Leukemia (1999) 13 , 230–240  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http:/ / www.stockton-press.co.uk/ leu Leukemic CD3 + LGL share functional properties with their CD8 + CD57 + cell counterpart expanded after BMT L Mollet 1 , B Fautrel 2 , V Leblond 3 , F Bergeron 1 , H Merle-Be ´ral 3 , E Baumelou 4 , P Hubert 1 , P Debre ´ 1 and B Autran 1 1 Laboratoire d’Immunologie Cellulaire et Tissulaire, Ba ˆtiment CERVI, CNRS-UMR 7627, 2 De ´partement de Rhumatologie, 3 De ´partement d’He ´matologie, Ho ˆ pital Pitie ´-Salpe ´trie ˆre, Paris; and 4 Service d’Oncologie et d’He ´ matologie, Centre Me ´dico-chirurgical Foch, Surenes, France Leukemic T-LGL (large granular lymphocyte) composed of clonal CD3 + TCR + CD8  CD57  cells were compared with olig- oclonally CD3  CD8 hi CD57  lymphocytes expanded after BMT. Leukemic CD3  CD8 hi CD57  LGL showed several phenotypic differences such as an upregulation of CD16 and adhesion mol- ecules (mainly CD11c, CD58 and CD54), activation markers and an exclusive CD45RA isoform expression. Unstimulated CD3  CD8  CD57  LGL from both leukemic and BMT donors spontaneously developed an ex vivo CTL-like CD3-redirected cytotoxicity but no NK cell activity. Different stimuli (PHA, PMA or rhIL-2) induced similar cytotoxic profiles after a 6-day culture involving a CD3-redirected lysis predominating over a low NK cell activity. However, culture of leukemic LGL with these stim- uli allowed either a 2 week persistence (PMA or rhIL-2) of CD8  CD57  LGL or their disappearance after 3 days (PHA). Furthermore, leukemic CD8 hi CD57  T lymphocytes produced an inhibitor of cytotoxic functions as previously described for BMT recipients’ CD8  CD57  cells. Thus, despite some pheno- typic differences between both cell sources, leukemic CD57  T-LGL display the same functional characteristics of cytotoxic effector and immunoregulatory T cells as CD8  CD57  T cells from BMT recipients which might represent their normal counterpart. Keywords: T-LGL leukemia; bone marrow transplantation; CD8 + CD57 + lymphocytes; cytotoxicity; immunomodulation Introduction Increased numbers of circulating large granular lymphocytes (LGL) were recognized as a distinct clinical entity in 1977, and this syndrome was characterized by clonal lymphocytosis and tissue invasion. 1 In light of the evidence that clonal LGL proliferations may be either of T cell origin and express the TCR-CD3 complex, or of NK cell origin and are CD3 - cells, the following terminology was adopted: (1) T-LGL leukemia, characterized by clonal CD3 + TCR + CD16 + CD57 + CD56 - LGL proliferation; (2) NK-LGL leukemia, characterized by clonal CD3 - TCR - CD16 + CD57 ± CD56 + LGL proliferation. 2 T- LGL leukemia follows a chronic course during which major features are neutropenia, anemia, bone marrow infiltration and mild to moderate splenomegaly. Multiple immune abnor- malities are frequent (presence of rheumatoid factor, and/or autoimmune antibodies, polyclonal hypergammaglobuline- mia and circulating immune complexes). Rheumatoid arthritis coexists in 28% of patients, who thus resemble patients with Felty’s syndrome. 3 As T-LGL expansions can display an het- erogeneous phenotype, 4–6 in this report we will focus on the non-aggressive and most frequent CD3 + CD16 + CD57 + T-LGL leukemia, ie negative for CD56 and positive for CD8. 3 Perforin, a cytolytic molecule responsible for pore forma- tion in target cells, and TiA-1, a membrane protein from cyto- Correspondence: B Autran, Laboratoire d’Immunologie Cellulaire et Tissulaire, Ba ˆt. CERVI, CNRS-UMR-7525, CHU Pitie ´-Salpe ´trie ˆre, 83 bld de l’Ho ˆpital, 75013 Paris, France; Fax: 33 1 42 17 74 90 Received 2 June 1998; accepted 13 October 1998 lytic granules, were both present in several cases of T-LGL leukemia 7,8 suggesting a lytic potential. Furthermore, FasL, predominantly expressed on and secreted by activated T cells, 9 is constitutively expressed by these leukemic cells and sera from LGL leukemic patients contain detectable levels of sFasL, while sera from healthy persons do not. 10,11 While no spontaneous NK cell activity is usually observed, 5 T-LGL leu- kemic cells can be stimulated after a 24–48 h culture by anti- CD3 Ab or rhIL-2 to acquire NK cell cytotoxicity against K562 target cells. 6,12,13 However, the capacity of their CD3 complex to transduce a signal for cytolysis has not been directly evalu- ated on sorted unstimulated CD8 + CD57 + leukemic LGL. An impairment of T cell proliferative responses to mitogens such as phytohemagglutinin A (PHA) 14 has also been reported as well as a defect in the formation of high-affinity IL-2 recep- tors, 12 despite constitutive expression of functional p75 inter- mediate-affinity IL-2 receptors. 13,15 However, these leukemic LGL cannot be considered as anergic cells since they can also proliferate in vitro after the combinative signal of anti-CD3 and either IL-2 or IL-4 16 and can be maintained in long-term cultures for as long as 1 or 2 months. 17 CD3 + LGL leukemia are also highly sensitive to the combined stimulation by anti- CD3 + rhIL-12, mediated by upregulation of IL-12 receptor upon anti-CD3 ligation. 18 Taken together, these results show that leukemic T-LGL can proliferate after activation through their CD3-TCR complex in the presence of exogenous lymphokines. Since leukemic CD3 + T-LGL usually display the CD57 marker, we postulated that their normal counterpart might be the CD3 + CD8 + CD57 + T lymphocytes. This normal subset is frequently expanded in vivo after chronic antigen-specific stimulation of CD8 + T cells due to viral infections (human immunodeficiency virus (HIV) or cytomegalovirus (CMV) ) or allogenic transplantations and displays the cytological charac- teristics of LGL. 19–23 In this case, these expanded cells are not clonal although a remarkable restriction of the TCR V usage by CD8 + CD57 + cells could be evidenced after BMT. 24 A simi- lar oligoclonality of the expanded peripheral CD8 + CD57 + sub- set has recently been confirmed in CMV-infected patients. 25 Both findings were suggestive of an antigen-driven skewing of the TCR repertoire in these cells. Thus, based on their cytolo- gical features, CD3 + CD8 + CD57 + T cells appear as the normal counterpart of the leukemic CD3 + CD57 + LGL, although their functional characteristics have never been compared until now. Our group and others showed that non-leukemic CD8 + CD57 + lymphocytes mediate immunoregulatory func- tions. Indeed, the CD8 + CD57 + cells, initially reported to down-modulate B cell differentiation and CD4 + T cell prolifer- ation, inhibit the CD8 + T cell or NK cell cytotoxic activity. 19,21,26–29 Our group further showed that CD8 + CD57 + T cells mediate this inhibitory activity by releasing a soluble 30 kDa glycoprotein (ICF). 19 In contrast, they can also mediate a killer cell activity after non-specific activation, 30–32 and we recently showed their capacity to develop a peptide-specific