Anti-thoracic duct lymphocyte globulin stimulates human megakaryocytopoiesis in vitro

Anti-thoracic duct lymphocyte globulin (ALG) therapy is effective in patients with aplastic anemia. We examined the effect of ALG on human megakaryocyte progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) in vitro. Normal human bone marrow mononuclear cells (MNC) were cultured in plasma clots with varying concentrations of ALG or non-immunized horse IgG. After 12 days of culture, significant megakaryocyte colony formation was observed in cultures containing ALG but not in cultures containing non-immunized horse IgG. The peak stimulatory effect seemed to occur with 10-25 micrograms/ml of ALG. When marrow MNC, depleted of adherent and T cells, were cultured in plasma clots with ALG, its stimulatory effect on megakaryocytopoiesis decreased markedly. Finally, it was demonstrated that ALG stimulated marrow MNC to produce a factor stimulatory for CFU-Meg. The in vitro megakaryocytopoietic stimulatory effect of ALG may be related to its clinical efficacy in some patients with aplastic anemia.     

Human umbilical cord blood-derived stromal cells: Multifaceted regulators of megakaryocytopoiesis

It has been demonstrated that stromal cell precursors exist in human umbilical cord blood. After being cultured in vitro, these cells are called human umbilical cord blood-derived stromal cells (hUCBDSCs). However, the role of hUCBDSCs in hematopoiesis is still unclear. We have previously shown that hUCBDSCs are superior to human bone marrow stromal cells (hBMSCs) at enhancing the expansion of megakaryocyte colony forming units (CFU-Meg). Based on this observation, we postulated that hUCBDSCs might promote megakaryocytopoiesis. To test this hypothesis, we developed a megakaryocyte/hUCBDSC co-culture model and a hematopoietic microenvironment injury model in nude mice. We explored the ability and mechanisms by which hUCBDSCs promoted the proliferation of megakaryocytes in vitro, and we also explored their capacity to restore the hematopoietic microenvironment in vivo. As expected, hUCBDSCs were more effective than hBMSCs at enhancing the proliferation of megakaryocyte lines from HEL cells and restoring megakaryocytopoiesis in a hematopoietic microenvironment injury model in nude mice. Thrombopoietin (TPO) and stromal cell derived factor-1 (SDF-1) are two of the key factors underlying this capacity. We also found that gap junction intercellular communication (GJIC) between HEL cells and hUCBDSCs might be partially absent. Our data provide the first evidence that hUCBDSCs play a regulatory role during megakaryocytopoiesis, which might be important for designing treatments for patients with megakaryocytic injury.     

Depletion of NK by cellular immunoadsorption.

The binding of human natural killer (NK) cells to their tumor cell targets was investigated by using monolayers of sensitive target cell lines. Monolayers of K562 and HSB, a myeloid and T cell line, respectively, were prepared on poly-L-lysine-coated plastic tissue culture dishes and briefly fixed with 0.2% formaldehyde. Freshly isolated peripheral blood lymphocytes (PBL) were incubated on the monolayers. Nonadherent PBL were then removed, after gentle agitation, by decanting and gently washing the monolayer. They were tested, along with unseparated controls, for NK activity in a short-term 51Cr release assay. PBL that were nonadherent to a tested monolayer had only 20 to 60% of the control cytotoxic activity. Our results suggest that NK recognition sites on the effector lymphocytes were able to interact with reciprocal determinants on the target cell monolayers, resulting in selective loss of NK effector cells from the PBL population. The specificity of the NK effector-target interaction was investigated by testing the ability of each monolayer to remove activity against both targets. These data imply heterogeneity with regard to recognition structure within the NK effector population as well as among the target cells.     

Cytolytic activity of mononuclear cells, T-lymphocytes and monocytes of the peripheral blood in patients with lung cancer

A comparative analysis of cytotoxic activity of mononuclear cells (MNC) from peripheral blood, T-lymphocytes and monocytes from patients with lung cancer has been performed. It has been shown that in 27% of cases MNC, T-lymphocytes and monocytes lyse freshly isolated autologous and allogenic tumor cells. In all the patients examined the effector cells were active in respect to culture cell line of lung adenocarcinoma (ACL). The decrease in NK activity of the cell population enriched by T-lymphocytes in comparison to the control group (p less than 0.05) was noticed. MNC and T-lymphocytes, in contrast to monocytes, had high killer activity identified by lectin-dependent cytotoxicity technique. The activity of the effector cells does not depend on the morphological structure of the tumor, but decreases with the disease progression. The results of the experiments show that in patients with lung cancer peripheral blood lymphocytes and monocytes are essential, independently functioning effectors involved in antitumor defense.     

Role of serotonin in the regulation of human natural killer cell cytotoxicity

Addition of serotonin to mixtures of target cells and natural killer (NK)-enriched human mononuclear cells (MNC) in a 4-hr 51Cr-release assay strongly augmented NK cell cytotoxicity (NKCC) vs K562, Chang, or Molt-4 target cells. The effect was dose dependent at serotonin concentrations of 10(-4) to 10(-7) M, expressed at several effector to target cell ratios, and required the presence of accessory monocytes. A 5-HT1-specific receptor agonist, 8-OH-DPAT, mimicked the enhancing properties of serotonin with similar potency. Equimolar concentrations of the mixed 5-HT1/5-HT2 receptor antagonist cyproheptadine, but not the 5-HT2-specific antagonist ketanserin, completely blocked the serotonin-induced NKCC enhancement. Monocyte/NK cell mixtures incubated with serotonin for 1 hr produced a soluble factor that could enhance the cytotoxicity of autologous, NK-enriched cells depleted of monocytes, which did not respond to serotonin alone. The factor displayed no IFN or IL 2 activity as judged by the lack of antiviral activity and inability to support the growth of an IL 2-dependent cell line. In the presence of monocytes, serotonin (10(-5) M) was considerably more effective than human IFN-alpha or IFN-gamma at optimal concentrations and was about equally effective as IL 2 at a final concentration of 50 U/ml in a short-term NK assay. The potency and efficacy for serotonin were similar to that earlier reported for histamine in monocyte-containing effector cells. The NKCC-enhancing effect of serotonin was additive to that induced by IFN-alpha, IFN-gamma, or IL 2, but not to histamine. The presented data suggest an earlier unrecognized, serotonin receptor-mediated regulation of human NK cells.     

Propagation of cytotoxic effectors from chronic myeloid leukemia patients and cloning of cytotoxic T cells

Summary Cytotoxic cells (CTCs) generated from peripheral blood lymphocytes of 5 chronic myeloid leukemia (CML) patients in remission on stimulation with autologous leukemic cells and allogeneic lymphocytes (3-cell assay), were propagated in vitro in interleukin-2 (IL-2)-containing medium and periodic stimulation with autologous leukemic cells, for a period of 4 to 6 months. During this period, the cells were assessed for phenotype and for cytotoxic responses in a 4-h ⁵¹Cr release microcytotoxicity assay. The CTCs continued to show specific lysis of autologous leukemic cells and bone marrow (BM) cells. However, the nonspecific lysis of natural killer (NK) targets and the proportion of cells showing NK phenotype (HNK-1 antigen) increased progressively on cultivation in IL-2-containing medium. Therefore cells showing CD8 phenotype and specific cytotoxic function were segregated by cloning CTCs under the condition of limiting dilution in the presence of allogeneic feeder cells and IL-2-containing medium. Three cytotoxic T cell (CTL) clones expressing CD3+, CD8+, and HLA DR+ phenotypes were obtained from CTCs of 2 CML patients. These clonoid populations, maintained in IL-2-containing medium and periodic antigenic stimulation with autologous leukemic cells, showed specific lysis of autologous leukemic cells and BM cells even at lower (10:1) effector:target ratios. They did not kill K562 (erythroblastoid leukemic NK target cell line) cells and autologous phytohemagglutinin-induced blasts. These clones apparently functioned in an MHC-restricted manner as they did not lyse allogeneic CML cells which would also express a similar set of maturation antigens if sensitization was, as it appeared, against these antigens. Finally, interaction of autologous BM cells with CTL clones reduced the colony forming potential of BM cells only to the extent of 18%–30%. The results therefore indicate that such CTL clones can possibly be used in adoptive immunotherapy as they showed minimal BM toxicity.     

New insights into the regulation of human megakaryocytopoiesis

It is apparent that multiple cellular stages and biologic processes can be identified during megakaryocytopoiesis that are potentially subject to control by hematopoietic growth factors and marrow accessory cell populations. Two classes of megakaryocyte progenitor cells, the colony forming unit-megakaryocyte (CFU-MK) and the burst forming unit-megakaryocyte (BFU-MK), have now been detected in normal human bone marrow cells. The BFU-MK by virtue of the greater cellular content of its resultant colonies and the delayed time of appearance of these colonies appears to be a more primitive progenitor cell with a greater proliferative potential than the CFU-MK. A number of hematopoietic growth factors including megakaryocyte colony stimulating factor, (MK-CSF), recombinant erythropoietin (EPO) and granulocyte macrophage colony stimulating factor (GM-CSF) are each capable of increasing cloning efficiency of human megakaryocyte progenitor cells. It is presently unknown whether these factors act directly on the CFU-MK or whether they stimulate marrow accessory cells to elaborate growth factors that influence CFU-MK proliferation. In order to answer this question, the effect of these growth factors on the cloning efficiency of a human megakaryocytic cell line, EST-IU, was examined. Each of these factors was capable of increasing leukemia cell colony formation. One can conclude from these studies that MK-CSF, EPO, and GM-CSF act directly on cells of the megakaryocytic lineage. The physiologic significance of the lineage nonspecific effects of EPO and GM-CSF on megakaryocytopoiesis is yet to be determined. On the basis of these observations, a model of human megakaryocytopoiesis was suggested. Several factors appear able to influence multiple steps in megakaryocytic development, whereas others influence only specific stages or cellular events occurring during megakaryocytopoiesis.     

Maturation and regulation of megakaryocytopoiesis.

The in vitro cloning technique for detecting megakaryocyte precursor cells was employed to compare stimuli known to influence megakaryocytopoiesis. Preparations of thrombopoietic stimulating factor (TSF) did not directly stimulate the growth of megakaryocyte colonies (CFU-m) but increased the frequency of CFU-m when TSF was added to the cultures with a constant amount of megakaryocyte colony stimulating factor. Platelets or platelet homogenates did not influence the frequency of CFU-m or the size of individual colonies. Analysis of cell surface properties of megakaryocytes obtained either by isolation from bone marrow or from in vitro colonies revealed species differences. The possibility that megakaryocytopoiesis and platelet release are regulated both within the marrow as well as by humoral factors is discussed.     

Enhancement of human natural killer cell cytotoxicity by serotonin: role of non-T/CD16+ NK cells, accessory monocytes, and 5-HT1A receptors

Serotonin (10(-4) - 10(-7) M) augmented natural killer cell cytotoxicity (NKCC) of human CD16+/non-T lymphocytes in vitro against the NK-sensitive target cells K 562 erythroleukemic, Molt-4 lymphoma, Chang liver cells, and against EBV-transformed Daudi B-lymphoblastoid target cells by a mechanism of action involving a prostaglandin-and IL-1-independent accessory function of monocytes. No evidence for the production of intermediary, NK-enhancing cytokines by serotonin was obtained, suggesting a cell-to-cell-mediated interaction between monocytes and NK cells as a plausible mechanism of action for the NK-augmenting effect. Monocytes recovered by counter-current centrifugal elutriation but not monocytes recovered by adherence reconstituted the effect of serotonin when added to nonadherent NK cells. NK-enhancing effects of serotonin were mimicked by two 5-HT1A-type serotonin receptor agonists, 8-OH-DPAT and (+)-ALK. The development of NKCC in response to serotonin could be resolved into (i) an induction phase, dependent on the presence of accessory monocytes and serotonin, and (ii) an effector phase, independent of the presence of monocytes or serotonin. Serotonin-activated MNC continued to exert augmented cytotoxicity for at least 8 hr after the removal of serotonin and monocytes. In several experiments, serotonin-activated NK cells killed greater than 75% of K 562 target cells even at low effector to target cell ratios and low baseline NKCC. We suggest that serotonin may have a role in nonspecific tumor defence by regulating an earlier unrecognized interplay between monocytes and NK cells.     

In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

Phorbolester-treated human lymphocytes are susceptible to natural killer cell-mediated cytolysis

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was found in this study to render normal human lymphocytes susceptible to allogeneic and autologous natural killer (NK) cell lysis. Both T and non-T cells became susceptible after culture for 3 days in the presence of 1 ng/ml TPA. Effector cells were nonadherent mononuclear cells of low density, enriched for large granular lymphocytes and HNK-1+ cells. Activation of effector cells with interferon increased lysis of TPA-treated lymphocytes. The present system may provide a new model to study NK cell target structures.     

Effect of histamine and histamine antagonists on natural and antibody-dependent cellular cytotoxicity of human lymphocytes in vitro

The in vitro effect of histamine and its antagonists, cimetidine and clemastine fumarate, on natural killer (NK) and antibody-dependent cellular Cytotoxicity (ADCC) activities of human lymphocytes was investigated. The histamine 1 (H1) antagonist, clemastine fumarate, and the histamine 2 (H2) antagonist, cimetidine, but not histamine alone, inhibited the NK and ADCC activities of lymphocytes when added directly to the mixture of effector and target cells in a ⁵¹Cr-release assay. This inhibition was proportional to the concentration of drugs added and was observed at various effector to target ratios against several targets. H1 and H2 antagonists also inhibited NK activities of T cells as well as Percoll-separated, NK-enriched effector cells. The inhibition was significantly reversed by histamine. In target binding assays, clemastine fumarate and cimetidine also decreased the target binding capacity of effector lymphocytes. Further, PBL precultured with histamine (10^?3–10^?4M) for 24 hr showed a significant decrease in their NK and ADCC activities. In coculture experiments, PBL precultured with histamine suppressed the NK activity of normal autologous effector lymphocytes. PBL precultured with histamine showed an increased number of OKT8⁺ cells, as estimated using monoclonal antibodies. The suppression of Cytotoxicity was not due to either direct toxicity, steric hindrance, crowding, or cell death, but by functionally viable suppressor cells. An immunoregulatory role for histamine in NK and ADCC reactions is proposed.     

Stimulation with autologous lymphoblastoid cell lines: lysis of Epstein-Barr virus-positive and -negative cell lines by two phenotypically distinguishable effector cell populations

Human lymphocytes, stimulated in vitro for 6 days with x-irradiated or glutaraldehyde-treated autologous Epstein-Barr (EB) virus-transformed lymphoblastoid cell lines (LCL), are cytotoxic for autologous and allogeneic EB+ LCLs as well as for several EB- cell lines that are also susceptible to lysis by interferon-activated natural killer (NK) cells. To determine whether the apparent nonspecific lysis mediated by LCL-stimulated cells is due to a mixture of effector cells directed against different target cells, advantage was taken of our recent finding that monoclonal antibody OKT8 reacts with human cytotoxic T lymphocytes but not with NK cells or NK-like cells generated in mixed leukocyte cultures. The depletion of OKT8+ cells from LCL-stimulated cultures by treatment with OKT8 and complement abolished or markedly depleted cytotoxicity against all EB+ target cells tested, whereas cytotoxicity against EB-, NK-sensitive cell lines including K562, MOLT-4 and HSB-2 was not or only minimally reduced. These results indicate that stimulation with autologous LCL results in the generation of OKT8+ cytotoxic T lymphocytes that lyse EB virus-transformed LCL and OKT8- NK-like cells that lyse EB-, NK-sensitive cells.     

Effect of 6-hydroxydopamine on murine hematopoietic stem cells: enhanced cytotoxicity on megakaryocyte colony forming units

We examined the effect of catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on murine committed megakaryocyte progenitor cells, the megakaryocyte-colony forming unit (CFU-Meg). More mature cells of the megakaryocyte series have the capacity for active uptake of catecholamines, and we speculated that the CFU-Meg would also take up 6-OHDA and be selectively killed. CFU-Meg were much more sensitive to the effects of this agent than were granulocyte-macrophage colony forming units (CFU-GM) or spleen-colony forming units. Co-incubation with catalase, which would destroy hydrogen peroxide generated extracellularly by the autoxidation of 6-OHDA, ablated 6-OHDA toxicity towards CFU-GM, but also significantly reduced the effect on CFU-Meg. Mazindol, a selective dopamine uptake inhibitor did not alter 6-OHDA effect on either CFU-Meg or CFU-GM. Finally, CFU-Meg were no more sensitive to incubation with hydrogen peroxide than were CFU-GM. These data suggest that CFU-Meg, unlike their more mature progeny, do not actively concentrate 6-OHDA, and the excess toxicity of this agent towards CFU-Meg is probably due to increased sensitivity to autoxidation products of 6-OHDA, other than hydrogen peroxide, generated extracellularly.     

Surface receptors and immune activity of purified human circulating mononuclear cells. III. The mechanisms of lysis, by lymphocytes and monocytes, of target cells in the lymphocyte-dependent and monocyte-dependent ADCC, NK, NOCC, and MICC cytotoxic reactions

The abilities of unfractionated mononuclear cells (MNC), monocytes (98-99% pure), and lymphocytes (98-99% pure) to carry out the lysis of target cells in the ADCC, NK, NOCC, and MICC assays were compared. Lymphocytes by themselves were able to lyse the CRBC (ADCC), K-562 (NK), and RRBC (MICC) target cells. The monocytes were very effective in the lysis of the CRBC (MICC) target cells. However, the lysis of two other target cells--RRBC (NOCC) and HRBC (ADCC)--required the simultaneous presence of both lymphocytes and monocytes in order to effect optimal lysis. Soluble factor(s) secreted by the cytotoxic cells capable of lysing the target cells were detected only in the NK assay. The activity of the soluble cytotoxic factor (NKCF) was only 25-40% of that exhibited by the cytotoxic NK cells and it was secreted by the cytotoxic cells after 48 hr of culture and not 24 hr of culture which is the usual assay condition. The NKCF was cytotoxic only to the NK target cells and not to the target cells used in the ADCC, NOCC, and MICC cytotoxic assays. Different classes of lymphocytes were cytotoxic in the monocyte-independent assays [ADCC (CRBC), NK (K-562), and MICC (RRBC)]. The null lymphocytes and the T lymphocytes were the primary cytotoxic cells in the ADCC and MICC assays, respectively, whereas the T, B, and null cells were almost equally cytotoxic in the NK assay. With respect to the monocyte-dependent assays [ADCC (HRBC), NOCC (RRBC), and MICC (CRBC)], the cytotoxic activity of any one class of lymphocytes failed to approach that of the unfractionated MNC. The T cells were the most cytotoxic; the B cells exhibited limited cytotoxic activity in only the ADCC assay and the null cells showed no cytotoxic activity. However, the combination of T and non-T cells and, to a lesser extent, T and B cells, exhibited much greater cytotoxic activity than the individual cells and together were as cytotoxic as the unfractionated MNC. It is concluded that, depending upon the selection of the target cells, lysis in the ADCC, NK, NOCC, and MICC assays may be effected by lymphocytes only, by monocytes only, by both monocytes and lymphocytes, or as a result of lymphocyte-monocyte collaboration. In the latter instance more than one class of lymphocytes must be present in order for maximum cytotoxic activity to be expressed.     

NK cell lysis of HIV-1-infected autologous CD4 primary T cells: requirement for IFN-mediated NK activation by plasmacytoid dendritic cells

In vivo, several mechanisms have been postulated to protect HIV-1-infected cells from NK surveillance. In vitro, previous research indicates HIV-1-infected autologous CD4(+) primary T cells are resistant to NK lysis. We hypothesized that NK lysis of HIV-1-infected target cells would be augmented by the presence of accessory cells and/or accessory cell factors. In this study, we show that stimulation of plasmacytoid dendritic cells (PDC) with the TLR9 agonist, CpG ODN 2216, triggered NK lysis of HIV-1-infected autologous CD4(+) primary T cells. PDC-stimulated NK lysis was dependent upon MHC class I (MHC-I) down-regulation on infected cells, and primary HIV-1 isolates that exhibited enhanced MHC-I down-regulation were more susceptible to NK-mediated lysis. PDC-stimulated NK lysis of HIV-1-infected autologous CD4(+) primary T cells was blocked by neutralizing Abs to type 1 IFN and was perforin/granzyme dependent. Overall, our data suggest that HIV-infected cells are not innately resistant to NK lysis, and that exogenous NK stimulation derived from PDC can trigger NK cytotoxicity against HIV-1-infected autologous CD4(+) primary T cells.     

Differentiation of NK-like cells from OKT3-, OKT11+, and OKM1+ small resting lymphocytes by culture with autologous T cell blasts and lymphokine

NK-like cells have been generated in vitro from a resting lymphocyte population of PBMC by 8 days culture with mitomycin C-treated autologous T cell blasts and lymphokine. The responder lymphocyte population was purified to the extent that it lacked classical NK cells, and lacked the precursors of MLC-derived NK-like cells and of lymphokine-activated killer cells. The NK-like cells were not generated when the responder lymphocytes were cultured with either T cell blasts or lymphokine alone. Thus, at least two signals are required for their activation. Metabolically inactive T cell blasts plus lymphokine were effective in stimulating the generation of NK-like cells, suggesting that a membrane determinant on the T cell blasts was involved in activation. The phenotype of the NK-like cells and their precursors was analyzed by monoclonal antibody and complement treatment. The phenotype of both precursor and effector cells was OKT3-, OKT11+, and OKM1+, with a distinct pattern of reactivity with OKT8 and Leu-7 for each individual donor tested. The NK-like cells were morphologically large granular lymphocytes, and they killed a variety of target cells. These studies show that signals provided by autologous T cell blasts and lymphokine are essential in triggering the differentiation of NK-like cells from appropriately purified resting lymphocytes. This mechanism of activation could occur in vivo, leading to the generation of NK cells subsequent to an antigen-specific T cell response.     

Human Antigen-Presenting Cells: Characterization of the Cells in the T-Lymphocyte Proliferative Response

Human antigen-presenting cells (APC) which present the antigen to T lymphocytes resulting in a T-lymphocyte proliferative response were found among peripheral mononuclear cells (MNC), by employing purified protein derivative (PPD) as soluble antigen. To assess the adherence capacity of human antigen-presenting cells, MNC were separated by plastic Petri dishes or nylon wool columns. Plastic nonadherent cells were almost equivalent to unseparated cells in antigen-presenting ability. Plastic adherent cells, however, showed better antigen-presenting ability than unseparated cells. On the other hand, cells passed over nylon wool columns showed essentially no ability to present PPD to T lymphocytes. Removal of phagocytic cells by carbonyl iron resulted in about 50–70% reduction in antigen-presenting ability. Carrageenan, which is known to be toxic to macrophages, had no effect on APC. By using both rabbit anti-human Ia-like antiserum and alloantiserum specific for HLA-DR phenotype and complement, it was shown that APC possessed Ia-like antigens, whereas they did not bear surface immunoglobulins. These results indicate that the human APC is probably a cell in the monocyte-macrophage lineage. Allogeneic MNC were used as APC in order to determine whether any genetic restriction exists between MNC as APC and responding T lymphocytes. Optimal stimulation was shown to require identity of mixed leukocyte reaction (MLR)-activating determinants between APC and T lymphocytes. It is, however, obscure whether an HLA-D region restriction exists in these combinations because PPD-pulsed allogeneic MNC lost their ability to elicit even MLR. It is possible that this failure to elicit MLR was caused by T lymphocytes among the MNC used as APC.     

T11/CD2 activation of cloned human natural killer cells results in increased conjugate formation and exocytosis of cytolytic granules

The T11 (CD2) antigen has been found to be an alternate pathway for antigen-independent activation of resting T cells. T11 triggering also results in activation of NK cells and enhancement of their cytolytic function. The present studies were carried out to further define the mechanisms whereby cytotoxicity is enhanced after T11 activation. A series of clonal human NK cell lines were analyzed after incubation with monoclonal anti-T112 and anti-T113 antibodies specific for different epitopes of the CD2 protein. Anti-T112/3 triggering resulted in increased cytotoxicity against a variety of target cells. Similar results were obtained with F(ab')2 fragments of anti-T112/3, indicating that this effect was not mediated through binding of FcR. The induction of cytotoxicity was found to be associated with increased formation of effector cell-target cell conjugates and with release of secretory granule-localized 35S-labeled proteoglycans. Both enhanced conjugate formation and cytotoxicity could be blocked by anti-lymphocyte function-associated antigen (LFA-1) mAb. Ultrastructural analysis of NK cells after T11 activation demonstrated increased adherence of effector cells to targets and other NK cells as well as a directional reorientation of cytoplasm and intracellular granules toward the area of contact between cells. Discharge of granules occurred into pockets bounded by closely apposed plasma membranes. In the presence of anti-LFA-1 and anti-T112/3, the close apposition and formation of pockets between effector cells and target cells did not occur but the cells exocytosed their intracellular granules. T11 activation of NK cloned cells also resulted in the formation of the homotypic conjugates and autocytotoxicity. As seen with resistant allogeneic targets, autocytotoxicity was mediated by F(ab')2 fragments of T112/3 antibodies and could be blocked by anti-LFA-1 antibody. Ultrastructural analysis of NK cloned cells after T11 activation confirmed the presence of homotypic conjugates with reorientation of effector cells toward one another and discharge of cytolytic granules into pockets formed between NK cloned cells. Taken together, these results indicate that T11-induced cytolytic function of NK cells is, in part, mediated through increased binding of effector cells and targets and that enhanced conjugate formation is at least in part mediated by the LFA-1 antigen. In addition, T11 activation results in the triggering of the cytolytic mechanism of NK cells and the exocytosis of cytolytic granules and their constituents.     

Regulation of cytolytic activity in CD3- and CD3+ killer cell clones by monoclonal antibodies (anti-CD16, anti-CD2, anti-CD3) depends on subclass specificity of target cell IgG-FcR

Anti-CD3 MAb can inhibit MHC-restricted cytolytic activity of CD3+ mature cytotoxic T cells. In particular effector-target cell combinations, however, anti-CD3 MAb enhance or induce cytolysis by cross-linking CD3+ effector and IgG-FcR+ target cells. Virtually all natural killer (NK) cells or NK cell-derived clones are CD3-4-8- but do express CD2 and CD16 (IgG-FcR) antigens. We have studied how these cell surface molecules are involved in the regulation of cytolytic activities. The addition of anti-CD2 MAb to effector and target cells was found to induce conjugate formation of the IgG-FcR+ target cells with the effector cell and nonspecific cytolysis of, for instance, the P815 mouse mastocytoma cells. Enhancement or induction of conjugate formation and cytolysis of IgG-FcR+, P815, U937, and Daudi cells was also accomplished by using anti-CD16 MAb (e.g., Leu-11c (B73.1) or CLB Fc-gran 1 (VD2) MAb). Some human and mouse tumor cell lines (K562, P815, and U937) appear to express distinct types of IgG-FcR, showing different affinities for distinct subclasses of MAb (e.g., IgG1, IgG2a), but another line (Daudi) expresses only one type of IgG-FcR preferentially binding IgG1 MAb. Here we demonstrate that IgG-FcR on the effector cells can act as activation sites because anti-CD3 as well as anti-CD16 MAb of IgG1 and IgG2a subclasses can induce lytic activity of target cells bearing the relevant IgG-FcR. These data demonstrate that induction of conjugate formation and cytolysis by MAb occur when the target cells bear IgG-FcR with "specificity" for those MAb. Thus, besides via CD3, cytolytic activity by mature T and NK cells also can be induced via the CD2 and CD16 antigens on these cells.