Kinetics of interaction of HLA-C ligands with natural killer cell inhibitory receptors

OBJECTIVE: To measure effectiveness, adverse event experience, and acceptability of the Food and Drug Administration-approved variant of levonorgestrel capsule implants in the United States through 5 years and to examine determinants of these outcomes. METHODS: In a prospective, multicenter study, 511 sexually active women selecting contraceptive implants were monitored four times in the 1st year, then semiannually through 5 years. Adverse events were elicited by query and physical examination, and their incidence was measured. Lifetable analyses computed pregnancy and other discontinuation rates. Cox regression models examined effects of age, parity, and preadmission desire for more children on continuation. Removal times were analyzed by analysis of variance. RESULTS: Three pregnancies occurred, yielding a 5-year cumulative rate of 1.3+/-0.8 per 100 users, an average annual rate of three per 1000 women, and an ectopic pregnancy rate of 0.6 per 1000 woman years. No pregnancies occurred to women weighing less than 79 kg. Prolonged or irregular menstrual bleeding, followed distantly by headache, weight gain, and mood changes, was the most frequent medical conditions leading to removal. Weight gain averaged 1 kg per year. Each annual continuation rate was above 80 per 100, for a cumulative 5-year rate of 39 per 100. Continuation was age-dependent, with younger women (younger than 25 years at entry) having lower 5-year continuation rates than older subjects (P < .01). Tissue trauma from deeply placed or poorly aligned implants or severe reactions to local anesthetic affected subjects in 3.1% of removals (nine cases). CONCLUSION: As measured by annual continuation rates of 80 per 100 or higher and annual pregnancy rates below one per 100, implant contraception in the United States was found to be highly acceptable and effective, year after year, regardless of the woman's age or family formation status. The cumulative 5-year pregnancy rate, 1.3 per 100, is comparable to that of tubal ligation.     

Function and specificity of human natural killer cell receptors

In humans, natural killer lymphocytes express HLA class I-specific inhibitory receptors belonging to at least two different molecular families. The first is represented by members of the Ig superfamily that are involved in the recognition of different groups of HLA class I alleles, and the second is represented by a molecular complex formed by CD94 and NKG2A that displays a broad specificity for various class I molecules including the 'non-classical' HLA-G molecules. In addition to the inhibitory receptors, a series of activating receptors has been identified. Some display the same specificities as the corresponding inhibiting receptors and can be viewed as HLA class I-specific activating receptors. Another group of activating receptors appear to be involved in the cytolytic activity against HLA-'negative' target cells. These receptors are clearly non-MHC specific and, under physiological conditions, their function is suppressed by the HLA class I-specific inhibitory receptors.     

Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity

Natural killer (NK) cells take up chylomicrons (CM), very low density (VLDL), low density (LDL), high density (HDL) and acetyl-modified low density (AcLDL) lipoproteins through different receptors, VLDL being the lipoprotein with the highest uptake and HDL the lowest. The uptake of LDL can be selectively blocked by the anti-LDL receptor, which does not affect the uptake of CM, VLDL, HDL and AcLDL. Although the uptake of lipoproteins assessed by flow cytometry using DiI is not very high, the lipoproteins are able to induce an increase in proliferative responses, VLDL, AcLDL and HDL being the most important ones with 12- and 17-fold increments, respectively. CM, VLDL and LDL at low concentrations increase NK cytotoxic activity, while HDL and AcLDL inhibit, in a dose-dependent fashion, the killing of NK cells against K562. These results suggest the presence of four different receptors that are responsible for the cytotoxic and proliferative responses observed.     

Control of self-reactive cytotoxic T lymphocytes expressing gamma delta T cell receptors by natural killer inhibitory receptors

The majority of peripheral blood gamma delta T cells in human adults expresses T cell receptors (TCR) with identical V regions (V gamma 9 and V delta 2). These V gamma 9 V delta 2 T cells recognize the major histocompatibility complex (MHC) class I-deficient B cell line Daudi and broadly distributed nonpeptidic antigens present in bacteria and parasites. Here we show that unlike alpha beta or V gamma 9- gamma delta T cells, the majority of V gamma 9V delta 2 T cells harbor natural killer inhibitory receptors (KIR) (mainly CD94/NKG2A heterodimers), which are known to deliver inhibitory signals upon interaction with MHC class I molecules. Within V gamma 9V delta 2 T cells, KIR were mainly expressed by clones exhibiting a strong lytic activity against Daudi cells. In stark contrast, almost all V gamma 9V delta 2 T cell clones devoid of killing activity were KIR-, thus suggesting a coordinate acquisition of KIR and cytotoxic activity within V gamma 9V delta 2 T cells. In functional terms, KIR inhibited lysis of MHC class I-positive tumor B cell lines by V gamma 9V delta 2 cytotoxic T lymphocytes (CTL) and raised their threshold of activation by microbial antigens presented by MHC class I-positive cells. Furthermore, masking KIR or MHC class I molecules revealed a TCR-dependent recognition by V gamma 9V delta 2 CTL of ligands expressed by activated T lymphocytes, including the effector cells themselves. Taken together, these results suggest a general implication of V gamma 9V delta 2 T cells in immune response regulation and a central role of KIR in the control of self-reactive gamma delta CTL.     

T cells expressing killer cell inhibitory receptors do not carry TCR for alloantigens

The role of T cells carrying the killer cell inhibitory receptor (KIR) is unknown. Here we investigate the allo-response of KIR+ CD3+ cells (KIR+ T cells). KIR+ T cells was found in 4.6-14.7% of the T cells from the PBMC of healthy individuals. Flow cytometry analysis showed that the percentage of KIR+ T cells in T cells from three individuals was decreased after stimulation with allogeneic PBMC. These findings suggest that KIR+ T cells do not respond to alloantigens. Furthermore, the analyses using purified KIR+ T cells also confirmed that KIR+ T cells do not respond to alloantigens.     

Natural killer activating receptors trigger interferon gamma secretion from T cells and natural killer cells

Proliferation of human CD4+ alphabeta T cells expressing a natural killer cell activating receptor (NKAR) has been shown to be enhanced, particularly in response to low doses of antigen, if the target cells present appropriate human class I major histocompatibility complex (MHC) molecules. Here, we show that NKAR also enhance proliferation and killing of target cells by subsets of CD8+ alphabeta and CD8+ gammadelta T cells, as well as by NK cells. Strikingly, interferon gamma secretion from all of these types of lymphocytes was markedly increased by interaction of the NKAR with their MHC class I ligands, independently of enhancement of proliferation. Thus, the recognition of class I MHC molecules by NKAR on both T cells and NK cells may provide a regulatory mechanism that affects immune responses through the secretion of interferon gamma and possibly other cytokines. It represents a signal for cytokine secretion alternative and/or augmentative to that through the T cell receptor.     

Genomic organization of a human killer cell inhibitory receptor gene

We have cloned a region of human chromosome 19q13.4 which contains multiple killer cell inhibitory receptor (KIR) loci. By random and directed sequence analysis of these KIR-specific clones, we deduced the genomic structure of KIR genes. A locus encoding a member of the NKAT-2 family of KIRs is presented here. The structure of the gene is reminiscent of loci of the Fc receptor gene family, and the two sets of genes may derive from a common ancestor. The KIR gene contains potentially nine exons. The first two exons encode the leader sequence, as in Fc receptor genes. The third exon encodes an untranslated pseudo exon specifying an immunoglobulin domain with an in-frame stop codon. Expressed cDNAs do not contain this exon. This finding is consistent with the hypothesis that certain KIR genes may have been derived from the duplication of a primordial three immunoglobulin domain structure with subsequent skipping of one exon to derive genes with two expressed immunoglobulin domains. Variation in numbers of immunoglobulin domains in different KIR genes is facilitated by conservation of splicing frame in respect to the codon triplet for each immunoglobulin domain.     

The human natural killer lymphocyte receptors for MHC class I molecules

OBJECTIVES: Angiomyolipoma (AML) is a benign renal tumor that may require treatment because of associated local complications. The present study was undertaken to evaluate the efficacy of nephron-sparing surgery (NSS) in the management of renal AML, with respect to the long-term preservation of renal function and absence of tumor recurrence. METHODS: From 1980 to 1997, 27 patients underwent NSS for treatment of renal AML. The clinical presentation, surgical approach, and outcome in these patients were analyzed. Surgical treatment for renal AML was indicated because of associated symptoms, size of 4.5 cm or greater, and/or suspicion of renal malignancy. RESULTS: In symptomatic patients (52%) the most common presenting signs or symptoms were pain (50%), retroperitoneal hemorrhage or shock (43%), hematuria (36%), hypertension (7%), palpable mass (7%), and anemia (7%). Two patients had tuberous sclerosis. Fifteen patients had a solitary functioning kidney (group I), 6 patients had an impaired contralateral kidney (group II), and 6 patients had a normal contralateral kidney (group III). All operations were performed in situ. There were no operative deaths. All operated kidneys functioned postoperatively, and no patient required dialysis. The mean postoperative serum creatinine level in groups I, II, and III was 1.81, 0.98, and 0.97 mg/dL, respectively. No patients have developed recurrent AML, related symptoms, or required dialysis with follow-up to 177 months (median 39). CONCLUSIONS: When surgical treatment for renal AML is indicated, NSS can be performed with a high success rate even in patients with a very large tumor involving a solitary kidney.     

Migratory response of human natural killer cells to lymphotactin

Lymphotactin (Lptn) is a new protein belonging to the C or gamma subfamily of chemokines with only two of the four cysteine residues. Lptn was reported to act specifically on T lymphocytes and not on monocytes and neutrophils. To understand better the spectrum of action of Lptn we have examined its ability to induce natural killer (NK) cell migration. Freshly isolated human NK cells as well as long-term cultured NK cells propagated in interleukin-2 (IL-2)-containing medium migrated in response to Lptn. Optimal activity was observed at concentrations ranging from 50 to 200 ng/ml, and the efficacy was comparable to that of MCP-1, the prototype of C-C chemokines. Migration in response to Lptn was chemotaxis rather than chemokinesis as determined in a checkerboard analysis. Migration of NK cells was comparable to that observed with T lymphocytes from the same donor, under the same experimental conditions. Finally, in contrast to other cytokines (IL-2 and IL-12) which in addition to chemotaxis augment NK cell adhesion to endothelial cells in vitro, Lptn did not affect the adhesiveness of NK cells to vascular endothelium.     

Human natural killer cells

Human natural killer (NK) cells comprise 10 to 15% of peripheral blood lymphocytes, characterized by their morphologic appearance of large granular lymphocytes (LGLs) and phenotype CD3- CD56+ CD16+ or CD16-. Functionally, these cells are defined by their ability to lyse target cells without prior sensitization and without restriction by major histocompatibility (MHC) antigens. These cells play an important role in immune defenses, especially after hematopoietic transplantation. They contribute to the defenses against virus-infected cells, graft rejection, and neoplasias; they also participate in the regulation of hematopoiesis through cytokine production and cell to cell interaction. In this mini-review we attempt to summarize the most relevant findings about these cells in terms of their origin and differentiation, their cell surface characteristics including activation and their cytolytic pathways. We have also provided a brief approach of their potential clinical use. Increased knowledge of NK cell differentiation, ontogeny and regulatory mechanisms may be of use for the planning of immunotherapeutic strategies.     

Effect of a Staphylococcus epidermidis-extracted slime factor on human natural killer cell activity

A slime factor produced by Staphylococcus epidermidis was a complex glycoconjugate extracted by the phenol extraction method. The potential stimulatory or inhibitory capacity of the phenol-extracted slime (PES) was tested on human natural killer cell cytotoxic activity. Various concentrations of the PES preparation were incubated with the effector cells 30 min before and during the assay period. The PES factor inhibited natural killer cell cytotoxic activity at a concentration of 250 micrograms/ml and at higher concentrations (p < 0.05). The inhibition of natural killer cell cytotoxic activity may probably be related to the complex composition of the slime substance.     

SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors

Activation of immune system cells via antigen-, Fc-, or natural killer cell-triggering-receptor stimulation is aborted by co-engagement of inhibitory receptors. Negative signaling by killer cell inhibitory receptors and related receptors depends on the Src homology 2 (SH2)-containing protein tyrosine phosphatase SHP-1. Using a combination of direct binding and functional assays, we demonstrated that the SH2 domain-containing leukocyte protein 76 (SLP-76) is a specific target for dephosphorylation by SHP-1 in T cells and natural killer cells. Furthermore, we showed that tyrosine-phosphorylated SLP-76 is required for optimal activation of cytotoxic lymphocytes, suggesting that the targeted dephosphorylation of SLP-76 by SHP-1 is an important mechanism for the negative regulation of immune cell activation by inhibitory receptors.     

Differential expression of natural killer cell markers: human versus baboon

The use of baboons as a model for the study of allo- and xenotransplantation has become increasingly important, but there are few studies on the basic immunological responses in baboons that might be relevant for a rejection reaction. In present study, the cell-surface phenotype, cytokine-induced activation and growth, and cytotoxicity of baboon and human natural killer (NK) and lymphokine-activated killer (LAK) cells were compared. A panel of murine monoclonal antibodies specific for human cell-surface markers expressed on lymphocytes was used to compare relevant baboon and human peripheral blood lymphocytes (PBL). Baboon PBL were 52.1+/-2.9% CD8+, 18.5+/-2.2% CD16+, 3.0+/-0.5% CD25+, and 5.5+/-1.8% CD69+. The corresponding proportions in humans were 23.8+/-7.1%, 12.8+/-3.2%, 4.5+/-1.0%, and 2.3+/-1.1%. In contrast to human PBL, less than 1% of baboon lymphocytes expressed CD56, CD57, and CD122 (interleukin [IL]-2Rbeta). Baboon lymphocytes showed NK cytotoxic activity against the human K562 and CEM cell lines, which was comparable to human NK activity. Depletion of baboon CD16+ or CD8+ cells led to dramatic decreases in NK cytotoxicity, and removal of both subsets completely abrogated NK activity. Incubation of baboon lymphocytes with human recombinant IL-2 for 1 week led to the appearance of CD56+ cells (11.3+/-2.8%). Most of the baboon CD56+ cells induced in culture were in S and G2 phases of cell cycle. Both baboon and human IL-2-activated lymphocytes were highly cytotoxic against the human LAK-sensitive cell line Daudi. Depletion of baboon CD8+ but not CD56+ cells significantly decreased LAK activity. These studies revealed differences in the NK system of humans and baboons that should be taken into consideration when analyzing immune responses to allo- and xenotransplantation in baboons.     

Antileukemia activity of a natural killer cell line against human leukemias

We describe here the in vitro and in vivo antileukemia activity of a recently described natural killer (NK) cell line (NK-92), which has features of human activated NK cells. The cytotoxic activity of rhIL2-dependent cultured NK-92 cells against primary patient-derived leukemic target cells [12 acute myelogenous leukemias (AMLs), 7 T acute lymphoblastic leukemias (T-ALLs), 14 B-lineage-ALLs, and 13 chronic myelogenous leukemias (CMLs)], human leukemic cell lines (K562, KG1, HL60, Raji, NALM6, TALL-104, CEM-S, and CEM-T) and normal bone marrow cells was measured in 51Cr-release assay (CRA). The patient-derived leukemias could be subdivided into three groups based on their sensitivity to NK-92 cells: insensitive (< or =19% lysis), sensitive (20-49% lysis), and highly sensitive (> or =50% lysis) at an E:T ratio of 9:1. Of 46 patient-derived samples, 24 (52.2%) were sensitive or highly sensitive to NK-92-mediated in vitro cytotoxicity (6 of 12 AMLs, 7 of 7 T-ALLs, 5 of 14 B-lineage-ALLs, and 6 of 13 CMLs). NK-92 cells were highly cytotoxic against all of the eight leukemic cell lines tested in a standard 4-h CRA. Normal human bone marrow hematopoietic cells derived from 18 normal donors were insensitive to NK-92-mediated cytolysis. In comparison with human lymphokine-activated killer cells, normal NK cells, and T cells, NK-92 cells displayed more powerful antileukemia activity against a patient-derived T-ALL as well as K562 and HL60 cells, both in in vitro CRA and in a xenografted human leukemia SCID mouse model. The NK-92 cells did not induce the development of leukemia in SCID mice after i.v., i.p., or s.c. inoculation. In adoptive transfer experiments, SCID mice receiving i.p. inoculations of human leukemias derived from a T-ALL (TA27) and an AML (MA26) that were highly sensitive to the cytolysis of NK-92 cells in vitro, as well as a pre-B-ALL (BA31) that was insensitive to the in vitro cytolysis of NK-92 cells, were treated by administration of NK-92 cells with or without rhIL2 (2 x 10(7) NK-92 cells i.p.; one dose or five doses). Survival times of SCID mice bearing the sensitive TA27 and MA26 leukemias were significantly prolonged by adoptive cell therapy with NK-92 cells. Some of the animals who received five doses of NK-92 cells with or without rhIL2 administration were still alive without any signs of leukemia development 6 months after leukemia inoculation. In contrast, survival of mice bearing the insensitive BA31 leukemia were not affected by this treatment. This in vitro and in vivo antileukemia effect of NK-92 cells suggests that cytotoxic NK cells of this type may have potential as effectors of leukemia control.     

Nitric oxide synthase pathway may mediate human natural killer cell cytotoxicity

The present study provides evidence that the human natural killer (NK) cell effector mechanism causing target cytolysis has a requirement for L-arginine. In a deficient medium (DM) containing only salts, buffer system and glucose, NK cell-mediated cytotoxicity was found to decrease by 70% as compared to that obtained in a complete medium (CM). However, adding L-arginine to such DM could restore the activity of NK cells to the normal level. Many other components of CM, such as serum, glutamine and vitamins did not improve NK cell-mediated killing in DM. When all amino acids except L-arginine were added to DM only a partial recovery of NK cell functional cytolysis was seen. L-arginine enhanced the NK cell activity in a dose-dependent manner. Additionally, the inhibitor of both inducible and constitutive nitric oxide synthase, N-monomethyl-L-arginine (L-NMMA) inhibited NK cytolytic activity in DM supplemented with L-arginine indicating participation of nitric oxide (NO). The results also show that the stimulatory effect of L-arginine on human NK cell-mediated cytotoxicity was accompanied by an increase in NO formation as determined by accumulation of nitrite and citrulline. L-NMMA gave a dose-dependent reduction in NO generation as well. The nitrite and citrulline production dose-dependently correlated with not only the concentration of L-arginine in the cultivation medium, but also the enhanced NK cell-mediated cytolysis. Taken together, these findings could define a L-arginine/NO-linked effector mechanism in human NK cells. Nitrite and citrulline were not formed when NK cell-mediated target cell killing took place in a L-arginine-free DM supplemented with additives. Thus, it appears as if human NK cells may cause target cell killing via both NO-dependent and -independent processes.     

Stromal progesterone receptors mediate the inhibitory effects of progesterone on estrogen-induced uterine epithelial cell deoxyribonucleic acid synthesis

The role of epithelial and stromal progesterone (P) receptors (PR) in the regulation of uterine epithelial DNA synthesis by P was investigated by analyzing the four types of tissue recombinants prepared with uterine stroma (S) and epithelium (E) from wild-type (wt) and PR knockout (PRKO) mice: wt-S + wt-E, PRKO-S + PRKO-E, wt-S + PRKO-E, and PRKO-S + wt-E. 17-Beta estradiol (E2) stimulated DNA synthesis in all four types of tissue recombinants. On the other hand, P inhibited E2-induced DNA synthesis only in tissue recombinants prepared with wild-type (PR-positive) stroma (wt-S + wt-E or wt-S + PRKO-E) but not knockout (PR-negative) stroma (PRKO-S + wt-E or PRKO-S + PRKO-E). These results clearly demonstrate that the inhibitory effect of P on uterine epithelial DNA synthesis is mediated by stromal PR. Epithelial PR is neither necessary nor sufficient for P inhibition of E2-induced epithelial DNA synthesis.     

Hematopoietic cells and radioresistant host elements influence natural killer cell differentiation

Radioresistant host elements mediate positive selection of developing thymocytes, whereas bone marrow-derived cells induce clonal deletion of T cells with receptors that are strongly autoreactive. In contrast to T cell development, little is known about the elements governing the natural killer (NK) cell repertoire, which, similar to the T cell repertoire, differs between individuals bearing different major histocompatibility complex (MHC) phenotypes. We have used murine bone marrow transplantation models to analyze the influence of donor and host MHC on an NK cell subset. We examined the expression of Ly-49, which is strongly expressed on a subpopulation of NK cells of H-2b mice, but not by NK cells of H-2a mice, probably because of a negative effect induced by the interaction of Ly-49 with Dd. To evaluate the effect of hematopoietic cell H-2a expression on Ly-49 expression of H-2b NK cells, we prepared mixed allogeneic chimeras by administering T cell-depleted allogeneic (B10.A, H-2a) and host-type (B10, H-2b) marrow to lethally irradiated B10 mice, or by administering B10. A marrow to B10 recipients conditioned by a nonmyeloablative regimen. Expression of H-2a on bone marrow-derived cells was sufficient to downregulate Ly-49 expression on both H-2a and H-2b NK cells. This downregulation was thymus independent. To examine the effect of H-2a expressed only on radioresistant host elements, we prepared fully allogeneic chimeras by administering B10 bone marrow to lethally irradiated B10.A recipients. B10 NK cells of these fully allogeneic chimeras also showed downregulation of Ly-49 expression. The lower level of H-2a expressed on H-2b x H-2a F1 cells induced more marked downregulation of Ly-49 expression on B10 NK cells when presented on donor marrow in mixed chimeras than when expressed only on radioresistant host cells. Our studies show that differentiation of NK cells is determined by interactions with MHC molecules expressed on bone marrow-derived cells and, to a lesser extent, by MHC antigens expressed on radioresistant host elements.     

A single amino acid in the p58 killer cell inhibitory receptor controls the ability of natural killer cells to discriminate between the two groups of HLA-C allotypes

To examine the structural basis for the specific recognition of the MHC class I allotypes HLA-Cw*0401 and HLA-Cw*0304 by the killer cell inhibitory receptors (KIR) cl42 and cl43, respectively, mutant KIR-Ig fusion proteins were tested by direct binding to cells transfected with single HLA-C alleles. The putative loop region at position 44-46 of KIR contained amino acids that were necessary for the discrimination between HLA-Cw*0401 and HLA-Cw*0304. Surprisingly, exchanging the methionine at position 44 in cl42 with the lysine at position 44 in cl43 was sufficient to switch the specificity of cl42 from HLA-Cw*0401 to HLA-Cw*0304, and vice versa. Thus, a single amino acid in the first Ig domain of these KIR determines their ability to discriminate between the two groups of HLA-C allotypes.     

Effect of eccentric exercise on natural killer cell activity

The effect of eccentric one-legged exercise on natural killer (NK) cell activity was studied in eight healthy males. To distinguish between local and systemic effects, blood samples were collected from veins in the exercising leg and resting arm. However, the results did not significantly differ between the leg and arm. To eliminate diurnal variations, the results were compared with a control group that did not exercise but had blood samples collected at the same time points. In the exercising group, plasma creatine kinase increased progressively during and up to 4 days after exercise. The percentage of CD16+ NK cells increased during exercise, which was paralleled by an increase in the NK cell activity per fixed number of blood mononuclear cells. The NK cell activity on a per NK cell basis did not change. The percentage of CD3+, CD4+, CD8+, CD19+, and CD14+ cells did not change significantly during exercise. The present study thus showed that eccentric exercise with a relatively small muscle mass (1 quadriceps femoris muscle) causes systemic effects on NK cells. It is suggested that the increase in plasma epinephrine during eccentric exercise is responsible for the observed increase in the percentage of CD16+ cells.     

Killer cell inhibitory receptor recognition of human leukocyte antigen (HLA) class I blocks formation of a pp36/PLC-gamma signaling complex in human natural killer (NK) cells

The killer cell inhibitory receptors (KIR) of human natural killer (NK) cells recognize human leukocyte antigen class I molecules and inhibit NK cell cytotoxicity through their interaction with protein tyrosine phosphatases (PTP). Here, we report that KIR recognition of class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adaptor protein (pp36) with phospholipase C-gamma in NK cells. In addition, we demonstrate that pp36 can serve as a substrate in vitro for the KIR-associated PTP, PTP-1C (also called SHP-1), and that recognition of class I partially disrupts tyrosine phosphorylation of NK cell proteins, providing evidence for KIR-induced phosphatase activity.