Blocking of CTL-mediated killing by monoclonal antibodies to LFA-1 and LYT-2,3. I. Increased susceptibility to blocking after papain treatment of target cells

It is now established that monoclonal antibodies (MAb) against LFA-1 and Lyt-2,3 antigens on cytolytic T lymphocytes (CTL) block killing function in the absence of C. It has been suggested that the blocking is inversely related to CTL-target affinity. In this report, we studied the effect of papain pretreatment of target cells, because papain is known to remove H-2 and to render target cells more resistant to allospecific CTL. CTL-target conjugate formation was weaker with papain-treated target cells (based on reduced post-dispersion lysis in dextran-containing medium). The concentration of MAb required to produce 40 to 60% inhibition of 51Cr release (2-hr assay) was reduced four to 29-fold for alpha LFA-1 and 64 to 114-fold for alpha Lyt-2,3. Papain, however, did not induce blocking by MAb to other CTL antigens such as Thy-1, H-2, and T200. Flow cytometric analysis confirmed that papain selectively removed more than 95% of H-2. In kinetic studies of removal and recovery, H-2 density and conjugate formation correlated well with each other. Sensitivity to blocking was not as well correlated, raising the possibility that an unidentified papain-sensitive target cell molecule other than H-2 plays an important role in CTL-target interaction.     

Mouse alloantibodies capable of blocking cytotoxic T cell function. V. The majority of I region-specific CTL are Lyt-2+ but are relatively resistant to anti-Lyt-2 blocking

In an attempt to solve the conflict concerning the correlation between the Lyt-2 phenotype of T cells subsets and the type of the MHC antigens involved in the recognition by T cells, class 2 (I region) antigen-specific CTL were studied for their Lyt phenotypes and the sensitivity to the blocking effects of anti-Lyt-2,3 antibodies. To avoid contamination by CTL to class 1 antigens such as Qa antigens, A.TH anti-A. TL attackers and A.TH anti-A attackers were tested on LPS blasts of the A strain and the A.TL stain, respectively. By using these combinations, it was shown that the majority of I region-specific killers were Thy-1+, Lyt-1+23+. Specific target cell lysis by these cells were, however, found to be far less sensitive to the blocking effects of various monoclonal antibodies to the Lyt-2,3 antigens than conventional class 1-specific CTL. This conclusion was drawn by directly comparing the sensitivity of the I region-specific and K region-specific killing by identical numbers of the same attacker cells (A.TH anti-A). No significant difference was seen between the primary and the hyperimmune CTL. Lyt-2-, Thy-1+ killer cells with I region specificity could be induced when Lyt-2-depleted A.TH responder cells were stimulated in vitro. Such Lyt-2- killer cells were not induced to the H-2K alloantigen.     

Differences between macrophage migration inhibitions by lymphokines and muramyl dipeptide (MDP) or lipopolysaccharide (LPS): migration enhancement by lymphokines

To investigate the repertoire of molecules which are associated with cytolytic T-lymphocyte (CTL)-mediated killing, function-blocking monoclonal antibodies (MAb) have been selected and characterized. Spleen cells from rats immunized with secondary mouse CTL were fused with mouse myeloma cells. Antibodies secreted by 2400 hybrid cultures were selected solely by their ability to block CTL-mediated killing in a mouse anti-rat xenogeneic system. Fifteen cultures with antibodies which blocked CTL-mediated killing were chosen for cloning and further characterized by immunoprecipitation and immunofluorescence flow cytometry. One group of five monoclonal antibodies recognized the Lyt-2,3 molecule of 35,000 M_r. The second group of six MAb recognized the LFA-1 antigen containing two subunits of 180,000 and 95,000 M_r. One MAb giving only partial inhibition of killing was an IgM anti-Thy-1. It strongly agglutinated CTL. The target antigens defined by three other MAb were not definitively identified. Competition in cell binding between anti-Lyt-2,3 and anti-LFA-1 MAb suggested that their blocking effect in cytolysis is due to binding to distinct and spatially separate molecules on effector cells. The results of direct screening for functional blockade support the important role of Lyt-2,3 and LFA-1 molecules in T-cell-mediated cytolysis.     

Functional distinctions between the LFA-1, LFA-2, and LFA-3 membrane proteins on human CTL are revealed with trypsin-pretreated target cells

We asked whether we could distinguish the roles of the human lymphocyte membrane proteins LFA-1, LFA-2, and LFA-3 in the function of CTL-mediated killing. Little is known about the functions of these molecularly distinct proteins beyond the facts that i) binding of a monoclonal antibody (MAb) to any one of them is sufficient to inhibit killing, ii) that in each case inhibition involves prevention of CTL-target cell conjugate formation, and iii) that MAb to LFA-1 and LFA-2 inhibit best when bound to the CTL, whereas anti-LFA-3 inhibits only when bound to the target cell. This latter is despite the fact that (in our test system) LFA-1 and LFA-3 are expressed both on the CTL and on the target. When the target cells were pretreated with trypsin, the sensitivity of CTL-mediated killing was affected in a different way for each site. Inhibition of anti-LFA-1 was increased by approximately 20-fold. Inhibition by anti-LFA-2 was unaffected. Inhibition by anti-LFA-3 was abolished. Trypsin did not remove the specific antigens recognized by the various CTL, HLA-A,B,C or HLA-DR. Nor did it remove LFA-1 from the target cell. It did, however, selectively remove LFA-3 from the target cell. These results indicate, for the first time, that LFA-1 and LFA-2 have functionally distinct roles. They suggest that an unidentified trypsin-sensitive target cell molecule, operationally designated the "trypsin-sensitive counter blocker" (TSCB), plays an important role in the function of LFA-1, possibly by providing a target cell binding site for LFA-1 on the CTL. The hypothesis that this TSCB is identical to LFA-3 (and the related possibility that LFA-1 and LFA-3 are mutual ligands) is not favored by our data, but is not excluded. Finally, the data indicate that the mechanisms by which MAb inhibit killing differ at the LFA-1 and LFA-3 sites. They are consistent with LFA-1 providing adhesion strengthening by binding to another site (the TSCB?) and with LFA-3 delivering an inhibitory signal when provoked with MAb.     

Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones

Human cytolytic T lymphocytes (CTL) clones and HLA-A2- and HLA-B7-transfected human, monkey, and mouse cell lines were used to investigate the basis for species-restricted antigen recognition. Most allospecific CTL clones obtained after stimulation with the human JY cell line (source of HLA-A2 and HLA-B7 genomic clones) recognized HLA antigens expressed in human and monkey cell lines but did not recognize HLA expressed in murine cells. By initially stimulating the responder cells with HLA-transfected mouse cells, two CTL clones were obtained that recognized HLA expressed in murine cells. Functional inhibition of these CTL clones with anti-class I monoclonal antibodies (MAb) indicated that clones reactive with HLA+ murine cells were of higher avidity than clones that did not recognize HLA+ murine target cells. MAb inhibition of accessory molecule interactions demonstrated that the LFA-1 and T8 surface molecules were involved in CTL-target cell interactions in all three species. In contrast, the LFA-2/CD2 molecule, previously shown to participate in a distinct activation pathway, was involved in the cytolysis of transfected human and monkey target cells, but not in the lysis of HLA+ murine cells. Thus transfection of HLA genes into different recipient species cell lines provides us with the ability to additionally delineate the functional requirements for allospecific CTL recognition and lysis.     

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.     

Lymphocyte-mediated cytolysis of allogeneic tumor cells in vitro. III. Enzyme sensitivity of target cell antigens.

Target tumor cells pretreated with high concentrations of papain or Pronase were resistant to lysis by cytotoxic T lymphocytes (CTL), whereas treatment with trypsin or neuraminidase had no protective effect. Parallel determinations of the H-2 content of target cells following enzyme treatment showed that approximately 80% of surface H-2 was removed by papain or Pronase, 40% by trypsin, and virtually none by neuraminidase treatment. Both susceptibility to lysis by CTL and content of surface H-2 after papain treatment were fully restored by 6 hr at 37 °C in nutrient medium. These findings suggest that lymphocyte-mediated cytolysis (LMC) determinants (target cell antigens bound by CTL) are sensitive to degradation by papain and Pronase but are resistant to the enzymatic action of trypsin and neuraminidase. That a similar pattern of enzyme sensitivity is shown by serologically defined H-2 antigens indicates that both functional classes, LMC and H-2, may have a structural association.     

T3 monoclonal antibody activation of nonspecific cytolysis: a mechanism of CTL inhibition

The T3 antigen is expressed on all cytotoxic T lymphocytes (CTL). Monoclonal antibodies (MAb) to the T3 antigen previously have been shown to inhibit CTL-mediated killing of cells expressing the relevant target antigens. The mechanism of T3 MAb inhibition, however, remains undefined. In this report, we describe a novel effect of the T3 MAb: the stimulation of allospecific CTL clones to kill target cells that do not express the relevant HLA antigens. The stimulation of nonspecific killing was seen only with MAb to the T3 antigen; MAb to other function-associated antigens (e.g., LFA-1, LFA-2, LFA-3, T4, T8, HLA-A,B,C, and DR) had no effect. T3 MAb stimulated nonspecific killing by CTL clones expressing both the T4+ and T8+ phenotype and by CTL clones specific for both class I and class II HLA alloantigens. Target cell susceptibility to T3 MAb stimulated killing was variable. CTL clones lysed some target cell lines very efficiently (e.g., K562, Daudi, and M124.1) but lysed other cell lines much less efficiently (e.g., 23.1, Mann, and L cells). In CTL-mediated cytotoxicity assays with target cells expressing the relevant HLA antigens, T3 MAb demonstrated the expected inhibition of cytolysis. Thus, the ability of T3 MAb to stimulate and inhibit CTL-mediated cytolysis suggests that both effects may be the result of a common mechanism of activation.     

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.     

CTL adhesion and antigen recognition are discrete steps in the human CTL-target cell interaction

Th initial step in cytolytic T lymphocyte (CTL)-mediated cytolysis involves target cell adhesion and antigen recognition. To investigate these initial events in the CTL-target interaction, we used HLA-A2- and HLA-B7-specific human CTL clones and HLA-typed B lymphoblastoid target cells. By using two different adhesion assays, we demonstrated antigen nonspecific CTL-target cell adhesion. To more precisely define the contribution of the antigen-specific receptor to CTL-target cell adhesion, we used the HLA-A2, HLA-B7, and mock transfected RD target cells. Consistent with the results when using B lymphoblastoid target cells, the CTL clones demonstrated equivalent adhesions to the RD target cells whether or not they expressed HLA-A2 or HLA-B7. These results suggested that CTL-target cell adhesion occurred independent of the T cell receptor. By using the calcium-sensitive dye Indo-1 and flow cytometry, we assessed CTL-target cell adhesion and CTL activation. Simultaneous measurement of adhesion and intracellular free calcium demonstrated that CTL-target cell adhesion alone did not activate CTL clones. Both CTL-target cell adhesion and the presence of the appropriate HLA target molecule were necessary for the efficient activation of human CTL. MAb inhibition studies indicated that antigen nonspecific adhesion is largely regulated by the LFA-1, CD2 (LFA-2/T11), and LFA-3 cell surface molecules. These antigen nonspecific cell-cell interaction molecules appear to play an important role in facilitating antigen recognition and subsequent target cell lysis.     

Heritable lymphocyte function-associated antigen-1 deficiency: abnormalities of cytotoxicity and proliferation associated with abnormal expression of LFA-1

The effect of heritable LFA-1 deficiency on T lymphocyte function was measured. After primary mixed lymphocyte stimulation, all six patients studied showed diminished allospecific T lymphocyte cytolytic and NK activity as compared with kindred and normal controls. MLR and mitogen-induced proliferative responses were consistently depressed. LFA-1-deficient, EBV-transformed B cell lines were poor stimulators of T cell responses. Primary cytolytic responses by lymphocytes from severely LFA-1-deficient patients (less than 0.2% of normal surface expression) were consistently more profoundly depressed than those by lymphocytes from moderately deficient patients (about 5% of normal surface expression). These results demonstrate the importance of LFA-1 in lymphocyte function. After repeated MLR restimulation, proliferative and cytolytic capacity improved and CTL lines could be established from all patients. Cytolysis by lines from one but not a second severe patient, and by four of four moderate patients, was inhibited by anti-LFA-1 MAb, and at 10-fold lower concentrations than required for inhibition of killing by control CTL lines. The locus of inhibition was on the target cell for the severely deficient CTL line, and on both the target and effector cells for moderately deficient CTL lines. In contrast, the locus of inhibition for normal CTL is on the effector cell. These findings show that LFA-1 can participate bidirectionally in cell interactions. The in vitro results are discussed in terms of the clinical findings in patients.     

Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences

Information was sought on the phenotype of lymphoid cells causing lethal graft-vs-host disease (GVHD) in irradiated mice expressing whole or partial H-2 differences. In all strain combinations tested, pretreating donor lymph node (LN) cells with anti-Thy-1 monoclonal antibodies (MAb) plus complement (C) abolished mortality. With GVHD directed to class I H-2 differences, pretreating LN cells with anti-Lyt-2 MAb prevented mortality, whereas MAb specific for Ly-1 or L3T4 cell surface determinants caused severe mortality. These data imply that lethal GVHD directed to class I H-2 differences is mediated by L3T4-, Lyt-2+ cells; this subset of T cells was shown previously to control GVHD directed to multiple minor histocompatibility antigens, i.e., antigens seen in the context of self-class I molecules. With whole H-2 differences, GVHD appeared to be controlled largely but not exclusively by L3T4+, Lyt-2-T cells. This T cell subset was also the predominant cause of GVHD directed to class II differences. With class II incompatibilities, depleting donor cells of L3T4+ T cells, either by pretreatment with anti-L3T4 MAb + C or by fluorescence activated cell sorter selection, greatly reduced but did not completely abolish GVHD. These data might imply that L3T4-, Lyt-2+ cells have some capacity to elicit anti-class II GVHD. A more likely possibility, however, is that the residual GVHD to class II differences observed with Lyt-2+-enriched cells reflected minor contamination with L3T4+ cells.     

Unexpected cell surface labeling in conjugates between cytotoxic T lymphocytes and target cells

Specific binding of target cells by cytotoxic T lymphocytes (CTL) is an example of tight interaction between two different cell types. The molecular events that occur at the cell membranes during these interactions are largely unknown. In the present report, we describe an electron microscopic immunostaining study made on CTL-target cell conjugates. Various membrane structures were labeled with monoclonal antibodies specific for structures possibly relevant to cytolysis (Lyt-2, LFA-1, and target cell class I major histocompatibility antigens) or probably unrelated to the cytolytic process (effector cell class I major histocompatibility antigens). Antibodies against Thy-1 were also used. Staining was achieved with immunoperoxidase or immunoferritin. With both techniques nonconjugated cells were either stained or not, depending on whether they bore the antigen corresponding to the antibody used. However, when conjugated to an antigen-bearing cell, a "non-antigen bearing" cell was labeled near the cell interaction area. No increased Fc receptor activity could be detected on bound cells near the interaction area. These data are consistent with the occurrence of limited exchange of membrane macromolecules between bound CTL and target cell.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). I. Analysis of bulk populations and establishment of Lyt-2+L3T4- and Lyt-2-L3T4+ bulk CTL lines

Murine allogeneic cytolytic T lymphocytes (CTLs), including long-term bulk CTL lines, were induced in I-region-incompatible combinations of strains in vitro in order to study the phenotypes of class II major histocompatibility complex (MHC) antigen-specific CTLs, as well as the possible functional involvement of accessory cell interaction molecules such as Lyt-2 and L3T4. This report shows that class II-specific allogeneic CTL populations consist of two types of T cells. Lyt-2+L3T4- (2+4-) and Lyt-2-L3T4+ (2-4+), in variable proportions depending on the strain combination, that in vitro bulk CTL lines with each of these phenotypes can be established, that the killing function of 2-4+ CTL is sensitive to the blocking effect of anti-L3T4 antibodies, suggesting functional involvement of this molecule in the CTL-target interaction, that anti-Lyt-2 antibodies fail to block killing by 2+4- cells, suggesting that such CTLs do not utilize this molecule in their killing function, and that while I-A-specific CTLs of both phenotypes are detectable, 2-4+ cells could not be detected among I-E-specific CTL populations.     

Specific T-cell-mediated killing of autologous lung tumour cells

The phenomenon that strong syngeneic T-cell-mediated cytotoxicity is observed if killer, stimulator, and target cells share H-2 histocompatibility antigens is called H-2 restriction. Here a syngeneic model system making use of hapten-coupled stimulator and target cells is used to explore whether H-2 restriction is absolute or not. Using TNP-coupled spleen or tumor cells as stimulator or target cells in syngeneic and allogeneic situations, it is shown that neither the induction step nor the effector step of TNP-dependent killing is H-2 restricted. By varying the experimental assay conditions more or less H-2-restricted, TNP-dependent killing can be observed. For instance, suboptimal coupling of TNP to targets may result in H-2-restricted killing. Similarly, the use of spleen cell targets as opposed to spleen blast cells or tumor cells may result in H-2-restricted lysis. In contrast optimal coupling of TNP to sensitive target cells and coupling of TNP to cells with certain H-2 haplotypes may lead to significant TNP-dependent killing which is not H-2 restricted. Since hapten-coupled cells lacking H-2 are neither stimulators nor targets these results suggest that the T-cell receptor recognizes TNP-modified H-2 antigens simply as nonself-H-2. Thus hapten coupling of syngeneic cells appears to lead to a histocompatibility antigen change similar to the situation in an allogeneic cytotoxic reaction. Experiments are presented which support this view showing that TNP-coupled and uncoupled syngeneic or allogeneic stimulator and target cells cross-react. For instance allogeneic sensitization may lead to killing on TNP-coupled targets syngeneic to the effector cells and TNP-coupled stimulator cells syngeneic to the effector cells may induce killing on uncoupled syngeneic targets. TNP-dependent cytotoxicity can therefore be envisaged as a kind of allogeneic reactivity due to modification of H-2 antigens by the TNP coupling. This conclusion may have bearing on other model systems in which syngeneic killing appears to be H-2 restricted. In support of this possibility it is shown that allogeneic sensitization may lead to priming of memory cells able to respond to minor histocompatibility antigens.     

In vitro T cell-mediated killing of Pseudomonas aeruginosa. II. The role of macrophages and T cell subsets in T cell killing

T lymphocytes from immune mice can adoptively transfer protection against infection with the extra-cellular Gram-negative bacterium Pseudomonas aeruginosa to nonimmune recipients, and in vitro, immune T cells are able to kill these bacteria. Earlier studies indicated that this killing is mediated by a bactericidal lymphokine. Those studies also showed that macrophages enhance this in vitro T cell killing but do not directly participate in the bacterial killing, nor do macrophages function to present antigen to T cells. The current studies demonstrate that the ability of macrophages to enhance T cell killing can be replaced by macrophage culture supernatants or by purified recombinant interleukin 1 (IL 1). In addition, the macrophage supernatant-induced enhancement can also be blocked by antibody to purified IL 1. These studies also demonstrate that the T cell subset that serves as the final effector cell in the killing process is the Lyt-1-, 2,3+, I-J+ phenotype.     

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.     

Functional characterization of LFA-1 antigens in the interaction of human NK clones and target cells

In the present studies we analyzed the role of LFA-1 antigens in the interaction between NK clones and target cells. The use of various cloned NK cell lines allowed us to analyze homogeneous populations of NK cells which ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotype and specificity. Indirect immunofluorescence with monoclonal antibodies against the alpha (MHM24) and beta (MHM23) chains of the LFA-1 antigen revealed similar patterns of positive reactivity with all NK clones. Both monoclonal antibodies exerted a significant blocking effect on NK cytotoxicity against target cells such as Molt-4 and CEM, whereas the inhibition was very weak against other targets such as K562 and HSB cells. Additive blocking effects were seen when both monoclonal antibodies MHM23 and MHM24 were added to the cytotoxicity assays. When we compared the inhibitory effect of MHM23 and MHM24 on uncultured peripheral blood NK cells and IL 2-activated NK cells, inhibition of cytotoxicity also was found to be primarily dependent on the individual target cells. Thus, the inhibitory activity of anti-LFA-1 antibody was shown to be independent of the phenotypic and functional heterogeneity of the NK clones, activated NK cells, and unstimulated NK cells utilized in these studies. These blocking effects were found to be independent of the LFA-1 antigen expression on the target cell membrane and inhibition occurred only when antibody was bound to the effector cells. Comparison of the effects of anti-LFA-1, anti-T3, and anti-clonotypic antibodies against a Ti-like structure of different NK clones with a mature T cell phenotype demonstrated that each of these antibodies acts on the effector cells in an independent and additive fashion. However, unlike T3 and NKTa antigen, LFA-1 antigen expression is not modulated by cell surface interaction with antibodies specific for this molecule.