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 51Cr-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°C but proceeding rapidly at 37°C. 51Cr release from tumor cells is demonstrable within 10 min at 37°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

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. Cardiovascular Research 1994;28:643-64

Spatial relationships of microtubuleorganizing centers and the contact area of cytotoxic T lymphocytes and target cells

ABSTRACT 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. A prominent manifestation of cell-mediated immunity is the lytic interaction of cytotoxic T lymphocytes (CTL) with appropriate target cells (TC). This process is believed to be relevant to virus, tumor, and transplantation immunity (4, 13, 42). The first step in CTL-mediated lysis is the binding of CTL and TC (conjugation) mediated through specific CTL cell surface receptor(s) and TC major histocompatibility complex determinants. Binding is followed by a lethal hit step delivered by the CTL, ultimately leading to lysis. Following lysis of the TC, effector CTL detach and can recycle to start a new lyti