Using fluorescein-labeled effector lymphocytes and tetramethyl rhodamine-labeled target cells a cytofluorometric method is described for the detection and quantification of lymphocyte-target cell ...cluster (conjugate) formation. Conjugation levels measured by cytofluorography correspond well with those scored microscopically. The method has so far been used successfully to monitor conjugate formation by specifically sensitized cytotoxic T lymphocytes (CTL). In preliminary experiments it has been applied to nonspecific, lectin concanavalin A-dependent conjugate formation by CTL, as well as to conjugates formed by hybridoma CTL. The procedure has potential application in other cell-cell interactions, relevant to immunology as well as to cell biology.
We have shown previously that the interaction between cytotoxic T lymphocytes (CTL) and ventricular myocytes, an in vitro model for heart transplant rejection, results in electrophysiological and ...morphological alterations indicative of overload of the intracellular Ca2+ (Ca2+i). Since these deleterious effects cannot be accounted for by increased L-type Ca2+ current (ICa,L), we hypothesize that Ca2+i overload due to Ca2+ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inositol trisphosphate (IP3) cascade. Patch-clamp and fura-2-fluorescence techniques were utilized to record transmembrane potentials and Ca2+i from ventricular myocytes bound to peritoneal exudate CTL (PEL). In ventricular myocyte-PEL conjugates (after 60 min), resting potential was reduced (compared with the nonconjugated state) from -80.9 +/- 0.7 to -59.9 +/- 2.5 mV, action potential amplitude from 139.5 +/- 1.4 to 80.6 +/- 1.7 mV and action potential duration to 50% repolarization (APD50) from 797 +/- 97 to 52 +/- 12 ms. The ratio of fluorescence at 340 and 380 nm (R340/380) increased from a control value (in nonconjugated myocytes) of 0.71 +/- 0.02 to 2.07 +/- 0.03, 30 min, after conjugate formation, and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 micrograms/ml), an antagonist of IP3-induced Ca2+ release from SR channels, or U-73122 (2 microM), a phospholipase C (PLC) inhibitor (drugs were included in the pipette solution), prevented PEL-induced morphological and electrophysiological alterations. Accordingly, heparin attenuated the PEL-induced increase in Ca2+i; after 60 min of PEL-myocyte interaction, R340/380 was 1.15 +/- 0.09 (compared with approximately 4.0 in the absence of heparin). The results indicate that CTL-mediated damage to ventricular myocytes is, at least partially, mediated by PLC activation and IP3-induced Ca2+ release from intracellular stores. Pharmacological targeting of IP3 in heart transplant rejection is thus suggested.
Tumor cells insensitive to lysis through the Fas and TNF pathways were injected either subcutaneously or into the peritoneal cavities of allogeneic perforin-less (P0) and perforin wild-type (P2) ...mice. In three of four cases, the tumors were rejected equally rapidly in both strains of mice. Rejection was accompanied by vigorous in vitro cytotoxicity in P2, but not in P0 mice. The rapid clearance of allografted cells in mice where all three known cytolytic pathways are seriously compromised raises important questions about the involvement of cell-mediated cytotoxicity, as defined by current assay techniques, in primary allograft rejection.
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.
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
α-
d-mannopyranoside (
αMM) 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.
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.
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