Molecular interactions required for triggering alloantigen-specific cytolytic T lymphocytes

Affinity-purified class I proteins in detergent solution can be directly immobilized on plastic by a simple dilution procedure. The immobilized Ag retains its native structure and will trigger specific, TCR-mediated degranulation of cloned CTL. Stimulation of the response is dependent on the surface density of Ag, and displays a critical threshold density below which response does not occur. Individual clones differ with respect to the threshold density required for activation, but these differences are not large. With one exception, the cloned lines examined respond to Ag densities comparable to that found on normal allogeneic cells, and critical threshold densities varied over about a fourfold range. Coimmobilization of alloantigen and nonantigenic class I protein of a different specificity has the effect of decreasing the threshold density of alloantigen required for response to occur. This augmentation is specific for class I, coimmobilized class II protein does not affect responses, and is very likely mediated by Lyt-2 (CD8) interaction with nonpolymorphic determinants on the class I protein. Thus, class I alloantigen is the necessary and sufficient ligand for activation of most allogeneic CTL clones, and both TCR and Lyt-2 interactions contribute to the response. The results described here for effector CTL are compared with those previously found in examining the ligand requirements for activation of precursor CTL.     

Calcium ionophore plus phorbol ester can substitute for antigen in the induction of cytolytic T lymphocytes from specifically primed precursors

Previous studies indicated that Ca++ ionophores and phorbol esters in synergy could substitute for the initial activation step of normal T lymphocytes or T cell clones leading to increased expression of receptors for the growth factor interleukin 2 (IL 2) and secretion of interleukins, with the mitogenic signal for T cell proliferation being dependent on the presence of IL 2. In this study, the question was addressed as to whether T lymphocytes activated through the Ca++ ionophore ionomycin and the phorbol ester 12-o-tetradecanoyl phorbol 3-acetate (TPA) also acquired the competence to kill relevant target cells. The results indicate that T lymphocytes from primed mice proliferate and lyse the relevant allogeneic target cells after in vitro stimulation with ionomycin plus TPA, and that T lymphocyte preparations enriched for a subpopulation bearing the Lyt-2 marker are dependent on exogeneous sources of IL 2 to proliferate and become competent killer cells, whereas preparations enriched for subpopulations bearing the L3T4 marker grow independently of exogenous IL 2.     

Increased ornithine decarboxylase activity and polyamine biosynthesis are required for optimal cytolytic T lymphocyte induction

The objective of the present investigation was to evaluate the requirement for increased ornithine decarboxylase (ODC) activity and polyamine biosynthesis in the induction of cytolytic T lymphocytes (CTL). In this regard, we have utilized alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO treatment completely abrogated Con A-induced NW T-cell ODC activity. Similarly, DFMO treatment reduced putrescine and spermidine biosynthesis 100 and 87% respectively by the end of a 48-hr incubation period. Polyamine depletion reduced the Con A-mediated polyclonal induction of CTL by 52 and 81% at 24 and 48 hr of culture, respectively. The effect of DFMO on CTL induction could be reversed by the addition of exogenous putrescine. These data indicate that the observed effects of DFMO on CTL induction were mediated through inhibition of polyamine biosynthesis. Therefore, increased ODC activity and polyamine biosynthesis are required for optimal CTL induction. Furthermore, polyamine depletion did not impair IL-2 production; however, IL-2-dependent proliferation was reduced. These data are the first to discriminate between the requirement for polyamines with regard to IL-2 responsiveness, rather than IL-2 production, during a primary T-cell mitogenic response.     

Accessory cells, dendritic cells, or monocytes, are required for the lymphokine-activated killer cell induction from resting T cell but not from natural killer cell precursors

In this study we have investigated the role of accessory cells in the development of lymphokine-activated killer cells (LAK) from highly purified human NK and small resting T cell progenitors. As accessory cells we used autologous, as well as allogeneic, monocytes, and dendritic cell enriched cells. Both NK and T cells were able to generate LAK activity, but their activation requirements were different. NK cells were activated merely by IL-2, and accessory cells did not enhance their lytic activity in the presence or absence of IL-2. Conversely, T cells were practically unresponsive to even high concentrations of IL-2 having a strict requirement for accessory cells for the development of lytic activity and proliferation. Accessory cells differed in their ability to activate T cells presumably depending on their ability to induce IL-2 synthesis, allogeneic dendritic cells being the most effective accessory cells and IL-2 synthesis stimulators. Allogeneic accessory cells could induce lytic activity in T cells even in the absence of exogenous IL-2. Thus, accessory cells play a central role in expanding the LAK effector cell population.     

Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes

Immunotherapy of cancer could be possible in cases in which competent effector T cells can be induced. Such an approach depends on expression of tumour-specific antigens by the tumour cells and on the availability of sufficient costimulatory support for activation of cytotoxic T lymphocytes. Here, a strategy for helper T cell recruitment for induction of tumour-specific cytotoxic immune responses is presented. Allogenic MHC class II molecules were introduced into tumour cells by cell fusion. These hybrid cells, when injected into mice, induced rejection of an established tumour. The contribution of CD4-expressing helper T cells in the induction phase and of CD8-expressing T cells in the effector phase of the immune response was demonstrated. The approach described could be applicable to cases in which a suitable tumour antigen is present but not identified; it employs regulatory interactions that govern physiological immune responses and is designed to be minimally invasive.     

Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients' malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-gamma, TNF-alpha, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.     

The induction of cytolytic T lymphocytes with syngeneic trinitrophenyl-coupled membranes.

Evidence is presented that trinitrophenyl-coupled tumor membranes are able to induce cytolytic T lymphocytes (CTL) when co-cultured with syngeneic spleen cells. These haptenated membranes stimulate spleen cells from naive and immune mice. The specificity of these CTL is determined by the H-2 antigens of the membranes used for stimulation.     

The induction of cytolytic T lymphocytes with purified plasma membranes.

Subcellular fractions were demonstrated to be effective in stimulating primary and secondary allogeneic CTL responses. 5' nucleotidase activity was used to assess purification of plasma membranes and stimulating activity was found to co-purify with the plasma membrane fraction of the cell. Electron micrographs of these purified plasma membranes showed the majority of the material to be in the form of vesicles of relatively heterogeneous size. The cytolytic T lymphocytes generated by incubation with purified plasma membranes demonstrated immunologic specificity.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.     

Three different erythrocyte surface molecules are required for spontaneous T cell rosette formation

We have obtained three anti-sheep erythrocyte (E) monoclonal antibodies (MAb) which block rosettes with human T cells. They also block rosettes with E from all the species we have tested, including rosettes with autologous E. They define three different minor components on the E surface: MAb N217 precipitates a single 42-kilodalton (kDa) chain and MAb N4 a single 14-kDa chain, and MAb N23 immunoprecipitates under nonreducing conditions a single band at approximately 220 kDa, which is resolved under reducing and alkylating conditions, in two bands migrating at approximately 110 kDa and approximately 220 kDa. Thus MAb N23 is likely to react with a complex made of covalently linked 100-kDa chains associated in a noncovalent fashion with approximately 220 kDa chains. In addition, we have observed a puzzling phenomenon, i.e., that binding the MAb N23 first increases, to a large extent, the amount of N4 epitopes which can be subsequently detected on E. This effect was not observed when N217 MAb or antiglycophorin (either monoclonal or polyclonal) antisera were first bound on E. Therefore the N23 and N4 molecules must interact on the E surface. This finding discloses the complex interactions between the T cell and E surface component that must occur in the process of rosette formation, including that with autologous E. These observations are of interest in view of the recent evidence that the CD2 molecule is involved both in adhesion and activation aspects of T cell functioning.     

Maturation of cytolytic T lymphocytes

We have studied the maturation of cytotoxic T-lymphocytes (CTL) following primary and anamnestic responses in vivo and in vitro. Parameters evaluated included: frequency of effector CTL, specificity of binding to and lysis of target cells, killing and recycling ability of individual CTL, and the avidity of effector-target conjugation. While the frequency of effector CTL in the peritoneal cavity of BALB/c mice immunized against leukemia EL4 of C57BL/6 origin increases from 0 to 35% in 11 days of priming, a paradoxically lower frequency has been observed usually after 2 degrees and repeatedly after 3 degrees immunizations both in the peritoneal cavity and in the spleen. The H-2 haplotype and H-2 sub-loci specificity of CTL is preserved upon repeated immunizations. Likewise, the rate of killing and recycling of individual CTL do not change throughout immunizations, suggesting that the cytolytic activity of individual effector CTL is discrete ("quantal") and not subject to maturation upon repeated immunizations. On the other hand, inhibition of conjugate formation and of lysis by antibodies against target major histocompatibility complex (MHC) or effector Lyt-2 determinants is consistently less effective with 3 degrees CTL, suggesting an increase in avidity of effector/target interaction upon repeated immunizations. A striking increase in apparent avidity has been observed during CTL priming in mixed lymphocyte reaction, as deduced from blocking by target cell MHC antibodies. These results suggest that alloimmune CTL undergo maturation with respect to their ability to interact with the target, and that the composition of the responding population is subject to moderate selective processes driven by repeated antigenic stimuli.     

The induction of cytolytic T lymphocytes with specificity for p-azophenylarsonate coupled syngeneic cells.

Conditions are described for diazonium linkage of the hapten p-azophenylarsonate (Ar) to mouse lymphocytes for the purpose of induction of syngeneic cytolytic T lymphocytes. These cytolytic effector cells do not lyse target cells that are coupled with an unrelated hapten, trinitrophenyl. Cell lysis can be blocked by the addition of anti-Ar antiserum. Only those target cells that share H-2 K or D end antigens with the stimulating cell population are susceptible to lysis. Several methods were used in an attempt to demonstrate the presence of the Ar cross-reactive idiotype on CTL induced in A/J mice.     

Alloantigen-specific cytotoxic and suppressor T lymphocytes are derived from phenotypically distinct precursors

Although Leu-2+ (OKT8+) T cells activated in the mixed lymphocyte reaction (MLR) mediate both alloantigen-specific cytotoxicity and suppression of alloantigen-induced proliferation, it is not known whether these functions derive from a single cell type or phenotypically distinct cells. This study was undertaken to examine the alloantigen-specific cytolytic and suppressor potential of two subpopulations of Leu-2+ cells distinguishable from one another on the basis of their binding to the monoclonal antibody 9.3. Leu-2+, 9.3+ and Leu-2+, 9.3- populations were purified from peripheral blood, cultured for 7 days with autologous helper/inducer (Leu-3+) cells and allogeneic non-T cells, and reisolated before testing for cytotoxicity and suppression. All detectable alloantigen-specific cytolytic activity was confined to the Leu-2+, 9.3+ subpopulation. Killing by this subset was specific for the HLA-A and B (class I) major histocompatibility complex (MHC) antigens of the priming cell. By contrast, suppression of proliferation was mediated predominantly by the Leu-2+, 9.3- cells, and suppression by this subpopulation was specific for the HLA-DR (class II) MHC antigens of the priming cell. The development of suppression by Leu-2+, 9.3- cells was unaffected by cyclosporin A (CsA), an agent shown previously to block the development of cytolytic but not suppressor cells in MLR. Alloactivated Leu-2+, 9.3+ cells were slightly inhibitory of fresh MLR, but this effect as well as the development of cytolytic cells was completely abrogated by CsA. These results indicate that suppressor and cytolytic Leu-2+ T cells activated in MLR are derived from distinct precursors separable by antibody 9.3.     

Anti-idiotypic B cells are required for the induction of suppressor T cells

A nylon wool-adherent, B cell-enriched population is required during the in vitro induction of third order effector suppressor T cells (Ts3). This B cell population expresses IgM and IgD and is devoid of conventional T cell markers such as Thy-1, L3T4, and Lyt-1. Treatment of the B cell population with anti-NP antibodies expressing the NPb idiotype and complement specifically eliminated the ability to generate Ts cell activity, suggesting that the critical B cells expressed anti-idiotypic receptors. To independently verify the role of anti-idiotypic B cells in the generation of Ts cells, B cells were panned on antibody-coated plates. The results demonstrated that only NPb idiotype-binding B cells could induce effector suppressor cells from naive T cell populations. The combined data demonstrate the role of Ig network interactions in the generation of Ts cells.     

Dissociation of differentiation and proliferation in the primary induction of cytolytic T lymphocytes by alloantigens

The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Accessory cell requirements for the generation of cytolytic T lymphocytes.

A system is presented that may simplify the study of accessory cell requirements for CTL generation. Cortisone resistant (CR) thymocytes containing alloreactive CTL precursors do not respond to allogeneic tumor cells unless non-T accessory cells are added to culture. In addition, splenic T cells do not respond to allogeneic tumor cells in the absence of non-T accessory cells. These accessory cells share several properties of macrophages.     

Histamine regulates the generation of human cytolytic T lymphocytes

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.     

Prerequisite for the induction of lymphokine-activated killer cells from T lymphocytes

Human blood mononuclear cells were separated into Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells by means of a combination of the Percoll gradient method and C-mediated cytolysis using mAb. When purified Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells were cultured with rIL 2 (500 U/ml) for 6 days in a medium supplemented with 10% FCS, Leu-11+7-NK cells responded at the maximum level and Leu-11-7+ cells responded moderately as shown by both cell-proliferation response and cytotoxic activity generated. On the other hand, Leu-11-7-T cells did not respond at all to rIL-2. However, when Leu-11-7-T cells were cultured with rIL-2 in a medium supplemented with 10% autologous serum, they showed considerable responsiveness to rIL-2. In addition, much greater response to Leu-11-7-T cells were produced by the addition of monocytes. Monocyte cytokines, neither IL 1, IFN-gamma, TNF, nor their combination were able to substitute for monocytes in the induction culture. In contrast, the response level of Leu-11+7- NK cells remained unchanged irrespective of supplementation with autologous serum to medium or the addition of monocytes to the culture. These results indicated that culture conditions in the experiments significantly affected the results as to determination of lymphokine-activated killer cell precursors, especially the result pertaining to the conversion of T lymphocytes to lymphokine-activated killer cells. Under appropriate conditions, not only NK cells but also T cells are important precursors of lymphokine-activated killer cells.