The coexpression of CD45RA and CD45RO isoforms on T cells during the S/G2/M stages of cell cycle.

The tyrosine phosphatase CD45 is alternatively spliced to generate isoforms of different molecular weights (180-220 kDa) which are differentially expressed on hematopoietic cells. Monoclonal antibodies reacting with either the 180-kDa (UCHL-1, CD45RO) or the 200- to 220-kDa (2H4, CD45RA) isoform have been used to subdivide T cell populations based on their expression of one or the other of these two epitopes. CD45RA T cells have "naive" characteristics of unresponsiveness to recall antigens and prominence in cord blood, while CD45RO T cells are considered "memory" T cells because they proliferate to recall antigens and increase following PHA activation of cord blood. However, we have recently demonstrated the expression of the CD45RA isoform on a subpopulation of CD45RO+ T cell clones, suggesting that CD45RA is not a universal marker for naive T cells. Using propidium iodide staining of the DNA to determine cell cycle stage, we now show that CD45RA expression is significantly higher on T cell clones during the S, G2, and M stages of cell cycle when compared to CD45RA expression on cells in Go and G1. Furthermore, CD45RA expression on cells undergoing mitosis is not limited to long-term activated T cell clones, as uncultured peripheral blood T cells in the S/G2/M phase express significantly more CD45RA. The percentage of T cells coexpressing CD45RA and CD45RO also increases following PHA activation, indicating that T cells in the process of division express both isoforms. These results suggest a potential role of the CD45RA isoform during the stages of cell cycle leading to mitosis.     

Distribution and functional analysis of a 120- to 130-kDa T-cell surface antigen

A monoclonal antibody (mAb), SPV-L14, was raised that detected a human T-cell surface antigen with a molecular weight (MW) of 120 kDa on resting and phytohemagglutinin-activated peripheral blood T lymphocytes (PBL). An additional band with a MW of 130 kDa could be precipitated with variable intensities from thymocytes, neoplastic T cells, and CD4+- or CD8+ T-cell clones. Based on their reactivity with SPV-L14 and a mAb directed against CD3, four subpopulations of CD2+ lymphocytes could be detected and their existence was confirmed at the clonal level. The majority (95%) of the CD3+ cells were SPV-L14+, whereas 5% were CD3+, SPV-L14-. Among cloned cell lines CD3-,SPV-L14- and CD3-,SPV-L14+ cells were found to exist. The CD3-,SPV-L14- and CD3-,SPV-L14+ clones were shown to have NK cell activity, indicating that the 120- to 130-kDa antigen is expressed heterogeneously on CD3- NK cell clones. In addition, neoplastic T cells representing these four subpopulations were shown to exist. Although the tissue distribution and the MW of the SPV-L14 target antigen strongly suggest that SPV-L14 reacts with an epitope on CD6, the SPV-L14 mAb did not react with resting or activated B cells or with malignant B cells. Blocking studies showed that SPV-L14 inhibited the proliferative response of PBL, induced by anti-CD3 mAb, but that SPV-L14 did not affect the proliferation induced by phytohemagglutinin. These results suggest that the 120- to 130-kDa MW antigen is associated with T-cell proliferation, depending on the mode of activation.     

Monoclonal antibody to a structure expressed on a subpopulation of rat CD8 T cell subsets.

We have developed a monoclonal antibody, RTS-1, which can divide a rat CD8 (+) peripheral T cell population into two functionally distinct subsets. The cell-surface structure defined by this antibody is a glycoprotein with a molecular weight of 220 kDa found to be a high molecular isoform of rat CD45 antigen. CD4 (+) T cells were not stained by RTS-1 antibody. The cytotoxic T cell-enriched population did not express RTS-1 epitope on the cell surface. CD8 (+) spleen cells as well as RTS-1(+)CD8(+)T cells exhibited strong inhibition on mitogen-induced immunoglobulin G production by rat B cells. Furthermore, RTS-1 antibody, but not the control antibody, abolished CD8(+)T cell-mediated inhibition of immunoglobulin G production by rat B cells. These data suggest that RTS-1 antibody recognizes a unique determinant of rat CD45 antigen that is expressed on a fraction of CD8(+) cells.     

The B cell-associated CD37 antigen (gp40-52). Structure and subcellular expression of an extensively glycosylated glycoprotein

The human B lymphocyte-associated CD37 antigen (gp40-52) has been characterized by the monoclonal antibody HD28. The CD37 antigen is strongly expressed on surface immunoglobulin positive B lymphocytes and weakly on a subpopulation of T lymphocytes and myeloid cells. The total molecular mass of the antigen ranges from approximately 40 to 52 kDa in B cell-derived leukemias and malignant lymphomas as well as in normal and anti-mu/B cell growth factor-activated tonsillar B cells. The polydisperse nature of the electrophoretic pattern of the CD37 antigen was found to be due to a microheterogeneity in its carbohydrate moiety. Biochemical analysis showed that the CD37 antigen derived from B cell-lines BJAB and LICR-LON-HMy2 consists of a single chain protein core of approximately 25 kDa to which two N-linked, complex carbohydrate antennae of various length are bound. The glycosylation of the molecule comprises about 50% of the total molecular mass. The molecule does not contain O-linked carbohydrate chains. In contrast, the non-Hodgkin's lymphoma cell line, OCI.LY1, which is growth-dependent on human serum, carries a CD37 antigen with an additional carbohydrate chain resulting in a total molecular mass of approximately 40 to 64 kDa. At the electron microscopy level, this cell surface-expressed antigen was found to be associated with intracellular vesicles. The subcellular distribution of the CD37 antigen may reflect a function of this antigen both at the cell surface and in the cytoplasm. We found that, both due to its peculiar biochemical structure and its ultrastructural distribution, the CD37 antigen closely resembles the 46-kDa species of the mannose 6-phosphate receptor. The implications of this possible congruence for the function of the CD37 antigen are discussed.     

Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system.

CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extracellular region of the membrane-bound human CD23 is processed into at least four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29, and 25 kDa. High levels of sCD23 are found in patients with allergy, certain autoimmune diseases, or chronic lymphocytic leukemia. Therefore, inhibition of the processing of membrane-bound CD23 to control the cytokine-like effects of sCD23 offers a novel therapeutic opportunity. While the 37-, 29-, and 25-kDa forms of sCD23 have been expressed previously as recombinant proteins, the 33-kDa form has not been purified and characterized. To further investigate the multiple roles of sCD23 fragments and to devise assays to identify potent small-molecule inhibitors of CD23 processing, we have produced the 33-kDa form of sCD23 using Chinese hamster ovary (CHO) and Drosophila S2 cells. The CHO-expressed 33-kDa protein was found to undergo proteolytic degradation during cell growth and during storage of purified protein, resulting in accumulation of a 25-kDa form. The Drosophila system expressed the 33-kDa sCD23 in a stable form that was purified and demonstrated to be more active than the CHO-derived 25-kDa form in a monocyte TNFalpha release assay.     

CD4+CD45RA+ and CD4+CD45RA- T cell subsets in man maintain distinct function and CD45RA expression persists on a subpopulation of CD45RA+ cells after activation with Con A.

We have previously shown that Con A-induced suppressor T cells belong to the CD45RA+ subset. After unseparated T cells are activated with Con A, CD45RA expression increases to a maximum (Day 2), and then decreases significantly, but does not disappear entirely (Day 9), while CD29 expression increases steadily. In the present study, we examined the fate of these cell surface molecules on isolated CD4+CD45RA+ and CD4+CD45RA- cells following activation with Con A, and their relationship to the regulatory functions of these subsets. After activation of CD4+CD45RA+ cells with Con A, CD45RO and CD29 antigen expression rapidly increases (greater than 90%). While CD45RA expression is downregulated, approximately 40% of the cells continue to express low-density CD45RA in a stable fashion through Day 21. Despite these phenotypic changes, cells originally CD45RA+ continue to suppress IgG synthesis and provide only minimal B cell help. Furthermore, when cells originally CD45RA+ were sorted on the basis of continued presence, or loss of CD45RA antigen 14 days after activation, both populations demonstrated potent suppression and minimal help. In contrast, after activation with Con A, CD4+CD45A- cells maintain stable phenotype and provide significant help and minimal suppression. Immunoprecipitation of the CD45RA antigen from Day 14 activated CD4+CD45RA+ cells confirms the continued presence of the 205-kDa isoform, but reveals a significant decrease in the 220-kDa isoform. These results suggest that after activation with Con A, cells originally CD45RA+ remain functionally distinct from cells originally CD45RA-, and that CD45RA antigen persists on a subpopulation of CD45RA+ cells after activation with Con A.     

Distinctive lack of CD48 expression in subsets of human dendritic cells tunes NK cell activation

CD48 is a glycosyl phosphatidylinositol anchor protein known to be virtually expressed by all human leukocytes. Its ligand, 2B4, is a signaling lymphocyte activation molecule-related receptor involved in NK cell activation. Because dendritic cells (DCs) are strong inducers of NK cell functions, we analyzed the expression of CD48 in different human DC subsets. We observed that monocytes differentiating in DCs promptly down-regulate CD48. Similarly, DCs isolated from inflamed lymph nodes generally do not express CD48. Plasmocytoid DCs do not express CD48 either, whereas myeloid DCs harbored in blood, bone marrow, and thymus express it. In addition, we showed that CD48 expression in DCs affects NK cell functions during NK/DC cross-talk, because NK cells obtained from normal donors and from X-linked lymphoproliferative disease patients are, respectively, triggered or inhibited by DCs expressing surface CD48. Remarkably, IFN-gamma production by lymph node NK cells, in contrast to blood NK cells, can be negatively modulated by 2B4/CD48 interactions, indicating a 2B4 inhibitory pathway in lymph node NK cells. Therefore, the CD48 deficiency of DCs harbored in inflamed lymph nodes that we report in this study might be relevant to successfully activate lymph node NK cells in the early phase of the immune response. Our results show that distinct subsets of human DCs, differently from all other mononuclear hemopoietic cells, specifically do not express CD48. Moreover, the expression of CD48 depends on the anatomic location of DCs and might be related to the tissue-specific 2B4 function (activating or inhibitory) of the NK cells with which they interact.     

Cutting edge: the NK cell receptor 2B4 augments antigen-specific T cell cytotoxicity through CD48 ligation on neighboring T cells

2B4 is expressed on all NK and a subset of memory/effector CD8(+) T cells. 2B4 binds to CD48 and activates NK cytotoxicity, but its function on CD8(+) T cells is not clear. Furthermore, two isoforms of 2B4 (2B4S and 2B4L) exist in mice but the role of individual isoforms is not known. To address these questions, we generated primary T cell cultures from L(d)-specific 2C/Rag2(-/-) TCR transgenic mice and transduced them with 2B4S or 2B4L. 2B4S- or 2B4L-transduced T cells showed greater cytotoxicity over control cells against CD48(+) and CD48(-) targets, suggesting that ligation of 2B4 by CD48 on target cells was not necessary for 2B4 function. Rather, 2B4/CD48 interaction on adjacent T cells appeared to be critical for cytotoxicity. Therefore, 2B4 functions as a costimulator of CD8(+) T cells in MHC-restricted cytotoxicity. We conclude that 2B4/CD48 interactions among T cells themselves can augment CTL lysis of their specific targets.     

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc     

The murine NK receptor 2B4 (CD244) exhibits inhibitory function independent of signaling lymphocytic activation molecule-associated protein expression

2B4 (CD244) is a receptor belonging to the CD2-signaling lymphocytic activation molecule family and is found on all murine NK cells and a subset of NKT and CD8+ T cells. Murine 2B4 is expressed as two isoforms (2B4 short and 2B4 long) that arise by alternative splicing. They differ only in their cytoplasmic domains and exhibit opposing function when expressed in the RNK-16 cell line. The ligand for 2B4, CD48, is expressed on all hemopoietic cells. Previous studies have shown that treatment of NK cells with a 2B4 mAb results in increased cytotoxicity and IFN-gamma production. In this report, we used CD48+/- variants of the P815 tumor cell line and 2B4 knockout mice to show that engagement of 2B4 by its counterreceptor, CD48, expressed on target cells leads to an inhibition in NK cytotoxicity. The addition of 2B4 or CD48 mAb relieves this inhibition resulting in enhanced target cell lysis. This 2B4-mediated inhibition acts independently of signaling lymphocytic activation molecule-associated protein expression. Imaging studies show that 2B4 preferentially accumulates at the interface between NK and target cells during nonlytic events also indicative of an inhibitory receptor. This predominant inhibitory function of murine 2B4 correlates with increased 2B4 long isoform level expression over 2B4 short.     

Ligand-induced association of surface immunoglobulin with the detergent insoluble cytoskeleton may involve alpha-actinin

B cell surface immunoglobulin (SIg) plays an important role in antigen recognition and cellular activation. Cross-linking of SIg by bivalent antibody converts it into a detergent insoluble state. The resultant SIg may be partially solubilized by incubating the detergent insoluble cytoskeleton in buffers that convert F actin to G actin. Immunoprecipitation of SIg from the detergent soluble fraction of [35S]methionine-labeled B cells results in the co-isolation of 112 kDa, 42 kDa, (actin), and three additional proteins in the 70- to 73-kDa molecular mass range, isoelectric point 4.8 to 5.6. Analysis of anti-Ig immunoprecipitates made after preclearing with anti-alpha-actinin showed complete depletion of the 112-kDa protein, suggesting that the 112-kDa protein is immunologically related to alpha-actinin. These immunoprecipitates also showed partial depletion of 70- to 73-kDa proteins and mu and delta heavy chains. After treatment of a rat B cells with anti-Ig, much of the Ig-associated 112-kDa protein and 70- to 73-kDa proteins became detergent insoluble, concomitant with most of the SIg. The migration of the SIg-associated 112-kDa and 70- to 73-kDa proteins from the detergent soluble fraction to the detergent insoluble fraction after ligand treatment, suggests that these proteins might be involved in linking SIg to the underlying cytoskeleton and could be involved in the transmission of mitogenic signals.     

A functional role for interleukin (IL)-4-driven cyclic amp accumulation in human b lymphocytes

Interleukin-4 (IL-4) regulates the expression of the 55-kDa alpha-subunit (CD25) of the IL-2 receptor complex in human B lymphocytes. This report suggests that the cAMP/protein kinase A (PKA) component of the IL-4 receptor signalling programme in human tonsillar B cells has a functionally important role in regulating expression of the CD25 gene by attenuating activity of a protein binding to a potent negative regulatory element (NRE) in the CD25 promoter; this effect can be mimicked by agents that elevate cAMP and blocked by inhibitors of PKA but not protein kinase C (PKC). In a B-cell line that fails to elevate cAMP, attenuate NRE-binding protein (NRE-BP) activity or express CD25 following IL-4 treatment, stimulation of cAMP accumulation by forskolin facilitates IL-4-mediated induction of both the endogenous gene and an exogenous reporter gene under the control of a minimal promoter/enhancer fragment of the CD25 gene.     

Peripheral murine CD3+, CD4-, CD8- T lymphocytes express novel T cell receptor gamma delta structures

A mAb directed against the CD3 molecule was used to identify a subset of CD3+, CD4-, CD8- T cells previously undefined in the peripheral lymphoid organs of the mouse. Biochemical analysis of CD3+, CD4-, CD8- splenocytes revealed that the vast majority of these cells express one of at least two distinct CD3-associated TCR gamma delta heterodimeric structures, but no detectable TCR alpha beta. One disulfide-linked heterodimer (77 kDa) is composed of two chains of 45 to 46 and 32 kDa. The latter chain was immunoprecipitated with an anti-TCR C gamma 1/C gamma 2 antiserum and was not glycosylated. An antiserum produced against a peptide corresponding to the C-terminal region of the predicted C gamma 4 gene product immunoprecipitated additional heterodimers (80 to 90 kDa). One heterodimer, composed of disulfide-linked 41- to 45-kDa protein (including a V gamma/C gamma 4 component), is expressed on a T cell hybridoma, DN-1.21, which was derived from fused splenic CD3+, CD4-, CD8- T cells. Another V gamma/C gamma 4-containing heterodimer is composed of disulfide-linked 46- to 47-kDa glycoproteins. These findings demonstrate that CD3+, CD4-, CD8- T cells present in the peripheral lymphoid organs express a variety of paired TCR gamma delta proteins. Unlike CD3+, CD4-, CD8- thymocytes, these cells express high levels of C gamma 4, but little, if any TCR alpha beta.     

Modification of CD43 and other lymphocyte O-glycoproteins by core 2 N-acetylglucosaminyltransferase

CD43, the major leukocyte sialoglycoprotein, is expressed onT lymphocytes in two predominant glycoforms. CD43 115 kDa isa pan T cell marker and is specifically recognized by the monoclonalantibody S7. CD43 130 kDa is associated with T cell activationand is specifically recognized by the monoclonal antibody 1B11.The thymoma EL-4 has been identified to express mainly CD43115 kDa and little or no CD43 130 kDa. Transfection of EL-4cells with core 2 ß16N-acetylgIucosaminyltransferase(C2GnT), an enzyme in the O-glycan biosynthesis pathway, resultedin an enhanced expression of the 1B11 epitope, CD43 130 kDa,and a loss of expression of the S7 epitope, CD43 115 kDa. Analysisof CD43 by SDS-PAGE revealed that CD43 in C2GnT transfectedEL-4 cells has a molecular weight of 125 kDa compared to 115kDa in nontransfected or control transfected EL-4 cells. SDS-PAGEanalysis of three other lymphocyte O-glycoproteins, CD44, CD45,and RPTPa., revealed that C2GnT expression resulted in a molecularweight increase of approximately 3–5 kDa for each of thesethree cell surface glycoproteins. Our data indicate that, whileCD43 may be a predominant substrate for C2GnT, other lymphocyteO-glycoproteins are also modified by this glycosyltransferase.Increased reactivity of cells with the monoclonal antibody 1B11,which specifically detects the expression of murine CD43 130kDa, may thus be a marker of increases in branching of O-linkedglycans generally. CD43 core 2 N-acetylglucosaminyltransferase lymphocyte glycoproteins     

Activation of T cells through a T cell-specific epitope of CD45.

The 180- and 190-kDa isoforms of CD45 are preferentially expressed on the helper inducer (memory) subset of CD4 cells. In order to generate monoclonal antibodies against the extracellular domains of these isoforms and determine whether they could regulate the function and activation of these cells, we developed a mAb, anti-4H2D, by immunizing Balb/c mice with an isogenic mouse pre-B cell line expressing the human 190-kDa CD45 isoform. Anti-4H2D reacts with approximately 60% of T cells, 70% of CD4 cells, and 60% of CD8 cells. The CD4 cell population defined by this mAb corresponds functionally and phenotypically to that defined by the CD45RO+CD29+ subset. Western blotting demonstrated that anti-4H2D reacts primarily with the 190-kDa isoform of CD45 and to a minor extent, the 205- and 180-kDa CD45 isoforms. Interestingly, this mAb reacted with only a subpopulation of mature thymocytes and peripheral T cells, despite the fact that the 190-kDa CD45 isoform, as well as CD45RO and CD29, is more widely distributed on cells of hematopoietic origin. The 4H2D epitope was neuraminidase sensitive, indicating that anti-4H2D reacts with a carbohydrate epitope which is present on only a subset of the T cells containing the 190-kDa CD45 isoform epitopes. Functional studies showed that soluble anti-4H2D augmented T cell proliferation induced by the CD2 and CD3 pathways, and treatment of T cells with this mAb up-regulated [Ca2+]i flux induced by both anti-CD2 and anti-CD3 mAbs. These results suggest that the 190-kDa CD45 isoform on human CD4 cells is heterogeneous and that the 190-kDa isoform recognized by anti-4H2D regulates the function and activation of CD4 helper T cells.     

Comparative biochemical and tissue distribution study of four distinct CD45 antigen specificities

Studies on the antigenic structure of the human CD45 glycoproteins using mAb have revealed the existence of four reactivity patterns defined specifically by distinct biochemical, cellular, and histochemical distributions. In addition to the two well characterized Ag specificities, CD45 and CD45R, present on the four glycoproteins (220, 205, 190, and 180 kDa) and on the 220-kDa member, respectively, we have identified two novel specificities. These have been defined by two different mAb, UCHL1 and PD7/26/16, that recognize an epitope exclusively expressed on the 180-kDa lowest molecular sized polypeptide and an epitope shared by the three higher molecular sized polypeptides (220, 205, 190 kDa) of the complex, respectively. It has been demonstrated that they are also part of the CD45 complex by immunoprecipitation, immunoblotting, and binding assays with purified CD45 molecules. Comparative analysis by flow cytometry and immunoperoxidase techniques also showed a distinct pattern of cell distribution for each specificity. The 180-kDa specificity is present on a subset of T cells but absent on B lymphocytes, whereas the 220-kDa specificity is mainly expressed by B cells and a subpopulation of T lymphocytes. On the other hand, the cell distribution of the epitope common to the three higher members is slightly different to the conventional pan-leukocyte CD45 specificity. Thus, certain CD45+ cell types such as cutaneous Langerhans' cells, sinusoidal lymph node macrophages, and a small subset of T cells in both lymph node and tonsil did not express the 220/205/190-kDa specificity. These results further support that CD45 glycoproteins constitute a very heterogeneous molecular complex with epitopes that are selectively expressed by different cell types and by T cells at different stages of maturation.     

Lymphokine-activated killer (LAK) cells. VI. NK1.1+, CD3+ LAK effectors are derived from CD4-, CD8-, NK1.1- precursors.

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1+, CD3+ lytically active subset. These lymphocytes were not derived from NK1.1+ precursors since NK1.1+ cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NK1.1+, CD3+ effectors were contained in a B cell-depleted CD4-, CD8-, NK1.1- splenic subset. Freshly obtained CD4-, CD8-, NK1.1- splenocytes were mostly CD3+, CD5+, B220-, had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4-, CD8-, NK1.1- splenocytes with IL2, for 6 days, resulted in the development of NK1.1+, CD3+, B220+ effectors 40% of which were CD5dim and 20-25% of which expressed TCR-V beta 8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1+ precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1+ cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1+ lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate that splenic NK cells do not express or acquire CD3; that the NK1.1+, CD3+ LAK effectors are derived from an NK1.1- precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.