Natural killer cell and granulocyte Fc gamma receptor III (CD16) differ in membrane anchor and signal transduction

CD16 is a low affinity Fc gamma R III expressed on granulocytes, macrophages and large granular lymphocytes, the mediators of antibody-dependent cellular cytotoxicity and NK. The occupancy of CD16 by aggregated IgG on large granular lymphocytes induces expression of activation markers, release of inflammatory mediators and triggering of effector functions such as antibody-dependent cellular cytotoxicity. Recently we and others described that CD16 is anchored to the membrane of granulocytes via a phosphatidylinositol glycan moiety. Here we show that the CD16 molecule expressed on NK cells, cultured monocytes, and lung macrophages is not phosphatidylinositol glycan moiety anchored. It is not released with phosphatidylinositol-specific phospholipase C, and after removal of N-linked carbohydrate is 5 to 7 kDa larger than the granulocyte CD16 molecule, strongly suggesting the presence of transmembrane and cytoplasmic protein domains. Redirected killing of hybridoma targets expressing anti-CD16 surface Ig shows that NK cell CD16 is unable to do so. These findings demonstrate that NK cell and granulocyte CD16 have different membrane anchors and indicate that the type of membrane anchor is an important biologic mechanism for regulating the functional capacity of surface receptors.     

Involvement of CD28 in MHC-unrestricted cytotoxicity mediated by a human natural killer leukemia cell line.

NK cells and certain CTL can recognize and lyse targets without restriction by the MHC. NK cells do not express CD3/TCR complexes and the membrane receptors participating in MHC-unrestricted cytotoxicity are largely unknown. We demonstrate that YT2C2, a human NK leukemia cell line, expresses the CD28 differentiation Ag and can spontaneously lyse both murine and human cell lines expressing B7, a B cell- activation Ag that is a ligand for CD28. The participation of CD28/B7 interactions in MHC-unrestricted cytotoxicity mediated by YT2C2 cells was demonstrated by correlation of target sensitivity with levels of B7 expression, inhibition of cytotoxicity by anti-CD28 or anti-B7 mAb, and by making both murine and human cell lines susceptible to YT2C2-mediated lysis by genetic transfection with expression vectors containing B7 cDNA. However, CD28/B7 interactions alone were insufficient to initiate cytotoxicity. mAb inhibition experiments and selection of CD54- (intercellular adhesion molecule-1) deficient B cell targets indicated that CD11a/18 (lymphocyte function-associated Ag-1) also cooperated in CD28/B7-dependent cytotoxicity. The requirement for both CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interactions in YT2C2-mediated MHC-unrestricted cytotoxicity was confirmed by demonstrating that efficient lysis of murine L cells required cotransfection with both B7 and intercellular adhesion molecule-1. These findings support the concept that MHC-unrestricted cytotoxicity may not be due to a unique receptor, but may result from interactions between an appropriate array of "adhesion" molecules with their ligands.     

Functional CD2 mutants unable to bind to, or be stimulated by, LFA-3.

To define epitopes on the CD2 (T11, the T cell erythrocyte receptor) molecule that are necessary for interaction with lymphocyte function-associated Ag-3 (LFA-3), we have expressed the human wild-type CD2 cDNA and mutant CD2 cDNA in a murine Ag-specific T cell hybridoma that responds to human HLA-DR Ag. Here we have expressed mutations at amino acid 91 and 92 of CD2 in the T cell hybridoma. The mutated CD2 molecules were functional in that pairs of anti-CD2 mAb that continued to bind were able to stimulate IL-2 production by the hybridomas. However, CD2 mutants with either the 91 or 92 amino acid substitution had lost the ability to bind to or be activated by either SRBC, which bear an LFA-3 homologue, or by murine L cells expressing human LFA-3. Unlike hybridomas expressing the wild-type CD2 molecule, there was no enhanced response to Ag stimulation. Taken together, these data suggest that the mutated CD2 molecules were no longer able to bind to, or to utilize, LFA-3 for activation. We have previously demonstrated that a mutation at amino acid 51 of CD2 results in loss of binding to LFA-3. Whether these two regions of CD2, discrete and separable by amino acid sequence, form one or more binding sites for LFA-3 remains to be determined.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Antigen-specific and antibody-mediated growth inhibition of suppressor T cell hybridomas. Roles of H-2 and the CD3-T cell receptor-alpha/beta complex

Eight different Ts cell hybridomas (including inducer (Ts1) and effector (Ts3) suppressor cells) specific for the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were tested for their ability to respond to Ag or anti-CD3 antibody in a growth-inhibition assay. Results suggest that the expression of the TCR-CD3 complex on Ts hybridomas is required for the Ag or anti-CD3-mediated growth inhibition. One of the CD3+, Ts hybridomas (CKB-Ts3-9.H3) was tested in detail; this CD4- effector suppressor cell hybridoma showed specific inhibition of growth in the presence of NP or NIP-coupled protein conjugates but not in the presence of other irrelevant hapten-protein conjugates. In addition, growth of this hybridoma was specifically inhibited by anti-CD3 and anti-TCR-alpha/beta antibodies but not by control hamster antibodies. In order to study the role of MHC molecules in Ag-mediated growth inhibition, Ts cell hybridomas were incubated with Ag (NP-keyhole limpet hemocyanin) in the presence of spleen cells from various H-2 congenic strains. The results suggest that the Ts hybridomas that express donor Ts-derived TCR beta-chain recognize Ag in an MHC-restricted manner, whereas the two Ts3 hybridomas that utilize BW5147-derived TCR-beta recognize Ag in H-2 unrestricted way. Co-incubation of anti-CD3 and anti-TCR-alpha/beta antibodies with specific Ag enhanced the Ag-mediated growth inhibition, whereas anti-LFA-1 antibody completely blocked the Ag-mediated effect. The combined data suggest that, like Th hybridomas, expression of CD3-associated-TCR complex is essential for the Ag responsiveness of Ts cell hybridomas.     

Relative contribution of CD2 and LFA-1 to murine T and natural killer cell functions.

We have examined the functional property of murine CD2 as an intercellular adhesion molecule by using five anti-murine CD2 mAb which were classified into two groups according to their mutual competition in binding to cell surface CD2. Hamster fibroblasts transfected with murine CD2 cDNA exhibited increased conjugate formation with a murine mastocytoma P815 which expresses the putative murine LFA-3 mRNA detected by cross-hybridization with human LFA-3 cDNA under conditions of low stringency. This increase in conjugate formation was abrogated by both groups of anti-CD2 mAb, although some differences in the extent of inhibition were observed at lower concentrations of the mAb. We then examined the involvement of CD2 in several murine T cell responses by using these mAb to abrogate CD2-mediated cellular interactions. Anti-CD2 mAb significantly inhibited mitogenic T cell responses induced by suboptimal doses of Con A and PHA. In the allogenic MLR response and in the Ag response of two KLH/I-Ak-specific Th cell clones, the inhibitory effect of anti-CD2 mAb was also greatest under suboptimal conditions, i.e., with lesser doses of the Ag. These results indicate that the contribution of CD2 as an accessory molecule is variable, depending on the Ag dose used for stimulation, and they suggest that CD2 is involved in the Ag response of murine T cells under the physiologic conditions where only a limited amount of Ag is available. We next examined the contribution of CD2 to MHC-restricted cytotoxicity by CTL and to MHC-unrestricted cytotoxicity by NK and lymphokine-activated killer cells. Only a marginal inhibition by anti-CD2 mAb alone was observed. Anti-lymphocyte function-associated Ag (LFA)-1 mAb alone exhibited greater inhibitory effects than anti-CD2 mAb in all of the cases tested. In most cases, however, substantial levels of cytotoxicity remained, even in the presence of both anti-CD2 and anti-LFA-1 mAb. These results indicate a minor contribution of CD2, as compared with LFA-1, to cytotoxicity by murine CTL, NK cells, and lymphokine-activated killer cells, and they reveal the presence of undefined cellular interaction pathways other than those mediated by CD2 and LFA-1.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

Partial deletions of the cytoplasmic domain of CD2 result in a partial defect in signal transduction

CD2 (T11, the T cell erythrocyte receptor or the SRBC receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, appears to play a role in T lymphocyte adhesion, signal transduction, and differentiation. Pairs of anti-CD2 mAb induce T cell proliferation, suggesting that CD2 may be an Ag-independent pathway of T cell activation. We have expressed the human CD2 and a number of cytoplasmic domain deletion mutants of CD2 in an Ag-reactive murine hybridoma. We have previously shown that a cytoplasmic domain deletion mutant, CD2 delta B, in which the carboxyl-terminal 100 amino acids have been deleted, is no longer capable of signaling through CD2. Here we have expressed a second cytoplasmic domain deletion mutant, CD2 delta S, in which the terminal 41 amino acids have been removed, including the region with greatest conservation between the mouse, rat, and human species. CD2 delta S+ hybridomas were able to respond to Ag and to LFA-3 plus an anti-CD2 mAb. Although the CD2 delta S+ hybridomas responded comparably to the wild-type CD2+ hybridomas to certain pairs of anti-CD2 mAb (e.g., MT110 + 9-1 mAb), these CD2 delta S+ hybridomas were markedly deficient in their ability to respond to other pairs of stimulatory anti-CD2 mAb (e.g., 9.6 + 9-1 mAb). These data suggest that the cytoplasmic domain may have several functional regions, as partial deletions of the cytoplasmic domain appear to result in partial defects in signal transduction.     

Human natural killer cell adhesion molecules. Differential expression after activation and participation in cytolysis.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.     

CD16 on human gamma delta T lymphocytes: expression, function, and specificity for mouse IgG isotypes.

We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human gamma delta T cells. CD16 is expressed by the majority of gamma delta T cells in peripheral blood and by part of the gamma delta T cell clones. The amount of CD16 expressed on gamma delta T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated gamma delta T cells. Like CD16 on CD3- CD16+ natural killer (NK) cells, CD16 on gamma delta T cells can act as an activation site triggering cytotoxic activity. CD16+ gamma delta T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of gamma delta T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of Fc gamma R+ target cells by CD16+ gamma delta T cell clones. The mouse IgG-isotype specificity of CD16 on gamma delta T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3- CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both gamma delta T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.     

CD27-mediated activation of murine NK cells

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-gamma production of freshly isolated NK cells and enhanced the proliferation and IFN-gamma production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-gamma-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Role of CD16 (Fc receptor III) and interleukin-2 in the reactivation of natural killer cells after inhibitory target cell contact.

Contact with natural killer (NK)-resistant monolayer targets is an inhibitory signal to NK cells. In this study, we have analyzed the effect of such effector/target cell interactions on the CD16 (FcRIII) expression on lymphocytes and the role of CD16 and interleukin-2 (IL-2) in the reactivation of their cytolytic machinery. Coculturing peripheral blood mononuclear cells with NK-resistant monolayer cells did not change the percentage of CD 16-positive effector cells, although this treatment effectively inhibited their cytotoxicity against NK-sensitive targets. The inhibited effector cells partially regained their activity by incubating for 24 h in medium supplemented with 10% fetal calf serum (FCS), whereas human albumin-, newborn calf serum- or human AB serum-supplemented media had no reactivating effect. Monoclonal class IgG1, IgG2a and IgM anti-CD16 antibodies [Abs; 3G8, CLB-CD16 (CLB-FcR gr1) and Leu 11b], and normal rabbit IgG (NR-IgG) prevented the FCS-mediated reactivation of cytotoxicity, whereas nonreactive control Abs significantly enhanced it. The detection of the CD16 antigen by the monoclonal anti-CD16 Abs Leu 11a and Leu 11c was blocked by the above anti-CD16 Abs and NR-IgG, while the expression of other NK cell-associated surface molecules (CD2, CD56) remained unchanged. Mere blocking of CD16, using a short-term incubation with anti-CD16 Abs, had an insignificant effect on endogenous NK activity, suggesting that CD16 is involved in NK cell (re)activation rather than in the killing process itself. In the presence of IL-2, inactivated effector cells also regained their killing activity. The IL-2-induced reactivation was not inhibited by anti-CD16 Abs. The results suggest that FCS-derived factors and soluble nonreactive immunoglobulins enhance the NK activity of down-regulated effector cells via CD16, and that CD16 and IL-2 receptors represent alternative independent pathways of NK cell reactivation.     

Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways

NK cells lyse certain tumor cell targets but the effector cell surface molecules responsible for this reactivity remain uncertain. The allotypic NK1.1 Ag is the most specific serologic marker on murine cells that display non-MHC-restricted cytolysis of tumor cell targets, but no function has been previously ascribed to this Ag. In this report, we demonstrate that, in the presence of a mAb specific for the NK1.1 Ag (mAb PK136), freshly isolated and IL-2-activated NK cells from C57BL/6 mice can be induced to lyse an otherwise resistant target cell, Daudi. This phenomenon is effector and mAb specific because NK cells derived from BALB/c mice do not express the NK1.1 Ag and cannot be triggered by mAb PK136. We demonstrate that IL-2 activated but not freshly isolated NK cells express the Ly-6 and VEA Ag, originally described as T cell activation Ag. Moreover, mAb specific for Ly-6 and VEA induce target cell lysis by IL-2 activated but not freshly isolated NK cells. These mAb effects are specific, concentration dependent, and display kinetics that are similar to spontaneous cytolysis of NK-sensitive targets. The Fc portion of the activating antibodies and only FcR bearing target cells participate in mAb-induced activation, consistent with the mechanism of redirected lysis. Finally, analysis of Daudi cells transfected with beta 2-microglobulin gene demonstrate that the expression of MHC class I Ag by the target cell does not affect its sensitivity to mAb-induced lysis by NK cells. These data demonstrate that the NK1.1 Ag is functionally active on both freshly isolated and IL-2-activated NK cells and that IL-2-activated NK cells possess additional pathways of specific stimulation.     

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

Participation of the CD27 antigen in the regulation of IL-2-activated human natural killer cells.

The CD27 Ag is expressed by the majority of resting T lymphocytes and appears to play a crucial role in T cell activation. We found that some resting peripheral blood NK cells also express CD27. Furthermore, CD27 expression was up-regulated on NK cells stimulated by IL-2. The cytolytic activity of IL-2-activated, but not resting, NK cells was inhibited by an anti-CD27 mAb (anti-1A4). However, anti-1A4 did not affect conjugate formation between IL-2-activated NK cells and tumor cell targets. In contrast, anti-1A4 inhibited CD2-mediated calcium mobilization and the serine esterase activity of NK cell granules. These inhibitory effects could be mediated in part by increase in intracellular cAMP levels induced by anti-1A4. Our results suggest that the CD27 Ag plays an important role in the regulation of activated NK cells.     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of lysis by cytotoxic T lymphocyte clones. Lytic activity and gene expression in cloned antigen-specific CD4+ and CD8+ T lymphocytes.

Cloned murine Th having properties of either Th1 or Th2 cells as well as CD8+ CTL were tested for the capacity to lyse: 1) nucleated target cells bearing Ag or coated with anti-CD3 mAb, or 2) SRBC target cells coated with anti-CD3 mAb in a short term 51Cr-release assay. The lysis of SRBC occurs by a mechanism that does not involve nuclear degradation but presumably does involve membrane damage. Three patterns were observed: CTL and some Th2 cells lysed efficiently nucleated target cells and SRBC coated with anti-CD3 mAb. Th1 and some Th2 T cells lysed nucleated target cells but did not lyse efficiently the SRBC coated with anti-CD3 mAb. Finally, some Th2 cells failed to lyse efficiently either nucleated or SRBC targets. We also examined these clones for their expression of N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity, and for the expression of perforin or CTLA-1 (granzyme B) mRNA. Total N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity expressed by CTL and Th2 clones tended to be higher than that of Th1 cells. Perforin mRNA and CTLA-1 mRNA were readily detectable in CTL and some Th2 clones. Expression of perforin and CLTA-1 mRNA correlated well with the capacity of these clones to lyse SRBC coated with anti-CD3 mAb. Our results show that some but not all Th2 clones have lytic characteristics similar to those of CD8+ CTL. Two mechanisms appear to contribute to their lytic process, one mechanism of lysis involves membrane damage that correlates with the expression of perforin mRNA; a second mechanism involves the induction of DNA degradation in the target cells. In contrast, some CD4+ effector cells appear to lack the capacity to lyse efficiently via the mechanism involving membrane damage and may only have the lytic activity associated with the capacity to induce DNA degradation.     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.     

Lysis of tumor cells by CD3+4-8-16+ T cell receptor alpha beta- clones, regulated via CD3 and CD16 activation sites, recombinant interleukin 2, and interferon beta 1

A small subpopulation (about 2%) of normal CD3+ human T lymphocytes lacks both CD4 and CD8 antigens. We have cloned these cells from peripheral blood lymphocytes (PBL) obtained from healthy individuals and from a patient with severe combined immunodeficiency. Six out of seven CD3+4-8-clones exert strong cytolytic activity against a variety of so-called NK-susceptible and -nonsusceptible tumor target cells. Their target cell specificity spectrum can virtually be as wide as that of CD3-NK cell-derived clones, with strong lytic capacity. Some of these clones also exert antibody-dependent cellular cytotoxicity (ADCC), a characteristic of NK cell-derived clones but not of CD3+4+ or CD8+ mature T cell-derived clones. Such CD3+ T cell clones do not express the CD16 (IgG Fc receptor) antigen, but as we demonstrate here, the CD16 antigen can be identified on CD3+4-8-clones. Both ADCC activity and CD16 antigen expression are lower in CD3+4-8- than in CD3- NK cell clones. Lytic activity of mature CD3+4+ or CD8+ and CD3- NK cell clones can be augmented, respectively, by anti-CD3 or anti-CD16 monoclonal antibodies (MAb), but that of CD3+4-8- clones are augmented by both MAb. Lytic activity of CD3+4+ or CD8+ clones is considerably enhanced after 3 hr of incubation with recombinant IL 2, as found for CD3- NK cells. Enhancement of lytic activity of allospecific CD3+4+ or CD8+ clones requires 18 hr of incubation. Thus, CD3+4-8-16+ cells share several features with CD3- NK cells. However, they express the CD3 antigen, which is characteristic for CD4+ or CD8+ mature T cells. Our results also indicate that although CD3+4-8- clones react with five preparations of anti-CD3 MAb tested, these clones do not express a classical CD3+/Ti alpha, beta antigen receptor complex. This is suggested by the finding that the CD3+4-8- clones do virtually not express the common epitope of the T cell receptor alpha, beta-chains as identified by the WT31 MAb. These CD3+4-8- lymphocytes may represent functionally mature lymphocytes of a distinct T cell subpopulation having a particular immune function.