Evaluation of CD8 T cell killing models with computer simulations of 2-photon imaging experiments

In vivo imaging of cytotoxic T lymphocyte (CTL) killing activity revealed that infected cells have a higher observed probability of dying after multiple contacts with CTLs. We developed a three-dimensional agent-based model to discriminate different hypotheses about how infected cells get killed based on quantitative 2-photon in vivo observations. We compared a constant CTL killing probability with mechanisms of signal integration in CTL or infected cells. The most likely scenario implied increased susceptibility of infected cells with increasing number of CTL contacts where the total number of contacts was a critical factor. However, when allowing in silico T cells to initiate new interactions with apoptotic target cells (zombie contacts), a contact history independent killing mechanism was also in agreement with experimental datasets. The comparison of observed datasets to simulation results, revealed limitations in interpreting 2-photon data, and provided readouts to distinguish CTL killing models.     

Antimelanoma activity of CTL generated from peripheral blood mononuclear cells after stimulation with autologous dendritic cells pulsed with melanoma gp100 peptide G209-2M is correlated to TCR avidity

Anchor residue-modified peptides derived from tumor-associated Ag have demonstrated success in engendering immune responses in clinical studies. However, tumor regression does not always correlate with immune responses. One hypothesis to explain this is that CTL resulting from such immunization approaches are variable in antitumor potency. In the present study, we evaluated this hypothesis by characterizing the activity of tumor-associated Ag-specific CTL. We chose an anchor residue-modified peptide from gp100, G209-2M, and used peptide-pulsed dendritic cells to generate CTL from PBMC of HLA-A2(+) normal donors. The specificities and avidities of the resulting CTL were evaluated. The results demonstrate that CTL generated by G209-2M can be classified into three categories: G209-2M-specific CTL which are cytotoxic only to G209-2M-pulsed targets; peptide-specific CTL which recognize both G209 and G209-2M peptides but not melanomas; and melanoma-reactive CTL which recognize peptide-pulsed targets as well as HLA-A2(+)gp100(+) melanomas. CTL that kill only peptide-pulsed targets require a higher peptide concentration to mediate target lysis, whereas CTL that lyse melanomas need a lower peptide concentration. Increasing peptide density on melanomas by loading exogenous G209 peptide enhances their sensitivity to peptide-specific CTL. High avidity CTL clones also demonstrate potent antimelanoma activity in melanoma model in nude mice. Injection of G209 peptide around transplanted tumors significantly enhances the antitumor activity of low avidity CTL. These results suggest that peptide stimulation causes expansion of T cell populations with a range of avidities. Successful immunotherapy may require selective expansion of the higher-avidity CTL and intratumor injection of the peptide may enhance the effect of peptide vaccines.     

The Kinematics of Cytotoxic Lymphocytes Influence Their Ability to Kill Target Cells

Cytotoxic lymphocytes (CTL) have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment – on average 21 hours – which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.     

Immunosensitization of melanoma tumor cells to non-MHC Fas-mediated killing by MART-1-specific CTL cultures

The discovery of human melanoma rejection Ags has allowed the rational design of immunotherapeutic strategies. One such Ag, MART-1, is expressed on >90% of human melanomas, and CTL generated against MART-1(27-35) kill most HLA A2.1(+) melanoma cells. However, variant tumor cells, which do not express MART-1, down-regulate MHC, or become resistant to apoptosis, will escape killing. Cytotoxic lymphocytes kill by two main mechanisms, the perforin/granzyme degranulation pathway and the TNF/Fas/TNF-related apoptosis-inducing ligand superfamily of apoptosis-inducing ligands. In this study, we examined whether cis-diaminedichloroplatinum (II) cisplatin (CDDP) sensitizes MART-1/HLA A2.1(+) melanoma and melanoma variant tumor cells to non-MHC-restricted, Fas ligand (FasL)-mediated killing by CTL. MART-1(27-35)-specific bulk CTL cultures were generated by pulsing normal PBL with MART-1(27-35) peptide. These CTL cultures specifically kill M202 melanoma cells (MART-1(+), HLA A2.1(+), FasR(-)), and MART-1(27-35) peptide-pulsed T2 cells (FasR(+)), but not M207 melanoma cells (MART-1(+), HLA A2.1(-), FasR(-)), FLU(58-66) peptide-pulsed T2 cells, or DU145 and PC-3 prostate cells (MART-1(-), HLA A2.1(-), FasR(+)). CDDP (0.1-10 microg/ml) sensitized non-MART-1(27-35) peptide-pulsed T2 to the CD8(+) subset of bulk MART-1-specific CTL, and killing was abolished by neutralizing anti-Fas Ab. Furthermore, CDDP up-regulated FasR expression and FasL-mediated killing of M202, and sensitized PC-3 and DU145 to killing by bulk MART-1-specific CTL cultures. These findings demonstrate that drug-mediated sensitization can potentiate FasL-mediated killing by MHC-restricted CTL cell lines, independent of MHC and MART-1 expression on tumor cells. This represents a novel approach for potentially controlling tumor cell variants found in primary heterogeneous melanoma tumor cell populations that would normally escape killing by MART-1-specific immunotherapy.     

Cytotoxic T lymphocytes induce different types of DNA damage in target cells of different origins

Nuclear changes may be important in the mechanism of CTL-mediated lysis. Rapid cleavage of target cell DNA into oligonucleosomes has been demonstrated as a very early event in CTL-mediated killing of murine hematopoietic targets. However, the results presented herein and by other investigators have shown that this extensive dsDNA fragmentation does not occur in all CTL targets. In terms of actual DNA damage, there is a wide range in the extent and type of DNA cleavage in various targets. Differences exist at both the species and the cell lineage level. The extent of DNA damage generally corresponds to the efficiency of lysis; thus, murine hematopoietic cells, which undergo dsDNA fragmentation, are killed more rapidly and at lower E/T cell ratios than are murine nonhematopoietic cells, which sustain single-stranded nicks. Experiments using cloned CTL demonstrate that the same effector cell kills both hematopoietic and nonhematopoietic targets, producing different types of DNA damage. These observations indicate that the fate of the target cell DNA is determined by the nature of the target cell and not by the CTL. We propose that DNA damage results from an enzyme pathway inherent to the target, which is activated by, not transferred from, the CTL.     

Emulsified Phosphatidylserine,Simple and Effective Peptide Carrier for Induction of Potent Epitope-Specific T Cell Responses

Background

To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability.

Methodology/Principal Findings

Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c⁺CD11b⁺MHCII⁺ conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo.

Conclusions/Significance

This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.     

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205⁺ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4⁺ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ⁺ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205⁺ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.     

Raltegravir and elvitegravir-resistance mutation E92Q affects HLA-B*40:02-restricted HIV-1-specific CTL recognition

Interplay between drug-resistance mutations in CTL epitopes and HIV-1-specific CTLs may influence the control of HIV-1 viremia. However, the effect of integrase inhibitor (INI)-resistance mutations on the CTL recognition has not been reported. We here investigated the effect of a raltegravir and elvitegravir-resistance mutation (E92Q) on HLA-B*40:02-restricted Int92-102 (EL11: ETGQETAYFLL)-specific CTLs. EL11-specific CTLs recognized E92Q peptide-pulsed and E92Q mutant virus-infected cells less effectively than EL11 peptide-pulsed and wild-type virus-infected cells, respectively. Ex vivo ELISpot analysis showed no induction of E92Q-specific T cells in chronically HIV-1-infected individuals. Thus, we demonstrated that EL11-specific CTL recognition was affected by the INI-resistance mutation.     

Induction of human cytotoxic T lymphocytes that preferentially recognise tumour cells bearing a conformational p53 mutant

 The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212–217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193–237 and peptides 186–194, 187–197, 188–197, 263–272, 264–272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio>1.5) at 26 °C, and five (187–197, 193–200, 217–224, 263–272 and 264–272) also stabilised the complexes at 37 °C. Peptides 188–197, 196–203 and 217–225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217–225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217–225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264–272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217–225; and CTL-264 using peptide 264–272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2⁺ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R → H) or 273 (R → H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy. Received: 23 March 2000 / Accepted: 22 June 2000     

Inhibition of the lytic phase of murine T-cell-mediated alloimmune cytotoxicity by a rat antiactivated T-cell antiserum

Antisera produced in rats by immunization with alloimmune murine C57Bl/6 anti-P815 splenic lymphocytes or purified T cells activated in vitro by coculture with phytohemagglutinincoated L-929 cells were found to inhibit the in vitro cytolytic action of in vivo and in vitro alloimmune C57Bl/6 anti-P815 cytotoxic T cells in a 4-hr chromium-51 release assay. The rat anti-murine-activated lymphocyte (anti-MAL) or antiactivated T-cell (anti-ATC) serum inhibited lysis in the absence of exogenously added complement activity and were not directly cytotoxic to CTL. Absorption of anti-MAL with target cells P815, L-929, EL-4, and normal C57Bl/6 lymphocytes removed a limited amount of the CTL-inhibitory activity. In contrast, lectin-activated alloimmune lymphocytes fully absorbed the inhibitory activity indicating these antisera preferentially recognize unique antigenic determinants associated with the activated CTL cell surface. The anti-ATC was found to block alloimmune lysis by CTL from several inbred mouse strains suggesting these antisera recognized antigenic determinants of a common lytic mechanism. A kinetic analysis of the inhibitory activity of the anti-MAL on the CTL reaction scheme revealed this antiserum inhibited lysis at a post-Ca²⁺-dependent step, presumably during the target cell lytic phase. This result suggests the rat antiserum can neutralize the CTL lytic mechanism.     

Investigating CTL Mediated Killing with a 3D Cellular Automaton

Cytotoxic T lymphocytes (CTLs) are important immune effectors against intra-cellular pathogens. These cells search for infected cells and kill them. Recently developed experimental methods in combination with mathematical models allow for the quantification of the efficacy of CTL killing in vivo and, hence, for the estimation of parameters that characterize the effect of CTL killing on the target cell populations. It is not known how these population-level parameters relate to single-cell properties. To address this question, we developed a three-dimensional cellular automaton model of the region of the spleen where CTL killing takes place. The cellular automaton model describes the movement of different cell populations and their interactions. Cell movement patterns in our cellular automaton model agree with observations from two-photon microscopy. We find that, despite the strong spatial nature of the kinetics in our cellular automaton model, the killing of target cells by CTLs can be described by a term which is linear in the target cell frequency and saturates with respect to the CTL levels. Further, we find that the parameters describing CTL killing on the population level are most strongly impacted by the time a CTL needs to kill a target cell. This suggests that the killing of target cells, rather than their localization, is the limiting step in CTL killing dynamics given reasonable frequencies of CTL. Our analysis identifies additional experimental directions which are of particular importance to interpret estimates of killing rates and could advance our quantitative understanding of CTL killing.     

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti‐tumor activity in vivo

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B‐cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL–tumor contacts in vivo. In the therapeutic settings of anti‐CD19 CAR T cells, the majority of CAR T cell–tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti‐tumor responses in vivo.     

Modulation of the recognition and lysis of EL4 tumor target cells by cytotoxic T lymphocytes

When the EL4 targets were harvested from the peritoneal cavity (in vivo), they had less than half as much cell-surface sialic acid as EL4 cells harvested from tissue culture (in vitro), apparently due to the presence of a neuraminidase activity in the peritoneal cavity. Both the recognition and the lysis of either EL4 in vivo or EL4 in vitro target cells by allogeneically primed cytotoxic T lymphocytes were enhanced upon removal of cell-surface sialic acid by neuraminidase treatment. However, even after neuraminidase treatment, there still remained a difference in the lytic profile when using EL4 targets that were harvested in vivo versus in vitro. Both conjugate formation between the target and the T cells and anti-H-2D^b adsorption by the target cells were unaffected by the culture conditions of the target line. However, antibody-induced capping and exocytosis of vesicles differed between the differently cultured target cells, suggesting that there was a membrane organizational difference between them that was detected by the cytotoxic T cells. These data are consistent with the idea that cell surface sialic acid as well as the membrane organization can influence T-cell recognition and lysis of target cells.     

Polyomavirus DNA is damaged in target cells during cytotoxic T-lymphocyte-mediated killing.

Target cell DNA damage is an early event in cytotoxic T-lymphocyte (CTL)-mediated killing. It has been hypothesized that this DNA damage may serve as one mechanism of destroying viral genetic material inside infected cells. We directly examined the fate of viral DNA in target cells during CTL-mediated lysis. Polyomavirus DNA in transfected murine P815 mastocytoma targets was digested along with cellular DNA into oligonucleosome-sized fragments, although intact forms, possibly virion-associated DNA, were also present. In infected BALB/3T3 murine fibroblasts, which normally undergo single-stranded nicks when killed by CTL, polyomavirus DNA was converted to relaxed forms in the presence of CTL. These results suggest that the fate of the viral DNA depends on the stage of the viral life cycle and corresponds to the fate of the host cell DNA. Cleavage of the viral genome prior to assembly may thus be an important mechanism in specific antiviral immunity.     

The use of tunicamycin to study the role of cell surface oligosaccharides in lymphocyte recognition

Cell surface N-linked sugars may play a role in target cell recognition by cytotoxic T lymphocytes (CTL). We have studied this role by treating tumor cell targets with tunicamycin, an effective inhibitor of N-linked glycosylation in mammalian cells. We determined a tunicamycin treatment protocol in which glycosylation was blocked and in which target cell killing by 5-day primary mixed lymphocyte reaction CTL was inhibited, yet in which cell viability was high and expression of major histocompatibility complex molecules was normal. It was found that tunicamycin-treated cells were killed only about one-half as well as untreated targets and that tunicamycin-treated target cells were less effective than untreated target cells as cold target competitors in cold target competition experiments. These observations suggest that for optimal killing, CTL require an interaction with the target cell that involves N-linked glycans on the target cell surface.     

Induction of cytotoxic T lymphocytes specific to malignant glioma using T2 cells pulsed with HLA-A2-restricted interleukin-13 receptor alpha 2 peptide in vitro

Interleukin-13 receptor α2 （IL-13Rα2） is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-1 3Rα2-specific cytotoxic T lymphocyte （CTL） induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen （HLA）-A2-restricted IL-1 3Rα2345-353 peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2^＋ glioma cells expressing IL- 1 3Rα2345-353, while HLA-A2 glioma cell lines that express IL-13Rα2345-353 could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-1 3Rα2345-353 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.     

Increased PRAME antigen-specific killing of malignant cell lines by low avidity CTL clones, following treatment with 5-Aza-2��-Deoxycytidine

The cancer testis antigen Preferentially Expressed Antigen of Melanoma (PRAME) is overexpressed in many solid tumours and haematological malignancies whilst showing minimal expression in normal tissues and is therefore a promising target for immunotherapy. HLA-A0201-restricted peptide epitopes from PRAME have previously been identified as potential immunogens to drive antigen-specific autologous CTL responses, capable of lysing PRAME expressing tumour cells. CTL lines, from 13 normal donors and 10 melanoma patients, all of whom were HLA-A0201 positive, were generated against the PRAME peptide epitope PRA(100-108). Specific killing activity against PRA(100-108) peptide-pulsed targets was weak compared with CTL lines directed against known immunodominant peptides. Moreover, limiting dilution cloning from selected PRAME-specific CTL lines resulted in the generation of a clone of only low to intermediate avidity. Addition of the demethylating agent 5-aza-2'-Deoxycytidine (DAC) increased PRAME expression in 7 out of 11 malignant cell lines including several B lineage leukaemia lines and also increased class I expression. Pre-treatment of target cells was associated with increased sensitivity to antigen-specific killing by the low avidity CTL. When CTL, as well as of the target cells, were treated, the antigen-specific killing was further augmented. Interestingly, one HLA-A0201-negative DAC-treated line (RAJI) showed increased sensitivity to killing by clones despite a failure of expression of PRAME or HLA-A0201. Together these data point to a general increased augmentation of cancer immunogenocity by DAC involving both antigen-specific and non-specific mechanisms.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. I. Resolution and characterization.

Addition of high molecular weight dextran to culture medium prevents the initiation of T lymphocyte-mediated killing by holding the cytolytic T lymphocytes (CTL) and target cells in suspension and preventing intercellular contact. Suspension in 10% dextran was used to interrupt the ongoing formation of adhesions between CTL and target cells already in contact in a centrifuged pellet. The results demonstrate that 1) firm adhesions form between CTL and target cells within 1 min at 37 degrees C; 2) once formed, these adhesions are stable at low temperature and are resistant to mechanical shearing forces; 3) these adhesions can be disrupted by EDTA; 4) immediately after the adhesions form, separation of the CTL from the target cells prevents lysis of the latter; 5) after incubation of targets adhering to CTL for an additional 6 min at 37 degrees C, removal of the CTL no longer prevents target cell lysis. Thus, target cells become "programmed" for subsequent lysis within a few minutes after contact with CTL, after which lysis occurs during the next several hours without further participation of the effector cell. At 15 degrees C, adhesions form 1/17 as fast as at 37 degrees C. Programming of target cells for lysis occurs 1/76 as fast at 15 degrees C as at 37 degrees C. Thus, the programming for lysis step is about 4-fold more temperature dependent than the adhesion step. In addition to being detected by subsequent target cell lysis in 10% dextran, the adhering cell clusters can be counted with low power microscopy. This permitted verification that EDTA separates the clusters after programming for lysis is complete. Moreover, the great majority of the clusters seen at 37 degrees C are antigen-specific. Knowledge of the cluster size distribution and the subsequent level of lysis permits the deduction that not less than 6% of the sensitized peritoneal cell populations used were CTL.