Study of the cellular origin of histamine release inhibitory factor using highly purified subsets of mononuclear cells

We have recently described a specific antagonist of histamine-releasing factors that inhibits histamine release from basophils and mast cells. This histamine release inhibitory factor (HRIF) is produced by PBMC upon stimulation with histamine as well as mitogens such as Con A. The objective of this study was to investigate the cellular origin of HRIF produced by PBMC. Monocytes, T cells, and B cells were isolated to 96 to 99% purity by a combination of plastic adherence, E rosetting, and negative selection with mAb (OKM1, OKT11, OKB7, OKT4, and OKT8) and C. Purified subpopulations were cultured with histamine or Con A and then the processed supernatants were assayed for the inhibition of HRF-induced histamine release from basophils. The results of this study suggest that the highest amount of HRIF is synthesized by B cells followed by T cells and monocytes. The B cell origin of HRIF was confirmed by abolishing the activity after incubation of the cells with OKB7 mAb and C. Both CD4- and CD8- T cells are capable of producing HRIF. In mixing experiments, the synthesis of HRIF by two different subpopulations has been less than additive. T + B cells produced most of the HRIF activity. Monocytes tended to suppress the synthesis of HRIF by B cells. The synthesis of HRIF by so many cell types suggests that a fine balance between HRIF and HRF may regulate the mediator release from basophils and mast cells.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

Potentiation of transmembrane signaling by cross-linking of antibodies against the beta chain of the T cell antigen receptor of JURKAT T cells.

Three monoclonal antibodies (mAb) 2D1, 3B9, and 3B12 were produced by immunizing BALB/c mice with JURKAT cells. These mAb induce comodulation of the TCR/CD3 complex expressed on JURKAT cells, but do not react with the CD3- JURKAT variant, J.RT3.T3.1. Immunoprecipitation studies with detergent-solubilized JURKAT cell lystes indicate that these mAb react with proteins having characteristics of the TCR molecules. Their low reactivity with peripheral blood mononuclear cells (PBMC) and lack of reactivity with other CD3+ T cell lines suggest that they may be anti-idiotypic mAb. Results from binding inhibition assays, reactivity with PBMC, and generation of transmembrane signals suggest that these three anti-TCR mAb recognized different epitopes on the TCR beta chain of JURKAT cells. Although the three mAb are capable of inducing the production of inositol phosphates and cytosolic free Ca2+ increase in JURKAT cells, their stimulatory capacities vary and are lower than that observed by anti-CD3 antibody (OKT3) stimulation. However, crosslinking these mAb with rabbit antimouse immunoglobulins potentiates the stimulatory response to comparable levels induced by OKT3. These mAb could be useful as tools to study V beta 8+ T cells in relation to antigen-specific activation.     

Identification of a histamine release inhibitory factor (HRIF) and an inhibitor of histamine releasing factor synthesis (IHS) produced by guinea pig lymphoid cells

Guinea pig spleen cells, thymocytes, and blood mononuclear cells have been shown to produce a histamine releasing factor (HRF) spontaneously or after stimulation with PHA or specific antigens. In this study we have shown that antigenic stimulation of spleen cells obtained from animals immunized with high doses of antigen suppresses the spontaneous HRF production. Likewise, stimulation with Con A of spleen cells also inhibits the spontaneous HRF production. When the supernatants from Con A- and antigen-stimulated cultures were added to fresh thymocyte cultures, a significant inhibition of HRF production was observed. These supernatants also inhibited HRF-induced histamine release from mast cells. We have identified an inhibitor of HRF synthesis (IHS) and also a histamine release inhibitory factor (HRIF) by gel chromatography. Virtually all mitogen- and antigen-stimulated culture supernatants elaborated the activities of HRF, IHS, and HRIF in various quantities depending on the dose of antigen and the kind of adjuvant used for immunization. IHS has a MW of 22K-35K and 10K. This cytokine also inhibits DNA synthesis by thymocytes. HRIF has a MW of 15K-20K and 10K. It inhibits both HRF- and antigen-induced histamine release from lung mast cells. These results suggest that lymphocytes produce a variety of factors which function to regulate histamine release from mast cells.     

Induction of suppressor cells to T- and B-cell proliferative responses and immunoglobulin production by monoclonal antibodies recognizing the CD3 T-cell differentiation antigen

Monoclonal antibodies (mAb's) recognizing the CD3 T-cell differentiation antigen induced the generation of suppressor cells. These cells inhibited (1) proliferative responses of human peripheral blood mononuclear cells (PBMC) to PHA and allogeneic cells in mixed leukocyte culture; (2) proliferative responses of purified E-rosette-negative cells to Staphylococcus aureus Cowans I; and (3) de novo immunoglobulin synthesis and secretion in the pokeweed mitogen (PWM)-induced differentiation system. Monoclonal antibodies recognizing other T-cell differentiation antigens (anti-Leu 2a, anti-Leu 3a, and anti-Leu 5) did not induce the generation of suppressor cells, even at very high antibody concentrations. Statistically significant differences were not observed in the ability of the OKT3 and anti-Leu 4 mAb's to induce suppressor cells. Monocytes were not required for the generation of anti-CD3-induced suppressor cells. F(ab')2 fragments of the OKT3 mAb's were equally effective when compared with intact antibody molecules in inducing suppressor cells, although they did not induce proliferative responses. Proliferation was not required for the induction of suppressor cells. Irradiation (2500 rad) of PBMC before incubation with the anti-CD3 mAb did not affect the generation of suppressor cells. Furthermore, anti-CD3-induced suppressor cells were radioresistant. Addition of recombinant IL-2 to the cultures of responding cells and suppressor cells did not reverse the suppression. In vitro treatment of anti-CD3-induced suppressor cells with either the OKT4 mAb plus complement or the OKT8 mAb plus complement partially decreased the suppression of proliferative responses of PBMC to PHA or allogeneic cells in mixed lymphocytes culture. However, treatment with both OKT4 and OKT8 mAb's plus complement or the OKT11 mAb plus complement completely abolished the suppression. These results suggest that the suppressor cells are of the T11+T4+T8- and T11+T4-T8+ phenotypes. In other experiments, T4+T8- and T8+T4- cells were isolated from PBMC treated for 48 hr with anti-CD3 mAbs. Both these two populations significantly inhibited proliferative responses of autologous PBMC to PHA and de novo immunoglobulin synthesis and secretion by mixtures of purified T4 and B cells from normal donors, in the PWM-induced differentiation system. These results demonstrate that anti-CD3-induced suppressor cells are of the T4 or T8 phenotype. Treatment of purified T4+T8- and T8+T4- cells with anti-CD3 mAb's resulted in the generation of suppressor cells, suggesting that the precursors of the anti-CD3-induced suppressor cells can belong to either of these two populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

HUMAN B CELL DIFFERENTIATION: DEPENDENCE ON INTERACTIONS WITH MONOCYTES AND T LYMPHOCYTES VIA CD40, CD80 (B7.1), AND THE CD2-LIGANDS CD48 AND CD58 (LFA-3)

B cell differentiation depends on cellular interactions with T lymphocytes and monocytes via adhesion molecules (AM). In order to characterize AM which are required for B cell differentiation immunoglobulin production using unseparated peripheral blood mononuclear cells (PBMC) was studied. Unstimulated human PBMC were cultured for 9 days with mAb directed at CD2/CD48, /CD58, CD59, CD5/CD72, CD11a—CD18/CD54, CD28/CD80, CD86, CD40/CD40L, or rat CD2 (control). B cell differentiation was quantified measuring IgM and in some cases IgA, IgG, and IgE production. IgM levels were significantly reduced by mAb against CD40, CD48, CD58 and CD80. The reduction was not due to isotype switching to IgA, IgG or IgE. The role of CD40, CD48, CD58 and CD80 was further investigated after depletion of different cell types. Depletion of monocytes and NK cells resulted in no detectable IgM production irrespective of added mAbs. In contrast, IgM production was still present after depletion of T cells and NK cells. Only mAb against CD80 and CD48 significantly reduced IgM production, the reduction of IgM production by anti-CD40 mAb was less than in the presence of T cells. Importantly, anti-CD58 mAb had no effect on IgM production after T cell and NK cell depletion. Taken together, the AM CD40, CD48, CD58, and CD80 are involved in Ig production of unseparated PBMCs. In this model of B cell differentiation only the AM CD58 depend on the presence of T cells while CD48 and CD80 help was found to be T cell independent.     

Production and properties of histamine-releasing factor of guinea pigs

The production of histamine-releasing factor (HRF) by human mononuclear cells has previously been reported. In this paper we describe the production of HRF by guinea pig spleen cells, thymocytes, and PBMC. Guinea pig lymphoid cells were cultured either alone or in the presence of mitogens (PHA and Con A) or specific Ag(OVA and keyhole limpet hemocyanin) and the dialyzed cell-free supernatant was tested for histamine-releasing activity on guinea pig lung mast cells and blood basophils. Lung mast cells were isolated by enzymatic digestion and partially purified by countercurrent elutriation and discontinuous Percoll gradient centrifugation. Guinea pig spleen cells, thymocytes, and PBMC spontaneously produced significant amounts of HRF. The production was enhanced upon stimulation with PHA or specific Ag in animals immunized with Ag in CFA. Two distinct species of HRF were identified with m.w. of 50,000 to 70,000 and 5000 to 8000 by gel chromatography. HRF is a trypsin- and chymotrypsin-sensitive heat-stable protein. It does not bind to Con A-Sepharose and its production is not inhibited by tunicamycin. HRF-induced histamine release from lung mast cells is a temperature-dependent process and is complete in 10 min at 37 degrees C. Intradermal injection of HRF caused an immediate ear-swelling reaction in guinea pigs. The most severe ear-swelling reactions did not resolve within 1 h, but instead evolved over a period of 12 to 24 h.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Intracellular activation signal requirements for the induction of IL-2 responsiveness in resting T cell subsets in humans

Activation signal requirements for the induction of the IL-2 responsiveness in purified subsets of human resting T cells, T4+ or T8+, have been investigated under the monocyte-depleted conditions. Substantial levels of IL-2 responsiveness were induced in T8+ cells by lectin, Con A, mAb directed against the CD3 Ag, OKT3, Ca2+ ionophore, ionomycin or phorbol ester, PMA. In contrast, none of these stimuli was by itself sufficient for the induction of IL-2 responsiveness in the T4+ subset. The latter cells could, however, be induced to respond to IL-2 by combinations of PMA plus either of Con A, OKT3, or ionomycin (but not any combination of Con A, ionomycin, and OKT3). These data indicate that induction of IL-2 responsiveness in the resting T4+ subset is more complex, possibly requiring two intracellular activation signals, increase in the concentration of intracellular Ca2+ and activation of protein kinase C, whereas either signal may directly trigger IL-2 responsiveness in the resting T8+ cells. The data further suggest that under optimal conditions, growth of both resting T4+ and T8+ subsets may be independent of monocytes.     

Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions by flow cytometric analysis

Rapid separation of large numbers of human peripheral blood mononuclear cells into fractions enriched for B lymphocytes, T lymphocytes, or monocytes was accomplished by counterflow centrifugal elutriation (CCE). The first fraction contained 98% of the platelets. Ten additional fractions containing subpopulations of mononuclear cells were collected by sequential increases in the flow rate while maintaining a constant centrifuge speed. Analysis of the fractions using monoclonal antibodies revealed that fraction 2, which was free of esterase-positive monocytes, was highly enriched for B cells. T lymphocytes (OKT3+) were the predominant cell type found in fraction 4. No enrichment for T-lymphocyte-helper (OKT4+) or -suppressor (OKT8+) subpopulations was observed in the lymphocyte containing fractions. Three fractions (7-9), highly enriched for esterase-positive cells, were predominantly OKM1+ monocytes with no evidence of selective separation of monocyte subpopulations. Thus, cell fractions enriched for B cells, T cells, and monocytes could be obtained, by utilizing CCE, in large enough quantities to enable analysis of their functional properties. Of particular interest was the ability to separate small, resting B lymphocytes from monocytes.     

Induction of cytotoxicity in human peripheral blood mononuclear cells by monoclonal antibody OKT3

It has been previously reported from this laboratory that incubation of PBMC with OKT3 generates potent cytotoxic lymphocytes that can be targeted by using antibody heteroconjugates consisting of anti-target cell antibody and OKT3. In the present study these conjugates were used to explore the kinetics of induction of cytotoxicity in PBMC and the subpopulations of lymphocytes involved. It was found that in addition to conjugate-dependent cytotoxicity, a considerable amount of conjugate-independent cytotoxicity was generated during OKT3 stimulation. Although the conjugate-dependent activity resided in the CD8+ population, the conjugate-independent cytotoxicity was found to be a function of CD4-/CD8- natural killer-like cells. Being largely CD3-, those cells were most likely activated by lymphokines produced by OKT3-stimulated CD3+ cells. They were capable of killing not only tumor cells but also autologous lymphocytes. The CD4+ cells of some donors were found to exhibit low but clearly demonstrable cytotoxicity. Induction of cytotoxicity was characterized as an early event in T cell activation, correlating with the kinetics of RNA synthesis. Cytotoxicity, interleukin 2 receptor expression, and DNA synthesis declined after 3 days of activation with OKT3, indicating the existence of as yet undefined regulatory mechanisms.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Lysis of fresh solid tumor targets in the presence of Con A is mediated primarily by Leu 7+ peripheral blood T lymphocytes: blocking by the anti-CD3 monoclonal antibody and comparison with recombinant interleukin 2-induced lysis by natural killer cells

We investigated the lysis of fresh human solid tumor cells by peripheral blood T lymphocytes in the presence of lectins and anti-CD3 monoclonal antibodies (mAb). Addition of certain lectins (Con A, PHA, or WGA) directly into the 4-hr 51Cr-release assay caused significant lysis of (P less than 0.001) noncultured solid tumor targets by enriched populations of granular lymphocytes (GL). Significant levels (P at least less than 0.001) of Con A- or PHA-dependent solid tumor lysis by GL-enriched lymphocytes were observed in 32 of 39 donors (82%) and 14 of 20 donors (70%), respectively. In contrast, the addition of other lectins (PNA, PWM, or LPS) or anti-CD3 mAb did not cause cytotoxicity. The levels of Con A-dependent lysis were comparable to those of interleukin 2 (IL-2)-induced lysis by Leu 11b+ natural killer (NK) cells. The presence of lectins at the effector phase, but not of recombinant IL-2 (rIL-2), was required for the lysis of solid tumor targets. Both Con A-dependent and rIL-2-induced lysis were totally inhibited by treatment of the effector cells with the lysosomotropic agent L-leucine methyl ester (LeuOMe). Effector cells responsible for Con A-dependent lysis of solid tumors expressed T3 (CD3), T8 (CD8), and Leu 7 antigens, but lacked T4 (CD4) and Leu 11 (CD16) antigens as determined by both negative and positive cell selection studies. Con A-dependent lysis was inhibited at the effector phase by anti-CD3 (OKT3 or anti-Leu 4) or anti-CD2 (OKT11) mAb. On the basis of their phenotype (Leu 7+ CD3+ CD8+ CD16-), we hypothesize that these effector cells may contain a population of cytotoxic T cells (CTL) generated in vivo against autologous modified cells that can lyse fresh solid tumor target cells under conditions where the recognition requirements for the CTL are bypassed by lectin approximation.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

Activated peripheral T lymphocytes undergo apoptosis when cultured with monocytes activated by HLA class II ligation

We treated PBMC with anti-MHC class II mAb known to inhibit T lymphocyte proliferation. Adherent cells from mAb-treated PBMC showed increased metabolic activity by the MTS assay that was not due to cell proliferation. PBMC cultured with solid-phase anti-class II mAb in chamber inserts inhibited, across a membrane, the proliferation of PBMC cultured with soluble anti-CD3 mAb. PBMC treated with both soluble mAb underwent apoptosis as shown by nucleosomal DNA fragmentation. The monocytes formed multinucleated giant cells as shown by fluorescent microscopy, and contained apoptotic bodies as shown by the TUNEL method and by electron microscopy. The apoptotic cells were identified as T cells by double-staining with anti-CD4/CD8-PE and annexin-V-FITC. Thus, MHC class II ligation stimulates monocytes to increase their metabolic activity, induce apoptosis of activated T lymphocytes, and phagocytize the apoptotic cells. TCR-mediated ligation of MHC class II may play a role in the downregulation of immune responses.     

Relationship between immune system and gram-negative bacteria: monocyte chemotaxis induced by supernatants from human peripheral blood OKT8+ lymphocytes stimulated with smooth and rough Salmonella strains

It has been recently reported that smooth (S) Salmonella typhimurium LT-2 and rough (R) mutants, S. minnesota R345 (Rb) and R595 (Re), spontaneously adhere to human peripheral blood mononuclear cells (PBMC). The binding is confined to T cells and is mediated by the lipopolysaccharide (LPS) moiety of the bacteria outer membrane. In this study, we have investigated the monocyte chemotactic responsiveness induced by supernatants recovered from human PBMC stimulated with either S or R Salmonella strains. All supernatants were able to trigger monocyte chemotaxis, even if to a different degree according to the bacterial strain used. These data were further supported by experiments which showed that stimulation of PBMC by purified homologous LPS led to the release of a chemotactic lymphokine (CLK) for human monocytes. Moreover, this CLK seems to be released by T cells, and in particular OKT8+ cells, since OKT8- PBMC failed to produce CLK.     

Effector activity of OKT4+ and OKT8+ T-cell subsets in lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The role of OKT4+ and OKT8+ T-cell subsets was studied in lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC was evaluated by detachment from the monolayer of [3H]thymidine prelabeled HEp-2 cells in a 24-hr assay with a concanavalin A (Con A) dose of 25 microgram/ml at effector:target cell ratios of 5:1, 25:1, and 50:1. Under these conditions but without Con A considerable natural cell-mediated cytotoxicity (NCMC) was not elicited; however, the cytotoxicity was significantly augmented in the presence of Con A (=LDCC) by sheep erythrocyte rosette-forming T lymphocytes and by both OKT4+ and OKT8+ T-cell fractions. LDCC activity by isolated OKT8+ T cells was superior to that by OKT4+ T cells and unfractionated T lymphocytes. By contrast, addition of either OKT4+ or OKT8+ T cells together with unfractionated T lymphocytes, or OKT4+ and OKT8+ T cells mixed at ratios of 1:1, 1:2, and 2:1, to target cells did not result in major differences in comparison of LDCC activities by these mixed effector cell populations with each other or with that by unfractionated T lymphocytes. Parallel studies were carried out to determine the effect of OKT4+ and OKT8+ T-cell subsets on the Con A-induced proliferation of peripheral blood mononuclear cells (PBMC). While OKT8+ T cells inhibited the mitogenic response to Con A, OKT4+ T lymphocytes had no major effect. A higher responsiveness of the OKT8+ to OKT4+ T-cell subset in LDCC to HEp-2 targets and in Con A-induced lymphocyte proliferation is suggested.     

Association of human lymph node homing receptor (Leu 8) with the TCR/CD3 complex.

Cross-linking of the human homologue of the murine MEL-14 lymph node homing receptor (Selectin-1, LECAM-1, Leu 8) on both T and B cells results in modification of cell function. To investigate this phenomenon, we performed studies to determine if the Leu 8 molecule influences T cell activation via the TCR/CD3 complex. In initial studies, we treated T cells with immobilized anti-CD3 (OKT3 mAb) in the presence or absence of immobilized Leu 8 mAb. We found that although Leu 8 mAb alone had no effect on T cell proliferation, this antibody markedly augmented immobilized OKT3 mAb-induced proliferation. In further studies, we immunoprecipitated surface radioiodinated T cell lysates with OKT3 and Leu 8 mAb to determine if molecules in the TCR/CD3 complex associate with Leu 8 molecules. Although Leu 8 mAb immunoprecipitated only a single protein of approximately 80 kDa from T cell lysates treated with Nonidet P-40 under reducing condition, it coimmunoprecipitated additional proteins of 48, 42, 28, 24, and 22 kDa from T cell lysates treated with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. These additional proteins were identified as the alpha-, beta-, gamma-, delta-, and epsilon-chains of the TCR/CD3 complex by one-dimensional and two-dimensional diagonal SDS-PAGE. Densitometric scanning showed that, on average, 18% of the TCR/CD3 complex associates with Leu 8. In a final study, we showed by immunoblotting analysis using anti-zeta peptide antibody that Leu 8 mAb coimmunoprecipitates the zeta-chain of CD3. These results indicate that the human lymph node homing receptor homologue (Leu 8) participates in the activation of T cells, probably via its association with the TCR/CD3 complex.