Human allospecific cytolytic T lymphocyte lysis of a murine cell transfected with HLA-A2

A variety of molecules are involved in the interaction of human allospecific cytolytic T lymphocytes (CTL) with target cells. Monoclonal antibodies specific for these molecules inhibit CTL-target conjugate formation and/or lysis. To further study recognition and lysis of targets by human CTL, we used a murine mastocytoma cell line transfected with the histocompatibility leukocyte antigen (HLA)-A2 gene (P815-A2+) as a target for human HLA-A2-specific CTL. We find that only a subset of human HLA-A2-specific CTL can lyse murine P815-A2+ cells, suggesting that the murine cells may lack one or more accessory molecules needed for CTL recognition and lysis.     

Target cell lysis by cytotoxic T lymphocytes redirected by antibody-coated polystyrene beads

Cytotoxic T lymphocytes were found to mediate rapid lysis of target cells not normally recognized in the presence of small polystyrene beads coated with a combination of anti-T3 and antitarget cell antibodies. Lysis was not seen with beads bearing one of these antibodies alone, nor with a mixture of two types of beads each coated with a single antibody. The effector cells mediating this lysis include long term allospecific human CTL, and both human and mouse CTL clones recognizing mouse class I MHC Kb Ag. TNP-modified mouse tumor cells, a human lymphoblastoid line, and human red cells were found to be good targets for this cytotoxicity. Polystyrene beads with diameters of 3 to 15 mu caused target lysis, with a dose-response curve which typically went through a maximum and declined at high bead numbers. Maximal bead-redirected lysis by CTL was less efficient than that mediated by soluble antibody heteroconjugates of the same two antibodies. Bead-redirected target lysis was calcium dependent. These results are interpreted as a form of bystander lysis induced by the beads, since the target cell membrane is not directly crosslinked to the region of CTL activation. These observations thus favor a mechanism of lysis involving the polarized secretion of a locally acting lytic agent by CTL.     

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.     

Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gp19K but not E3 14.7K

Early region E3 of adenovirus (Ad) appears to encode proteins involved in the interaction of the virus with the host immune system. The E3 region 19-kDa glycoprotein (gp19K) binds to class I MHC Ag in the endoplasmic reticulum and inhibits their transport to the cell surface; it has been proposed that this protects virus infected cells from lysis by CTL. We have found that the E3 14.7-kDa protein (14.7K) inhibits lysis of infected cells by TNF, and here we show that it also protects cells from lysis by lymphotoxin, which has been implicated as a mediator of CTL lysis. We have developed a method for producing CTL specific for human Ad2 and Ad5 in mice, in order to test directly which of the genes in the E3 region protect infected cells from lysis by virus specific CTL. The presence of the E3 region inhibits both the induction of Ad-specific CTL in culture and the lysis of infected target cells by these CTL. The inhibition varies between different mouse strains, with almost complete inhibition in C57BL/10 (H-2b) mice, partial inhibition with BALB/c (H-2d) and little or no inhibition with C3H (H-2k); results were similar for Ad2 and Ad5. By using a panel of E3 deletion mutants, inhibition of target cell lysis by Ad5 specific CTL was mapped exclusively to the gp19K gene. The 14.7K gene had no effect on CTL lysis despite its ability to protect cells against lysis by lymphotoxin. gp19K was synthesized abundantly in mouse cells by mutants retaining the gp19K gene; some mutant forms of the protein were synthesized but were nonfunctional. These data support the hypothesis that gp19K can protect Ad infected cells against lysis by virus specific CTL.     

Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.

We examined the ability of human anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL) to lyse autologous human fibroblasts infected with HSV. In contrast to HSV-infected human Epstein-Barr virus-transformed B cells (LCL), which were lysed by HLA-restricted anti-HSV CTL, autologous fibroblasts infected with HSV were resistant to lysis. This resistance was not due to a lack of infectivity or production of HSV proteins since greater than 90% of the cells were infected and expressed abundant levels of viral proteins. HSV-infected human fibroblasts were also tested for susceptibility to lysis by alloantigen-specific CTL. Although allogeneic LCL and uninfected allogeneic fibroblasts were killed, human fibroblasts infected with HSV demonstrated a time-dependent resistance to lysis by alloantigen-specific CTL. HSV-infected human fibroblasts were not resistant to all forms of cell-mediated cytotoxicity since they were sensitive to antibody-dependent cellular cytotoxicity. Although one may suspect that the resistance of HSV-infected human fibroblasts to anti-HSV CTL and alloantigen-specific CTL-mediated lysis was due to a lack of major histocompatibility complex expression, Confer et al. (Proc. Natl. Acad. Sci. USA 87:3609-3613, 1990) previously demonstrated that incubation of human natural killer and lymphokine-activated killer cells with monolayers of human fibroblasts infected with HSV "disarmed" the killers in that they were unable to lyse sensitive target cells. We extend their results and show that incubation of anti-HSV CTL or alloantigen-specific CTL with uninfected fibroblasts did not affect their lytic activity, whereas CTL incubated with HSV-infected fibroblasts for 2 to 6 h rendered the CTL incapable of lysing their normally sensitive target cells. Indeed, human fibroblasts infected for merely 2 h with HSV were able to profoundly inhibit the cytotoxic activity of alloantigen-specific CTL. Thus, HSV-infected human fibroblasts are not inherently resistant to lysis by anti-HSV CTL or alloantigen-specific CTL, but rather contact of CTL with HSV-infected fibroblasts resulted in inactivation of the CTL. The inactivation of CTL appears to be HSV specific since incubation of alloantigen-specific CTL in sandwich assays with fibroblasts infected with HSV type 1 (HSV-1) or HSV-2 resulted in inactivation, whereas incubation of CTL with fibroblasts infected with adenovirus or vaccinia virus had no effect. Further, although incubation of alloantigen-specific CTL in sandwich assays with HSV-infected fibroblasts resulted in inhibition of CTL activity, exposure of CTL in Transwell cultures to cell-free supernatant from HSV-infected fibroblasts did not mediate this inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of monoclonal antibodies specific for the T cell receptor complex by Fc receptor-mediated CTL lysis

Monoclonal antibodies (mAb) directed at the T cell receptor complex (TcR) on cloned T cells have generally been identified by their ability to inhibit the clone's antigen-specific function. Because such inhibition is highly dependent on antibody concentration and affinity, detection of anti-clonotypic antibodies to murine alloreactive T cells has been very difficult. In this report, an alternative method is described on the basis of the ability of antibodies specific for the TcR complex to activate T cells in an antigen-independent manner. The assay is based upon the observation that soluble antibodies to human T3 promote lysis of irrelevant, Fc receptor-positive targets by a human CTL line. By using this approach, an anti-TcR mAb has been identified among a panel of murine mAb generated against an alloreactive CTL clone. Induction of lysis by soluble anti-TcR mAb has been shown to require both the expression of Fc receptors on the target cell and conjugate formation between the effector and the target cell. This assay provides a screening procedure that is much more sensitive than inhibition of function, and it preferentially detects antibodies specific for cell surface molecules involved in T cell activation.     

Temperature requirements and kinetics of T cell signaling. Velocity of triggering correlates with functional effects of anti-CD3 mAb

Antibody reactive with the CD3 complex on the surface of T lymphocytes can either: inhibit CTL lysis of target cells expressing Ag; or redirect CTL to lyse target cells expressing FcR in the absence of Ag expression. To investigate these phenomena we examined the effect of anti-CD3 mAb on two indicators of CTL activation, the release of esterase and target cell lysis. Esterase release by long term allo-reactive human CTL in response to target cells (JY or HLA transfected K562 cells) was found to be Ag specific and correlate with target cell lysis. Addition of anti-CD3 to either JY targets or K562 cells expressing FcR resulted in a high level of esterase release. Triggering of esterase release was found with both soluble intact and Fab fragment of anti-CD3 in the absence of cells expressing measurable FcR. This apparent FcR-independent triggering of esterase release occurred at 37 degrees C but not at 24 degrees C. In contrast esterase activity was released from CTL at both 24 and 37 degrees C in response to intact target cells, JY or K562 cells plus intact anti-CD3 mAb. Addition of anti-CD3 mAb, at a level capable of blocking target cell lysis by greater than 50%, resulted in an initial velocity of esterase release almost twice that found in response to JY target cells. With a low level of anti-CD3 mAb, able to block JY lysis by approximately 10%, the initial rate of esterase release was much slower than that found in response to target cells. In contrast when FcR+ cells, K562, were added along with a low level of anti-CD3 the initial velocity of esterase release was about twofold more than the velocity of esterase release triggered by soluble anti-CD3 alone. These results indicate that soluble antibody can trigger long term active CTL and the velocity of this triggering correlates with anti-CD3-mediated inhibition as well as redirected lysis.     

Gene mapping and somatic cell hybrid analysis of the role of human lymphocyte function-associated antigen-3 (LFA-3) in CTL-target cell interactions

LFA-3 is expressed on a wide variety of human cell lines, including those which have been used as recipients for gene transfer of human class I gene products, whereas a murine counterpart is either absent or significantly different such that the anti-LFA-3 monoclonal antibody (MAb) does not bind. By using a somatic cell genetic approach, we demonstrate that LFA-3 is not a major histocompatibility complex-encoded molecule, and that its gene locus maps to human chromosome 1. When LFA-3 and HLA-A2 are coexpressed on the mouse cell surface, anti-LFA-3 MAb interfered with specific recognition and lysis of these target cells by human CTL capable of lysing HLA-A2-expressing mouse transfectants. A significant contribution of the LFA-3 molecule to CTL reactivity was not observed, however, because the presence of LFA-3 did not restore recognition by CTL clones previously found incapable of lysing HLA-A2-expressing mouse transfectants, nor was it required by those human CTL that could lyse mouse cell transfectants. Thus, we have used genetic techniques to demonstrate that LFA-3 may serve a role in CTL-target cell interactions at the target cell level, but is not a molecule absolutely required for human allospecific CTL recognition of HLA antigens expressed on mouse cells. We suggest that LFA-3 may not participate directly in CTL function under normal circumstances, but delivers a more general inhibitory signal only when provoked by bound MAb.     

Anti-T cell receptor antibodies fail to inhibit specific lysis by CTL clones but activate lytic activity for irrelevant targets

In this report, we describe the functional effects of anti-T cell receptor antibodies on a panel of MHC-restricted, influenza virus-specific CTL clones. Approximately 25 to 30% of these clones are recognized by KJ16-133, an anti-T cell receptor monoclonal antibody presumably specific for products of the V beta 8 gene family, and an antibody with similar specificity, F23.1. In contrast to most previous reports, both KJ16-133 and F23.1, over a wide range of antibody concentrations, fail to inhibit the antigen-specific effector function of these CTL. Instead, the antibodies activate the CTL to kill without regard for the MHC haplotype of the target cells or the presence of the appropriate viral antigen. This anti-T cell receptor antibody-induced cytolysis by our clones does not appear to be mediated by Fc receptors on target cells. Nuclear destruction of target cells as a result of antibody-induced lysis suggests that it occurs via a mechanism similar to antigen-specific lysis by CTL. In addition, both soluble bivalent F23.1 and F23.1 coupled-Sepharose beads are able to induce the secretion of interferon-gamma from these CTL clones.     

Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.     

Synergistic in vitro and in vivo anti-tumor effect of daunomycin-anti-96-kDa melanoma-associated antigen monoclonal antibody CL 207 conjugate and recombinant IFN-gamma

The mAb CL 207 recognizes a 96-kDa melanoma-associated Ag. The latter is not modulated by antibody but is highly susceptible to induction by immune IFN-gamma on human carcinoma and melanoma cells. The mAb CL 207 does not mediate C- and cell-dependent lysis of human carcinoma and melanoma cells. Conjugation of daunomycin with mAb CL 207 causes a slight reduction of its immunoreactivity and affinity but does not affect its serologic specificity or the toxicity of daunomycin. In combination with IFN-gamma, the daunomycin-mAb CL 207 conjugate displays a selective in vitro and in vivo toxic effect on tumor cells that express the 96-kDa MAA. The synergistic effect of IFN-gamma and daunomycin-mAb CL 207 conjugate is specific. The in vivo toxicity is influenced by the interval between injection of human tumor cells into nude mice and that of IFN-gamma and daunomycin-mAb CL 207 conjugate. The present results suggest that the 96-kDa melanoma-associated Ag may be a useful model to investigate the combined use of IFN-gamma and mAb for the selective destruction of tumor cells.     

Structure of the gene of tum- transplantation antigen P35B: presence of a point mutation in the antigenic allele.

Mutagen treatment of P815 tumour cells produces tum- variants that are rejected by syngeneic mice because they express new transplantation antigens. These 'tum-' antigens elicit a cytolytic T lymphocyte (CTL) response but no detectable antibody response. The DNA of tum- variant P35 was transfected into P815 cell line P1.HTR. Transfectants expressing tum- antigen P35B were identified on the basis of their ability to stimulate anti-P35B CTL. This was repeated with a cosmid library and a cosmid carrying the sequence encoding antigen P35B was recovered from a transfectant expressing the antigen. Gene P35B is 6 kb long and contains 11 exons. The sequence shows no homology with the previously identified tum- gene P91A nor with any gene presently recorded in the data banks. The antigenic allele of gene P35B differs from the normal allele by a point mutation located in exon 5. This mutation, which replaces a Ser by an Asn residue, was shown by site-directed mutagenesis to be responsible for the expression of the antigen. A synthetic decapeptide covering the sequence surrounding the tum- mutation rendered P815 cells sensitive to lysis by anti-P35B CTL. Surprisingly, the homologous peptide corresponding to the normal sequence of the gene had the same effect, indicating that this tum- mutation does not exert its effect by generating the aggretope or the epitope of the antigenic peptide. As observed previously with gene P91A, we found that fragments of gene P35B containing only exons 4 and 5, which were cloned in non-expression vectors, transferred efficiently the expression of the antigen.     

An epitope in the V1 domain of the simian immunodeficiency virus (SIV) gp120 protein is recognized by CD8+ cytotoxic T lymphocytes from an SIV-infected rhesus macaque.

Cytotoxic T-lymphocyte (CTL) responses against the external envelope glycoprotein (gp120) of the simian immunodeficiency virus (SIV) were studied in a rhesus macaque infected with SIVmac/239. CD8+ T cells enriched from concanavalin A-stimulated peripheral blood mononuclear cells lysed autologous target cells infected with recombinant vaccinia virus vectors expressing the SIVmac/239 or SIVsm/H4 envelope protein, which share approximately 80% identity in amino acid sequence. A CD8+ CTL line derived by limiting dilution culture of the concanavalin A-stimulated lymphocytes was also specific for the envelope proteins of both SIV isolates. Mapping studies revealed that this cell line recognized an epitope between amino acids 113 and 121 (CNKSETDRW) in the V1 domain of gp120. Amino acid substitutions are observed at positions 116 and 120 among viruses of the SIVsm/mac/human immunodeficiency virus type 2 group, and thus synthetic peptides representing these variants were tested for the ability to sensitize target cells for lysis by the CTL line. Autologous target cells sensitized with a synthetic peptide representing the SIVmac/239 sequence were efficiently killed. In contrast, recognition of target cells was reduced or abolished when peptides representing the amino acid substitutions at position 116 or 120 of other SIVmac, SIVsm, SIVmne, or SIVstm strains were tested. Further studies of CTL responses against this epitope could provide insights into mechanisms of variability within the gp120 V1 domain and its importance in evasion of immunity in infected or vaccinated monkeys.     

Clonal analysis of cytolytic T lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for Lyt-2/3 function

The lysis by allospecific cytolytic T lymphocytes (CTL) of the BALB/c lymphoma ST-4.5, a cell line that can be induced by interferon-gamma (IFN-gamma) to express increased amounts of major histocompatibility complex (MHC) class I antigens, was investigated. Culture of ST-4.5 in IFN-gamma increased the surface expression of Kd molecules from originally low levels and Dd from undetectable amounts by approximately fivefold as determined by fluorescence-activated cell sorter (FACS) analysis, whereas the levels of several other antigens (Ld, I-Ad, Thy-1, Lyt-2, L3T4, and LFA-1) were not affected. The lysis of ST-4.5 by Dd- and Ld-specific CTL clones correlated with the expression of those antigens on target cells as determined by both FACS and biochemical analysis. Lysis of ST-4.5 by CTL clones specific for Kd antigen fell into two distinct groups: those that could lyse targets cultured either normally or in IFN-gamma, and those that could only lyse targets that had been precultured in IFN-gamma. The apparent sensitivity to antigen exhibited by the Kd-specific CTL clones predicted their sensitivity to inhibition of target lysis by anti-Lyt-2/3 antibody. Those CTL clones that were only active against ST-4.5 expressing higher amounts of surface antigen (resulting from IFN-gamma preculture) were readily inhibited by anti-Lyt-2/3 antibody, whereas those CTL capable of lysing normally cultured targets having lower amounts of surface antigen were heterogeneous in their sensitivity to anti-Lyt-2/3; some were inhibitable, whereas others were resistant. In addition, another CTL clone that was resistant to inhibition by anti-Lyt-2/3 alone was readily inhibited by a synergistic combination of anti-Lyt-2/3 plus anti-Kd (but not anti-Dd or Ld) antibodies. These results indicate that CTL antigen receptor sensitivity to (or affinity for) antigen and the level of specific antigen expression by the target cell may both be important criteria in assessing Lyt-2/3 molecule function in CTL-mediated cytolysis. The function of recognition-associated molecules such as Lyt-2/3 may be to strengthen and increase the number of receptor-ligand binding events that facilitate CTL-target membrane interactions that lead to the lysis of the target cell.     

H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells

Long-term syngeneic mouse cytolytic T lymphocyte (CTL) clones were obtained from DBA/2 (H2d) mice immunized with P815 (H2d) cells transfected with cloned human class I histocompatibility genes, HLA-CW3 or HLA-A24. Three distinct patterns of specificity were defined on P815 HLA transfectant target cells. One clone lysed HLA-CW3 but not -A24 transfectants, and a second lysed HLA-A24 but not -CW3 transfectant target cells. The third clone lysed P815 targets transfected with either HLA gene. None of the CTL clones lysed L cells (H2k) transfected with the same HLA genes or human targets that expressed these HLA specificities. Several lines of evidence indicated that recognition of HLA transfectants by these CTL clones was H2 restricted. First, lysis of P815 HLA transfectants could be inhibited by anti-H2Kd monoclonal antibody. In addition, the anti-P815-HLA CTL clones could lyse a (human X mouse) hybrid target that expressed both HLA class I and H2Kd antigens, but not a clonal derivative that no longer expressed H2Kd. The most direct evidence for H2-restricted recognition of P815-HLA transfectants by the syngeneic CTL clones was obtained by double transfection of mouse L cells (H2k) with both HLA and H2 class I genes. L cells transfected with HLA and H2Kd genes were susceptible to lysis by the same CTL clones that lysed the corresponding P815-HLA transfectant targets. Thus under certain conditions, CTL recognition of xenogeneic class I histocompatibility gene products can be restricted by other class I gene products.     

Susceptibility of cytotoxic T lymphocyte (CTL) clones to inhibition by anti-T3 and anti-T4 (but not anti-LFA-1) monoclonal antibodies varies with the "avidity" of CTL-target interaction

To explore the role of the T3, T4, and LFA-1 molecules in high and low "avidity" interactions between SB2-specific cytotoxic T lymphocyte (CTL) clones and their targets, monoclonal antibody-mediated inhibition of cytotoxicity has been studied in experiments that vary the "avidity" of interaction in three different ways. 1) Previous results have been extended with respect to different CTL clones assayed on the same SB2-positive target cells. Differences between clones in susceptibility to anti-T3 inhibition paralleled variations in anti-T4 inhibition, and both correlated inversely with the "avidity" of the effector-target interaction (inferred previously from studies of conjugate dissociation). 2) A high "avidity" clone, 8.4, was identified that lysed not only SB2-positive cells but also cross-reacted on a few SB2-negative cells. Cold target inhibition studies confirmed the cross-reaction, and together with conjugate dissociation studies, indicated that cross-reaction to be of lower "avidity" than the specific recognition of SB2. Cross-reactive lysis was much more susceptible to inhibition by anti-T3 and anti-T4 than was specific lysis. 3) Anti-T3 and anti-T4 blocking was analyzed in the presence of anti-Ia antibody to reduce the amount of Ia antigen available on the target. Anti-T3 and anti-T4 antibody blocking was more efficient after the addition of anti-Ia antibody concentrations that (by themselves) produced minimal inhibition of lysis. As a control, anti-LFA-1 antibody blocking was analyzed in each of these three experimental systems that compare interactions of different "avidity"; minimal variation was observed in the efficiency of inhibition by anti-LFA-1. Thus, anti-T3 and anti-T4 inhibition correlates inversely with the "avidity" of that CTL-target interaction, but anti-LFA-1 inhibition does not.     

Regulation of influenza virus-specific cytotoxic T cell responses by monoclonal antibody to a human T cell differentiation antigen

The results in this report indicate that the OKT3 monoclonal antibody, which is specific for a human T cell differentiation antigen present on 90 to 95% of peripheral T cells, can exert several effects that regulate the generation and expression of human influenza virus-immune cytotoxic T lymphocytes (CTL). The OKT3 antibody, but not OKT1 or OKT11 (which bind to all peripheral T cells), is able to inhibit anti-influenza CTL effector cell activity. An F(ab')2 preparation of OKT3 IgG were as effective as whole IgG for the inhibition of CTL effectors, indicating that the inhibitory activity of the antibody was not a function of the Fc portion of the molecule. OKT3 IgG and OKT3 F(ab')2 fragments (but not OKT4, OKT8, or OKI were able to inhibit the generation of anti-influenza CTL. The culture of human lymphoid cells with OKT3 in the presence or absence of influenza virus induced radioresistant cells that could suppress the CTL response of fresh autologous lymphocytes to influenza. These results suggest that T cell functions can be regulated by signals that are initiated by the binding of antibody to cell surface molecules that may not be related to the T cell antigen-specific receptor(s).     

Surface immunoglobulins as targets for anti-immunoglobulin-dependent cell-mediated lysis of B cells

Human K cells are able to lyse human lymphoblastoid B cells in the presence of specific anti-human immunoglobulin isotype antibodies. The human lymphoblastoid B-cell line DAUDI (surface mu- and kappa-chains positive) was lysed in the presence of anti-mu and anti-kappa antisera, or IgG fraction of these antisera, and B-cell-depleted human lymphocytes. Lysis was not induced by anti-isotypic antisera alone or human lymphocytes alone. Lysis was not induced by antisera directed at isotypes which were not expressed on the DAUDI cells. The human lymphoblastoid B-cell line RAJI, which does not express surface membrane isotypes, could not be lysed by the anti-isotype-dependent cell-mediated mechanism. Lysis of DAUDI cells by this mechanism was mediated by Fc gamma receptor-bearing human non-B lymphocytes and required an intact Fc piece in the inducing anti-isotypic antibody. These observations are discussed as a possible role for K cells in the regulation of immune responses by interaction between anti-idiotypic antibodies and idiotype-bearing cells.     

Susceptibility of astrocytes to class I MHC antigen-specific cytotoxicity

Cell-mediated immune mechanisms contribute to tissue injury within the central nervous system (CNS) in a number of experimental diseases, including experimental allergic encephalomyelitis and some viral infections, and may mediate lesion formation in multiple sclerosis. We investigated the conditions under which murine astrocytes can become susceptible targets of cytotoxic T cells. We demonstrate that mouse astrocytes in vitro can be susceptible targets of class I major histocompatibility complex (MHC)-specific cytotoxicity mediated by L3 cytotoxic T lymphocytes (CTL). Expression of appropriate class I MHC antigen on the astrocytes is a requirement, because only cells bearing the H-2d phenotype are susceptible to lysis by L3 cells. BALB/c-H-2dm2 astrocytes lacking the specific determinant recognized by L3 cells are not susceptible to lysis. Astrocyte lysis can, however, occur under culture conditions in which MHC antigen expression is immunocytochemically low or undetectable. Cytolysis can be inhibited by pretreatment of the effector L3 cells with either anti-Lyt-2 monoclonal antibody (mAb) or anti-clonotypic mAb and by preincubation of the glial target cells with an appropriate anti-H-2 antibody (anti-H-2Ld). mAb to lymphocyte function-associated antigen does not inhibit cytotoxicity of the L3 clone against glial cells. Knowledge regarding the role of CTL within the CNS, including the surface molecules involved in glial cell lysis, could further the development of immunotherapies designed to effect immune reactivity within the CNS.     

Human monocyte-mediated cytotoxicity: the use of Ig-bearing hybridomas as target cells to detect trigger molecules on the monocyte cell surface

We recently reported the preparation and characterization of a monoclonal antibody, 32.2, specific for the high-affinity Fc receptor (FcR) for IgG on human monocytes. We have utilized the hybridoma cell line producing this antibody as a target for monocyte-mediated cytotoxicity. The hybridoma was selected for stable sublines that expressed high quantities of surface 32.2 immunoglobulin (Ig) through flow cytometry. Monocyte-mediated cytotoxicity, with these sublines used as targets, was evaluated with the use of a 51Cr-release assay. It was found that monocytes could efficiently lyse the hybridoma cells (HC 32.2) bearing surface Ig directed to the high-affinity FcR. Consistent with the specificity of the 32.2 antibody for an epitope on the high-affinity receptor outside of the ligand binding site, human IgG did not block monocyte killing of HC 32.2. In contrast, monocytes could not mediate lysis of hybridoma cells bearing high levels of antibody directed to other monocyte cell surface molecules, in particular, class I MHC molecules, the C3bi receptor, and the My 23 antigen. The effect of IFN-gamma on the ability of monocytes to mediate lysis of the 32.2 Ig-bearing hybridomas was also assessed. Monocytes cultured in the absence of IFN-gamma could lyse the hybridoma line expressing high levels of 32.2 Ig as efficiently as monocytes cultured in the presence of IFN-gamma. However, untreated monocytes were less able than IFN-gamma-treated monocytes to kill HC 32.2 expressing lower levels of Ig. Thus, IFN-gamma may enhance the efficiency of monocyte-mediated antibody-dependent killing under conditions where limited antibody is available on the target. These studies demonstrate that the high-affinity FcR on monocytes can act as a cytotoxic trigger molecule for killing of tumor cell targets and that this trigger does not require specific binding to the Fc binding epitope. These results further encourage possible clinical application of the 32.2 monoclonal antibody in tumor therapy.