Inhibition of the death receptor pathway by cFLIP confers partial engraftment of MHC class I-deficient stem cells and reduces tumor clearance in perforin-deficient mice.

NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist. By analyzing the anti-apoptotic effects of cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein (cFLIP) overexpression, we investigated the possible role of death receptor-induced apoptosis in NK cell-mediated cytotoxicity. In the absence of perforin, we found that cFLIP overexpression reduces lysis of tumor cells by NK cells in vitro and in vivo. In addition, perforin-deficient NK cells were impaired in their ability to acutely reject cFLIP-overexpressing TAP-1 knockout stem cells. These results emphasize the importance of NK cell death receptor-mediated killing during BMC grafts in the absence of perforin.

Potential pathways for regulation of NK and T cell responses: differential X-linked lymphoproliferative syndrome gene product SAP interactions with SLAM and 2B4.

SAP, the gene that is altered or absent in the X-linked lymphoproliferative syndrome (XLP), encodes a small protein that comprises a single SH2 domain and binds to the cell-surface protein SLAM which is present on activated or memory T and B cells. Because defective NK cell activity also has been reported in XLP patients, we studied the SAP gene in NK cells. SAP was induced upon viral infection of SCID mice and shown to be expressed in NK cells by in vitro culturing in the presence of IL-2. Moreover, SAP was expressed in the NK cell lines YT and RNK 16. Because SLAM, the cell-surface protein with which SAP interacts, and 2B4, a membrane protein having sequence homologies with SLAM, also were found to be expressed on the surfaces of activated NK and T cell populations, they may access SAP functions in these populations. Whereas we found that 2B4 also binds SAP, 2B4-SAP interactions occurred only upon tyrosine phosphorylation of 2B4. By contrast, SLAM-SAP interactions were independent of phosphorylation of Y281 and Y327 on SLAM. As CD48, the ligand for 2B4, is expressed on the surface of Epstein-Barr virus (EBV)-infected B cells, it is likely that SAP regulates signal transduction through this pair of cell-surface molecules. These data support the hypothesis that XLP is a result of both defective NK and T lymphocyte responses to EBV. The altered responses may be due to aberrant control of the signaling cascades which are initiated by the SLAM-SLAM and 2B4-CD48 interactions.

Functional defects of NK cells treated with chloroquine mimic the lytic defects observed in perforin-deficient mice.

NK cells are the primary effectors mediating acute rejection of incompatible bone marrow cell grafts. To reduce rejection, we evaluated the ability of chloroquine (CHQ) to prevent perforin-dependent NK cell activity. Perforin is a key cytotoxic component released from the lytic granules of activated NK cells. Generation of functional perforin requires an acidic protease activity that occurs in the secretory, lytic lysosomes. Our hypothesis was that CHQ, a lysosomotropic reagent, would raise the pH of the acidic compartment in which perforin is processed and thereby block perforin maturation and cytotoxicity. We have measured NK cytotoxicity in vivo by clearance of YAC-1 tumor cells from the lungs and by rejection of incompatible bone marrow transplants and in vitro by cytolysis of YAC-1 and Jurkat cells. The engraftment of bone marrow cells was monitored by recolonization of the spleen with hemopoietic cells from transplants of MHC class I-deficient bone marrow cells into lethally irradiated recipient mice. Transplant rejection was compared in two inbred strains of mice: 129, which apparently use perforin-dependent cytotoxicity, and C57BL/6, in which rejection can be perforin-independent. CHQ treatment reduced NK cell activity in 129 mice in which perforin is important for mediating rejection. CHQ affected the fraction of NK cell cytolysis that was Fas independent. In addition, we found that CHQ prevents perforin processing by LAK cells in vitro. These data indicate that CHQ may impair rejection of incompatible bone marrow transplants and other functions mediated by NK and cytotoxic T cells.

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants.

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.

Gene structure of the murine NK cell receptor 2B4: presence of two alternatively spliced isoforms with distinct cytoplasmic domains.

The NK cell receptor 2B4 is expressed on the surface of all murine NK cells and a subset of T cells. Ligation of 2B4 with monoclonal antibodies increases target cell lysis and IFN-gamma production. 2B4 is the high-affinity counter-receptor for CD48 in mice and humans. 2B4-L is a member of the CD2 subgroup of the immunoglobulin supergene family, which includes CD48, LFA-3, CD84, Ly9 and SLAM. Here we describe 2B4-S, a second 2B4 isoform, and the genomic structure of the 2B4 gene. 2B4-S is identical to the 5' end of 2B4-L, differing only at the 3' end, corresponding to a portion of the cytoplasmic domain and the 3' untranslated sequence. Both 2B4-L and 2B4-S are expressed on IL-2-activated NK cells. The genomic clone of 2B4 reveals that the two cDNA clones are products of alternative splicing. Since they differ only in a portion of the cytoplasmic domain, it is likely that they transduce different signals.

Cutting edge: expression of functional CD94/NKG2A inhibitory receptors on fetal NK1.1+Ly-49- cells: a possible mechanism of tolerance during NK cell development.

Fetal liver- and thymus-derived NK1.1+ cells do not express known Ly-49 receptors. Despite the absence of Ly-49 inhibitory receptors, fetal and neonatal NK1.1+Ly-49- cells can distinguish between class Ihigh and class Ilow target cells, suggesting the existence of other class I-specific inhibitory receptors. We demonstrate that fetal NK1. 1+Ly-49- cell lysates contain CD94 protein and that a significant proportion of fetal NK cells are bound by Qa1b tetramers. Fetal and adult NK cells efficiently lyse lymphoblasts from Kb-/-Db-/- mice. Qa1b-specific peptides Qdm and HLA-CW4 leader peptide specifically inhibited the lysis of these blasts by adult and fetal NK cells. Qdm peptide also inhibited the lysis of Qa1b-transfected human 721.221 cells by fetal NK cells. Taken together, these results suggest that the CD94/NKG2A receptor complex is the major known inhibitory receptor for class I (Qa1b) molecules on developing fetal NK cells.

Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4.

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and gamma-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.

Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vitro systems.

In the last few years, the routine development of knockout and transgenic mice and the ease with which rare progenitor populations can be isolated from hematopoietic organs and cultured in vitro has facilitated significant advances in understanding the lineage and development of natural killer (NK) cells. Fluorescence-activated cell sorter analyses have identified a common lymphoid progenitor capable of giving rise to NK, T, and B cells, confirming the lymphoid origin of NK cells. Knockout and transgenic mouse models have pointed to an absolutely critical role for signals sent through the interleukin (IL)-2/15 receptor beta (CD122) chain and common gamma (gamma c) chain for NK development. Such signals are likely relayed inside the cell by the tyrosine kinase Jak3, which associates with gamma c. Recently developed IL-15 and IL-15 receptor alpha knockout mice have pinpointed IL-15 as the mediator of this signal. Other mouse models have indicated an unexpected role for flt3 ligand in early NK-cell development as well as minor roles for stem cell factor and IL-7 in expanding NK-cell progenitor numbers. Finally, in vitro culture systems have proven useful in identifying the point in NK development at which each of these signals is critical.

Development of self-recognition systems in natural killer cells.

Differentiation of NK cells is bone marrow dependent. Although all the factors necessary for NK differentiation are yet to be fully characterized, IL-15 has emerged as the most likely candidate that drives terminal differentiation of NK cells. Other cytokines are needed for the expansion and maintenance of the progenitor population. Although the in vivo role for IL-15 cannot be established without the generation of either IL-15 or IL-15R alpha deficient mice, in vitro data suggests that it is responsible for the generation of lytic, NK1.1+ cells from immature progenitors. So far, it has not been possible to obtain Ly-49+ cells from marrow or fetal-derived progenitor cells in vitro. Stromal cells along with cytokines may be necessary to induce expression of Ly-49 on NK1.1+ cells. Expression of the NK receptors seems to be a sequential process with expression of IL-2/15R beta on progenitor cells occurring first followed by the expression of NK1.1 and then probably Ly-49. The same sequence seems to hold true in vivo as well, Ly-49 surface expression on splenic NK1.1+ cells is first detected 4-6 days after birth, and the frequency of cells expressing Ly-49 receptors reaches adult levels by days 20-24. Despite the lack of expression of Ly-49 receptors by fetal NK1.1+ as well as bone marrow derived NK1.1+ cells, they are able to distinguish between MHC class Ihi and class Ilo targets. This suggests that these NK1.1+Ly-49- cells express non-Ly-49 class I receptors. Efforts in the future need to be focused on elements responsible for the expression of Ly49 on these NK1.1+ cells in order to establish an in vitro system in which establishment of the Ly-49 repertoire can be studied.

Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates.

We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)-, c-kit+, Sca2+, interleukin (IL)-2/15Rbeta (CD122)- marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5-6 d followed by IL-15 alone for an additional 4-5 d expanded the starting population 30-40-fold and gave rise to a virtually pure population of NK1.1+, CD3- cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9-11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

Synergistic stimulation of avian I kappa B alpha transcription by rel and fos/jun factors.

Rel/NF-kappa B transcription factors and I Kappa B alpha function in an autoregulatory network. Avian I kappa B alpha transcription is increased in response to both c-Rel and v-Rel. This study shows that I kappa B alpha transcription is synergistically stimulated by Rel and AP-1 factors (c-Fos and c-Jun). However, the response to v-Rel and the AP-1 factors was not as vigorous as that of c-Rel and AP-1. A 386 bp region of the I kappa B alpha promoter (containing two NF-kappa B and one AP-1 binding sites) was shown to be both necessary and sufficient for response to both Rel factors alone or Rel factors in conjunction with the AP-1 proteins. In addition, an imperfect NF-kappa B binding site was found to overlap the AP-1 binding site. Mutation of either of the NF-kappa B binding sites or the AP-1 binding site dramatically decreased the response of the I kappa B alpha promoter to Rel proteins alone or Rel and AP-1 factors. Overexpression of c-Rel or v-Rel resulted in the formation of DNA binding complexes associated with the imperfect NF-kappa B binding site which overlaps the AP-1 site. v-Rel associated with the imperfect NF-kappa B site stronger than c-Rel, and overexpression of v-Rel also resulted in the formation of a v-Rel containing complex bound to a consensus AP-1 site. These studies address the difference in c-Rel and v-Rel's ability to synergistically stimulate I kappa B alpha expression in conjunction with the AP-1 factors.

Structure and regulation of the gene encoding avian inhibitor of nuclear factor kappa B-alpha.

The Rel/NF-kappa B family of transcription factors exist in the cytoplasm as inactive complexes in association with an inhibitory protein called I kappa B-alpha. We have isolated a clone containing the avian I kappa B-alpha gene from a chicken genomic library. Avian I kappa B-alpha is devoid of any recognizable promoter elements, i.e., TATA and CAAT boxes; however, the 5'-UTR of the gene contains the initiator elements frequently found in TATA-less genes. Avian I kappa B-alpha contains seven putative Rel/NF-kappa B binding sites. A CAT reporter construct containing the 5' upstream region of I kappa B-alpha was expressed when transfected into cells which produce I kappa B-alpha. This construct, however, was not expressed in cells in which I kappa B-alpha activity was not induced, indicating that the regulatory elements which promote I kappa B-alpha expression are contained within 1000 nt of the transcription start site. Southern analysis suggests that I kappa B-alpha is present as a single-copy gene per haploid genome and is expressed in avian hematopoietic tissues, as well as lymphoid cells transformed by avian reticuloendotheliosis virus (REV-T).

Avian I kappa B alpha is transcriptionally induced by c-Rel and v-Rel with different kinetics.

The Rel/NF-kappa B family of transcription factors participates in the regulation of genes involved in defense responses, inflammation, healing and regeneration processes, and embryogenesis. The control of the transcriptional activation potential of the Rel/NF-kappa B proteins is mediated, in part, by their association with inhibitory proteins of the I kappa B family. This association results in the cytoplasmic retention of these factors until the cell receives a proper stimulatory signal. The I kappa B alpha gene is a target for regulation by the Rel/NF-kappa B proteins and is in fact upregulated in response to Rel/NF-kappa B activation. A naturally occurring oncogenic variant of the Rel/NF-kappa B family, v-rel, transforms avian lymphocytes, bone marrow cells, monocytes, and fibroblasts. Avian I kappa B alpha expression is upregulated in cells transformed by v-Rel. Avian I kappa B alpha is also upregulated in fibroblasts overexpressing c-Rel and oncogenic variants of c-Rel. c-Rel, a carboxy-terminally truncated variant of c-Rel, and v-Rel are all able to directly transactivate the expression of the avian I kappa B alpha gene. However, c-Rel was the most potent activator of this gene, and the induction of I kappa B alpha expression showed faster kinetics in cells overexpressing c-Rel than in those overexpressing v-Rel. The regulation of I kappa B alpha induction by the Rel proteins was shown to be dependent on a 362-bp region of the I kappa B alpha promoter that contains two potential NF-kappa B binding sites and one AP-1-like binding site. Results of electrophoretic mobility shift assays using these NF-kappa B binding sites indicate that major changes in the profile of DNA binding complexes in fibroblasts overexpressing v-Rel correlated temporally with the kinetic changes in v-Rel's ability to activate the expression of the I kappa B alpha gene.

Transformation of avian fibroblasts overexpressing the c-rel proto-oncogene and a variant of c-rel lacking 40 C-terminal amino acids.

The v-rel oncogene was derived from the c-rel proto-oncogene, which encodes a transcriptional activator. Expression of v-rel transforms avian hematopoietic cells and fibroblasts. Here we report that overexpression (via a replication-competent retroviral vector) of full-length c-Rel as well as a 40-amino-acid, carboxy-terminal deletion construct of c-Rel (c-Rel delta) resulted in the morphological transformation of chicken embryo fibroblasts (CEFs). Subcellular localization of Rel polypeptides in these transformed cells as determined by immunofluorescence and immunoprecipitation revealed their presence in both the nucleus and the cytoplasm, with the majority of Rel polypeptides showing cytoplasmic localization. Cytoplasmic localization could be due to interaction with I kappa B molecules, and in fact, the overexpression of c-Rel or the C-terminal deletion construct of c-Rel resulted in an increase in the levels of mRNA encoding the avian I kappa B protein pp40 and the avian homolog of the NF-kappa B protein, p105. However, expression of v-Rel resulted in the induction of pp40 mRNA only. While c-Rel was a weak activator of kappa B-mediated transcription of a reporter construct in transformed CEFs, v-Rel and c-Rel delta were transcriptional repressors. However, in spite of these differences, all of these proteins resulted in the transformation of CEFs.