Effector-target Cell Interaction Research Articles

Efficient Combinatorial Adaptor-Mediated Targeting of Acute Myeloid Leukemia with CAR T-Cells

Introduction: Chimeric Antigen Receptor (CAR) T-cell therapies are remarkably efficient in treating B- and plasma-cell malignancies by targeting cell-of-origin antigens, eradicating both tumor cells as well as healthy B- and plasma-cell counterparts. Their loss can be compensated by immunoglobulin substitution until they regenerate from hematopoietic stem and progenitor cells (HSPCs). However, similar on-target, off-tumor cell-of-origin elimination would be detrimental in the context of HSPC-derived malignancies, such as acute myeloid leukemia (AML) or myelodysplastic neoplasia (MDS), as the CAR T-cell activity might lead to terminal ablation of hematopoiesis. In principle, CAR T-cells could be used to abrogate both, AML cells as well as HSPCs, followed by HSPC transplantation, ensuring hematopoietic recovery after termination of CAR T-cell activity and possibly without genotoxic preconditioning. This approach could be particularly beneficial for patients unable to tolerate radiation and high-dose chemotherapy. In order to address this challenge, we developed adaptor-mediated CAR T-cells displaying the single-chain variable fragment E2, AdFITC(E2), that binds to fluorescein conjugated to antigen-binding adaptors in diabody format (Db-FM). Such tagged adaptors, used as single linkers or in combination, would then act as safety switch, modulating CAR T-cell activation, proliferation, and lytic activity. Results: We generated second-generation AdFITC(E2)-CAR T-cells and multiple diabody-based adaptors against selected AML antigens. We then tested the diabodies targeting CD33 and CD117 with respect to their ability to mediate CAR T-cell biocidal activity as single adaptors or in combination. Combinatorial staining with CD117 Db-FM and CD33 Db-FM, each added at saturating concentration for the cognate antigen, resulted in higher fluorescein decoration of target cells, with more than two-fold increased fluorescence intensity detected by flow cytometry upon combined staining, thus enhancing AdFITC(E2)-CAR T-cell target density. In vitro cytotoxicity assays of healthy-donor-derived AdFITC(E2)-CAR T-cells, both against cell lines (MOLM14-CD117 +GFP +Luc +) as well as patient-derived AML blasts, revealed that dual adaptor use significantly improved tumor cell lysis compared to equimolar concentration of single adaptors. To further investigate the basis for increased killing by dual targeting, we performed live-cell imaging of effector-target cell interactions in microwells. The addition of both CD117 and CD33 Db-FM to the co-culture of MOLM14-CD117 +GFP +Luc + and AdFITC(E2)-CAR T-cells resulted in faster tumor cell lysis (measured by influx of PI) which correlated with a more rapid, durable engagement time of CAR T-cells with target cells. We next tested CAR T-cell biocidal activity in therapeutic xenogeneic mouse models engrafted with MOLM14-CD117 +GFP +Luc + where AdFITC(E2)-CAR T-cells in combination with CD117 or CD33 Db-FM administered as single adaptors were as efficient as direct CAR T-cells against the same antigens. Injection of AdFITC(E2)-CAR T-cells and both CD117 and CD33 Db-FM effectively inhibited tumor growth, outperforming monotherapies, and leading in some cases to full AML elimination in bone marrow and blood as shown by bioluminescence and terminal flow cytometry analysis (Figure 1). Conclusions: We here tested an adaptor CAR T-cell approach using multiple adaptors to on-off modulate and enhance CAR T-cell activity, focusing on AML as a relevant disease model with high clinical need for therapeutic improvement. The high heterogeneity in antigen expression on AML cells supports a combinatorial targeting strategy, individualized based on the respective AML immunophenotype. Due to their relatively small molecular weight (~55kDa), diabody-based adaptors are rapidly cleared from the body via the kidneys allowing for rapid control over AdFITC-CAR T-cell on-off activity prior to HSPC transplantation. We envision that this approach has the clinical potential to improve CAR T-cell safety profiles by enhancing the specificity towards target cells, while ameliorating the side-effects against healthy tissues expressing only one antigen of the combination.

An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip

Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.

Anti-myeloma activity and molecular logic operation by Natural Killer cells in microfluidic droplets

Immune-targeted therapies that activate effector lymphocytes such as Natural Killer (NK) cells are currently being investigated for the treatment of Multiple myeloma (MM), the second most common form of hematological cancer. However, individual NK cells are highly heterogeneous in their cytolytic potential, making it difficult to detect, quantify and correlate the outcome of dynamic effector-target cell interactions at single cell resolution. Here, we present a microfluidic bioassay platform capable of activity-based screening of cellular and molecular immunotherapies. We identified distinct functional signatures associated with NK-MM cell interaction. The addition of immunomodulatory drug lenalidomide altered responses of NK-susceptible MM cells but not that of NK-tolerant MM cells. Antitumor cytotoxicity was significantly increased by the blockade of PD1/PDL1 axis as well as the clinically relevant cell line NK92, which were used to construct molecular logic functions (AND and NOT gates). A predictive agent-based mathematical model was developed to simulate progressive disease states and drug efficacy. The findings of the current study validate the applicability of this microfluidic cytotoxicity assay for immunotherapy screening, biocomputation and for future employment in detection of patient-specific cell response for precision medicine.

Leukemia Related Co-Stimulation / Co-Inhibition Predict T-Cell Attack of Acute Lymphoblastic Leukemia Mediated By Blinatumomab

Refractory B-precursor acute lymphoblastic leukemia (ALL) remains an unsolved therapeutic challenge. Various T-cell immunotherapies are promising options in relapsed/refractory B-ALL, like the CD19/CD3-bispecific T-cell engaging antibody Blinatumomab. Until now it has not been possible to determine critical factors for T-cell attack against leukemia that decide on in vivo response or non-response to treatment. Immune-checkpoint molecules regulate immune escape of malignant cells and antibody blockade of these inhibitory pathways enhances antitumor immune responses. Therefore, we investigated the role of co-stimulatory and co-inhibitory molecules for effector-target cell interactions and influence on T-cell attack against leukemia. CD19+ lymphoblast lines, primary pediatric B-ALL bone marrow blasts (n=10) and physiologic CD19+ CD10+ pre-B bone marrow precursors from healthy bone marrow were screened for surface expression of 20 different co-signaling molecules. Surface expression of PD-L1, PD-1, LAG3, CD40, CD86, CD27, CD70 and HVEM revealed differences in stimulatory and inhibitory profiles of pediatric ALL blasts as compared to physiologic cells. Pediatric ALL patients refractory to Blinatumomab-treatment (n=4) as well as patients with relapsed leukemia (n=7) showed increased expression of PD-L1 on blasts. Expression of exhaustion markers PD-1 and TIM-3 was significantly higher on patients' T cells as compared to healthy donors and is induced by T-cell attack against blasts. Blinatumomab-mediated T-cell function was examined in healthy donors as compared to pediatric patients with ALL through analysis of proliferation and effector function. Significant differences in Blinatumomab-induced T-cell function were found to be target-cell dependent and correlated to expression of co-signaling molecules on target cells. Blockade of inhibitory PD-1-PD-L and CTLA-4-CD80/CD86 interactions could further enhance effector T-cell function of healthy donors and patients whereas blockade of co-stimulatory CD28-CD80/86 interactions resulted in reduced T-cell effector and proliferation potential. Combined treatment with Blinatumomab and PD-1 blocking antibody Pembrolizumab was feasible and induced an anti-leukemic immune response in a 12 year old patient with refractory ALL. In conclusion, we show that regulation of T-cell activation and inhibition by co-signaling molecules guides the efficacy of T-cell attack against ALL. Inhibitory interactions between leukemia-induced checkpoint molecules on T cells and their counterparts on ALL regulate in vivo resistance to T-cell immunotherapy and will guide future therapeutic interventions. DisclosuresOff Label Use: Pembrolizumab.

Histological Assessment of Cutaneous Acute Graft-Versus-Host Disease in a Preclinical Swine Model of Hematopoietic Cell Transplantation and Vascularized Skin Flap Tolerance

Abstract 1894Graft-versus-host disease (GVHD) remains a frequent complication of hematopoietic cell transplantation with skin being a principal target organ. Murine models have provided some insight into the mechanisms of this complex disease process. However, mouse skin differs from human skin, and results of studies in rodents may not translate well to the clinic. Miniature swine is a well-recognized animal model for preclinical studies of skin including dermal toxicology, transdermal drug delivery and wound healing. Unlike skin of rodents, dogs or non-human primates, porcine skin is similar to human skin in terms of structure of epidermal rete ridges (Figure 1A), hair follicle structure and density, and presence of sweat glands and subcutaneous fat.We have developed a miniature swine model of haploidentical hematopoietic cell transplantation that results in stable stem cell engraftment, hematopoietic chimerism and donor skin flap tolerance without GVHD. Swine leukocyte antigen (SLA)ad animals receive 100cGy total body irradiation and T cell depletion using porcine CD3 immunotoxin prior to cytokine mobilized peripheral blood hematopoietic cell transplantation from a haploidentical SLAac donor. Daily cyclosporine is administered at doses adjusted to maintain serum levels between 400–800 ng/ml starting one day prior to transplantation and continued for 30 days with taper until day 45.Mixed hematopoietic cell chimerism persists in these animals and donor vascularized skin flaps are accepted without the need for maintenance immunosuppression regardless of whether they are placed at the time of hematopoietic cell transplantation or greater than 100 days later. Despite the initial resistance to GVHD in this model, acute cutaneous GVHD could be induced through infusion of a high dose of donor leukocytes (DLI) including 15 million donor T cells/kg recipient body weight administered 463 days post hematopoietic cell transplantation and 314 days post vascularized donor skin flap transplantation. Conversion to full donor hematopoiesis was observed between 3–4 weeks post DLI. At this time a mild skin rash was evident in both donor and host skin which appeared to progress specifically within host skin. The presence of both donor and host skin on a tolerant animal whose peripheral blood consists of a mixture of both donor and host derived hematopoietic cells provides a novel model to evaluate specific targets of acute GVHD.In this study we assess histologically the manifestations of acute GVHD in both donor and host skin and compare it to typical histological manifestations of acute GVHD observed in patients. Specifically, recipient skin developed superficial lymphoid infiltrates that progressed to confluence along the dermal-epidermal junction by day 21. These infiltrates were associated with intraepidermal migration, prominent basal cell layer keratinocyte apoptosis, and satellitosis virtually indistinguishable from grade 3–4 human cutaneous acute GVHD (Figure 1B). Superficial portions of hair follicles were involved, as also seen in human disease. Of interest, mild disease was detected in the donor skin grafts by day 62 post-DLI, where selective lymphoid infiltration in the tips of the epidermal rete ridges and in association with target cell apoptosis/satellitosis was documented, a finding remarkably similar to early targeting events in humans.These findings emphasize the value of the porcine model for translational studies of cutaneous GVHD. Preliminary data suggest differences in cellular targeting between recipient and donor skin whereby the latter may be triggered by the presence of chimeric recipient-derived hematopoietic elements that have populated donor grafts. Further studies are now possible to explore the effector-target cell interactions in this clinically-relevant model of human cutaneous GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

AbstractAlthough membrane phospholipid phosphatidylinositol-4,5bisphosphate (PIP2) plays a key role as signaling intermediate and coordinator of actin dynamics and vesicle trafficking, it remains completely unknown its involvement in the activation of cytolytic machinery. By live confocal imaging of primary human natural killer (NK) cells expressing the chimeric protein GFP-PH, we observed, during effector-target cell interaction, the consumption of a preexisting PIP2 pool, which is critically required for the activation of cytolytic machinery. We identified type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) α and γ isoforms as the enzymes responsible for PIP2 synthesis in NK cells. By hRNA-driven gene silencing, we observed that both enzymes are required for the proper activation of NK cytotoxicity and for inositol-1,4,5-trisphosphate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5KIs, we found that lytic granule secretion but not polarization resulted in impaired PI5KIα- and PI5KIγ-silenced cells. Our findings delineate a novel mechanism implicating PI5KIα and PI5KIγ isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca2+ response and lytic granule release.

Hypoxia attenuates effector–target cell interaction in the airway and pulmonary vascular compartment

Leucocyte infiltration is known to play an important role in hypoxia-induced tissue damage. However, little information is available about hypoxia and interaction of effector (neutrophils) with target cells (alveolar epithelial cells, AEC; rat pulmonary artery endothelial cells, RPAEC). The goal of this study was to elucidate hypoxia-induced changes of effector-target cell interaction. AEC and RPAEC were exposed to 5% oxygen for 2-6 h. Intercellular adhesion molecule-1 (ICAM-1) expression was determined and cell adherence as well as cytotoxicity assays were performed. Nitric oxide and heat shock protein 70 (HSP70) production was assessed in target cells. Under hypoxic conditions enhanced ICAM-1 production was found in both cell types. This resulted in an increase of adherent neutrophils to AEC and RPAEC. The death rate of hypoxia-exposed target cells decreased significantly in comparison to control cells. Nitric oxide (NO) concentration was enhanced, as was production of HSP70 in AEC. Blocking NO production in target cells resulted in increased cytotoxicity in AEC and RPAEC. This study shows for the first time that target cells are more resistant to effector cells under hypoxia, suggesting hypoxia-induced cell protection. An underlying mechanism for this phenomenon might be the protective effect of increased levels of NO in target cells.

Ultrastructural characterization of effector-target interactions for human neonatal and adult NK cells reveals reduced intercellular surface contacts of neonatal cells

Limitations in neonatal natural killer (NK) cell responses may be associated with the less efficient newborn capacity to solve viral infections. Although these limitations have been extensively reported they are poorly characterized. Making use of the major histocompatibility complex (MHC) class I negative cell line K562, the parameters required for the initial events involved in neonatal NK/target cell interactions were determined and compared with adult blood NK cell/target cell interactions. Ultrastructural characterization of effector-target cell interactions revealed that neonatal NK cells are more strongly activated upon contact with K562 cells than adult blood NK cells. Furthermore, the neonatal capacity to establish contacts, in particular extensive contacts, is significantly reduced when compared with adult blood NK cells. However, no significant differences were found either in the cell surface expression levels or activation state of LFA-1, which could account for the reduced intercellular contacts. Because extensive contacts are crucial for effective immunologic synapse formation, these data suggest that a limited or nonsustained positive signaling may occur on neonatal NK cells, restricting their NK cell-mediated lysis capacity.

The carcinoembryonic antigen (CEA) modulates effector-target cell interaction by binding to activated lymphocytes.

We and others have shown that the carcinoembryonic antigen (CEA) modulates the susceptibility of human colorectal carcinoma cells to cytotoxic lymphocytes. We now demonstrate that the density of the CEA molecules on the tumor cell surface has a determining influence on its protective function. In contrast, CEA released by tumor cells has no protective effect for CEA-negative cells. To elucidate the responsible mechanism, we analyzed the binding properties of CEA to lymphokine activated killer (LAK) cells. In agreement with our observation that only membrane-bound CEA provides protection, we found that intercellular contact between LAK cells and CEA-expressing tumor cells is required for binding of CEA to LAK cells. Using FACScan analysis, we demonstrate the presence of CEA on lymphocytes co-cultured with CEA-transfected cells but not after co-culture with their parental cells and after incubation with soluble CEA. Interestingly, following overnight co-culture the amount of bound CEA on LAK cells was identical regardless of adherence on tumor cells or loss of contact with tumor cells. This indicates that CEA is released from the tumor cells after binding to LAK cells. Our results suggest that tumor cells can modulate effector cell adhesion by regulating the turnover rate of CEA on the tumor cell membrane.

Expression of the intercellular adhesion molecule-3 (ICAM-3) in human renal tissue with relation to kidney transplants and various inflammatory diseases.

Adhesion molecules are important for immune regulatory mechanisms concerning antigen presentation, lymphocyte activation, localisation and migration as well as effector-target cell interactions in inflammatory processes. The immunohistochemical expression of ICAM-3, a recently cloned new member of the immunoglobulin family which also binds leucocyte function antigen 1 (LFA-1), was examined in 80 needle core biopsies from 35 renal allografts, 7 patients with mesangioproliferative glomerulonephritis, 5 patients with extracapillary glomerulonephritis, 4 patients with interstitial nephritis and 5 patients with diabetic nephropathy and 20 normal kidneys. In all types of lesions ICAM-3 was constitutively expressed on the majority of infiltrating leucocytes without detectable upregulation or presentation on possible target structures during inflammation indicating its possible role to be mainly in initiation of the inflammatory response.

Granzyme B and perforin can be used as predictive markers of acute rejection in heart transplantation

We have investigated perforin and granzyme B expression in graft-infiltrating lymphocytes of patients who underwent heart transplantation. Those proteins are commonly present in the cytoplasmic granules of cytotoxic T lymphocytes and are released upon effector-target cell interaction. From 28 patients 103 endomyocardial biopsies were obtained and examined by histology and immunocytochemical analysis using relevant monoclonal antibodies. We found that "high" biopsy histological grades were associated with perforin and granzyme B expression in graft-infiltrating lymphocytes of patients with acute severe rejection crisis. In contrast, these markers were not detected in patients without rejection or during graft stabilization. Interestingly, in patients with mild rejection and "low" histological grades, two groups could be distinguished with a differential expression of the two intracytoplasmic proteins. The presence of perforin and granzyme B-expressing cells was found to be predictive of rapid progression to severe rejection, so that this situation required additional treatment; in contrast, their absence seemed to correlate with a good graft outcome without additional treatment. Moreover, perforin and granzyme B expression seemed to be down-regulated by immunosuppressive drugs, which coincided with graft stabilization. In conclusion, our data suggest that detection of granzyme B and perforin in graft-infiltrating lymphocytes might be helpful for routinely monitoring heart transplant patients.

Hepatocellular expression of lymphocyte function—associated antigen 3 in chronic hepatitis

T lymphocyte-mediated cytolytic immune reactions are considered a major cause of hepatocyte injury in chronic viral and autoimmune hepatitis. To further investigate local immune responses, we studied the expression of lymphocyte antigens and cell-cell interaction molecules known to be involved in effector-target cell interactions by light and electron microscopy in liver biopsy specimens from patients with chronic viral and autoimmune hepatitis. CD8+ lymphocytes were found to be the predominant population of cells in the inflammatory infiltrate in chronic hepatitis B and non-A, non-B hepatitis. In contrast, CD4+ cells constituted a comparably higher proportion of cells and were more numerous than CD8+ cells in chronic autoimmune hepatitis. In both viral and autoimmune hepatitis, a substantial portion of lymphocytes expressed activation antigens such as T11/3 (CD2R) and IL-2-R (CD25). Lymphocyte function-associated antigen-3 (CD58), which mediates lymphocyte adhesion and activation and is the natural ligand of the CD2/T11 lymphocyte surface receptor, could be demonstrated on endothelial cells and hepatocytes. Hepatocellular lymphocyte function-associated antigen-3 expression in chronic hepatitis showed membranous and cytoplasmic staining of hepatocytes and had a positive correlation with the degree of inflammatory activity. These results suggest that effector-target interactions between hepatocytes and lymphocytes mediated by the lymphocyte function-associated antigen-3/CD2 pathway play a role in chronic inflammatory liver disease. Possible functional consequences of this interaction include enhancement of antigen-specific immune reactions and antigen-independent mechanisms of T cell activation, which may contribute considerably to the degree of inflammatory activity and tissue damage in chronic hepatitis.

Hierarchy of cytotoxic T cell clones: II. Intraclonal triggering of lysis by hapten-specific CTL

Cells from clones of anti-hapten marine cytotoxic T lymphocytes (CTL) can act as both target and effector cells, but will not lyse members of the same clone. The effect of haptenation on the cytolytic activity of anti-fluorescein (FL) and anti-trinitrophenol (TNP) CTL clones was examined. Treatment of anti-FL clones with fluorescein isothiocyanate or anti-TNP clones with trinitrobenzene sulphonic acid induces these clones to kill in an antigen-independent fashion. Targets killed by the haptenated CTL included syngeneic and allogeneic B lymphocyte blast cells, P815, YAC-1 and in one case human GM 4072 tumor cells. The importance of CD8 and T cell receptor (TCR) occupancy is demonstrated by the ability to block autotriggering by antibody directed against Ly 2 and the TCR. The results demonstrate that effects other than antigen recognition of the target play a role in the final outcome of effector-target cell interactions and provide a mechanism which could lead to autodestruction and immunosuppression particularly in some types of viral infection.

An inhibitor specific for the mouse T-cell associated serine proteinase 1 (TSP-1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells.

We have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T-cell specific serine proteinase 1 (TSP-1) for its effect on the cytolytic potential of cloned cytotoxic T-cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H-D-Pro-Phe-Arg-chloromethyl-ketone (PFR-CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule-associated TSP-1 activity after extraction with Triton X-100. Furthermore, PFR-CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR-CK inhibited in a dose-dependent manner both TSP-1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high-salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule-associated proteinase TSP-1, which probably becomes active upon exocytosis following effector-target cell interactions.

Cell-mediated cytotoxicity: A highly sensitive and informative flow cytometric assay

Determination of target cell lysis by cytolytic effectors has typically been achieved by two methods: the release of various markers from the cell, as in 51chromium release assays and the uptake of markers into the cell, as in trypan blue uptake in single cell/conjugate binding assays. Problems associated with these assays might include: (1) poor uptake, (2) nonspecific release, (3) poor statistics, (4) length of assays, or (5) subjectivity. These difficulties prompted the development of a new sensitive flow cytometric assay employing two fluorochromes. PKH-1, a fluorochrome which fluoresces in the green, binds to the cytoplasmic membrane and does not leak or transfer, is used to identify the target cell population. Propidium iodide fluoresces in the red and is used to detect non-viable cells. Use of these two fluorochromes and two parameter analysis allows for identification of four subpopulations in the sample: live effectors, dead effectors, live targets and dead targets. By enumeration of these subpopulations the following information can be calculated: (1) the percent target lysis, (2) effector-to-target cell ratios, (3) viability of the effector cells at the termination of the assay, and (4) viable effector to target cell ratios. The results show that PKH-1 labeling of target cells had no effect on effector-target cell interactions. Excellent correlation was found between this method and the chromium assay, however, due to earlier detection of the lytic event, this method provides a distinct time advantage over current methods.

Interaction between CD4 and class II MHC molecules mediates cell adhesion

The CD4 glycoprotein is expressed on T-helper and cytotoxic lymphocytes which are restricted to class II major histocompatibility complex (MHC) antigens on target cells. Antibody inhibition studies imply that CD4 acts to increase the avidity of effector-target cell interactions. These observations have led to the speculation that CD4 binds to a monomorphic class II antigen determinant, thereby augmenting low affinity T-cell receptor-antigen interactions. However, no direct evidence has been presented indicating that CD4 and class II molecules interact. To address this issue, we have used a vector derived from simian virus 40 (SV40) to express a complementary DNA (cDNA) encoding the human CD4 glycoprotein. When CV1 cells expressing large amounts of the CD4 protein at the cell surface are incubated with human B cells bearing MHC-encoded class II molecules, they are bound tightly to the infected monolayer, whereas mutant B cells which lack class II molecules fail to bind. Furthermore, the binding reaction is specifically inhibited by anti-class II and anti-CD4 antibodies. Thus, the CD4 protein, even in the absence of T-cell receptor-antigen interactions, can interact directly with class II antigens to function as a cell surface adhesion molecule.

Oxygen-reactive metabolites are not detected at the effector-target interface during natural killing.

Oxygen-reactive metabolites are not detected at the natural killer (NK)-target cell interface in quantities comparable to those seen for other effector-target cell interactions. A novel luminol-coated target cell chemiluminescence assay is described in which luminol is conjugated to the target cell surface with the bifunctional crosslinker 3,3'-dithiobis(propionic acid N-hydroxysuccinimide ester) (DSP). This modification of the basic chemiluminescence assay precludes exclusion of the detection system from the tightly occluded intercellular junction, a possible deficiency in previous investigations. Luminol conjugation does not affect NK-mediated conjugate formation or cytolysis. As NK activity is enriched by a standard series of effector fractionation procedures, chemiluminescence generated against labeled target cells diminishes. Residual chemiluminescence in the most highly NK-active effector fraction is ablated upon antibody and complement depletion of MO2+ cells. This indicates that monocyte contamination is the source of luminol-detectable oxygen-reactive metabolites.

The Role of Serologically Defined Epitopes on Mumps Virus HN‐Glycoprotein in the Induction of Virus‐Dependent Cell‐Mediated Cytotoxicity

The importance of virus structural proteins for the induction of virus-dependent cellular cytotoxicity (VDCC) was studied by means of monoclonal antibodies raised in mice against mumps virions. Antibodies against the viral glycoprotein bearing the haemagglutination and neuraminidase activities (HN) inhibited VDCC but not the natural cytotoxicity (NK) displayed by the lymphocytes in the absence of virus. Antibodies to the fusion factor, the membrane protein or the nucleoprotein were inactive. These results confirmed our previous conclusion, that the only viral component required for VDCC induction is the HN protein. To clarify the role of this protein in VDCC further, the inhibitory activity of 13 HN-specific monoclonals, all of IgG isotype and directed against 9 distinct determinants, was studied in detail. Seven antibodies reacting with 3 different determinants of the peptide moiety of the HN protein were strongly inhibitory. The remaining antibodies, specific for 5 additional peptide epitopes, had intermediate or weak inhibitory effects. One carbohydrate specific anti-HN antibody was inactive although its antigen-binding capacity was of the same magnitude as that of a good inhibitory antibody. The anti-HN antibodies inhibited VDCC regardless of their IgG subclass. Moreover, VDCC inhibition was not correlated with the capacity of the antibodies to inhibit haemagglutination, haemolysis, neuraminidase activity, or the infectivity of the virus. These results suggest that full expression of VDCC requires the interaction of more than one of the serologically defined structures of the HN polypeptide with virus receptors on the lymphocytes and probably also on the target cells. These structures may be different at least in part from those involved in other known biological activities of the virus. Treatment of lymphocytes with virus increases both the number of target-binding cells (TBC) and the number of cytotoxic effector cells. However, when treated under conditions which gave optimal VDCC inhibition, none of the inhibitory antibodies reduced the virus-mediated increase in TBC. This indicates either that the anti-HN antibodies decreased the efficiency of effector-target cell interaction necessary for VDCC induction, or that they blocked a post-binding step required for triggering of cytotoxicity.

An exoglycosidase-sensitive triggering site on NK cells which is coupled to transmethylation of membrane phospholipids.

Glycosidic enzymes were used as probes to analyze the mechanism of NK cell-mediated cytotoxicity. Pretreatment of nylon wool-enriched CBA/J spleen cells, a murine NK clone, or human peripheral blood lymphocytes (PBL) with alpha-mannosidase, an exoglycosidase, led to a marked dose-dependent inhibition of NK lytic activity against YAC-1.2 or K562 tumor cells. Maximal inhibition occurred after a 60-min pretreatment of murine effectors at 37 degrees C, and the kinetics of NK inhibition by alpha-mannosidase was similar to the reported kinetics for enzymatic activity. Released hexose was detected chemically in the supernatant of mouse spleen cells treated with NK inhibitory dose of alpha-mannosidase, and inactivation of enzymatic function with EDTA reversed the NK inhibitory effect. These results suggest that alpha-mannosidase inhibited NK function by virtue of its enzymatic action. Culture of human PBL for 20-hr after treatment with this enzyme led to a greater than 70% recovery in NK lytic function. Recovery was blocked by incorporating tunicamycin, a glycosylation inhibitor of asparagine-linked glycoproteins, into the culture medium. These results suggest that the alpha-mannosidase-sensitive site may be de novo synthesized glycoprotein. Neuraminidase, beta-galactosidase, endo-beta-N-acetylglucosaminidase-D and H, and peptide-N-glycosidase treatments did not inhibit human NK cell lysis of K562 cells. Pretreatment of nylon wool-enriched CBA/J spleen cells or Percoll-enriched human LGL with alpha-mannosidase did not influence their capacity to bind YAC 1.2 target cells or K562 target cells, respectively, Ca++ pulse experiments revealed that the alpha-mannosidase-sensitive site on the NK cells was involved after target-effector binding but before the Ca++ influx. Pretreatment of effector cells with this enzyme which normally occurs after effector-target cell interaction. These results suggest that the phospholipid methylation reaction is coupled to the alpha-mannosidase-sensitive site on the NK cells. By analogy to other physiologic systems, such as histamine release in mast cells, the triggering of phospholipid methylation in the NK cells may serve as a mechanism for signal transduction across the plasma membrane.

Defective interleukin-1 production by natural killer cells of patients with cancer.

Large granular lymphocytes (LGLs) from patients with malignant disease and from controls were activated by endotoxin or K562 cells, and the supernatants assayed for interleukin-1 (IL-1) activity. Normal LGLs produced significant amounts of IL-1, the activity of which could be neutralized by anti-human IL-1 antiserum. In patients with advanced cancer depressed IL-1 production was observed, which generally correlated with the degree of cytotoxicity produced by the LGLs. Prior treatment of the LGLs with interferon increased production of IL-1 by both control and patient cells. It is suggested that LGLs coming into contact with K562 cells produce IL-1, which is important in the effector-target cell interaction. The decreased cytotoxic activity of LGLs from cancer patients could be related to a defect in IL-1 production, an effect which can be partially corrected by in vitro interferon treatment.