Class I MHC-binding characteristics of the 129/J Ly49 repertoire.

The Ly49 family of NK cell receptors and its MHC-binding characteristics have only been well characterized in C57BL/6 (B6) mice. Previous studies have shown that 129/J mice express unique Ly49 genes that are not found in the B6 strain. Screening of a 129/J cDNA library led to the discovery of 10 distinct full-length Ly49-related coding sequences (Ly49e, g, i, o, p, r, s, t, u, and v). Although 129/J mice share identical class I MHC (K(b) and D(b)) transcripts with B6 mice, only one Ly49 is identical in the two strains (Ly49E). In addition to the previously characterized Ly49P, two new activating Ly49 proteins were discovered, Ly49R and U. The MHC specificity of the total 129/J Ly49 repertoire was evaluated with soluble class I MHC tetramers and found to be distinct compared with the B6 Ly49 repertoire. Ly49V bound to many types of class I MHC, suggesting that Ly49V(+) NK cells may monitor host cells for a global down-regulation in MHC levels. An activating receptor, Ly49R, was shown to bind soluble class I molecules to a moderate degree, a result not previously observed for other activating Ly49 proteins. Furthermore, tetramer-binding results were confirmed functionally with cytotoxicity assays using sorted 129/J NK cells. This study shows that the Ly49 repertoire and its MHC-binding characteristics can be very different among inbred mouse strains. Ly49 divergence should be considered when using 129-derived embryonic stem cells for the production of gene-targeted mice, especially when an immune or NK-derived phenotype is under scrutiny.

Activating Ly-49D NK receptors: expression and function in relation to ontogeny and Ly-49 inhibitor receptors.

Developmental changes in the repertoire of activating Ly-49 family members have not been examined previously. In the present study, we have examined the expression and function of the activating Ly-49s (D and H) from birth through 8 weeks of age. We demonstrate that 1) activating Ly-49s are expressed early, 2) their expression intensity is not different from adult NK cells, and 3) activating receptors are functional. Examination of the inhibitory Ly-49s also demonstrated functional capacity immediately upon expression. To examine the kinetics of expression of the repertoire of activating Ly-49 members, we utilized five- and six-color flow cytometric analyses of NK cells from birth through adulthood. Previous studies examining the inhibitory Ly-49 repertoire have proposed that expression is regulated by the product rule. Our results indicated that Ly-49D, which recognizes H-2Dd, had a discordantly high coexpression of the inhibitory Ly-49s that recognized H-2Dd (Ly-49A and Ly-49G2). The product rule of Ly-49 expression does not explain the coexpression of selected activating and inhibitory receptors. This high level of coexpression of H-2Dd recognizing activating and inhibitory Ly-49s suggests an in vivo selection or regulated coexpression.

Identification of the Ly49L protein: evidence for activating counterparts to inhibitory Ly49 proteins.

Previous studies have indicated that NK cells from different strains of inbred mice may express distinct Ly49 repertoires. Screening of NK cells from the CBA/J mouse for inhibitory and activating Ly49s revealed a novel DAP12-associated receptor that was immunoprecipitated with the Ly49G-specific mAb 4D11. Degenerate primers were designed to amplify and clone Ly49 cDNAs from CBA/J NK cells. A novel activating Ly49 cDNA was identified, which bears strong homology to the partially sequenced Ly49l gene found in C57BL/6 mice. Transfection of Ly49l into a DAP12+ cell line and subsequent immunoprecipitation experiments showed that Ly49L is likely the activating Ly49 detected by the 4DD11 antibody in CBA/J NK cells. Antibody-mediated cross-linking of Ly49L induced DAP12 phosphorylation, providing evidence that Ly49L is a functional activating receptor. Comparison of the extracellular domains of Ly49 family members indicates that all known activating members have an inhibitory counterpart with a highly related extracellular region.

Synergistic effects of in vivo depletion of Ly-49A and Ly-49G2 natural killer cell subsets in the rejection of H2(b) bone marrow cell allografts.

Subsets of murine natural killer (NK) cells exist that express the Ly-49 family of molecules that recognize different major histocompatibility complex (MHC) determinants. Bone marrow transplantation studies were performed to examine the in vivo functions of 2 of these subsets. Subsets of Ly-49A and Ly-49G2 NK share specificity for the same MHC class 1 ligand, D(d), binding of which results in an inhibitory signal to the NK cell but allows them to lyse H2(b) targets in vitro. We therefore examined the ability of these subsets to reject H2(b) bone marrow cell allografts in lethally irradiated mice. Surprisingly, depletion of Ly-49A(+) NK cells in BALB/c or B10.D2 mice (both H2(d)) had no effect on the rejection of H2(b) BMC. However, Ly-49A depletion did partially abrogate the ability of B10.BR (H2(k)) mice to reject H2(b) allografts. Although depletion of either Ly-49A(+) or Ly-49G2(+) NK cells alone had no effect on the ability of B10.D2 mice to reject H2(b) BMC, depletion of both subsets dramatically and synergistically abrogated rejection. Studies with various B10 congenic mice and their F(1) hybrids indicate that this synergy between Ly49A and Ly4G2 depletion occurs in every instance. Thus, Ly-49A(+) NK cells appear to play a role in the rejection H2(b) bone marrow allografts, but, in most strains of mice studied, Ly-49G2(+) NK cells must also be eliminated. The putative roles of these NK cell subsets in clinical transplantation remains to be elucidated. (Blood. 2000;95:3840-3844)

Structure/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK receptors.

Murine NK cells express Ly-49 family receptors capable of either inhibiting or activating lytic function. The overlapping patterns of expression of the various receptors have complicated their precise biochemical characterization. Here we describe the use of the Jurkat T cell line as the model for the study of Ly-49s. We demonstrate that Ly-49D is capable of delivering activation signals to Jurkat T cells even in the absence of the recently described Ly-49D-associated chain, DAP-12. Ly-49D signaling in Jurkat leads to tyrosine phosphorylation of TCRzeta and requires Syk/Zap70 family kinases and arginine 54 of Ly-49D, suggesting that Ly-49D signals via association with TCRzeta. Coexpression studies in 293-T cells confirmed the ability of Ly-49D to associate with TCRzeta. In addition, we have used this model to study the functional interactions between an inhibitory Ly-49 (Ly-49G2) and an activating Ly-49 (Ly-49D). Ly-49G2 blocks activation mediated by Ly-49D in an immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent manner. In contrast, Ly-49G2 was incapable of inhibiting activation by the TCR even though human killer cell inhibitory receptor (KIR) (KIR3DL2(GL183)) effectively inhibits TCR. Both the ability of Ly-49G2 to block Ly-49D activation and the failure of Ly-49G2 to inhibit TCR signaling were confirmed in primary murine NK cells and NK/T cells, respectively. These data demonstrate the dominant effects of the inhibitory receptors over those that activate and suggest an inability of the Ly-49 type II inhibitory receptors to efficiently inhibit type I transmembrane receptor signaling in T cells and NK cells.

Ly-49 receptor expression and functional analysis in multiple mouse strains.

We present data on the strain distribution and functional characteristics of the Ly-49 receptors A, C/I, D, and G2 on DX5+ natural killer (NK) cells. We have examined tyrosine phosphorylation of the Ly-49 molecules, regulation of NK cytotoxic functions, and in vivo marrow rejection capability. The flow cytometry results demonstrate a diverse and complex pattern of expression of the Ly-49 receptors in the 11 strains examined. The vast majority of NK cells express Ly-49s, although some NK1.1+ CD3+ cells also express these receptors. The results of our functional analysis indicate that H-2Dd was able to inhibit the function of Ly-49G2+ NK cells, not only in B6 mice, but also by NK cells derived from several haplotypes. The examination of Ly-49 receptor tyrosine phosphorylation, which is a biochemical measure of inhibitory function, was consistently observed in the 11 mouse strains examined. In contrast, analysis of Ly-49D function suggests its expression appears to be more restricted and that H-2Dd is an activating ligand for this receptor. In addition, the in vivo examination of both inhibitory (Ly-49G2) and activating (Ly-49D) receptors demonstrated regulatory roles of these class I binding receptors in marrow transplantation.

IL-13 production by NK cells: IL-13-producing NK and T cells are present in vivo in the absence of IFN-gamma.

In this study, we demonstrate that human NK cells, human NK clones, the human NK cell line (NK3.3), and a population of murine NK cells can produce the type 2 cytokine IL-13 in response to IL-2 or phorbol myristate acetate plus ionomycin. IL-2 rapidly induced new IL-13 mRNA and protein synthesis in the NK3.3 cell line. Six of 12 human NK clones tested produced IL-13 protein in response to IL-2 or phorbol myristate acetate and ionomycin. Intracellular analysis revealed that approximately 2% of human peripheral NK cells produced IL-13 protein in response to IL-2. Isolated NK cells from SCID and RAG-2 knockout (-/-) mice that lack T and B cells as well as normal mice also can produce IL-13 mRNA and protein in response to IL-2. We hypothesized that in the absence of IFN-gamma, IL-13-producing NK cells may predominate in vivo. Utilizing IFN-gamma knockout (-/-) mice as a model system, IL-2-activated liver NK and T cells expressed 10-fold more IL-13 and IL-5 mRNA and protein than normal controls following IL-2 treatment in vitro. These results suggest that in the absence of IFN-gamma, an IL-13- and IL-5-producing NK and T cells predominate in vivo. The existence of this cell type has important implications in innate immunity given that the balance between IFN-gamma and IL-13/IL-5-producing NK cells may influence the early development of a cell-mediated or humoral immune response.

Molecular mechanisms of immune-mediated lysis of murine renal cancer: differential contributions of perforin-dependent versus Fas-mediated pathways in lysis by NK and T cells.

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-gamma or TNF-alpha, and the combination of IFN-gamma + TNF-alpha synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-gamma and TNF-alpha are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-gamma and TNF-alpha.

The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells.

The Ly-49 gene families are class I-recognizing receptors on murine NK cells. Most Ly-49 receptors inhibit NK cell lysis upon recognizing their target class I ligands. In this report we have examined the ability of Ly-49A and Ly-49G2 to regulate T cell functions on CD3+ cells, primarily the subset that also expresses NK-1.1 and/or DX5. The majority (>50%) of T cells that express Ly-49 molecules also coexpress NK-1.1 and/or DX5, although some NK-1.1- and/or DX5-/CD3+ cells express Ly-49 molecules. Lysis of target cells by IL-2-cultured T cells expressing Ly-49A and G2 was enhanced by Abs specific for Ly-49A and G2 as well as by Abs to class I (H-2Dd alpha1/alpha2). Murine T cells also were cultured in the presence of targets that express (H-2Dd) which is inhibiting for the Ly-49A and G2 receptors. These cells were examined for a coincident increase in cytokine production (IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF). Abs to Ly-49A and G2 or their respective class I ligands blocked the negative signals mediated via the Ly-49 receptors and increased IFN-gamma and granulocyte-macrophage CSF production after interaction of these T cells with H-2Dd-expressing tumor targets. Furthermore, an EL-4 T cell line expressing both Ly-49A and G2, when treated with mAb YE148 and 4D11, demonstrated reduced cytokine production and calcium mobilization. These results demonstrate for the first time that Ly-49 class I binding receptors, previously thought to be restricted to mouse NK cells, can mediate important physiological functions of T cell subsets.

The Ly-49 family: regulation of cytokine production in murine NK cells.

Most proteins encoded by members of the Ly-49 gene family are class I-recognizing receptors on murine natural killer (NK) cells. Class I recognition by Ly-49 receptors usually results in inhibition of NK cell lysis of target cells. However, NK cells function not only in a lytic capacity, but also can mediate cytokine production. In this report we have demonstrated the ability of Ly-49A and Ly-49G2 to inhibit production of cytokines by NK cells by showing that specific antibodies against these gene products stimulate cytokine production. Murine NK cells were cultured in the presence of P815 (H2-Dd), and supernatants were analyzed for the production of interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocytemacrophage colony-stimulating factor (GM-CSF). NK cell populations were sorted for Ly-49A+ or Ly-49G+ subsets, and these subsets were analyzed for their ability to alter cytokine induction by target cell interaction. In the presence of target cells expressing the appropriate class I molecules, Ly-49A and G2 were found to inhibit cytokine induction by NK cells. Examination of mRNA for IFN-gamma and GM-CSF indicated that Ly-49 receptors increased mRNA levels of NK cells. These results demonstrate that class I binding of these NK receptors can inhibit production of important physiological cytokines, in addition to the regulation of cytotoxic activity.

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.

Fas involvement in human NK cell apoptosis: lack of a requirement for CD16-mediated events.

Propriocidal regulation of T cells refers to apoptosis induced by interleukin-2 (IL-2) activation with subsequent antigen receptor stimulation. We previously reported that natural killer (NK) cells also exhibit propriocidal death. Cell death can be induced following occupancy of the Fc gamma RIII (CD16) receptor when NK cells were pretreated with IL-2, IL-12, or IL-15. Here we show other triggering receptors on NK cells such as CD44, anti-NK-receptor antibodies, and pharmacological activation can result in the cell death signal. Requirement for cell interactions indicated that cell contact was required; however, unlike cell-mediated lysis, extracellular calcium was not required. Like T cells, the process of cell death for NK cells was receptor-induced apoptosis. Activation-induced apoptosis of T cells is mediated by members of the tumor necrosis factor (TNF) cytokine superfamily. We examined the involvement of TNF receptor family members or Fas in this rapid cell death. Antibody directed against Fas, TNFR60, TNFR80, LTBR, and LT alpha failed to inhibit receptor-induced death. Therefore, NK cells appear to demonstrate a rapid apoptotic episode when CD16 is cross-linked, but the mechanism of this apoptosis is quite different than was observed in T cells with CD3. The direct examination of the Fas pathway on activated NK cells revealed that susceptibility required longer treatment times and IL-2 activation. This susceptibility was paralleled by increased Fas-ligand expression. Therefore, NK cells can demonstrate an apoptotic response to CD16, CD44, NK receptors, and Fas. The enumeration of ligands capable of eliciting NK cell death and the in vivo relevance of this observation require further study.

Bone marrow and thymus expression of interferon-gamma results in severe B-cell lineage reduction, T-cell lineage alterations, and hematopoietic progenitor deficiencies.

Interferon-gamma (IFN-gamma) is an immunoregulatory lymphokine that is primarily produced by T cells and natural killer cells. It has effects on T-cell, B-cell, and macrophage differentiation and maturation. We have developed transgenic mice that express elevated levels of IFN-gamma mRNA and protein by inserting multiple copies of murine IFN-gamma genomic DNA containing an Ig lambda-chain enhancer in the first intron. The founder line carrying eight copies of this transgene has eightfold to 15-fold more IFN-gamma-producing cells in the bone marrow and spleen than do nontransgenic littermates. Transgenic mice show a pronounced reduction in B-lineage cells in the bone marrow, spleen, and lymph nodes. In addition, single positive (CD4+,CD8- and CD4-,CD8+) thymocyte numbers are increased twofold, yet the number of splenic T cells is reduced by 50%. There is also a twofold to threefold decrease in the frequency and total number of myeloid progenitors in the bone marrow. Granulomatous lesions and residual degenerating cartilaginous masses are also present in the bones of these mice. Overall, our data show that the abnormal expression of IFN-gamma in these transgenic mice results in multiple alterations in the immune system. These animals provide an important model to examine the role of IFN-gamma expression on lymphoid and myeloid differentiation and function.

The Ly-49D receptor activates murine natural killer cells.

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Cellular and molecular mechanisms of IFN-gamma production induced by IL-2 and IL-12 in a human NK cell line.

Interferon-gamma (IFN-gamma) is an important immunoregulatory protein produced predominantly by T cells and large granular lymphocytes (LGL) in response to different extracellular signals. In particular, two interleukins (ILs), IL-2 and IL-12, have been shown to be potent inducers of IFN-gamma gene expression in both T cells and LGL. Although it has been reported that there are some T cell lines that produce IFN-gamma in response to IL-2 and IL-12 stimulation, there has as yet been no report of a natural killer (NK) cell line that responds in a similar manner. In this report we present evidence that the cell line NK3.3 derived from human NK cells, responds to both IL-2 and IL-12, as measured by increases in IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) cytoplasmic mRNA and protein expression. In addition, when used together IL-2 and IL-12 synergized in the induction of IFN-gamma and GM-CSF and this synergy was attributed to an increased accumulation and stability of the IFN-gamma and GM-CSF mRNAs. To investigate the signaling pathways involved in the gene induction, five inhibitors, cyclosporin A (CsA), transforming growth factor-beta, cycloheximide, genistein, and staurosporine A, were used in analyzing the effects of IL-2 and IL-12 on NK3.3 cells. The results suggest that activation of protein kinase C, but not new protein synthesis, is required for IL-2 induction of IFN-gamma and GM-CSF cytoplasmic mRNA. In contrast, IL-12 induction of IFN-gamma cytoplasmic mRNA appears to only partially depend on activation of protein kinase C. Furthermore, both transforming growth factor-beta and genistein, a tyrosine kinase inhibitor, could suppress IL-2 and IL-12 signaling but CsA was generally inactive. It also was observed that suppression of cytokine gene expression by these agents was independent of the inhibition of proliferation. In addition, IL-2 but not IL-12 induced nuclear factors NF-kappa B and AP1, and regulation of the nuclear levels of these two DNA binding protein complexes is correlated with IFN-gamma and GM-CSF gene expression. These data indicate that IL-2 and IL-12 may have distinct signaling pathways leading to the induction of IFN-gamma and GM-CSF gene expression, and that the NK3.3 cell line may serve as a novel model for dissecting the biochemical and molecular events involved in these pathways.

Characterization of a non-granule associated pore-forming protein in agranular lymphocytes.

Recently, two populations of small lymphocytes (SL) have exhibited non-major histocompatibility complex (MHC) restricted lysis. Recent studies by numerous laboratories have demonstrated that resting T cells triggered through CD3 and CD28 costimulations can result in immediate, non-MHC restricted killing. Our recent studies with CD3-, CD56+ SL demonstrated that although these cells contained no cytoplasmic granules detected with electron microscopy, they mediated potent NK and ADCC activity. In the present study, we have used a Western blotting technique that allows for the detection and quantitation of total cellular levels of pore-forming protein (PFP). We have found that freshly isolated peripheral non-granulated lymphocytes (both CD3+ and CD3-) contain PFP. In addition, CD3-, CD56+ SL contain levels of PFP similar to those of the highly granular CD3- LGL. In search of non-granule PFP, we exploited the rat NK (RNK) cell lines as a source of other potential cytotoxic factors. A membrane associated PFP, based on Western blotting, was isolated from rat RNK cells. Unlike PFP isolated from granules, this PFP was active after culture in Ca(2+)-containing medium. However, the lytic activity isolated from the non-granule PFP of RNK cells was inhibited by monoclonal antibodies to PFP. Collectively, these studies indicate that PFP is present in small agranular lymphocytes (both NK and T cells) and that it is not stored in large cytoplasmic granules. The implication of our results for the acquisition and activation of lytic ability in NK and T cells will be discussed.

CD3+, CD56+ non-MHC restricted cytotoxic T lymphocytes in two fraternal AIDS patients: a case report.

We describe two brothers with hemophilia and AIDS, with an unusually large percentage of CD3+, CD56+ lymphocytes. They experienced no major complications with opportunistic infections (OI) and infrequent secondary infections, even though they had nearly 0% CD4 lymphocytes for 3 years. Both patients died in 1991 of progressive cardiomyopathy. The patients' lymphocytes were immunophenotyped by flow cytometry and analyzed for functional cytolytic activity against K562 and HIV infected HUT 78 cell lines. Single color CD4 counts were 2-9% for 4 years. Dual color CD4 counts at our laboratory demonstrated 0-1% CD4 for the last 6 months. When CD3+ lymphocytes were examined, both patient 1 and patient 2 demonstrated a significantly higher proportion and absolute number of CD3"bright"+, CD56+ double-positive cells, 47% and 22%, respectively, compared to other HIV-positive children with hemophilia (< or = 2%). Functional studies with the K562 target cell line demonstrated the highest natural killer (NK) lymphocyte activity in patient 1 that could not be augmented by in vitro addition of IL-2, whereas patient 2 had no NK activity unless IL-2 was added. Functional studies with HIV-infected HUT-78 cells demonstrated patient 2 had cytolytic activity against HIV-infected cells and patient 1 had high nonspecific cytolytic activity even against uninfected HUT-78 cells, whereas controls had minimal activity to HUT-78 cells or HIV-infected HUT-78 cells. The case report raises a speculative question requiring a larger database, whether the anti-HIV activity and/or unusual clinical course without typical O.I. of some AIDS patients may be related to the presence of CD3"bright"+, CD56+ lymphocytes of their immune system.

The presence in a mouse T cell line of a 97-kDa protein kinase C (PKC) with characteristics similar to known members of the novel PKC subgroup and its possible role in lymphocyte gene expression.

The receptor for tumor-promoting phorbol esters has been shown to be the Ca+2/phospholipid dependent enzyme protein kinase C (PKC). There are two major groups of PKC, the conventional PKC isotypes alpha, beta I, beta II, gamma) and the novel Ca+2-independent PKC (delta, epsilon, zeta, eta). Phorbol esters previously have been demonstrated to increase human IFN-gamma gene expression after treatment of a murine T cell line (Cl 9) that has been transfected with human IFN-gamma genomic DNA. In contrast, treatment with Ca+2 ionophore alone or in combination with phorbol ester did not enhance IFN-gamma production in a synergistic manner above the level obtained with phorbol ester treatment alone. To determine whether the lack of effect of Ca+2 ionophore is due to a defect in PKC, we compared the level of PKC autophosphorylation in the mouse T cell line (Cl 9), a mouse epidermal cell line (JB6), and purified rat brain PKC by in vitro kinase assays. The results demonstrate that instead of the expected 80-kDa autophosphorylated PKC band seen in purified rat brain PKC or mouse JB6 cell lysates, only a novel 97-kDa Ca+2-independent phosphoprotein was observed in Cl 9 cells. To ascertain if there was any nucleic acid sequence similarity to PKC epsilon, we hybridized Cl 9 poly(A+) RNA with a cloned fragment of the PKC epsilon gene and observed two hybridizing RNA bands (4.4 and 4.0 kb). Our results suggest that the 97-kDa phosphoprotein is similar to, but not identical with, PKC epsilon and is the major PKC expressed in the Cl 9 murine T cell line. These data suggested that the 97-kDa PKC may be responsible for the induction of both the transfected human IFN-gamma gene and the endogenous murine IL-2R alpha-chain.

Direct evidence for release of pore-forming protein during NK cellular lysis.

In the present study, we have examined the exocytosis model for cellular lysis. Using monoclonal antibodies reactive with human pore-forming protein (PFP), we examined the localization of PFP at the interaction site of natural killer (NK) cells and the NK tumor targets K562 and Molt-4 as well as during antibody-dependent cellular cytotoxicity. Following the interaction of effector-target cell contact, an increased frequency of PFP was detected on the effector surface, in the micro-environment, and on the target surface of the interaction site. This temporal deposition of PFP was paralleled by loss of target cell integrity and release of chromium. In addition, selective deposition of PFP was seen at the interaction site of the target cell compared to other target cell regions. Collectively, these results are consistent with the exocytosis model and further support the hypothesis that PFP is one of the secreted moieties involved in NK cellular cytotoxicity.

Relationship of large and small CD3- CD56+ lymphocytes mediating NK-associated activities.

We have defined a population of CD3-, CD56+ small lymphocytes (SLs) that exhibit the same phenotype and lytic capacity as natural killer (NK) cells. NK cells characteristically express the surface markers CD16 and CD56, mediate non-major histocompatibility complex (MHC)-restricted lysis, and have been equated with CD3- large granular lymphocytes (LGLs). In the present study we extended the observation that CD3-, CD56+ SLs can mediate NK- and antibody-dependent cellular cytotoxicity activity by studying the activation signals and lytic mechanisms that might be utilized by CD3-, CD56+ SLs in comparison to CD3- CD56+ LGLs. Our results show that CD3- SLs, similar to CD3- LGLs, exhibited activated killing in response to interleukin-2 (IL-2). In addition, after IL-2 activation, the CD3- SLs exhibited morphologic changes, including increases in size and granularity, and both morphologically and phenotypically became virtually indistinguishable from CD3- LGLs. Similar to CD3- LGLs, CD3- SLs could be directly activated by IL-2 alone to secrete significant quantities of interferon-gamma (IFN-gamma) and to express IL-2 receptor (IL-2R) p55. Examination of serine esterases and pore-forming protein (PFP) demonstrated that these cells exhibited a cytoplasmic distribution of perforin, which, unlike that of CD3- LGLs, was not associated with dense cytoplasmic azurophilic granules. Serine esterase levels were similar. However, after IL-2 activation PFP was concentrated in dense cytoplasmic granules, similar or identical to the situation in CD3-, CD56+ LGLs. These CD3-, CD56+ subsets appear to represent a continuum of activated cells that might represent various states of maturation of NK cells.