CELLULAR IMMUNOLOGY 166, 113–122 (1995) Autogeneic but Not Allogeneic Earthworm Effector Coelomocytes Kill the Mammalian Tumor Cell Target K562 1 EDWIN L. COOPER,* , †ANDREA COSSARIZZA,* MICHAEL M. SUZUKI,† STEFANO SALVIOLI,* MIRIAM CAPRI,* DANIELA QUAGLINO,* AND CLAUDIO FRANCESCHI* *Department of Biomedical Sciences, Section of General Pathology, University of Modena, via Campi 287, 41100 Modena, Italy; and †Laboratory of Comparative Immunology, Department of Neurobiology, University of California, Los Angeles, California 90095-1763 Received April 11, 1995; accepted July 17, 1995 ner of the CTL (2). These two cell types, NK and CTL, share cytolytic effector mechanisms and NK cells ex- Earthworm coelomocytes have been used as effector press the z-chain of the CD3–TCR complex (3). cells against the human tumor target, K562. To first Invertebrates spontaneously kill various targets with- assess the viability of effectors, incorporation of [ 3 H]- out evidence that they possess clonal-specific immuno- thymidine was tested and was higher in autogeneic (A B A, self) than in allogeneic (A B B, nonself) coelomo- cytes such as CTLs (4 – 11), suggesting that NK-like activ- cytes. A B A showed significantly greater numbers in ity is present. Earthworms are an inexpensive, noncon- S, G2, or M phases than A B B coelomocytes. When A troversial model, useful in analyzing cellular and B A or A B B were cultured, no significant cell killing humoral immunodefense because of the presence of at occurred in either, as measured in a 4-hr 51 Cr release least two major different coelomocyte populations (12) assay. A B A but not A B B killed K562 target cells. previously revealed by velocity sedimentation (13). Their Cytotoxicity was dependent upon membrane binding responses are considered (19) by some to be adaptive, between small, electron-dense coelomocytes and tar- induced, and anticipatory (14, 15), in contrast to an alter- gets; it was enhanced by adding PHA. The heat labile native proposal that their responses are nonadaptive, supernatant from A B A but not from A B B killed natural, and nonanticipatory (16). Evidence for adapti- K562 targets after cultivation for 10 min at 22°C, but vity in earthworm cellular immunity includes (1) acceler- not immediately after washing. Recognition of, bind- ated graft rejection (17 – 20); (2) adoptive transfer by cells ing to, and killing of foreign cells in a natural killer from primed donors (21) (revealing short-term memory cell-like reaction may reflect natural immunity in and specificity); (3) independent second-set and third earthworms.  1995 Academic Press, Inc. party rejection times (22); and (4) specific in vivo incorpo- ration of [ 3 H]thymidine in a small cell fraction in response to xenografts but not to autografts nor wounds (23). INTRODUCTION Except for microscopic analyses in vivo, especially during graft rejection (24), evidence for direct and inti- Cell-mediated immunity involves reactions of host mate cell contact has been limited and is only beginning cells to potentially pathogenic organisms and infected, to emerge as defined in this recently developed in vitro tumor, or nonself cells. Both lymphocytes and macro- assay system that allows potential effectors to confront phages can act as effectors without depending primar- self/nonself antigens. Here, we report that earthworm ily upon antibodies. From the viewpoint of evolution, coelomocytes (leukocytes) effect NK-like activity natural killer (NK) cells are more primitive than cyto- against the erythromyeloid human tumor cell line toxic T lymphocytes (CTL), which deploy the complete K562. Lytic activity occurs when autogeneic coelomo- CD3 – T cell receptor (TCR) complex. Although NK cells cytes (A B A) from single earthworms are cocultured have been recently shown to possess clonally distrib- with K562 targets, but not when allogeneic coelomo- uted recognitive mechanisms (1), NK cells still may be cytes (A B B) from more than one earthworm are used. akin to unknown progenitors, permitting them to kill a variety of targets in a major histocompatibility complex MATERIALS AND METHODS (MHC)-nonrestricted manner. Thus, the NK cell, ac- cording to one hypothesis, is an evolutionary forerun- Source and Care of Wild Populations of Earthworms Adult sexually mature (outbred) Eisenia fetida were 1 This paper is dedicated to the memory of Professor Eva Lotzova ´, editor-in-chief of Natural Immunity. purchased from Colombini (Modena, Italy), where they 113 0008-8749/95 $12.00 Copyright  1995 by Academic Press, Inc. All rights of reproduction in any form reserved.