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Asthma and allergy are characterized by dysregulation of inflammatory responses toward Th2 responses and high serum levels of IgE. IgE plays a role in the effector phase by triggering the degranulation of mast cells after antigen-crosslinking but its role in the induction of helper T cell differentiation is unknown. We have previously shown lymphotoxin is required for maintaining physiological levels of serum IgE which minimize spontaneous Th1-mediated airway inflammation, suggesting a physiological role for IgE in the regulation of T helper cell differentiation. We describe the mechanism in which IgE modulates inflammation by regulating dendritic cell cytokine production. Physiological levels of IgE suppress IL-12 production in the spleen and lung, suggesting IgE limits Th1 responses in vivo. IgE directly stimulates dendritic cells through FcgammaRIII to suppress IL-12 in vitro and influences APC to skew CD4+ T cells toward Th2 differentiation. We demonstrate a novel role for IgE in regulating differentiation of adaptive inflammatory responses through direct interaction with FcgammaRIII on dendritic cells.

Previously we reported that the administration of human (h) lymphotoxin (h-LT) markedly protected NOD mice from insulin-dependent diabetes mellitus (IDDM) partly by affecting the generation phase of anti-islet effector cells, probably in the thymus. In this study, we investigated the effect of h-LT on the signal transduction of the mouse thymocytes by observing c-Fos expression in the thymocytes by using a flow cytometer. The intensity of c-Fos expression in whole thymocytes was significantly lower in the female NOD with a high incidence of diabetes than that in the male NOD mice with a low incidence of diabetes and than that in normal mice (P < 0.0001). The low c-Fos expression in the female NOD thymocytes was most prominent in CD3low thymocytes. c-Jun expression of the CD3low thymocytes was also lower in the female NOD mice. Administrations of h-LT, h-TNF, and h-IL-2, which has been reported to prevent IDDM in NOD mice by systemic administration, significantly up-regulated c-Fos expression in CD3low thymocytes. From these results, it is assumed that a relationship may exist between the high diabetes incidence and the defective c-Fos expression in female NOD mice and between the prevention of IDDM and the amelioration of the defective c-Fos expression with h-LT in female NOD mice.

Acute thymic involution is known to be induced under conditions of physical stress, bacterial infections, and malignancies. It is speculated that glucocorticoids, tumor necrosis factor (TNF), and other factors may act as mediators for the thymic involution under such conditions. It was herein investigated whether either lymphotoxin (TNF beta) or estrogen could induce thymic involution without the help of glucocorticoids. Interestingly, both lymphotoxin or estrogen alone induced profound thymic involution even in adrenalectomized mice. In contrast to glucocorticoids, which induce lymphocytopenia throughout the organs, lymphotoxin and estrogen did not induce lymphocytopenia in the peripheral organs. More importantly, lymphotoxin and estrogen rather stimulated extrathymic T cells in the liver and other organs. These results suggest that lymphotoxin and estrogen per se might be important regulators of immune systems.

Recent studies suggest that tumor necrosis factor (TNF) family members such as TNF alpha and lymphotoxin alpha beta (LT alpha 1 beta 2) are important in the development of follicular dendritic cells (FDCs) and maintenance of FDC function. In this study we used FDC-like cells (FDC-LC) cultured from normal human tonsil and investigated the effects of TNF and LT alpha 1 beta 2 on expression of adhesion molecules and the production of cytokines and chemokines. TNF and LT alpha 1 beta 2 both increased the expression of VCAM-1 and ICAM-1 on FDC-LC, In addition, IL-4 with LT alpha 1 beta 2 synergistically increased the expression of VCAM-1, but not ICAM-1, Cytokine IL-6 and IL-15 mRNAs were induced following stimulation with TNF and LT alpha 1 beta 2. These two cytokines were present in FDC-LC supernatants by ELISA and increased following TNF and LT alpha 1 beta 2 stimulation. We also examined FDC-LC for chemokines, which affect B cells, including IL-8, SDF-1, MIP3 beta/ELC, and BCA-1/BLC, SDF-1 mRNA and protein were expressed by FDC-LC, and following stimulation with TNF and LT alpha 1 beta 2, decreases in both were observed. Therefore, TNF and LT alpha 1 beta 2 which are produced by activated B cells, increased the expression of adhesion molecules and cytokines from FDC-LC, potentially providing key signals to support germinal center B cell survival and differentiation, (C) 2000 Academic Press.

Human lymphokine-activated killer (LAK) cells cultured with interleukin-2 express membrane-associated lymphotoxin (mLT) on the cell surface. mLT-lacking (mLT-) LAK cells, which are generated by culturing without IL-2 for 24 hr, fully retained natural killer (NK) and LAK activity. However, they partially lost their killing activity against various tumor cells when compared to mLT-expressing (mLT+) LAK cells. An anti-LT but not anti-tumor necrosis factor (TNF) antibody somewhat suppressed the killing activity of mLT+ LAK cells against these tumor cells. However, an anti-LT antibody did not alter NK or LAK activity of mLT+ LAK cells. Anti-CD18 (β chain of LFA-1) and anti-CD54 (ICAM-1) monoclonal antibodies, by inhibiting LAK cell binding to targets, suppressed the killing activity of mLT+ LAK cells. During the process in the cytotoxic assay, mLT+ but not mLT- LAK cells upregulated ICAM-1 expression on target tumor cells, the effect of which was marginally inhibited by anti-TNF and anti-IFN-γ but not by anti-LT antibodies. The upregulation of ICAM-1 expression elicited by LAK cells on target tumor cells was not simply caused by diffused soluble factors but required membrane contact of LAK and target cells. Paraformaldehyde-fixed mLT+ but not mLT- LAK cells up-regulated ICAM-1 expression on target cells. These findings indicate that mLT+ but not mLT- LAK cells up-regulate ICAM-1 expression on target tumor cells via undetermined membrane factor(s), and this effect appears to be at least partially responsible for the higher killing activity of mLT+ LAK cells than that of those lacking mLT in vitro.

Rats of the W/F strain were immunized with DNP-Ascaris conjugates using complete Freund's adjuvant (CFA), Al(OH)3 gel (alum), or B. pertussis vaccine as adjuvants. Cell-mediated immunity was assessed by lymphotoxin in vitro and by delayed hypersensitivity in vivo. IgE and IgG antibody determinations were made on serum pools obtained at various times during the primary and secondary responses. Although delayed hypersensitivity appeared earlier than lymphotoxin, these two parameters correlated during the primary but not during the secondary response. The discrepancies suggested that different cells may be responsible for these two phenomena. Antibody production was influenced by the adjuvant used. CFA led to IgG antibody responses to both hapten and carrier but not to IgE antibody production. The use of B. pertussis resulted in both IgE and IgG antibody production. In the case of alum, anti-hapten antibodies appeared during the primary response while anti-carrier antibodies of both IgE and IgG classes were detected after booster. The results indicated that cell-mediated immunity, IgE, and IgG antibodies appeared independently in an ordered, temporal sequence, and that these responses were not mutually exclusive but were under strong modulatory influences of the various adjuvants used.

When the plasma-membrane protein content of L cells was assayed after 16 hr of treatment with human α-lymphotoxin (LT) or control buffer, the LT-treated cells showed a considerably higher protein content than the control cells. Cells treated for 16 hr with LT also showed an increase in plasma-membrane protein synthesis, measured by pulse labeling the experimental and control populations with equimolar concentrations of [ 14 C]- or [ 3 H]leucine, but showed no increase in total cellular protein synthesis. Target-cell plasma-membrane protein content was further assessed by measuring labeling of cell surface protein with 125 I. Cells treated with LT for 24 hr (but not those treated for 4 or 8 hr) and microsomal fractions of cells treated with LT for 16 hr showed much higher levels of iodination than control cells. SDS-PAGE and radioautography of the iodinated preparations revealed no apparent differences in band pattern between LT-treated and control cells.

Lymphocytes from murine spleens released granulocyte colony stimulating activity (CSA), macrophage migration inhibition factor and lymphotoxin from 24–96 hr after stimulation with phytohemagglutinin (PHA). Cytochalasin B at 5 μg/ml completely inhibited the release of migration inhibition factor and lymphotoxin but significantly increased the release of CSA.

One-way mixed lymphocyte cultures employing human adenoid or peripheral blood lymphocytes activate lymphotoxin (LT)-secreting cells. Kinetic analysis of lymphocytes stimulated in mixed culture demonstrates that LT is secreted before the onset of DNA synthesis, but that maximum levels of LT secretion are reached simultaneously with maximum levels of DNA synthesis. Although the response of peripheral leukocytes is qualitatively similar to the response of adenoid-derived lymphocytes, unexplained high nonspecific background levels of toxic material(s) obscure early events in the former response. While cytochalasin B reversibly inhibits LT secretion, mitomycin C treated cultures are still capable of LT secretion. The results suggest that a population of cells exists, which does not require DNA synthesis to develop into effector cells. The requirement for DNA synthesis for the maximal development of effector cells may reside in a separate helper cell population as postulated by the two cell model of the mixed lymphocyte reaction. © 1975 Academic Press, Inc. All rights reserved.

The effect of antilymphocyte globulin (ALG) on lymphocyte function in the presence or absence of complement was investigated by comparing (i) ALG-induced blast-cell transformation, (ii) lymphotoxin production and (iii) its effect on PHA-induced blast-cell transformation, and on (iv) lymphotoxin production and (v) its cytolytic effect on lymphocytes. The most striking observation was that even extremely low doses of ALG were able to suppress significantly the lymphotoxin production without affecting other lymphocyte functions or cell viability; this effect was found to be independent of complement. From the comparisons of the mitogen-induced lymphocyte function in the presence of varying doses of ALG and PHA it can be concluded that ALG exerts its inhibitory effect on lymphokine production most likely by interfering with mitogen recognition rather than lymphokine secretion.

The interaction of cytotoxin with target cells to produce target cell lysis is not understood. Herein reported is the identification of an elutable target mouse fibroblast L cell component which when added to other sensitive L cells enhances their sensitivity to cytotoxin, or which when added to L cells rendered relatively insensitive to cytotoxin by incubation in a lipid depleted media, largely restores their sensitivity to cytotoxin. Extraction of the L cell component is carried out by either trypsinization, scraping or by an incubation of 4–6 hr with the monolayer L cells. The elutable component enhances the susceptibility of target cells to cytotoxin rather than by directly affecting cytotoxin as its effect is dependent on an incubation period with the cells prior to the addition of cytotoxin.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.

Human lymphotoxin (LT)-producing T-cell hybridomas were constructed by fusing concanavalin A-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated human acute lymphatic leukemia cells. LT secretion from these hybridomas was considerably enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA) and concanavalin A or PMA alone. A study using cloned hybrid lines revealed that PMA/Con A acted directly on the LT-producing clones. Furthermore, PMA/Con A stimulated A-B9-24, one of the cloned hybridomas, and secreted fourfold larger amounts of LT under serum-free conditions than under serum-containing conditions. However, MIF MAF and LT-producing cloned hybrid line E10–20 secreted rather decreased amounts of MIF MAF when stimulated with PMA, while the LT secretion from the same hybridoma was enhanced with PMA.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [ 3 H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [ 3 H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.

This study explores, by kinetic analysis, the movement of calcium among cellular pools of target cells in which cytotoxicity is induced by human lymphotoxin, and evaluates the requirement for calcium in this reaction. We employed the kinetic model of Borle to quantitate flux rates and pool sizes. It was found that the rate of flux between the surface (plasma membrane-glycocalyx) compartment and the intracellular compartment was greatly increased. The size of the total exchangeable intracellular calcium pool was not altered, but there was an apparent decrease in the size of the surface calcium pool. This latter phenomenon may be related to the blebbing and exofoliation of plasma membranes under the influence of the lymphokine. Lymphotoxin-induced cytotoxicity is observed in calcium-free medium and over a range of calcium concentrations. These results argue against cell death due to a massive inrush of calcium into the cell under these circumstances.

Sensitized allogeneic aggressor lymphocytes caused a marked loss of plasma-membrane protein in target L cells of a subline sensitive to lymphocyte- or lymphotoxin-induced lysis. By sodium dodecyl sulfate-polyacrylamide electrophoretic analysis, this loss was shown to be general and not restricted to specific membrane fractions. Target L cells of a subline partially resistant to attack by lymphocytes or lymphotoxin showed little or no loss of membrane protein after incubation with sensitized lymphocytes. These observations suggest that target-cell plasma-membrane damage plays an important role in lymphocyte-mediated cytolysis.

Multiple families of cytotoxic molecules [Lymphotoxin (LT)] have been identified in phytohemagglutinin (PHA-P) activated human lymphocyte supernatants and lymphocyte homogenates, using gel filtration chromatography on Sephadex G-150. These macromolecules have molecular weights of 80–90,000, 50,000, and 10–15,000 daltons and have been termed LT 2 , LT 2 and LT 3 , respectively. They are secreted by cells from a variety of lympboid tissues, i . e ., tonsil, adenoid, and peripheral blood. The kinetics of appearance of the cytotoxins indicate that all three are present within 16 hr after lymphocyte activation. However, while LT 1 and LT 2 persist in these cultures through day 5, LT 3 is not detectable after day 3. These molecules can also be detected when either PHA or concanavalin A are employed as the stimulating agent. Moreover, the relative amounts of LT 1 , LT 2 and LT 3 activity in a given supernatant vary dramatically from culture to culture. Extracellular levels of LT accumulate and peak by 4 to 5 days in culture, however, intracellular levels of LT reach a maximum on day 3 and decrease to very low levels on day 5. Mitogen-stimulated lymphocytes at 3 days contain intracellular levels of LT which are several logs higher than that detectable in unstimulated cells. This observation suggests that both the biosynthesis and secretion of lymphotoxin is governed by a regulatory control process(es).

Serum-free culture supernatants of Con A-stimulated pig leukocytes contain cytotoxins (PCT) which have a fast-acting lytic effect on a variety of murine lymphomas. Mathematical analysis revealed that the cytodestructive reaction which leads to the immediate release of 51 Cr from labeled target cells follows “single hit”/“first order” kinetics. The release of labeled compounds was found to be size dependent, such that low molecular isotope ( 86 Rb) was released at faster rates than high molecular 51 Cr-labeled complexes. The 51 Cr release was strongly reduced by lowering the temperature to 4 °C. PCT-mediated target cell lysis could be competed by cold target cells and the factor could be absorbed by intact cells, plasma membranes and artificial liposomes. Neither the presence of EDTA or of various monosaccharides, nor exposure of target cells to trypsin, metabolic inhibitors, and cytoskeletal antagonists altered the target cells susceptibility to PCT-mediated lysis. Labeling of target cell DNA and subsequent exposure of these target cells to PCT revealed that target cell DNA is degraded into low-molecular-weight split products which is similar to that seen during T-cell mediated target cell lysis. The analysis of the lytic event mediated by PCT thus revealed similarities to T-cell mediated target cell lysis. PCT was clearly distinct from the well-known cytolytic agents lymphotoxin (LT), tumor necrosis factor (TNF), or complement (C′). Taken together, the described characteristics make PCT a unique-acting cytolytic cytokine with anticancer activity and suggest further research concerning its mode of action and its possible therapeutic application.

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (T G+ ) or IgM (T M+ ), or the absence of both IgG and IgM receptors (T G−M− ). When restimulated with alloantigen of the same derivation, T G+ , T M+ , and T G−M− cells yielded almost equal amounts of cytotoxin. Anti-α-lymphotoxin serum neutralized most of this cytotoxic activity indicating that α-lymphotoxin (α-LT) constituted most of this activity. Although T G−M− cells function as effectors in allogeneic cytotoxicity, T G+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by α-LT. Whether T M+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, T G+ , T M+ , and T G−M− cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-α-LT. The T G+ cells incorporated less thymidine than the T M+ or T G−M− cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in T M+ cells, where positive and negative results have been reported.

Biological and physicochemical properties of keyhole limpet hemocyanin (KLH)-induced guinea pig lymphotoxin (LT) were compared to those of LT induced by another antigen, ovalbumin, or by the mitogen, phytohemagglutinin (PHA). Mitogen- and antigen-induced LT had similar colony inhibitory activities toward guinea pig and rat tumorigenic cells but none toward nontumorigenic cells. Conversely, human nontumorigenic but not tumorigenic cells were inhibited by the three LT preparations. The molecular weight of each LT was 45,000 daltons on gel filtration chromatography but sucrose density gradient ultracentrifugation indicated greater heterogeneity. The KLH-, ovalbumin-, and PHA-induced LTs were resolved into multiple peaks upon preparative column isoelectric focusing (IEF) with IpH's in the range of 4.5 to 5.3. Analytic IEF of the KLH-induced LT revealed the presence of three distinct biologically active peaks with IpH at 4.77, 5.02, and 5.20. LT activity was stabilized during preparative IEF by inclusion of 0.1% polyethylene glycol (4000 molecular weight) in the ampholine gradient. This resulted in 90% recovery of LT activity with a concomitant removal of 96% of the KLH used as the antigenic stimulus for LT production. Fractionation of LT activity during diafiltration and IEF when exogenous proteins were included as protective agents altered the number and the isoelectric characteristics of the peaks obtained suggesting that LT was being bound by the added protein. Colony inhibitory (cytostatic) and 3 H-release (cytolytic) LT activities copurified during fractionation. Thus, this study demonstrated that high yields (approximately 50% of the starting material) of LT, free of antigen or exogenous serum proteins, can be obtained by a combination of diafiltration and IEF performed in the presence of polyethylene glycol.

The molecular class of Golden Syrian hamster lymphotoxin produced in vitro and in vivo was determined by size-exclusion high-performance liquid chromatography using silica-based protein separation columns eluted with a 0.1 M sodium phosphate, pH 7.4 buffer containing 0.1% M r 4000 polyethylene glycol. Lymphotoxin cytolytic activity was quantitated in the column effluent by measuring the ability of the fractions to lyse alpha-L929 cells as indicated by [ 3 H]TdR release. Lymphotoxin activity induced by an 8- or 24-hr or 5-day phytohemagglutinin stimulation of peritoneal leukocytes, by 24-hr phytohemagglutinin-coated alpha-L929-cell stimulation of peritoneal leukocytes, or by 24-hr phytohemagglutinin stimulation of spleen cells occurred in the M r range of 20,000–56,000, with major components in the 35,000–50,000 beta-lymphotoxin region. No activity was present in the complex (greater than 200,000) region and only minimal activity was detectable in the alpha (70,000–160,000) and gamma (12,000–20,000) regions. In vivo -induced lymphotoxin, obtained by peritoneal lavage 48 hr after intraperitoneal administration of phytohemagglutinin, was entirely beta-lymphotoxin and was not detectable in the plasma. Lymphotoxin produced in vitro and injected simultaneously with the gamma-emitting radio-nuclide 99m technetium, inhibited in vivo development of radiation-induced transplacental carcinogenesis. Thus, Syrian hamster lymphotoxin with antitumor activity consists of glycoproteins with isoelectric points of 4.8–5.2, M r of 20,000–56,000, and major in vitro and in vivo forms in the beta-lymphotoxin range.

Lymphokines were detected in the sera of 16 strains of inbred mice sensitized intravenously with cell walls of Mycobacterium bovis strain BCG and challenged subsequently intravenously with old tuberculin (OT). Variations occurred between the strains in the types and quantities of six lymphokines studied, namely, chemotactic factor (CF), type II interferon (IF II), lymphotoxin (LT), migration inhibitory factor (MIF), mitogenic factor (MF), and skin reactive factor (SRF). The times for maximum release of the lymphokines in the different strains were similar for MIF, IF II, SRF, and MF, but differed for CF and LT. The degree of activity of MIF and IF II generally paralleled one another in the different strains but such parallelism did not occur for the other four lymphokines. Each of the 16 strains was a high responder for at least one of the lymphokines, indicating that in sensitized mice the release or presence in the circulation of lymphokines in response to a specific antigen is a selective process. Thus, each strain may have an individual combination of lymphokines, interactions of which may determine the types of pathway utilized in an immunological response.

Actinomycin-treated mouse L cells and human HeLa cells are sensitive indicators of lymphotoxin activity in supernatant fluids of mitogen-stimulated lymphocytes. Without actinomycin, various strains of these cells are 10–200 times less sensitive. The concentration of actinomycin used amplifies the toxic effect of LT but is not itself cytotoxic. Actinomycin-treated indicator cells permit detection of LT activity where toxicity is not often found, as in supernatants of mixed lymphocyte cultures and of cell-mediated cytotoxicity reactions. This assay makes available to any investigator a sensitive indicator of LT activity.

Concanavalin A (Con A) activates nonimmune human lymphocytes in vitro to undergo transformation, DNA synthesis, and lymphotoxin (LT) secretion. LT secretion is inhibited (within minutes) when free and membrane-bound Con A are removed by washing or incubation with the competitive inhibitor, α-methyl mannoside. LT secretion can be reinitiated by addition of fresh Con A. While LT can be rapidly regulated, blast transformation and cellular DNA synthesis are under less restrictive control. Although they appear to be related, LT secretion and lymphocyte transformation seem to be regulated by independent control mechanisms. These studies indicate that recognition and contact of lymphocyte membrane sites initiate as well as regulate the efferent or destructive phase of cell-mediated immune (CMI) reactions. A model of how lymphocytes could employ LT in specific and nonspecific cytodestructive CMI reactions is presented.

Surface-associated lymphotoxin (LT) molecules have been identified on mitogen-activated human lymphocytes employing heterologous anti-α-LT serum in vitro . These membrane-associated LT molecules are present on PHA- or Con A-activated lymphocytes but do not appear to be expressed on unstimulated cells. Furthermore, these molecules were detected primarily on activated T lymphocytes, with little detectable on activated B- or null-cell populations. The removal of surface LT-bearing lymphocytes, using anti-α-LT serum + C′, does not dramatically affect the capacity of the remaining cells to release LT after mitogen restimulation. In addition, the presence of toxic molecules on the surface of activated lymphocytes suggests that these materials may be expressed in an inactive, noncytotoxic form.

Freshly isolated human T lymphocytes were demonstrated to produce lymphotoxin (LT) after mitogenic stimulation with phytohemagglutinin (PHA). In contrast, freshly isolated B lymphocytes, stimulated with two B-cell mitogens [pokeweed mitogen (PWM) and Staphylococcus protein A (Staph A)] did not produce the lymphokine, although thymidine incorporation was increased in these cells. We also examined a series of nine continuous human lymphoid-cell lines with B-cell markers and observed the spontaneous release of either large or small amounts of cytotoxin, or none at all. Cytotoxin from one of the productive cell-lines (H4218) was compared in detail with that obtained from PHA-stimulated, freshly isolated human lymphocytes. The behavior of the two cytotoxins was found to be identical in respect to migration on polyacrylamide gel, neutralization with rabbit anti-human α-LT serum, ultracentrifugation on 5–30% sucrose gradients, and stability for 15 min at 75 °C. Observation of these identical parameters strongly suggests that the α-LT elaborated by PHA-stimulated, freshly isolated human lymphoid cells is the same as the cytotoxin obtained from the continuous human lymphoid-cell line H4218. Thus α-LT may also be produced in quantity from continuous lymphoid-cell lines by mass tissue-culture techniques, which are more readily applicable to large-scale production than is purification from freshly cultured human lymphoid tissues. Notably, in cultures of freshly isolated human lymphoid cells, only T cells, and not B cells, generated lymphotoxin. However, continuous human lymphoid-cell lines with B-cell markers can also secrete this lymphokine.

The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assessed in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-γ, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect.

Cytotoxic activity (lymphotoxin (LT)) present in supernatants from lectin stimulated human lymphocytes in vitro is composed of a heterogeneous system of biological macromolecules which can be separated into multiple classes and subclasses on the basis of their molecular weight and charge. These studies further characterize a large molecular weight human LT class, termed complex (MW >200,000 d), which elutes in the void volume off Sephadex G-150 or Ultrogel AcA 44. Immunological studies on the complex, employing various rabbit anti-LT class and subclass antisera, revealed this material is a macromolecular assemblage of the smaller MW α, β, γ LT classes and subclasses. Furthermore, the reactivity of this material with anti-human Fab′ 2 (IgG) indicates these smaller molecular weight LT components can associate with immunoglobulin or Ig-like molecules. The materials present in the LT complex class appear to be noncovalently associated, since conditions of high ionic strength dissociate certain small MW LT components, while low ionic strength buffers may cause these components to reaggregate with the complex. When subjected to velocity sedimentation on sucrose gradients or gel filtration on Ultrogel AcA 22, LT complex activity elutes as several discrete peaks of activity in the 200,000 to 1,000,000 MW range. These findings suggest the concept that LT molecules can form discrete and specific macromolecular structures which contain the smaller MW LT classes. Moreover, these structures can also associate with immunoglobulin-like molecules to form secondary LT-Ig complexes. This may have important biological significance in explaining how nonspecific cell toxins could play a role in specific or nonspecific cell lytic reactions in vitro .

Mouse lymphotoxin (LT) was assayed by release of tritium ( 3 H) from mouse fibrosarcoma cells (7943) labeled with [ Methyl - 3 H]thymidine. Incorporation of [ Methyl - 3 H]thymidine into the cells resulted in morphological alterations and an increase in the fragility of the cells. Although the percentage of 3 H that was released in either the presence or absence of LT increased with the amount of incorporated [ 3 H]thymidine, the concentration range over which LT induced increasing 3 H release was the same, indicating that the sensitivity of the assay was not altered. A lag between the action of lymphotoxin and 3 H release was found. Within 24 hr LT initiated the events that led to subsequent 3 H release; however, release did not begin until 30 hr after incubation with LT. The release of 3 H from target cells could be increased by deoxyribonuclease (DNase) or decreased by undiluted lymphotoxin. Both of these effects were on the release of 3 H from LT-damaged cells and not on the activity of LT. Treatment of labeled cells with LT and DNase for 72 hr released as much as 92% of the incorporated thymidine; however, during this time the cell number increased four-fold, suggesting some cells are refractory to LT. These results indicate that care must be used in estimating cell destruction by 3 H release from cells labeled with [ Methyl - 3 H]thymidine.

Antigens induce sensitized lymphocytes to undergo mitosis and to secrete soluble products, termed lymphokines, which modulate the immune response. Plant lectins are known to act as polyclonal lymphocyte mitogens and, in some cases, stimulate lymphocytes to produce lymphokines. In an effort to explore the relationship of specific cell surface glycoconjugates to the induction of mitosis and the production of lymphokine activities we have examined the ability of the mitogenic lectins, concanavalin A and Wistaria floribunda mitogen, and the nonmitogenic hemagglutinin from Wistaria floribunda seeds to stimulate the production of macrophage migration inhibition factor (MIF), macrophage chemotactic factor (CF), and lymphotoxin (LT). Concanavalin A causes lymphocytes to produce MIF and LT but no detectable CF activities. W. floribunda mitogen induces lymphocytes to produce soluble substances which exhibit all three lymphokine activities. The nonmitogenic W. floribunda agglutinin causes lymphocytes to produce MIF and CF but no observable LT activity. Within the sensitivity of the assays employed, the results indicate that mitogenesis is not a corequisite of the expression of either macrophage migration inhibition factor or lymphocyte-derived chemotactic factor but it may be associated with the induction of lymphotoxin. It is also apparent that the expression of each lymphokine activity is independent of the expression of the other lymphokines studied.

The purpose of this paper is to present evidence for the suitability of a lymphotoxin (LT) assay as an in vitro correlate of cell-mediated immunity (CMI) to a hapten-carrier conjugate known to stimulate a high IgE antibody response. This would be used in a study of the factors influencing the relationship(s) between CMI and IgE antibody responses to the same antigen. Antigen-induced LT was assayed on actinomycin-inhibited, chromium-51-labeled L929 fibroblasts, using supernatants obtained from spleen and lymph node cells of animals immunized with 2,4-dinitrophenyl-Ascaris conjugates. Although LT was present at all times tested, beginning at Day 10, its concentration varied with time after immunization, adjuvant used, and cell type assayed. The induction of LT was T-cell dependent and conjugate specific. LT was produced by nonadherent cells. Adherent cells from immunized animals produced L-cell cytotoxin in the absence of antigen stimulation when tested before Day 10.

When normal murine spleen cells were treated with anti-theta serum and complement, they failed to produce LT or synthesize cellular DNA when stimulated in vitro with PHA. Theta-positive cells were responsible for LT production in spleens removed from X-irradiated and bone marrow- or thymus-reconstituted animals. Finally, spleens from congenitally athymic Nu/Nu mice failed to produce LT when stimulated with pokeweed mitogen or phytohemagglutinin.

The susceptibility of L cells in spinner and monolayer culture to lysis by lymphocyte-produced cytotoxin (“lymphotoxin”) is very similar. These observations disprove the suggestion that the cytotoxin works by detaching monolayer-adapted L cells which subsequently die in suspension.

Phytohemagglutinin (PHA)-stimulated cells were suspended in 10% dextran to prevent cellular aggregation. Cells suspended in dextran did not respond to PHA by any of the five parameters tested: [ 3 H]leucine, [ 3 H]uridine, and [ 3 H]thymidine incorporation into acid-insoluble products, lymphotoxin synthesis, or morphologic transformation. Dextran itself was not cytotoxic to the lymphocytes. However, one major technical problem is attaining a proper dextran “control.” The difficulty in culturing lymphocytes in dextran without simultaneously causing some cell dispersion yielded dextran “control” values that showed less stimulation than cells without dextran. These results suggest that cell contact is necessary for PHA-induced transformation as judged by both DNA synthesis and lymphotoxin secretion. This technique now provides an experimental system for biochemically assessing the effects of antigens and mitogens without the attendant problems caused by cell clustering.

An effective method for the concentration of cultured medium to detect small amounts of lymphotoxin is described. A microassay for cytotoxicity modified from the method of Takasugi and Klein has been performed using L cells (mouse fibroblast subline 5b) as nonspecific target cell. Activated human lymphocytes produce lymphotoxin in serum-free media, but activity is low compared to culture in media with higher serum concentrations. Human lymphotoxin is stable for three weeks at 3 °C, and no loss of activity is observed after dialysis or lyophilization. Some nonlymphotoxin protein is removable during dialysis. A “concentration assay” of human lymphotoxin and human lymphotoxin-like activity induced by purified protein derivative was performed using lymphocytes cultured in medium containing 2% serum. This method markedly increased the sensitivity of the microassay for lymphotoxin induced by phytohemagglutinin and purified protein derivative. Lymphocyte-mediated cytotoxicity induced by thyroid antigen(s) in patients with chronic thyroiditis was observed. After concentration of supernatants, significant cytotoxic activity was obtained in patients, but not in normal subjects. Lymphocytemediated cytotoxicity, presumably mediated by soluble human lymphotoxin-like cytotoxic factor, may play an importan role in human autoimmune thyroiditis.

The high-molecular-weight (MW) complex class (MW = greater than or equal to 200,000) of lymphotoxin (LT) may be involved in the process of lymphocyte-mediated cytolysis. This LT class was produced by concanavalin A-stimulated human adenoid and tonsil lymphocytes in vitro and purified approximately 1000 fold by a scheme employing lectin affinity chromatography on concanavalin A-Sepharose, negative hydrophobic affinity chromatography on phenyl-Sepharose, and molecular sieving on Ultrogel AcA 44. A cell-lytic subunit, termed C-alpha toxin, was dissociated from this partially purified complex LT (Cx) preparation. The identification of C-alpha toxin as an alpha 2 LT form was established by its comigration with the alpha 2 LT form during native polyacrylamide gel electrophoresis (PAGE), molecular sieving, and isoelectricfocusing. In addition, C-alpha toxin was neutralized by antiserum raised against purified alpha 2 LT, and the latter was neutralized by antiserum raised against C-alpha toxin. Furthermore, preliminary evidence indicates that both alpha 2 LT and C-alpha toxin are composed of peptides with apparent MW of approximately 69,000. Additional data indicate the Cx LT form is composed of alpha 2 LT and other subunits.

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [ 3 H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro -transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal 3 H-release activities of mitogen (PHA) 1 -stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. 3 H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces 3 H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by 3 H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.

Serum was found to play an essential role in cytotoxin-mediated target cell lysis. The degree of cytotoxic activity on target L cells was directly related to the concentration of sera in the media incubating the L cells. Furthermore, when ether-ethanol-extracted sera were used in the incubation media, almost no cytotoxic activity was found, indicating that a lipid component of sera, removed by the extraction procedure, might be involved in the action of cytotoxin. The effects of increased sera concentrations and extracted sera occur primarily in the initial 24-hr incubation of L cells (prior to the addition of cytotoxin) rather than when cytotoxin is added, indicating that serum acts by influencing target cell susceptibility to cytotoxin rather than on cytotoxin itself.

A series of Lyt 2 + , trinitrophenyl (TNP)-specific T-cell lines are shown to lyse 51 Cr-labeled target cells in an antigen-specific, H -2-restricted fashion in a 4-hr assay. These cells also produce lymphotoxin, in addition to other factors, upon stimulation with TNP-haptenated syngeneic splenocytes. A technique for introducing macromolecules into the cytoplasm of fibroblasts by inducing the cells to pinocytose the molecule in hypertonic medium, and then lysing the newly formed pinocytic vesicles with a mild hypotonic shock was used to assess the role of soluble mediators in the cytotoxic T lymphocyte (CTL)-mediated lytic process. The technique itself has little effect on cell growth rate or viability. A minimum of 24 hr, and more frequently 48–72 hr is required for lymphotoxin to manifest it's lethal effect when it is merely included in the culture medium of growing fibroblasts. In contrast, supernatant fluids from the Lyt 2 + cells kill 51 Cr-labeled fibroblasts in a dose-dependent fashion during a 4-hr assay when they are rapidly internalized via the osmotic procedure. The data serve as preliminary evidence of a role for soluble mediators such as lymphotoxin in T-cell-mediated lysis, and suggest that the cytotoxic-T-cell lethal hit may include a mechanism for rapidly internalizing a toxin into appropriate target cells.

Previous studies showed that the tumor-promoting phorbol ester 12- O -tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-γ) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-γ and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-γ during a multistep purification procedure.

An endogenous inhibitor for human lymphocyte RNA, protein, and DNA synthesis contained in the extracts of cultured human lymphoid cells (PGLC-33H) is described. The cell extract was found to inhibit RNA, protein and DNA synthesis of autologous cultured lymphoid cells (PGLC-33H) as well as heterologous PHA stimulated human peripheral lymphocytes but not that of nonlymphoid cells (HeLa cells, human embryonic lung, human embryonic skin and muscle and WI-38). Extracts of human peripheral lymphocytes were also found to inhibit the DNA synthesis of cultured lymphoid cells (PGLC-33H) and PHA stimulated human peripheral lymphocytes but not that of nonlymphoid cells (HeLa cells and human embryonic lung). Similarly prepared extracts of long term cultured skin and muscle (H 1 S) cells did not inhibit the DNA synthesis of the cultured lymphoid cells. No evidence of cytotoxicity was detected, and the inhibition was reversible. The inhibitory activity of the extract was found to be thermolabile, nondialyzable, DNase resistant, and sensitive to trypsin. The inhibitor seems to be different from lymphotoxin.

The question of nonthymus-derived lymphocyte-mediated cytotoxicity was investigated with T and B cell subpopulations separated from the blood of normal donors. Mononuclear cells, T cells (E-RFC), and cell preparations enriched for B cells (non-E-RFC) by depletion of E-RFC gave negligible cytotoxic responses when incubated with either human melanoma or lung fibroblast target cells. In contrast, EAC and ZC rosetting cells separated from this same B-rich population consistently gave cytotoxic responses which were not dependent on either antibody or phagocytic cells. The cytotoxic effector cells appeared to be nonthymus-derived lymphocytes as characterized by C3 receptor rosetting and presence of surface membrane immunoglobulin on the majority of cells. In addition, supernatants from EAC-RFC cultures contained lymphotoxin (LT) activities which were eightfold higher than those of control E-RFC cultures. These findings suggest the existence of a nonthymus-derived cell cytotoxic effector mechanism, induced by the binding of membrane C3 receptors, which is independent of antibody.

Experiments were undertaken to investigate the abilities of human T and B lymphocytes to produce the lymphokines interferon (IF) and lymphotoxin (LT). Sheep erythrocyte rosette-forming cells (E-RFC) were separated from nonrosetting cells by sedimentation on Ficoll-Hypaque gradients. Unseparated peripheral blood mononuclear cells and T-cell-enriched and B-cell-enriched populations were assayed for lymphokine production and proliferative response after stimulation with the T-cell mitogen, phytohemagglutinin (PHA), or the B- and T-cell mitogen, staphylococcal phage lysate (SPL). PHA induced LT production and cell proliferation primarily in the E-RFC-enriched population. In contrast, SPL preferentially induced LT production in the E-RFC-depleted population. SPL stimulated cell proliferation and induced equivalent IF production in both populations. Thus, PHA and SPL have differential and contrasting effects in terms of the induction of LT production and cell proliferation in T-cell and B-cell populations. In the same experiments there was no population specificity in induction of IF production. Therefore, the potentials to produce LT and IF appear to be inherent in both cell populations with their in vitro expression being dependent upon the nature of the stimulus.

In a previous study it was reported that human α-interferons (IFN) caused a significant enhancement of human lymphotoxin (LT)-induced in vitro killing of human target cells. This synergistic effect was dose dependent and was demonstrable on normal and tumor cell targets. The effects of IFNs from human and several animal species on human LT-induced cell killing of human, mouse, and rabbit target cells are examined. In addition to enhancement of IFN, a new finding was made showing protective effects of IFN on human LT activity. IFN-induced enhancement or protection depended on the particular IFN:target cell combination, with the highest degree of enhancement being observed in the homologous human combination. In this latter case, IFN-induced enhancement was blocked by antiserum to IFN. While a role for other soluble factors cannot be ruled out, the results suggest that, in the homologous human system, enhancement of LT activity was mediated by IFN. These results are discussed in relationship to previously observed enhancing and protective effects of IFN in natural killer cell systems.

This study was designed to determine if sodium metaperiodate (NaIO 4 )-treated lymphocytes secrete lymphokines and if these lymphokines are similar to those obtained from mitogen- or antigen-stimulated lymphocytes. A brief exposure of CBA spleen cells to NaIO 4 induced the secretion of significant amounts of migration inhibitory factor (MIF). This MIF had a molecular weight range between 30,000 and 58,000, and was stable when heated at 56 °C for 30 min, but unstable at 80 °C. These characteristics are similar to those previously reported for mitogen- and antigen-induced MIF. In addition, NaIO 4 induced the secretion of lymphotoxin (LT) from CBA and Balb/c spleen cells, as well as from guinea pig lymph node cells. NaIO 4 was compared to the other inducers in regard to the quantity of LT secreted. Supernatant derived from NaIO 4 -treated mouse spleen cells contained less LT than supernatants derived from concanavalin A- or phytohemagglutinin-treated cells, but contained more activity than those supernatants derived from lipopolysaccharide-treated cells. CBA spleen cells secreted significantly more LT than Balb/c spleen cells after NaIO 4 stimulation. NaIO 4 -stimulated CBA spleen cells secreted LT in cultures with or without serum, but stimulated Balb/c spleen cells secreted LT only in serum-containing cultures. The advantages of NaIO 4 as an inducer of lymphokines, as opposed to other mitogens or antigens, is the brief exposure of this agent to the cells after which the NaIO 4 is removed, and the lymphokines can be obtained free from the inducer.

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.

T helper cells reactive to myelin basic protein are clearly implicated in the pathogenesis of murine EAE. We have developed a T cell line, BML-1 that (1) is reactive to the encephalitogenic amino terminal nonapeptide (1-9NAC) of MBP, (2) is I-Au restricted, and (3) induces relapsing EAE in B10.PL (H-2u) mice. Measurement of the lymphokine profile of BML-1 revealed secretion of IL-2, interferon-gamma and lymphotoxin but not IL-4. This profile is consistent with the Th1/DTH subtype. Coculture of BML-1 with MBP-primed B cells shows that BML-1 does not provide significant helper function in vitro. In addition, BML-1 secretion of interferon-gamma was found to inhibit LPS-induced anti-MBP antibody responses. This suggested that anti-MBP antibodies may not be necessary for induction of EAE. Sera from mice, in which severe disease was induced with the 1-9NAC peptide and Bordetella pertussis, showed no development of serum antibodies to MBP. These data show that MBP-reactive Th cells of the Th-1/DTH subtype can induce EAE and do not provide Th function for anti-MBP responses and that serum anti-MBP antibodies are not found in peptide 1-9NAC-induced disease. T cell lines specific for encephalitogenic epitopes and characterized for lymphokine secretion will provide a useful tool for understanding the role of T cells in the induction of EAE.

A hybridoma, F133, that produces macrophage activation factor (MAF) after mitogen stimulation was developed by fusing the AKR-derived BW5147 thymoma with alloantigen-stimulated C3H/HeJ splenocytes. F133 supernatants were shown to contain MAF, migration inhibition factor, and a factor capable of suppressing the plaque-forming response to sheep erythrocytes but not lymphotoxin, interleukin II, or interferon. Both concanavalin A (Con A) and phytohemagglutinin (PHA) induced MAF production by F133. Time course and dose-response experiments showed that maximal concentrations of MAF were present 48 hr after stimulation with either 1.5 μg/ml Con A or 6 μg/ml PHA. F133 and normal splenocyte MAF preparations shared physicochemical properties in that heating at 100 °C for 30 min abolished MAF activity while 56 °C for 30 min or 100 °C for 2 min had little effect. In addition, both MAF preparations were dependent on the presence of lipopolysaccharide for macrophage activation and each was inactivated by pH 4.0 or pH 10 treatment while pH 6.0 and pH 8.0 had little effect. Also, pretreatment of both MAF preparations with either trypsin or chymotrypsin inactivated MAF activity.

The reaction of serum from rabbits immunized with human brain (RHB) with human lymphocytes has been measured by cytotoxicity, membrane immunofluorescence, and inhibition of lymphocyte function (mitogen-induced blastogenesis and lymphotoxin production). RHB kills most of the T-cells in the blood, but no reaction with B-cells or monocytes was detected. The remaining cells are reduced in their ability to respond to mitogen by lymphotoxin production and/or secretion. Treated PBL respond normally to PHA and PWM by incorporation of [ 3 H]TdR, but the response to Con A is inhibited. The effect on lymphotoxin release is not dependent on cell death, as it occurs in the absence of added complement. RHB inhibits spontaneous E rosette formation by T-cells; again the effect does not depend on cell death but it requires approximately 10% normal serum—fresh or heated! The synergistic effect of normal and immune serum is apparently due to steric or charge effects of binding of some complement component(s), since normal serum depleted by incubation with antigen-antibody complexes supports rosette inhibition by rabbit anti-human brain serum very poorly, and purified Clq partially restores the inhibition. All of the measured properties of RHB are induced by immunization and can be produced by treating cells and washing away the serum components which do not adhere. We conclude that there is a small subpopulation of human lymphocytes, probably T-cells, which lacks the brain antigen and responds by DNA synthesis but not lymphotoxin to PHA and PWM stimulation. The majority of T-cells, including those responsive to Con A, bear an antigen(s) cross-reactive with brain; this antigen is not identical to the receptor for sheep red blood cells.