Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc.     

Mechanism of cell-mediated cytotoxicity at the single cell level. II. Evidence for first-order kinetics of T cell-mediated cytolysis and for heterogeneity of lytic rate.

The kinetics and rate of T cell-mediated cytolysis was assessed by measuring the times required for lysis of isolated target cells by single cytotoxic lymphocytes. Single target cell lysis was determined microscopically by observing trypan blue uptake as a function of time of incubation of effector-target conjugates in agarose. Lysis of EL-4 target cells by alloimmune peritoneal exudate lymphocytes was initiated without a lag and was essentially complete at 2 hr. Both zero-order and first-order kinetics equations were analyzed for fit to the 0 to 2 hr lysis values. Statistically, the zero-order kinetic function could be rejected (p greater than 0.05), but the first-order kinetics function (p less than 0.01) could not. This strong evidence for first-order kinetics of T cell-mediated cytolysis implies that within each CTL-target cell population, cytolysis occurs exponentially as a random decay process and that one event in the entire process of cytolysis is rate limiting. The first-order equation was then applied to measurements of the rate of cytolysis in many different individual effector-target cell combinations. Significant differences in the lytic rate were apparent when either the effector or target cell were varied, with the rate constants spanning a 5-fold range. The heterogeneity of lytic rates is consistent with the hypothesis that lytic efficiency is a function of both the effector and target cells used.     

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.     

Feline cytotoxic large granular lymphocytes induced by recombinant human IL-2

Large granular lymphocytes (LGL) have been characterized phenotypically and functionally as cytotoxic T lymphocytes, NK cells or lymphokine-activated killer cells. The most prominent morphologic feature of LGL is large cytoplasmic granules that are thought to contain the molecules responsible for cell lysis. In this study, we describe the morphologic and functional characteristics of IL-2-dependent cytotoxic lymphocytes derived from feline PBL. Stimulation of feline PBL with Con A followed by culturing in 50 U of gibbon monkey IL-2 human rIL-2 induced long term lymphocyte cultures. These lymphocytes are cytotoxic for the feline leukemia virus-induced T cell lymphoma (FL74), in a 4-h 51Cr release assay. All cell lines are either constitutively cytotoxic for FL74 cells, or cytotoxic in a lectin-dependent cell cytotoxic assay, the latter being a characteristic of low passage cultures. In contrast, no cell lines express self lysis or lysis for other lines. [3H]TdR uptake showed that 1 U of human rIL-2 produces a 50% maximal proliferative response by feline lymphocytes suggesting a high degree of homology between the ligand binding sites of feline and human IL-2R. Feline cytotoxic lymphocytes possess abundant cytoplasm containing large azurophilic granules characteristic of LGL. These granules are bound by a bilipid membrane and contain numerous smaller membrane-bound vesicles 50 to 60 nm in diameter. A model is proposed, whereby subsequent to binding of LGL to target cell the large granules fuse to the LGL plasma membrane and release the small vesicles into the binding pocket. The vesicles then transport the lytic molecules directly and selectively to the target cell membrane.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VIII. Kinetics of lysis of target cells bound by more than one cytotoxic T lymphocyte

We measured the effects of having multiple cytotoxic T lymphocytes (CTL) bound to one target cell by using the single-cell cytotoxicity in agarose assay. We found that even though there is variability in the time at which individual target cells are lysed, we can identify a general trend: the mean rate of lysis increases with the number of CTL bound per target cell, reaching a maximum when the CTL-target cell ratio is three. Combining a quantitative model for the rate of lethal hitting in multicellular conjugates with a multi-event model for the rate of target cell disintegration, we developed a new multistage kinetic model for predicting the rate of target cell lysis in multiple lymphocyte-target cell conjugates. The variability in the time at which target cells are hit and the variability in the time until they disintegrate are incorporated into the model. By analyzing our measured data in the context of the multistage kinetic model, we were able to estimate via nonlinear least squares regression the target cell disintegration rate, but not the lethal hitting rate. Lethal hitting appeared to be too fast, when compared with disintegration, to significantly affect the time of target cell lysis. By using previously determined values of the lethal hitting rate for single lymphocyte-target cell conjugates and by postulating that lymphocytes act independently of each other in delivering lethal hits, we were able to estimate the rate at which target cells are hit in multiple-lymphocyte single target cell conjugates. By using this estimate of the lethal hitting rate and the regression estimate of the disintegration rate, the multistage kinetic model gave a quantitative fit to our data. From this analysis, we found that the rate at which a target cell disintegrates after being lethally hit increases with the number of CTL per conjugate. This result is quite surprising, because once the first hit has been received, a target cell can disintegrate in a killer cell-independent manner. Under the conditions of our experiment, it appears as if target cell disintegration is not killer cell-independent. Furthermore, our analysis of the time course of target cell disintegration suggests that the process is not governed by simple first order kinetics, but rather by a more complex multistep mechanism.     

A population balance model of enzymatic lysis of microbial cells

A model is proposed for enzymatic lysis of microbial cells based on number balances over the distribution of cell-wall mass in a population of cells. Analytical solutions to the population balance equations were obtained by the method of characteristics for simple reaction kinetics. The model has been used to analyze the following cases of lysis in a nonhomogeneous cell population: wall hydrolysis with cell rupture and product release, the effect of a distribution of lysis rates, and lysis of two-layer cell walls. Rate expressions for the reactions of lysis can be derived from bulk-phase experiments; the distributions of cell size and product content can be measured independently by flow cytometric techniques. The population model also provides an explanation for the initial lag seen in lysis kinetics for virtually any initial distribution. The model demonstrates patterns of lysis and product recovery for heterogeneous populations of cells and also applies to the more general problem of soluble-enzyme reactions with heterogeneous solid substrates.     

Natural cytotoxic cells and tumor necrosis factor activate similar lytic mechanisms

The lytic activity of natural cytotoxic (NC) cells has several characteristics which clearly distinguish it from other cell-mediated lytic activities and from most soluble cytolytic factors. An exception is the lytic activity mediated by tumor necrosis factor (TNF). In this paper, we report a detailed comparison of NC and TNF lysis of target cells which are used as prototype NC targets or TNF targets, and show that the two cytolytic activities have very similar, if not identical, lytic mechanisms. We present data showing that target cells which are NC-sensitive are also TNF-sensitive and that target cells which are NC-resistant are also TNF-resistant. Moreover, cells selected either in vivo or in vitro for NC resistance are selected for TNF resistance, and cells selected for TNF resistance are selected for NC resistance. The analysis of the kinetics of 51Cr release mediated by NC cells or by TNF show that both activities affect similar kinetics, in that there is no cell lysis for several hours after targets and effectors first interact. However, NC and TNF lytic activities can be distinguished. By using the cell lines 10ME or B/C-N as targets, it can be shown that whereas NC-mediated lysis is dependent on protein synthesis, TNF-mediated lysis is not. We also show that targets which are resistant to NC-mediated lysis because they express a protein synthesis-dependent resistance mechanism also require protein synthesis to resist TNF-mediated lysis, suggesting that the same resistance mechanism protects cells against both NC cells and TNF. Together, these data strongly support the hypothesis that NC cells and TNF activate the same lytic mechanism within target cells and that TNF may mediate the lytic activity of NC effector cells.     

Enhancement of redirected target cell lysis by cytotoxic T lymphocytes in the presence of cytochalasin B

The cytochalasins are known secretogogues. Their function as such is examined in light of the granule exocytosis model for lymphocyte-mediated cytotoxicity. Cytochalasin B is found to enhance target cell lysis by cytotoxic T lymphocytes when antibody-coated polystyrene beads are used to bridge the cells. The pattern of lysis is found to be biphasic in its dependence on cytochalasin B. Secretion of the enzyme BLT-esterase from the effector cells parallels the cytochalasin concentration-dependent pattern of lysis. Cytochalasin D is also able to enhance lysis but at concentrations less than cytochalasin B. Cytochalasin B does not inhibit binding of breads to the effector cell. This is shown by the ability of fluorescent beads coated with antibody to bind with an appropriate specificity to cells. These studies indicate that cytochalasin B is not strictly inhibitory for the induction of target cell lysis but can enhance lymphocyte-mediated lysis at low drug concentrations. These results are compatible with the interpretation that target cell lysis is mediated through a secretion process from cytotoxic T lymphocytes.     

Susceptibility to lysis by natural killer and natural cytotoxic cells is independent of the mitotic stage of the target cell cycle

Natural cell-mediated cytotoxicity (NCMC) against a number of target cells is mediated by at least two distinct effector populations, with natural killer (NK) and natural cytotoxic (NC) cells being the predominant in the murine system. The studies described in this report examine the role that the phase of the mitotic cycle of the target cell has on its susceptibility to lysis by NC and NK cells. We show that neither the kinectics nor the magnitude of NC cell lysis is altered when assayed using target cells which have been enriched for G1, S, or G2 + M stages of the cell cycle. Similarly, NK cell lysis by fresh or poly-IC augmented effector cells was not effected by target Cell Cycle.     

EMT impairs breast carcinoma cell susceptibility to CTL-mediated lysis through autophagy induction

Epithelial to mesenchymal transition (EMT) has become one of the most exciting fields in cancer biology. While its role in cancer cell invasion, metastasis and drug resistance is well established, the molecular basis of EMT-induced immune escape remains unknown. We recently reported that EMT coordinately regulates target cell recognition and sensitivity to specific lysis. In addition to the well-characterized role for EMT in tumor phenotypic change including a tumor-initiating cell phenotype, we provided evidence indicating that EMT-induced tumor cell resistance to cytotoxic T-lymphocytes (CTLs) also correlates with autophagy induction. Silencing of BECN1 in target cells that have gone through the EMT restored CTL susceptibility to CTL-induced lysis. Although EMT may represent a critical target for the development of novel immunotherapy approaches, a more detailed understanding of the inter-relationship between EMT and autophagy and their reciprocal regulation will be a key determinant in the rational approach to future tumor immunotherapy design.     

A multistage model for the action of cytotoxic T lymphocytes in multicellular conjugates

We propose a multistage stochastic model to explain data on the kinetics of target cell lysis by cytotoxic T lymphocytes in multicellular conjugates. A novel feature of our model is that we explicitly consider both the lethal hitting stage and the target cell disintegration stage of the cytolytic process. Further, we allow for the possibility that target cell disintegration is itself a complex process composed of many events. The comparison of our model with the data of other investigators suggests that cytotoxic T cells deliver lethal hits at random to undamaged target cells. Having received a lethal hit, the target cell disintegrates over a variable length of time. The disintegration times of target cells from different conjugates appear to be randomly distributed and to be consistent with a model in which disintegration occurs by at least two major, sequential, rate-limiting events. For conjugates containing one lymphocyte and multiple target cells, the mean rate at which a lethally hit target cell disintegrates is found to be independent of the total number of target cells in the conjugate. Our model predicts that in such multicellular conjugates, individual target cells lyse one by one, on average at approximately 30-min intervals, thus agreeing closely with previously reported experimental observations.     

The role of HLA antigens in the control of the cytotoxic T-cell response to Epstein-Barr virus: a family study

Epstein-Barr (EB) virus-specific, HLA-restricted cytotoxic T-cell populations have been generated in vitro from each member of a family by cocultivating peripheral blood mononuclear cells with autologous EB virus-transformed B cells, the resulting effector cells being expanded as interleukin 2-dependent T-cell lines. The cytotoxicity of each of these effector populations was tested on a large panel of EB virus-transformed target cells of known HLA type, so that the particular HLA antigens which acted as restricting elements for each cytotoxic population could be identified. There was a consistent pattern within the family of preference for certain HLA class I antigens as restricting elements of the virus-specific T-cell response. Extensive functional analysis showed that, in addition to the virus-specific lysis, each effector population mediated a cross-reactive lysis against target cells prepared from certain HLA-mismatched individuals. This cross-reactivity appeared to be directed against HLA class I alloantigens and occurred irrespective of the EB virus genome status of the target cells. Effector T-cell lines derived from different family members but with virus-specific lysis predominantly restricted through the same HLA antigen showed similar patterns of concomitant allo-cross-reactivity. This suggests that antigenic mimicry of virally altered self by alloantigens is a genuine phenomenon which may be important in channelling the human cytotoxic T-cell response to a virus through preferred self-HLA determinants.     

Production and characterization of T-cell clones specific for trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self)

Using serial antigenic challenge as the method of selection and stimulation, continuous lines of cytotoxic T-lymphocytes (CTL) directed against TNBS-modified syngeneic spleen cells (TNP-self) have been generated. Spleen cells from C3H/HeJ (H-2k) mice were primed in vitro with autologous spleen cells modified with TNBS, and subsequently cloned by limiting dilution and in soft agar in the presence of IL2. These CTL clones grew continuously in medium supplemented with IL2 and in the presence of antigen. They are antigen specific and H-2 restricted in their target cell recognition. They all express Thyl and Lyt2 surface markers. None of the clones exhibit natural killer (NK) cell activity. All CTL clones tested so far are restricted in their target cell recognition to H-2Kk-TNP and none were found to be restricted to H-2Dk-TNP. These findings demonstrate at the clonal level the H-2K/D restriction of TNP-self specific CTL. These clones provide tools that may facilitate an understanding of the development and regulation of antigen specific CTL. They may also serve as models useful towards an understanding of the mechanism of lysis by CTL.     

Thymic lymphomas mediate non-MHC-restricted, TNF-dependent lysis of the murine sarcoma WEHI-164

Tumor necrosis factor (TNF), lyses a range of sensitive tumor targets and has been shown to be the mediator of natural cytotoxic (NC) activity first described in our laboratory. In this report, we identify two thymic lymphoma cell lines which lyse the prototype NC target WEHI-164 and share characteristics of NC. R1.1E and L5178-27av lyse the WEHI-164 sarcoma in 18-hr 51Cr release assays via a TNF-dependent, non-MHC-restricted (R1.1E) mechanism although they do not constitutively produce TNF. NC- and TNF-resistant variants of WEHI-164 are resistant to lymphoma-mediated lysis. Expression of the ganglioside GD3 by the lymphomas correlates with their relative levels of lysis. Thus, GD3, which is known to have a role in T cell activation may be involved in recognition or triggering for TNF-dependent cell-mediated lysis.     

Multi-functionality of allospecific cytotoxic T-lymphocytes in vitro

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.     

Polyfunctionality of Allospecific Cytotoxic T-Lymphocytes in in vitro Systems

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.     

T lymphocyte-mediated cytolysis: dissociation of the binding and lytic mechanisms of the effector cell.

To determine functional relationships between the cytotoxic T lymphocyte (CTL) receptor for target binding and the lytic mechanism, we have studied the reaction between two immunized CTL populations (AalphaB and BalphaA), both at the population and the single-cell level. When studied at the population level, the reaction of AalphaB with BalphaA (bidirectional system) resulted in formation of AalphaB/BalphaA conjugates and bidirectional cytolysis. However, when the viability of cells in individual AalphaB/BalphaA conjugates was analyzed, unidirectional instead of bidirectional lysis occurred. These results indicate that under conditions that are conducive to lysis, binding of a potentially lytic cell to its target does not necessarily result in target lysis. Short heat treatment of CTL (44 degrees C, 10 min) totally abolished their lytic activity, without affecting their capacity to bind specifically, thus dissociating the binding from the lytic activity of the CTL. The cytolytic activity is probably associated with, or triggered by the CTL-binding unit. The binding unit, on the other hand, appears to be a functional receptor of the CTL, which is involved in but not sufficient to bring about lysis.     

Analysis of rat natural killer cytotoxic factor (NKCF): mechanism of action and relationship to other cytotoxic/cytostatic factors

Natural killer cytotoxic factor (NKCF) has been proposed as one of the factors that mediates lysis induced by natural killer (NK) cells. Recently, an excellent source of NKCF has been found to be the rat large granular lymphocyte (LGL) tumor (RNK) cell line. In this study, the kinetics of lysis of the NK-sensitive, tumor target YAC-1 by the RNK-NKCF was analyzed and found to parallel that seen with NK cell-mediated killing. RNK-NKCF was also capable of killing the NK-resistant target cell, MBL-2, over a longer time period. This study utilized monoclonal antibodies (mAbs) prepared against granule protein, previously termed "anti-NKCF mAbs." These mAbs established the nature of RNK-NKCF as compared to other known cytotoxic factors in combination with studies that show that RNK-NKCF causes both 51Cr release and nuclear degradation. Antibody inhibition experiments have verified that RNK-NKCF is unique from tumor necrosis factor (TNF), leukoregulin, or complement. Anti-NKCF mAbs were capable, however, of neutralizing the RNK cell granule activity against YAC-1 tumor target cells. Based on these results, the ability of anti-NKCF mAbs to neutralize the cytolytic function of pore-forming protein (PFP), a component of these granules, was analyzed. In these experiments, the antibodies were found to inhibit the hemolytic activity of granules. Interestingly, the antibodies were effective in inhibiting the activity of unbound granule proteins as well as those bound to sheep red blood cell (SRBC) targets. Further studies to examine the target lysis requirements demonstrated that in contrast to PFP, the RNK-NKCF was able to lyse the tumor target in the absence of calcium. In addition, treatment of targets with RNA and protein synthesis inhibitors indicated that the mechanism of lysis of NKCF is quite unique from other defined cytotoxic moieties.     

Cell-mediated lympholysis: A receptor-associated lytic mechanism

The mechanism of the lytic event in cell-mediated lympholysis is as yet incompletely understood. Two major hypotheses have been proposed: (1) lysis is a nonspecific result of the intimacy created between killer and target by specific antigen-receptor binding, and (2) lysis is effected by the killer cell receptor itself or by a closely linked cell surface component.Two populations of cytotoxic effector cells were coincubated, one relevantly sensitized to the second and the second irrelevantly sensitized to a third party; in some experiments the third party cells were also added. If target cell lysis occurred because of killer-target intimacy, the relevantly sensitized killers should have been lysed when incubated with cytotoxic targets. Relevantly sensitized killers were not lysed when coincubated with either cytotoxic targets alone or with cytotoxic targets and their appropriate third party targets.These findings suggest that the lytic event in cell-mediated lympholysis is accomplished by the receptor itself, or by a geographically closely linked cell surface component.     

Synergy between subpopulations of normal mouse spleen cells in the in vitro generation of cell-mediated cytotoxicity specific for "modified self" antigens.

Responding lymphoid cells cultured in vitro with irradiated trinotrophenyl (TNP)-modified syngeneic spleen cells develop direct cell-mediated cytotoxicity which is specific for target cells bearing both the TNP moiety and histocompatibility determinants of the modified sensitizing cell. Two subpopulations of normal mouse spleen cells have been shown to synergize in the in vitro generation of specific cell-mediated cytotoxicity to these "modified self" antigens. The synergizing populations are nylon wool column-adherent and column-nonadherent fractions of normal mouse spleen. When mixtures of these two cell populations are cultured in vitro with irradiated TNP-modified syngeneic spleen cells, greater cytotoxicity is generated in the two populations sensitized separately. The synergizing cell in the column-adherent population is resistant to lysis by rabbit anti-mouse brain serum, is distinct from the cytotoxic effector T lymphocyte, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by peritoneal cells. These results suggest that it is a non-T cell which may be distinct from the macrophage.