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ST2 is a member of the IL-1 receptor family and IL-33 was recently identified as its natural ligand. The IL-33/ST2 pathway regulates Th1/Th2 immune responses in autoimmune and inflammatory conditions, but the role of ST2 signaling in tumor growth and metastasis has not been investigated. We aimed to investigate whether ST2 gene deletion affects tumor appearance, growth, and metastasis, and antitumor immunity in an experimental metastatic breast cancer model. Deletion of ST2 in BALB/c mice bearing mammary carcinoma attenuated tumor growth and metastasis, which was accompanied by increased serum levels of IL-17, IFN-γ, and TNF-α and decreased IL-4. Tumor-bearing ST2-/- mice had significantly higher percentages of activated CD27high CD11bhigh NK cells, CD69+ and KLRG- NK cells and higher cytotoxic activity of splenocytes, NK cells, and CD8+ T cells in vitro. A significantly higher number of NK cells expressing IFN-γ were found in ST2-/- mice compared with WT recipients. In vivo depletion of CD8+ or NK cells revealed a key role for NK cells in enhanced antitumor immunity in ST2-/- mice. We report for the first time that suppressed breast cancer progression and metastasis in mice lacking ST2 corresponds mainly with enhanced cytotoxic activity of NK cells, and increased systemic Th1/Th17 cytokines., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK and T-cell subsets and recognizes members of the classical cadherin family. KLRG1 is widely used as a lymphocyte differentiation marker in both humans and mice but the physiological role of KLRG1 in vivo is still unclear. Here, we generated KLRG1-deficient mice by homologous recombination and used several infection models for their characterization. The results revealed that KLRG1 deficiency did not affect development and function of NK cells examined under various conditions. KLRG1 was also dispensable for normal CD8+ T-cell differentiation and function after viral infections. Thus, KLRG1 is a marker for distinct NK and T-cell differentiation stages but it does not play a deterministic role in the generation and functional characteristics of these lymphocyte subsets. In addition, we demonstrate that E-cadherin expressed by K562 target cells inhibited NK-cell reactivity in transgenic mice over-expressing KLRG1 but not in KLRG1-deficient or WT mice. Hence, the inhibitory potential of KLRG1 in mice is rather weak and strong activation signals during viral infections may override the inhibitory signal in vivo.

The mouse killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor known to be expressed on a subset of NK cells and antigen-experienced CD8 T cells. Here, we have characterized expression of KLRG1 on CD4+ T cells from normal mice. While a polyclonal TCR repertoire suggests thymic origin of KLRG1+ CD4+ cells, KLRG1 expression was found to be restricted to peripheral CD4+ T cells. Based on phenotypic analyses, a minority of KLRG1+ CD4+ cells are effector/memory cells with a proliferative history. The majority of KLRG1+ CD4+ cells are, however, bona fide Treg cells that depend on IL-2 and/or CD28 and express both FoxP3 and high levels of intracellular CD152. KLRG1-expressing Treg are contained within the CD38+ subset but are only partially overlapping with the CD25+ CD4+ Treg subset. In functional assays, KLRG1+ CD4+ cells were anergic to TCR stimulation with respect to proliferation, and sorted KLRG1+ CD25+ CD4+ cells were equal or superior to KLRG1+ CD25- CD4+ cells, which were more potent than KLRG1- CD25+ CD4+ cells in suppressing responder cell proliferation. Together, our results demonstrate that KLRG1 expression defines novel and distinctive subsets of senescent effector/memory and potent regulatory CD4+ T cells.

In this issue of the European Journal of Immunology (EJI), Stewart et al. report about a population of gammadelta-T cells expressing an extensive repertoire of NK cell receptors, and the presence of non-rearranged germline TCRdelta transcripts in conventional NK cells. These findings and other recent studies highlight the similarities of NK cells and T cells and the problems in discriminating between these two lineages.

High mobility group box protein 1 (HMGB1) is a ubiquitous nuclear protein that can be actively released from the cell in certain conditions thereby mediating cytokine-like function. While nuclear HMGB1 modulates the transcriptional activity of cells, extracellular HMGB1 partially acts via binding to the receptor for advanced glycation end products (RAGE), which is highly expressed in lung tissue. Therefore, we studied the impact of food-derived advanced glycation end products (AGEs), the Maillard reaction products, on the lung expression of HMGB1. Feeding rats with AGE-rich diet, containing either bread crust or coffee beverage, resulted in an upregulation of HMGB1 mRNA and protein especially in those animals receiving bread crust diet. The expression of RAGE was not influenced. Moreover, we revealed a positive correlation between an increased lung AGE level and HMGB1 protein expression in both animal groups receiving either bread crust or coffee extract but not in the control group. In contrast, the ageing-related AGE accumulation was not associated with an increased level of HMGB1 protein in lung tissue from senescent (100 wk) compared to young-adult (24 wk) rats. Our data suggest a physiological role of food- but not ageing-associated AGEs in the regulation of the HMGB1 expression in lung.

Natural killer (NK) cells have not previously been precisely identified or characterized in cattle or any other ruminant species. We have generated a monoclonal antibody against bovine NKp46, which is expressed exclusively by NK cells in man. NKp46+ cells comprised 1-10% of blood mononuclear cells in cattle, and did not stain with antibodies against CD3, CD4, TCR1, B cell or granulocyte markers. The majority of the NKp46+ cells expressed CD2, and a variable fraction also expressed CD8. The tissue distribution of NKp46+ cells in cattle was compatible with the tissue distribution of NK cells in other species. Bovine NKp46+ cells had typical, large granular lymphocyte morphology, and proliferated vigorously in response to bovine IL-2 for a limited number of cell divisions. IL-2-activated NKp46+ cells killed the bovine kidney cell line MDBK. This cytotoxicity was inhibited by preincubation with antibody against NKp46. In a redirected lysis assay, IL-2-activated NKp46+ cells killed the FcgammaR+ target cell line P815 after preincubation with antibody against NKp46. Together, these data indicate that bovine NKp46 is anactivating receptor and demonstrate the existence of a subset of leukocytes in cattle that, in terms of surface markers, morphology and function, represent NK cells.

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.

The cognate NK-DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte-derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig-like receptors (KIR). Consistent with these data, the expression of HLA-E (i.e. the cellular ligand of CD94/NKG2A) was down-regulated in iDC. On the other hand, HLA-B and HLA-C down-regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A(+)KIR(-) NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA-E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA-E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.

Target cell lysis by natural killer cells is inhibited by killer cell immunoglobulin-like receptors (KIR) that bind major histocompatibility complex class I molecules. Many lymphocyte receptors, including KIR, become enriched at the interface with ligand-bearing cells. The contribution of the enrichment to inhibitory signaling has not been determined. We now describe a KIR variant with enhanced green fluorescent protein (EGFP) at the N terminus that can mediate inhibitory signaling, but its enrichment is markedly reduced. This receptor is only slightly weaker at inhibiting lysis than the same KIR tagged with EGFP in the cytoplasmic tail, even though the latter enriched as extensively as wild-type KIR. A slight defect was also detected in the ability of the receptor to reduce adhesion to target cells and for binding of a soluble counterpart to cell surface HLA-C. Our findings suggest that the strength of the interaction required to readily detect receptor enrichment exceeds that required for signaling.

Natural killer (NK) cell recognition and function in humans is regulated by multiple cell surface receptors. The "on" signal leading to NK cell triggering is primarily mediated by natural cytotoxicity receptors (NCR). Analysis of NK cells in primate animal models is of particular relevance because NK cells may play an essential role in host defenses against infections. We analyzed Macaca fascicularis PBMC and in vitro-derived NK cell populations and clones by cytofluorometry, using a wide panel of mAb, and by cytolytic activity assays. In addition, RT-PCR strategy and transient transfections were used to isolate M. fascicularis NCR. NCR-specific mAb reactivity (anti-NKp46 and anti-NKp30) was present on M. fascicularis PBMC and on NK cell cultures. Macaque NCR were functional in both redirected killing and in mAb-mediated masking assays. Cloning of macNKp46 and macNKp30 NCR homologous genes showed a high sequence similarity (86 % and 88 %, respectively) with their human counterparts. Attempts at identifying NKp44 surface reactivity and at cloning the macaque homologue were unsuccessful. NKp46 and NKp30 NCRs, but not NKp44, are highly conserved in M. fascicularis NK cells. This suggests the possibility of a staged appearance of the NCR during phylogenesis and provides a useful tool for the study of natural immunity correlates of protection in primate SIV/SHIV infection models.

The leukocyte-associated Ig-like receptor-1 (LAIR-1), a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motif (ITIM) is expressed on acute myeloid leukemia (AML) blasts isolated from peripheral blood or bone marrow of 17 patients (2 M0, 3 M1, 5 M2, 2 M4 and 5 M5 according to French, American and British classification). Further, we provide evidence thatLAIR-1 engagement inhibits granulocyte-monocyte colony-stimulating factor (GM-CSF)-induced proliferation of AML blasts. Indeed, leukemia cells stimulated with GM-CSF were blocked in the G0/G1 phase of the cell cycle and underwent apoptosis within 4 days after the engagement of LAIR-1. Remarkably, LAIR-1 was functional also in AML blasts which do not express CD33, mainly M4 and M5. Importantly, the LAIR-1 ligation led to a strong inhibition of both GM-CSF receptor-mediated intracellular calcium increases, phosphorylation and activation of Akt1/protein kinase B alpha, a substrate of the phosphatidylinositol-3 kinase. This last inhibitory effect was prevented by a synthetic peptide spanning the ITIM portion of LAIR-1, suggesting the involvement of SHP-1 phosphatase in LAIR-1-mediated inhibitory signal. Altogether, these findings indicate that the engagement of LAIR-1 can down-regulate GM-CSF-mediated survival and proliferation of AML blasts, suggesting an additional therapeutic approach to the treatment of AML patients.

Whereas the majority of NKT cells in the mouse express an alpha beta TCR (NKTalpha beta cells), a small subset of NKT cells express a gamma delta TCR (NKTgamma delta). Here we have systematically analyzed the phenotype, TCR repertoire and activation status of NKTgamma delta cells in the thymus, liver, spleen and bone marrow of normal C57BL/6 mice. Our data indicate that NKTgamma delta cells segregate in a tissue-specific manner according to these parameters. While most NKTgamma delta cells in the thymus and liver have a recently activated CD62L(lo) phenotype and a TCR repertoire that is heavily biased to Vgamma1.1 and Vdelta6.3, the majority of NKTgamma delta cells in the spleen and bone marrow are CD62L(hi) and have a much less biased TCR repertoire. Moreover, expression of NK markers is high on NKTgamma delta cells in spleen and bone marrow but low in thymus and liver. Collectively our results reveal a tissue-specific segregation of NKTgamma delta cells that is strikingly similar to that recently described for CD1d-dependent and Cd1d-independent NKTalpha beta cells. We therefore propose that chronic TCR activation by tissue-specific endogenous ligands is a generic property of NKT cells of both the alpha beta and gamma delta lineages.

In this study we present new differential characteristics of NK cells expressing CD56 surface antigen in low (CD56dim) or high (CD56bright) density. In contrast to CD56bright NK cells CD56dim cells express killer cell immunoglobulin (Ig)-like receptors (KIR) such as CD158a, CD158b, and NKB1. However, c-type lectin-like receptors (KLR) CD94/NKG2 and CD161 are present on both subsets. The ability to form conjugates with susceptible targets is approximately twice as strongly pronounced in CD56dim vs. CD56bright NK cells. Last but not least, granules of CD56dim cells contain about tenfold more perforin and granzyme A enabling potentially more effective cytolysis compared to CD56bright NK cells. On the other hand, CD56bright NK cells are superior in producing the proinflammatory cytokines IFN-gamma (28.5% vs. 20.8%, p<0.05) and TNF-alpha (28% vs. 15.8%, p<0.001). The different NK cell populations retained their specific phenotype in vitro during culture in the presence of IL-2 contradicting that they simply display different stages of maturity. Taken together our data support the view that CD56bright cells are specialized NK cells that regulate immunological response mechanisms rather by cytokine supply than by their cytotoxic potential. The poor cytolytic capacity of CD56bright NK cells can be explained by weak ability in forming conjugates with target cells and low contents of perforin and granzyme A in their granules.

Natural killer (NK) cells destroy virus-infected and tumor cells without prior antigen stimulation. The NK cell cytotoxicity is regulated in large part by the expression of NK cell receptors that are able to bind major histocompatibility complex (MHC) class I glycoproteins. NK cells also express lysis triggering receptors specific for non-MHC ligands, including NKp30, NKp44, NKp46 and CD16. However, the nature of their ligands, recognized on target cells, is undefined. We have recently shown that the NKp46 protein, but not the CD16 protein, recognizes the hemagglutinin (HA) of influenza virus (IV) and the hemagglutinin-neuraminidase (HN) of Sendai virus (SV), and that the recognition of HA from IV requires the sialylation of NKp46 oligosaccharides. We have also demonstrated that binding of NKp46 to HA of IV is required for lysis of cells expressing the corresponding glycoproteins by a substantial subset of NK clones. Here we show that NKp44, but not NKp30, can also recognize the HA of both IV and SV and that the recognition of IV HA requires the sialylation of the NKp44 receptor in a similar way to that of NKp46. SV infection of 721.221 cells expressing MHC class I proteinsresulted in the abrogation of the inhibition by NK clones expressing high levels of NKp44. In addition, the binding of NKp44 to HA improves the ability of some NK clones to lyse IV infected cells.

The ability of NK cells to kill a wide range of tumor or virally infected target cells as well as normal allogeneic T cell blasts appears to depend upon the concerted action of multiple triggering NK receptors. In this study, using two specific monoclonal antibodies [(mAb) MA152 and LAP171], we identified a triggering NK receptor expressed at the cell surface as a dimer of approximately 80 kDa (NKp80). NKp80 is expressed by virtually all fresh or activated NK cells and by a minor subset of T cells characterized by the CD56 surface antigen. NKp80 surface expression was also detected in all CD3- and in 6 / 10 CD3+ large granular lymphocyte expansions derived from patients with lymphoproliferative disease of granular lymphocytes. In polyclonal NK cells, mAb-mediated cross-linking of NKp80 resulted in induction of cytolytic activity and Ca2+ mobilization. A marked heterogeneity existed in the magnitude of the cytolytic responses of different NK cell clones to anti-NKp80 mAb. This heterogeneity correlated with the surface density of NKp46 molecules expressed by different NK clones. The mAb-mediated masking of NKp80 led to a partial inhibition of the NK-mediated lysis of appropriate allogeneic phytohemagglutinin-induced T cell blasts, while it had no effect on the lysis of different tumor target cells, including T cell leukemia cells. These data suggest that NKp80 recognizes a ligand on normal T cells that may be down-regulated during tumor transformation. Molecular cloning of the cDNA coding for NKp80 revealed a type II transmembrane molecule of 231 amino acids identical to the putative protein encoded by a recently identified cDNA termed KLRF1.

Natural killer (NK) cells survey potential targets using an array of receptors specific for major histocompatibility complex class I molecules. In mice, members of the Ly49 receptor gene family are expressed on overlapping subsets of NK cells and on CD1-restricted NK1 T cells. Here we characterize a population of memory cytotoxic (CD8(+)) T lymphocytes which also express inhibitory Ly49 family members. This cell population increases steadily with age; by 11 months, over one third of memory CD8(+) T cells express Ly49 molecules. These cells appear to express a normal TCR repertoire, and share several traits with previously activated T cells. Analysis of mutant mouse strains reveals that normal development of these cells depends upon the presence of the transporter associated with antigen presentation (TAP), classical class I molecules, and class II molecules. As a functional consequence of Ly49 expression, we demonstrate that T cell receptor-mediated activation of CD8(+) T cells is inhibited by Ly49 interactions with cognate class I molecules. We hypothesize that conventional memory CD8(+) T cells initiate Ly49 expression as a means of dampening an immune response and / or inhibiting T cell autoreactivity.

NK and T lymphocytes share various cell surface receptors, including NK receptors for MHC class I molecules (NKR). NKR include killer cell Ig-like receptors (KIR) and lectin-like dimers which are composed of the invariant CD94 associated with a variety of NKG2 molecules. The combination of KIR and CD94/NKG2 dimers expressed on NK and T cell subsets defines a repertoire of MHC class I recognition. Engagement of NKR by cognate MHC class I molecules governs T and NK cell activation. We investigated the NKR distribution on NK and T cell subsets from uninfected and HIV-infected individuals, according to the clinical status, the absolute numbers of CD4+ T cells as well as the plasmatic viral load of the patients. We show that the KIR distribution on NK cells is not affected by HIV-1 infection, whereas the absolute numbers of T cells expressing specific KIR members (CD158b, p70) transiently increase in early stages of HIV infection. By contrast, the percentages of NK and T cells which express CD94 dimers increase in parallel with the disease. These results document a differential regulation of KIR and CD94 lectin-like dimers during the course of a chronic viral infection in humans and further suggest that both types of NKR are independently regulated.

Our understanding of the biology of human natural killer (NK) cells has significantly advanced in recent years upon identification of a family of NK cell-expressed genes that encode killer cell inhibitory receptors (KIR). Individual KIR can selectively bind various HLA class I allotypes and consequently transduce inhibitory signals that block NK cell lysis of ligand-bearing target cells. A distinct subset of related and linked genes express truncated versions of KIR that are otherwise highly homologous in amino acid sequence. Interestingly, these receptors appear to transmit stimulatory signals into NK cells and have been termed killer cell activating receptors (KAR). In this report, we demonstrate that recognition of HLA-Cw3 by the p50 KAR, NKAT8, can potentiate the cytotoxic response of appropriate NK cell clones. Specific cross-linking of this KAR with a monoclonal antibody resulted in intracellular calcium mobilization, protein tyrosine phosphorylation, and phosphorylation of the MAP kinases, ERK1 and ERK2. In addition, we identified a KAR-associated disulfide-linked dimer of a 13-kDa protein that was absent in the Jurkat T cell line and is predicted to participate in these activation signaling events. Upon treatment of NK cells with pervanadate, the disulfide-linked p13 and additional proteins of 25, 30, 37 and 50-95 kDa were identified as KAR-associated tyrosine phosphoproteins. Importantly, p13 was inducibly tyrosine phosphorylated upon cross-linking of NKAT8, which strongly suggests that the associated p13 provides KAR with appropriate cytoplasmic structure to couple with tyrosine kinase-mediated signaling effectors.

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six-eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2p) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3 zeta 1 ITAM with a non-peptidic linker made up of gamma-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.

Conserved sequences within gene families permit the design of consensus primers that match several members of a given class of homologous genes. Polymerase chain reaction (PCR) products obtained with such consensus primers were characterized by restriction mapping or single-strand conformation polymorphism (SSCP) analysis, using precast polyacrylamide minigels and automated silver staining. Examples for the electrophoretic distinction of consensus amplificates are presented in the fields of guanylyl cyclase expression studies and in the determination of B-cell clonality in human blood samples. Guanylyl cyclase expression in inner ear tissues of guinea pigs was investigated by reverse transcription PCR using consensus primers with specificity for the subclass of particulate guanylyl cyclases. The resulting PCR products were assigned to three representatives of this group by restriction mapping. The consensus PCR approach enabled the detection of an unexpected receptor type, namely guanylyl cyclase C, in the inner ear. The distinction by SSCP analysis of denatured consensus amplificates was appropriate for the identification of clone-specifically rearranged immunoglobulin heavy chain genes of B-lymphocytes. Genomic DNA isolated from blood samples of leukemia patients served as the template for the consensus amplification of clone-specific VDJ rearrangements. Rapid distinction and re-identification of consensus PCR products was achieved by SSCP analysis for regular antigen receptor rearrangements and for t(14; 18) translocations. The potential of these procedures for detecting leukemia or lymphoma clones when monitoring minimal residual disease was assessed.

Macrophage scavenger receptors are trimeric integral membrane glycoproteins which have been implicated in various macrophage functions including uptake of oxidized lipoprotein and the serum-dependent, divalent cation-independent adhesion of macrophages to tissue culture-treated plastic. In this study we have used a recently defined monoclonal antibody (2F8) which recognizes murine macrophage scavenger receptor, to explore its expression in lymphoid and non-lymphoid organs of the normal adult. Scavenger receptor was detected in the red pulp and marginal zone of normal adult mouse spleen, medulla of the thymus and subcapsular region of lymph nodes. Kupffer cells in the liver, alveolar macrophages in the lung and lamina propria macrophages in the gut all reacted with 2F8 monoclonal antibody. The antigen was not detected on any non-macrophage cells, with the exception of sinusoidal endothelial cells in the liver. In the spleen, lymph node and liver, scavenger receptor antigen expression was associated specifically with phagocytic cells which had taken up colloidal carbon. To examine macrophage adhesion in a context relevant to the interactions occurring within lymphoid and non-lymphoid organs, and the contribution of macrophage scavenger receptor to this adhesion, we designed an assay of macrophage adhesion to frozen tissue sections. Adhesion to most tissues was high and uniform in the absence of any chelating agents. The chelation of Ca2+ and Mg2+ revealed specific patterns of macrophage adhesion in lymphoid and non-lymphoid organs which was completely inhibited by 2F8. The ability of this antibody to block the EDTA-resistant adhesion correlated with tissue expression of the antigen in some tissues. Unlike adhesion to tissue culture-treated plastic, macrophage scavenger receptor-dependent adhesion of macrophages to frozen tissue sections did not exhibit an absolute requirement for exogenous fetal bovine serum indicating the presence of an endogenous ligand for scavenger receptor within the tissues. We propose that macrophage scavenger receptor is a candidate homing or retention molecule for macrophage localization within ligand-rich tissues.

In T lymphocytes, CD2 forms part of a loosely associated membrane complex which includes the T cell receptor (TcR) for antigen, the CD3 subunits, CD4 or CD8, CD5 and the protein tyrosine kinases p56lck and p59fyn. The interaction of CD2 with tyrosine kinases in this complex provides a possible mechanism for transmembrane signal transduction by CD2. We have investigated whether the interaction of CD2 with the kinases is dependent on other known members of the complex, or whether an independent association can be observed. Using in vitro kinase assays with immune complexes precipitated from cell lysates, we demonstrate that CD2 can associate with p56lck and p59fyn in a rat thymoma line that does not express CD4 or CD8, and in a TcR-negative Jurkat cell line. In TcR-positive Jurkat cells that express rat CD2, interaction of CD2 with p56lck and p59fyn was clearly seen, but it was absent in cells where the cytoplasmic tail of CD2 is truncated, indicating that the interactions are mediated by the cytoplasmic region of CD2. Furthermore, using cells expressing CD2 molecules with partial truncations in the cytoplasmic domain, we show that the association of CD2 with p56lck is progressively lost as the cytoplasmic domain is shortened, and that the capacity of the mutants to associate with p56lck correlates with their capacity to transduce transmembrane signals.

Lipopolysaccharide (LPS) stimulates mononuclear phagocytes to synthesize and secrete immunoregulatory and inflammatory molecules such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha). LPS forms complexes with either the serum protein termed LPS-binding protein or a serum factor, septin. These complexes are more stimulatory than LPS alone. The myeloid differentiation antigen CD14 is known to be the receptor for such complexes. In the present study, by using a monocytic cell line, we demonstrate the release of two different soluble forms of CD14 (sCD14) which are secreted by different mechanisms. We show that the two sCD14 forms differ in their electrophoretic mobility, two-dimensional gel electrophoretic patterns, sensitivity to endoglycosidases and peptide maps. One of the sCD14 molecules, apparent molecular mass 48 kDa, was found in supernatants of both surface iodinated and [35S]methionine biosynthetically labeled cells. The other sCD14 molecule (56 kDa) was found labeled only in supernatants of [35S]methionine-labeled cells. Furthermore, purified 48 kDa sCD14 enhanced the LPS-induced TNF-alpha and IL-6 release by the monocytic cells suggesting that a cell-surface signal transducer molecule may be involved in signaling. The data suggest a possible novel role for sCD14 in the monocyte response to LPS.

The characteristics of soluble interleukin-6 receptor (sIL-6R) in murine sera were examined. To investigate a relationship between serum sIL-6R level and autoimmune diseases, quantitative analysis of serum sIL-6R in MRL/lpr mice was performed by an enzyme-linked immunosorbent assay. The serum sIL-6R level in MRL/lpr mice of both sexes was below the detection limit (< 1.0 ng/ml) at 8 weeks of age, but it increased in accordance with age and reached 42 +/- 9.3 ng/ml in female and 31 +/- 13 ng/ml in male mice at 30 weeks of age. In MRL/+ mice, although an age-associated increase in serum sIL-6R level was observed, it was much less extensive than that in MRL/lpr mice. Elevated serum sIL-6R level at the age of 30 weeks was observed in female and male (NZB x NZW)F1 mice (32 +/- 10 ng/ml and 17 +/- 5.0 ng/ml, respectively), and male BXSB/Mpj Yaa mice (42 +/- 18 ng/ml), suggesting that elevated serum sIL-6R in aged mice is one of the characteristics of autoimmune-prone mice. Quantitative analysis of serum IL-6 in MRL/lpr revealed that the serum sIL-6R level correlated well with the serum IL-6 level. We also showed that sIL-6R in the sera from MRL/lpr mice could mediate the IL-6 functions through the IL-6 signal-transducing receptor component gp130, suggesting that elevated production of sIL-6R may partly contribute to development of autoimmune disease in MRL/lpr mice.

The expression of interleukin (IL)-1 beta, IL-6 and their respective receptors has been studied in the rat brain before and up to 24 h after injury. Messenger RNA transcripts of these four genes were detected by in situ hybridization (ISH) in different structures of the intact brain. The distribution was very similar for IL-1 beta, IL-6 and IL-6 receptor (IL-6R). The expression of IL-1R was more widespread. Within hours after injury, an increased expression of IL-1 beta, and thereafter of IL-6 was documented. The expression of IL-1R and IL-6R was also increased. This expression was bilateral and not restricted to the injured area. Within 24 h, all ISH patterns had returned to normal. The molecular data were confirmed by protein data. Indeed, the distribution of IL-6 (detected by immunocytochemistry) agreed with the ISH patterns for IL-6. Furthermore, extracellular fluid was collected by microdialysis at the site of the lesion during 12 h and successive fractions were assayed for the presence of bioactive IL-1 and IL-6. Increases in IL-1 and later in IL-6 levels were detected. The rapid and concomitant increased expression of IL-1 beta, IL-6 and their receptors after injury stresses their possible early role in inflammatory mechanisms also in the brain, before any recruitment of inflammatory cells from remote nervous and not nervous areas.

Deregulation of the proto-oncogene MYC by specific chromosomal translocations has been shown to be essential but not sufficient for the development of Burkitt's lymphoma (BL). To identify other genes which either mark important steps in tumorigenesis or which reflect the cellular differentiation state of BL cells we have compared tumor cells to immortalized lymphoblastoid B cells by subtractive hybridization. We have identified a complementary DNA clone which encodes a novel member of the superfamily of GTP-binding (G) protein-coupled receptors, designated BLR1. The corresponding mRNA is expressed in BL and lymphatic tissues but not in other cell lines either of the B cell lineage or of other hematopoietic or non-hematopoietic origin. This exclusive expression of BLR1 and the oncogenic potential of this receptor class supports the hypothesis that BLR1 exerts a regulatory function in BL lymphomagenesis and/or B cell differentiation. Moreover, the protein sequence is highly related to that of receptors for the cytokine interleukin (IL)-8 and other neutrophil chemoattractants. We conclude that BLR1 may represent a potential candidate involved in the process of physiologic trafficking, cell-cell interactions, and activation of mature B lymphocytes in lymphatic tissues.

The T cell surface molecule CD2 forms, with its counter-receptor CD58 (LFA-3), a powerful adhesion/activation pathway. There is some evidence that CD2 might bind more than a single ligand. Chinese hamster ovary cells (CHO) expressing human CD59 after cDNA transfection (CD59+CHO) form rosettes with human T cells; these rosettes are inhibited in a dose-dependent fashion by the CD59 monoclonal antibody (mAb) H19 and by the CD2 mAb O275 known to block natural rosettes, but not by the CD2R mAb D66, a poor rosette blocker. CD2+CHO transfectants form rosettes with human erythrocytes; these rosettes are inhibited by the CD59 mAb H19 in addition to CD2 mAb O275 and CD58 mAb; murine thymoma cells expressing human CD2 form rosettes with CD59+CHO cells that again are blocked by CD59 H19 and by CD2 O275 mAb. In a marked contrast with H19, two others CD59 mAb, YTH 53.1 and MEM 43, which react with a different epitope on CD59, led to a 50%-70% increase of the number of cells forming rosettes. In addition to rosette experiments, the binding of 125I-labeled CD59 molecules to CD2+CHO cells was specifically inhibited by unlabeled CD59 molecule and CD2 mAb. Furthermore, the binding of CD59 molecules to resting T cells induced expression of CD2R epitopes. These results directly show that CD2 binds CD59 and that subtle molecular changes occur upon binding.

Interferon-gamma (IFN-gamma) is produced by activated T lymphocytes and plays a regulatory role in immune responses. The nature and location of cells that express the IFN-gamma receptor (R) and respond to this lymphokine are not well documented. The distribution of human IFN-gamma-R (HuIFN-gamma-R) was, therefore, investigated in situ by immunohistochemistry, using affinity-purified rabbit polyclonal antibodies directed against the extracellular domain of the receptor. In lymphoid organs, IFN-gamma-R expression is restricted to the B cell areas of lymph nodes, adult and fetal spleen, tonsils, appendix, and mucosa-associated lymphoid tissue of the small bowel. Macrophages and other reticular cells in lymphoid tissues and other organs are strongly positive for IFN-gamma-R, whereas its expression was consistently negative in the cortical and medullary thymocytes. Two-color flow cytofluorometric analysis of blood, lymph node, tonsil, spleen and thymus cells confirms that most B lymphocytes are IFN-gamma-R positive, whereas T lymphocytes are negative. However, after in vitro activation, peripheral blood T cells become IFN-gamma-R+. In non-lymphoid organs, IFN-gamma-R is expressed on endothelial cells of the medium- and small-size vessels. In epithelial tissues, high expression of IFN-gamma-R is detected on trophoblastic epithelium, glandular cells of stomach, ileum and colon, lung alveolar cells, salivary duct cells, renal tubular cells, and endometrial mucosa cells. Hepatocytes are weakly positive, while squamous epithelial cells are negative. The distribution of the HuIFN-gamma-R is discussed in view of the known functions of IFN-gamma.

Myeloma is one of the interleukin (IL)-6-related diseases to which abnormal expression of IL-6 has been reported to be linked. We examined the in vivo inhibitory effect of anti-human IL-6 receptor (IL-6R) antibody on human myeloma cell growth in mice. SCID mice were subcutaneously inoculated with solid tumor of the myeloma cell line S6B45 in which human IL-6 was acting as an autocrine growth factor. Ten intraperitoneal administrations of 100 micrograms of the anti-human IL-6R antibody PM1 at 48-h intervals strongly inhibited the growth of S6B45 cells when the administration started 24 h after tumor inoculation. The tumor growth inhibition in vivo was also observed by administration of the anti-human IL-6 antibody MH166 using the same procedure as for PM1. The inhibitory effect of PM1 was not significant when the administration started 5 or more days after tumor inoculation. This work indicates that anti-human IL-6R antibody, as well as anti-human IL-6 antibody inhibits human myeloma growth in vivo, and provides an animal model for testing the therapeutic value of agents such as antibodies to human IL-6, IL-6R and gp130, an IL-6R-associated signal transducer, in the treatment of human myelomas.

This paper reports on cDNA coding for the 80-kDa murine IL6 receptor (mIL6R) that was cloned from a mouse liver cDNA library. Human hepatoma Hep3B cells transfected transiently or stably with an expression vector carrying the entire coding region for mIL6R become responsive to mouse IL6 (mIL6). We monitored response to the cytokine through the transcriptional activation of a co-transfected IL6-inducible human C-reactive protein (CRP) promoter; response to mIL6 is lost upon treatment of the cells with increasing amounts of a monoclonal antibody to mIL6R. mIL6R mutants have been generated in the carboxy-terminal portion of the molecule. Their functional analysis in hepatoma cells shows that the intracytoplasmic domain of the receptor is not absolutely essential to IL6 signal transduction (i.e. CRP promoter activation), but that the last 40 amino acids contribute to maximal IL6 response in these cells.

The T cell-derived cytokine interleukin (IL) 4 is known to increase the proliferative response of T cells stimulated with IL 1. IL 4 is also an autocrine growth factor for type II T helper cells (Th2) cells. In the present studies, we examined the effect of murine recombinant IL 4 on the expression of the IL 1 receptor type I (IL 1RtI) in murine Th2 cell lines at the mRNA and surface level. Using a specific anti-murine IL 1RtI monoclonal antibody and flow microfluorometry, we found that IL 4 increased the surface expression of IL 1RtI in a dose-dependent manner. In D10S cells, a subline of the Th2 cell line D10.G4.1, 50-500 pg/ml IL 4 up-regulated the receptor 1.8- to 3.2-fold (p less than 0.05). This up-regulation was also seen at the mRNA level. The effect was not due to increased stability of the mRNA, since IL 4 did not modify the half-life of IL 1RtI mRNA. IL 4 also up-regulated IL 1RtI on CDC25 cells, another Th2 cell line. However, we did not observe an effect of IL 4 on gene expression of IL 1RtI in BALB/c 3T3 fibroblasts. IL 2 and IL 4 showed an additive effect in up-regulating IL 1RtI and D10S cells. These studies indicate that IL 4 up-regulates IL 1RtI in murine Th2 cells by increasing gene expression for IL 1RtI without affecting mRNA stability. Thus, IL 4 production by Th2 cells may amplify the immune response via up-regulation of IL 1RtI.

The relationship of dendritic cells (DC) isolated from the peripheral blood to those of lymphoid tissue is, in terms of maturation and function, incompletely understood. In our present study, we have explored the molecular basis of adhesion of T cells to blood DC. Analysis of the expression of adhesion receptors on the cell surface of blood DC revealed that these cells express lymphocyte function-associated antigen (LFA)-1 (CD11a/18), ICAM-1 (CD54), LFA-3 (CD58) and CD44, but are very late antigen (VLA)-4 (CD49d) and vascular cell-adhesion molecule (VCAM)-1 negative. The LFA-1 pathway was found to play a key role in T cells-blood DC adhesion; monoclonal antibodies (mAb) against both LFA-1 and ICAM-1 strongly inhibited adhesion between those cells. Moreover, a T cell clone from an LFA-1-deficient patient showed poor binding to blood DC. The important role of LFA-1 in T cell-blood DC adhesion was also supported by the metabolic energy and divalent cation dependence of the interaction. mAb against LFA-3 and CD2 did not inhibit T cell-blood DC binding. In contrast to the strong inhibition by antibodies to LFA-1 and ICAM-1, antibodies to CD44 enhanced conjugate formation between T cells and blood DC. Together, our results show that the LFA-1/ICAM-1 pathway plays a central role in T cell-blood DC adhesion, a situation like that in T cell adhesion to lymphoid DC. However, unlike lymphoid DC, blood DC do not express VCAM-1 nor use LFA-3 for T cell binding.

Acute P. falciparum malaria is associated with loss of in vitro T cell responsiveness to antigenic stimulation, and with high plasma levels of soluble interleukin 2 receptor (IL 2R). In the present study peripheral T cells from acute P. falciparum malaria patients from a malaria-endemic area of Sudan were analyzed for expression of cell surface antigens associated with T lymphocyte adhesion, activation and maturation. The results were compared to results from T cells obtained from the same donors either before the attack, or during convalescence. Most donors showed a remarkable loss of T cells with high expression of the surface marker LFA-1 (CD11a/CD18) during the clinical episode, in addition to the functional changes described above. Two donors that did not show phenotypic changes were furthermore characterized by having an unabated proliferative response and normal plasma IL 2R levels. All peripheral CD3+ T lymphocytes expressed LFA-1, which had a clearly bimodal distribution on these cells. The T cell subpopulation having high LFA-1 expression (LFA-1++) was composed of both memory and unprimed T cells, according to their expression of CD45RA and CD45R0. Analysis of expression of membrane-bound IL 2R (CD25) and ICAM-1 (CD54) did not reveal in vivo activated T cells in the peripheral blood of the patients. Taken together, these data suggest that circulating T cells recognizing parasite antigens are temporarily withdrawn from peripheral circulation during P. falciparum malaria.

A polypeptide chain for the mouse interleukin 5 receptor (IL5R) was purified from detergent-lysed B13 cells, a mouse IL5-dependent pre-B cell line. Purification was by a single immunoaffinity chromatographic step using an anti-mouse IL5R monoclonal antibody, R52. Internal amino acid sequence was obtained from four trypsin-generated peptides. All peptides were found to be present in the published amino acid sequence of a mouse IL3R and the mouse IL3R-like protein deduced from the cDNA. This indicates that the mouse IL5R and the mouse IL3R have a homologous polypeptide in common and suggests that the specificity of these lymphokine receptors is mainly generated by association with another ligand-specific polypeptide chain.

The phenotype of intraepithelial lymphocytes (IEL) from the small intestine of adult rats was studied by flow cytometry. Using appropriate monoclonal antibodies the expression patterns of the T cell receptor alpha/beta (TcR2), CD2, the alloantigen RT6 and several other T cell antigens were analyzed. The vast majority of rat IEL expressed TcR2 which is in contrast with data reported for the mouse. The comparison of IEL with lymph node cells revealed major phenotypic differences. Whereas CD2 was present on virtually all lymph node T cells it was found on only less than 5% of IEL. The T cell-specific differentiation antigen RT6 present on only a fraction of lymph node cells was found on about 99% of IEL demonstrating uniform expression with an approximately tenfold higher density. Identity of the detected molecule with RT6 was proven by using congenic controls and by the demonstration of phosphatidylinositol linkage to the IEL membrane. About 86% of IEL expressed CD8 but a substantial proportion of these cells co-expressed the CD4 molecule (34%). Two-color analysis revealed that the CD4+CD8+ double-positive subset completely lacked CD45RB suggesting that they represent memory cells. In the CD4-CD8+TcR2+ subset there was a remarkable heterogeneity of CD5 expression. A substantial number of these cells did not express CD5 despite high density of TcR2. Phenotypic peculiarities found on all or most IEL such as the lack of CD2 and the increased expression of RT6 indicate that the intestinal epithelial environment exerts strong effects on the development and maturation of these cells.

The characteristics of the interleukin (IL) 5-reactive splenic B cell population of C57BL/6 nu/nu mice, with respect to IL 5/IL2 reactivity, cell surface phenotype, VH gene family usage, autoreactivity and the structure of the IL5 receptor (IL5R), were analyzed. It was found that 2%-4% of splenic B cells express relatively high levels of IL 5R as determined by the binding of the anti-IL 5R monoclonal antibody R52.120. Over 90% of the splenic B cells that mature to IgM secretion upon activation with IL5 are comprised in this small subpopulation of B cells. Moreover, the vast majority of splenic B cells that mature to IgM-secreting cells when activated by IL2 also reside in this IL5R+B cell population. The cell surface phenotype of the IL5R+ splenic B cells is IgM+, B220+, Ly-1- and IL2R p55-. Upon activation with IL5 this cell surface phenotype changes, in that a vast majority of the B cells then express the p55 chain of the IL2R, whereas the level of IL5R decreases. VH gene family usage in the IL5-activated splenic B cells was analyzed by in situ hybridization. VH gene family usage was found to be random and not different from the VH genes expressed in LPS-activated B cells. Hybridoma collections from IL5-activated splenic B cells and LPS-activated B cells were screened and compared for the production of autoantibodies and antibodies directed against the haptens (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and 2,4,6-trinitrophenyl (TNP). In both collections high, but not significantly different frequencies of autoantibody-(32% IL5, 31.4% LPS) and of anti-hapten antibody (27.8% IL5, 18.6% LPS)-producing hybridomas were found. The structure of the IL5R on IL5-activated B cells was analyzed by 125I-labeled IL5 binding and cross-linking. About 100 high-affinity (10(-11) M) and 1000 low-affinity (10(-9) M) IL5-binding sites are present on IL5-activated splenic B cells, and both high- and low-affinity IL5R are similar to those expressed on the IL5-dependent B13 cell line. Cross-linking of 125I-labeled IL5 to the receptors on IL5-activated B cells revealed one major IL5-binding protein of 45-50 kDa molecular mass and another minor binding protein of 130-140 kDa. The same IL5-binding proteins are present on the IL5-dependent B13 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

The species swine provides the only example for CD2+ and CD2- subsets of Ig-CD4-CD8- lymphocytes with the propensity for homing to lymphoid tissue (Saalmüller et al., Eur. J. Immunol. 1989. 19: 2011). That the CD2-CD4-CD8- lymphocytes are bare of marker molecules that typify T lymphocytes raised the question of whether or not this cell type is descended from the T lymphocyte differentiation lineage. It is documented that expression of a phylogenetically conserved external epitope of T cell receptor gamma/delta subdivides porcine CD2- lymphocytes into an epitope 86D+ minor and an 86D- major subset. Expression of distinct forms of the T cell receptor gamma/delta, disulfide-bonded N-glycosylated surface heterodimers of under reducing conditions 38/40 and 37/40 kDa, respectively, hallmarks the CD2-86D+ and CD2-86D- subsets both as T lymphocytes.

By sequential immunization with a variety of different interleukin (IL) 1 receptor (IL 1R)-bearing cells and by generating a large number of clones from a single fusion experiment, we have managed to produce two monoclonal antibodies which react with the lymphocyte IL 1R. The antibodies also react with the mouse fibroblast line, 3T3, but we have not yet defined their reactivity with cells carrying low-affinity receptors. Immunoprecipitation allows the IL 1R to be isolated from EL4 6.1 cell membrane preparations and shows that the IL 1R exists as a complex. The antibodies clearly interfere with IL 1 responses both in vitro and in vivo. These are the first anti-mouse IL 1R antibodies to be described which clearly and profoundly affect the immune response.

Latex particles coated with human IgA were used to detect the presence in human blood of lymphocytes capable of binding IgA. It was necessary to culture the lymphocytes in medium without IgA before the assay. A small percentage of both T and non-T cells had IgA receptors. The receptors were distinct from those binding to IgG or IgM.

It is shown here that a minor subpopulation of the human thymus, representing 2-3% of the whole thymocyte population, can suppress proliferation of syngeneic or allogeneic thymocytes to various stimuli. In contrast, this suppressor cell population has no influence on the proliferative response of peripheral blood lymphocytes. This subpopulation was defined by its cell surface phenotype as CD3+, CD1-, 4-, 8- cells. Its suppressive activity is generated after concanavalin A (Con A) stimulation, but not after stimulation with phytohemagglutinin. In addition, the suppressive activity is not modified by extensive washes of Con A-stimulated cells with 1-O-methyl-alpha-D-glucopyranoside, a specific sugar for Con A. This suppressor cell population does not exert any detectable cytotoxic activity, nor does it act by consuming available interleukin 2 (IL 2). Rather, it prevents IL 2 receptor expression on thymic target cells. Since Con A might activate T cells by binding to molecules physiologically involved in T cell activation, particularly the CD3-T cell receptor complex, these data could be relevant to the regulation of normal thymic differentiation.

The MRC OX-22 monoclonal antibody recognizes a restricted determinant of the rat leukocyte-common antigen (L-CA, CD45), which is expressed on most peripheral T cells and all B cells. In contrast only 2%-3% of thymocytes are OX-22+ and these are now shown to be mostly of the immature CD4-CD8- (double-negative, DN), phenotype with very few of the mature phenotype cells being OX-22+. Analysis of immunoperoxidase sections suggests that the DN OX-22+ cells are located in the cortex. Among the DN cells about 60% are OX-22+ and a similar percentage are positive for CD2 antigen. Double staining showed that OX-22+CD2-, OX-22+CD2+, and OX-22-CD2+ populations can be defined and that these three sets account for approximately 95% of the DN cells. Measurement of the thymopoietic activity of DN subsets showed that OX-22+CD2- and OX-22+CD2+ cells have regenerative capacity whilst OX-22-CD2+ cells do not.

Fifteen T cell clones with different (specific, antibody-dependent or natural killer-like) cytolytic activity were derived from mixed lymphocyte culture activated T cells and analyzed for their morphological characteristics and, in some instances, for their surface markers. All of the five cytolytic clones analyzed by electron microscopy possessed numerous electron-dense granules and in some instances multivesicular bodies, with or without an electron-dense matrix, that are the putative precursors of the granules. In addition, light microscopy examination of semithin sections of other ten cytolytic clones showed that the large majority of the cells in each clone had numerous toluidine blue-stained cytoplasmic granules. It is of note that nine clones without detectable cytolytic activity analyzed by electron microscopy did not possess granules and presented the features of large agranular blasts. Ten cytolytic clones were analyzed for different surface markers including rosette formation with sheep erythrocytes (E rosettes), receptors for the Fc portion of IgG or IgM ((Fc gamma R and Fc mu R) and a group of antigens recognized by monoclonal antibodies including Ia, 4F2, OKT8 or OKT4. All the clones were E-rosette+, Ia+ and 4F2+. Expression of Fc gamma R was restricted to the clones active in antibody-dependent cell-mediated cytotoxicity. Expression of OKT8 and OKT4 antigens was variable; in particular, five clones were OKT8+/OKT4-, whereas the other five expressed the OKT8-/OKT4+ phenotype. It is noteworthy that the ultrastructural features of cytolytic T cell clones are similar to those of large granular lymphocytes, known to be the only lymphoid cells in normal peripheral blood which possess cytolytic activity. Thus, it is possible that the presence of electron-dense granules may represent a morphological marker for all human cytolytic lymphocytes.

Highly purified blood platelets from man and rat could be induced into cytotoxic effectors against schistosome larvae by an IgE-dependent mechanism. Such a process implied the existence of a receptor for the Fc part of IgE on the surface of these blood elements. Normal platelets, incubated in the serum of infected individuals as well as in the IgE-rich serum from asthmatic patients, showed similar capabilities. Flow cytofluorometric analysis evidenced that the platelets bearing IgE receptors represented a subpopulation (20%), the percentage of which was significantly increased (up to 50%) in rats or patients with high levels of circulating IgE. Radiolabeled IgE, whose binding was specifically inhibited by an excess of unlabeled IgE or by anti-Fc epsilon receptor antibody, allowed the demonstration that the receptor for this isotype on the platelet surface was saturable. The binding of increasing amounts of IgE followed a bimodal curve, with less than 1000 sites per platelet showing an affinity coefficient of 3.3 X 10(7) M-1 at low concentrations, and a Ka of 7.8 X 10(5) M-1 for higher concentrations. Beyond their interest in the demonstration of cytotoxic properties of thrombocytes, these observations place emphasis on the potential role of the platelets in immediate-type allergic reactions by their direct interaction with IgE antibody molecules, through a specific receptor.

Local graft-vs.-host reaction (GVHR) was induced in rats by injecting parental cells into young F1 recipients. As a consequence of antigenic stimulation in the course of developing GVHR in the responding lymph nodes, the number of interleukin 2-receptor (IL 2R)-bearing T cells increased from less than 1% up to 10% of the total population. The IL 2R-bearing cells were located mainly in the T cell areas of the reactive lymph nodes. As assessed by the determination of the GVHR indices, treatment of the recipients with anti-T-helper subset-specific mAb (W3/25) or with anti-IL 2R mAb (ART-18) inhibited the GVHR. In parallel, the number of IL 2R-bearing cells was reduced to the normal levels. W3/25 mAb treatment changed the helper/suppressor subset ratio and reduced the number of circulating lymphocytes in the peripheral blood. In contrast, ART-18 mAb treatment did not induce any detectable changes in the subset distribution and it did not affect the number of circulating lymphocytes. The results demonstrate the key role that the IL 2R-positive cells play in the proliferative phase of acute GVHR, and favor the use of anti-IL 2R mAb as selective immunosuppressive agents.

The presence of Fc receptors (FcR) on rabbit peripheral blood leukocytes is demonstrated using rosette formation with an ox erythrocyte-antibody (EoxA) complex. The receptor is specific for the Fc fragment of IgG (neither IgM nor F (ab')2 anti-Eox mediates rosette formation) that is antigen-bound (aggregated rabbit IgG inhibits the rosette formation only transiently). The receptor is species-specific: guinea pig IgG/Eox, goat IgG/Eox and sheep IgG/Eox complexes do not show rosette formation, and goat IgG aggregates do not inhibit rosette formation. The origin of the target erythrocytes is of importance. Sheep erythrocytes are not useful, and within Eox large differences between donors were found. Rosette formation was only inhibited by pretreatment of the rosette-forming cells with homologous immune complexes, whereas the size of the antigen greatly influenced the degree of inhibition. The rabbit FcR is pronase-resistant, unlike the human and murine RcR. The interaction of IgG and the FcR is not inhibited by isolated C gamma 3 domains. Further evidence for the requirement of the whole Fc region was obtained in experiments where inhibition of the rosette formation was observed using antisera directed to the C gamma 3 and the C gamma 2 domain, respectively. Anti-Fab antiserum did not inhibit rosette formation. Results are discussed in relation to the mechanism of allotypic suppression.

We have used short-term, high-density cultures to demonstrate that interleukin 2 (IL 2) in picomolar amounts causes entry of approximately 2% of thymocytes from 3-month-old rats into mitosis. Newborn and fetal animals show a higher response reflecting a greater proportion of cells which have been shown to express IL 2 receptors at this age. In vivo administration of nanogrammes of IL 2 or injection of rats with syngeneic spleen cells which had been stimulated in vitro with concanavalin A to release IL 2 were also shown to increase the proliferation of both thymus and bone marrow cells. This suggests that IL 2, in amounts which could be produced by peripheral lymphoid tissue during immune responses, could act to increase the turnover of lymphocytes in bone marrow and thymus.

Thymocytes contain a small population of light (o = 1.067), large cells with high cytolytic potential against natural killer target as well as against IgG- or IgM-coated erythrocytes and nucleated cells, thus displaying natural killer and antibody-dependent cellular cytotoxicity functions, but not exhibiting T killer cell capacity. As far as characterized, the cells show T cell characteristics: they are nonadherent, nonphagocytic, surface immunoglobulin-negative, C3 receptor-negative, partly Fc gamma receptor-positive, carry the receptor for Helix pomatia A agglutinin, and are, at least partly, cortisone-resistant and Thy-1-positive. The implications of this finding with respect to the lineage of natural killer and killer cells and their interdependence will be discussed briefly.

The composition of the T lymphocyte population in swine is special in that in addition to the CD4-CD8+ subpopulations, CD4-CD8- and CD4+CD8- and CD4+CD8+ subpopulations are prominent in the peripheral circulating as well as in the resident T lymphocyte pools. Since the same phenotypes are characteristic of thymic populations, it was asked whether the unusual distribution in swine may result from an emigration of thymic precursor phenotypes to the periphery. This explanation was refuted, as all thymic subpopulations were found to express CD1, albeit with differences in antigen density, whereas all extrathymic subpopulations lack CD1. The cellular distribution of CD2 in swine is without precedent among all species studied. Whereas in sheep and cattle the extrathymic CD4-CD8- subpopulation is known to entirely lack CD2 and to have a low propensity for homing to lymphoid tissues, the CD4-CD8- subpopulation in swine splits into CD2+ and CD2- subsets, both of which do reside in lymphoid tissues. While CD2+CD4-CD8- T lymphocytes are rare in the circulating pool, this subset accumulates in spleen and lymph nodes. This may indicate a role for CD2 in homing. Thus the species swine is immunologically unique, not only because of having CD1-CD2+CD4+CD8+ T lymphocytes in the periphery, but also with regard to subdivision and homing behavior of its CD4-CD8- T lymphocyte subpopulation.

After activation by anti-mu antibody human B cells acquire the ability to proliferate in the presence of recombinant interleukin 2 (IL 2), a 20-kDa mol. mass B cell growth factor (BCGF) and a high mol. mass BCGF (50-kDa BCGF). An anti-IL 2 receptor (IL 2R) monoclonal antibody inhibits the IL 2-dependent proliferation without affecting that induced by BCGF. B cells expressing the IL 2R after anti-mu antibody activation (IL 2R+ cells) were separated from those not expressing IL 2R (IL 2R- cells). IL 2 stimulated the proliferation of only IL 2R+ cells whereas the 20-kDa BCGF acted on both IL 2R+ and IL 2R- cells. Importantly, the 50-kDa BCGF supported the proliferation of IL 2R- cells whereas it was inactive on IL 2R+ cells. Thus, the B cell subset responding to the 50-kDa BCGF after anti-mu antibody activation is distinct from that responding to IL 2.