CD4 is involved in a post-binding event in the cytolytic reaction mediated by human CD4+ cytotoxic T lymphocyte clones

CTL lyse their target cells in discrete phases. First, the CTL bind to the target cell in a Mg2+-dependent manner followed by a Ca2+-dependent cytolytic phase. In the present study, we investigated the role of CD4 in the different phases of the cytolytic reaction mediated by human CD4+ class II MHC-specific CTL clones by using a single cell assay. It was found that the anti-CD4+ mAb OKT4A, which blocks cytotoxic reactions by CD4+ CTL clones as measured with a 51Cr release assay, only marginally affects the formation of conjugates. It appeared that OKT4A more strongly blocked the post-binding phase of the cytolytic reaction. In contrast, anti-leukocyte function-associated mAb strongly blocked the formation of conjugates but not the subsequent lytic phase. As was found previously with CD8+ CTL clones, anti-TCR mAb generally did not affect the formation of conjugates. One exception was noted. The activity of a CD4+ CTL clone, HY-640, could not be blocked by OKT4A, but was affected by an anti-TCR mAb. This anti-TCR mAb could partly reduce the formation of conjugates between HY-640 cells and their specific target cells. These results suggest that this clone has a high affinity TCR, which can contribute to the formation of conjugates. Although preincubation of the CTL clones with OKT4A only marginally affects the number of conjugates upon subsequent mixture with target cells, it was observed that incubation at 37 degrees C of preformed conjugates with OKT4A markedly reduced the number of conjugates. This dissociation of preformed conjugates was optimal only after 2 h of incubation. In contrast, an anti-leukocyte function-associated mAb induced a much more rapid dissociation of preformed conjugates.     

Inhibition of cytotoxic T cell lysis by anti-CD8 monoclonal antibodies: studies with CD3-targeted cytolysis of nominal antigen-negative targets

The function of the CD8 molecule in lympholysis mediated by cytotoxic T cells was investigated by examining possible contributions of ligands on the target cell to the inhibition of lysis observed with CD8-specific mAb. In order to evaluate a variety of target cells, including those not expressing the nominal Ag (NA) for which the CTL was specific, lysis was effected by cross-linking the CTL and the target cells with anti-CD3 mAb. Such CD3 redirected cytotoxicity was demonstrated to be inhibited by anti-CD8 mAb when low anti-CD3 mAb concentrations were used. The possibility that inhibition by anti-CD8 mAb resulted for competition for the FcR between the anti-CD3 mAb and anti-CD8 mAb was eliminated by targeting TNP-modified cells with an antibody heteroconjugate prepared from Fab fragments of anti-CD3 and anti-DNP antibodies. Inhibition of the lysis of target cells not expressing NA including those deficient in class I expression, demonstrated that neither NA nor class I expression was required for anti-CD8 mAb inhibition. Whether the anti-CD8 mAb inhibition required CD8 Ag interaction with any ligand on the target cell was further investigated by measuring exocytosis of enzyme granule from CTL activated with CD3-coated poly-styrene beads. CD8-specific mAb inhibited such CTL activation in this target cell-free system. A CD8(+), MHC class II-specific CTL clone, was used to show differential inhibition by anti-CD8 mAb, depending on the target cell, therefore providing evidence that anti-CD8 mAb binding does not generate an absolute off signal. These data are consistent with the hypothesis that anti-CD8 mAb affect the lytic process independent of the recognition of a ligand on the target cell by CD8.     

Human T cell clones exerting multiple cytolytic activities show heterogeneity in susceptibility to inhibition by monoclonal antibodies

Human cytotoxic T cell clones (CTL) were obtained by limiting dilution after in vitro priming against an allogeneic Epstein Barr virus (EBV)-transformed B cell line (B-LCL) BSM. Three OKT3+, OKT8+ E rosette-forming (RFC) but EA gamma-RFC- clones with cytotoxic activity against the stimulator cell and one "non-cytolytic" clone were expanded for over 50 generations and further characterized. Clone G9 showed allospecific lysis of Cw3+ lymphocytes and B cell lines. Three cytolytic clones (G9, D11, and A3) showed cytotoxicity to the stimulator B-LCL, to the human plasma cell leukemia-derived line LICR-LON-HMY2 and to short-term cultured melanoma cells (O-mel). Four other EBV-transformed B-LCL unrelated to the stimulator B-LCL were not lysed. These clones also exerted cytotoxic activity against NK-sensitive target cells (TC), e.g., the erythroleukemia cell line K562. Other NK-sensitive TC, e.g., lymphoma-derived Daudi cells, were killed provided they were pretreated with phytohemagglutinin (PHA). Cytolytic activity against the B-LCL cell LICR-LON and O-mel, but not against K562 or PHA-treated target cells, was inhibited by monoclonal anti-HLA ABC antibodies (MCA). The cytolytic activities of OKT3+,8+ clones G9 and A3 but not that of OKT3+,8+ clone D11 were inhibited by OKT8. Another MCA, 13.3, directed against the murine glycoprotein T-200, inhibited the cytolytic activity of clone D11 against K562 but not against the stimulator cells. Clone G9 was not inhibited by MCA 13.3. The four clones, including the OKT4+ "non-cytotoxic" clone K12, exerted lytic activity against TC that are normally resistant to lysis provided these TC were pretreated with PHA. The TC specificity range of the clones was confirmed by cold target inhibition experiments. A correlation between blocking of lytic activity by cold TC and the percentage of conjugate formation with the particular cold TC was observed. Because these clones also show differential susceptibility to inhibition of lysis by various MCA, it is concluded that human cytotoxic T cell clones can exert multiple lytic activities, i.e., the operationally defined lytic mechanisms differ at least at certain stages of the lytic cycle.     

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.     

Evidence for an association between CD8 molecules and the T cell receptor complex on cytotoxic T cells

The T cell differentiation molecule CD8 is thought to play an important role in class I major histocompatibility complex-restricted T cell activities but the precise function of this molecule is unknown. To explore this question, we have studied several CD3+, CD8+ class I alloantigen-specific cytotoxic T lymphocyte (CTL) lines and clones. The ability of these CTL to proliferate as well as to lyse specific targets was inhibited by either anti-CD3 or anti-CD8 monoclonal antibodies. Exposure of CTL to relevant but not irrelevant target cells induced the rapid (less than 1 hr) disappearance of approximately 20 to 30% of CD3 and CD8 molecules from the cell surface. The modulation of these molecules became maximal at 6 to 12 hr and recovered thereafter in parallel. Treatment of CTL with anti-CD8 prevented alloantigen-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD8. Incubation of CTL with the combination of anti-CD3 and goat anti-mouse Ig also resulted in modulation of CD8. In contrast, the expression of other CTL surface antigens, such as CD2 (Leu-5, T11) and HLA-DR, was not reduced by any of these manipulations. These results suggest that CD8 molecules are associated with the CD3/antigen receptor complex on the surface of CTL, and may play a direct role in antigen-induced modulation and cross-linking of the T cell receptor.     

Characterization of anti-CD8-resistant cytolytic T lymphocytes induced by multivalent cross-linking of CD8 on precursor cells

The lytic activity of most CD8+ MHC class I allospecific CTL generated in vitro can be inhibited by anti-CD8 antibodies. Such inhibition has led to hypotheses that CD8/class I interactions normally contribute to the triggering of CTL with low or moderate avidity Ag-specific TCR by providing those CTL with auxiliary binding avidity. However, CD8 has also been proposed to play an active signaling role in T cell activation. We have recently reported that multivalent cross-linking of CD8 on CTL precursors in MLC does appear to mediate activation signals, and induces the generation of CD8+ MHC class I allospecific CTL whose lytic activity cannot be blocked by anti-CD8 antibodies. In our present study, we have further characterized such anti-CD8 uninhibitable effector cells. These CTL are resistant to blocking of their lytic function by anti-Lyt-3 mAb as well as anti-Lyt-2 mAb, but remain sensitive to blocking by anti-LFA-1 mAb, indicating that they do use non-CD8 cell adhesion molecules during target cell recognition and lysis. As a consequence of mAb-induced multivalent CD8 cross-linking during their generation, anti-CD8 uninhibitable CTL significantly reduce their cell surface expression of CD8, which permits their identification and facilitates their purification from heterogeneous MLC populations. Such anti-CD8 uninhibitable effector cells can be maintained as stable CTL lines, in the absence of anti-CD8 mAb after the initial induction period. The in vitro generation of anti-CD8 uninhibitable CTL, which may be highly enriched for cells bearing high affinity TCR, could represent a new experimental approach to studies of TCR gene usage and repertoire, as well as a potentially important strategy for the deliberate generation of high affinity effector cells for adoptive immunotherapy.     

Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL

Destruction of virus-infected cells by CTL is an extremely sensitive and efficient process. Our previous data suggest that LFA-1-ICAM-1 interactions in the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse mediate formation of a tight adhesion junction that might contribute to the sensitivity of target cell lysis by CTL. Herein, we compared more (CD8(+)) and less (CD4(+)) effective CTL to understand the molecular events that promote efficient target cell lysis. We found that abrogation of the pSMAC formation significantly impaired the ability of CD8(+) but not CD4(+) CTL to lyse target cells despite having no effect of the amount of released granules by both CD8(+) and CD4(+) CTL. Consistent with this, CD4(+) CTL break their synapses more often than do CD8(+) CTL, which leads to the escape of the cytolytic molecules from the interface. CD4(+) CTL treatment with a protein kinase Ctheta inhibitor increases synapse stability and sensitivity of specific target cell lysis. Thus, formation of a stable pSMAC, which is partially controlled by protein kinase Ctheta, functions to confine the released lytic molecules at the synaptic interface and to enhance the effectiveness of target cell lysis.     

A murine cytotoxic T lymphocyte clone from the intestinal mucosa that is antigen specific for proliferation and displays broadly reactive inducible cytotoxic activity

Thy-1+, Lyt-1-,2+, asialo GM1+ cytotoxic T lymphocyte (CTL) clones have been isolated from the intestinal mucosa of mice primed with alloantigens. Two different types of cytotoxic clones have been obtained. The first type is functionally similar to most splenic and lymph node-derived CTL clones in that they are strictly antigen specific with respect to proliferation and cytolytic activity. The second type of CTL clone has several unique characteristics. Although these clones are also antigen specific with regard to proliferation, they are not cytolytic under standard growth conditions in medium containing 4% rat concanavalin A-induced spleen cell supernatant. After culture for 4 days in the presence of high concentrations of interleukin 2, cells become activated and exhibit broad lytic potential. Moreover, during the activation process, these CTL begin to express a murine T cell surface antigen, CT-1, which is associated with activated cytotoxic cells. The findings reported in this report should now allow us to precisely define, both phenotypically and functionally, specific lymphocyte populations that make up the gut-associated lymphoid tissues. These data also describe a new type of effector CTL that differs from other cytotoxic cells reported to date, because it is antigen dependent for proliferation, but requires signals mediated by lymphokines for lytic activation.     

Differences in surface phenotype between cytolytic and non-cytolytic CD4+ T cells. MHC class II-specific cytotoxic T lymphocytes lack Leu 8 antigen and express CD2 in high density

A number of cell surface molecules are differentially expressed on functionally distinct subsets of CD4+ T cells. However, to date CD4+ T cells capable of becoming CTL have not been shown to be phenotypically distinct from other CD4+ T cells, and in the current study we examined the ability of Leu 8+ and Leu 8- CD4+ subpopulations to become cytotoxic effectors after their stimulation with allogeneic lymphoblastoid cell lines. Although CD4+, Leu 8+ cells proliferated more vigorously than CD4+, Leu 8- cells in primary cultures stimulated with allogeneic LCL, the CD4+, Leu 8- population was the major source of cytotoxic effectors, killing targets with specificity for their class II MHC alloantigens. In most subjects, CD4+ precursors of CTL were distinguished not only by their lack of Leu 8 expression but also by their relatively high density of CD2, LFA-1, and LFA-3, molecules known to mediate non-specific cell-to-cell adhesion and postulated to be markers of immunologic memory. The absence of Leu 8 does not appear to be a reliable memory cell marker, however, because Leu 8+ as well as Leu 8-, CD4+ cells from PPD skin test positive subjects responded to the recall Ag, PPD. During 3 mo of continuous culture with allogeneic stimulators, Leu 8- cells retained their cytolytic activity and remained unreactive with anti-Leu 8 mAb, whereas Leu 8+ cells remained non-cytolytic and reactive with anti-Leu 8, suggesting that under the conditions used the Leu 8 phenotype is relatively stable. PHA or anti-CD3 mAb enhanced non-specific killing by alloantigen-stimulated CD4+,Leu 8- lines but failed to unmask any cytolytic potential in CD4+,Leu 8+ lines. We conclude that MHC class II-specific cytolytic CD4+ T cells can be distinguished from non-cytolytic CD4+ cells on the basis of their surface phenotype, and that most CD4+ CTL are contained within the Leu 8- subpopulation.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation.

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.     

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.     

The lymphocyte function-associated antigen (LFA)-1 and CD2/LFA-3 pathways of antigen-independent human T cell adhesion

Human cytotoxic T lymphocyte clones form conjugates with both antigen-positive and antigen-negative lymphoblastoid cells. Conjugates with antigen-negative targets form as rapidly, and are almost as frequent, as those with antigen-positive targets; both types are strong. Monoclonal antibodies against lymphocyte function-associated antigen (LFA)-1, CD2, and LFA-3 (or their Fab fragments) each consistently inhibit conjugate formation, but only partially; mixes of alpha LFA-1 with either CD2 monoclonal antibodies or alpha LFA-3 cause complete inhibition. Our previous studies have demonstrated two distinct pathways of antigen-independent conjugate (AIC) formation, one involving LFA-1 and the other involving CD2/LFA-3. The present studies showing supra-additive inhibition with mixes of Fab indicate that at least a major fraction of the conjugates involve T cells which utilize both pathways. Preincubation studies (and restricted expression for CD2) demonstrate that in the CD2/LFA-3 pathway, CD2 is critical on the effector and LFA-3 on the target and that in the LFA-1 pathway, LFA-1 is critical on the effector. Analysis of conjugate formation by primary allosensitized T cells confirms the critical findings made with T cell clones. Among a panel of antigen-negative "target" cell lines tested, there is wide variation in the number of AIC formed with cytotoxic T lymphocyte clones; this variation correlates partially with differences in level of expression of LFA-3. Both pathways of adhesion are utilized in AIC formation with all five targets tested, but there was variation between targets in the relative contribution by each pathway. Studies of inhibition of lysis (rather than conjugate formation) support the relevance of the two-pathway model to the lytic process as a whole. These studies demonstrate the general involvement of two pathways of adhesion in human T cell interactions: one involving T cell LFA-1 and the other involving T cell CD2 binding to target cell LFA-3.     

Antigen specific and MHC nonrestricted cytotoxicity of T cell receptor alpha beta+ and gamma delta+ human T cell clones isolated in IL-4

IL-4 has been shown to act as a growth factor for human T cells. In addition, IL-4 can enhance CTL activity in MLC, but blocks IL-2 induced lymphokine activated killer cell activity in PBL. In our study, the cloning efficiencies, Ag-specific CTL activity and non-MHC-restricted cytotoxicity of CTL clones generated in IL-2 were compared to those generated in IL-4. In a first experiment, T cells were stimulated with the EBV-transformed B cell line JY and cloned 7 days later with feeder cells and either IL-2 or IL-4. In a second experiment, stimulation of the T cells was carried out in the presence of IL-2 plus anti-IL-4 antibodies or IL-4 plus anti-IL-2 antibodies in order to block the effects of IL-4 and IL-2, respectively, produced by the feeder cells. Although the cloning efficiencies in the second experiment were lower than those obtained in the first experiment, the cloning efficiencies obtained with IL-2 or IL-4 were similar in both experiments. The overall proportion of TCR alpha beta+ T cell clones cytotoxic for the stimulator cell JY established in IL-2 or IL-4 were comparable. A striking difference between the clones obtained in IL-2 or IL-4 was that a large proportion of the clones obtained in IL-4 expressed CD4 and CD8 simultaneously, whereas none of the clones isolated in IL-2 were double positive. Also gamma delta+ T cell clones could be established with IL-4 as a growth factor. TCR gamma delta+ T cell clones isolated in either IL-2 or IL-4 were CD4-CD8- or CD4-CD8+, but the proportion of CD4-CD8+ clones isolated in IL-4 was higher. Interestingly, one TCR gamma delta+ clone isolated in IL-2 was CD4+CD8-. Most of the TCR alpha beta+ and TCR gamma delta+ CTL-clones isolated in IL-2 lysed the NK cell sensitive target cell K562. In contrast, only a small proportion of the TCR alpha beta+ or TCR gamma delta+ CTL clones isolated in IL-4, lysed K562. One TCR gamma delta+ T cell clone (CD-124) isolated in IL-4 and subsequently incubated in IL-2 acquired lytic activity against K562.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of the role of leukocyte function-associated antigen-1 in activation of human influenza virus-specific T cell clones

The role of leukocyte function-associated Ag-1 (LFA-1) in intercellular adhesion is well documented. Previously, we demonstrated that the LFA-1 molecule (CD11a/CD18) can also regulate the induction of proliferation of peripheral blood T cells. In these studies, we observed opposite effects of antibodies against CD11a (LFA-1-alpha-chain) or CD18 (LFA-1-beta-chain). Here, we determined the effects of anti-CD11a and anti-CD18 mAb on proliferation of cloned influenza virus-specific T cells. Anti-CD18 mAb had similar inhibiting effects on the proliferative response of T cell clones induced by immobilized anti-CD3 mAb as it had on the response of peripheral blood T cells. In contrast to its costimulatory effect on resting peripheral blood T cells, anti-CD11a mAb did not increase the proliferation of cloned T cells. Similar differences in effects of anti-CD11a and anti-CD18 mAb were observed when proliferation of the T cell clones was induced by immobilized anti-TCR mAb. When proliferation was induced by influenza virus presented by monocytes as APC, both anti-CD11a and anti-CD18 mAb inhibited T cell proliferation. However, when EBV-transformed B cells were used as APC, neither anti-CD11a nor anti-CD18 mAb inhibited proliferation. These results demonstrate that the effects of antibodies against CD11a (LFA-1-alpha) or CD18 (LFA-1-beta) on T cell proliferation depend on 1) the stage of activation of the T cells, 2) the activation stimulus and its requirement for intercellular adhesion involving LFA-1, and 3) the type of cell used to present Ag.     

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.     

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.     

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.     

Cytolytic T lymphocytes: an overview of their characteristics

Cloned T cells have been useful for assessing the lytic potential of distinct T cell subsets and for determining the relative contribution of different effector mechanism involved in the lytic process. Alloreactive CD8⁺ murine T cell clones and cloned murine CD4⁺ T_H1 and T_H2 T cells reactive with nominal antigen (ovalbumin) lysed nucleated target cells bearing antigen or coated with anti-CD3 monoclonal antibody in a short term⁵¹Cr-release assay. These clones were also evaluated for their ability to lyse efficiently sheep erythrocyte (SRBC) target cells coated with anti-CD3 mAb by a mechanism (presumably involving membrane damage) that does not involve nuclear degradation. Three patterns of lysis were observed: CD8⁺ and some CD4⁺ T_H2 effector cells lysed efficiently nucleated target cells and anucleated SRBC coated with anti-CD3 mAb. However, CD4⁺ T_H1 (and a few T_H2) T cells which lysed nucleated target cells bearing antigen or coated with anti-CD3 mAb didnotlyse efficiently the SRBC coated with anti-CD3 mAb. One CD4 bearing T_H2 cell failed to lyse efficiently either nucleated target cells or anucleated SRBC coated with anti-CD3 mAb. These results indicate that both T_H1 and T_H2 clones have lytic capabilities. Furthermore, they suggest that some but not all T_H2 murine T cell clones have lytic characteristics similar to those of conventional CD8⁺ CTL. However, it is not certain how these patterns of lysis of target cellsin vitro relates to the capacity of CTL to lyse such target cellsin vivo.