Regression of established murine carcinoma metastases following vaccination with tumour-associated antigen peptides.

The cure of micrometastases following surgery is the major goal of cancer immunotherapy. We have recently isolated tumour-associated antigen (TAA) peptides, MUT 1 and MUT 2, derived from a mutated connexin 37 gap-junction protein, from the malignant 3LL-D122 murine lung carcinoma. We now report that synthetic MUT 1 or MUT 2 induces effective antitumour cytoxic T lymphocytes. Peptide vaccines protect mice from spontaneous metastases of 3LL-D122 tumours. Moreover, peptide vaccines reduce metastatic loads in mice carrying pre-established micrometastases. Tumour-specific immunity was primarily mediated by CD8+ T cells. This is the first evidence that peptide therapy may be effective in treatment of residual tumours and provides a rationale for the development of peptide vaccines as a modality for cancer therapy.

Localization of aggregated cell surface antigens of target cells bound to cytotoxic T lymphocytes.

The redistribution of aggregated cell surface antigens of target cells bound to cytotoxic T lymphocytes was investigated. It was found that cap formation induced by antibody always occurred toward the site of binding. It is suggested that the polar localization of capped target cell surface determinants is a consequence of cytotoxic T-lymphocyte target cell interaction.

Nature of lymphocyte-tumor interaction. A general method for cellular immunoabsorption.

The binding of sensitized lymphocytes to tumor cells that leads to tumor cell lysis in vitro has been investigated using poly-L-lysine-fixed tumor cell monolayers and lymphocytes obtained from the anatomical site of tumor allograft rejection. The results show that magnesium is an important prerequisite for this interaction and that the extent of lymphocyte-tumor cell binding depends upon temperature as well as pH. Binding can occur in the absence of serum, although serum factors are necessary for the completion of the cytolytic process. The poly-L-lysine technique is applicable to the formation of confluent monolayers with virtually any normal or neoplastic cell type, including those that are otherwise nonadherent to surfaces. Cells immobilized by this technique can be used for the specific immunoabsorption and subsequent recovery of effector lymphocytes sensitized against transplantation or tumor cell antigens.

Cellular immunoabsorbents in transplantation immunity. Specific in vitro deletion and recovery of mouse lymphoid cells sensitized against allogeneic tumors.

Mouse lymphoid cells, sensitized against tumor allografts, can be deprived of the immunoreactive cells by in vitro absorption with specific fibroblast monolayers. Populations of lymphocytes so depleted are less effective in retarding tumor growth in vivo and in lysing tumor cells in vitro. Moreover, the adsorbed immunoreactive cells can be recovered specifically and are subsequently efficient in inhibiting tumor growth in vivo and in killing tumor cells in vitro. Further evidence is presented for the suggestion that the destruction of target cells in vitro by sensitized lymphoid cells is truly representative of the mode of destruction of grafted cells in vivo.

Rejection of ascites tumor allografts. I. Isolation, characterization, and in vitro reactivity of peritoneal lymphoid effector cells from BALB-c mice immune to EL4 leukosis.

Peritoneal exudate cells (PEC), obtained after the rejection of EL4 leukemia by BALB/c mice, are much more effective in the specific in vitro destruction of (51)Cr-labeled EL4 cells than are spleen, thymus, lymph node, or peripheral blood lymphocytes. The presence of a large number of effector cells at the site of graft rejection is reflected in the potent cytolytic activity seen in vitro. Effector cells temporarily lose cytolytic reactivity when treated with trypsin but regain reactivity with time. This recovery occurs in normal as well as in immune serum. The destructive reactivity of PEC is increased when macrophages are removed. The remaining population of nonadherent PEC is composed primarily of small- to medium-sized lymphocytes. Complex tissue culture media are not needed, but there is a definite requirement for serum. The required serum component is heat stable, nondialyzable, and is not consumed during the reaction. The use of an ascites allograft system made these observations possible and permitted the isolation of those host cells intimately associated with rejection.

Rejection of ascites tumor allografts. II. A pathway for cell-mediated tumor destruction in vitro by peritoneal exudate lymphoid cells.

A pathway for cell-mediated tumor destruction in vitro by immune peritoneal exudate lymphoid cells has been proposed. The union of lymphocytes and tumor cells precedes the formation of an intermediate phase leading to lysis. The initial interaction is only partially temperature dependent. The cytolytic process per se is highly temperature dependent, being negligible at 25 degrees C but proceeding rapidly at 37 degrees C. (51)Cr release from tumor cells is demonstrable within 10 min at 37 degrees C and can be reversibly arrested by cooling. Once initiated, lysis is largely independent of additional interactions and continues at almost full rate for 30 min. The effector cells are not lysed and appear to be free to enter into further effector cycles.

Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells.

Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC) is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL-TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.

Tumor immunity in vitro: destruction of a mouse ascites tumor through a cycling pathway.

Lymphocytes in peritoneal exudate from BALB/(c) mice immunized against ascites leukemia EL4 are uniquely efficient at destroying (51)chromiumlabeled EL4 cells in vitro. The lytic process depends upon the number of lymphocyte-tumor cell interactions. Efector lymphocytes are not inactivated as a result of lethal contact but can interact repeatedly with tumor cells.

T-cell-mediated cytotoxicity.

There are two competing theories to explain the mechanism(s) by which cytolytic T lymphocytes kill target cells: granule exocytosis of a pore-forming protein, and contact-induced internal disintegration. Accumulated evidence supports alternative pathways in lymphocytoxicity, possibly reflecting distinct effector functions expressed by different killer cells and cells at different stages of activation.

Effects of purified perforin and granzyme A from cytotoxic T lymphocytes on guinea pig ventricular myocytes.

OBJECTIVE: Involvement of cytotoxic T lymphocytes (CTL) in heart transplant rejection as well as in viral myocarditis is well established, but the precise mechanisms whereby infiltrating CTL damage the myocardium are unknown. The aim of the study was to investigate how CTL derived perforin, the serine protease granzyme A, and the combination of both, damage guinea pig ventricular myocytes. METHODS: Action potentials and membrane currents were recorded by means of the whole cell configuration from guinea pig ventricular myocytes. RESULTS: Resembling the effects of CTL derived lytic granules, perforin caused gradual myocyte shortening and contracture, leading to complete loss of the rod shaped morphology and to cell destruction. These changes were preceded by shortening of action potential duration and reduction of resting potential and action potential amplitude, followed by complete inexcitability. Granzyme A alone was ineffective, but accelerated the deleterious effects of perforin on the morphological and electrophysiological properties of myocytes. The effects of perforin were further evaluated by measuring membrane currents by means of the whole cell voltage clamp. Perforin induced discrete changes in membrane current, reminiscent of single ion channels, with large conductance and open time of up to several seconds. Linear regression analysis of the channel I-V relations resulted in a conductance of 890 pS and a reversal potential of -7.6 mV. These results suggest that perforin induces large non-selective channels, which can account for most of the observed adverse effects. CONCLUSIONS: As CTL participate in the immunological rejection of the transplanted heart, it is conceivable, but remains to be shown, that part of this damage is inflicted by perforin containing lytic granules.

Fluorescence depolarization as an early measure of T lymphocyte stimulation.

We have used the Cellscan, an apparatus capable of measuring optical properties of individual cells, to study changes in fluorescence polarization associated with T cell stimulation. We show that the fluorescence polarization of human peripheral blood lymphocytes (PBL) labeled with fluorescein diacetate (FDA) is markedly reduced upon exposure to the mitogenic lectins phytohemagglutinin (PHA), concanavalin A (ConA), or to phorbol esters. Methyl alpha-D-mannopyranoside (alphaMM) is able to reverse the depolarizing effect induced by ConA as long as the cells are not committed to proliferate. H7 and staurosporin, both inhibitors of protein kinase C (PKC), inhibit the depolarization induced by PHA. The mitogen-induced depolarization is dependent on metabolic energy. The results support the use of fluorescence depolarization of FDA-labeled PBL, monitored by the Cellscan, as a sensitive means of measuring early lymphocyte stimulation.

A rapid method for generating cytotoxic effector cells in vivo.

A method is described for the production of highly cytotoxic effector T lymphocytes. The cells can be raised in vivo in healthy animals in 4--5 days, thus combining the short time period of sensitization obtained in MLC or radiation GVH, without any of the attendant risks of these two methods (infection or loss of cultures or animals). DNA synthesis and blastogenesis are maximal 4 days after sensitization and are insignificant by day 6. 20--30% of the effector cells from conjugates with the sensitizing cell strain on days 4 through 7. Cytotoxicity was specific for cells of the sensitizing strain; blast cells are cytotoxic at the peak of the reaction (day 4) but small lymphocytes are cytotoxic on subsequent days.

Competitive inhibition of cytotoxic T lymphocyte-target cell conjugation. A direct evaluation of membrane antigens involved in cell-mediated immunity.

A method to determine directly the antigenic similarity of target cells (TCs) involved in cytotoxic T lymphocyte (CTL)-TC interactions is described. When fluorescently labeled TC (FL-TCs) are bound (conjugated) to CTLs in the presence of antigenically similar but unlabeled TCs, the percentage of CTL-TC conjugates containing unlabeled TCs corresponds linearly to the number of unlabeled TCs in the TC population. This assay is specific, permits direct observation of individual CTLs and TCs which interact to form conjugates, can be completed within 10 min, eliminates complications attributable to CTL recycling, and the degree of competition observed fits that theoretically expected. The results of this paper provide the theoretical basis for "cold" (unlabeled) target inhibition of radioactive assays of lymphocyte-mediated cytolysis.

Is the presence of serologically defined target cell antigens sufficient for binding of cytotoxic T lymphocytes?

Target cell parameters that coincide with specific binding (conjugation) of target cells to cytotoxic T lymphocytes were examined. It was demonstrated that concentrations of formaldehyde (0 to 1.2%) which do not affect the capacity of target cells to exclude dyes and incorporate sodium chromate, or their susceptibility to cellular and humoral immune lysis did not affect target cell conjugation. Treatments with higher formaldehyde concentrations (1.6 to 3.2%) caused similar decreases in all of the above-mentioned target cell parameters. Formaldehyde-treated (over 0.1%) targets that could not cap alloantigens or incorporate macromolecular precursors could still conjugate with cytotoxic T lymphocytes. Quantitative changes of serologically detected antigens alone cannot account for the decrease in binding of formaldehyde (greater than 1.2%)-treated target cells to cytotoxic T lymphocytes, since absorption of lytic alloantibodies by formaldehyde-fixed targets was not affected over the fixative range used (0 to 3.2%). From this study, it appears that certain target cell properties related to membrane structure and function and not the apparent expression of serologically defined antigens alone are essential for binding of targets by cytotoxic T lymphocytes. Additionally, it was shown that target cell susceptibility to immune lysis is retained, as long as their ability to bind to cytotoxic T lymphocytes is not affected.

Enucleated cytotoxic T lymphocytes bind specifically to target cells in vitro.

We report the isolation of enucleated particles (cytoplasts) from mouse cytotoxic T lymphocytes (CTLs) which, like intact CTLs, can specifically bind to target cells (TCs) in vitro. CTL-enriched populations were enucleated by centrifugation on a Ficoll density gradient at 31 C in the presence of cytochalasin B. The resulting cytoplasts which consist of 99% of enucleated particles retained about 40% of the nucleated cells' protein but less than 1% of their DNA. The presence of cell surface membrane antigens Thy 1.2 and H-2 on the cytoplasts indicated that their surface membranes originated from the plasma membranes of the intact cells. Electron microscopy of cytoplasts revealed two types of particles, one (type a) contained cytoplasmic organelles such as mitochondria, Golgi apparatus, microfilaments, and microvilli, while the second (type b) contained little cytoplasm and no microfilaments or microvilli. The evidence presented suggests that only type a cytoplasts can bind specifically to TCs. The specific binding to TCs by type a and not type b cytoplasts shows that while the CTL nucleus or nucleus-associated structures affected by enucleation are not essential for specific CTL receptor activity, cytoskeletal structures and activities related to these structures must be preserved.

Specific lysis of antigenically irrelevant cells by cytotoxic T lymphocytes upon insertion of appropriate antigens into the target cell plasma membranes.

The importance of the membrane milieu to functional presentation of target cell (TC) antigens to cytotoxic T lymphocytes (CTL) was investigated by examining the interaction of CTL with TC plasma membrane (PM) fractions, in isolated form or integrated into antigenically irrelevant TC. Isolated ascitic vesicles, microsomes, and purified PM, containing serologically defined alloantigens that have been implicated as the relevant TC antigens, effectively, yet nonspecifically, inhibited the binding and lysis of TC by CTL. The same PM fractions, when inserted into antigenically irrelevant TC via vesicles containing Sendai virus components, rendered the TC susceptible to CTL-mediated lysis directed against the inserted antigens. These findings suggest that CTL interact specifically with TC determinants only when they are embedded in the proper membrane environment.

The cytolytic T lymphocyte and its mode of action.

While the binding step of cytolytic T lymphocyte (CTL) target cell interaction resulting in conjugate formation is a well-characterized event, there seems to be more than one mechanism whereby lymphocytes kill the target. In recent years, infliction of complement (C)-like "holes" (I.D. 10-20 nm) on the target cell membrane, believed to be produced by the Ca2+-dependent lytic protein(s) perforin/cytolysin of secretory lytic granule origin has been proposed to be the mechanism of lymphocytotoxicity. More recent evidence, however, suggests that Ca2+-dependent exocytosis of lytic granules (where detectable) is not involved in lymphocyte-mediated cytolysis. Furthermore, neither formation of C-like "holes" in targets exposed to CTL, nor higher-than-background levels of lytic granules, perforin or BLT-esterases, have been detected in highly potent, peritoneal exudate CTL (PEL) derived directly from the animal or in cytocidal PEL-hybridomas. Hence exocytosis of perforin and formation of the above pores may apply to certain effector cells, particularly those grown in vitro in IL-2, but not to in vivo primed CTL such as PEL. On the other hand, work from this laboratory with Ca2+ probes has shown that lysis induced by CTL such as PEL-not involving lytic granules, perforin or formation of the above "holes"-is preceded by a marked prelytic elevation of cytosolic Ca2+ in the target. CTL-induced target cell membrane perturbation--a direct result of receptor-mediated effector-to-target interaction or through a membrane-bound or secreted effector component(s)--may be responsible for triggering the prelytic influx of Ca2+ from external sources, or its mobilization from internal stores in the target. We propose that CTL-induced, persistent elevation of cytosolic Ca2+, above a critical level, rather than formation of 10-20 nm pores, is responsible for the catastrophic prelytic events observed in the target, such as bleb formation, metabolic exhaustion and DNA degradation, ultimately leading to lysis.

Activation of influenza-specific memory cytotoxic T lymphocytes by Concanavalin A stimulation.

Traditionally, the in vitro activation of virus-specific memory cytotoxic T lymphocytes (CTLs) has been achieved by stimulating the CTLs with antigen-presenting cells (APCs) infected with an appropriate virus or pulsed with virus-specific antigenic peptides. Here, we describe the utilization of the polyclonal activator Concanavalin A (ConA) for in vitro restimulation of memory CTLs from virus-primed mice. Using this simple method, the activation of splenocytes with ConA for 3 days (i) eliminates the need to stimulate with virus-pulsed APCs and (ii) generates CD8+ CTLs that exhibit virus specificity and MHC-restricted lytic activity similar to CTLs obtained by conventional viral restimulation. In vitro ConA stimulation of splenocytes from BALB/c mice primed with the A/Texas/77 or A/Japanese/57 strain of influenza virus and from C57L/J mice infected with the A/Texas strain, generated CTLs with specific lytic activity. Hence reactivation of memory CTLs by this method is a general phenomenon rather than a mouse or viral strain-specific one. The ConA stimulation method used here had a recall of long-term (1 year) memory CTLs that effectively lysed virally infected targets. Further ConA-stimulated effector lymphocytes from virally primed animals have been shown to recognize and subsequently lyse target cells pulsed with virus or virus-derived peptides. The ConA reactivation of specific anti-viral CTLs may facilitate (i) studying anti-viral CTL responses and (ii) identifying of viral epitopes when unknown or when appropriate viral stimulation is impossible.

Effect of oxidation on the conformation of cell surface H-2 antigens detected by monoclonal antibodies.

Periodate treatment of lymphoid cells is known to render them susceptible to nonspecific lysis by cytotoxic T lymphocytes. To detect alterations in cell surface molecules as a result of such treatment, we investigated the binding of antibodies to oxidized and nonoxidized lymphoid cells and found that oxidation selectively decreases the binding of some anti-H-2 and anti-beta 2m antibodies. This effect was not caused by loss of H-2 molecules from the cells, since the rate of exchange of cell surface beta 2m by exogenously added beta 2m was unchanged, and neither H-2 nor beta 2m molecules could be detected in the supernates of oxidized cells. Our findings suggest that oxidation alters the conformation of H-2 antigens in such a way as to reduce antibody binding. Such conformational changes may further affect the recognition of histocompatibility antigens by cytotoxic T cells.

Intraoperative measurement of the elastic modulus of the vocal fold. Part 1. Device development.

Although the concept of manipulating laryngeal biomechanics to improve vocal function is not new, there has been a recent proliferation in surgical techniques used to affect laryngeal function. These include methods which increase the stiffness of the vocal folds, medialize the vocal folds, alter the pitch by changing the tension of the vocal folds, and augment the tissues using injection of alloplastic materials. Despite these new and possibly revolutionary methods, no means are presently available to surgeons to intraoperatively evaluate and optimize results of a surgical intervention. This study involved the development of a device to measure the in vivo elastic modulus of the vocal folds.