Antibody 3G8, specific for the human neutrophil Fc receptor, reacts with natural killer cells

Antibody 3G8 reacts with the receptor for the Fc fragment of aggregated IgG present on the majority of neutrophilic granulocytes and on a small proportion of lymphocytes. In this report, we compare the pattern of reactivity of antibody 3G8 on peripheral blood lymphocytes (PBL) and on polymorphonuclear leukocytes (PMN) with that of antibody B73.1, which reacts with the Fc receptor of natural killer (NK) cells or with a molecule functionally associated with it. We show that 3G8 reacts with the same PBL subset detected by antibody B73.1 and is responsible for virtually all NK cytotoxic activity. The lymphocyte subset recognized by the two antibodies has the morphology of large granular lymphocytes and includes neither B nor T cells. Our results indicate that NK cells and PMN express the same Fc receptor for immune complexes, and that B73.1 and 3G8 recognize on the same receptor two distinct epitopes that are preferentially expressed on NK cells and on PMN, respectively.     

Functional effects of a monoclonal antibody directed against a distinct epitope on 4F2 molecular complex in human peripheral blood mononuclear cell activation.

The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1 beta but not IL-2 or interferon-gamma were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43+ cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43+ cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation.     

Lymphokine-activated killer (LAK) cells. VI. NK1.1+, CD3+ LAK effectors are derived from CD4-, CD8-, NK1.1- precursors.

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1+, CD3+ lytically active subset. These lymphocytes were not derived from NK1.1+ precursors since NK1.1+ cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NK1.1+, CD3+ effectors were contained in a B cell-depleted CD4-, CD8-, NK1.1- splenic subset. Freshly obtained CD4-, CD8-, NK1.1- splenocytes were mostly CD3+, CD5+, B220-, had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4-, CD8-, NK1.1- splenocytes with IL2, for 6 days, resulted in the development of NK1.1+, CD3+, B220+ effectors 40% of which were CD5dim and 20-25% of which expressed TCR-V beta 8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1+ precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1+ cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1+ lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate that splenic NK cells do not express or acquire CD3; that the NK1.1+, CD3+ LAK effectors are derived from an NK1.1- precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.     

Characterization and utilization of a monoclonal antibody inhibiting porcine natural killer cell activity for isolation of natural killer and killer cells

A mAb, porcine NK-inhibitory mAb (PNK-I) that inhibits porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) has been developed. PNK-I acts at the level of the effector cell and inhibition of NK activity is independent of complement. Inhibitory effects are seen against various human and murine NK-susceptible targets. Addition of PNK-I antibody up to 60 min after assay initiation was effective at inhibiting NK activity. Furthermore PNK-I does not inhibit E:T conjugation and inhibits during the Ca2(+)-dependent phase of NK cytolysis. PNK-I Ag is present on virtually all PBL showing a bimodal distribution with 74% "dim" and 15% "bright" by flow cytometry. Monocytes and granulocytes stain with an intermediate intensity with greater than 90% and 95% staining positively, respectively. F(ab')2 fragments of PNK-I antibody show identical staining and functional activity as the whole molecule indicating that PNK-I acts independently of FcR. PNK-I immunoprecipitates molecules of molecular mass of 166, 155, 95 kDa under reducing and nonreducing conditions. PNK-I appears to be recognizing an epitope on a CD18 molecule. The CD18 molecule (beta-chain of CD11a,b,c) is ubiquitous on the surface of leukocytes and is implicated in a variety of cellular functions. Dim and bright populations were sorted and assessed functionally for NK and ADCC activity. It is demonstrated that PNK-I+ bright lymphocytes contain all detectable NK and ADCC activity in porcine PBL. Furthermore PNK-I+ bright lymphocytes contain the cytokine responsive NK cells capable of stimulation by IL-2, porcine NK-activating factor, and porcine natural killer-enhancing mAb. PNK-I+ dim cells were devoid of all baseline as well as inducible NK and ADCC activity. Giemsa stain of sorted populations show PNK-I+ bright cells containing the large granular lymphocytes whereas dim are devoid of these. Two color analysis show that PT4+ cells are PNK-I+ dim whereas PT8+ lymphocytes are divided between PNK-I+ bright and dim populations. Our results indicate that we are able to isolate all active as well as inducible NK and ADCC effector cells from porcine PBL based on relative Ag expression of CD18. Therefore quantitative as well as qualitative antigen expression is important in NK/ADCC-mediated cytotoxicity.     

A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody.

In the present study we describe a novel functional cell surface molecule, designated as Kp43, which is expressed among leukocytes by NK cells, TCR-gamma/delta + T lymphocytes, and some CD8+ CD56+TCR-alpha/beta + T cell clones. The Kp43 Ag is a 70-kDa disulfide-linked dimer, which migrates in SDS-PAGE under reducing conditions as a single 43-kDa band. Two-color immunofluorescence staining of fresh PBL revealed that only a fraction of CD16+, and of TCR-gamma/delta + T lymphocytes expressed the Ag. The analysis of TCR-alpha/beta + T cell clones showed that a small proportion (2 out of 20) weakly expressed Kp43 together with the CD8 and CD56 molecules. By immunoperoxidase staining of different tissues the anti-Kp43, reactivity was detected exclusively in lymphoid organs, where a minority of scattered cells was stained, and in some liver sinusoidal cells. Essentially all NK cells acquired Kp43 when stimulated with a B lymphoblastoid cell line. By contrast, the pattern of distribution of Kp43 remained stable upon in vitro culture of T-gamma/delta lymphocytes, thus delineating two subsets according to its expression. In lymphokine-activated killer populations, obtained by culturing either PBL or NK cells with high concentration of IL-2, most CD16+ and CD56+ cells became Kp43+. The Kp43-specific mAb inhibited the IL-2-dependent proliferative response of cultured NK and TCR-gamma/delta + T cells without affecting their non-MHC-restricted cytotoxicity. The partial inhibitory effect, which was mediated as well by pepsin digested F(ab')2 fragments, was lost upon reduction to Fab. The anti-Kp43 mAb did not interfere with the specific binding of IL-2 to its surface receptors. Altogether the data point out that the Kp43 dimer is involved in the regulation of the IL-2-dependent proliferative response of NK cells and a subset of TCR-gamma/delta + T lymphocytes.     

Suppressor cell function of human granular lymphocytes identified by the HNK-1 (Leu 7) monoclonal antibody

The HNK-1 (Leu 7) differentiation antigen defines a subpopulation of human granular lymphocytes with natural killer (NK) and K cell function. In this study, we investigated whether HNK-1+ cells, identified with the monoclonal antibody and purified with a fluorescence-activated cell sorter (FACS), could function as suppressor cells. The results demonstrated that purified HNK-1+ cells efficiently suppressed both PWM-induced IgG production by B cells and T cell proliferation in mixed lymphocyte reactions (MLR). Manifestation of this suppressor cell activity required immune complex activation and was partially sensitive to 2000 rad irradiation. This suppressor cell activity was predominantly mediated by a subset of HNK-1+ cells that have previously been shown to have maximum NK function and lack expression of the E rosette (ER) receptor and T cell antigens (e.g., T3 and T8). Thus, HNK-1+ER- cells suppressed a MLR by an average 52%; HNK-1+ER+ were one-half as efficient, causing an average 23% suppression. For comparison, we also examined the characteristics of Leu 2a+ suppressor T lymphocytes. In contrast to HNK-1+ cells, unactivated Leu 2a+ cells suppressed both B and T cell responses. This suppressor activity was not augmented by immune complex activation and was absolutely radio-sensitive in PWM assays. HNK-1+ cells, especially the HNK+ER- subset, can therefore mediate suppressor cell function in addition to their spontaneous cytotoxic function. Furthermore, some of their suppressor cell properties are distinct from those attributed to other types of suppressor lymphocytes.     

Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-leu-8

Previous studies have shown that monoclonal anti-Leu-8 antibody identifies functionally distinct subpopulations within both the Leu-2 (T8+) and Leu-3 (T4+) lineages of human T lymphocytes. We now report in detail on the tissue distribution of the Leu-8 antigen and on extensive functional studies of T cells subsets distinguished by their expression or lack of expression of this marker. Leu-8 is present on a wide variety of hematologic cells, including granulocytes, T and B lymphocytes, monocytes, and null or NK cells. Within lymph nodes and tonsils, Leu-8 is absent from both B and T cells within germinal centers but is present on nearly all paracortical lymphocytes. Leu-8 is present on most but not all EBV-transformed B cell lines, reflecting its presence on a subset of normal peripheral blood B cells. None of six malignant T cell lines tested were Leu-8+, whereas most circulating T cells are Leu-8+. Although standard immunoprecipitation techniques failed to demonstrate any specific bands on SDS polyacrylamide gels, the antigenic determinant recognized by anti-Leu-8 is protein or protein-associated, because brief treatment of target cells with pronase abrogated binding of anti-Leu-8. Both Leu-3+8+ and Leu-3+8- cells proliferated in response to several soluble antigens and to autologous and allogeneic non-T cells. Nonetheless, nearly all of the helper T cells for PWM- and AMLR-induced PFC were contained within the Leu3+8- subset. Optimal suppression of the PWM-induced PFC response required both Leu-2+8+ and Leu-2+8- cells, and irradiation of either subset with 3000 R abrogated the capacity of the recombined subsets to effect suppression. In contrast to help for B cell differentiation, both Leu-3+8+ and Leu-3+8- cells were capable of amplifying the development of allospecific T killer cells; precursor and effector T killer cells could be found within both Leu-2+8+ and Leu-2+8- subpopulations. The correlation between Leu-8 phenotype and selected immune functions of T cells (and B cells; see companion paper) indicates that anti-Leu-8 distinguishes important immunoregulatory T and B lymphocyte subsets in man.     

Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1.

Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo.     

Human natural cytotoxic lymphocytes: definition by a monoclonal antibody of a subset which kills an anchorage-dependent target cell line but not the K-562 cell line

A monoclonal mouse antibody (HNC-1A3) which defines a subset of human lymphocytes with natural cytotoxic activity was produced and studied. HNC-1A3+ cells represent 12 +/- 3% of peripheral blood mononuclear leukocytes. When sorted out using a fluorescence-activated cell sorter, they consist of 60% small lymphocytes, 35% large (predominantly agranular) lymphocytes, and 5% monocytes. They contain 30 +/- 6% E-rosette-forming cells, 6 +/- 1% OKT4+ cells, 17 +/- 6% OKT8+ cells and less than 2% OKT10+ or Leu-7 (HNK-1)+ cells. They are responsible for most of the natural cytotoxic activity against the MA-160 prostatic adenoma cell line but mediate an insignificant amount of cytotoxicity against the NK prototype target K562 cell line. Conversely, Leu-7+ cells which mediate most NK activity against K562 are weakly active against MA-160. Our data suggest a heterogeneity among leukocytes mediating natural cytotoxicity, with restricted specificities for the recognition sites on target cells.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

Identification and characterization of murine DNAM-1 (CD226) and its poliovirus receptor family ligands

The leukocyte adhesion molecule DNAM-1 (CD226) is a member of the immunoglobulin superfamily and constitutively expressed on the majority of CD4+ and CD8+ T lymphocytes, natural killer (NK) cells, monocytes/macrophages, and a subset of B lymphocytes. The poliovirus receptor (PVR; CD155) and its family member nectin 2 (CD112) have recently been identified as the ligands for DNAM-1. Interaction of DNAM-1 with the ligands induces NK cell- and CD8+ T cell-mediated cytotoxicity and cytokine secretion. However, in vivo function of the receptor-ligand interaction has remained unclear. Here, we identified murine DNAM-1 and PVR homologues that physically and functionally bind each other. We demonstrated that ligand binding of murine DNAM-1 induced a costimulatory signal in antigen-specific CD8+ T cells. These results should provide a useful animal model to explore a role of DNAM-1 in immune responses in vivo.     

Phenotypic and functional deficiency of natural killer cells in patients with chronic fatigue syndrome

Natural killer (NK)3 cells are large granular lymphocytes that appear to play a significant role in the host's defense against viral infection. We performed an extensive phenotypic and functional characterization of NK cells on 41 patients with the chronic fatigue syndrome (CFS), or "chronic active Epstein-Barr virus infection" syndrome, and on 23 age- and sex-matched asymptomatic control subjects in an attempt to further characterize this illness. These studies demonstrated that a majority of patients with CFS have low numbers of NKH1+T3- lymphocytes, a population that represents the great majority of NK cells in normal individuals. CFS patients had normal numbers of NKH1+T3+ lymphocytes, a population that represents a relatively small fraction of NK cells in normal individuals. When tested for cytotoxicity against a variety of different target cells, patients with CFS consistently demonstrated low levels of killing. After activation of cytolytic activity with recombinant interleukin 2, patients were able to display increased killing against K562 but most patients remained unable to lyse Epstein-Barr virus-infected B cell targets. Additional cytotoxicity experiments were carried out utilizing anti-T3 monoclonal antibody to block killing by NKH1+T3+ cells. These experiments indicated that the NK cell that appears to be responsible for much of the functional activity remaining in patients with CFS belongs to the NKH1+T3+ subset, which under normal circumstances represents only approximately 20% of the NK cell population.     

Heterogeneity of human T4+ inducer T cells defined by a monoclonal antibody that delineates two functional subpopulations

A monoclonal antibody termed anti-T4 that detected approximately 60% of peripheral blood T lymphocytes was shown to define the human inducer population. In the present study, we characterized three additional monoclonal antibodies, anti-T4A, anti-T4B, and anti-TQ1, that were reactive with a similar percentage of T lymphocytes. Anti-T4A, anti-T4B, and anti-T4 delineated identical cell populations, while those defined by anti-TQ1 differed in several respects: 1) Anti-TQ1 stained a minority (less than 7%) of thymocytes, whereas the other antibodies stained a majority (80%); 2) Anti-TQ1 reacted with 70 to 85% of T4+ lymphocytes, but also stained 50% of T cells within the T4- (T8+) cytotoxic/suppressor subset; 3) The antigen defined by anti-TQ1 was not restricted in its expression to T cells; it defined a fraction of normal B and null lymphocytes as well as non-T cell lines. In vitro studies indicated that the subpopulations of T4+ T lymphocytes delineated by anti-TQ1 were functionally distinct. Although T4+TQ1+ and T4+TQ1- T cells proliferated in an equal fashion to soluble antigen and alloantigen, only the T4+TQ1+ subset was responsible for maximal proliferation in autologous MLR. This T4+TQ1+ subset contained a population of lymphocytes reactive with the previously defined JRA autoantibody. In contrast, the T4+TQ1-, but not the T4+TQ1+, subset provided the majority of T cell help for B cell immunoglobulin production in a pokeweed-driven system. We conclude that the subpopulation of T4+ inducer cells responsible for maximal helper activity in T-B interactions is restricted to a minor subpopulation of T4+ lymphocytes.     

Natural killer-like cells in the sheep: functional characterization and regulation by pregnancy-associated proteins

Natural killer (NK) cells represent an important component of the innate immune system. In ruminants there are few reports regarding presence or characterization of NK cells. Although absence of expression of major histocompatibility complex proteins on ovine trophoblast makes it potentially a target for NK cells, little is known about regulation of NK cells by products of pregnancy in sheep. Objectives of the present study were to determine whether cells with characteristics of NK cells exist in preparations of ovine peripheral blood lymphocytes (PBL) and endometrial epithelial cells (EEC) and to determine regulation of such cells by two pregnancy-associated molecules with immunoregulatory properties (ovine uterine serpin [OvUS] and interferon-tau [IFN-tau]). Ovine PBL and EEC lysed a putative NK target cell, the BHV-1 infected D17 cell, and lysis by both types of cells was neutralized by antibody against a molecule called function-associated molecule (FAM) expressed on NK cells of several species. Moreover, inhibitors that interfere with perforin-mediated lysis blocked NK-like activity of PBL. The NK-like lytic activity of PBL and EEC was inhibited by OvUS, whereas ovine and bovine IFN-tau significantly enhanced NK-like activity of PBL. In conclusion, NK-like activity present in preparations of ovine PBL and EEC is mediated by FAM(+) cells, is dependent on processes that involve perforin processing, and is regulated by OvUS and IFN-tau. Inhibition of NK-like activity of PBL and EEC by OvUS is consistent with a role for OvUS in protecting the conceptus from maternal cytotoxic lymphocytes. Stimulation of lysis by IFN-tau implies the existence of other inhibitory mechanisms during early pregnancy to prevent NK cell-mediated destruction of the conceptus.     

The T cell triggering molecule Tp103 is associated with dipeptidyl aminopeptidase IV activity

Tp103 is a 103-kDa T cell activation molecule that defines an alternative activation signal for human T lymphocytes. It is absent from or present in only low amounts on resting T cells but is expressed strongly after activation. Cross-linking of Tp103 via a mAb CB.1 leads to triggering of functional activities in preactivated CD3+ T lymphocytes. By using mAb CB.1 in immunohistology we found that Tp103 is expressed in epithelial cells of various tissues, such as kidney, prostate, epidermis and on endothelia of liver, spleen, lungs, and most vessels, and in bile duct canaliculi in the liver. We found a carcinoma cell line expressing Tp103 and could precipitate a 110-kDa molecule from the surface of these cells. We considered several known molecules of similar distribution and molecular mass for identity with Tp103 and show here that Tp103 is probably identical to the proteolytic enzyme dipeptidyl aminopeptidase IV. When we purified Tp103 by affinity chromatography, typical dipeptidyl aminopeptidase IV activity copurified with Tp103. On activated T cells the enzymatic activity associated with Tp103 is expressed on the outside of the cell. We show that mAb CB.1 recognizes the same molecule as the anti-CD26 mAb anti-Ta1. The anti-Ta1 mAb was found to have T cell-activating activity too, but to differ in its requirements for triggering of T lymphocytes.     

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Murine natural killer cells express the Ly24 (Pgp-1) marker on their surface

The Ly24 (Pgp-1) marker is expressed on some, but not all, mature T lymphocytes. It has recently become apparent that the development of Ly24- T lymphocytes is dependent on the presence of an intact thymus and that virgin Ly24- T cells rapidly acquire this marker upon antigenic or mitogenic stimulation. Although natural killer (NK) cells can develop and function in the absence of an intact thymus, some NK cell subsets express certain markers normally associated with T lymphocytes. The experiments in this report were undertaken to determine if NK cells express Ly24 and whether such an expression could be used to delineate distinct NK cell subsets. We found that mature functional NK cells expressed the Ly24 marker as defined by the monoclonal antibody 9F3. Double-color fluorescence analysis using C57BL/6 splenocytes (whose NK cells express the NK1.1 marker) showed all the NK1.1+ cells to be Ly24+ as well. For C3H/HeN (an NK1.1- strain), double-color fluorescence analysis utilizing asialo GM1 and Ly24 revealed a distinct subset positive for both markers and containing most of the functional NK cell activity. Whereas the Ly24 marker did not illuminate an NK cell subset, these findings demonstrate that this determinant can be useful for the further characterization and isolation of NK cells.     

CD3/Ti gamma A: a functional gamma-receptor complex expressed on human peripheral lymphocytes

We have recently developed a mAb designated anti-Ti gamma A, which was found to immunoprecipitate from the well characterized CD3+ TCR alpha/beta- F6C7 fetal clone a CD3-associated disulfide-linked gamma-glycoprotein. This antibody recognizes approximately 3% of adult peripheral lymphocytes and delineates a CD2+ CD3+ TCR alpha/beta- CD4- NKH1- subset where expression of CD8 appears to vary widely from one individual to another. In the present study, we have used anti-Ti gamma A mAb to assess whether gamma-chains expressed on these adult lymphocytes are used as functional R. The two activities which have been associated thus far with TCR gamma+ cells, that is, IL-2-dependent proliferation and non-MHC-restricted cytotoxicity, were investigated here by using either resting or activated Ti gamma A+ lymphocytes. On the resting state, these cells (which appear as a very homogeneous population of granular lymphocytes) mediate little if any NK activity that could not be augmented by anti-Ti gamma A mAb. In contrast, after initial stimulation by PHA plus rIL-2 and subsequent culture in the presence of IL-2, activated Ti gamma A+ lymphocytes were strongly lytic against a series of conventional NK target cell lines. This cytotoxic function was either blocked or enhanced by anti-Ti gamma A mAb, depending upon experimental conditions. With respect to proliferation, it was possible to induce responses of resting Ti gamma A+ lymphocytes with antibody-coated CNBr beads only in the presence of exogenous IL-2, whereas, in culture, the same cells proliferated directly and secreted IL-2 after treatment by anti-Ti gamma A beads. Taken together, these data demonstrate that a major subset of circulating CD3+ TCR alpha/beta- lymphocytes use protein products of T cell gamma rearranging genes as functional R structures.     

Age-associated alterations in human natural killer cells. 2. Increased frequency of selective NK subsets

We have analyzed the peripheral blood lymphocytes from healthy volunteers (20 to 94 years) for the expression of natural killer (NK) cell surface markers, NK activity, and B-cell proliferative response. An increase (2- to 3.5-fold) in relative percentage and absolute number of lymphocytes expressing Leu-7 (HNK-1) or Leu-11a (CD 16) antigen was found in the elderly group (greater than 80 years) as compared to young adults (less than 40 years). A two-color immunofluorescence analysis revealed that the age-associated increment was both progressive and selective; the actual increase occurred in Leu-7+11a+ and Leu-7+11a- populations (subsets with variable and weak NK activity) but not in the Leu-7-11a+ (most active) subset. There is a corresponding decrease in the 7-11a- cells. The ratios of 7+11a+/7-11a+ and 7+11a-/7-11a+ cells doubled with advancing age. Linear regression analysis suggests that the 7-11a+ cells are highly preserved through human senescence and the ratio of 7+11a- cells to the most stable subset, 7-11a+, could expand nearly 100-fold from birth to old age. Further analysis of Leu-7+ cells for the coexpression of Leu-11c (an epitope of Leu-11a) confirmed a similar pattern of changes in 7+11c+ and 7+11c- NK subsets with advancing age. The frequency of Leu-11+ (epitopes 11a+ or 11c+), but not of the subsets of 7+ phenotype (7+11a- or 7+11c-), correlates well with the NK activity (spontaneous killing of K562 tumor cell line). The 7+11c+ cells may directly or indirectly be responsible for the increase in NK activity observed with a majority of aged donors. The inverse relationship observed between the mitogenic response of lymphocytes to pokeweed mitogen (PWM) and the initial frequency of 7+11a-, but not other phenotypes, raises a potential functional significance for the expansion of the 7+11a-(7+11c-) subset. These age-associated NK phenotypic changes provide a cellular basis for our observations on age-associated increase in NK activity and decrease in mitogenic response to PWM.