Eur. J. Immunol. 1989.19: 1669-1675 Allosuppression zyxwv in zyxwvuts vitro is caused by cytotoxic T lymphocytes 1669 z Lawrence Rozendaal., Steven T. Pals', Marco Schilham., Cornelis J. M. Melief" and Ernst Gleichmann' Central Laboratory of the Netherlands Red Cross Blood Transfusion Service., Division of Pathology', Vrije Universiteitand Division of Immunology', Erasmus Universiteit, Rotterdam, and Division of Immunology", Netherlands Cancer Institute, Amsterdam and Division of Immunology', Medical Institute of Environmental Hygiene at the University of Diisseldorf, Diisseldorf Allosuppression of B cells in vitro by graft-vs.-host reaction-derived T cells is caused by cytotoxic T lymphocytes* An acute graft-vs.-host reaction (GVHR) was induced by i.v. injection of lo8 lym- phoid cells from C57BL110 (B10) donors (H-zb") into adult non-irradiated (B10 z x DBA/2)FI mice (H-2b'd). Previous experiments have established that spleen cells obtained from such GVHFl mice suppress the primary antibody response of normal F1 spleen cells to sheep red blood cells. This type of suppression was termed "allosup- pression", and it was shown to be mediated by Ly-2+ T cells of donor origin that react against H-2 antigens of the host. It was unclear, however, whether the actual mechan- ism of allosuppression was due to suppressive factors generated by donor T cells or whether the latter killed the F1 B cells. Here, we show that for their suppressive effect GVHFl spleen cells need direct cell contact with F1 spleen cells; no suppressive effect was measured in a double-chamber culture system in which the GVHFl spleen cells were separated from the responding normal F1 spleen cells by a cell-tight membrane filter. The suppressive effect only affected cells expressing the appropriate H-2 class I alloantigen; in mixed cultures of irradiated F1 spleen cells and GVHFl spleen cells with third-party B cells the antibody response of the third-party B cells was not suppressed. GVHFl spleen cells showed cytotoxic T lymphocyte (CTL) activity specific for the allogeneic F1 H-2 antigen. The suppressive effect of the GVHFl spleen cells did not differ from that exerted by cloned CTL specific for MHC class I alloanti- gens; cloned CTL suppressed the primary antibody response of murine spleen cells without affecting the response of third-party B cells added to the cultures. The com- bined findings show that "allosuppression" in vitro is not due to suppression of F1 B cells. but to a direct killing of these cells by alloreactive CTL. 1 Introduction Injection of T lymphocytes from the B10 parental strain into non-irradiated F1 hybrid mice induces GVH disease (GVHD). Development of acute GVHD is this experimental setting requires (a) an H-2 incompatibility in the F1 hybrid recipient which involves both class I and class I1 loci and (b) existence in the donor cell inoculum of both Ly-2-positive and -negative T cells [l]. After a transient phase of lymphoid stimulation during the first week of GVH reaction (GVHR), acute GVHD is associated with so-called suppressive pathological symp- toms, such as pancytopenia and hypogammaglobulinemia [l-31. During the latter phase of acute GVHD, activation of Ly-2- donor allohelper T (Th) cells that react against the class I1 incompatibility antigens in the F1 recipient [4] is eclipsed by the activation of alloreactive Ly-2' donor T cells that react against the class I incompatibility antigens [2-41. About 3 days after activation of the alloreactive Ly-2' donor T cells is first detectable, the lymphohemopoietic system of mice suffering from acute GVHD (GVHFl mice) becomes hypoplastic [ l , 4-61. The conclusion that Ly-2+ T cells of [I 76511 zyxwvu ____~ ____ ~ * This work was supported by a grant from the Dutch Foundation for Pure Scientific Research (ZWO-FUNGO). Correspondence: Lawrence Rozendaal, 5 Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406, 1006 AK Amsterdam, The Netherlands Abbreviations: BDF,: (C57BL/lOScSn X DBA/2)FI GVHD GVH disease GVHR: GVH reaction GVHF,: F1 hybrid mice with acute GVHD donor origin are the effector cells that cause the pancytopenia and, hence, suppression of the immune system of GVHFl mice is based on three different pieces of evidence: (a) the well established suppressive effect of alloreactive Ly-2+ T cells on in vitro immune responses [3, 6-91; (b) the requirement of Ly- 2' alloreactive T cells in the donor cell inoculum in order to induce acute GVHD [lo, 111 and (c) the increased activity of Ly-2+ T cells in vivo, briefly preceding and coinciding with the depletion of the immune system of GVHFl mice [4]. When spleen cells obtained from GVHFl mice were added to cultures of normal F1 spleen cells mounting a primary antibody response to SRBC, a strong suppression of the response was noted. Since this phenomenon is the opposite of allohelp it was termed allosuppression [ 121. The requirements for allosup- pression in vitro were (a) presence among the GVHFl spleen cells of parental strain Ly-2' T cells and (b) existence of the MHC class I alloantigens identical to those of the F1 host on the normal spleen cells [2, 9, 121. However, the same require- ments have to be met for a reaction of parental strain CTL. It remained an unresolved question whether allosuppression was actually due to T, cells or to CTL (2, 6-9, 13-17]. Theoreti- cally, T, and CTL differ in that zyx T, cells suppress the function of immunocompetent cells by means other than cytotoxicity [2,9, 13, 14, 181, whereas CTL suppress the function of target cells by killing them [17, 191. CTL require direct cell contact with their target cells, whereas T, cells are thought to operate by the secretion of soluble suppressive factors [20, 21). Yet another mechanism that might account for the phenomenon of allosup- pression is the consumption of lymphokines required for an optimal immune response [22, 231. In that case, suppression should affect different types of B cells regardless of their H-2 make-up. zyxwv 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989 0014-2980/89/0909-1669$02.50/0