function reflects down-regulation and/or dysfunction of triggering receptors involved in natural cytotoxicity.

Corticosteroids are known to inhibit NK cell functions. However no information is available on whether such inhibition may affect the expression and/or the function of receptors involved in NK cell activation. In an attempt to analyze this point, we studied peripheral blood NK cells isolated from pediatric patients undergoing allogeneic BM transplantation. NK cells were analyzed before, during and after methylprednisolone administration to treat acute graft-versus-host disease. In NK cells freshly isolated from peripheral blood during methylprednisolone treatment, the surface expression of activating receptors, particularly NKp46 and NKp30, was consistently reduced. Such impaired expression could also be detected after 5 days of culture in IL-2. Such cultured NK cells also failed to express the IL-2-inducible NKp44 receptor. Accordingly, cytotoxicity against different tumor target cell lines was sharply reduced. The effect on NK cells isolated from healthy individuals and cultured in the presence of methylprednisolone was also analyzed. A similar inhibitory effect occurred in the expression of activating NK receptors. In addition, a sharp impairment of NK cytotoxicity against different tumor target cell lines or immature DC was detected.

Severe herpes virus (HSV-2) infection in two patients with myelodysplasia and undetectable NK cells and plasmacytoid dendritic cells in the blood

Background: How the immune system contains herpes simplex virus (HSV) infections is partly understood. T cells from infected persons proliferate in response to HSV antigens in vitro and may control local relapse rather than primary infection. NK cells have been involved in the control of experimental infections. A potentially important, as yet unexplored, population of interest might be the plasmacytoid dendritic cells (PDC), which contrary to monocytes, produce very high amounts of the major antiviral molecules, type-I interferon (IFN) following interaction with HSV. Objectives: Measure type-I IFN production, PDC, and NK cells in patients with unusually severe HSV infections. Study design: Two female patients of 33- and 50-year-old, respectively were referred because of severe disseminated HSV2 infection and myelodysplastic marrow. One patient had leukaemia and a primary HSV2 infection whereas the other had systemic lupus erythematosus (SLE) and a chronic HSV2 infection. The following studies were performed at various time points over 18 months: analysis of the lymphocytes and PDC subsets phenotype, lymphocyte proliferation assays to recall antigens; generation of NK cells in cultures, and production of type-I IFN in serum and by HSV-infected and by sendai virus (SV)-infected blood cells. Results and conclusions: PDC and NK cells were undetectable in the blood of both patients and NK cells could not be generated in culture at the time of ongoing infection. PBMC failed to produce IFN after infection with HSV contrasting with a normal T cell proliferation to HSV antigens in patient 1. Our observation suggests that innate immunity, through NK cells and PDC may control HSV infections, and together with IFN-producing capacity, should be investigated in patients with unusually severe HSV infections.

Comparisons of hematopoietic support by adipose-derived and bone marrow-derived stroma

Adipose-derived stromal (AdS) cells are multipotent mesodermal progenitors with developmental potentials similar to those ascribed to bone marrow-derived stromal (MdS) cells. The bone marrow mesenchyme supports hematopoietic stem cells and progenitors in vivo and in vitro. The AdS are readily accessible from subcutaneous fat but have not been fully characterized for hematopoietic support. We now directly compare the relative capacities of allogeneic AdS and MdS to support hematopoiesis. In total, 4 preparations of AdS and 4 preparations of MdS were used to complete these studies. Support for primitive hematopoietic progenitors was monitored in 5-week long-term culture (LTC) assays. CD34+ CD38neg cells were purified from umbilical cord blood (UCB, n = 8) to establish duplicate cultures with 100 hematopoietic progenitors per well in standard Dexter-type media. LTC cultures on MdS gave rise to 67.1 ± 19.8 CFU after 5 weeks. Parallel cultures on AdS gave rise to 26.6 ± 11.4 CFU. These frequencies approach statistical significance (p = 0.054). Importantly, however, 30% of the cultures initiated on AdS failed to support LTC. Thus, 100 CD34+ CD38neg cells may define a limit-dilution for LTC activity on AdS cells. In contrast, all cultures initiated on MdS supported LTC. In shorter-term assays, parallel cultures were established using 10,000 CD34+ linneg UCB cells for 12 days. The cultures were established in DMEM/F12 media containing 10% serum. No exogenous cytokines were used. The results are tabulated below. AdS and MdS supported similar expansions of total hematopoietic (CD45+) cells. However, cultures established on MdS maintained higher percentages of CD34+ cells over the 12 day time course. Not surprisingly, there was a strong association between the percentages of CD34+ cells and the frequencies of CFU present at day 12 (AdS, r = 0.81; MdS, r = 0.85). However, without exogenous cytokines these stroma maintained but did not expand the total content of CFU. Finally, both AdS and MdS encouraged the outgrowth of lymphoid progenitors as monitored by the presence of CD7+ CD34neg cells as well as by the growth of NK (CD56+) cells in secondary cultures. Higher percentages of CD7+ cells were noted on AdS than on MdS. In total, these data suggest that AdS and MdS provide similar hematopoietic support, although subtle distinctions were noted. These studies provide a model that can be manipulated to optimize hematopoietic expansion ex vivo. TableHematopoietic Support in Short-term CultureCD45+ Cell ExpansionPercent CD34+, Day 12CFU/100 Input CellsCFU ExpansionPercent CD7+, Day 12AdS3.78 ± 1.1 fold17.4 ± 5.1 %12.2 ± 2.8 CFU0.90 ± 0.3 fold28.6 ± 5.6 %MdS3.4 ± 1.3 fold28.7 ± 6.5 %16.3 ± 3.7 CFU1.07 ± 0.27 fold19.5 ± 4.2 %P0.1270.0020.0620.3660.003n88886Values reported are the average ± SEM, P values are by t-test.

Differential expansion of umbilical cord blood mononuclear cell–derived natural killer cells dependent on the dose of interleukin-15 with Flt3L

Objective We investigated the effect of interleukin-15 (IL-15) with Flt3 ligand (Flt3L) on the expansion and activation of NK cells derived from umbilical cord blood mononuclear cells (UCB-MNCs). Materials and Methods UCB-MNCs were cultured at 1 to 100 ng/mL of IL-15 + Flt3L (10 ng/mL) compared with 1 to 500 ng/mL of IL-2 + Flt3L (10 ng/mL). Cultured cells were assessed for surface marker and we calculated absolute number of NK cells and T cells. The cytotoxic activity was analyzed with purified NK cells. Results After 2 weeks culture with 5 ng/mL of IL-15 + Flt3L, the fold inductions of absolute number of NK cells significantly increased to 20.9-fold±9.3-fold of the number of NK cells on day 0 ( p<0.05), with 24.4-fold±16.1-fold of T cells. But with 50 ng/mL of IL-15 + Flt3L, fold induction of NK cells decreased to 5.1-fold±3.9-fold, while T cells showed 34.8-fold±18.7-fold (n = 8). The proportion of NK vs T cells showed to be significantly higher (1.61±0.91) with 5 ng/mL of IL-15 than with 50 ng/mL of IL-15 (0.12±0.03). Such proportional change of NK/T cells could not be observed with IL-2. Immunophenotypes of CD56, CD16, LFA1, CD94, CD8, and perforin of cultured NK cells with 10 ng/mL of IL-15 + Flt3L showed the same pattern of those with 50 ng/mL of IL-2 + Flt3L. Cytotoxic activity against K562 of cultured NK cells resulted in the same level as adult peripheral blood (PB)-derived NK cells. Conclusions Higher induction of NK cells derived from UCB-MNCs was achieved by low dose (5 to 10 ng/mL) rather than high dose (>50 ng/mL) of IL-15.

Independent control of Ly49g alleles: implications for NK cell repertoire selection and tumor cell killing.

A novel murine NK cell-reactive mAb, AT8, was generated. AT8 recognizes Ly49G from 129/J, BALB/c, and related mouse strains, but does not bind to Ly49G(B6). Costaining with AT8 and a Ly49G(B6)-restricted Ab (Cwy-3) provides the first direct evidence that Ly49G protein is expressed from both alleles on a significant proportion of NK cells from four different types of F(1) hybrid mice. The observed level of biallelic Ly49G expression reproducibly followed the product rule in both freshly isolated and cultured NK cells. Surprisingly, the percentage of NK cells expressing both Ly49G alleles could be dramatically increased in vitro and in vivo through IL-2R- and IFN receptor-dependent signaling pathways, respectively. Unexpectedly, Ly49G(B6+) NK cells in an H-2(d), but not H-2(b), background were more likely to lyse D(d+) and Chinese hamster ovary tumor cells than Ly49G(BALB/129+) NK cells. Furthermore, Ly49G(B6+) NK cells also proliferated to a higher degree in response to poly(I:C) than NK cells expressing a non-Ly49G(B6) allele in an H-2(d), but not H-2(b), background. These results suggest that Ly49G(B6) has a lower affinity for H-2D(d) than Ly49G(BALB/129), and the genetic background calibrates the responsiveness of NK cells bearing self-specific Ly49. Other H-2D(d) receptors on the different Ly49G(+) NK cell subsets were unequally coexpressed, possibly explaining the disparate responses of Ly49G(B6+) NK cells in different hybrid mice. These data indicate that the stochastic mono- and biallelic expression of divergent Ly49G alleles increases the range of MHC affinities and the functional potential in the total NK cell population of heterozygous mice.

KIR2DL4 (CD158d) genotype influences expression and function in NK cells.

The expression and function of the NK cell receptor KIR2DL4 are controversial. Two common alleles of the transmembrane domain of KIR2DL4 exist. The 10A allele with 10 adenines at the end of the transmembrane exon encodes a full length receptor, whereas the 9A allele has only 9 adenines resulting in a frame shift which in turn generates a stop codon early in the first cytoplasmic exon. The possibility that the 10A and 9A alleles might result in differences in expression and function of KIR2DL4 was explored using mAbs to KIR2DL4. Transfection experiments with cDNA from the 10A and 9A alleles revealed significant membrane expression only with the protein encoded by the 10A allele. Analysis of peripheral blood NK cells demonstrated that only in subjects with at least one 10A allele was cell surface expression of KIR2DL4 detectable, and then only on the minor CD56(bright) NK cell subset. The major CD56(dim) NK cell subset did not cell surface express KIR2DL4 but, interestingly, did so after in vitro culture. Functional analysis using cultured NK cells in redirected lysis assays demonstrated that KIR2DL4 is an activating receptor for NK cells with at least one 10A allele. No significant activity was detected for NK cells generated from subjects homozygous for the 9A allele. These data show that genotype influences cell surface expression and function of KIR2DL4 which may account for reported differences in KIR2DL4 expression and function.

Use of interleukin-15 for preparation of adherent NK cells from human peripheral blood: comparison with interleukin-2

To search the possibility of utilizing interleukin-15 (IL-15) in preparation of adherent human natural killer (A-NK) cells, recombinant human IL-15 (rhIL-15) or rhIL-2 (500 u/ml of each cytokine) were added to purified human NK cell culture in 24-well plastic plate. The cytokine-induced adherent ratio was calculated by percentage of A-NK cell in whole NK cells. The cytotoxicity of NK cells (NA- or A-NK cells) was examined by 4-h 51Chromium release assay, the surface markers of NK cells were checked by flow cytometry, and the cytokines were analyzed by reverse transcript (RT)-PCR and ELISA method. RhIL-15-induced adherence of human NK cells into plastic was higher than IL-2 when harvesting the A-NK cells at each hour point from hr 1 to hr 12. IL-15- and IL-2-induced adherent ratio peaked to 36.67% and 27.73% at hr 1, and the IL-15-induced adherent ratio was around two folds higher than IL-2-induced group at hrs 2, 3, 4, 5, 6, 7, and 8. The IL-15 group expanded more rapidly than IL-2 during 2 weeks' culture. IL-15- and IL-2-A-NK cells exerted similar levels of higher cytotoxic potentials. A-NK cells were characterized with phenotypes of CD3 −CD16 +CD56 + (more than 93%) in the presence of IL-2 or IL-15 stimulation. CD54, an intracellular adhesion molecule (ICAM), was also continuously expressed in A-NK cells (more than 85%) induced by each cytokine. Interestingly, IL-15 stimulated relatively low level of expression of CD18, a beta2 integrin molecule related to lymphocyte apoptosis in A-NK cells (11.45%), whereas IL-2 exerted a strong effect on CD18 expression (87.54%). IL-11b was only expressed at A-NK cell induced by IL-2 (49.56%), IL-15 did not exert any stimulating effect on CD11b expression. All A-NK cells expressed high levels of interferon gamma (IFNγ) after stimulation with IL-2 or IL-15. In contrast to IL-2, IL-15 did not stimulate gene expressions of type 2 cytokines (e.g. IL-4, IL-6, IL-10 and IL-13) in A-NK cells. The results indicate that rhIL15 is possibly a stronger stimulator for A-NK cell preparation by improving adherence and proliferation through inhibiting apoptosis by down-regulating the expression of CD18 and type 2 cytokines.

NK Cell Receptors of the Orangutan (Pongo pygmaeus): A Pivotal Species for Tracking the Coevolution of Killer Cell Ig-Like Receptors with MHC-C

CD94, NKG2, Ly49, and killer cell Ig-like receptor (KIR) expressed by orangutan peripheral blood cells were examined by cloning and sequencing cDNA from a panel of individuals. Orthologs of human CD94, NKG2A, D, and F were defined. NKG2C and E are represented by one gene, Popy-NKG2CE, that is equidistant from the two human genes. Several Popy-CD94, NKG2A, and NKG2CE alleles were defined. Popy-Ly49L is expressed in cultured NK cells and has a sequence consistent with it encoding a functional receptor. Orangutan KIR corresponding to the three KIR lineages expressed in humans and chimpanzees were defined. Popy-KIR2DL4 of lineage I is the only ortholog of a human or chimpanzee KIR, but in all individuals examined, the transcripts of this gene produced premature termination, either in the D2 domain or at the beginning of the cytoplasmic domain. Ten Popy-KIR3DL and one Popy-KIR3DS of lineage II are all closely related, but represent the products of at least two genes. The two Popy-KIR2DL and four Popy-KIR2DS of lineage III also represent two genes, both being more related to KIR2DS4 than to other human and chimpanzee KIR of lineage III. The Popy-KIR2D include ones predicted to be specific for the C1 epitope of MHC-C, but none specific for C2. This correlates with the observation that all orangutan MHC-C allotypes examined have the C1 motif.

Primary immune responses by cord blood CD4(+) T cells and NK cells inhibit Epstein-Barr virus B-cell transformation in vitro.

Epstein-Barr virus (EBV) transformation of B cells from fetal cord blood in vitro varies depending on the individual sample. When a single preparation of EBV was simultaneously used to transform fetal cord blood samples from six different individuals, the virus transformation titer varied from less than zero to 10(5.9). We show that this variation in EBV transformation is associated with a marked primary immune response in cord blood samples predominately involving CD4(+) T cells and CD16(+) CD56(+) NK cells. After virus challenge both CD4(+) T cells and NK cells in fetal cord blood cultures expressed the lymphocyte activation marker CD69. The cytotoxic response against autologous EBV-infected lymphoblastoid cell line (LCL) targets correlated with the number of CD16(+) CD69(+) cells and was inversely correlated with the virus transformation titer. Although NK activity was detected in fresh cord blood and increased following activation by the virus, killing of autologous LCLs was detected only following activation by exposure to the virus. Both activated CD4(+) T cells and CD16(+) NK cells were independently able to kill autologous LCLs. Both interleukin-2 and gamma interferon were produced by CD4(+) T cells after virus challenge. The titer of EBV was lower when purified B cells were used than when whole cord blood was used. Addition of monocytes restored the virus titer, while addition of resting T cells or EBV-activated CD4(+) T-cell blasts reduced the virus titer. We conclude that there are primary NK-cell and Th1-type CD4(+) T-cell responses to EBV in fetal cord blood that limit the expansion of EBV-infected cells and in some cases eliminate virus infection in vitro.

Book Review

Haematopoietic and lymphoid cell culture. Margaret J. Dalman and Jonathan R. Lamb. Cambridge University Press, 2000. Pp. 188. £55·00. ISBN: 0-521-62969-1 (paperback). This is a most succinct and clearly written handbook describing the characteristics and methods of isolation and culture of the major types of haematopoietic cells. The cell types discussed are embryonic stem cells, dendritic cells, murine thymic explant cultures, T cells, B lymphocytes, monocytes and macrophages, NK and LAK cells, and, lastly, mast cells and basophils. An obvious omission, however, are multipotent haematopoietic stem cells and their progenitors. Each chapter is devoted to each of the above cell types and written by appropriate experts in the field. The chapters are well laid out and structured, each with a clear and comprehensive introduction summarizing the origin, localization, development, phenotypic features, culture requirements and functions of each cell type. This is followed by a detailed and practical summary of methodologies for their isolation and in vitro culture. The book includes helpful explanatory diagrams and well-presented photographs of the various cells types grown in culture. The chapter on embryonic stem cells describes the conditions for growth, aggregation and differentiation of embryonic stem cells and analysis of differentiated progeny. For dendritic cells, their generation in culture from mouse bone marrow in human peripheral blood monocytes is discussed, as well as an assessment of the migratory properties. The chapter on thymic explant cultures discusses the culture of murine, fetal and newborn thymic organ, and the reaggregate thymus organ cultures, to assess the interaction of isolated lymphoid cells and thymic stromal cells as an in vitro model of the thymus. The chapter on T cells discusses the isolation and expansion of T cell lines and clones. For B lymphocytes, the culture of early bone marrow-derived B cells, and mature B lymphocytes from spleen and the peritoneal cavity in human and mouse, are described. In addition, flow cytometry and molecular techniques for phenotypic analysis are included. The chapter on monocytes and macrophages describes the isolation and culture of these cells from a variety of tissues in mouse and human, and the chapter on NK and LAK cells describes the identification, culture and cloning of murine and human NK cells. Finally, the culture of mast cells and basophils in mouse and human is detailed, together with the growth of mast cells from human CD34+ progenitor cells. Protocols for the detection of these two cell types are also included. In conclusion, this handbook is an invaluable and easily readable reference. Its attractively compact size makes it easily accessible not only on book shelves, but it will also slip easily in to briefcases and laboratory coat pockets. It is just the type of eagerly sought after book that can easily sprout legs and wander off into neighbouring offices and pockets!

Characteristics of tumor infiltration by adoptively transferred and endogenous natural-killer cells in a syngeneic rat model: implications for the mechanism behind anti-tumor responses.

Interleukin-2-activated, cultured NK cells (A-NK) cells were adoptively transferred into a syngeneic rat liver-tumor model. The kinetics of tumor infiltration by NK cells, originating either from adoptively transferred or from endogenous sources, the localization of these cells in the tumor, and their interactions with extracellular-matrix proteins were studied by immunohistochemistry and transmission-electron microscopy. The adoptive transfer of A-NK cells via the hepatic artery and s.c. injections of IL-2 into rats bearing subcapsularly induced CC531 liver tumors, but also IL-2 monotherapy, resulted in a significant increase of the number of NK cells both at the tumor border and in the tumor center. The majority of tumor-infiltrating NK cells was present in the tumor stroma and only occasionally was an NK cell observed in a tumor nodule in direct contact with tumor cells. Observations by electron microscopy suggested that matrix proteins, abundantly present in the tumor stroma but absent in the tumor nodules, provide a substrate for migration of infiltrating cells, whereas tight structures of matrix proteins surrounding tumor nodules provide a barrier for establishment of direct NK-cell-to-tumor-cell-contact. Our results suggest that direct NK-cell-to-target-cell-contact-mediated lysis is of minor importance for attaining an anti-tumor effect in this model. We hypothesize that treatment of tumor-bearing rats with A-NK cells and/or IL-2 initiates a cascade of events (e.g., secretion of tumor-killing cytokines and/or infiltration of other immune cells) ultimately leading to tumor regression.

Immunoregulatory Roles of IL-10 in Innate Immunity: IL-10 Inhibits Macrophage Production of IFN-γ-Inducing Factors but Enhances NK Cell Production of IFN-γ

AbstractIn our study of the immunoregulatory roles of IL-10 in innate immunity, nonantigenic phagocytosable chitin particles were administered i.v. to IL-10-deficient (knockout (KO)) mice or KO mice pretreated with anti-NK1.1 or anti-IFN-γ Abs. The results established that chitin treatment of KO mice increased superoxide anion release from alveolar macrophages (Mφ) to a level much higher than that in wild-type (WT) mice. The results also suggested that the NK cell is the source of IFN-γ that is primarily responsible for this alveolar Mφ priming. To further study the roles of IL-10-inhibiting chitin-induced IFN-γ production, we used spleen cell cultures. The experiments showed that IL-12, IL-18, and TNF-α, which were produced by chitin-stimulated Mφ, contributed to the IFN-γ-inducing activity of chitin. Our results established that exogenous IL-10 inhibited chitin-induced IFN-γ production in spleen cell cultures from both KO and WT mice. Exogenous IL-10 also inhibited IL-12 and TNF-α production by chitin-stimulated Mφ. Exogenous IL-10 decreased IL-12- or IL-18-induced IFN-γ levels in KO but not in WT NK cell cultures. However, exogenous IL-10 enhanced IFN-γ levels when NK cells were stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures. Our in vitro data indicate that IL-10 has differential effects on chitin-induced IFN-γ production. However, the inhibitory effects of endogenous IL-10 appear to be dominant in the chitin-induced alveolar Mφ priming response in vivo.

Selective inhibition of human and mouse natural killer tumor recognition using retroviral antisense in primary natural killer cells: involvement with MHC class I killer cell inhibitory receptors.

The natural killer tumor recognition (NK-TR) protein has been shown to be a necessary component for the killing of NK-sensitive and virus-infected targets by the rat RNK-16 cell line. Class I-recognizing killer cell inhibitory receptors (KIR) have been found in the human (p58; NKAT family) and mouse (Ly-49 family). The principal functional characteristic of these receptors is their ability to block NK cell lysis by recognition of selected class I molecules on target cells. In the present study, we examined whether abrogation of NK-TR expression by retroviral infection of primary human or mouse NK cells with virus-producing antisense NK-TR also would demonstrate loss of non-MHC-restricted killing and whether the NK-TR was associated with KIR function in humans or with Ly-49 in the mouse. Using short term culture of fresh human or mouse NK cells, antisense NK-TR-treated NK cells demonstrated strong selective reduction of NK cytotoxicity. NK-TR was necessary for lytic activity even when KIR function was blocked by Ab in experiments involving NK3.3 lysis of HLA.cw3-expressing targets or killing of Dd targets by Ly-49A+ or Ly-49G2+ mouse NK cells. These studies extend our previous studies in rat NK cell lines to demonstrate that primary mouse and human NK cells require NK-TR for non-MHC-restricted lysis of tumor and virus-infected targets. In addition, the reversal of KIR or Ly-49 inhibition of NK cell lysis requires NK-TR expression for cellular killing in both human and mouse.

Interleukin-12-stimulated natural killer cells can activate human macrophages to inhibit growth of Mycobacterium avium.

Interleukin-12 (IL-12) is a critical cytokine that affects many of the biological functions of NK cells and T cells. We have previously shown that both human and murine NK cells are important in host defense against Mycobacterium avium complex and act by secreting cytokines that induce macrophages to inhibit the growth of intracellular M. avium. To define the role of IL-12 in M. avium complex infection, we stimulated human NK cells with recombinant human IL-12 at 0.01 to 1 ng/ml for 24 h and used the tissue culture supernatant to treat human monocyte-derived macrophage monolayers infected with M. avium. IL-12 had no direct effect on M. avium-infected macrophages, but culture supernatant from IL-12-treated NK cells activated macrophages to inhibit the growth of intracellular M. avium in a dose-dependent manner. Stimulation of NK cells with IL-12 in combination with tumor necrosis factor alpha (TNF-alpha) or IL-1 increased the ability of supernatant from NK-cell culture to limit M. avium growth within macrophages, compared with that of culture supernatant from IL-12-treated NK cells. Results with supernatant from nonstimulated NK cells were similar to those with supernatant from untreated controls. Treatment of supernatant from IL-12-stimulated NK cells with anti-TNF-alpha, anti-granulocyte-macrophage colony-stimulating factor, but not anti-gamma interferon antibodies decreased the ability of NK-cell supernatant to induce anti-M. avium activity in infected macrophages. Treatment of macrophage monolayers with anti-transforming growth factor beta antibody before adding supernatant from IL-12-stimulated NK cells was associated with an increase of anti-M. avium activity compared with that of supernatant from IL-12-treated NK cells. These results suggest that IL-12 has a role in host defense against M. avium and that the effect of IL-12 is dependent chiefly on TNF-alpha and granulocyte-macrophage colony-stimulating factor.