Cytotoxic T cell activation by class I protein on cell-size artificial membranes: antigen density and Lyt-2/3 function

The role of antigen surface density, and its relationship to the function of the Lyt-2/3 complex, in recognition and triggering of allospecific cytotoxic T lymphocyte (CTL) precursors has been studied by using a novel type of Class I protein-bearing artificial membrane. The cell-size membranes, termed pseudocytes (artificial cells), can be handled like cells but have a well-defined and easily quantitated surface composition. Class I antigen on these membranes stimulated generation of secondary in vitro allogeneic CTL responses as effectively as allogeneic spleen cells, provided that lymphokines were added to the cultures. Antigen density on the pseudocyte surfaces could be varied over a wide range and quantitated by papain cleavage and fluorescence-activated cell sorter analysis. Recognition and triggering of precursor CTL was found to be dramatically dependent on the surface density of antigen and displayed a marked threshold density requirement, below which little or no response occurred. Examination of the effects of anti-Lyt-2 antibody on responses to pseudocytes provided direct evidence for a reciprocal relationship between antigen density and susceptibility to antibody blockade. The results strongly suggest that antigen density is likely to have important biological consequences in control of immune responses. They also show that if Lyt-2/3 functions by interaction with a ligand, then that ligand is the Class I protein.     

Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2+ T cells with cytolytic and protective activity

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.     

Lyt-2- T cell-independent functions of Lyt-2+ cells stimulated with antigen or concanavalin A

Approximately 30% of cytolytic Lyt-2+ clones from primed mice are able to proliferate autonomously, i.e., independent of IL 2 derived from Lyt-2- cells after antigenic stimulation. H-2K- or -D-restricted induction of Lyt-2+ cells to autonomous proliferation requires Ia+ stimulator cells. A strict correlation was observed between the ability of Lyt-2+ clones to proliferative autonomously and to induce DH. Eventually, the growth of all Lyt-2+ cytolytic clones becomes dependent on exogenous IL 2, and their ability to induce DH is lost. Small Lyt-2+ cells can also be induced in primary cultures by antigen or concanavalin A to proliferate in the absence of exogenous IL 2. The frequency of autonomously proliferating small Lyt-2+ cells is the same as that of small Lyt-2+ cells proliferating in the presence of exogenous IL 2. IL 2 derived from Lyt-2- cells can augment proliferation of Lyt-2+ cells, but is not obligatory.     

Characteristics of the T-lymphocytes interacting with hematopoietic stem cells: the expression of Lyt-3 antigen

The lymph node cells were treated with monoclonal anti-Lyt-3 antibodies (anti-Lyt-3) and complement. Their interaction with colony-forming units (CFU) of bone marrow was studied. Anti-Lyt-3 did not affect the activity of T-lymphocytes, which changed the pattern of syngenic CFU differentiation in spleen of irradiated mice. On the contrary, the activity of T-lymphocytes, which inactivated proliferation and differentiation of non-syngenic CFU in spleen, was decreased. In the peritoneal cavity of irradiated mice antibodies did not affect the activity of T-lymphocytes which suppressed the colony-formation of non-syngenic stem cells and switched off the activity of T-lymphocytes that stimulated syngenic CFU colony-formation. The subpopulation characteristics of regulatory and effector T-cells has been analysed.     

Therapy of murine leukemia with cyclophosphamide and immune Lyt-2+ cells: cytolytic T cells can mediate eradication of disseminated leukemia

Several animal models have been developed in which the adoptive transfer of specifically immune syngeneic T cells has been shown to mediate the eradication of established tumors. In adoptive chemoimmunotherapy (ACIT) of disseminated FBL leukemia with cyclophosphamide and immune T cells, the major effector T cell has been shown to be a noncytolytic Lyt-1+2- T cell that mediates its therapeutic effect without the participation of CTL. Because studies in other models have suggested that CTL can mediate an anti-tumor effect, the efficacy of Lyt-2+ T cells rendered highly cytolytic before adoptive transfer in ACIT of disseminated FBL was examined. The results demonstrated that such CTL had a detectable but limited therapeutic effect in the treatment of FBL. Because this limited activity of transferred purified CTL might have reflected a requirement for helper T cells to produce IL 2 for promotion of the in vivo survival and proliferation of the CTL, the effect of administering IL 2 to tumor-bearing hosts after transfer of CTL was examined. A dose of IL 2 previously shown to induce in vivo proliferation of transferred T cells rendered CTL that were minimally effective alone curative in ACIT of FBL leukemia. Thus, either lymphokine-producing T cells or the lymphokines produced by these cells are necessary for the full expression of the in vivo therapeutic potential of CTL.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Effects of beta-2 microglobulin anti-sense oligonucleotides on sensitivity of HER2/neu oncogene-expressing and nonexpressing target cells to lymphocyte-mediated lysis.

The mechanism by which HER2/neu overexpressing tumor cells resist NK, LAK, and LDCC cytotoxic lymphocytes was investigated. Resistance was not explained by a delay in kinetics of lysis, concurrent resistance to TNF, or a diminished expression of the transferrin receptor. HLA-class I expression, however, was markedly elevated compared to HER2 nonexpressing targets suggesting a reason for resistance. To test the role of class I, we selectively decreased expression by incubation of targets with beta-2 microglobulin anti-sense oligonucleotides. Anti-sense-treated HER2+ targets, displaying levels of class I comparable to HER2- targets, were still markedly resistant to cytotoxic effectors. Down-regulation of class I expression in HER2- carcinoma cells also had no effect on sensitivity to cytotoxicity by anti-sense treatment of Raji and U937 targets resulted in enhanced sensitivity to NK and LAK effectors but not to T cells mediating LDCC. These data indicate resistance to cytotoxicity in HER2-expressing targets cannot be solely explained by heightened expression of class I. The data also support the concept that class I expression regulates sensitivity to NK and LAK cells (but not LDCC effectors) in selected targets.     

The phenotypic characteristics of T-lymphocytes that interact with hematopoietic stem cells: the expression of the antigens Lyt-1, Lyt-2 and L3T4]

The role of T-lymphocytes, bearing antigens Lyt-1, Lyt-2 and L3T4, in regulation of the functional activity of blood-forming precursor cells of syngen and non-syngen origin was investigated. The treatment of cells of murine lymphatic nidi with monoclonal antibodies to the above antigens and with the complement did not abolish the capacity of T-lymphocytes of controlling proliferation and differentiation of syngen and allogen blood-forming precursor cells. The subpopulation characteristics of lymphocytes interacting with the stem cells is discussed.     

Relationship between surface H-2 concentration, size of different target cells, and lysis by cytotoxic T cells

We have examined the parameters of target cell cross-sectional area and surface H-2 concentration in relation to their susceptibility to cytotoxic T-cell-mediated lysis, using a series of commonly used murine target cells of the H-2k haplotype. We used a sensitive immunoferritin labeling technique and electron microscopy to estimate relative cell diameter and H-2 concentration combined with standard 51Cr-release assays for cytotoxicity. We found that susceptibility to cytotoxic T-cell lysis was not related consistently to either factor alone, but was related to a combination of the two, such that above a certain value for the product of the two factors, no further increase in cytotoxic T-cell efficiency was seen. The information presented here should be of value to workers seeking to select a target cell type that will maximize the sensitivity of 51Cr-release-based assays for the detection of cytotoxic T cells.     

Clonal analysis of antigen-specific interactions between T cells and genetically engineered B cells

In order to investigate T cell-B cell interactions we constructed monoclonal, antigen-specific T- and B-cell populations. The Ia+ B-cell lymphoma A20-2J was transfected with trinitrophenyl (TNP)-specific heavy (mu) and light (kappa) chain Ig genes. A hapten-carrier complex (TNP-keyhole limpet hemocyanin (KLH)) bound to the surface Ig expressed on the transfectant and was presented to carrier-specific T-cell hybridoma clones at markedly low doses of antigen (0.01 microgram/ml) and in an Ia-restricted fashion. Two responses were elicited in the responding T-cell clones: (i) high levels of IL-2 secretion (320 units/ml), and (ii) cytotoxicity directed against the antigen-presenting B cell. This cytotoxicity was inhibited by D-mannose and was directed against innocent bystander cells, unlike cytotoxicity mediated by NK cells or alloreactive cytotoxic T lymphocyte. Helper and cytotoxic functions were often present in different T-cell hybridomas but some clones exhibited both activities. One representative T-cell hybridoma exhibited strong helper function for TNP-primed splenic B cells as detected in a plaque-forming cell assay, but was cytotoxic toward antigen-presenting B cells. Such monoclonal assay systems for studying cognate interactions of heterogeneous T cells and specific antigen-presenting cells will provide us with valuable new approaches for the study of antigen-specific T-cell regulation of B-cell activation in immune responses.     

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.     

Studies on the mechanism of lymphocyte-mediated cytolysis. IX. Relationships between antigen recognition and lytic expression in killer T cells.

The relationship between antigen recognition and lytic expression by killer T cells was studied by co-culturing two effector cell populations. When antigen recognition was bidirectional (e.g., b anti-d cells cultured with d anti-b) there was a loss of lytic activity in both populations. In contrast, when antigen recognition was unidirectional (e.g., a anti-d co-cultured with d anti-b) then the loss of lytic activity only occurred in that direction; i.e., there was a marked decline in the d anti-b activity but no change in the a anti-d population. These studies suggest: i) that mere proximity to a killer cell does not lead to target cell death; ii) that accommodation of the T cell's antigen receptor is necessary for the cell to express its lytic potential; and iii) there is direct linkage between the T cell's antigen receptor site and its killing mechanism.     

Activation of human cytolytic cells through CD2/T11. Comparison of the requirements for the induction and direction of lysis of tumor targets by T cells and NK cells

We studied the mechanisms whereby human T cells and NK cells are activated and directed to lyse tumor targets through the CD2 (T11/E-rosette) Ag. Using two cloned NK lines, we showed that these cells, as had previously been shown for T cells, could be directed to lyse an "NK-resistant" tumor target in the presence of antibody heterodimers. These heterodimers consisted of a (mAb) to CD2 (anti-T11(2) or anti-T11(3] linked to a mAb recognizing the tumor cell (J5, anti-CALLA). However, distinct differences between NK cells and T cells were observed with regard to the requirements for such directed lysis: first, only one epitope of CD2 on NK cells (either T11(2) or T11(3] needed to be recognized by the antibody heterodimer in order for directed lysis to occur, whereas for T cells both T11(2) and T11(3) epitopes had to be recognized. Second, in confirmation of previous data with monomeric anti-T11(2) or anti-T11(3) antibody, heterodimers constructed with these reagents enhanced conjugate formation between NK cells and tumor targets, whereas no such enhancement was seen with T cells. All types of heterodimer directed lysis were dependent on the adhesion molecule LFA-1, as an anti-LFA-1 antibody-blocked lysis. Third, whereas in T cells lysis mediated through CD2 appeared to be regulated by CD3 but not vice versa, all types of lysis by NK cells appeared to be regulated through CD2. Finally we showed that F(ab')2 fragments of the anti-T11(2) and anti-T11(3) antibodies could activate NK cells, but were unable to activate T cells either as cloned cytolytic lines, or in populations of PBL. The implications of our findings with regard to the role of CD2 in the activation of cytolytic cells is discussed.     

Subpopulations of mouse Lyt-2+ T cells defined by the expression of an Ly-6-linked antigen, B4B2

The production and characterization of a rat mu,kappa monoclonal anti-mouse T cell subset antibody, B4B2, is reported in this paper. B4B2 typing of lymphoid tissues of commonly used inbred mouse strains revealed two types of reactivity patterns. They can be characterized as C57BL/6-like (B6-like) or C3H/He-like (C3H-like). Among B6-like strains, B4B2 recognizes 5 to 10% of spleen cells, 30 to 50% of bone marrow cells, and less than 2 to 3% of thymocytes. In C3H-like strains, B4B2 reacts with less than 1% of spleen cells, 2 to 8% of bone marrow cells, and less than 1% of thymocytes. B4B2 recognizes a T cell subset differentiation antigen expressed by B6-like strains but not by C3H-like strains. Typing of BXH recombinant inbred strains showed linked expression of B4B2 and the Ly-6 antigen. The expression of B4B2 antigen appears to be under codominant control as the median fluorescence distribution of B4B2+ cells in C57BL/6 was approximately twice that of (C57BL/6xC3H)F1. B4B2 was shown to react with approximately 40 to 50% of Lyt-2+ T cells and less than 1% of L3T4+ T cells. No staining of resting or activated B cells by B4B2 was detected. The ratio of B4B2+:Lyt-2+ cells was similar for resting T cells and activated T cells obtained from mitogen-stimulated cultures or mixed lymphocyte cultures. In neonatal spleen, substantially more B4B2+ than Lyt-2+ cells were found. With increasing age, however, a rapid decline in B4B2+ cells and a corresponding increase of Lyt-2+ cells was observed. By approximately 1 mo of age, the relative proportion of these subsets had reversed so that Lyt-2+ cells became more numerous than B4B2+ cells.     

Subsets of Lyt-2+ cells defined by differential expression of the 9F3 antigen: alterations in mice of the lpr/lpr genotype

Recently, we found that a novel murine cell surface antigen recognized by the 9F3 monoclonal antibody demonstrates T cell heterogeneity in normal mice and in congenic strains homozygous for the lymphoproliferation- and autoimmunity-inducing lpr gene. The objective of the present work was to further characterize this heterogeneity by studying the distribution of the 9F3 marker among Lyt-2+ T cells. By using dual parameter flow cytofluorometry analysis, at least two subsets of peripheral Lyt-2+ cells differing in 9F3 expression could be distinguished. In MRL/Mp-++, C57BL/6, and C3H/HeJ mice, 9F3- and 9F3+ cells accounted, respectively, for 65 to 80% and 20 to 35% of the whole Lyt-2+ population. Interestingly, in lpr/lpr-bearing congenic strains, a three- to fivefold selective increase in the frequency and absolute number of 9F3+ Lyt-2+ cells was found to take place during aging. Functional analysis of Lyt-2+ cells isolated by panning revealed that in +/+ mice, these cells respond better to phytohemagglutinin and concanavalin A than in lpr/lpr mice, indicating that phenotypic changes of Lyt-2+ cells correlate with functional changes. Further evidence for the functional relevance of 9F3-defined Lyt-2+ cell heterogeneity was provided by the study of sorted cells from +/+ mice, showing that 9F3- cells exhibit higher mitogenic reactivities than 9F3+ cells. This finding suggests that 9F3+ Lyt-2+ cells in +/+ and lpr/lpr mice are functionally homologous. Together, these data indicate that, in addition to the known expansion of a population of abnormal Lyt-2-T cells, mice of the lpr/lpr genotype have an alteration of their Lyt-2+ cell population, characterized by an imbalance of 9F3-defined subsets and decreased mitogenic reactivities.     

Role of L3T4 antigen in the activation of various functions of Lyt-1+2- T cells against vaccinia virus

The present study defines assay systems for vaccinia virus-reactive Lyt-1+2- T cells mediating various functions and investigates the positivity of L3T4 antigen on these Lyt-1+2- T cells as well as the role of L3T4 antigen in the activation of these T cells with respect to their functions. C3H/He mice were immunized against vaccinia virus by inoculating viable virus intraperitoneally (i.p.). Anti-vaccinia virus reactivity in lymphoid cells from these immunized mice was assessed by proliferative response, helper T cell activities involved in cytotoxic T lymphocyte (CTL) and B cell (antibody) responses, delayed type-hypersensitivity (DTH) response, and production of lymphokines such as interleukin 2 (IL2) and macrophage-activating factor (MAF). The results demonstrate that all of the above anti-vaccinia virus responses were mediated by Lyt-1+2- T cells and that these Lyt-1+2- T cells expressed L3T4 antigens on their cell surfaces. Moreover, such anti-vaccinia Lyt-1+2- T cell responses were inhibited in the presence of anti-L3T4 antigen antibody. These results indicate that there is a reciprocal relationship between Lyt-2 and L3T4 markers, and that L3T4 antigen is closely related to the activation of various functions of anti-vaccinia virus Lyt-1+2- T cells.     

Protein-antigen specific Ia-restricted cytolytic T cells: analysis of frequency, target cell susceptibility, and mechanism of cytolysis

We previously showed that cloned, antigen-specific, Ia-restricted L3T4a+ T cell lines can be cytolytic for antigen-pulsed B cell lymphoma targets. Such cells can also, under different experimental conditions, activate B cells to proliferate and secrete immunoglobulin. In the present experiments, we show that this functional phenotype is a common one among a panel of cloned T cell lines. In keeping with this finding, freshly isolated, antigen-activated lymph node T cells show similar functional properties. Such cytolytic L3T4a+ T cells differ from classical H-2K/D-restricted cytolytic T cells in two distinct ways. First, Ia-restricted cytolytic T cells can kill bystander targets, whereas H-2K/D-specific cytolytic T cells do not. Second, in testing a panel of target cells by using lectin-mediated cytolysis, Ia-restricted cytolytic clones reveal large differences in target cell susceptibility, whereas all targets are similarly susceptible to H-2K/D-specific killer cells. Finally, evidence is presented that both direct and bystander killing effected by L3T4a+ T cells are mediated by the same soluble factors, in that there is a strong positive correlation of these two activities for individual cloned lines. The relevant mediators appear to be lymphotoxin and IFN-gamma, although the latter molecule by itself is not cytolytic on our target lines.     

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.