In vitro determination of phagocyte activity by luminol- and lucigenin-amplified chemiluminescence

Amplified chemiluminescence (CL) detects most sensitively biologically important reactive oxygen species (ROS) which are generated by phagocytes by the respiratory burst permitting the determination of cell activity in vitro. Different murine phagocyte populations were used in combination with various ROS-catabolizing enzymes and some of their inhibitors to determine the possible advantages of one of the two main presently used amplifiers, i.e. luminol and lucigenin. Lucigenin appeared to react mainly with the first of the generated ROS the superoxide anion radical (O-.2) and thus records cell activity via the respiratory burst much more reliable than luminol. The more commonly employed luminol reacts mainly with hydrogen peroxide (H2O2) and probably the singlet oxygen (1O2) which result in photon emission. However, it seems not to react with the hydroxyl radical (OH.). The dependence of luminol-amplified CL upon the generation of the chain reaction intermediate H2O2 and its three main catalysts catalase, myeloperoxidase and glutathione makes this reaction prone to different artifacts if cell activity is to be determined. Lucigenin-amplified CL offers great advantages to study cell activating or inhibiting properties of drugs and kinetics in vitro because of the biological relevance of O-.2 determination, its sensitivity, reproducibility and ease in handling.     

Macrophage oxidative metabolism and intracellular Toxoplasma gondii

We explored the mechanisms by which Toxoplasma gondii avoids destruction by the oxidative metabolism of normal macrophages. Unelicited murine peritoneal macrophages were infected with T. gondii and exposed to different experimental conditions. As endpoints we used measurement of hydrogen peroxide (H2O2) release and intracellular reduction of nitroblue tetrazolium dye (NBT). Three main observations were made. Firstly, different T. gondii preparations (live or dead, opsonized or not) failed to trigger the respiratory burst. Combined challenges also showed that a primary T. gondii infection was able to block H2O2 release triggered by heat-killed (HK)-Candida albicans. The H2O2 release, however, once triggered by HK-C. albicans, was not inhibited by a subsequent challenge with T. gondii. Secondly, when a respiratory burst was obtained in T. gondii-infected macrophages--for instance by stimulation with phorbol myristate acetate (PMA)--the toxic oxygen metabolites (as determined by the NBT reduction test) did not seem to reach the vacuoles containing the parasite. Thirdly, when a respiratory burst occurred in T. gondii-infected macrophages, the intracellular development of T. gondii did not seem to be affected. In conclusion, we hypothesize that T. gondii is not damaged by the macrophage oxidative metabolism because the parasite fails to encounter toxic oxygen metabolites. The killing of intracellular T. gondii, as it is commonly observed in activated macrophages, does not appear oxygen-dependent.     

Reactive forms of oxygen and chemiluminescence in phagocytizing rabbit alveolar macrophages.

Chemiluminescence (CL), superoxide anion (O2-) production, and particle uptake were measured to determine the role of antibacterial substances in the chemiluminescent response associated with phagocytosis in rabbit alveolar macrophages (AM). Exposure of AM to zymosan particles induced both CL and the production of extracellular O2-. CL is inhibited by superoxide dismutase, an enzyme which catalyzes the conversion of O2- to hydrogen peroxide (H2O2), by catalase, an enzyme which destroys H2O2, and by the hydroxyl radical (.OH) scavengers, benzoate and ethanol. Superoxide dismutase and catalase probably exert their effects in the extracellular fluid. CL can also be produced by the addition of NaO2 or H2O2 to zymosan in a noncellular system. The chemiluminescent response occurs before particle uptake is complete, which also indicates that CL occurs in the extracellular fluid. These results suggest that CL induced by zymosan in AM is due to the extracellular reaction between various reactive forms of oxygen and zymosan.     

Activation of macrophages by Entamoeba histolytica extracts in mice

The effect of Entamoeba histolytica extracts on the production of inflammatory macrophages and the release of hydrogen peroxide (H2O2) and superoxide (O2-) from these cells was examined in C57BL/6 mice. Two different strains of E. histolytica, either virulent (IP:0682:1) or nonvirulent (DKB), were used in this study. The number of macrophages recovered from the peritoneal cavity of mice treated 5 days previously with 150 micrograms of either strain of amoebic extracts was significantly higher than in the saline-treated groups. Macrophages from mice treated with 150 micrograms of the IP:0682:1 strain of amoebic extracts exhibited a powerful burst of oxidative metabolis when triggered with phorbol myristate acetate (PMA). Extract from the non-virulent strain was much less effective in activating macrophages for the production of oxidative metabolites. Thus, a crude extract preparation from E. histolytica, particularly the virulent strain, induces a strong macrophage inflammatory response in which the cells also produce high levels of H2O2 and O2-.     

The stimulation of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils by phorbol myristate acetate, opsonized zymosan and IgG2-containing soluble immune complexes

The kinetics of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils were determined following in vitro stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ) and soluble immune complexes of guinea-pig IgG2 (SIC). Superoxide production was recorded as chemiluminescence (CL) arising from the reductive cleavage of lucigenin. With PMA, both macrophages and neutrophils displayed a two-phase response consisting of a rapid initial burst of CL, which preceded ligand ingestion, followed by a plateau in the CL response which persisted for more than 30 min. By contrast, OZ induced a slow progressive increase in CL in both phagocytes which was consistent with the development of an oxidative burst concomitant with ingestion. The phagocytes differed in their responses to SIC, the macrophages displaying CL kinetics similar to those observed with PMA, whereas the neutrophils responded in the manner observed with OZ. The relationship between disparity in the patterns of macrophage and neutrophil CL responses to SIC and differences in their expression of Fc receptors for IgG2 (Coupland & Leslie, 1983) is discussed.     

Macrophage antimicrobial functions in a chicken MHC chromosome dosage model.

Macrophages respond to certain inflammatory signals with a marked increase in respiratory burst and the production of reactive oxygen intermediates; these metabolites play an essential role in the destruction of invading microorganisms. In this study, macrophage antibacterial inflammatory responses were compared among chickens having two (disomic), three (trisomic), or four (tetrasomic) copies of the major histocompatibility complex (MHC)-encoding microchromosome (B15 haplotype). Phorbol myristate acetate (PMA)-stimulated superoxide anion (O2-) production by cross-linked dextran (Sephadex)-elicited peritoneal macrophages was measured at early (4 h), intermediate (24 h), and late (42 h) stages of the inflammatory response using ferrocytochrome c reduction. Significantly elevated O2- production was observed for trisomic versus disomic macrophages during both early and intermediate stages of the inflammatory response. Late in the response, tetrasomic macrophages produced a significantly higher level of O2- than disomic cells. When PMA was used to trigger hydrogen peroxide (H2O2) production, no significant genotype difference was found for any stage of the inflammatory response. Phagocytosis of heat-killed Salmonella enteritidis by macrophages differed among the three genotypes: trisomic macrophages were superior to disomic cells during early inflammation, no genotypic difference was observed at the intermediate stage, and disomic cells had greater phagocytic capacity than aneuploid macrophages late in the response. Likewise, when S. enteritidis was cultured with macrophages to induce oxygen intermediate secretion, H2O2 production followed a kinetic pattern among the genotypes similar to that observed for bacterial phagocytosis. Endogenous superoxide dismutase (SOD), catalase, and glutathione peroxidase (GP) activities were determined for the macrophages during intermediate and late inflammatory stages. Tetrasomic macrophages had reduced SOD activity at the late stage, no significant difference was observed in catalase activity among genotypes at either time point, and trisomic macrophages had enhanced GP activity compared to disomic cells at both time points. These results indicate that differences in MHC gene dosage are associated with differences in chicken macrophage activation for the acquisition of selected antibacterial functions.     

Lucigenin-dependent chemiluminescence as a new assay for NAD(P)H-oxidase activity in particulate fractions of human polymorphonuclear leukocytes

The enzyme responsible for the respiratory burst in human neutrophils is an oxidase that catalyzes the reduction of oxygen to superoxide anion (O-2). Superoxide anion production may be measured by chemiluminescence (CL) in the presence of lucigenin (10,10'-dimethyl-9,9'- biacridinium dinitrate). We established an assay of the oxidase, by measuring the CL of particulate fractions of PMN in the presence of lucigenin . This CL required the addition of NAD(P)H and was very low in fractions of resting cells. In particulate fractions of PMNs stimulated with PMA selectively, the NADPH-dependent CL was found to be increased. CL was linear with protein concentrations up to 100 micrograms and was shown to be at least 10 times more sensitive for the detection of O-2 than the assay based on the spectrophotometric determination of superoxide mediated cytochrome c reduction. CL was abolished by inactivating the enzyme at 56 degrees C.     

Activation of macrophages from aging mice by detoxified lipid A.

A detoxified derivative of endotoxic lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), which is capable of inducing nonspecific resistance against several infectious organisms, was tested for its capacity to activate peritoneal macrophages (M phi) from young and immunodeficient aging BALB/c and C3H/HeN mice. Superoxide generation and hydrogen peroxide release by M phi from aging mice were elevated following intraperitoneal injection with 25 micrograms of LPS or MPL, although they did not reach the peak levels achieved in LPS or MPL-treated young mice. Nitroblue tetrazolium reduction (NBT) by peritoneal M phi from aging C3H/HeN mice treated with MPL was higher than that in control aging mice, equalling that from MPL-treated young mice. LPS, its toxic counterpart, however, failed to increase NBT reduction in either group. MPL enhanced lysozyme activity in M phi from both aging and young C3H/HeN mice above initial control levels. On the other hand, LPS suppressed lysozyme activity in M phi from young, but not aging mice. Phagocytosis of Candida albicans by M phi from BALB/c mice was increased in both groups when stimulated by MPL, but not LPS. Similarly, MPL enhanced the ability to kill Candida in both aging and young BALB/c mice. This effect was not seen with LPS. Thus, a detoxified derivative of LPS was found capable of activating the respiratory burst, NBT reduction, elevating lysozyme activity, as well as phagocytosis and killing of Candida in murine peritoneal M phi from both young and aging mice.     

NADH-oxidase,NADPH-oxidase and myeloperoxidase activity of visceral leishmaniasis patients

It is believed that the enhanced capability of activated macrophages to resist infection is related to the remarkable increase in the production of oxygen metabolites in response to phagocytosis. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. Low NADH-oxidase, NADPH-oxidase and myeloperoxidase activity were observed in monocytes of patients with active visceral leishmaniasis as compared with healthy controls. These results suggest that low NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for persistence of Leishmania parasites in visceral leishmaniasis.     

Respiratory burst activity is impaired during phagocytosis of gelatinized fixed erythrocytes by inflammatory macrophages

The ability of immune and nonimmune opsonized gelatin-coated particles to stimulate respiratory burst activity by inflammatory macrophages was studied. The uptake and phagocytosis of⁵¹Cr-labeled gelatin-coated fixed erythrocytes, opsonized with either specific IgG or purified plasma fibronectin, was measured in monolayer cultures of rat inflammatory peritoneal macrophages. Respiratory burst activity was evaluated in monolayers of rat inflammatory peritoneal macrophages by measuring: (1) luminol-dependent chemiluminescence and (2) the production of¹⁴CO₂ from the oxidation of [1-¹⁴C] glucose. Uptake of opsonized gelatin-coated, fixed erythrocytes resulted in no stimulation of chemiluminescence and only a limited stimulation of [1-¹⁴C] glucose oxidation. Respiratory burst activity produced by phorbol myristate acetate was not inhibited during the uptake of opsonized gelatincoated particles. These data suggest that metabolic processes associated with macrophage respiratory burst activity may not be coupled to the ingestion of opsonized gelatin-coated fixed erythrocytes.     

Entamoeba histolytica proteins modulate the respiratory burst potential by murine macrophages.

Reactive oxygen intermediates are important components of macrophage microbicidal mechanisms and pathogenesis of parasitic disease. The purpose of the present study was to investigate the effect of virulent Entamoeba histolytica (strain HM1-IMSS) on respiratory burst potential of macrophages. Pretreatment of elicited peritoneal macrophages (EPM) with crude soluble amoebic proteins from 1 to 6 hr was found to prime EPM for enhanced O2 and H2O2 release in response to phorbol myristate acetate (PMA) in a dose-dependent manner, whereas pretreatment with the same concentrations of the non-pathogenic E. histolytica-like Laredo strain was without priming effect. Low molecular weight (MW) amoebic proteins (27,000-67,000) purified by Sephacryl-200 column chromatography and subfractionated by diethylaminoethyl cellulose chromatography were 10-fold more potent than crude amoebic proteins in priming EPM for an enhanced respiratory burst potential. Both crude and purified amoebic proteins inhibited the priming effect of lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and antagonized the stimulating effect of PMA. Amoebic proteins by themselves were incapable of stimulating EPM respiratory burst. These findings demonstrate that amoebic proteins are capable of modulating the respiratory burst response of macrophages, suggesting an important role for them in the immunoregulation and pathogenesis of amoebiasis.     

Oxygen free radical production by mouse peritoneal macrophages as a function of age

The ability of thioglycollate-elicited peritoneal macrophages (PM) from young and senescent C57BL/6J mice to produce oxygen free radicals was assessed by luminol-dependent chemiluminescence (CL) after introduction of phagocytic stimuli. A significant age-dependent variation in the CL response was detected. A 2-fold increase in the oxygen reactive species was produced by senescent PM in response to latex and zymosan stimulation; but, the capacity to ingest latex and zymosan A particles did not vary significantly between PM from young and senescent mice. Peritoneal macrophages from both age groups responded much more vigorously to opsonized zymosan. The response of the PM from young mice was, however, 2.8-fold higher than that of old ones. There was no age-related difference in oxygen free radical production after stimulation with phorbol myristate acetate (PMA). Also, no age-dependent differences were found in the relative contribution of the various oxygen reactive species O2.-, OH., 1O2 and H2O2) to the overall oxidative burst, with latex zymosan A or PMA.     

Deficient intracellular killing of bacteria by murine alveolar macrophages

Microbiologic methods were used to assess the in vitro phagocytosis and intracellular killing of various species of bacteria by freshly isolated murine peritoneal and alveolar macrophages. Peritoneal macrophages showed effective phagocytosis of opsonized Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Listeria monocytogenes, and moderate ingestion of Staphylococcus aureus and Escherichia coli. Alveolar macrophages were poor in phagocytosing opsonized S. pyogenes, S. aureus, and E. coli; ingestion of S. pneumoniae, P. aeruginosa, and S. epidermidis was moderate. Peritoneal macrophages killed 40 to 80% of these bacteria intracellularly, but alveolar macrophages showed almost no intracellular killing of bacteria. To find out whether there is a correlation between the poor bactericidal activity of alveolar macrophages and the oxygen-dependent microbicidal mechanisms of these cells, we determined the uptake of oxygen and the release of superoxide anion and hydrogen peroxide by macrophages at rest and after stimulation with phorbol myristate acetate (PMA) or opsonized S. aureus. Upon exposure to these stimuli, peritoneal macrophages, but not alveolar macrophages, showed an increased uptake of oxygen and release of superoxide anion and hydrogen peroxide. Because alveolar macrophages contain surface active material (SAM), we investigated the phagocytosis and intracellular killing of bacteria and the release of hydrogen peroxide by peritoneal macrophages pretreated with SAM. The results showed reduced phagocytosis and impaired intracellular killing of S. epidermidis by these macrophages. The release of hydrogen peroxide by SAM-pretreated peritoneal macrophages upon stimulation with PMA or opsonized S. aureus was equal to that of the control peritoneal macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine peritoneal macrophages activated by the mycobacterial 65-kilodalton heat shock protein express enhanced microbicidal activity in vitro.

After an intraperitoneal (i.p.) injection of purified protein derivative, peritoneal macrophages from mice infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) show an enhanced respiratory burst, inhibit the intracellular proliferation of Toxoplasma gondii, and kill Listeria monocytogenes more efficiently than peritoneal macrophages from normal mice. One of the immunodominant antigens of Mycobacterium spp. is the 65-kDa heat shock protein (Hsp 65), and in the present study, we determined whether injection of this protein into mice leads to activation of their peritoneal macrophages. After an i.p. injection of Hsp 65, peritoneal macrophages from BCG-infected CBA/J mice also released more H2O2, inhibited the proliferation of T. gondii, and killed L. monocytogenes faster than peritoneal macrophages from normal mice, although Hsp 65 was less effective than purified protein derivative. When normal mice were injected with Hsp 65 suspended in saline after a booster injection with Hsp 65, their macrophages did not display enhanced antimicrobial activity, indicating that an adjuvant was required for a cellular immune response against Hsp 65. In the present study, the adjuvant dimethyl dioctadecylammonium bromide (DDA) was preferred because it contains no endotoxin or mycobacterial antigens and because it has been reported that DDA does not induce the production of gamma interferon. Peritoneal macrophages from C57BL/6 and CBA/J mice that had received a subcutaneous injection of Hsp 65 suspended in DDA followed by an i.p. booster injection of Hsp 65 suspended in saline were activated, as indicated by the enhanced production of H2O2, inhibition of the intracellular proliferation of T. gondii, and increased rate of intracellular killing of L. monocytogenes in vitro relative to that by resident peritoneal macrophages and peritoneal macrophages obtained from mice that had received ovalbumin instead of Hsp 65. The rate of phagocytosis of L. monocytogenes was not affected by Hsp 65 treatment. Despite the in vitro expression of enhanced microbicidal activity of peritoneal macrophages, no difference in the growth of L. monocytogenes in the liver and spleen between Hsp 65-treated and control mice was found.     

In vitro NADH-oxidase,NADPH-oxidase and myeloperoxidase activity of macrophages after Tinospora cordifolia (guduchi) treatment

It is believed that the enhanced microbicidal and tumoricidal capability of activated macrophages is related to the remarkable increase in the production of oxygen metabolites. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. It was observed that the macrophage cell line J774A.1 when treated with Tinospora cordifolia (guduchi) and LPS showed enhanced NADH-oxidase, NADPH-oxidase and myeloperoxidase production as compared to macrophages treated with medium alone. The direct drug treatment to J774A cells showed activation as assessed by biochemical assays. These results suggest that high NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for tumoricidal and microbicidal properties via macrophage activation.     

Pretreatment with phorbol myristate acetate inhibits macrophage activity against intracellular protozoa

To further document the role of toxic oxygen intermediates in mononuclear phagocyte antiprotozoal activity, microbicidal macrophages were depleted of the capacity to generate superoxide anion (O-2) and hydrogen peroxide (H2O2) by pretreatment with phorbol myristate acetate (PMA), a soluble agent which triggers the macrophage respiratory burst. Treating cells for 90 min with 200 ng/ml of PMA inhibited the extracellular release of both O-2 and H2O2 by 90% upon subsequent restimulation with either PMA or opsonized zymosan. This effect persisted for 48 h, and could not be reversed by the addition of lymphokine. Intracellular nitro-blue tetrazolium reduction by PMA-treated cells was also inhibited by 66-95% upon rechallenge with either PMA or inert or viable particulate agents. In parallel, PMA pretreatment abolished or markedly impaired the ability of normal, lymphokine-stimulated, and in vivo activated macrophages to kill three diverse protozoa--Toxoplasma gondii, Leishmania donovani, and Trypanosoma cruzi. These studies illustrate an additional technique for investigating the antiprotozoal effects of macrophage-derived O-2 and H2O2 and reemphasize the importance of an intact respiratory burst mechanism in killing of intracellular parasites.     

In vitro NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of macrophages after Tinospora cordifolia (guduchi) treatment

It is believed that the enhanced microbicidal and tumoricidal capability of activated macrophages is related to the remarkable increase in the production of oxygen metabolites. Both the production of H2O2 and the oxidation of NAD(P)H are directly dependent upon NAD(P)H-oxidase. It has been established that the respiratory burst is due to activation of NAD(P)H-oxidase localised in the plasmalemma. Myeloperoxidase is believed to be involved in augmenting the cytotoxic activity of H2O2. It was observed that the macrophage cell line J774A.1 when treated with Tinospora cordifolia (guduchi) and LPS showed enhanced NADH-oxidase, NADPH-oxidase and myeloperoxidase production as compared to macrophages treated with medium alone. The direct drug treatment to J774A cells showed activation as assessed by biochemical assays. These results suggest that high NADH-oxidase, NADPH-oxidase and myeloperoxidase activities may account for tumoricidal and microbicidal properties via macrophage activation.     

Inhibition of various functions in murine peritoneal macrophages by aflatoxin B1 exposure in vivo.

Aflatoxin B1 (AFB1) has been known to impair specific and nonspecific immunity. In the present study, we tested various functions of murine peritoneal macrophages that were isolated and stimulated with LPS after AFB1 (400 microg/5 ml/kg) was administered every other day for 2 weeks. AFB1 decreased phagocytosis and the production of superoxide anion (O2-) and hydrogen peroxide (H2O2), compared to those of corn oil-treated control group. In addition, the production of NO and TNF-alpha was decreased in macrophages of AFB1-treated mice. In vitro antitumor activity of in vivo AFB1-treated macrophages was reduced against target cell, L929. Taken together, these results suggested that AFBI might have the immunosuppressive effect on macrophages after in vivo exposure, which was related to the antitumor activity reduction.     

The mechanism of action of lymphokines. VIII. Lymphokine-enhanced spontaneous hydrogen peroxide production by macrophages.

Oil-elicited guinea-pig peritoneal macrophages (MPs) cultured for 2-3 days in medium containing supernatant of concanavalin A-activated lymphocytes (lymphokine, LK) generated large amounts of hydrogen peroxide (H2O2), as detected by the horseradish peroxidase (HRP)-dependent oxidation of phenol red, in the absence of further stimulation. H2O2 production increased with the duration of exposure to LK and was evident at high dilutions of supernatant (1/64). Parallel cultures of MPs in medium or a supernatant of non-activated lymphocytes also increased their H2O2 production during culture but levels at all time intervals were significantly lower than those measured in LK treated cultures. The marked increase in H2O2 production was associated with only a moderate increment in superoxide (O-2) liberation and this was not specific for LK treated cells. Detection of LK-dependent H2O2 production was dependent on ongoing pinocytosis during the assay. This and other arguments suggest that the HRP-phenol red assay, as applied here, detects H2O2 generation occurring at the level of intracellular vesicles and it is concluded that LK elicits H2O2 production that is limited to the intracellular compartment. H2O2 is, apparently, derived by non-enzymatic dismutation of O-2 taking place within the cell; LK treatment of MPs also resulted in a significant reduction in catalase activity.     

Chemiluminescence and immune cell activation. I. Early activation of rat thymocytes can be monitored by chemiluminescence measurements.

Immediately after the addition of concanavalin A, rat thymocytes respond, in the presence of luminol, with a burst of chemiluminescence (CL) that can be conveniently monitored in an ordinary liquid scintillation spectrometer. Peak CL is reached after 50 sec. Addition of catalase suppresses 65% of the CL, suggesting that H2O2 generation may be its major source. CL with different kinetic characteristics can also be generated by the calcium ionophore A23187. Our rat thymocytes contain approximately 0.1% endogenous macrophages. Bone marrow-derived rat macrophages also respond to concanavalin A or A23187 stimulation with a burst of CL. However, the kinetic properties of CL as well as the inhibition of CL by catalase in these cells differ markedly from those of thymocyte preparations, suggesting that the major portion of the CL in rat thymocytes actually originates in T lymphocytes. Because CL measurements allow the monitoring of very early events in cell activation and because of the simplicity of the technique, CL measurements may become a useful method for the study of lymphocyte activation, of macrophage-lymphocyte interactions, as well as for the rapid screening for specificity and reactivity of T lymphocyte populations.