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While it is generally accepted that natural killer (NK) cells, by killing tumor cells in the circulation, represent a first line of defense against metastases, their therapeutic activity against established tumors has been limited. In this review, we describe studies to improve the therapeutic effectiveness of activated NK cells in both animal models and clinical trials to better understand the biological problems that limit their effectiveness.

Development of post-transplant lymphoproliferative disease (PTLD) is a major complication of organ transplantation. While immune mechanisms seem to play a major role in the development of PTLD, how the immune system contributes to the process of PTLD development or its regression remains unknown. Between 1990-1994, 303 organ transplant recipients were enrolled into a prospective study designed to analyze risk factors for PTLD. Using a nested case-control design, 9 PTLD and 18 control patients were matched for age, EBV serological status at the time of transplantation, and, in most cases, for the type of transplanted organ. The immunologic profiles of both groups were compared prior to and following transplantation. Immune measures included absolute numbers of lymphocytes and of subsets of T, B and natural-killer (NK) cells as well as spontaneous NK-cell and in vitro generated LAK-cell activities. A consistent trend for higher levels at baseline as well as following transplantation for almost all immune parameters was observed in patients who developed PTLD. A high absolute count of activated NK cells (CD56+ DR+) at baseline was found to be a significant predictor of PTLD development. The immunologic profile of patients who developed PTLD was consistent with pre- as well as post-transplant chronic immunologic stimulation, and not immunosuppression. In the PTLD group, 3 patients had pre-transplant autoimmune hepatitis and one had primary biliary cirrhosis, which suggests that the underlying presence of certain autoimmune disorders in organ transplant recipients might predispose to PTLD development.

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-alpha, LT-beta, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-alpha1beta2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-alpha1beta2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.

We recently reported that human NK cells express, in addition to CD16 [Fcgamma receptor (FcgammaR) IIIA], a second type of FcgammaR, namely CD32 (FcgammaRII). Molecular characterization of CD32 transcripts expressed by highly purified NK cells revealed that they predominantly express products of the FcgammaRIIC gene. Using stable Jurkat transfectants we have analyzed the functional properties of two FcgammaRIIc-specific isoforms isolated from NK cells, namely FcgammaRIIc1 and FcgammaRIIc3, which differ in their cytoplasmic tails. The ligand binding specificity for both murine and human IgG isotypes was found to be similar to that observed for FcgammaRIIb isoforms. Immunoprecipitation studies of FcgammaRIIc isoforms expressed in Jurkat cells revealed a protein of around 40 kDa for FcgammaRIIc1, and a protein of around 32 kDa for FcgammaRIIc3. Signal transduction studies performed on FcgammaRIIc1-expressing Jurkat cells indicated that this molecule is functional, i. e. capable of Ca2+ mobilization and activation of Lck, Zap-70 and Syk protein tyrosine kinases, although the CD3 zeta chain was not found to functionally associate with FcgammaRIIc1. In contrast, FcgammaRIIc3 transfectants showed an impaired ability of this molecule to mobilize Ca2+, but activation of Lck was detected following activation via FcgammaRIIc3. These studies demonstrate the functional activity of FcgammaRIIc isoforms and suggest that the presence of CD32, in addition to CD16, on NK cells may have functional relevance.

Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3-4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1+ with high NK activity and 5%-10% were NK1.1- B220+. With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1- B220+, not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660+/-292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1- and Sca-1+ c-kit+ lineage- subpopulations at a frequency of 1/(156+/-52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.

Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.

Copyright 2000 S. Karger AG, Basel

A family was identified with 5 of 6 siblings and 3 other immediate family members who had developed chronic fatigue syndrome (CFS) as adults. All 8 met criteria for the CFS case definition as recommended by the Centers for Disease Control and Prevention. Sixty-eight blood samples were obtained over a period of 2 years from 20 family members (8 affected, 12 unaffected) and 8 normal controls. All blood samples were tested for NK activity in 4-h 51Cr-release assays and for the number of circulating CD3-CD56(+) and CD3-CD16(+) by flow cytometry. NK activity of the affected immediate family members (cases, n = 8) was significantly lower (P = 0.006, two-sided) than that of the concurrently tested normal controls. The results for unaffected family members were intermediate between these two groups, and the pairwise comparison of unaffected family members to either cases or controls showed no statistically significant difference (P = 0.29, two-sided). No differences were seen between the groups in the absolute number of CD3-CD56(+) or CD3-CD16(+) lymphocytes in the peripheral blood. Familial CFS was associated with persistently low NK activity, which was documented in 6/8 cases and in 4/12 unaffected family members. In the family with 5 of 6 siblings who had documented CFS, 2 of their offspring had pediatric malignancies. Low NK activity in this family may be a result of a genetically determined immunologic abnormality predisposing to CFS and cancer.

IFNs were first described as potent antiviral agents 40 years ago, and recombinant IFN-alpha2a and IFN-alpha2b were approved for the treatment of hairy cell leukemia just 11 years ago. Today, alpha-IFNs are approved worldwide for the treatment of a variety of malignancies and virologic diseases. Although the exact mechanism of action of IFN-alpha in the treatment of such diseases is not fully understood, many advances have been made in the characterization of the physicochemical and diverse biological properties of this highly pleiotropic cytokine. Here we review recent developments in our understanding of the antiviral and immunoregulatory properties of IFN-alpha, the nature of the multisubunit IFN-alpha receptor, and the molecular mechanisms of signal transduction. Where available, we have included comparative data on recombinant alpha-IFNs derived from both naturally occurring and nonnaturally occurring synthetic genes. We also review clinical data and data on the side effects and antigenicity of different sources of recombinant alpha-IFNs in humans. These latter topics are of clinical interest, because they may potentially affect the efficacy of these various products. Hopefully, what is already known about IFN will prompt further exploration into the mechanism(s) of action of IFN-alpha and thus deliver new applications for this prototypic cytokine, whose full therapeutic potential is yet to be realized.

Recombinant alfa interferons (IFN-alpha s) are approved worldwide for the treatment of a variety of cancers and diseases of virologic origin. A series of recent advances in the molecular characterization of recombinant IFN-alpha s have allowed the determination of the three-dimensional IFN-alpha 2b structure by high-resolution x-ray crystallography. We review here recent developments in our understanding of the molecular and physicochemical properties of recombinant IFN-alpha, including our current state of knowledge of the IFN-alpha gene family and the multiple species of human leukocyte IFN. Based on the reported three-dimensional structure of IFN-alpha 2b, we propose a molecular model for the IFN-alpha 2b receptor complex and predict models for the naturally occurring subtypes IFN-alpha 1 and IFN-alpha 8, as well as the synthetic, non-naturally occurring consensus IFN. Such models provide molecular insights into the mechanism of action of IFN-alpha.

A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2-dependent NK-92 and IL-2-independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 and neor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/10(6) cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.

While CD4+ T-cell counts in the blood of HIV-infected individuals gradually decrease, there is a parallel increase in the number of blood CD8+ T cells such that the total number of T cells remains essentially constant for several years (1). The basis and significance of this phenomenon are not known. Based on a statistical analysis of longitudinal T-cell counts from the Transfusion Safety Study (TSS) database and on theoretical considerations, we evaluate several alternative models, including versions of the "blind homeostasis" (BH) hypothesis (1-3). At issue is the nature of the homeostatic regulation of lymphocytes and its apparent failure in HIV infection. The most plausible explanation for the conservation of total blood T-cell numbers while subset ratios change is that CD4+ and CD8+ T cells compete for a limited access to the blood compartment. Such interaction between the subsets implies, in particular, that changes in the number of CD4+ T cells occurring in other tissues cannot be reliably inferred from those observed in the blood. We reiterate propositions made earlier (4) that much of the apparent "depletion" of CD4+ lymphocytes during the asymptomatic phase of HIV infection may be attributed to redistribution between the tissues and the blood compartment.

Human natural killer (NK) cells were thought to express only FcgammaRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcgammaR, ie, FcgammaRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcgammaRII from NK cells derived from several normal individuals that may represent four different products of the FcgammaRIIC gene. One transcript (IIc1) is identical with the already described FcgammaRIIc form. The other three (IIc2-IIc4) appear to represent unique, alternatively spliced products of the same gene, and include a possible soluble form. Analyses of the full-length clones have revealed an allelic polymorphism in the first extracellular exon, resulting in either a functional open reading frame isoform or a null allele. Stable transfection experiments enabled us to determine a unique binding pattern of anti-CD32 monoclonal antibodies to FcgammaRIIc. Further analyses of NK-cell preparations revealed heterogeneity in CD32 expression, ranging from donors lacking CD32 expression to donors expressing high levels of CD32 that were capable of triggering cytotoxicity. Differences in expression were correlated with the presence or absence of null alleles. These data show that certain individuals express high levels of functional FcgammaRIIc isoforms on their NK cells.

Evidence has been reviewed which indicates that NK cells play a role in the control of metastasis dissemination. Both activation of endogenous NK cells in a tumor-bearing host and adoptive transfer of ex vivo activated NK cells may be therapeutically beneficial. The small number of phase I/II clinical trials of AIT with A-NK cells performed in patients with cancer so far does not allow firm conclusions, except to ascertain the feasibility and a lack of toxicity of this form of therapy. Although numerous trials have been performed with BRMs, many of which are known to upregulate NK activity in vivo, a general lack of correlations between clinical responses or survival and upregulated NK activity in the peripheral blood has dampened enthusiasm for biological therapies. However, these clinical trials have been confined largely to patients with advanced metastatic disease. It is highly likely that tumor-induced immunosuppression plays a crucial role in neutralizing the benefits of BRM therapy, and that levels of effector cell activation sufficient for metastasis elimination are seldom achieved in this clinical setting. On the other hand, administration of BRMs in the adjuvant setting could be more effective and when combined with monitoring for effector cell functions might perhaps provide a better guide for achieving the levels of endogenous NK activity necessary for elimination of remaining or occult metastases. An improved understanding of NK cell biology in cancer patients is likely to serve as a positive reinforcement for design of a new generation of clinical trials incorporating novel approaches to NK cell mediated cancer therapy.

Purpose: To compare the toxicity, pharmacokinetics, and efficacy seen in ovarian cancer patients treated with escalating doses of intraperitoneal (I.P.) interleukin-2 (IL-2) by two different infusion schedules., Patients and Methods: Forty-five patients were sequentially entered onto a phase I/II study in groups of four at fixed dosage tiers of 6 x 10(4), 6 x 10(5), 6 x 10(6), and 3 x 10(7) IU/m2/d in either of two schedules: (A) intermittent weekly infusions of 24 hours' duration; or (B) alternating continuous 7-day infusions followed by 7-day intervals without therapy. Eligibility criteria included > or = six courses of prior platinum-based chemotherapy and laparotomy-confirmed persistent or recurrent ovarian cancer., Results: Forty-one eligible patients received I.P. IL-2 and were assessable for toxicity, but six patients were not assessable for response, which left 35 patients assessable for response. Significant locoregional dose-limiting toxicity was seen with the 7-day infusions (including bowel perforation), with 600,000 IU/m2 as the maximum-tolerated dose (MTD), but catheter infection was the only significant complication seen with the 24-hour infusions, for which an MTD was not established. Among 35 assessable patients, there were six laparotomy-confirmed complete responses (CRs) and three partial responses, for an overall response rate of 25.7% (nine of 35). The median survival time of the cohort was 13.7 months and the overall 5-year survival probability was 13.9%. For the nine patients who demonstrated responses (six on the 24-hour infusion and three on the 7-day infusion), the median survival time has not been reached (range, 27 to 90+ months)., Conclusion: I.P. IL-2 is better tolerated as a weekly infusion as compared with a 7-day infusion and demonstrates evidence of possible long-term efficacy in a modest number of patients. A randomized trial is indicated to determine if the prolonged survival seen in this study is a due to I.P. IL-2 therapy or other factors that cannot be controlled for in a single-arm study.

alpha-Interferons (IFN-alphas) have been shown to have pleiotropic effects on most components of the immune system. The most studied effect of IFN-alphas on the immune system is their ability to augment natural killer cell activity, an effect that is mediated by a complex variety of mechanisms. Both naturally occurring and recombinant IFN-alphas have shown differences of as much as 100-fold in their ability to activate natural killer activity. Although consensus IFN, a synthetic, nonnaturally occurring hybrid IFN, has been reported to have a high natural killer stimulatory capacity relative to its antiviral activity, the available data and experimental design do not permit a definitive conclusion. In summary, it is clear that the complex immunomodulatory effects of IFN-alphas are likely to contribute to their therapeutic activity.

The microbial immunostimulant OK-432 has been studied intensively in preclinical systems and has shown promise as an anticancer agent in trials that have been conducted over the past 20 years in Japan. To date, no systematic dose response evaluation of this agent has defined its dose-limiting toxicity or immunobiological activity. A phase IA study has been conducted in 25 patients with metastatic cancer at the University of Pittsburgh Cancer Institute Melanoma Center, establishing 30 KE as the maximal tolerable dosage, on the basis of cutaneous reactions. Subsequently, 48 patients with resected high-risk melanoma participated in a phase IB study of OK-432. This study has evaluated the immunomodulatory activity of OK-432 at five dosages ranging from 1 KE to 20 KE, administered ID twice weekly for 3 months. A formal analysis of the treated population in comparison to the randomized control group has been conducted, and profound immunological effects have been defined in the group of patients treated with OK-432. Patients who participated in this trial had a significant depression of OK-432-inducible cytokine production (interleukin-1 beta, interferon gamma, and tumor necrosis factor alpha) at baseline. Treatment with OK-432 reversed this deficit for interferon gamma (IFN gamma) production in a dose-dependent manner, and mitigated the inhibition for interleukin-1 (IL-1) across all dosage groups. The impact of OK-432 upon other immunological functions of the treated cohorts is more variable, with durable suppression of mononuclear cell superoxide production, and in vitro cytotoxicity to tumor. Immunological characteristics of the entire cohort demonstrate a strong and significant correlation of elevated blood CD16+ cell counts and natural killer activity with early tumor progression and death due to melanoma. Favorable prognosis is associated with monocyte capacity to produce tumor necrosis factor (TNF), and polymorphonuclear leukocyte formylmethionyl-leucylphenylalanine-inducible superoxide release. This study reveals several new immunological correlates of tumor progression and lethal outcome in resected high-risk melanoma. It demonstrates that the depressed IL-1, TNF, and IFN gamma release associated with melanoma may be mitigated by treatment with OK-432. This study has defined treatment and dose response patterns of immunomodulation associated with one of the most complex immunological agents yet evaluated in phase IB trials, in a well-defined population of high-risk patients with resected melanoma.

The effects of methionine enkephalin (met-enkephalin) on human immune function are reviewed. This pentapeptide functions to upregulate, or enhance, immune function in the majority of donor samples at low doses and suppresses at high doses. The influence of this molecule is shared by the central nervous, neuroendocrine, and immune systems. Cells from each of these systems possess receptors for met-enkephalin and have the ability to process met-enkephalin from its prohormone, proenkephalin A. Studies have shown that this molecule is capable of enhancing immune function in patients with cancer or AIDS. It is proposed that this molecule be classified as a cytokine.

The objective of this study was to study the antitumor, host toxicity, and immunomodulatory effects of recombinant interferon-alpha 2b (IFN) in patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). Seventy-one patients with recurrent or metastatic SCCHN were entered into a phase II noncomparative randomized trial of IFN at two dosage schedules. Eligible patients with histologically proven SCCHN were randomized to receive low-dose IFN, 6 x 10(6) U/m2 daily x 3 every 4 weeks or high-dose IFN, 12 x 10(6) U/m2, 3 x/week. Pretreatment levels of natural killer (NK) activity, CD3, CD4, CD5, CD8, CD16, CD19, CD56, DR, and the CD4/CD8 ratio were evaluated for any relationship with survival. The toxicity encountered in patients receiving low-dose IFN was for the most part mild to moderate. With high-dose IFN, toxicity was greater with significantly more episodes of grade 3 and 4 toxicity encountered. Dosage reduction was required in the majority of patients receiving high-dose IFN. Of the four lethal complications, only one was thought to be possibly associated with therapy. Of the 32 evaluable patients receiving low-dose IFN, there were 1 complete response, 1 stable disease, 24 patients with progressive disease, and 6 unevaluable. Of the 29 evaluable patients taking high-dose IFN, there were 2 complete responses, 7 with stable disease, 16 with progressive disease, and 4 patients were unevaluable. Median survival in the two arms was similar (6.2 months). Because it was postulated that a more prolonged exposure to IFN might be needed for it to be effective, patients receiving > or = 6 weeks of therapy were evaluated. Median survival in that subset was 10 and 12 months for patients receiving low- and high-dose IFN, respectively. None of the immune parameters tested was a significant predictor of survival when evaluated in all cases entered into study regardless of therapy duration. No difference in baseline NK activity was noted between patients who received < 6 or > or = 6 weeks of IFN (p = 0.90). However, among the 35 patients who received > or = 6 weeks of therapy, a high baseline NK activity was a significant predictor of the duration of survival (p = 0.04). IFN was well tolerated in patients with recurrent or metastatic SCCHN. The higher incidence of toxicity encountered in the high-dose arm could be ameliorated by reducing the dose 50%. In patients receiving 6 or more weeks of therapy, elevated baseline NK activity was associated with increases in survival, suggesting that IFN may play an immunomodulatory role. Although the overall response rates were low, disease stabilization was noted, suggesting an antiproliferative, noncytotoxic role of IFN in this group of heavily pretreated patients.

Adhesive interactions mediated by cell surface receptors have been shown to induce signal transduction pathways that regulate changes in cellular function. We have reported recently that fibronectin (FN) receptors, alpha4beta1 and alpha5beta1 integrins, on NK cells transduce transmembrane signals leading to tyrosine phosphorylation of 60-, 70-, and 120-kDa proteins. In the current study, we have identified a 120-kDa phosphoprotein as the focal adhesion kinase (p125FAK), a structurally unique nonreceptor protein tyrosine kinase that localizes to focal adhesions. Activity of p125FAK was induced by adhesion of NK cells to plastic-immobilized FN, by cross-linking of cell surface-bound FN or FN fragments, FN120 or FN40, with anti-FN mAb, or by cross-linking of alpha4beta1 or alpha5beta1 integrins with alpha-chain-specific Abs. We also observed that enhanced in vitro kinase activity was associated with immunoprecipitates of alpha4beta1 or alpha5beta1 integrins from lysates of FN-adherent NK cells as compared with BSA-treated NK cells. In addition to p125FAK activity, FN-induced kinase activity was also found to be mediated by Fyn, Lyn, and Zap-70, as assessed by in vitro phosphorylation of the immunoprecipitated kinases in the presence of [gamma-32P]ATP. Clustering of FN receptors on NK cells by agonists such as immobilized FN or alpha4- or alpha5-specific Abs also induced association of Fyn and Zap-70 with p125FAK. Our observations indicate that activation and phosphorylation of p125FAK as well as Zap-70 and certain kinases of the src family play an important role in formation of active signaling complexes in response to triggering via beta1 integrins on NK cells. These results also suggest the existence of cross-talk or points of convergence between the beta1 integrin-mediated and other receptor-signaling pathways.

Cytophilia is one of the biologic properties of the Fc region of immunoglobulins operationally defined as the ability of an antibody molecule to attach to certain cell types before combination with antigen. By attachment of cytophilic antibody to membrane Fc receptors, cells become "armed" with antibodies and able to interact specifically with soluble or particulate antigens. In contrast to this cytophilia, the so-called opsonic antibody binds to FcR of various cell types only after it has complexed with antigen. In spite of knowledge of cytophilic properties of immunoglobulins for more than 30 years and an impressive accumulation of facts regarding the structure and function of cell-surface FcR binding IgG, IgE, or IgA molecules, less progress has been made in understanding the molecular basis of cytophilia. Our extensive studies of the structural and functional aspects of the interaction of IgG in monomeric form with human natural killer cells, via the type IIIA IgG Fc receptor (Fc gamma RIIIA), provide a focal point for revisiting this major pathway of direct and continuous communication between the humoral and cellular compartments of the immune system.

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.

We defined a novel constitutive mechanism of cell-mediated cytotoxicity, utilized by nonactivated human peripheral blood NK cells against a variety of tumor cell targets resistant to secretory (i.e., perforin/granzyme-mediated) NK cell killing. Untreated NK cells rapidly induced membrane damage and necrosis in K562 target cells (as determined by 51Cr release assay and confirmed by transmission electron microscopy; TEM), in the absence of DNA fragmentation and apoptosis (as assessed by [3H]thymidine release assay and TEM). Chelation of extracellular Ca2+ or paraformaldehyde fixation completely abrogated NK cell secretory activity and necrotic killing. In contrast, NK cells with either normal or impaired secretion consistently exhibited cytotoxicity against a wide variety of solid tumor cell lines in 1-h [3H]thymidine release, but not in 4-h 51Cr release, assays. Thus, the cytotoxicity was attributable to a nonsecretory, cell membrane-mediated mechanism. It appeared to selectively induce DNA fragmentation and apoptosis without plasma membrane damage and necrosis. This was further demonstrated by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and confirmed by TEM. Among defined populations of immune cells, only NK cells showed an inherent ability to rapidly induce apoptotic death in solid tissue-derived malignant cells. This study demonstrates that NK cells are unique immune cells constitutively endowed with multiple mechanisms of destroying abnormal cells.

Influenza A virus (FLU) is an important pathogen in humans. Although many features of the antiviral immune response have been elucidated in murine and human models of disease, little is known about the role of NK cells, which provide natural, innate immunity. The effects of experimental intranasal FLU (H1N1) inoculation on NK cells and other immune parameters were studied in 18 healthy, adult volunteers during the acute and convalescent phases of infection. Peripheral blood mononuclear cells (PBMNC) were assayed at baseline and on Postinoculation Days 1, 3, 4, 6, 7, 23, and 44. FLU infection and pathophysiologic upper airway responses were documented in all subjects, and there was no mortality. During both the acute (Days 1-3) and the convalescent (Days 23 and 44) stages of the FLU infection, significant increases in NK activity and decreases in the number of activated NK cells were observed. Reductions in the absolute number of T lymphocytes and in PBMNC proliferation to FLU virus antigen and mitogen were also observed. The current investigation extended those findings to include reductions in the number of CD4+ and CD8+ T lymphocytes and increases in the number of activated T lymphocytes. These results document that FLU infection was accompanied by enhancement of natural immunity and, as expected, suppression of most of the other measured parameters of cellular immunity. The normal response to FLU infection in humans may involve sequential modulation of the different components of the cellular immune system.

The Fc gamma receptor (R)IIIA (CD 16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via Fc gamma RIIIA, purified NK cells were cultured for 2-3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to Fc gamma RIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2R alpha, ICAM-1), cytotoxicity, cytokine production (IL-1 beta, IFN-gamma and TNF-alpha) and release of soluble IL-2R. Thus, mIgG binding to Fc gamma RIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2R alpha expression, cell proliferation and cytokine release.

Natural killer (NK) cells have been recognized as effector cells responsible for the elimination of blood-borne metastases. Newer evidence suggests that NK cells play an important role in the control of solid tissue metastases. NK cells have the ability to kill a broad variety of fresh or cultured "NK-resistant" tumor cell targets by mechanisms that are not dependent on perforin or granzyme secretion. Also, a subset of interleukin 2 (IL-2)-activated NK cells, which are called A-NK cells, is capable of extravasation, movement in solid tissues, localization to the sites of metastases, and killing tumor cells in situ. Studies in experimental animals and in cancer patients indicate that systemic adoptive immunotherapy with A-NK cells and IL-2 is a tolerable and promising treatment for advanced solid tissue as well as hematologic neoplasms. In this article, antitumor functions of A-NK cells in vitro and in vivo are reviewed, and the mechanisms responsible for their antitumor effects are considered.

An in vivo model of liver metastasis induced by human gastric carcinoma was established in nude mice and used for locoregional or systemic immunotherapy with a subset of human A-natural killer (NK) cells defined previously. A single intrasplenic (i.s.) delivery of A-NK cells (1 x 10(7)) and interleukin 2 (IL-2; 60,000 international units, twice a day for 5 days, i.p.) to animals with 3-day established liver metastases, but not IL-2 alone, resulted in rapid (within 24 h) elimination of the majority of metastases and significantly improved survival. A single i.s. or i.v. transfer of these effector cells and IL-2 significantly prolonged survival of the mice with 3-day established metastases (P < 0.03 and P < 0.02, respectively) compared with untreated mice. Using 51Cr-labeled A-NK cells, it was determined that, at best, 75% of 1 x 10(7) cells delivered i.s., and up to 50% of those delivered i.v. were found in the liver 30 min-4 h later. Using image analysis with Di-O dye-labeled A-NK cells, 60-100% of A-NK cells delivered i.s. or i.v. were detected in the liver 24 h later. By light microscopy, 3-day liver metastases were mostly intravascular, but some had already begun to spread into liver tissue. When rhodamine- or Di-O dye-labeled A-NK cells were injected i.s. or i.v. to study their distribution in the liver, they were detectable by confocal fluorescence microscopy in tumor-free tissue and in association with tumor cells 12-24 h after transfer. No evidence for selective localization of A-NK cells to liver metastases was obtained; many A-NK cells were randomly distributed in tissue and not associated with visible metastases. However, confocal fluorescence and electron microscopy showed some A-NK cells to be in cell-to-cell contact with tumor cells, both in the blood vessel and liver tissue. These results indicate that a majority of human A-NK cells transferred i.s. or i.v. to mice with liver metastases have the capacity to migrate to the liver and to enter liver tissue and tumor metastases in vivo. The presence of these effector cells even in a modest number in the liver leads to elimination of most, but not all, metastases and to significantly prolonged survival of animals treated with A-NK cells and IL-2.

Previous studies have indicated that interaction of Fc gamma RIIIA on natural killer (NK) cells with various immunoglobulin ligands or monoclonal antibodies (mAbs) can have either stimulatory or inhibitory effects on cytotoxic activity, but the basis for such divergent functional effects has been unclear. We report here that stimulation of NK cells via Fc gamma RIIIA by monoclonal anti-human CD16 (3G8), monomeric IgG (mIgG), or dimeric IgG (dIgG), used either alone or cross-linked by secondary Ab (goat anti-mouse IgG or goat anti-human IgG), resulted in different phosphotyrosine protein patterns. These results suggest that distinct substrates are involved in signaling pathways activated via various agonists of the same triggering surface molecule. Three protein tyrosine kinases, i.e., LCK, LYN, and SYK, were activated by occupancy of the Fc gamma RIIIA, and only LCK activity showed a divergence in effects induced by the various ligands, with strong autophosphorylation induced by mIgG upon cross-linking. We observed no ligand-induced activation of p59fyn, p60c-src, or p62c-yes, src-related protein tyrosine kinases which are expressed in NK cells. Activity of phosphatidylinositol 3-kinase (PI 3-kinase) induced by receptor-specific antibodies or IgG ligands had different kinetics while the level of cytoplasmic free calcium was greatest upon 3G8-induced stimulation. Although the changes in kinase activities associated with Fc gamma RIIIA-mediated regulation of NK cells are complex, it appears that the patterns induced varied with the nature of the ligand and the direction of the regulation of NK activity.

In the past year, a subset of natural killer cells designated 'A-NK cells' has been characterized. These immune cells appear to be able to enter solid tissues, migrate to sites of metastasis and eliminate malignant tissue cells, but spare normal tissue cells. They appear to be ideal surveillance cells, readily capable of upregulating antitumor functions in response to local activation signals.

Various anti-Fc gamma RIII (CD16) monoclonal antibodies (mAbs) are shown here to have positive or negative modulatory effects on human NK cells. Thus, 3G8 mAb (IgG1) triggered a dose-dependent augmentation of NK activity in 67% (23/34) of individuals tested, who were designated as responders. All four IgG1 anti-CD16 mAb tested (BL-LGL/1, B73.1, Leu11c, and 3G8) were stimulatory for NK cells isolated from responders, whereas six non-IgG1 anti-CD16 mAbs were either inhibitory or had no significant effects on NK activity. The upregulation of NK activity in responders was not attributable to an increase in either the conjugate formation or the delivery of the lethal hit to target cells. This mAb-mediated up-regulation of NK activity was shown to be associated with a recycling capacity higher than that of controls and with enhanced release of cytokines by activated NK cells. Anti-CD16 mAb inhibited binding of either monomeric or polymeric IgG to Fc gamma RIIIA on NK cells. Also, mAb 3G8 or its F(ab')2 fragments decreased or reversed inhibition of NK activity induced by monomeric IgG (mIgG). Our data indicate that regulation of NK activity via the Fc gamma RIIIA is influenced by dose-dependent interactions between cytophilic mIgG and anti-CD16 mAb of IgG1 isotype.

To determine the role of NK cells in regulation of tissue growth, the phenotype and function of liver-resident NK cells were studied after 70% partial hepatectomy in rats. The process of liver regeneration was generally completed by day 14. In contrast, the number of liver-resident NK cells (NKR-P1bright) was restored as early as day 3 after partial hepatectomy. However, spontaneous functions of liver-resident NK cells, including killing of YAC-1 and P815 targets, Ab-dependent cellular cytotoxicity, and redirected killing via NKR-P1, were continuously suppressed throughout the entire period of liver regeneration (from 3 h to 14 days). Augmentation of NK cytotoxicity against P815 targets and induction of NK cell adherence to plastic following 24 h of IL-2 stimulation showed a similar pattern of suppression. However, IL-2-induced augmentation of YAC-1 killing, proliferation and generation of adherent NK cells, and LAK activity in 5- to 7-day cultures were found to be suppressed only during the first 24 h and increased between days 2 and 7 after hepatectomy. Sorted NK cells (> or = NKR-P1bright) from liver-resident mononuclear leukocytes 24 h after partial hepatectomy showed the same pattern of suppression as unsorted mononuclear leukocytes. In contrast to liver-resident NK cells, no significant changes were detected in peripheral blood or spleen NK cells of rats following partial hepatectomy. Of particular interest, in normal liver, hepatocytes were resistant to NK lysis, while resident NK cells were cytotoxic for various NK-sensitive targets. In contrast, during the early period of liver regeneration, when hepatocytes were sensitive to lysis by liver-resident NK cells of normal rats, NK cells obtained from regenerating liver tissues were unable to mediate cytotoxicity. At the final phase of liver regeneration (days 7-14 after hepatectomy), both resistance of hepatocytes to killing by NK cells and cytotoxicity of liver-resident lymphocytes against hepatocytes from regenerating liver were simultaneously restored. In vivo depletion of NK cells by injection of rats with anti-NKR-P1 mAb resulted in a significant augmentation of liver regeneration subsequent to partial hepatectomy. Our data suggest that liver-resident NK cells may be involved in regulation of the extent of liver regeneration.

In this paper, we provide evidence that the vitronectin receptor (VNR) alpha v beta 3 is expressed on human NK cells. The presence of this VNR on freshly purified NK cells was demonstrated by flow cytometry analysis, as well as biochemically, after 125I-labeled surface lactoperoxidase labeling and immunoprecipitation. mAbs LM142 and LM609 specific for alpha v and alpha v beta 3, respectively, precipitated a heterodimer of alpha- and beta-chains with approximate molecular masses of 155 and 110 kDa under nonreducing conditions. Under reducing conditions, there was an apparent decrease in the molecular mass of the alpha-chain, which is likely to result from the release of a protein of 20 to 30 kDa linked by internal disulfide bond to the alpha v-chain. Integrin alpha v beta 3 expressed on NK cells became functional, i.e., was able to bind its ligand, vitronectin (VN), only after cellular activation or when costimulation with an additional signal was provided. Thus, NK cells adhered to plastic-immobilized VN only after IL-2 activation, and RGD-containing synthetic peptides or mAbs specific for alpha v beta 3 complex inhibited this binding. To assess the role of the VNR in signal transduction, anti-beta 3 mAb was used to cluster the VNR on NK cells and, thereby, mimic the process that occurs during formation of adhesive contacts. Cross-linking of VNR on fresh NK cells stimulated phosphorylation on tyrosine residues of several intracellular proteins. The major increase in tyrosine phosphorylation was observed in proteins of approximate molecular masses of 75 and 120 kDa. Therefore, signal transduction by the VNR on NK cells induced activation of intracellular protein kinases. Ligand engagement of the VNR on NK cells also costimulated cytokine production and proliferation of NK cells. Binding of NK cells to plastic-immobilized VN served as a costimulus with either anti-Fc gamma RIII or IL-2 to produce IFN-gamma, TNF-alpha, and cell proliferation. Our findings suggest that occupancy and subsequent clustering of VNRs play a role in the activation and function of human NK cells.

Natural killer (NK) cells express receptors that bind to the Fc portion of immunoglobulin molecules (FcR), and their function can potentially be modulated positively or negatively by such receptor:ligand interactions. This review discusses recent developments in the understanding of expression of various forms and isoforms of FcRs by NK cells, and the sequelae of binding of physiologic ligands. Particular attention is paid to the significance of FcR:Ig interactions in both activating and inactivating NK cells under various conditions.

Human NK cells can be separated into two functionally distinct subpopulations based on the ability to rapidly respond to IL-2 by adherence to solid surfaces. To determine functions of the NK cell subsets in solid tumor tissues, adherent (A) and nonadherent (NA) NK cells were evaluated for their ability to infiltrate multicellular tumor spheroids in vitro, to kill carcinoma (CA) cell targets in these spheroids, and to mediate antitumor activity in vivo. A-NK cells were less cytolytic than NA-NK cells against CA targets in single cell suspensions or in monolayers. However, A-NK cells showed a significantly better ability than NA-NK cells to infiltrate tumor tissues and kill tumor cells in spheroids of human squamous cell CA of the head and neck or breast CA. Perilesional delivery of human A-NK cells and IL-2 resulted in regression of established human squamous cell carcinoma of the head and neck tumors growing subcutaneously in immunosuppressed nude mice. Similarly, in a xenograft model of human gastric CA metastatic to liver of nude mice, a single intrasplenic injection of A-NK cells in combination with i.p. infusions of IL-2 significantly reduced the number of established hepatic metastases (p < 0.007) and prolonged survival of the mice (p < 0.003). In contrast, NA-NK cells were ineffective in either of the in vivo xenograft tumor models. These findings demonstrate that A-NK cells represent a biologically unique and important subset of NK cells that, in contrast to the rest of NK cells, function as effector cells in solid tumor tissues and, consequently, have a great antitumor therapeutic potential.

We have recently begun investigating the effects of prolactin (PRL) on the function, or growth and differentiation of rat NK cells. Initially, we sought to determine the expression of a receptor for PRL (PRLr) on rat NK cells and to determine the effects of interleukin 2 (IL-2) on its expression. By a combination of reverse transcription/polymerase chain reaction (RT/PCR) and Southern blotting, we were able to identify a PRLr specific transcript using RNA isolated from highly purified, IL-2-activated NK (A-NK) cells. Using primers specific for unique portions of the major forms of PRLr transcripts in liver and ovary, it was determined that the NK cell PRLr has some homology with the major form of transcript for PRLr found in rat liver. By flow cytometric and 251-PRL binding analyses, we determined that A-NK cells expressed detectable levels of PRLr internally. Surface PRLr was not detectable by flow cytometry on freshly isolated NK cells from normal rats, and incubation of NK cells with rIL-2 did not induce detectable surface PRLr. Similarly, incubation of NK cells with PRL was not found to induce expression of the alpha chain of the IL-2 receptor (IL-2ra; CD25) on NK cells or other lymphoid cells. However, we were able to detect surface expression of PRLr on NK cells from bromocriptine-treated rats. Taken together, these data indicate that both cultured and freshly isolated populations of NK cell express PRLr.

Studies have shown that the administration of anti-Ly-6E.1 monoclonal antibody (mAb) to tumor-bearing mice augments their T-cell-dependent functional activities, splenic natural killer (NK) cell activity, and antitumor resistance. The effect of the mAb on splenic NK cell activity resembles that of biological-response modifiers, which involves enhanced large granular lymphocyte (LGL) development in the bone marrow (BM) from pre-NK cells, and their subsequent migration and localization in peripheral organs. We analyze here the effect of anti-Ly-6E.1 mAb on the IL-2-dependent generation of NK cell activity in short-term cultures of mouse BM cells. The results indicate that an increase in the population of LGLs paralleled the mAb-mediated augmentation of IL-2-induced generation of NK cell activity in the cultures. Although pre-NK and T cells present in the BM expressed Ly-6 antigens, the augmentation of NK cell generation appears to be due to the mAb interacting with the T cells, not the pre-NK cells, resulting in increased synthesis of tumor necrosis factor-alpha, which in turn enhanced the interleukin-2-dependent development of NK cell activity.

We have previously reported that CD7 expressed on resting human NK cells is a signal-transducing molecule, which upon ligation with mAb induces a rapid increase in cytoplasmic free calcium, secretion of IFN-gamma, and augmented NK activity against K562 targets. We now demonstrate that Ab-mediated clustering of CD7 molecules on NK cells results in enhanced phosphorylation on tyrosine residues of intracellular proteins of 60, 70, 80, 97, and 120 kDa. In the presence of genistein, a specific inhibitor of protein tyrosine kinase, the enhanced level of tyrosine phosphorylation was blocked, indicating that CD7 may induce signaling via activation of tyrosine kinases. Cross-linking of CD7 or CD16 molecules with primary and secondary Abs, as well as stimulation of NK cells with phorbol ester (PMA) or with calcium ionophore A23187 also induced beta 1 integrin-mediated adhesion of these cells to fibronectin (FN)-coated plastic surfaces. In contrast, cross-linking of CD2 expressed on the surface of NK cells had no significant effect on NK cell adhesion to FN. This adhesion was not associated with up-regulation of expression of alpha 4 beta 1 or alpha 5 beta 1 FN receptors on NK cells, but it required an intact cytoskeleton. The CD7-induced adhesion to FN was mediated by alpha 4 beta 1 and alpha 5 beta 1 integrins, as it was partially blocked by FN connective segment-1 peptide (EILDVPST), the alpha 4 beta 1-binding domain, as well as by RGD-containing peptides, the alpha 5 beta 1-binding domain, but not by EILEVPST or RGE control peptides. NK cell binding to FN was also partially inhibited by mAb to alpha 4, alpha 5, and beta 1 integrins. The mechanism by which cross-linking of CD7 or CD16 on NK cells induced adhesion to FN appeared to involve both protein tyrosine kinase and protein kinase C, because this adhesion was blocked in the presence of either genistein or a protein kinase C inhibitor, staurosporin. Our data demonstrate that signals transduced via triggering of either CD7 or CD16 molecules are involved in the regulation of the functional activity of beta 1 integrins on NK cells.

To better understand the events occurring during immunotherapy of liver metastases with effector cells, we have developed a clinically relevant animal model in which both effector-tumor cell interactions and survival can be evaluated. A cell line of human gastric carcinoma (HR) metastatic to the liver has been established from a patient's liver biopsy. HR cells (10 x 10(6)) injected intrasplenically metastasize into the liver of immunosuppressed nude mice, with micrometastases detectable histologically by day 4 and macrometastases by day 7. The animals subsequently develop ascites and die between days 30 and 40 after tumor injection. To investigate early metastatic events in the liver, HR cells were transduced with a plasmid containing both the lacZ gene under the control of the CMV promoter and NeoR gene. Transfectants selected for neomycin resistance were lacZ gene positive and stained blue in the presence of a beta-galactosidase substrate, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). These transfectants (HRLZ) remained lacZ gene positive for at least 25 passages in vitro. Injected intrasplenically, an HRLZ clone grew invasively in nude mice and formed liver metastases comparably to parental tumor cells. The number and localization of blue X-Gal-positive tumor cells were followed in liver tissues of animals sacrificed at various times, from 1 h to 28 days postinjection of HRLZ cells. HRLZ cells were seen in liver blood vessels and sinusoids within 1 h after injection, and the progressive growth of micrometastases and macrometastases could be followed with precision by X-Gal staining. On day 3 after injection of HRLZ cells, numerous micrometastases were established containing 12-16 tumor cells. When these 3-day established HRLZ micrometastases were treated by the intrasplenic infusion of interleukin 2 (IL2)-activated human natural killer (NK) cells selected by IL2-induced adherence to plastic (A-NK) and systemic IL2, nearly all liver micrometastases were eliminated within 24 h after a single transfer of A-NK cells (P < 0.001). This xenogeneic model was also used for adoptive immunotherapy of 7-day established liver macrometastases with human A-NK cells injected intrasplenically and exogenous IL2 given i.p. A significant decrease in the number of hepatic metastases and the weight of livers (P < 0.003) in comparison with those of control mice was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of the adhesion molecule CD44 in the development of NK cells was analyzed in a mouse long-term bone marrow culture system. After 4 wk of culture (day 0), recombinant human IL-2 was added and 13 days later the cells generated were shown to have substantial cytotoxic activity against YAC-1 and to be enriched for NK cells, as assessed for NK-1.1 phenotype by flow cytometric analysis. Physical separation between stroma and precursors partially inhibited proliferation and, consequently, a lower number of cytotoxic cells were produced. Similar results were obtained when an anti-CD44 mAb was added together with IL-2 at day 0. The disruption of hyaluronic acid (HA), one of the ligands of CD44, by hyaluronidase or the competition for the binding of CD44 by soluble HA added with IL-2 on day 0 inhibited both proliferation and development of cytotoxicity to a greater degree than did anti-CD44. These results indicate that interaction of CD44 with HA plays an important role in the development of pre-NK cells into cytotoxic effector cells.

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.

A human cytotoxic-T-lymphocyte (CTL) line capable of killing autologous tumor (AuTu) cell targets was established from peripheral-blood lymphocytes of a patient with squamous-cell carcinoma of the tongue. The cultured CTL were CD3+CD8+CD11b-HLA-DR+T cell receptor (TCR) alpha/beta+. When tested in 4-hr 51Cr-release assays against various lines of squamous-cell carcinoma of the head and neck (SCCHN) and a variety of non-squamous human tumor and normal cell targets, the CTL were found to lyse the autologous SCCHN cell line (PCI-50) and 7 allogeneic SCCHN lines: PCI-1, -2, -4A, -4B, -13, -30 and -38. Of these tumor cell lines, PCI-13, -30 and -38 shared HLA-A2 locus with the AuTu, PCI-50, while PCI-4A and -4B shared HLA-B44 with AuTu. Lysis of AuTu (A2+B44+), PCI-13 (A2+B44-) and PCI-4B (A2- B44+) by the CTL was efficiently inhibited by monoclonal antibodies (MAbs) to CD3, CD8, TCR alpha/beta or the major-histocompatibility-complex (MHC)-class-I antigens. MAbs to HLA-A2 antigens inhibited lysis of PCI-50 or PCI-13 targets by the CTL. In cold-target inhibition assays, unlabeled PCI-4B or PCI-13 cells inhibited CTL lysis of AuTu targets. The CTL incubated in the presence of the HLA-A2+ SCCHN PCI-50 or -13, but not an HLA-A2+ gastric carcinoma, produced TNF-alpha, IFN-gamma and GM-CSF. The CTL were tested for their TCR V beta gene expression by polymerase chain reaction (PCR). At week 10 in culture, the time of the highest AuTu cytotoxicity mediated by the CTL line, V beta 6 was expressed by 26% of T cells. Three clones, obtained by limiting dilution from 10-week CTL and selected for high cytotoxicity against AuTu, were found to be V beta6+. Further analysis of the specificity of these clones indicated lytic activity against PCI-13 (A2+B44-), but not PCI-4B (A2-B44+) targets. In 16-week cultures, which retained AuTu cytotoxicity as well as V beta 6 expression, TCR V beta 2 was also expressed at high frequency (29%), and AuTu-reactive clones were found to be V beta 2+. Our results indicate that at least 2 different CTL populations (V beta 6+ and V beta 2+) are able to recognize SCCHN-associated antigen(s) and that the V beta 6+ T cells are HLA-A2 restricted, while V beta 2+ T cells may be HLA-B44 restricted.

Aminopeptidase (AP) activity on rat natural killer (NK) cells was found to have the following characteristics: (1) the activity was surface associated and not secreted, as determined by extracellular location of product and by the cessation of hydrolysis of substrate upon removal of the cells from the medium. (2) The activity was linear with respect to time and cell number. (3) The enzymatic activity on splenocytes and on the NK leukemia cell line CRNK-16, but not on IL-2 activated NK (A-NK) cells, was sensitive to trypsin treatment. (4) The AP activity on intact cells had a broad pH dependency with optimal activity at slightly alkaline pH but lower activity at acidic pH. (5) There was a preference for neutral substrates and essentially no activity towards acidic substrates. (6) Enzymatic activity was inhibited in the presence of the AP inhibitors bestatin and amastatin, and in the presence of the chelator, 1,10 phenanthroline, indicating the involvement of a metalloprotease. (7) Culture of A-NK cells with bestatin resulted in a decrease in cytotoxicity against YAC-1 and P815 targets. Amastatin treatment caused only a slight decrease in cytotoxicity against YAC-1 targets, but a significant decrease in cytotoxicity against P815 targets. (8) Treatment of A-NK cultures with specific inhibitors of APases caused an increase in expression of CD2 (an increase from 20-80% with bestatin and an increase from 25-35% in the presence of amastatin). These results provide the first evidence for the existence of APases on the surface of NK cells and suggest a role for these enzymes in the regulation of cytotoxic activity and of CD2 surface expression.

Natural killer (NK) cells, the CD3- CD56+ CD16+ subset of peripheral blood lymphocytes, have long been known to be involved in non-major histocompatibility complex-restricted natural immunity to virally infected and malignant target cells. The association of abnormalities in NK cell numbers or functions with a broad spectrum of human diseases has been more clearly defined in recent years as a result of the improved knowledge of NK cell physiology and advances in monitoring of NK cell functions in health and disease. The ability to reliably measure changes in NK activity and/or numbers during the course of disease or response to treatment has focused attention on the role of the NK cell in disease pathogenesis. The improved understanding of NK cell deficiency in disease has opened a way for therapies specifically designed to improve NK cell function. The therapeutic use of biologic response modifiers capable of augmenting NK cell activity in vivo and of adoptive transfer of highly enriched, activated autologous NK cells in diseases such as cancer and AIDS is being evaluated. The importance of NK cells in health and the consequences of NK cell deficiency or excess are likely to be more extensively monitored in the future.

Fluorescently labeled, adoptively transferred interleukin (IL)-2 activated natural killer (A-NK) cells have the ability to selectively accumulate within established pulmonary or hepatic metastases, binding to tumor cells and/or to microvascular endothelial cells. A-NK cells have also been shown to exert antimetastatic therapy in animal models and in the clinic. Transfection of genes for cytokines or possibly other molecules has the potential to improve the therapeutic potency and efficacy of the effector cells. Gene transfection to induce autocrine production of IL-2 and/or other cytokines is expected to augment their antimetastatic activities, while avoiding toxicity from systemic administration of high doses of cytokines. An alternative or complementary strategy for gene therapy is to transfect A-NK cells with genes for cytotoxic molecules, to selectively target them to metastatic sites.