Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.     

Evaluation of the ability of N-terminal fragment of lethal factor of Bacillus anthracis for delivery of Mycobacterium T cell antigen ESAT-6 into cytosol of antigen presenting cells to elicit effective cytotoxic T lymphocyte response

We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine.     

Measurement of CTL-induced cytotoxicity: The caspase 3 assay

Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the ⁵¹Cr release assay. However, measurement of target cell lysis by ⁵¹Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the ⁵¹Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.     

Granzyme B-induced cell death exerted by ex vivo CTL: discriminating requirements for cell death and some of its signs

Granzyme B (gzmB) of cytotoxic T lymphocytes (CTL) is essential for recovery from intracellular pathogens, but the molecular basis of its action is still unresolved. Here, we analyzed gzmB-mediated death pathways under physiological conditions using ex vivo virus-immune CTLs that express perf and gzmB, but not gzmA (gzmB(+)CTL). We show that gzmB(+)CTL abrogate target cell proliferation most likely by inducing cell death, independent of caspases and mitochondrial signaling. In addition, the data reveal that gzmB(+)CTL independently induce pro-apoptotic processes either via caspase-3/-7, leading to plasma membrane perturbance and ROS production or via Bid/Bak/Bax, resulting in cytochrome c release and that both pathways elicit loss of DeltaPsi(m). Our data provide evidence for a pleiotropic pro-apoptotic function of gzmB presumably to counteract evasion strategies of pathogens and to control tumors.     

Signal strength controls the rate of polarization within CTLs during killing

Cytotoxic T lymphocytes (CTLs) are key effector cells in the immune response against viruses and cancers, killing targets with high precision. Target cell recognition by CTL triggers rapid polarization of intracellular organelles toward the synapse formed with the target cell, delivering cytolytic granules to the immune synapse. Single amino acid changes within peptides binding MHC class I (pMHCs) are sufficient to modulate the degree of killing, but exactly how this impacts the choreography of centrosome polarization and granule delivery to the target cell remains poorly characterized. Here we use 4D imaging and find that the pathways orchestrating killing within CTL are conserved irrespective of the signal strength. However, the rate of initiation along these pathways varies with signal strength. We find that increased strength of signal leads to an increased proportion of CTLs with prolonged dwell times, initial Ca²⁺ fluxes, centrosome docking, and granule polarization. Hence, TCR signal strength modulates the rate but not organization of effector CTL responses.     

Granzyme B activity in target cells detects attack by cytotoxic lymphocytes

Lymphocyte-mediated cytotoxicity via granule exocytosis operates by the perforin-mediated transfer of granzymes from CTLs and NK cells into target cells where caspase activation and other death pathways are triggered. Granzyme B (GzB) is a major cytotoxic effector in this pathway, and its fate in target cells has been studied by several groups using immunodetection. In this study, we have used a newly developed cell-permeable fluorogenic GzB substrate to measure this protease activity in three different living targets following contact with cytotoxic effectors. Although no GzB activity is measurable in CTL or NK92 effector cells, this activity rapidly becomes detectable throughout the target cytoplasm after effector-target engagement. We have combined the GzB substrate with a second fluorogenic substrate selective for caspase 3 to allow both flow cytometry and fluorescence confocal microscopy studies of cytotoxicity. With both effectors, caspase 3 activity appears subsequent to that of GzB inside all three targets. Overexpression of Bcl-2 in target cells has minimal effects on lysis, NK- or CTL-delivered GzB activity, or activation of target caspase 3. Detection of target GzB activity followed by caspase 3 activation provides a unique readout of a potentially lethal injury delivered by cytotoxic lymphocytes.     

Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8(+) T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA(-/-) or gzmB(-/-) mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, Delta Psi(m) loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA(-/-) but not from gzmB(-/-) mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA(-/-) or gzmB(-/-) mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.     

Mimovirus: a novel form of vaccine that induces hepatitis B virus-specific cytotoxic T-lymphocyte responses in vivo

CD8(+) cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine-mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg(28-39), with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg(28-39)-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

CD8+ T cell responses against TAP-inhibited cells are readily detected in the human population

Target cell recognition by CTLs depends on the presentation of peptides by HLA class I molecules. Tumors and herpes viruses have adopted strategies to greatly hamper this peptide presentation at the important bottleneck, the peptide transporter TAP. Previously, we described the existence of a CD8(+) CTL subpopulation that selectively recognizes such TAP-deficient cells in mouse models. In this study, we show that the human counterpart of this CTL subset is readily detectable in healthy subjects. Autologous PBMC cultures were initiated with dendritic cells rendered TAP-impaired by gene transfer of the viral evasion molecule UL49.5. Strikingly, specific reactivity to B-LCLs expressing one of the other viral TAP-inhibitors (US6, ICP47, or BNLF2a) was already observed after three rounds of stimulation. These short-term T cell cultures and isolated CD8(+) CTL clones derived thereof did not recognize the normal B-LCL, indicating that the cognate peptide-epitopes emerge at the cell surface upon an inhibition in the MHC class I processing pathway. A diverse set of TCRs was used by the clones, and the cellular reactivity was TCR-dependent and HLA class I-restricted, implying the involvement of a broad antigenic peptide repertoire. Our data indicate that the human CD8(+) T cell pool comprises a diverse reactivity to target cells with impairments in the intracellular processing pathway, and these might be exploited for cancers that are associated with such defects and for infections with immune-evading herpes viruses.     

Cloned cytotoxic T lymphocyte target cells fail to induce early activation events in effector cytotoxic T lymphocytes

We have explored further the basis for resistance of cloned cytotoxic T lymphocytes (CTLs) to cell-mediated cytotoxicity. We find that most cloned CTLs recognized as specific target cells by other cloned CTLs used as effector cells fail to activate three early events that may be critical in triggering lysis in the effector CTLs: Ca2+ influx, microtubule organizing center (MTOC) reorientation, and serine esterase release. To the extent that any or all of these events are involved in activation or expression of the lytic pathway in effector CTLs, our results suggest that in addition to being inherently resistant to cytotoxic granule extracts, many CTLs are also unable to induce lytic function in other (effector) CTLs. We have found one CTL clone that can respond to recognizable cloned CTL target cells with at least MTOC reorientation and serine esterase release, although the target CTLs are still not lysed. In this case, the resistance of the target CTL to lysis may be due solely to its resistance to cytoplasmic granule contents.     

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.     

CTL-mediated killing of intracellular Mycobacterium tuberculosis is independent of target cell nuclear apoptosis

Two subsets of human CTL have been defined based upon phenotype and function: CD4(-) CD8(-) double-negative (DN) CTL lyse susceptible targets via Fas-Fas ligand interaction and CD8(+) CTL via the granule exocytosis pathway. CD8(+) CTL, but not DN CTL, can mediate an antimicrobial activity against Mycobacterium tuberculosis-infected target cells that is dependent on cytotoxic granules that contain granulysin. We investigated the role of nuclear apoptosis for the antimicrobial effector function of CD1-restricted CTL using the caspase inhibitor N:-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. We found that DN CTL-induced target cell lysis was completely dependent on caspase activation, whereas the cytolytic activity of CD8(+) CTL was caspase independent. However, both DN and CD8(+) CTL-induced nuclear apoptosis required caspase activation. More important, the antimicrobial effector function of CD8(+) CTL was not diminished by inhibition of caspase activity. These data indicate that target cell nuclear apoptosis is not a requirement for CTL-mediated killing of intracellular M. tuberculosis.     

CD8alpha+ dendritic cells selectively present MHC class I-restricted noncytolytic viral and intracellular bacterial antigens in vivo

CD8alpha(+) dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8alpha(+) DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8alpha(+) DCs are the principal DC subset that initiate CD8(+) T cell immunity.     

Expression and function of synaptotagmin VII in CTLs

The Ca(2+) sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca(2+)-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII(+/+) controls, Syt VII(-/-) animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII(-/-) mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII(+/+) or Syt VII(-/-) CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.     

Epitope-dependent avidity thresholds for cytotoxic T-lymphocyte clearance of virus-infected cells

Cytotoxic T lymphocytes (CTLs) are crucial for immune control of viral infections. "Functional avidity," defined by the sensitizing dose of exogenously added epitope yielding half-maximal CTL triggering against uninfected target cells (SD(50)), has been utilized extensively as a measure of antiviral efficiency. However, CTLs recognize infected cells via endogenously produced epitopes, and the relationship of SD(50) to antiviral activity has never been directly revealed. We elucidate this relationship by comparing CTL killing of cells infected with panels of epitope-variant viruses to the corresponding SD(50) for the variant epitopes. This reveals a steeply sigmoid relationship between avidity and infected cell killing, with avidity thresholds (defined as the SD(50) required for CTL to achieve 50% efficiency of infected cell killing [KE(50)]), below which infected cell killing rapidly drops to none and above which killing efficiency rapidly plateaus. Three CTL clones recognizing the same viral epitope show the same KE(50) despite differential recognition of individual epitope variants, while CTLs recognizing another epitope show a 10-fold-higher KE(50), demonstrating epitope dependence of KE(50). Finally, the ability of CTLs to suppress viral replication depends on the same threshold KE(50). Thus, defining KE(50) values is required to interpret the significance of functional avidity measurements and predict CTL efficacy against virus-infected cells in pathogenesis and vaccine studies.     

A General Functional Response of Cytotoxic T Lymphocyte-Mediated Killing of Target Cells

Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.     

A General Functional Response of Cytotoxic T Lymphocyte-Mediated Killing of Target Cells

Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.     

Expansion and contraction of the cytotoxic T lymphocyte response—An optimal control approach

The kinetics of the cytotoxic T lymphocyte (CTL) response against intracellular pathogens has been found to have many stereotypical features that appear to be programmed early in the infection. We explain these findings here in terms of CTL response kinetics that minimize the probability that a pathological symptom will occur in association with the infection and its eradication. We assume that both the infection and the CTLs contribute to this pathology. We find that contraction kinetics is influenced by the relative pathogenicities of infection and CTLs, as well as on the virulence of the infection and the efficiency of the CTLs, but not by the magnitude of expansion or the dose and duration of infection. Our analysis explains the finding that the duration of the CTL expansion is highly stereotypical, with the maximum expansion of the CTL response dependent on the dose of the infection. Finally, we show that the stereotypical nature of CTL kinetics relies upon stringent regulation of the rates at which CTLs proliferate and die.