Effects of stress on lysability of tumor targets by cytotoxic T cells and tumor necrosis factor

The effects of stress on four tumor cell lines are analyzed in view of the possibility that stress protects tumor cells against immune attack. We show that stress causes resistance to CTL and TNF in two cell lines. Induction of resistance is time dependent and reversible and not due to failure of killer cells to interact with stressed targets. It is shown that stress induces stress proteins concomitant with induction of resistance to killer cells and TNF. Moreover experiments are presented suggesting that resistance to either immune effector is due to independent mechanisms. The conclusion that stress can induce mechanisms in targets that interfere with the action of TNF as well as with target lysis following a lethal hit by CTL is discussed.     

Antitumor immunity and cellular cancer therapies

The identification of tumor specific antigens has provided important advance in tumor immunology. It is now established that specific cytotoxic T lymphocytes (CTL) and natural killer cells infiltrate tumor tissues and are effector cells able to control tumor growth. However, such a natural antitumor immunity has limited effects in cancer patients. Failure of host defenses against tumor is consecutive to several mechanisms which are becoming targets to design new immunotherapeutic approaches. CTL are critical components of the immune response to human tumors and induction of strong CTL responses is the goal of most current vaccine strategies. Effectiveness of cytokine therapy, cancer vaccines and injection of cells improving cellular immunity have been established in tumor grafted murine models. Clinical trials are underway. To day, interest is particularly focused on cell therapy: injected cells are either "ready to use" effector cells (lymphocytes) or antigen presenting cells able to induce a protective immune reaction in vivo (dendritic cells). The challenge ahead lie in the careful optimization of the most promising strategies in clinical situation.     

The rationale and practice of CTL therapy against cancer in the mouse and human

It is essential to investigate and elucidate the immune response especially T cell response to either syngeneic or autologous tumor for establishing a rational immunotherapy of cancer. We reported that major immune effector cells capable of inducing tumor regression are cytotoxic T lymphocytes (CTL). We found that there are at least two distinct CTL subsets directed to syngeneic tumor. One CTL subset which is selectively induced by syngeneic solid tumor is independent from CD4 positive helper T cells but requires a soluble factor (s) released from macrophage-like accessory cells designated killer T cell activating factor (KAF) in its induction and generation directed to the homologous tumor. The other CTL subset which is usually induced by syngeneic tumor of hematocytic origin is dependent on CD4 positive helper T cells in its induction. On the basis of our findings regarding the induction and activation mechanism of CTL to syngeneic tumors in the mouse, we have investigated the mechanisms of human CTL generation to autochthonous tumor in peripheral blood mononuclear cells of cancer patients. It was found that the nature of human CTL and its generation to autochthonous tumor are similar to those of murine CTL to syngeneic solid tumor. We are now establishing a rational cancer specific immunotherapy utilizing intravenous passive cell transfer of in vitro activated CTL to autochthonous tumor into an original cancer patient.     

IL-2 during in vitro priming promotes subsequent engraftment and successful adoptive tumor immunotherapy by persistent memory phenotypic CD8(+) T cells

Adoptive T cell tumor immunotherapy potentially consists of two protective components by the transferred effector cells, the immediate immune response and the subsequent development of memory T cells. The extent by which adoptively transferred CD8(+) CTL are destined to become memory T cells is ambiguous as most studies focus on the acute effects on tumor shortly following adoptive transfer. In this study we show that a substantial fraction of the input CTL develop into memory cells that reject a s.c. tumor challenge. The use of exogenous IL-2 or a combination of IL-2 and IL-4, but not solely IL-4, during the ex vivo culture for the CTL inoculation was necessary for efficient development of CD8(+) memory T cells. Thus, an important component of adoptive immunotherapy using CTL is the production of CD8(+) Ag-specific memory cells which is primarily favored by IL-2 receptor signaling during ex vivo generation of the effector CTL.     

Down-modulation of CXCR3 surface expression and function in CD8+ T cells from cutaneous T cell lymphoma patients

Viruses can escape destruction by the immune system by exploitation of the chemokine-chemokine receptor system. It is less established whether human cancers can adopt similar strategies to evade immunologic control. In this study, we show that advanced cutaneous T cell lymphoma (CTCL) is associated with selective and efficient inactivation of CXCR3-dependent T cell migration. Our studies demonstrate that this alteration is at least in part due to CXCR3 down-regulation in vivo by elevated serum levels of CXCR3 ligands. The T cell population most affected by this down-regulatory mechanism are CD8+ cytotoxic effector T cells. In CTCL patients, cytotoxic effector T cells have strongly reduced surface CXCR3 expression, accumulate in peripheral blood, but are virtually absent from CTCL tumor lesions, indicating an inability to extravasate into lymphoma tissue. CTCL-associated inactivation of effector cell recruitment may be a paradigmatic example of a new type of immune escape mechanisms shielding the neoplasm from a tumoricidal attack.     

A Biased Competition Theory of Cytotoxic T Lymphocyte Interaction with Tumor Nodules

The dynamics of the interaction between Cytotoxic T Lymphocytes (CTL) and tumor cells has been addressed in depth, in particular using numerical simulations. However, stochastic mathematical models that take into account the competitive interaction between CTL and tumors undergoing immunoediting, a process of tumor cell escape from immunesurveillance, are presently missing. Here, we introduce a stochastic dynamical particle interaction model based on experimentally measured parameters that allows to describe CTL function during immunoediting. The model describes the competitive interaction between CTL and melanoma cell nodules and allows temporal and two-dimensional spatial progression. The model is designed to provide probabilistic estimates of tumor eradication through numerical simulations in which tunable parameters influencing CTL efficacy against a tumor nodule undergoing immunoediting are tested. Our model shows that the rate of CTL/tumor nodule productive collisions during the initial time of interaction determines the success of CTL in tumor eradication. It allows efficient cytotoxic function before the tumor cells acquire a substantial resistance to CTL attack, due to mutations stochastically occurring during cell division. Interestingly, a bias in CTL motility inducing a progressive attraction towards a few scout CTL, which have detected the nodule enhances early productive collisions and tumor eradication. Taken together, our results are compatible with a biased competition theory of CTL function in which CTL efficacy against a tumor nodule undergoing immunoediting is strongly dependent on guidance of CTL trajectories by scout siblings. They highlight unprecedented aspects of immune cell behavior that might inspire new CTL-based therapeutic strategies against tumors.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Molecular characteristics of immunogenic cancer cell death

Apoptotic cell death is initiated by a morphologically homogenous entity that was considered to be non-immunogenic and non-inflammatory in nature. However, recent advances suggest that apoptosis, under certain circumstances, can be immunogenic. In particular, some characteristics of the plasma membrane, acquired at preapoptotic stage, can cause immune effectors to recognize and attack preapoptotic tumor cells. The signals that mediate the immunogenicity of tumor cells involve elements of the DNA damage response (such as ataxia telangiectasia mutated and p53 activation), elements of the endoplasmic reticulum stress response (such as eukaryotic initiation factor 2alpha phosphorylation), as well as elements of the apoptotic response (such as caspase activation). Depending on the signal-transduction pathway, tumor cells responding to chemotherapy or radiotherapy can express 'danger' and 'eat me' signals on the cell surface (such as NKG2D ligands, heat-shock proteins and calreticulin) or can secrete/release immunostimulatory factors (such as cytokines and high-mobility group box 1) to stimulate innate immune effectors. Likewise, the precise sequence of such events influences the 'decision' of the immune system to mount a cognate response or not. We therefore anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.     

hsp70: Nuclear concentration during environmental stress and cytoplasmic storage during recovery

The intracellular distribution of the major Drosophila heat-shock protein hsp70 was determined by indirect immunofluorescence with monoclonal antibodies. During heat shock the protein concentrates strongly in nuclei while a small quantity remains cytoplasmic. During recovery hsp70 leaves the nuclei and becomes distributed throughout the cytoplasm. With a second heat shock it is rapidly transported back into the nucleus. Nuclear translocation depends not on the temperature per se, but on the physiological state of the cell since it also occurs after exposure to an anoxic atmosphere at normal temperatures. We also provide evidence that hsps protect cells from the toxic effects of anoxia, as well as heat, and conclude that nuclear translocation of hsp70 is related to its function in protecting the organism from both forms of environmental stress.     

Suppressor T lymphocyte induction by a factor released from cultured blastocysts

For the analysis of immunologic escape mechanisms of embryos during the implantation period in mice, the effects of culture supernatant of blastocysts on in vitro responsiveness to alloantigen of mice was investigated. Blastocyst-cultured conditioned medium was prepared by culturing late blastocysts of outbred ICR mice for 5 days. The addition of culture supernatant containing four or eight blastocysts to allogeneic mixed lymphocyte culture inhibited both the MLR responses and the generation of cytotoxic T lymphocytes (CTL). Preincubation of the culture supernatant with lymphocytes syngeneic to the responder cells of MLR induced potent suppressor cell activity in the MLR. The supernatant did not inhibit the activity of CTL at the effector phase, but preinduced suppressor cells obtained by incubation of splenocytes with the supernatant showed almost complete suppression of CTL activity at the effector phase. Both of the suppressor cells, active on MLR and at the generation phase of CTL as well as active at the effector phase, had a surface phenotype of Thy-1+ and Ig-. The suppressive material could be extracted from the eight-cell stage of fertilized ova or blastocysts but not from unfertilized ova, indicating that the production of the factor(s) is dependent on the stages of early embryogenesis. These results suggest that the active induction of suppressor T lymphocytes by the factor(s) released from implanted embryos is one of the protective mechanisms from maternal immunologic attack.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

Thermal acclimation in Arabidopsis lyrata: genotypic costs and transcriptional changes

Frost and heat events can be challenging for sessile organisms that cannot escape thermal extremes. However, adverse effects of thermal stress on fitness may be reduced by pre‐exposure to cold or heat, a process known as acclimation. To understand the ecological and evolutionary implications of acclimation, we investigated (1) the reduction in performance due to stress pre‐exposure, (2) the magnitude of increased leaf resistance to subsequent stress, (3) the costs of acclimation and (4) the genes differing in expression due to stress pre‐exposure. Plants of Arabidopsis lyrata were raised under three treatments of pre‐exposure: bouts of frost, bouts of heat or constant temperature. Resistance of leaves to subsequent frost and heat stress was then measured by electrolyte leakage. RNA‐seq analysis was performed to examine the genes differentially expressed between stress‐pre‐exposed and control plants. Pre‐exposure to stress during growth decreased plant size and increased leaf resistance to subsequent stress independent of whether pre‐exposure was to frost or heat. But the highest increase in leaf resistance to frost was found after pre‐exposure to frost (as a trend) and in leaf resistance to heat after pre‐exposure to heat. No evidence for costs of acclimation was detected. RNA‐sequencing suggested that acclimation by frost and heat pre‐exposure was caused by distinct mechanisms: modification of the chloroplast membrane and modification of the cell wall and membrane, respectively. Our results suggest that thermal resistance is a labile complex of traits, strongly affected by the previously experienced stress environment, with undetermined costs.     

Opposing effects of IL-2 in tumor immunotherapy: promoting CD8 T cell growth and inducing apoptosis

Tumors often induce specific CTL responses, but these are usually ineffective at eliminating the growing tumor. The T cell growth factor IL-2 has potential for expanding and prolonging CTL responses, and there is considerable interest in using this cytokine in combination with other immunotherapeutic agents that target T cell responses. Using adoptive transfer of OT-I CD8 T cells specific for OVA(257-264) peptide, and E.G7 tumor cells transfected with OVA, we have examined the effects of IL-2 on the generation and maintenance of a CTL response to the tumor. Administration of IL-2 during the initial phase of the response, clonal expansion, and development of effector function, had no effect on the number of CTL generated or the control of tumor growth. In contrast, a short 2-day time course of low-dose IL-2 at the peak of clonal expansion or at later times resulted in prolonged and expanded responses by the OT-I CTL, with concomitant decrease in tumor load and extension of survival. However, when IL-2 administration was more prolonged, as is often the case in clinical trials, the therapeutic benefit was lost due to elimination of the tumor-specific CTL, at least in part through induction of apoptosis. These results demonstrate that use of IL-2 for tumor immunotherapy is very much a double-edged sword and strongly suggest that more limited time and dose regimens may substantially improve its clinical efficacy when it is used in conjunction with approaches that target CTL responses.     

Influence of interferon γ on modulation of Fas expression by human colon carcinoma cells and their subsequent sensitivity to antigen-specific CD8+ cytotoxic T lymphocyte attack

The inability of certain neoplastic populations to undergo Fas-mediated death by immune effector mechanisms may confer a selective survival advantage, which may contribute to tumor escape. In this study, we examined the role of Fas-mediated lysis in a human-antigen (Ag)-specific cytotoxic T lymphocyte (CTL)/colon carcinoma cell model, and the regulation of the lytic phenotype by interferon γ (IFNγ). Previously, we have identified mutated ras peptides reflecting the valine-for-glycine substitution at position 12 as unique HLA-A2-restricted, CD8⁺ CTL neo-epitopes. Peptide-specific CTL, established from both normal and carcinoma-bearing individuals, lysed in vitro a HLA-A2⁺ primary colon adenocarcinoma cell line, SW480, harboring the naturally occurring ras mutation. Pretreatment of SW480 cells with IFNγ was necessary to promote efficient Ag-specific CTL killing, although the mechanisms by which IFNγ influenced the lytic outcome remains to be elucidated. Here, we show, by phenotypic analysis of SW480 cells, a significant up-regulation of HLA-A2, ICAM-1 and Fas molecules after IFNγ pretreatment, which paralleled their sensitivity to lysis with anti-Fas stimuli. Moreover, nearly half of the lytic response to IFNγ-treated SW480 cells was inhibited by neutralizing anti-Fas or anti-Fasligand (FasL) mAb, revealing for the first time an important functional role for Fas/FasL interactions in carcinoma cell killing by human Ag-specific CTL. mAb against HLA-A2, ICAM-1, the αβ T cell receptor (TCR) and Fas molecules inhibited lysis; however, if these CTL were preactivated to express functional FasL and then used as effectors, only anti-Fas mAb efficiently blocked lysis. IFNγ also increased pro-caspase-3 protein expression and its subsequent activation in SW480 cells following Ag-specific CTL attack. Peptide-based caspase inhibitors blocked both caspase-3 activation and CTL-mediated lysis. Overall, these data suggested that IFNγ (a) facilitated both Ag-dependent and Ag-independent events as a prerequisite for efficient CTL/target interactions, FasL up-regulation and triggering of Fas-dependent, as well as Fas-independent lysis (perforin); and (b) enhanced or restored a Fas-sensitive phenotype in SW480 cells, reflecting modulation of cell-surface and intracellular elements of the Fas pathway. Thus, IFNγ may play an important role in the regulation of a human neoplastic cell death phenotype, which may have implications for our understanding of the processes of both tumor evasion and tumor regression following Ag-specific CTL attack. Received: 20 December 1999 / Accepted: 1 February 2000     

Differential role of Fas/Fas ligand interactions in cytolysis of primary and metastatic colon carcinoma cell lines by human antigen-specific CD8+ CTL

We have previously identified mutated ras peptides reflecting the glycine to valine substitution at position 12 as HLA-A2-restricted, CD8+ CTL neo-epitopes. CTL lines produced against these peptide epitopes lysed the HLA-A2+ Ag-bearing SW480 primary colon adenocarcinoma cell line, although IFN-gamma treatment of the targets was necessary to achieve efficient cytotoxicity. Here, we compared the lytic phenotype of the SW480 cell line to its metastatic derivative, SW620, as an in vitro paradigm to further characterize the nature of a HLA class I-restricted, Ag-specific CTL response against neoplastic cell lines of primary and metastatic origin. Although both colon carcinoma cell lines were lysed by these Ag-specific CTL following IFN-gamma pretreatment, the mechanisms of lysis were distinct, which reflected differential levels of sensitivity to the Fas pathway. Whereas IFN-gamma pretreatment rendered SW480 cells sensitive to both Fas-dependent and -independent (perforin) pathways, SW620 cells displayed lytic susceptibility to Fas-independent mechanisms only. Moreover, pretreatment of SW480 cells with the anti-colon cancer agent, 5-fluorouracil (5-FU), led to enhanced Fas and ICAM-1 expression and triggered Ag-specific CTL-mediated lysis via Fas- and perforin-based pathways. In contrast, these phenotypic and functional responses were not observed with SW620 cells. Overall, these data suggested that 1) IFN-gamma and 5-FU may enhance the lytic sensitivity of responsive colon carcinoma cells to immune effector mechanisms, including Fas-induced lysis; 2) the malignant phenotype may associate with resistance to Fas-mediated lysis in response to Ag-specific T cell attack; and 3) if Ag-specific CTL possess diverse lytic capabilities, this may overcome, to some extent, the potential "escape" of Fas-resistant carcinoma cells.     

Induction of immunogenic apoptosis by blockade of epidermal growth factor receptor activation with a specific antibody

Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.     

Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL

Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte x Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.     

Tumor antigen-specific T helper cells in cancer immunity and immunotherapy

Historically, cancer-directed immune-based therapies have focused on eliciting a cytotoxic T cell (CTL) response, primarily due to the fact that CTL can directly kill tumors. In addition, many putative tumor antigens are intracellular proteins, and CTL respond to peptides presented in the context of MHC class I which are most often derived from intracellular proteins. Recently, increasing importance is being given to the stimulation of a CD4+ T helper cell (Th) response in cancer immunotherapy. Th cells are central to the development of an immune response by activating antigen-specific effector cells and recruiting cells of the innate immune system such as macrophages and mast cells. Two predominant Th cell subtypes exist, Th1 and Th2. Th1 cells, characterized by secretion of IFN- and TNF-, are primarily responsible for activating and regulating the development and persistence of CTL. In addition, Th1 cells activate antigen-presenting cells (APC) and induce limited production of the type of antibodies that can enhance the uptake of infected cells or tumor cells into APC. Th2 cells favor a predominantly humoral response. Particularly important during Th differentiation is the cytokine environment at the site of antigen deposition or in the local lymph node. Th1 commitment relies on the local production of IL-12, and Th2 development is promoted by IL-4 in the absence of IL-12. Specifically modulating the Th1 cell response against a tumor antigen may lead to effective immune-based therapies. Th1 cells are already widely implicated in the tissue-specific destruction that occurs during the pathogenesis of autoimmune diseases, such as diabetes mellitus and multiple sclerosis. Th1 cells directly kill tumor cells via release of cytokines that activate death receptors on the tumor cell surface. We now know that cross-priming of the tumor-specific response by potent APC is a major mechanism of the developing endogenous immune response; therefore, even intracellular proteins can be presented in the context of MHC class II. Indeed, recent studies demonstrate the importance of cross-priming in eliciting CTL. Many vaccine strategies aim to stimulate the Th response specific for a tumor antigen. Early clinical trials have shown that focus on the Th effector arm of the immune system can result in significant levels of both antigen-specific Th cells and CTL, the generation of long lasting immunity, and a Th1 phenotype resulting in the development of epitope spreading.     

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.