Stimulation of autologous and allogeneic human T cells by B cells occurs through separate B-cell antigen systems.

Studies were performed to determine the cell types involved in autologous mixed lymphocyte reactions (AMLR). Separation of peripheral blood leucocytes from normal donors into T-cell and B-cell enriched populations and subsequent co-culture of T cells and mitomycin-C inactivated non-T cells resulted in increased DNA synthesis by the responding T cells. Procedures such as removal of plastic-adherent or phagocytic cells enriched the B-cell content of the stimulator population and also enhanced stimulation of both autologous and allogeneic T cells. Velocity sedimentation separation of peripheral blood leucocytes into large and small lymphocyte fractions showed that cells which stimulate in MLR and AMLR are found in the small cell fraction and that large cells, morphologically monocytes, do not stimulate either reaction. Studies with anti-B-cell antisera obtained from multiparous women showed that the antigens of B cells which stimulate AMLR are distinct from those which stimulate MLR.     

Activated human T cells can present alloantigens but cannot present soluble antigens

Human T cells, when activated by antigen or mitogen, express Ia antigens. We have examined the capacity of activated T cells to stimulate autologous and allogeneic T cells and their ability to present soluble antigen. Interleukin 2-dependent T-cell lines (TCL), free of accessory cells, were used for antigen-presenting cells. These activated T cells were potent stimulators in an autologous mixed lymphocyte reaction (AMLR), more so than autologous irradiated non-T mononuclear cells. Activated T cells were also able to stimulate proliferation of allogeneic T cells in the absence of any other accessory cells, and this stimulation was blocked by anti-Ia antibodies. Resting unstimulated T cells were unable to stimulate autologous or allogeneic responses. Thus, activated T cells were able to present self antigens and alloantigens. However, activated T cells could not present soluble antigens to autologous T cells or to antigen-specific TCL even if exogenous interleukin 1 was added to cultures. The ability of activated T cells to stimulate an AMLR in vitro may reflect an important immunologic amplification mechanism in vivo. The ability of activated T cells to present alloantigens but not soluble antigens suggests an inability to process antigen, and this may provide further insights into the complexities of antigen presentation.     

Activity of a partially purified human BCGF on murine assays for B-cell stimulatory factors. I. BCGF II-like activity of human BCGF

To further characterize a human B-cell growth factor (BCGF) produced by phytohemagglutinin (PHA) P-stimulated peripheral blood T cells, a partially purified preparation of this material was tested in a number of murine assays for B-cell stimulatory factors (BSF). Human BCGF lacked murine BSF-1 activity as assessed via the induction of polyclonal proliferation of anti-IgM-stimulated murine B cells; however, this material consistently augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1. Human BCGF also induced proliferation in unstimulated murine B cells, and augmented the proliferative response of dextran sulfate activated murine B cells. Human BCGF is therefore capable of causing proliferation of unstimulated and activated murine B cells, and by these criteria closely resembles murine BCGF II. In contrast to murine BCGF II, however, human BCGF failed to stimulate proliferation or immunoglobulin (Ig) secretion by murine BCL1 B lymphoma cells. A murine analog of this human BCGF showing the same pattern of biological responses was found in concanavalin A-stimulated supernatants of the murine MB2.1 T-cell line and D9-Cl T-cell hybridoma. The active component of the human BCGF preparation was not due to contaminating PHA, interleukin 1, interleukin 2; interferon-gamma, or endotoxin. Comparison between the above human BCGF and a commonly used source of murine BCGF II, i.e., supernatant from antigen-stimulated D10.G4.1 T cells, provided information suggestive of BCGF II heterogeneity. Both human BCGF and D10.G4.1 supernatant caused proliferation of unstimulated and dextran sulfate-stimulated murine B cells; however, only the human BCGF preparation augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1, and only the D10.G4.1 supernatant stimulated BCL1 cell proliferation and immunoglobulin secretion. The data therefore indicate that the different assays for BCGF II used in this study respond to different factors, and suggest the existence of two BCGF II-like activities.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

Regulation of the growth of Epstein-Barr virus-infected B cells. I. Growth regression by E rosetting cells from VCA-positive donors is a combined effect of autologous mixed leukocyte reaction and activation of T8+ memory cells

Regression of B cell proliferation in co-cultures of EBV-infected B cells (BEBV) and autologous T cells at 1:4 ratio was studied. 3H-TdR incorporation was used to measure proliferation by the participating lymphocyte populations and a 51Cr release assay was used to document the generation of cells capable of killing autologous EBV-transformed B lymphoblastoid cell lines (LCLEBV). EBV-infected B cells cultured alone transformed to blasts by culture day 10, and continued to proliferate throughout the 22 day observation period. When EBV-infected B cells were co-cultured with E rosetted cells from VCA-positive donors, there was a characteristic proliferative response on day 10 (an augmented autologous mixed lymphocyte reaction; AMLR), followed by the development of T8+ cells capable of killing autologous LCLEBV, as well as over 90% suppression of EBV growth by day 22 as assessed by 3H-TdR incorporation, and confirmed in a visual outgrowth assay. Negative and positive selection techniques were used to define the regulatory components in the T cell population. Depletion of T8+ cells from the blood lymphocytes of VCA-positive donors did not significantly reduce the 10 day proliferative response, but the subsequent development of cytotoxic cells and the regression of BEBV outgrowth was not observed. Thus, the circulating T8+ cells are required for the subsequent appearance of autologous LCLEBV cytotoxicity and BEBV growth regulation. However, when the responder population consisted only of T8+ cells, the augmented AMLR response was absent, cytotoxic cell development was weak or absent, and there was no regression of EBV outgrowth. Therefore, the cells participating in the AMLR, as well as T8+ memory cells from VCA-positive donors, are necessary for the control of the in vitro EBV infection. Growth regression is dependent on the proliferation of the regulatory T cells. Mitomycin C treatment of fresh E rosetting cells or those exposed to BEBV for up to 10 days in culture abrogates growth regression and the subsequent appearance of LCLEBV killer cells. However, E rosetting cells exposed to BEBV for 14 days or more already have developed the ability to kill LCLEBV and no longer need to proliferate to induce growth regression when cultured with newly infected BEBV. These results lend additional support to the view that the control of EBV-induced B cell expansion requires a AMLR-dependent clonal amplification of EBV-specific, T8+ cytotoxic cells.     

Lymphocyte transformation induced by autologous cells. III. Lymphoblast-induced lymphocyte to stimulation does not correlate with EB viral antigen expression or immunity.

Human mitogen-induced and cell line B lymphoblasts stimulate the proliferation of allogeneic and autologous lymphocytes in culture. The role in thes reaction of EB viral determinants on the stimulating cells and immunity of the lymphocyte donor to the EB virus has been studied. The stimulatory capacity of cultured cell line lymphoblasts is not inhibited by incubating lymphoblasts with antisera to EB viral determinants. Cultured cell line B lymphoblasts stimulate as much thymidine incorporation by lymphocytes from donors with or without immunity to the EB virus. Further, a B lymphoblast cell line (U-698) which lacks the EB viral genome stimulated as much lymphocyte proliferation as did B lymphoblasts with the EB genome. Cultured T lymphoblast cell lines do not stimulate allogeneic lymphocyte proliferation. These cells appear to lack the determinants which stimulate lymphocyte transformation. No evidence was found that cultured cell line T lymphoblasts suppressed allogeneic lymphocyte proliferation. Mitogeninduced lymphoblasts from EB-immune and non-immune subjects stimulated the proliferation of autologous lymphocytes comparably. It is concluded that neither immunity to the EB virus nor expression of EB viral antigens on mitogen-induced on cell line lymphoblasts is necessary for the stimulation of lymphocyte proliferation.     

Induction of T-cell proliferation and enhancement of NK activity by supernatants from Con A-stimulated human peripheral blood mononuclear cells: a new lymphokine

Supernatants from human peripheral blood mononuclear cells activated by Con A contain a factor(s) that stimulates blastogenic activity of normal human peripheral blood mononuclear cells. This Con A supernatant (CAS) contains stimulatory activity for E-rosette positive lymphocytes (T cells) and requires adherent cells for stimulation of T-cell proliferation. CAS does not contain detectable amounts of IL-2 as determined by its inability to support CTLL cell growth. Nor does it contain IL-1 or interferon. Examination of functional activity of lymphocytes stimulated for 3 days by CAS revealed that NK activity is augmented. This supernate does not appear to have any direct effect on B-cell function, although it induces suppression of polyclonal PWM stimulation of immunoglobulins. Thus, CAS appears to contain a new cytokine with immunomodulating potential.     

The alpha chain,not the beta chain of HLA-DR antigens participates in activation of T cells in autologous mixed lymphocyte reaction

Using antisera against the alpha, the beta or the complex of both chains of HLA-DR antigens, we have studied the role of individual chains of HLA-DR antigens in activation of T cells in autologous mixed lymphocyte reaction (AMLR). Alpha chain-specific antibody, not anti-beta chain serum prevented T cells from acquiring responsiveness to interleukin-2 (IL-2), suppressed the production of 1L-2, and inhibited the T cell proliferative response in both primary and secondary AMLR cultures. However, proliferation of already activated IL-2 reactive T cells supported by IL-2 was not affected by any of the four different types of anti-DR sera used. Fifty to sixty percent of T cells activated by AMLR or by PHA possessed DR antigens and functioned well as stimulator cells in secondary AMLR cultures. Moreover, the stimulatory activity of these DR-positive T cells was suppressed by the anti-alpha chain, not by the beta chain-specific antibody. Since continuous proliferation of T cells requires IL-2 and since nonactivated T cells are not sensitive to IL-2 and are unable to absorb this growth factor, we conclude the following: (1) The alpha, not the beta chain of HLA-DR antigens seems to be the structure responsible for enabling resting T cells to respond to IL-2 and induce production of IL-2 in AMLR. (2) Once T cells have acquired responsiveness to IL-2 and the growth factor has been produced, there is no further requirement for HLA-DR antigens, but the availability of IL-2 determines the level and extent of proliferation of IL-2 sensitive T cells.     

Autologous mixed-lymphocyte reaction in man. XVII. In vitro effect of ion channel-blocking agents on the autologous mixed-lymphocyte response

The in vitro effect of ion channel-blocking agents verapamil (V), 4-aminopyridine (4AP), tetraethylammonium (TEA), and quinine (Q) was examined on the proliferative response of human peripheral blood T lymphocytes in the autologous mixed-lymphocyte reaction (AMLR). All the above channel blockers in a dose-dependent manner inhibited the AMLR. Tetramethylammonium (TMA), an analog of TEA that does not block K+ channel currents, did not inhibit the AMLR. 4AP at 1 mM/ml concentration inhibited the expression of IL-2 receptors, as defined by monoclonal antibody anti-Tac, on T-cell activated in the AMLR. In vitro addition of recombinant interleukin 2(rIL-2) completely corrected the inhibition of the AMLR by channel blockers. Furthermore, the concentrations of ion channel blockers required for blocking 50% response of T cells in the AMLR was much lower than that reported for 50% block of T-cell proliferation in response to phytohemagglutinin or in allogeneic mixed-lymphocyte culture (MLC). These data suggest a role of ion channels in T-cell functions and show that the AMLR provides a more sensitive system, as compared to lectin stimulation or MLC, to examine any immunosuppressive effects of ion channel-blocking agents in disease states where they are used as therapeutic modalities.     

Spontaneous proliferation in unfractionated spleen cell cultures: autologous mixed-lymphocyte reactions (AMLR) which can be differentially regulated by prostaglandins and lymphokines

The present studies were undertaken to define the contribution of the autologous or syngeneic mixed-leukocyte reactions (AMLR/SMLR) to the cellular proliferation observed in unfractionated spleen cell cultures. Proliferation was studied in whole, untreated 6-day murine spleen cell cultures supplemented with syngeneic serum. These cultures exhibited relatively low but significant levels of cellular proliferation as measured by uptake of radioactive thymidine ([3H]TdR). Treatment of spleen cells with monoclonal anti-Thy 1.2 antibody and complement before culture, the addition of specific anti-I-A monoclonal antibodies to the cultures or removal of Ia+ adherent cells before initiation of culture all inhibited the proliferative response significantly. Thus, the autologous proliferation of untreated and unfractionated spleen cells manifests the main characteristics of the AMLR/SMLR, namely, its dependence on T (responder) and Ia+ (stimulator) cells and specific inhibition by anti-I-A antibodies. A marked augmentation in cellular proliferation was observed in unfractionated spleen cell cultures treated for the initial 24 hr of culture with 5 X 10(-6) M indomethacin, an inhibitor of prostaglandin synthesis. Conversely, the addition of 7 X 10(-9) M prostaglandin E1 (PGE1) to these cultures depressed cellular proliferation. This suppression of autologous splenic cell proliferation induced by PGE1 could be partially reversed by the addition of concanavalin A-induced lymphokine (LK) preparations early in the culture. These findings indicate that (a) the proliferation of unfractionated spleen cell cultures occurring in the absence of exogenous stimulatory signals is due largely to an ongoing AMLR, and (b) biologically active mediators with opposing influences, namely, prostaglandins and immunostimulatory LK, participate in the regulation of the AMLR.     

Effects of monoclonal antibodies against lymphocyte surface antigens on interleukin 2 excretion by Epstein-Barr virus-specific human T-cell hybridomas

Monoclonal antibodies were used as probes to study the role of cell surface antigens in the response of Epstein-Barr virus (EBV)-specific human T-T hybridomas to autologous EBV-infected B lymphoblasts. Somatic cell hybrids were generated by fusing EBV-primed peripheral blood T lymphocytes with a mutant clone of the JM human T-lymphoblastoid-cell line. When exposed to autologous EBV-infected B lymphoblasts, the resulting hybrid clones released Interleukin 2 into the culture medium. Incubation of the EBV-infected B cells with two monoclonal antibodies against human Ia-like molecules blocked their ability to trigger the hybridomas. Under the same conditions, monoclonal antibodies against beta 2-microglobulin, and a 45,000 MW surface antigen common to EBV-infected B lymphoblasts, did not alter the capacity of the B cells to stimulate the hybridomas. None of four monoclonal antibodies against surface antigens on the T-cell hybridomas impaired their responsiveness to EBV-infected B lymphoblasts. These results suggest the possibility that naturally occurring or exogenously administered antibodies against Ia molecules might interfere with T-cell regulation of EBV-induced B-cell activation.     

Interleukin 2 induces proliferation of normal "resting" human T cells in the absence of other known external stimulation

Interleukin 2(IL-2) is known to stimulate the progression of activated T cells from G1 through the rest of the cell cycle. We have demonstrated that addition of purified recombinant human IL-2 (rIL-2) to fresh normal human peripheral blood mononuclear cells (PBM), which were IL-2 receptor (Tac) negative by FACS analysis, stimulated marked proliferation of the PBM. IL-2-induced proliferation was also observed with umbilical cord blood mononuclear cells. Monocyte depletion of PBM resulted in a marked reduction of rIL-2-induced proliferative response which could be restored by adding back autologous irradiated monocytes but not by interleukin 1. The T cells preincubated with rIL-2 showed a five to six times enhanced autologous mixed-lymphocyte reaction (AMLR) compared to controls. The rIL-2-induced proliferative response of PBM was inhibited in a concentration-dependent fashion by preincubation of PBM with an anti-HLA-DR framework monoclonal antibody. The proliferating cells were shown by two-color flow cytometric analysis to be primarily Leu-1+ and Leu-4+ T cells (both leu-3+ and Leu-2+ subsets); however, 6 to 19% of responding cells had surface markers for B cells or NK cells. The data demonstrate that rIL-2 can induce proliferation of "resting" human T cells. The phenomenon may be related to a monocyte-dependent AMLR which induces IL-2 receptors and IL-2 responsiveness in a subset of T cells.     

Role of class II histocompatibility antigens in Staphylococcus aureus protein A-induced activation of human T lymphocytes

The capacity of peripheral blood monocytes and B lymphocytes to support staphylococcal protein A (SpA)-induced proliferation of autologous and allogeneic T cells, as well as the role of major histocompatibility complex (MHC) class I and II molecules in this activation process, were investigated. Highly purified peripheral T lymphocytes did not proliferate in response to SpA, but their response was reconstituted by both irradiated (or mitomycin C-treated) monocytes and B lymphocytes. The effect of B cells on the SpA-induced T-cell response could not be explained by a contamination of residual accessory cells because long-term continuous B-cell lines restored SpA-induced T-cell DNA synthesis as effectively as did monocytes. Support of SpA responsiveness by B cells could not be accounted for by polyclonal binding of SpA to cell surface immunoglobulins, since the ability of SpA-unreactive and SpA-reactive B cells was comparable. The cells from two human leukemic lines--K562 and Raji--showed the same ability in supporting the pokeweed mitogen-induced T-cell response, but the class II-positive Raji cells were much more effective than class II-negative K562 cells in restoring the T-cell responsiveness to SpA. Monoclonal antibodies specific for monomorphic determinants of MHC class II antigens, as well as their F(ab')2 fragments, consistently inhibited the SpA-induced proliferative response, whereas antibodies specific for MHC class I antigens were without effect. The antibodies specific for class II antigens appeared to act at the level of accessory cell, since pretreatment with these antibodies inhibited the ability of SpA-pulsed monocytes or Raji cells to present SpA to autologous or allogeneic T lymphocytes, respectively. These data indicate that either monocytes or normal and lymphoblastoid B cells can act as accessory cells for the proliferative response of human T cells to soluble SpA and that monomorphic determinants of MHC class II molecules play an important role in this activation process.     

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.     

Stimulation of mouse B cells by a factor that coisolates with T-cell proteoglycan

We have isolated a factor that copurifies with chondroitin sulfate proteoglycan secreted by mouse splenocytes and some murine T-cell hybridomas. This factor will stimulate proliferation and plaque-forming cell differentiation of B lymphocytes from mouse spleens, even after T cells have been depleted (less than 2% Thy 1.2-bearing cells). Adherent macrophages enhance the activity of this factor, but their function can be replaced in macrophage- and T-cell-depleted populations by small concentrations of a protein mitogen from Salmonella typhimurium. The stimulatory fraction contains chondroitin sulfate, a major protein which has a molecular weight of 74,000 and a minor moiety at 50,000. Stimulatory activity of this material is destroyed by (i) boiling, (ii) mild alkali treatment, and (iii) protease digestion. It is unaffected by RNase and chondroitinase treatments, suggesting that the factor is a protein. Our data define a new B-cell stimulatory substance(s) and suggest that it may be associated with chondroitin sulfate proteoglycan secreted by immune cells.     

Regulation of T cell proliferation by cloned interferon-alpha mediated by Leu-11b-positive cells

The autologous T lymphocyte proliferative response (AMLR) induced by a B lymphocyte-enriched non-T, nonadherent cell population (NT, NAC) and by a macrophage-enriched population were both suppressed by the addition of a cloned interferon-alpha (IFN-alpha Con1) directly to the cultures. Preincubation of the stimulating NT, NAC with IFN-alpha Con1 resulted in comparable suppression. In contrast, preincubation of the macrophages with IFN-alpha Con1 resulted in significant augmentation of T cell proliferation. Depletion of Leu-11b-positive cells from the NT, NAC exposed to IFN-alpha Con1 restored the autologous T cell response. Addition of IFN-alpha Con1 activated Leu-11b-positive cells, isolated from the NT, NAC population, was suppressive of the AMLR. Although NK cytotoxicity was irradiation sensitive, suppression of the AMLR by IFN-alpha Con1-activated NT, NAC was resistant, suggesting that different subsets of cells or mechanisms by the same cells may have been responsible. These observations may offer insights into the potential role of cells with the NK phenotype, Leu-11b, and IFN in contributing to immuno-regulatory changes observed in clinical states associated with elevated concentrations of IFN.     

The role of dendritic cells as stimulators of minor lymphocyte-stimulating locus-specific T cell responses in the mouse. I. Differential capacity of dendritic cells to stimulate minor lymphocyte-stimulating locus-reactive T cell hybridomas and the primary anti-minor lymphocyte-stimulating locus mixed lymphocyte reaction

The response of T cells to minor lymphocyte-stimulating locus (Mls) determinants remains poorly understood with respect to the antigenic determinants responsible for T cell stimulation and the types of APC capable of stimulating the response. In this report, we demonstrate that highly purified dendritic cells (DC) as well as B cells have the capacity to stimulate Mls-specific responses. Unseparated spleen cells, purified DC, resting B cells, and activated B cells were compared for their capacity to stimulate several Mls-reactive T cell hybridomas. Whereas the entire panel of Mls-reactive T cell hybridomas was stimulated strongly by unseparated spleen cells and activated B cells, the hybridomas responded only weakly to purified DC or resting B cells. Activation of resting B cells with either B cell stimulatory factor-1 (1 day pre-treatment) or LPS/dextran (2 or 3 day pre-treatment) greatly augmented their Mls-stimulatory capacity. In contrast, the Mls-stimulatory capacity of DC was not augmented by a 1-day pre-treatment with either B cell stimulatory factor-1 or supernatant from the DC-induced primary anti-Mls-MLR. In the primary anti-Mls-MLR, both purified DC and LPS/dextran-stimulated B blasts were found to elicit vigorous T cell proliferative responses. Much weaker responses were elicited by unseparated spleen cells. The stimulation of the primary anti-Mls-MLR by purified DC was further confirmed by producing Mls-specific T cell clones which were preferentially stimulated by DC. Autologous (Mlsb) DC were found to markedly enhance the primary anti-Mls-MLR response to small numbers of Mlsa B blasts. Thus, DC possess other "accessory cell" properties that augment the primary anti-Mls-MLR despite the predicted low level of Mls determinant expression on DC based on the results obtained with Mls-reactive hybridomas. Possible accessory cell properties of DC relevant to this phenomenon are discussed.     

Regulation of B lymphocytes by natural killer cells. Role of IFN-gamma

Using a co-culture system of fractionated B cells and highly purified NK cells, we have demonstrated direct interactions between B lymphocytes and NK cells. B cells are able to stimulate the production of IFN-gamma by NK cells. This stimulatory ability is restricted to a subpopulation of large, presumably in vivo activated B lymphocytes. The secreted IFN-gamma in turn inhibits polyclonally induced B cell proliferation. Small resting B cells neither stimulate IFN-gamma production nor are they measurably affected by NK cells.     

Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE): II. Functional Properties of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions

Counterflow centrifugal elutriation (CCE), a technique which separates cells by size and density, was used to separate human peripheral blood mononuclear cells into fractions enriched for T lymphocytes, B lymphocytes, and monocytes. These morphologically and phenotypically distinct fractions were analyzed for their ability to respond in several functional assays. B-Cell-enriched fractions devoid of monocytes did not proliferate nor produce significant quantities of lymphokines in response to antigens. These B cells did proliferate to anti-IgM antibodies but not to anti-IgD antibodies. B-Cell fractions served as stimulators of the autologous mixed-lymphocyte reaction (AMLR). T-Lymphocyte fractions were unable to respond to antigen challenge, but both proliferation and lymphokine production could be restored by the addition of monocytes. Monocyte fractions produced PGE₂, displayed chemotaxis, and functioned as stimulators in the AMLR. Thus, CCE appears to be a useful technique for reproducibly obtaining highly enriched subsets of human peripheral blood mononuclear cells with unique phenotypic and functional properties. These isolated populations can consequently be used to identify the independent and collaborative roles of the cells in immunological events.     

Interferon gamma modulates the ability of autologous non-T cells to stimulate T cells to produce and respond to interleukin 2

The interactions of T-cell receptor with self-Ia antigen on non-T cells induced IL-2 production and IL-2 receptors on the cell surface and thus responsiveness to IL-2 of T cells in autologous mixed-lymphocyte reaction (AMLR). Four-day-cultured autologous non-T cells lost their ability to stimulate T cells to produce and respond to IL-2 with concurrent decrease of HLA-DR and HLA-DQ antigen expressed on the cell surface. Culturing of non-T cells with 500 U/ml of recombinant interferon gamma (IFN-gamma) maintained their stimulating ability which was otherwise lost. Treatment of non-T cells with monoclonal anti-HLA-DR or anti-HLA-DQ antibody before mixture with T cells abrogated their ability to induce IL-2 production and IL-2 responsiveness of T cells. The combined data suggested that Ia antigen expressed on non-T cells is modulated by IFN-gamma, which increases the ability of non-T cells to stimulate autologous T cells to produce and respond to IL-2.