Phorbol esters inactivate the lytic apparatus of cytotoxic T lymphocytes

Treatment of cloned murine CTL with phorbol myristate acetate (PMA) initiates the loss of lytic potential of these cells. The process is relatively rapid (t 1/2 = 2.2 +/- 0.7 hr), occurs independently of protein synthesis, and can be largely reversed within 3 hr by incubation in normal medium with or without the addition of supernatants from activated lymphocytes. Examination of PMA-treated or untreated CTL in assays that separate binding and post-binding events suggests that the lytic apparatus itself is a major target of the PMA-initiated event.     

A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.     

Human natural killer cells and cytotoxic T lymphocytes require cell surface carbohydrate determinants for lytic function

Cloned and uncloned populations of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) were treated with tunicamycin, an antibiotic that inhibits N-linked glycosylation, in order to study the potential role of cell surface carbohydrate determinants in lytic function. It is shown that tunicamycin-treated NK and CTL effector cells lose killer function in a dose-dependent manner. This effect is reversible; cells washed free of tunicamycin begin to recover their killer activity within 2 to 3 days after initial treatment. Conjugate experiments indicate that killer-target cell binding is not affected by tunicamycin treatment of the NK cells. It is also shown that tunicamycin treatment of target cells does not significantly affect their ability to be lysed by NK or CTL effector cells. These studies provide evidence that carbohydrate determinants are important in the lytic mechanism of both CTL and NK cells, rather than in specific effector-target cell binding.     

A 15-kDa interferon-induced protein is derived by COOH-terminal processing of a 17-kDa precursor

An interferon-induced 15-kDa protein is synthesized from a precursor of higher molecular weight; the precursor contains 165 amino acids (17 kDa), whereas the stable product (15 kDa) contains 156 amino acids. The stable 15-kDa form is derived from the precursor 17-kDa form by the removal of eight amino acids from the COOH terminus and the methionine from the NH2 terminus. The existence of the precursor 17-kDa protein can be demonstrated after brief periods of in vivo labeling with [35S]methionine and by translation of mRNA in vitro.     

Activation of cytotoxic function in T lymphocytes.

The requirements for activation of cytotoxic function in mouse T lymphocytes were investigated. Initial generation of cytotoxicity in normal lymphocytes was equal in magnitude with either Con A or specific alloantigen, and in either case required DNA synthesis. Cytotoxic function in MLC-primed cells could also be regenerated by Con A, the magnitude and target specificity of the cytotoxicity thus generated being indistinguishable from that recalled by specific alloantigen. Cytotoxicity could also be regenerated by third party-stimulating cells; however, the cytotoxicity evoked by third party cells was always specific only for target cells of the original stimulating cell H-2 genotype. The data presented suggest that there are a number of ways to activate cytotoxicity in effector T cells, and are most consistent with a model for T cell triggering that minimizes a strict informational function of antigen-receptor interactions.     

Imaging of lytic granule exocytosis in CD8+ cytotoxic T lymphocytes reveals a modified form of full fusion

Here we imaged the exocytosis of lytic granules from human CD8⁺ cytotoxic T lymphocytes using rapid total internal reflection microscopy, Lamp-1 tagged with mGFP to follow the fate of the lytic granule membrane, and granzyme A, granzyme B or serglycin tagged with mRFP to follow the fate of lytic granule cargo. Lytic granules were released by full fusion with the plasma membrane, such that the entire granule content for all three cargos visualized was released on a subsecond time scale. The behavior of GFP-Lamp-1 was, however, more complex. While it entered the plasma membrane in all cases, the extent to which it then diffused away from the site of exocytosis varied from nearly complete to highly restricted. Finally, the diffusion properties upon release of the three cargos examined put an upper limit on the size of the macromolecular complex of granzyme and serglycin that is presented to the target cell.     

Solid tumor-targeted infiltrating cytotoxic T lymphocytes retained by a superantigen fusion protein

Successful immune-mediated regression of solid tumors is difficult because of the small number of cytotoxic T lymphocytes (CTLs) that were traffic to the tumor site. Here, the targeting of tumor-specific infiltrating CTLs was dependent on a fusion protein consisting of human epidermal growth factor (EGF) and staphylococcal enterotoxin A (SEA) with the D227A mutation. EGF-SEA strongly restrained the growth of murine solid sarcoma 180 (S180) tumors (control versus EGF-SEA, mean tumor weight: 1.013 versus 0.197 g, difference  = 0.816 g). In mice treated with EGF-SEA, CD4⁺, CD8⁺ and SEA-reactive T lymphocytes were enriched around the EGFR expressing tumor cells. The EGF receptors were potentially phosphorylated by EGF-SEA stimulation and the fusion protein promoted T cells to release the tumoricidal cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Intratumoral CTLs secreted cytolytic pore-forming perforins and granzyme B proteins near the surface of carcinomas, causing the death of many tumor cells. We additionally show that labeled EGF-SEA was directly targeted to the tumor tissue after intravenous (i.v.) injection. The findings demonstrate that antibody-like EGF-SEA plays an important role in arresting CTLs in the solid tumor site and has therapeutic potential as a tumor-targeting agent.     

IL2-PE664Glu, a new chimeric protein cytotoxic to human-activated T lymphocytes

To produce a molecule that will kill activated T cells as well as lymphomas and leukemias expressing interleukin 2 (IL2) receptors, we have created a recombinant chimeric protein in which IL2 is attached in peptide linkage to a truncated mutant form of Pseudomonas exotoxin (PE) (Lorberboum-Galski, H., FitzGerald, D.J.P., Chandhary, V.K., Adhya, S., and Pastan, I. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1922-1926). Although this molecule was very active on rodent cells, it had lower activity on some human cell types. A new chimeric protein termed IL2-PE664Glu has been constructed that is extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes. The chimeric gene encoding this protein was constructed by fusing a cDNA clone for human interleukin 2 to the 5' end of a mutated cDNA encoding a full-length PE molecule. Four amino acids in domain I of PE were changed thus decreasing its nonspecific toxicity. IL2-PE664Glu is a much more active cytotoxic molecule for primate and human-activated T cells than IL2-PE40 which is a chimeric protein that was found to be an effective immunosuppressive agent in rodent models. Our results indicate that IL2-PE664Glu should be evaluated as an immunosuppressive agent for the treatment of human immune disorders in which activated T cells expressing the IL2 receptor are prominent.     

Increased Phosphorylation of a 17-kDa Protein Kinase C Substrate (P17) in Long-Term Potentiation

Hippocampal long-term potentiation (LTP) is a persistent increase in the efficacy of synaptic transmission, which is widely thought to be a cellular mechanism that could contribute to learning and memory. Studies on the biochemical mechanisms underlying LTP suggest the involvement of protein kinases in both LTP induction and maintenance. In this report we describe an LTP-associated increase in the phosphorylation in vitro of a 17-kDa protein kinase C (PKC) substrate protein, which we have termed P17, in homogenates from the CA1 region of rat hippocampal slices. This LTP-associated increase in phosphorylation was expressed independent of significant levels of free Ca2+, as phosphorylation reactions were performed in the presence of 500 microM EGTA. The increased phosphorylation of P17 was substantially inhibited by PKC(19-36), a selective inhibitor of PKC. These data support the model that persistent PKC activation contributes to the maintenance of LTP and implicate P17 as a potential target for PKC in the CA1 region of the hippocampus.     

Conversion of specific to nonspecific cytotoxic T lymphocytes

The type of cytotoxic T lymphocyte (CTL) generated during a mixed leukocyte culture has been shown to depend on the purity of interleukin 2 (IL 2) used to stimulate growth. Antigen-specific CTL requiring both antigen and IL 2 to proliferate or express cytotoxicity (designated Type I in this work) are generated and maintained with highly purified IL 2. In the presence of IL 2 alone, they persist in a quiescent, noncytotoxic state, and on its removal they rapidly die. A second type of CTL (Type II), of nonspecific cytotoxicity, and dependent only on IL 2 for growth, is generated by exposure to an impure lymphokine preparation containing IL 2. Type I cells are T lymphocytes which are nongranular and nonadherent, whereas Type II cells are larger, adhere to plastic, and contain many granules. Cloned Type I cells are converted to Type II under three conditions: by low levels of IL 2 in the presence of a crude lymphokine preparation; by high levels of IL 2 in the presence of antigen; or by high levels of IL 2 in the presence of the phorbol diester PMA. Conversion results from an effect of the growth conditions on individual cells, and not from selection of a minor population.     

Resistance of HIV-infected cells to cytotoxic T lymphocytes

Although cytotoxic T lymphocytes (CTLs) are important for controlling HIV, CTLs are not effective at eradicating HIV infection. Recent studies have revealed a combination of factors that together make HIV-infected cells resistant to CTLs and make anti-HIV CTLs ineffective. These factors likely contribute to prolonged survival of infected target cells, which in turn increases the probability of antigenic variation and immune escape.     

Expression and function of asialo GM1 in alloreactive cytotoxic T lymphocytes

In the present study we examined asialo GM1 (AsGM1) expression and its function in alloreactive cytotoxic T lymphocytes (CTL). We consistently found that the cytotoxic activity of bulk culture-derived allo-CTL was susceptible to the treatment of anti-AsGM1 (alpha AsGM1) plus complement. To further determine whether the expression of AsGM1 was maintained in CTL, we examined cloned T cells. The expression of AsGM1 in the T cell clones was assessed by their susceptibility to lysis by alpha AsGM1 plus complement and the reduction or abrogation of their cytotoxic activity by this treatment. It was found that, with one exception, all Lyt-2+, Thy-1+ CTL clones were AsGM1+ (seven out of eight), independent of their class specificity (class I or class II). In contrast, all Thy-1+, L3T4+ CTL (2) or helper T cell (4) clones AsGM1-. These findings suggested that there was a close association between the expression of AsGM1 and the expression of Lyt-2. The cytotoxic reaction of the anti-class I MHC CTL clones that expressed AsGM1 was blocked by alpha AsGM1 or alpha Lyt-2 antibody. The Lyt-2+, AsGM1+ anti-class II MHC CTL clone-mediated lysis was inhibited by alpha AsGM1. Addition of AsGM1 in micelle form (AsGM1-M) alone also blocked the cytotoxic reactions. Addition of other structurally similar but antigenically different glycolipids or other non-AsGM1-containing liposome preparations did not affect CTL-mediated cytotoxicity. Furthermore, adding both alpha AsGM1 and AsGM1-M together at proper doses inhibited the blocking effect (deblocking) of either alone, and other structurally similar glycolipids did not inhibit the blocking. The deblocking was specific, since AsGM1-M did not affect the blocking by alpha Lyt-2. These findings indicate that not only is AsGM1 expressed in a majority of Lyt-2+ CTL clones, but it may also be involved in the CTL- target interaction to mediate lytic reaction.     

Treatment of human colon carcinoma cell lines with anti-neoplastic agents enhances their lytic sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes

Certain anti-neoplastic agents at subtoxic doses may exert immunomodulatory effects, which alter the expression of specific tumor cell surface molecules. We reasoned that potential increases in tumor cell surface markers, such as those important for facilitating effector-target contact, as well as triggering cell death pathways, might then improve antigen (Ag)-specific T-cell-mediated tumor cytolysis. Here, in a human colon carcinoma cell model in vitro, we examined whether the anti-neoplastic agents 5-fluorouracil (5-FU), CPT-11 or cisplatin (CDDP) could upregulate the expression of specific tumor cell surface markers, which may then enhance productive lytic interactions between CD8+ CTL and Ag-bearing tumor cells. Based on our earlier studies, IFN-gamma treatment was included as a control for sensitization to CTL-mediated lysis. Pretreatment of the SW480 primary colon carcinoma cell line with IFN-gamma, 5-FU, CPT-11 or CDDP enhanced ICAM-1 and Fas expression, resulting in Ag-specific CTL-mediated lysis involving Fas-dependent and -independent mechanisms. In contrast, pretreatment of the SW620 metastatic isolate, derived from the same patient, with IFN-gamma, CPT-11 or CDDP, but not 5-FU, enhanced ICAM-1 expression, resulting in Ag-specific CTL-mediated lysis via Fas-independent mechanisms only. Flow cytometric-based assays were then developed to measure the effects of drug treatment on caspase signaling and apoptosis incurred by tumor targets after interaction with CTL. We found that the lytic enhancement caused by drug treatment of SW480 or SW620 targets was accompanied by an increase in caspase-3-like protease activity. A peptide-based caspase inhibitor abrogated CTL-mediated apoptosis, suggesting that "chemomodulation" involved regulation of the caspase pathway. These results revealed for the first time an important role for components of the caspase pathway, such as caspase-3-like proteases, in the sensitization of human colon carcinoma cells by anti-neoplastic agents to Ag-specific CTL. Thus, certain anti-neoplastic agents may display unique immunoregulatory properties that facilitate human colon carcinoma death by engaging the lytic capacity of Ag-specific CTL, which may have implications for chemoimmunotherapy strategies.     

Induction of cytotoxic T lymphocytes in vivo with protein antigen entrapped in membranous vehicles.

Intravenous administration of APC such as splenocytes loaded with a soluble protein Ag has been shown to prime for an Ag-specific CTL response. It is thought that the APC directly presents loaded Ag in a MHC-restricted manner. However, it is demonstrated in this study that allogeneic splenocytes, MHC-free RBC, and even synthetic lipid vesicles (liposomes) after loading with OVA can elicit an OVA-specific and MHC-restricted CTL response. Biodistribution studies of these Ag-associated vehicles showed that the liver, spleen, and lung were the major organs responsible to scavenge these carriers, suggesting that the monocyte-macrophage system was involved in the Ag presentation for CTL. Depletion of macrophages by a specific macrophage killer, Cl2MDP, containing liposomes, abolished the CTL induction by immunization with OVA Ag carried by these vehicles except the induction by syngeneic splenocytes. Thus, the syngeneic splenocytes present Ag directly to the T cells, but other membranous vehicles carry the Ag to the host APC including macrophages, which then present it to the T cells. These results indicate that formulation of an Ag in membranous/colloidal vehicles may be a way to prime for a CTL response.     

Phosphorylation of a 27-kDa protein correlates with survival of protein-synthesis-inhibited MCF-7 cells

Summary Previously, we have shown that IGF-1, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and aurintricarboxylic acid (ATA) protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). We proposed that phosphorylation of a putative cellular protein(s) may be involved in this survival mechanism. In the present study we investigated the ability of several agents to induce phosphorylation of cellular proteins and correlated this ability to their survival effect. We found that TPA, ATA, and IGF-1 increased the degree of phosphorylation of a 27-kDa protein in a dose- and time-dependent manner in CHX-treated MCF-7 cells. The ED₅₀ values observed were 25 ng/ml, 40 μg/ml and 15 ng/ml for TPA, ATA, and IGF-1, respectively. The effect was measured upon 10 min of cell treatment with each agent; it reached maximum at 60 min and thereafter decreased continuously to control levels. The 27-kDa protein was found in the cytosolic fraction as a phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphoprotein was resolved into two isoforms with pI 5.7 and 5.9. Such characteristics were observed for the small molecular weight heat shock protein HSP27. Indeed, a single band of 27 kDa was detected immunologically with rabbit polyclonal anti-human HSP27. The inactive phorbol ester αTPA, epidermal growth factor (EGF), and 8-bromoadenosine 3′5′-cyclic monophosphate (Br-cAMP) did not increase phosphorylation of the 27-kDa protein. Cell survival was measured by exposure of the CHX-pretreated cells to increasing concentrations of the various agents for 60 min, followed by a further incubation for 48 h in the presence of CHX only. TPA, ATA, and IGF-1 were found to enhance cell survival, whereas αTPA, EGF, and Br-cAMP did not. Our results indicate a correlation between phosphorylation of a 27-kDa protein, probably HSP27, and enhanced cell survival, suggesting a role for this phosphoprotein in the survival mechanism.     

Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotoxic T lymphocytes

Telomere length is abnormally short in the CD8(+) T-cell compartment of human immunodeficiency virus type 1 (HIV-1)-infected persons, likely because of chronic cell turnover. Although clonal exhaustion of CD8(+) cytotoxic T lymphocytes (CTL) has been proposed as a mechanism for loss of antigen-specific responses, the functional consequences of exhaustion are poorly understood. Here we used telomerase transduction to evaluate the impact of senescence on CTL effector functions. Constitutive expression of telomerase in an HIV-1-specific CTL clone results in enhanced proliferative capacity, in agreement with prior studies of other human cell types. Whereas the CTL remain phenotypically normal in terms of antigenic specificity and requirements for proliferation, their cytolytic and antiviral capabilities are superior to those of control CTL. In contrast, their ability to produce gamma interferon and RANTES is essentially unchanged. The selective enhancement of cytolytic function in memory CTL by ectopic telomerase expression implies that loss of this function (but not cytokine production) is a specific consequence of replicative senescence. These data suggest a unifying mechanism for the in vivo observations that telomere lengths are shortened in the CD8(+) cells of HIV-1-infected persons and that HIV-1-specific CTL are deficient in perforin. Telomerase transduction could therefore be a tool with which to explore a potential therapeutic approach to an important pathophysiologic process of immune dysfunction in chronic viral infection.     

Normal lytic granule secretion by cytotoxic T lymphocytes deficient in BLOC-1, -2 and -3 and myosins Va, VIIa and XV

Melanocytes and cells of the immune system share an unusual secretory mechanism which uses the lysosome as a regulated secretory organelle. Recently, a number of the proteins required for these 'secretory lysosomes' to undergo exocytosis have been identified. These include Rab27a, Lyst, Rab geranyl geranyl transferase and the adapter protein complex AP-3. Patients lacking any of these proteins are characterized by the rare combination of albinism and immunodeficiency, revealing roles for these proteins in both melanocyte and immune cell secretion. In order to ask how far the link between albinism and immunodeficiency extends we have examined cytotoxic T-lymphocyte (CTL) secretion from two BLOC-3-deficient patients and seven different mouse models of Hermansky-Pudlak syndrome, all of which display defects in pigmentation and platelet function. We find that CTL function is normal in HPS patients and pale-ear mice deficient in BLOC-3, pallid, muted and sandy mice deficient in BLOC-1, ruby-eye mice deficient in BLOC-2 and buff mice deficient in Vps33a. Similarly, the unconventional myosins, Va, VIIa and XV, which can act as effectors for Rab27a in some cell types, are not required in CTL. These results reveal differences in the protein machinery required for biogenesis and/or secretion of lysosome-related organelles in CTL and melanocytes.     

A T cell receptor-associated GTP-binding protein triggers T cell receptor-mediated granule exocytosis in cytotoxic T lymphocytes

To study the subcellular events occurring after T cell activation we used cloned human CTL permeabilized with alpha-toxin of Staphylococcus aureus. This method of permeabilization leads to stable transmembrane channels that permit the introduction of small molecules into the cell but preserves the cellular structures and macromolecular contents of the CTL. We used the exocytosis of CTL-specific serine esterases as a marker of T cell activation. The TCR-activated exocytosis is functioning in such permeabilized CTL. Introduction of the membrane impermeable guanosine nucleotide-binding protein (G-protein) activating GTP-analog GTP gamma S into CTL triggers exocytosis if Ca2+ is present. For optimal exocytosis ATP is required. The G-protein inactivating GDP-analog GDP beta S inhibited exocytosis triggered via the TCR-CD3 complex but not that triggered by activating the protein kinase C. If the protein kinase C was depleted in CTL by overnight incubation with phorbolester, the response to GTP-gamma S was reduced by more than 50%. These experiments demonstrate the presence of a G-protein involved in TCR-mediated CTL triggering. In the sequence of signaling steps this G-protein is localized after TCR-triggering but before the formation of the protein kinase C-activating phosphoinositol breakdown product diacylglycerol in the sequence of signaling steps.     

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.     

Serine and tyrosine phosphorylation of 28- and 35-kDa proteins of human T lymphocytes stimulated by streptococcal M protein

Purified polypeptide fragments of certain surface M proteins of group A streptococci stimulate blastogenesis and the differentiation of cytotoxic T lymphocytes of normal human lymphocytes. The biochemical basis of lymphocyte stimulation by a type M5 protein polypeptide fragment (pep M5) was investigated. Optimal blastogenic doses of pep M5 or phytohemagglutinin stimulated the phosphorylation of several cellular proteins. However, pep M5 but not phytohemagglutinin induced the phosphorylation of 28- and 35-kDa proteins. The 28-kDa protein was shown to be phosphorylated only at serine residues, whereas the 35-kDa protein was phosphorylated only at tyrosine residues. Stimulation of peripheral blood lymphocytes with pep M5 caused a two-fold increase in the CD8+ and CD4+ 4B4+ subpopulations of T lymphocytes. The phosphorylation of the 28-kDa protein appeared to be confined to the CD4+ T cell subpopulation.