Biological, biochemical, and immunological properties of allogeneic effect factor (AEF)

WORKSHOP

ON

LYMPHOCYTE

333

MEDIATORS

that the receptor on T-cells is not a classical Ia antigen or the genes coding for the Ia product of GRF and for the receptor are different. These results suggest that the il~ z&o induction of T-helper cells is controlled by at least two genes, both located in the I-,4 subrrgion, one is expressed in M and the other in certain T-cells. NMF, which is obtained from M incubated without antigen, is not genetically restricted. N&IF can generate T-helper cells in the absence of M provided particulate antigen is present. Further studies showed that NMF interacts directly with Tz-cells (insensitive to ATx), whereas GRF binds to T1-cells (sensitive to ATx, insensitive to ALS) rendering them able to activate Ty-cells. (Supported by Grant 3.386.74 of the Swiss National Funds.) EGdeme Help.

for a B-Cell Subset Resporhe DAVID

D. WOOD, Merck

Institute,

to Plkrificd B-Ccl1 Actiztathg Rahway, New Jersey.

Factor

and Not

T-Cell

Tests were conducted to determine whether the murine B-cells which are stimulated to secrete IgM by the human monocyte product “B-cell activating factor” (BAF) are the same cells stimulated by T-cell help. When the BAF effect was compared in the presence and absence of T cells, the magnitude of the BAF effect was found to be similar (J. Itatrmknol. 114, 1094, 1975), suggesting that BAF may stimulate a B-cell subset distinct from the T-cell responsive subset. Studies of the Poisson distribution of responses to heterologous erythrocytes by limiting numbers of murine B-cells supported the possibility that the BAF-responsive and T-cell-responsive B-cells were independent subsets. Further, it was found that the BA4Fresponsive cells occurred more frequntly than did th Igl,I-secreting cells in unstimulated cultures, suggesting that B.;ZF does not simply stimulate the background. The subset hypothesis was independently supported by the observation that nude splenocyte cultures subjected to a lethal pulse of [“Hlthymidine after stimulation with antigen and educated T-cells still responded to B:ZF but not to readdition of educated T-cells. These results suggest that two B-cell subsets exist; one is stimulated to IgRi production in the presence of a T-dependent antigen and BAF, while the other requires antigen and T-cell help. Attempts to prove the hypothesis by physically separating the subsets are in progress. Detection

of Synergiziq

FARRAR,

WILLIAM

Helper J. KOOPUAN,

Factors

dztirimJ the A+ttiBody

JOOST J. OPPENHEIX

, AND

Respome JANET

is Vitro.

FULLER-B•

NAR,

JOHN

J.

NIH,

Bethesda, Maryland. The purpose of this study was to examine the mechanisms by which soluble helper factors enhance the i+t vitro anti-SRC PFC response of “T-cell-deficient” mouse spleen cells. Xenoa supernatant from t\\-o-\vay human MLC. enhances the geneic reconstitution factor (XRF), PFC response of SRC-immunized T X B mouse spleen cells. XRF-mediated restoration of the PFC response was shown to be dependent on (1) synergizing early and late acting helper components, and (2) the mandatory coparticipation of the residual T- or pre-T-cells in the “B-cell population.” Sephadex G-75 fractionation of XRF revealed two helper peaks which individually exhibited minimal activity, but which together fully restored the response. The low molecular weight component (11,000 to 13,000 daltons) was shown to be: (1) early acting, (2) chromatographically inseparable from lymphocyte activating factor (XLAF), and (3) generated in the absence of allogeneic stimulation. The high molecular weight peak (40,000 to 75,000 daltons) (1) contained at least two factors which synergized with XLAF and (2) presumably contained the late acting helper component. The results suggest that induction of the PFC response was dependent on XLAF-mediated activation of the residual T-cells which subsequently provided an initiator of helper function to the E-cells and a second XRF component of higher molecular weight which included terminal differentiation of the FFC precursors. liiological,

Biocltr~~~ical, and Im~r~unologicnl Prngcrtics of /!llogeneic Effect Fnctor (AEF). H. I
The properties of a biologically active lymphocyte cultures of alloantigen-activated

factor obtained from short-term i?z rho mixed T-cells and appropriate target cells, termed allo-

334

WORKSHOP

ON

LYMI’IIOCYTE

MEDIATORS

geneic effect factor (AEF), were reviewed. The salient features of AEF include: (1) its nonspecific biological activity on B-lymphocyte triggering and differentiation in z&o; (2) its capacity to enhance certain T-cell function ilz z&o; (3) the presence on it of Ia antigenic determinants ; (4) its association with ,%microglobulin ; and (5) its apparent bicomponent structure. It is clear that AEF is not a product of macrophages, nor are macrophages (in appreciable quantities) required for its production in vitro. Moreover, the presence of both activated T-cells and target cells are required for its production in vitro. What is not yet clear is the precise cellular origin(s) of the active moieties since recent evidence has suggested that AEF may, at least in part, be a product of B-lymphocytes in the target and/or responder cell population. Interaction. BRUCE H. LITTMAN, Human Mitogenic Factor: An Example of T-B Lymphocyte AND JOHN R. DAVID, The Medical College of Virginia, Richmond, Virginia, and Robert B. Brigham Hospital, Harvard Medical School, Boston, Massachusetts. Mitogenic factor is produced by T-lymphocytes stimulated by specific antigen or mitogen. It has previously been reported that human B-lymphocytes cannot proliferate in response to specific antigen although they do proliferate in response to mitogenic factor. B-lymphocytes may therefore be used to assay for mitogenic factor activity even in the presence of antigen. This example of T-lymphocyte control of a B-lymphocyte response has been studied. We have confirmed that mitogenic factor, unlike macrophage migration inhibitory factor (MIF) and some other mediators, is exclusively a T-lymphocyte product. Mitogenic activity is found after 20 hr of culture with specific antigen and maximal production occurs after 48-72 hr. Ten times less mitogenic factor is required to stimulate B-lymphocytes than T-lymphocytes with the peak response occurring after 3-4 days of culture. The magnitude of the B-lymphocyte response to mitogenic factor is also much greater than the T-lymphocyte response to ihe same preparation. The molecular weight of the active material was estimated to be 24,000 by gel filtration. There is evidence that mitogenic factor production by T-lymphocytes requires monocytes. Further, preliminary evidence indicates that the production of, or response to, mitogenic factor is regulated by a soluble suppressor substance. T-Cell Mediator Induction of Non-T-Cell O’NEILL, University of Texas Health Texas.

Cytotoxicity. Science Center,

BRUCE F. MACKLER AND PEGGY Dental Science Institute, Houston,

Evidence was found indicating a T-cell-derived mediator which induced non-T-cell-mediated nonspecific cytotoxicity. Lymphoid subpopulations from normal human donors were assessed for cytotoxicity. No cytotoxicity was obtained with either mononuclear cells or T-cells separated by sheep E rosetting. In contrast, the remaining non-E-rosetting cells were cytotoxic for autologous and allogeneic target cells in “Cr release and direct microcytotoxicity assays in trays. Activation of non-E-RFC cytotoxicity required continued binding of sheep E to T-cells and did not involve removal of suppressor cells. The E-RFC incubated at 4, 10, and 18°C released mediators which induced non-T cytotoxic effector cells; incubation at 37°C caused dissociation of E-RFC and cessation of mediator production. The E-rosetting supernatants (ERS) contained neither MF, PIF, LT, nor MIF activities. These cytotoxic non-T-cells were esterase negative, nonphagocytic, glass-adherent, C3 receptor-bearing cells with lymphoid morphology (presumably B-lymphocytes). The nonspecific cytotoxicity appeared to be mediated no evidence of antibody was found. These by LT released by ERS-activated non-T-cells; results suggested that T-cells released mediators (ERS) which activated non-T-cells to produce lymphotoxin (LT). (Supported by Grants DE-04210 and DE-04172.) Characterization PHILIPPA University

and Mode of Action of a Nonspecific T-Cell-Derived Helper Mediator. MARRACK, JOHN W. KAPPLER, LEE W. HARWELL, AND DANIEL M. KELLEP, of Rochester.

The properties of a nonspecific mediator (NSM) which replaces helper T-cells in tlrc in z&o response of mouse B-cells to red blood cell antigens (RBC) were investigated. NSMs with indistinguishable properties were produced in cultures containing macrophages