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The genetic defect in X-linked lymphoproliferative syndrome (XLP) is the Src homology 2 domain-containing protein SAP. SAP constitutively associates with the cell surface molecule, signaling lymphocytic activation molecule (SLAM), and competes with SH2-domain containing protein tyrosine phosphatase-2 (SHP-2) for recruitment to SLAM. SLAM exhibits homology with the mouse cell surface receptor 2B4. The human homologue of 2B4 has now been identified. It is recognized by the c1.7 mAb, a mAb capable of activating human NK cells. Human 2B4 became tyrosine phosphorylated following pervanadate-treatment of transfected cells and recruited SHP-2. SAP was also recruited to 2B4 in activated cells. Importantly, the 2B4-SAP interaction prevented the association between 2B4 and SHP-2. These results suggest that the phenotype of XLP may result from perturbed signaling not only through SLAM, but also other cell surface molecules that utilize SAP as a signaling adaptor protein.

T cell activation represents a balance between positive and negative signals delivered via distinct cell surface molecules. Many cytoplasmic protein tyrosine phosphatases are involved in regulating cellular responses by antagonizing the action of protein tyrosine kinases. CD148 is a receptor-type protein tyrosine phosphatase expressed by all human mononuclear cells. We have investigated the effect of CD148 on TCR-mediated activation of human T cells. Overexpression of wild-type, but not a phosphatase-deficient, CD148 in Jurkat T cells inhibited TCR-mediated activation, evidenced by reduced expression of the early activation Ag CD69, inhibition of tyrosine phosphorylation of many intracellular proteins including the critical protein tyrosine kinase ZAP-70, and impairment of mitogen-activated protein kinase activation. Taken together, these results suggest that CD148 is an important phosphatase involved in negatively regulating the proximal signaling events during activation of Ag-specific T cells.

Following ligation of the TCR and costimulatory molecules such as CD28, T cells proliferate and secrete cytokines. Several other cell surface molecules have been identified that are capable of augmenting activation mediated via the TCR. These include CD2, CD27, CD40 ligand, and signaling lymphocytic activation molecule. Here, we have characterized the expression and function of CD148, a recently identified receptor-type protein tyrosine phosphatase. CD148 is expressed at low levels on resting T cells, but is up-regulated following in vitro activation. Cross-linking CD148 with immobilized anti-CD148 mAb induced vigorous proliferation of anti-CD3 mAb-activated, highly purified peripheral blood T cells in an IL-2-dependent, cyclosporin A-sensitive manner. This effect was greatest after 8 days of in vitro culture, suggesting that this molecule is involved in the latter stages of a T cell response. CD148-induced proliferation was significantly greater for CD8+ T cells than for CD4+ T cells. Thus, CD148 is a receptor-type protein tyrosine phosphatase involved in the activation of T lymphocytes.

Several members of the Ly-49 receptor family inhibit NK cell-mediated lysis of targets expressing appropriate MHC class I molecules. Ly-49D and Ly-49H, two Ly-49 molecules that lack immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains, associate with mouse DAP12, a molecule that possesses an immunoreceptor tyrosine-based activation motif (ITAM). Cotransfection of either Ly-49D or Ly-49H with DAP12 induces surface expression of both Ly-49 and DAP12. The Ly-49/DAP12 complex was coimmunoprecipitated from the transfected cells, demonstrating a physical association of DAP12 with Ly-49D or Ly-49H in the plasma membrane. Stimulation of transfectants with Abs recognizing either Ly-49D or Ly-49H results in cellular activation, as assessed by induction of tyrosine phosphorylation of multiple cellular substrates.

NK cells and T cells express killer cell inhibitory receptors (KIR) recognizing polymorphic MHC class I molecules. Although prior studies have established that MHC class I can protect normal and transformed hematopoietic cells from NK cell lysis, the role of MHC class I on the recognition of solid tumors has been controversial. In this study, we investigated whether interactions of KIR with their ligands on melanoma tumor cells could inhibit tumor cell lysis by NK and gamma delta T cell clones. Ligation of the NK cell receptor KIR3DL1 by HLA-Bw4 allotypes resulted in inhibition of cytotoxicity against HLA-B*4403-transfected melanomas as well as against melanomas endogenously expressing HLA-Bw4 allotypes. Similarly, interactions of KIR2DL2 or KIR2DL3 (KIR2DL2/3) with HLA-Cw3-related allotypes on melanomas resulted in decreased tumor cell lysis. We also investigated whether signaling via KIR affected melanoma recognition by CTL. Introduction of KIR3DL1 molecules into HLA-A*0201-restricted gp100-specific CTL resulted in inhibition of lysis of gp100+ melanomas co-expressing HLA-A*0201 and HLA-Bw4 allotypes. These results suggest that disrupting interactions of KIR with their ligands on tumor cells in vivo may enhance antitumor responses mediated by both innate and adaptive immune effector cells.

Interferon regulatory factor-1 (IRF-1) is a member of a family of transcription factors that regulate an array of genes involved in cell growth, differentiation, and death. Analysis of cytokine expression by stimulated CD4+ cells from IRF-1(-/-) and IRF-1(+/+) mice revealed that IRF-1 deficiency resulted in an elevated production of Th2-related cytokines and a compensatory decrease in the expression of naive cell- and Th1-related cytokines. The altered cytokine profiles of IRF-1(-/-) cells could be explained, in part, by a shift in the representation of subsets of CD4+ cells; IRF-1(-/-) mice exhibited a decreased percentage of naive cells (a major source of IL-2) but increased numbers of memory or effector cells (the source of Th2-related cytokines). We analyzed purified, phenotypically matched memory/effector cells from IRF-1(-/-) and IRF-1(+/+) mice and found that the increased Th2:Th1 cytokine ratio was still evident in the IRF-1(-/-) group, thus suggesting that IRF-1 is involved in the polarization of the cytokine repertoire in CD4+ cells. Our data indicate that IRF-1 plays an important role in the maintenance of CD4+ cell subset homeostasis and in the expression of cytokines by naive and memory/effector cells.

Prior studies demonstrated that NK cells isolated from adult peripheral blood kill the HLA-A-, HLA-B-, and HLA-C-deficient B lymphoblastoid cell line 721.221, but many are unable to kill 721.221 cells transfected with HLA-G, a molecule expressed preferentially on fetal cytotrophoblasts. To determine the biologic relevance of this recognition, we established NK cell clones from the placenta and demonstrate that these NK cells also were unable to kill 721.221 cells expressing HLA-G. Recognition of HLA-G by NK cells was prevented in the presence of anti-CD94 mAb, implicating CD94/NKG2 as the predominant inhibitory NK cell receptor for HLA-G used by decidual NK cells. In contrast, mAbs against the killer cell inhibitory receptors recognizing HLA-Cw3-related, HLA-Cw4-related, or HLA-Bw4 ligands did not affect NK cell killing of the HLA-G transfectants.

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.

Allotypes from four divergent HLA-B families (B8, B15, B16, and B27) were compared for their inhibition of cytolysis by NK cells expressing the NKB1 receptor. Allotypes differing solely at the Bw4/Bw6 region were examined as were a more divergent subset of B15 allotypes. The capacity to interact with NKB1 correlated precisely with possession of a Bw4 sequence motif at residues 77-83, whereas no correlation was made with the peptide-binding specificities of two Bw4 and four Bw6 allotypes of the B15 family. HLA-B allotypes having four different Bw4 motifs were examined and all interact with NKB1. In contrast, HLA-A allotypes, which have a Bw4 motif identical with one of those present in HLA-B, do not. Mutation at leucine 82 and arginine 83, the residues common to Bw4 motifs, shows they contribute to NKB1 interaction but are not essential. Three types of polymorphism are implicated in formation of the ligand recognized by NKB1: ones shared by Bw4 motifs; ones distinguishing Bw4 motifs; and ones outside the Bw4/Bw6 region that distinguish HLA-B from HLA-A.

NKG2 is a small family of type II transmembrane proteins possessing extracellular C-type lectin domains expressed primarily on NK cells. The function of these proteins is unknown. We have developed mAbs that recognize NKG2 family members on Western blots. Examination of cell extracts from NK cell clones demonstrates that individual NKG2 family members are expressed on subsets of NK cells. Because the anti-NKG2 mAb do not react with the cell surface Ag, signaling function was studied by generating cell lines that express a chimeric receptor consisting of a cytoplasmic and transmembrane domain from NKG2-A or NKG2-C fused to the extracellular segment of mouse NKR-P1C (NK1.1 Ag). Transfectants of the rat NK cell line, RNK-16, were tested for the effect of anti-NK1.1 mAb on cytolytic activity against the FcR+ target cell lines, P388D1 and P815. The anti-NK1.1 mAb stimulated lytic activity and calcium mobilization in the NK cell line expressing the NKG2-C/NKR-P1C chimeric receptor. By contrast, anti-NK1.1 inhibited the lytic activity and failed to stimulate a calcium response in cells expressing the NKG2-A/NKR-P1C chimeric receptor. Immunoprecipitation experiments demonstrated that the inhibitory cytoplasmic tyrosine phosphatase SHP-1 selectively associates with the NKG2-A/NKR-P1C chimeric receptor, but not with the stimulatory NKG2-C/NKR-P1C receptor. These data indicate that NKG2-A and NKG2-C deliver, respectively, inhibitory and activating transmembrane signals in NK cells.

CD94 receptors expressed on NK cells have been implicated in the recognition of certain HLA class I allotypes. We now demonstrate that CD94 glycoproteins form disulfide-bonded heterodimers with the NKG2A/B, NKG2C, and NKG2E glycoproteins. NKG2A/B possesses two immunoreceptor tyrosine-based inhibition motif (ITIM) sequences in its cytoplasmic domain, which may be responsible for the inhibitory function of these receptors, whereas other NKG2 proteins lack ITIMs and may potentially transmit positive signals. Structural heterogeneity in the NKG2 gene family and the formation of heterodimers with CD94 provides for the creation of a diverse NK cell repertoire.

MHC class I glycoproteins possess an invariant site for N-linked oligosaccharide addition at position 86 of the heavy chain. For human HLA-A, -B, and -C class I glycoproteins, position 86 is the only site of N-linked glycosylation. Comparison of the size and relative abundance of oligosaccharides associated with nine HLA-A, -B, or -C allotypes isolated from EBV-transformed B cell lines and mixtures of HLA-A, -B, and -C allotypes isolated from pooled PBLs revealed a very restricted set of structures. Allotypes encoded by the HLA-A and -B loci have two predominant glycan structures that were almost exclusively di-sialylated. In contrast, HLA-C allotypes have four glycan structures, comprising those associated with HLA-A and -B and two additional glycans. Identical oligosaccharides were present on different allotypes of a class I HLA locus, and in particular, HLA-C allotypes defining two inhibitory specificities for NK cells were shown to possess the same set of oligosaccharides. The uniformity of oligosaccharide structure associated with different HLA-A, -B, and -C products and the relative lack of heterogeneity for any given allotype are unusual features for a mammalian glycoprotein. Particularly striking is that such conserved oligosaccharide structures juxtapose the major regions of amino acid sequence variation within the Ag recognition site, including the polymorphisms of the alpha 1 helix that determine the inhibitory ligands for human NK cells.

The expression of certain MHC class I allotypes by potential target cells can inhibit NK cell-mediated cytotoxicity. We recently identified the NKB1 surface Ag, expressed on T and NK cell subsets, as a putative inhibitory receptor for HLA-B class I molecules possessing the Bw4 serologic epitope. NKB1 is a 70-kDa glycoprotein that after deglycosylation migrates as a 50-kDa protein as determined by SDS-PAGE. A cDNA encoding the NKB1 receptor was cloned from a NKB1+T cell cDNA library by expression in COS-7 cells using the anti-NKB1 mAb DX9. NKB1 is a member of the lg superfamily containing three lg-like domains in the extracellular region and is related to the recently identified family (p58/NKAT) of human NK and T cell surface molecules that appear to function as inhibitory receptors for HLA class I.

Human NK cells produce IFN-gamma, TNF-alpha, and granulocyte macrophage-CSF when stimulated with susceptible target cells or through the CD16 and CD94 cell surface molecules. This study reports that NK cells also produce IL-5, a cytokine typically produced by Th2 cells, which mediates mobilization and differentiation of eosinophils. Polyclonal NK cell populations and NK cell clones produce IL-5 when stimulated to proliferate with gamma-irradiated MM-170 melanoma cells or JY B-lymphoblastoid cells and rIL-2. IL-5 is produced in cultures generated from freshly isolated NK cells (primary cultures) and when quiescent NK cells from primary cultures are restimulated to proliferate (secondary cultures). Production of IL-5 is on average 8.8-fold greater in secondary cultures compared with primary cultures (n18), suggesting that the ability of NK cells to produce IL-5 matures during primary stimulation. IL-5 secretion, particularly in primary cultures, is augmented by IL-4 and is inhibited by IL-12 and IL-10. By contrast, IL-4 and IL-12 have the reverse effects on IFN-gamma secretion. Cultured NK cells that no longer secrete cytokines can be restimulated to do so with either phorbol 12, 13 dibutyrate and ionomycin or with susceptible target cells in the presence of rIL-2. IL-5 production in these cultures occurs only when NK cells are in an exponential growth phase, whereas IFN-gamma, TNF-alpha, and granulocyte macrophage-CSF are produced also by stimulation of quiescent cells, although to a lesser extent. Furthermore, cytokine production is unrelated to the cytolytic activity of NK cells. In conclusion, proliferating human NK cells have the potential to produce IL-5 with secretion regulated by IL-4, IL-10, and IL-12.

NK cells lyse hematopoietic cells that lack expression of MHC class I molecules on the cell surface. Transfection of certain MHC class I negative cell lines with MHC class I genes renders these cells resistant to NK cell-mediated cytotoxicity. Recently, we described an NK cell receptor, NKB1, that inhibits NK cells from killing target cells expressing Bw4-reactive HLA-B molecules (-B*2705, -B*5101, -B*5801). In this study, we have demonstrated that another structurally distinct NK cell membrane glycoprotein, HP-3E4, is involved in the recognition of certain polymorphic HLA-C molecules (-Cw*0401 and -Cw*1503). NK cell clones co-expressing both the NKB1 and HP-3E4 receptors fail to lyse targets expressing HLA-Cw*0401 and -B*5801, but are able to kill the transfectants in the presence of mAbs against both receptors. These studies demonstrate that a single NK cell clone may possess multiple structurally distinct receptors for different polymorphic HLA class I molecules that function independently.

Signals initiated through both the TCR complex and CD28 are required for optimal activation of T lymphocytes. Recently, it has been demonstrated that CD28 interacts with two different ligands, designated CD80 (B7/B7-1) and CD86 (B70/B7-2). We have produced stable transfectants that express CD80, CD86, or both ligands and have examined their ability to costimulate T cell proliferation, cytokine production, and the generation of CTL. When we used small, resting human peripheral blood T cells as responders, both CD80 and CD86 transfectants efficiently costimulated anti-CD3 mAb-induced proliferation and the secretion of IL-2 and IFN-gamma. Additionally, both CD80 and CD86 transfectants were able to generate functional CTL. The magnitude and kinetics of these responses were similar, which indicates that both ligands provide efficient costimulatory signals. Because many APCs coexpress both CD80 and CD86, we compared the ability of anti-CD80 and anti-CD86 mAbs to inhibit allogeneic MLR stimulated with B lymphoblastoid cell lines and showed that it is necessary to inhibit interactions with both ligands to optimally block CD28-dependent proliferation. Given the limited homology of CD80 and CD86, it was surprising that the binding of CD28-Ig fusion protein to CD80 and that to CD86 transfectants were essentially indistinguishable. Binding of CTLA-4-Ig fusion protein to both transfectants also was quite similar, but was of higher affinity than CD28-Ig binding. Results from these studies indicate that both CD80 and CD86 are potent and similar costimulators of T lymphocytes. Therefore, the role of CD80 and CD86 in an immune response may be determined primarily by their differential expression on APC.

In rodents, the NKR-P1 family of glycoproteins are preferentially expressed on NK cells and have been implicated in NK cell function. In this study, we describe the characterization and cloning of a human homologue. Human (h)NKR-P1A cDNA was cloned from a NK cell cDNA library by expression in COS7 cells with the use of the DX1 mAb. hNKR-P1A is a type II membrane glycoprotein with characteristic properties of the C-type lectin superfamily. Comparison of the predicted amino acid of human NKR-P1A with rat and mouse NKR-P1 indicates 46% homology. NKR-P1A is on human chromosome 12, the syntenic of mouse chromosome 6, where the murine NKR-P1 genes are located. All rat NK cells express NKR-P1; however, hNKR-P1A is present on only a subset of human NK cells. Although rodent T cells only infrequently express NKR-P1, hNKR-P1A is present on approximately 25% of adult peripheral blood T cells, including both CD4+ and CD8+ T cells, and is expressed preferentially on adult T cells with a "memory" antigenic phenotype. The anti-hNKR-P1A mAb failed to affect lysis of NK-sensitive targets; however, the spontaneous cytotoxicity mediated by certain NK cell clones against the murine P815 cell target was blocked by anti-hNKR-P1A mAb. Our findings demonstrate that NKR-P1A is a human homologue of the rodent NKR-P1 genes and suggest that this molecule may be involved in NK cell function.

CD40 is a membrane differentiation antigen constitutively expressed on B cells that induces B cell growth and Ig synthesis after ligation with anti-CD40 mAb or with the recently identified CD40 ligand (CD40L). CD40L is rapidly induced on T cells after activation with anti-CD3 mAb or mitogens. While CD40-CD40L interactions are clearly beneficial to B cells, we speculated that a reciprocal costimulation of T cells might also occur. We have used genetic transfection to demonstrate that interactions between human small, resting T cells and CD40+ murine transfectants substantially augmented anti-CD3 induced T cell proliferation and resulted in the generation of CTL. T cell proliferation costimulated by CD40 was IL-2 dependent. The ability of CD40+ transfectants to costimulate T cell proliferation was specific in that VCAM-1+, CD54+, CD72+, CD56+, CD31+, and fas+ transfectants in the same host cells were inactive. CD4+ T cells preferentially responded to CD40 costimulation, whereas CD8+ T cells were substantially less reactive. By contrast, costimulation with B7 transfectants induced equivalent proliferation in the CD4+ and CD8+ T cell subsets. In addition, adult naive and memory T cells, as well as cord blood T cells, were responsive to CD40. These findings suggest that the CD40-CD40L costimulation pathway may allow for selective expansion of CD4+ T cells after interaction with CD40-bearing APC. The relatively restricted expression of CD40 on APC, as well as on medullary and cortical thymic epithelium, indicates a possible role for this interaction in T cell differentiation and activation.

Small, resting human peripheral blood T cells are able to mediate anti-CD3 redirected lysis against murine P815 cells transfected with human B7, a ligand of CD28. We demonstrate that cytotoxicity is mediated by preexisting cytotoxic effectors within the small, resting "memory" T cell population and by the de novo generation of additional CTL within both the "memory" and "virgin" T subsets. This conclusion is based on analysis of the kinetics of the response and the effects of metabolic inhibitors on the generation of CTL function. Memory CD45RO+ T cells demonstrated cytotoxicity within 4 h of coculture with anti-CD3 mAb and B7+ P815 cells and cytolysis was only partially prevented by inhibitors of protein synthesis. By contrast, virgin CD45RO- T cells demonstrated anti-CD3-induced lysis against B7+ P815 targets only after 6 or 8 h of coculture and cytotoxicity was completely prevented by inhibiting protein synthesis. Induction of cytotoxicity was B7 dependent in that parental P815 cells and P815 cells transfected with CD72 and vascular cell adhesion molecule-1, ligands for T cell-associated membrane receptors CD5 and very late activation antigen-4, respectively, did not initiate cytotoxicity. Our studies also revealed cooperation between the CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 pathways in the generation of CTL from small, resting T cells. However, after CTL generation, the CD28-B7 interaction was not required for cytotoxic effector cell function. These observations may have important physiologic implications because this would permit activated CTL to lyse targets in vivo that do not express B7, after the CTL were generated by APC that do express B7 or possibly other costimulatory molecules.

A subset of CD8+ alpha beta-TCR/CD3+ T lymphocytes in adult human peripheral blood lacks expression of CD28, a membrane receptor for the B7/BB1 B cell differentiation Ag that is involved in T cell activation. CD28-8+ T cells were not observed in the thymus and were present at only low frequency in cord blood, suggesting that these cells may represent a type of "memory" population. Consistent with this interpretation, CD28-8+ T cells were morphologically large, granular lymphocytes and expressed CD54 (intercellular adhesion molecule-1), CD58 (lymphocyte function-associated Ag-3 (LFA)), and high levels of CD11a (LFA-1), but did not express Ag associated with acute activation (e.g., HLA-DR, CD25, CD69). Freshly isolated CD28-8+ T lymphocytes mediated potent anti-CD3 redirected cytotoxicity against FcR-bearing targets, demonstrating that the CD3/TCR complex is functional and that these cells possess cytolytic activity. However, the anti-CD3-induced proliferative response of CD28-8+ T cells was substantially less than CD28+8+ T cells and this deficiency could not be overcome by addition of exogenous IL-2. A large panel of T cell clones were produced from the CD28+8+ and CD28-8+ T cell populations by single cell sorting using a flow cytometer. CD28 expression was stable on clones derived from CD28+8+ T lymphocytes, whereas CD28 expression was quite variable and apparently reexpressed on some clones generated from the CD28-8+ T cell population. As with the freshly isolated cells, CD28-8+ T cell clones were cytotoxic, but anergic to anti-CD3- induced proliferation and could not be costimulated using B7 or CD58 (LFA-3) murine L cell transfectants. These results indicate that CD28- and CD28+ T cells may play different roles in an immune response.

NK cells have been defined as CD3-, CD16+, and/or CD56+ lymphocytes that mediate MHC-unrestricted cytotoxicity against certain tumors and virus-infected cells. Although CD3 epsilon transcripts have been detected in some NK clones, it has generally been thought that NK cells do not express CD3 proteins other than zeta which is associated with CD16 (Fc gamma RIII). We demonstrate that adult peripheral blood NK cell lines and clones express cytoplasmic CD3 epsilon proteins, but not CD3 delta or gamma. CD3 epsilon proteins were detected by immunoprecipitation, Western blot analysis, and immunofluorescence using antiserum directed against the cytoplasmic domain of CD3 epsilon. Although resting, adult peripheral blood NK cells have essentially undetectable levels of CD3 epsilon protein, expression was increased substantially after activation. In contrast to adult NK cells, NK cell clones established from human fetal liver express CD3 gamma, delta, and epsilon protein subunits that associate and form CD3 epsilon, gamma and CD3 epsilon, delta complexes in the cytoplasm, but are apparently unable to be transported to the cell surface. These results indicate that expression of CD3 gamma delta epsilon subunits is not restricted to T lymphocytes and supports the possibility that NK and T cells may be derived from common origins.

NK cells and certain CTL can recognize and lyse targets without restriction by the MHC. NK cells do not express CD3/TCR complexes and the membrane receptors participating in MHC-unrestricted cytotoxicity are largely unknown. We demonstrate that YT2C2, a human NK leukemia cell line, expresses the CD28 differentiation Ag and can spontaneously lyse both murine and human cell lines expressing B7, a B cell- activation Ag that is a ligand for CD28. The participation of CD28/B7 interactions in MHC-unrestricted cytotoxicity mediated by YT2C2 cells was demonstrated by correlation of target sensitivity with levels of B7 expression, inhibition of cytotoxicity by anti-CD28 or anti-B7 mAb, and by making both murine and human cell lines susceptible to YT2C2-mediated lysis by genetic transfection with expression vectors containing B7 cDNA. However, CD28/B7 interactions alone were insufficient to initiate cytotoxicity. mAb inhibition experiments and selection of CD54- (intercellular adhesion molecule-1) deficient B cell targets indicated that CD11a/18 (lymphocyte function-associated Ag-1) also cooperated in CD28/B7-dependent cytotoxicity. The requirement for both CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interactions in YT2C2-mediated MHC-unrestricted cytotoxicity was confirmed by demonstrating that efficient lysis of murine L cells required cotransfection with both B7 and intercellular adhesion molecule-1. These findings support the concept that MHC-unrestricted cytotoxicity may not be due to a unique receptor, but may result from interactions between an appropriate array of "adhesion" molecules with their ligands.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.

Two genes encode the CD16 low affinity IgG FcR. CD16-I (Fc gamma RIII-1) is expressed on PMN as a phosphatidylinositol-glycan anchored glycoprotein. CD16-II (Fc gamma RIII-2) is expressed on NK cells and macrophages as a transmembrane glycoprotein associated with CD3 zeta or Fc epsilon RI-gamma. NK cells spontaneously release soluble CD16-II from the cell surface and this is enhanced by activation with phorbol ester. In this study, we demonstrate that a metalloprotease is involved in the spontaneous and PMA-induced release of CD16-II from NK cells. 1,10-phenanthroline, an inhibitor of Zn(2+)-dependent metalloproteases, efficiently inhibits CD16-II release. 1,7-phenanthroline, an inactive analogue that doesn't chelate Zn2+ or other divalent metal cations, and inhibitors of serine proteases do not affect spontaneous or PMA-induced release of CD16-II. Murine P815 mastocytoma cells transfected with human CD16-II cDNA shed membrane CD16, and 1,10-phenanthroline inhibits this process. P815 transfectants expressing CD16-II molecules with truncated cytoplasmic domains also release soluble receptors, indicating that the cytoplasmic segment of CD16-II is not required for interaction with the protease or the cytoskeleton. By contrast, 1,10-phenanthroline does not inhibit PMA-induced release of CD16-I glycoprotein from PMN, indicating a different mechanism of release for this phosphatidylinositol-glycan anchored molecule. Prior studies have demonstrated that NK cells are activated via the inositol phosphate pathway after engagement of CD16-II by immune complexes or Ig-coated tumor cell targets. A membrane metalloprotease with substrate specificity for CD16-II that is activated by PKC stimulation may provide a mechanism for releasing the immune complex or target from the effector cells and halting signal transduction.

The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N-CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell-mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets.

Two genes encode Fc gamma RIII (CD16), a low affinity FcR for IgG. CD16-I is expressed as a phosphatidylinositol glycan-anchored membrane glycoprotein on neutrophils, whereas CD16-II is a transmembrane-linked glycoprotein on NK cells. Membrane anchoring is determined by codon 203. Site-directed mutation of codon 203 and transient expression of these cDNA in COS-7 cells indicated that Phe, Ile, Leu, and Val permit transmembrane expression, whereas Ser, Thr, Tyr, Asn, Gly, Ala, Asp and Lys enable phosphatidylinositol-glycan attachment. Thus, the involvement of amino acid 203 in membrane anchoring cannot be explained simply on the basis of size, charge, or polarity of the amino acid side groups at this site. Efficient expression of CD16-II in COS-7 cells requires co-transfection with either CD3 zeta or Fc epsilon RI-gamma. Truncation of the cytoplasmic segment of CD16 failed to affect association with CD3 zeta. CD3 zeta and Fc epsilon RI-gamma with truncated cytoplasmic segments were also able to facilitate membrane expression of CD16-II, implicating the transmembrane segments as the interaction site between CD16-II and CD3 zeta or Fc epsilon RI-gamma. Prior studies have suggested that the acidic residue in the CD3 zeta transmembrane segment may be important for the association of CD3 zeta complexes. Although site-directed mutation of CD3 zeta-Asp36 to Glu, Leu, or Val retained the ability to permit membrane expression of CD16-II, quantitatively the wild-type CD3 zeta-Asp36 provided optimal levels of expression, consistent with conservation of this amino acid in mouse and human CD3 zeta.

The expression of secretory component (SC), the epithelial receptor for polymeric Ig, was enhanced by the addition of human rIFN-gamma or rIL-4, as revealed by the binding of radiolabeled polymeric, J chain-containing IgA or anti-SC antisera to the human colonic adenocarcinoma epithelial cell line HT-29. In combination, these cytokines exhibited a synergistic effect, and the potentiating effect of IL-4 was inhibitable by polyclonal anti-IL-4 antisera. Because the binding of radiolabeled polymeric IgA (pIgA) to HT-29 cells was inhibited by unlabeled pIgA or a polyclonal anti-SC reagent, but not by IgG, monomeric IgA, or Fab alpha fragments, we conclude that the receptor involved in the increased binding of pIgA is indeed SC. These data suggest that the expression of SC on human epithelial cells and the subsequent binding of pIgA (produced in mucosal tissues and glands by subepithelial plasma cells) is regulated by lymphokines such as IL-4 and IFN-gamma that are presumably derived from T cells found in abundant numbers in these tissues. These findings demonstrate a novel pathway of interaction between T cell products and epithelial cells that may result in enhanced translocation of large amounts of locally produced pIgA through epithelial cells into external secretions.

The Leu-19 (NKH-1) antigen is expressed on human peripheral blood NK cells and a subset of peripheral blood cytotoxic T lymphocytes that kill "NK-sensitive" tumor cell targets without major histocompatibility complex restriction. In the present study, we demonstrate that the Leu-19 (NKH-1) antigen is also expressed on most interleukin 2 (IL 2) dependent T cell lines and clones that have been maintained in long term culture. The Leu-19 (NKH-1) antigen expressed on an antigen-specific, class I directed cytotoxic T lymphocyte cell line was an approximately 200,000 to 220,000 dalton protein, similar to Leu-19 (NKH-1) protein expressed on natural killer cells and KG1a, an immature stem cell leukemia cell line. Furthermore, Leu-19 (NKH-1) was expressed on both CD4+ and CD8+ IL 2 dependent T cell clones, and was present on both cytotoxic and non-cytotoxic T cell clones. Thus expression of Leu-19 (NKH-1) antigen on cultured cell lines does not directly correlate with cytotoxic function, antigenic specificity, or cell lineage.

AKR/Gross leukemia virus-induced tumor reactive cytotoxic T lymphocyte (CTL) clones were derived from C57BL/6 spleen cells. Analysis of their specificity pattern was performed by using a panel of target cells such as E male G2 and AKR.H-2bSL1 (susceptible tumors to polyclonal anti-AKR/Gross virus CTL), and cl. 18-5 and cl. 18-12 (insusceptible variant sublines derived from AKR.H-2bSL1). Several of these CTL clones were selected for further study. Lysis of Gross cell surface antigen-positive tumor cells by these clones was restricted by the H-2Kb molecule. The cell surface phenotype of these clones was Thy-1.2+, Lyt-2.2+, L3T4-, a phenotype consistent with that of polyclonal anti-AKR/Gross CTL, suggesting that they were of conventional CTL origin. According to their fine specificity pattern, the CTL clones were divided into two major groups (A and B) which were further subdivided into five and three subgroups, respectively. The specificity of group A clones was essentially the same as that of the standard polyclonal CTL population except for a variable level of natural killer-like activity by some of the CTL clones. That is, group A clones did not efficiently lyse the insusceptible variant tumors nor any of Friend-Moloney-Rauscher-positive tumors tested, but they showed strong lytic activity to susceptible tumors and iododeoxyuridine-treated insusceptible variants. Thus, their CTL activity appeared to be strictly directed to Gross cell surface antigen-positive tumors that are susceptible to polyclonal anti-AKR/Gross virus CTL. In contrast, group B clones could lyse both susceptible and insusceptible variant tumors and also a Friend virus-induced tumor (FBL3). Therefore, as defined by these CTL clones, at least two distinct antigenic systems (A and B), each with several antigenic determinants, appeared to be present. Because recent findings suggested that most of the polyclonal anti-AKR/Gross virus CTL activity appeared to be directed to N-ecotropic proviral determinants, we further investigated the nature of these two antigenic systems by use of additional target cells including lipopolysaccharide (LPS)-stimulated spleen cell blasts from AKXL recombinant inbred strains and retrovirus-infected fibroblasts. Group A clones could lyse all LPS blasts derived from AKXL recombinant inbred strains containing the AKV-1 proviral genome, but lysed only very insufficiently or did not lyse AKV-1-negative blasts containing the AKV-3 and/or AKV-4 provirus.(ABSTRACT TRUNCATED AT 400 WORDS)

Leu 23 is a human cell surface differentiation Ag expressed very early during lymphoid cell activation. Resting T lymphocytes do not express Leu 23. However, expression of Leu 23 glycoprotein can be induced on the Jurkat T leukemia cell line within a few hours after stimulation with soluble anti-CD3 mAb or Con A. After removal of the stimulus Leu 23 is expressed for greater than 30 h. A total of 20 to 30% of resting human thymocytes was unexpectedly stained by anti-Leu 23 mAb. Biochemical analysis revealed that the Leu 23 Ag expressed on thymocytes is a phosphorylated disulfide-linked dimer composed of 28- and 32-kDa subunits. Within the thymus, Leu 23 is preferentially expressed on the CD3bright, mostly CD4+ CD8- or CD4- CD8+ subpopulations. By contrast, CD3dim CD4+CD8+ thymocytes expressed only very low levels of Leu 23, and CD7+ CD3- CD4- CD8- thymocytes were Leu 23-. However, Leu 23 was induced on the CD3dim thymocytes by in vitro culture of thymocytes with anti-CD3-conjugated Sepharose. The transition from CD3dim to CD3bright within the thymus may be an important differentiation stage and influence selection of the antigenic repertoire. Therefore, the ability to detect subpopulations within the thymus that express Leu 23 Ag may help to identify thymocytes activated in vivo, possibly by the functional engagement of their CD3/TCR.

Resistance to the BALB/c plasmacytoma MPC-11 by F1 hybrids between BALB/c and four C57BL/10 congenic resistant strains was abrogated or reduced by 450 rads total body irradiation but was unaffected by intravenous injection of 3 to 4 mg of silica. The results are consistent with the idea that hybrid resistance to MPC-11 depends on an active immune response and is different from Hh-1 controlled hybrid resistance.

The functional and phenotypic characteristics of cells in human peripheral blood that mediate "natural killer" (NK) cytolysis have been examined with the use of multiparameter flow cytometry analysis and cell sorting. Essentially, all lymphocytes expressing NK and ADCC activity reacted with the anti-Leu-11a monoclonal antibody. The Leu-11a antigen was expressed on cytotoxic large granular lymphocytes (LGL), neutrophils, and basophils, but was not present on B cells, mitogen-activated T lymphoblasts, or Leu-1+ and Leu 4+ resting T cells. Anti-Leu-11a antibody selectively inhibited the binding of FITC heat-aggregated IgG complexes to granulocytes and LGL, and it may recognize a type of Fc receptor on these cells. Two-color FACS cell sorting indicated the existence of four lymphocyte subsets defined by the expression of Leu-11a and Leu-7 antigens. The Leu-11a+, -7- cells were highly active in 4-hr NK assays with the use of 51Cr-labeled K562 as the target. In contrast, the Leu-11a-, -7+ cells demonstrated weak activity and the Leu-11a-, -7- cells demonstrated no activity. The function of the Leu 11a+, -7+ cells varied considerably among several individuals examined. Multiparameter analysis with the use of two-color flow cytometry was used to determine the relationship between the expression of these NK-associated antigens and T and B cell-associated markers. These data indicate that considerable heterogeneity exists within human peripheral lymphocytes with regard to cell phenotype and function, but that several defined cellular subsets can be clearly revealed by using multiparameter FACS analysis and sorting.

Highly purified recombinant human interleukin 2 (rIL 2) markedly augments the natural killer (NK) cell-mediated cytotoxicity of peripheral blood lymphocytes. In this study, we examined the cellular and metabolic basis of rIL 2-mediated activation of human lymphocyte subpopulations expressing the NK cell-associated surface antigens Leu 7 and Leu 11. All rIL 2-responsive cytotoxic NK cells were found within the subset of lymphocytes expressing the Leu 11 marker, an antigen associated with the Fc-IgG receptor on human NK cells. Cells lacking the Leu 11 antigen, including cells expressing another NK cell-associated marker, Leu 7, did not express NK cell-mediated cytotoxicity either before or after rIL 2 treatment. By contrast, rIL 2 augmented the NK activity of both Leu7-,11+ and Leu 7+,11+ subpopulations. Activation of Leu 11+ NK cells resulted from a direct effect of rIL 2 on these cells and neither required nor was amplified by the presence of T lymphocytes. Enhanced NK cell-mediated cytotoxicity occurred within 4 hr after exposure to rIL 2, and was blocked by the protein synthesis inhibitor cyclohexamide, but not by the DNA synthesis inhibitor mitomycin C or 1500 rad of x-irradiation. Neither Tac antigen, a high-affinity receptor for IL 2, nor other activation markers, such as transferrin receptor or HLA-DR antigen, were detectable on a significant proportion of Leu 11+ cells, either before or after incubation with rIL 2 for 48 hr. In addition, saturating concentrations of antibodies to each of these markers had no effect on the enhancement of NK activity by rIL 2. Finally, preliminary experiments with neutralizing antibodies to gamma- and alpha-interferons also failed to prevent rIL 2 enhancement of NK cell-mediated cytotoxicity, suggesting that rIL 2 does not mediate its effect via release of these cytokines.

Four T cell clones, isolated from Peyer's patches of keyhole limpet hemocyanin (KLH)-primed BALB/c mice, were selected on the basis of their ability to help IgA responses by TNP-KLH-primed BALB/c mouse B cells. Two were KLH-dependent both in terms of their own proliferative response and in terms of their help for that of B cells. The other two were autoreactive and helped B cells proliferate independently of the presence of Ag. Both primed and unprimed B cells proliferated to some extent when helped by the KLH-reactive clones in the presence of high concentrations of either KLH or TNP-KLH. Much higher proliferation was, however, induced when primed, but not unprimed, B cells were exposed to the T cells in the presence of low concentrations of TNP-KLH but not KLH, i.e., under conditions favoring direct, cognate interaction between the T and B cells. Only modest IgM, and no IgG or IgA plaque-forming cell (PFC) responses were generated by TNP-primed B cells upon interaction with either autoreactive T cells in the absence of Ag or KLH-reactive T cells in the presence of high concentrations of KLH. For high IgM responses as well as for the appearance of IgG and IgA PFC responses, TNP-KLH was required whatever the source of the T cell help. The isotype ratios depended on the TNP-KLH concentration; IgA responses were highest and IgM responses lowest at the lowest TNP-KLH concentrations suggesting that the precursors of the IgA PFC have higher average affinity for TNP than the precursors of IgM PFC. Overall, the results are compatible with the idea that the precursors of IgA and IgG PFC and many of the precursors of IgM PFC in the long term primed B cell populations used in these experiments require engagement of their Ag-receptors before they express sufficient class II Ag and/or receptors for "switch" and differentiation factors for cognate interaction with T cells leading to PFC responses.

In the present study we have identified and characterized three subpopulations of peripheral blood NK cells based on the surface expression of CD56 and CD16. We have designated these subsets CD16neg, CD16dim, and CD16bright according to the relative surface density of CD16. The CD16bright subset comprised about 10% to 15% of PBL, whereas the CD16dim and CD16neg subsets comprise less than 1% of the total lymphocytes. A detailed characterization of these subsets revealed both similarities and differences. The three subsets shared a great deal of phenotypic similarity, expressing CD2, CD7, CD11b, CD38, CD45R, CD18, and the p75 IL-2R on the majority of the cells in each subset. There were, however, several prominent phenotypic differences, particularly in the expression of CD57, CD11c, CD44, CD25, Leu-8, L263, and L265. The CD16neg cells were morphologically large agranular lymphocytes and demonstrated low levels of non-MHC restricted cytolysis of NK-sensitive tumor lines. The CD16dim and CD16bright subsets were large granular lymphocytes and revealed potent cytotoxicity against NK-sensitive targets. All subsets demonstrated IL-2-dependent activation and proliferation; however, the CD16dim and CD16neg subsets were preferentially responsive to very low concentrations of rIL-2. Although rIL-4 effectively inhibited the IL-2-induced cytolytic activation of all three NK cell subsets, only the CD16bright cells showed rIL-4 inhibition of IL-2 dependent proliferation. Cytokine transcription was also differentially regulated in the NK cell subsets after rIL-2 activation. Although TNF-alpha was equally transcribed in each subsets, IFN-gamma and serine protease-HF were preferentially transcribed in the CD16bright NK cells. Based on these results, we propose that these NK cell subsets represent portions of the NK cell differentiation pathway present in the peripheral blood.

Genetic control of hybrid resistance to the BALB/c plasmacytoma MPC-11 was investigated. The results indicate that a single dominant autosomal gene or gene complex, which segregates independently of H-2 and the coat color c and b-loci, controls resistance to this tumor. This gene has the same strain distribution pattern in the CXB Bailey recombinant inbred strains as three unlinked genes, H-2, Ly-4, and Ea-4. It is possible, therefore, that it could be linked to either of the latter two loci. Strains that carry a positive allele for resistance are C57BL/10 and all of its congenic resistant partners tested, C57BL/6, C57L, C57BL/Ks, AKR, and DBA/1. BALB/c and its congenic resistant partners are presumed to carry a negative allele of the gene for resistance to MPC-11. Strains such as SJL, DBA/2, and A and its congenic resistant partners, which form susceptible hybrids with BALB/c, could carry either the negative allele of the gene for resistance, like BALB/c, or could carry both a positive allele of the gene and some other gene conferring susceptibility on the hybrids. Heterozygosity within the H-2 complex increases resistance only in the presence of this non-H-2 linked gene for resistance, and the effect maps to the left of the H-2D region.

NK cells are directly activated by rIL-2 and subsequently undergo rIL-2-dependent proliferation in vitro. Herein, we report that rIL-4 is a potent regulator of human NK cells. Although rIL-4 had no effect on the cytotoxic activity of resting NK cells, it was capable of inhibiting in a concentration-dependent manner the rIL-2-induced cytolytic activation of NK cells against NK cell-resistant tumor cell targets. rIL-4 acted directly on NK cells and did not require accessory cells. rIL-4-induced inhibition of NK cell activation was specific for rIL-2 in that activation of NK cell cytolysis by IFN-alpha was not affected. These results represent the first direct evidence that rIL-2 and IFN-alpha activate NK cells by different pathways. rIL-4 also effectively blocked the rIL-2-dependent proliferation of NK cells. The results presented in this study clearly demonstrate that rIL-4 is a potent regulator of IL-2-dependent mechanisms of NK cell activation and proliferation and thus may play an important physiologic role in vivo.

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.

Mice were induced to produce IgA antibodies against ferritin after oral immunization. Such antibodies were detected by immunofluorescence in plasma cells in the intestinal mucosa as well as in secretory sites located elsewhere, such as the lactating mammary gland, salivary gland, and respiratory tract. The observation suggested that cells immunized locally via the gut could home to distant secretory sites. To confirm this hypothesis, lymphocyte transfer studies were done with mesenteric node (MN) versus peripheral node (PN) cells from orally immunized donors into nonimmunized recipients. IgA anti-ferritin cells from MN homed to exocrine targets, whereas IgM and IgG anti-ferritin cells homed to PN. The findings overall support the concept of a generalized and interrelated secretory immune system.

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.

A subpopulation of cells in murine mesenteric lymph nodes, about 15% of those synthesizing DNA at any given time, homes specifically to the gut and mesenteric nodes of syngeneic recipients within 1 day of i.v. transfer. In contrast, cells from Oeyer's patches or peripheral lymph nodes do not. A large proportion of the B blasts which home to the small intestine has surface Ig, but lacks complement receptors. Thy-1-positive T blasts home to the gut to a lesser extent than B blasts. However, it is probable that equal fractions of B and T blasts home to mesenteric nodes. Homing is not affected by measures calculated to interfere with the combination of cell surface IgA and secretory component.

Expression of transferrin receptors (TFR) is required for lymphocyte proliferation. Treatment of lymphoblastic leukemia cell lines with phorbol diester tumor promoters decreases proliferation and induces differentiation. Among changes induced by phorbol diesters is decreased cell surface expression of TFR. To elucidate effects of phorbols on lymphocyte growth and differentiation, we examined TFR expression by measuring 125I-transferrin binding, levels of TFR mRNA by Northern analysis and dot-blot hybridization, and rates of TFR gene transcription by nuclear run-on experiments in CCRF-CEM lymphoblastoid T cells treated with PMA or phorbol dibutyrate. Cell surface expression of TFR was decreased 60 to 85% within 2 min of exposing cells to phorbols and remained decreased for 96 h. Steady state levels of TFR mRNA decreased to less than 30% of control after 48 h. After treating cells with actinomycin D, estimated TFR mRNA t 1/2 was 2.7 h and was unaltered in phorbol-treated cells. Levels of TFR mRNA were not affected by treatment of cells with cycloheximide in either control or phorbol-treated cells. Therefore, post transcriptional mRNA processing by protein factors did not account for decreased TFR mRNA in phorbol-treated cells. Compared to baseline levels, rates of TFR gene transcription in PMA-treated cells increased up to two-fold during the initial 6 h of culture, then decreased over the ensuing 12 h to less than 10% of baseline values. This pattern was not seen in control cultures. Therefore, regulation of TFR gene transcription is a consequence of treating CEM cells with phorbol diesters. Cell surface expression of TFR in phorbol-treated lymphoblastoid T cells may be mediated in part at the level of gene transcription.

Tuftsin is a physiologic tetrapeptide, which has recently been shown to possess immunoadjuvant properties including the stimulation of macrophage and granulocyte phagocytosis, migration, bactericidal, and tumoricidal activities. Tuftsin has also been reported to possess in vivo immunologically mediated anti-tumor potential. To determine the potential role of tuftsin as an antineoplastic immunoadjuvant, the in vitro effects of tuftsin on murine natural cell-mediated cytotoxicity were studied. We observed that in vitro treatment of mouse splenic effector cells with synthetic tuftsin induced a pronounced enhancement of natural killer cell (NKC) cytotoxicity against the T cell lymphoma Yac-1. The magnitude of NKC enhancement was directly dependent upon the concentration of tuftsin employed, with maximum NKC stimulation observed at tuftsin concentrations of 50 to 100 microgram/ml. The tuftsin induced enhancement of NKC activity was not strain specific, since equivalent stimulation was seen in CBA/J, C56BL/10, and DBA/2 mice. Elimination of macrophages, monocytes, T cells, and immunoglobulin-bearing cells had no effect on the dose-dependent tuftsin stimulation of natural cell-mediated cytotoxicity; thus the characteristics of the effector cells activated by tuftsin were consistent with those reported for NKC. We also observed that treatment of splenic effector cells with tuftsin prolonged the cytotoxic capabilities of these cells beyond 18 hr.

Thymocytes undergo a complex series of phenotypic and genotypic changes during maturation in the thymus. This dynamic process involves qualitative and quantitative changes in the expression of certain cell surface differentiation antigens. In this study, we have directly examined the relationship of T cell differentiation antigen expression on normal human thymocytes by using multi-color immunofluorescence and multi-parameter flow cytometric analysis. The results from these studies have provided new insights into the complexity of antigen expression during thymic maturation and suggest that the CD3/T cell antigen receptor complex is expressed early in the development of thymocytes. Direct quantitative measurements of antigen expression by using multi-parameter flow cytometric analysis also suggest quantitative co-regulation of certain antigens (e.g., CD3 and CD5) during thymic maturation.

The Leu 23 Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer expressed on the surface of activated T cells, B cells, and NK cells. Resting, unstimulated peripheral blood T cells are Leu 23-, but 20 to 30% of normal thymocytes constitutively express Leu 23. Triggering of the CD3/TCR complex by mAb induced the expression of Leu 23 within 2 h after stimulation. Leu 23 was still detectable on the cell surface 48 h after the stimulus was removed. The induction of Leu 23 strictly correlated with the extent of CD3/TCR cross-linking on the surface of T cells. Anti-CD3-coupled beads, but not soluble IgG or IgM mAb, were able to induce Leu 23 expression on peripheral blood T cells. Soluble anti-CD3 mAb were sufficient to induce Leu 23 on the Jurkat T cell line, but a direct relationship was found between CD3 cross-linking valency and Leu 23 expression. RNA and protein synthesis were both required for Leu 23 induction. Moreover, only CD3/TCR-mediated signals able to stimulate PKC and maintain elevated intracellular calcium ion concentration for greater than 60 min resulted in Leu 23 induction. These findings suggest that the extent of CD3/TCR cross-linking influences the persistence of elevated intracellular calcium ion concentration and PKC activation, and that this in turn determines the commitment of the T cell to activate gene transcription programs necessary for Leu 23 expression. Induction of Leu 23 represented the first detectable cell surface marker after triggering via the CD3/TCR and thus provides an ideal indicator to monitor the very early events in T cell activation.