Generation of a lymphocyte growth factor by treatment of human cells with neuraminidase and galactose oxidase.

Supernates of neuraminidase and galactose oxidase (NAGO)-treated lymphocytes induce blastogenesis in nonproliferating cells harvested 7--14 d after treatment with mitogen or alloantigen and in cells incubated with mitogen for 7--14 d but not in freshly isolated peripheral blood lymphocytes9 Virtually all the growth factor is produced by NAGO-treated cells during the first 24 h of incubation, and no increase in factor activity is detected upon further cell culture. Serum is not required for growth factor production. NAGO-primed medium induces generation of specific cytotoxic T cells from mixed lymphocyte culture (MLC) memory cells to approximately the same extent as that induced by allogeneic cells (stimulating cells in the primary MLC). NAGO-primed medium provides a useful reagent for isolation and characterization of lymphocyte growth factors and other lymphokines.

Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors.

Septic shock results from excessive stimulation of the host immune system, especially macrophages, by lipopolysaccharide (LPS), or endotoxin, which resides on the outer membrane of bacteria. Protein tyrosine kinase inhibitors of the tyrphostin AG 126 family protect mice against LPS-induced lethal toxicity. The protection correlates with the ability of these agents to block LPS-induced production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in macrophages as well as LPS-induced production of TNF-alpha in vivo. Furthermore, this inhibitory effect correlated with the potency of AG 126 to block LPS-induced tyrosine phosphorylation of a p42MAPK protein substrate in the murine macrophage.

Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with end-organ resistance to 1,25-dihydroxyvitamin D.

Lectin-induced DNA synthesis by peripheral mononuclear cells from 17 normal donors was inhibited (40-60%) by 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) at physiological concentrations (10(-10)-10(-9) M). The lymphocytes acquire specific receptors for 1,25(OH)2D3 upon activation by the lectins. This process precedes the inhibitory effect of 1,25(OH)2D3. We studied lymphocytes from six patients from four different kindreds with the syndrome of hereditary end-organ resistance to 1,25(OH)2D (the so-called vitamin D-dependent rickets type II). In five patients (three kindreds) peripheral blood mononuclear cells did not acquire receptors for 1,25(OH)2D3 upon phytohemagglutinin-induced activation. Moreover, in contrast to normal lymphocytes, the mitogenic stimulation of these patients' lymphocytes by phytohemagglutinin and concanavalin A was not inhibited by 1,25(OH)2D3. Activated lymphocytes of the sixth patient from a fourth kindred exhibited normal binding of [3H]1,25(OH)2D3 but the hormone failed to inhibit the mitogenic stimulation. A similar pattern of the vitamin D effector system was previously observed in fibroblasts cultured from skin biopsies of the same group of patients. The conclusions from these findings are: (a) the inhibition of mitogenic stimulation by 1,25(OH)2D3 is mediated by specific functional receptors to the hormone; and (b) the receptors for 1,25(OH)2D3 in mononuclear cells are probably controlled genetically by the same mechanisms as the effector system in well-characterized target organs of the hormone, such as intestine and kidney.

Tyrphostin AG 556 improves survival and reduces multiorgan failure in canine Escherichia coli peritonitis.

Tyrosine kinase-dependent cell signaling is postulated to be a pivotal control point in inflammatory responses initiated by bacterial products and TNF. Using a canine model of gram-negative septic shock, we investigated the effect of tyrosine kinase inhibitors (tyrphostins) on survival. Animals were infected intraperitoneally with Escherichia coli 0111: B4, and then, in a randomized, blinded fashion, were treated immediately with one of two tyrphostins, AG 556 (n = 40) or AG 126 (n = 10), or with control (n = 50), and followed for 28 d or until death. All animals received supplemental oxygen, fluids, and antibiotics. Tyrphostin AG 556 improved survival times when compared to controls (P = 0.05). During the first 48 h after infection, AG 556 also improved mean arterial pressure, left ventricular ejection fraction, cardiac output, oxygen delivery, and alveolar-arterial oxygen gradient compared to controls (all P < or = 0.05). These improvements in organ injury were significantly predictive of survival. Treatment with AG 556 had no effect on clearance of endotoxin or bacteria from the blood (both P = NS); however, AG 556 did significantly lower serum TNF levels (P = 0.03). These data are consistent with the conclusion that AG 556 prevented cytokine-induced multiorgan failure and death during septic shock by inhibiting cell-signaling pathways without impairing host defenses as determined by clearance of bacteria and endotoxin.

Dimethylthiourea inhibition of melanoma cell growth in vitro and in vivo.

The effect of dimethylthiourea (DMTU), an agent known as a hydroxyl radical scavenger, was determined on growth and differentiation of the B16 murine melanoma cell line. DMTU inhibited melanoma cell growth in vitro and induced changes in the morphology of melanoma cells. Prolonged treatment of cells with DMTU resulted in an increase in melanin content. DMTU-treated melanoma cells had a decreased capacity to form tumors in syngeneic mice. Systemic administration of DMT to C57BL/6J mice inoculated with melanoma cells resulted in a delay in tumor appearance and a prolongation of survival. The doses of DMTU used did not cause any apparent toxic effects. A potential therapeutic role for DMTU in the treatment of melanoma is suggested.

A monoclonal antibody against a human B lymphoblastoid cell line induces tumor regression in mice.

We have developed a monoclonal antibody (BAT) to Daudi B lymphoblastoid cell line membranes. The antibody was selected for its ability to stimulate lymphocyte proliferation. Splenocytes of BALB/c or C57BL mice given i.v. injections of 10 micrograms/mouse of BAT exhibited increased proliferation and cytotoxic activity. A single i.v. administration of BAT monoclonal antibody 2 weeks after B16 melanoma cell inoculation resulted in a striking antitumor effect as manifested by the elimination of lung metastases and prolonged survival of the treated mice. BAT monoclonal antibody was also effective in the regression of tumors in mice bearing 3LL (Lewis lung carcinoma) and MCA-105 (fibrosarcoma). Transfer of 10(7)-10(8) splenocytes from mice that had been given injections of BAT to B16- or 3LL-inoculated recipients led to a reduction of lung metastases. Splenocytes from B16-inoculated mice that were cured by BAT were more effective than those from mice treated with BAT alone against recipients bearing either B16 or 3LL tumors. The antitumor activity of BAT is related to its immunostimulatory properties.

Inhibition of phorbol ester-mediated interleukin-2 production by cellular differentiating agents.

Lymphocyte proliferation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is inhibited by agents known to induce differentiation in murine erythroleukemia cells and other cell lines. In the present study, we determined the cellular targets for the action of TPA among murine thymocyte subpopulations, the phase of blastogenesis that is activated by the tumor promoter, and the phase that is inhibited by the differentiating agents. Mouse thymocytes were fractionated into populations bearing receptors for peanut agglutinin (PNA; PNA-positive cells) and populations lacking such receptors (PNA-negative cells). TPA is comitogenic for lectin-treated, unfractionated thymocytes and PNA-negative thymocytes but not for PNA-positive thymocytes. PNA-negative cells, a minor population in unfractionated thymocytes, are therefore the cellular targets for the comitogenic activity of TPA. TPA induces the production of interleukin-2 (IL-2) in lectin-treated PNA-negative populations but not in PNA-positive cells. The differentiating agents inhibit TPA-mediated proliferation of unfractionated and PNA-negative, lectin-treated thymocytes. In contrast, IL-2-mediated proliferation of lectin-treated thymocyte subpopulations is resistant to inhibition by these agents. Inhibition appears to be related to decreased production of IL-2, since the differentiating agents inhibit IL-2 production by both PNA-negative thymocytes and by a human leukemic cell line.

Tyrphostin 4-nitrobenzylidene malononitrile reduces chemotherapy toxicity without impairing efficacy.

In mice, 4-nitrobenzylidene malononitrile (AG1714), which belongs to the tyrphostin family, reduced toxicity induced by doxorubicin and cisplatin without impairing their antitumor efficacy. AG1714 reduced mortality induced by doxorubicin and cisplatin. It prevented, in a dose-dependent manner, cisplatin-induced nephrotoxicity as assessed by measurement of serum creatinine and blood urea nitrogen levels. The protective effect of AG1714 was most pronounced on its administration 2 h before cisplatin. AG1714 also prevented doxorubicin-induced myelosuppression as assessed by the scoring of bone marrow nucleated cells and colony-forming units. Cisplatin-induced small intestinal injury was also protected by AG1714 as assessed by histopathological analysis. In vitro, AG1714 reduced cisplatin-induced apoptosis in a murine fibroblastic cell line (A9) and did not affect doxorubicin-induced apoptosis of B-16 melanoma cells. In contrast to its protective effect against mortality and injury of normal tissues induced by chemotherapy, AG1714 did not impair its antitumor activity and in some tumor models enhanced it. This was evident by using the murine tumors B-16 melanoma, Lewis lung carcinoma, and methylcholanthrene-induced fibrosarcoma and the human tumors SK-28 melanoma and human ovary carcinoma xenografts in nude mice. Experiments in which low and high doses of cisplatin and doxorubicin were administered to tumor-bearing mice demonstrated that AG1714 reduced mortality of high-dose chemotherapy and increased its therapeutic index. AG1714 could provide a novel, useful tool to improve chemotherapy by allowing dose intensification.

Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore.

UNLABELLED: Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH)2D3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. IN CONCLUSION: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH)2D3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.

A phase II clinical trial of adoptive immunotherapy for advanced renal cell carcinoma using mitogen-activated autologous leukocytes and continuous infusion interleukin-2.

Forty patients with metastatic, recurrent, or unresectable renal cell carcinoma were entered into a study of the therapeutic efficacy of adoptive immunotherapy using periodate (IO4-) and interleukin-2 (IL2)-activated autologous leukocytes and continuous infusion low-dose IL2. Patient survival was also examined. The first 15 consecutive patients were enrolled in protocol A without an IL2 priming phase and the following 25 patients were entered in protocol B where a 5-day priming phase was initiated before leukapheresis. A maintenance regimen consisted of either 3 x 10(6) units of recombinant interferon-alpha (rIFN-alpha), three times per week only or together with leukapheresis and infusion of IO4-/IL2-activated cells and 2 days of continuous infusion IL2 per month. Thirty-four patients completed the protocol treatment. Four patients were removed from the study owing to rapid tumor progression and two patients died while receiving treatment. The clinical response rate was 22%: two patients had a complete response and five patients had a partial response. Among the 25 patients who had no clinical response, 11 patients had either a mixed response or stabilization. Neither response, response duration, nor site response correlated with the total dose of IL2 administered or the number of activated killer cells infused. Patients who received maintenance therapy had longer survival times than patients who did not receive such therapy. All toxicity and side effects associated with IL2 treatment were transient and resolved after discontinuation of the drug. Patients on maintenance therapy tolerated both rIFN-alpha and monthly infusions of activated killer cells and IL2 well. This study confirms the concept of adoptive immunotherapy as a new treatment approach for advanced renal cell carcinoma and suggests that maintenance therapy may prolong survival time.

Mononuclear cells from human neonates are partially resistant to the action of 1,25-dihydroxyvitamin D.

1,25-Dihydroxyvitamin D [1,25-(OH)2D] inhibits mitogen-induced proliferation of lymphocytes by a receptor-mediated mechanism. Peripheral blood lymphocytes may serve as a model for detecting hereditary defects in the response of classical target organs to 1,25-(OH)2D. Delayed bone mineralization and deficient intestinal calcium absorption are common in low birth weight formula-fed infants. The defect in calcium absorption exists despite normal or even elevated serum 1,25-(OH)2D levels, suggesting partial end-organ resistance to the hormone. We assessed the response to 1,25-(OH)2D of activated mononuclear cells obtained from cord blood of fullterm and preterm infants and from peripheral blood of adults. We found that the inhibitory effect of 1,25-(OH)2D on mitogen-induced [3H]thymidine incorporation was significantly less [mean, 34 +/- 8% (+/- SE)] in mononuclear cells from neonates (independent of gestational age) compared to mononuclear cells from adults (66 +/- 5%; P less than 0.001). This difference in the inhibitory effect was not due to a smaller number of high affinity receptors for 1,25-(OH)2D in activated cord blood lymphocytes. We conclude that the coupling between the receptors for 1,25-(OH)2D and the biological response in neonates is less efficient than that in adults.

1,25-dihydroxyvitamin D3 and agents that increase intracellular adenosine 3',5'-monophosphate synergistically inhibit the mitogenic stimulation of human lymphocytes.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], like the immune response modulators prostaglandin E2 (PGE2) and histamine, inhibits mitogen-induced proliferation of human peripheral blood mononuclear cells. 1,25-(OH)2D3 acts synergistically with PGE2 and histamine to inhibit lymphocyte mitogenesis. This is apparent at a wide concentration range of 1,25-(OH)2D3 (3 X 10(-11)-10(-8) mol/L). Cholera toxin, forskolin, and isobutylmethylxanthine, which like PGE2 and histamine increase intracellular concentrations of cAMP, also act synergistically with 1,25-(OH)2D3 in this system. Culture of mitogen-stimulated adherent cell-depleted mononuclear cells with PGE2 increases the number of high affinity binding sites for 1,25-(OH)2D3. This finding may account for the synergistic interaction between the two agents.

Hemin stimulation of cAMP production in human lymphocytes.

Hemin stimulates cAMP production in human peripheral blood mononuclear cells (PBMC). The kinetics are similar to that of hormone-induced cAMP generation, namely a rapid effect followed by a desensitization phase. Several experimental findings suggest that prostaglandins do not mediate this effect. First, macrophage depleted T and B cells purified by erythrocyte-rosetting were as responsive as unfractionated PBMC to hemin. Second, indomethacin, an inhibitor of prostaglandin synthesis, and meclofenamate, a prostaglandin E2 receptor antagonist, had no effect on hemin stimulated cAMP production. In addition, propranolol, a beta-adrenergic receptor antagonist, had no effect on hemin-stimulated cAMP production. We also examined structural analogues of hemin. Among the metalloporphyrins (Fe, Ni, Co, Zn and Sn) and protoporphyrin IX tested only hemin (Fe-protoporphyrin) was active in stimulating cAMP production. No correlation was found between the ability of metalloporphyrins to stimulate cAMP production and their ability to generate H2O2. The data indicate that hemin stimulates cAMP production by directly affecting lymphocytes and that prostaglandins do not mediate this effect.

1,25-Dihydroxyvitamin D3 enhances prostaglandin E2 production by monocytes. A mechanism which partially accounts for the antiproliferative effect of 1,25(OH)2D3 on lymphocytes.

Partial removal of monocytes from human peripheral blood mononuclear cells, or the addition of indomethacin, reduced the antiproliferative effect of 1,25(OH)2D3 on mitogen-stimulated mononuclear cells. Addition of 1,25(OH)2D3 (1 nM) to mitogen-stimulated mononuclear cells caused a 2-4-fold increase in prostaglandin E2 production during the second day of culture. The inhibitory effect of 1,25(OH)2D3 on lymphocyte proliferation is greatly augmented up to 7-fold in the presence of prostaglandin E2. We conclude that monocytes are involved in the inhibitory effect of 1,25(OH)2D3 on the mitogenic stimulation of human lymphocytes and that their action is probably mediated by prostaglandins.

Anti-proliferative effects and phenotypic alterations induced by 8-hydroxyquinoline in melanoma cell lines.

The effect of the transition metal chelator, 8-hydroxyquinoline (8-HQ), was examined on the growth and phenotype expression of B16 mouse melanoma cells. Micromolar concentrations of 8-HQ inhibited the growth of B16 cells as well as human melanoma cell lines. Removal of 8-HQ from the culture medium restored normal cell growth. Growth inhibition by 8-HQ was accompanied by phenotypic alterations that included changes in cell morphology, increased production of melanin and enhanced activities of the enzymes gamma-glutamyl transpeptidase and NADPH cytochrome c reductase. These changes might be associated with a better differentiated phenotype.

A cell line with unusual characteristics from an ovarian carcinoma patient: modulation of sensitivity to antitumour drugs.

A cell line (GZL-8) was established by cloning from ascitic fluid of an untreated ovarian carcinoma patient. The cells grew rapidly, accumulated lipids and showed chromosomal alterations. One of the marker chromosomes showed characteristics of a Y-like chromosome. This unusual finding was confirmed by DNA hybridisation using specific probes to the Y chromosome. The cells stained with fluorescent antibodies to desmoplakin and cytokeratins 8, 18, 19, and weakly with vimentin but not with desmin. The presence of epithelial membrane antigen, human milk fat globulin, alpha-lactalbumin, alpha-fetoprotein, placental alkaline phosphatase and oestrogen receptor-related antigen was demonstrated by indirect immunoperoxidase staining, but no CA-125 antigen could be detected. The cells showed positive reaction with antibodies to P-glycoprotein. The function of the P-glycoprotein transport system was demonstrated by the rhodamine-123 release test. The cells were initially responsive to doxorubicin, and to high concentrations of cisplatin. Growth inhibition by doxorubicin, especially at low doses was enhanced by the addition of verapamil or tamoxifen. This was shown by the soft agar clonogenic assay, by direct cell counting and by the MTT reducing test. Our results show that combination between drug and sensitivity modulators may be of potential clinical value in ovarian cancer.

Decreased macrophage-mediated suppression of lymphocyte activation in chronic renal failure.

Prostaglandin-dependent adherent cell suppressor activity was assessed in patients with end-stage renal insufficiency. Proliferative responses of uremic peripheral blood mononuclear cells to optimal concentrations of phytohemagglutinin and concanavalin A were impaired. Responses to the galactosyl-directed lectins, soybean agglutinin and peanut agglutinin, were, however, normal or supranormal. The addition of 1 microgram/ml of indomethacin, to cell cultures resulted in relatively less potentiation of blastogenic responses to the galactosyl-directed lectins in cells from uremic patients (soybean agglutinin, p less than 0.02; peanut agglutinin, p less than 0.05). Similarly, depletion of adherent cells markedly enhanced blastogenesis induced by the galactosyl-directed lectins in normal cell cultures, whereas the effect was much less pronounced (soybean agglutinin, p less than 0.02; peanut agglutinin, p less than 0.02) in uremic cells. Reduced activity of the adherent cell suppressor system in patients with renal failure might be associated with altered sensitivity of uremic lymphocytes to soluble mediators of suppression. The lymphocytes of uremic patients, depleted of adherent cells, were relatively resistant to the inhibitory action of prostaglandin E1 (0.001 microgram/ml, p less than 0.05, and 0.01 microgram/ml, p less than 0.02) on galactosyl-directed, lectin-induced mitogenesis. In contrast, dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-5) M), and 3-isobutyl-1-methyl xanthine (20 micrograms/ml) inhibited both control subject and patient cultures to the same extent. Prostaglandin E1 in combination with methyl isobutyl xanthine produced, in adherent-cell-depleted control subjects, levels of cyclic AMP that were significantly higher than in cells from uremic patients (p less than 0.05). Thus, depressed adherent cell suppressor activity in patients with renal failure may result in part from impaired generation of cyclic AMP by lymphocytes.

Adoptive immunotherapy for stage IV renal cell carcinoma: a novel protocol utilizing periodate and interleukin-2-activated autologous leukocytes and continuous infusions of low-dose interleukin-2.

In a pilot study involving 13 patients with advanced stage IV renal cell carcinoma, anti-tumor effects and toxicity of a novel form of adoptive immunotherapy were determined. The protocol utilizes infusions of autologous mononuclear leukocytes treated with the oxidizing mitogen sodium periodate (IO4-) and cultured in medium containing human recombinant interleukin-2 (IL-2), and continuous infusions of low-dose IL-2 (mean +/- SD dose = 39.5 +/- 8.6 X 10(3) U/kg/24 hours). Leukocytes (5 to 10 X 10(9] were removed by leukapheresis three times per week, mononuclear cells were separated, activated with IO4- and cultured in medium containing IL-2 (500 U/ml) for 48 to 72 hours. The cells were re-infused following the next leukapheresis procedure. IL-2 was administered five days per week. Treatment was continued for two three-week cycles. An increase in peripheral blood mononuclear cells bearing the natural killer cell (NK) surface marker, Leu 11, an increase in NK- and antibody-dependent cell-mediated cytotoxicity, and a slight increase in spontaneous cytotoxicity for non-NK targets were noted. Regressions (more than 50 percent decrease in tumor mass) of pulmonary, liver, bone, or soft tissue metastases were induced in six patients. Severe fluid retention did not develop in any patient and no patient required treatment in the intensive care unit. Five of the patients who showed a response have experienced a relapse at 5.2 +/- 1.0 (mean +/- SD) months. These observations indicate that IO4-/IL-2-activated killer cells plus continuous infusions of low-dose IL-2 can result in regression of metastatic renal cell carcinoma.

Antibodies to neuroblastoma cell line proteins in patients with schizophrenia.

The presence of antibodies against neural antigens was investigated in the serum of patients with schizophrenia, major depression and normal controls. Different immunological abnormalities, humoral and cellular, were reported in schizophrenia and major depression. The pathogenesis of schizophrenia is multifactorial. An autoimmune mechanism was suggested as a possible factor. We tested the serum of 26 patients with schizophrenia, eight patients with major depression and 22 normal controls. The serum samples were tested for antibody binding to protein extracts of IMR-32 neuroblastma cell line using Western blot analysis. Immunoglobulins of eight patients with schizophrenia (30.71%) reacted with a protein of 80-85 kDa. Serum samples from subjects of other groups did not react with this protein. Sera of all patients with major depression but one, and all normal controls reacted with HSP 60 kDa to different extent. This is an apparent discrepancy with the findings of Kilidireas et al. [Kilidireas, K., Latov, N., Strauss, D.H., Gorig, A.D., Hashim, G.A., Gorman, J.M., Sadig, S.A., 1992. Antibodies to the human 60 kDa heat shock protein in patients with schizophrenia. Lancet 340, 569-572.] who demonstrated the presence of antibodies against HSP 60 kDa in 44% of patients with schizophrenia tested and 8% of normal subjects. HSP 60 kDa is an antigen of many pathogens and antibodies against it might be a result of an infection and cannot be a good indicator for an autoimmune process. The presence of antibodies against a protein of 80-85 kDa should be investigated as a possible specific indicator.

Detection of alloimmune memory cells by chemical modification of the cell surface with mitogenic oxidizing agents.

The mitogenic oxidizing agents, neuraminidase and galactose oxidase (NAGO), and sodium periodate (IO-4) were used to induce the differentiation of human alloimmune memory cells. NAGO or IO-4 treatment of peripheral blood mononuclear (PBM) cells obtained from 10 sensitized potential allograft recipients resulted in the induction and augmentation of cytolytic activity to a D locus-defined lymphoblastoid cell panel (B cell panel) and to a HLA-disparate peripheral blood lymphocyte cell panel (PBL cell panel). The acquisition of cytolytic activity was determined in a 4-hr 51Cr release assay. Treatment of in vitro-primed PBM cells (alloimmune memory cells generated in primary long-term mixed lymphocyte cultures) obtained from normal subjects with NAGO or IO-4 also resulted in the induction of specific secondary cytolytic activity. In contrast, NAGO or IO-4 treatment of unprimed PBM cells from normal subjects did not result in the induction of cytolytic activity despite the extensive proliferation induced by such treatment. The strikingly similar results observed with PBM cells from sensitized patients and with in vitro-primed PBM cells suggest that in vivo- and in vitro-generated alloimmune memory cells can be detected by chemical modification of the cell surface induced by NAGO or IO-4. Furthermore, our findings indicate that alloantigen-independent activation of memory cells can be accomplished by treating the memory cells with the mitogenic oxidizing agents.