Identification of amino acid residues important for ligand binding to Fas.

The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas-FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages.

Human monocytes undergo spontaneous apoptosis upon culture in vitro; removal of serum from the media dramatically increases the rate of this process. Monocyte apoptosis can be significantly abrogated by the addition of growth factors or proinflammatory mediators. We have evaluated the role of the endogenous Fas-Fas ligand (FasL) interaction in the induction of this spontaneous apoptosis and found that a Fas-immunoglobulin (Ig) fusion protein, an antagonistic anti-Fas monoclonal antibody and a rabbit anti-FasL antibody all greatly reduced the onset of apoptosis. The results indicate that spontaneous death of monocytes is mediated via an autocrine or paracrine pathway. Treatment of the cells with growth factors or cytokines that prevented spontaneous apoptosis had no major effects on the expression of Fas or FasL. Additionally, monocyte-derived macrophages were found to express both Fas and FasL but did not undergo spontaneous apoptosis and were not sensitive to stimulation by an agonistic anti-Fas IgM. These results indicate that protective mechanisms in these cells exist at a site downstream of the receptor-ligand interaction.

A phase I study of CR002, a fully-human monoclonal antibody against platelet-derived growth factor-D.

OBJECTIVE: To investigate the safety, pharmacokinetics (PK), binding activity and immunogenicity of CR002, a human monoclonal antibody (mAb) directed against platelet-derived growth factor-D (PDGF-D), administered as a single intravenous (i.v.) infusion over a range of doses. SUBJECTS: 40 healthy male subjects received increasing doses of CR002 at 0.3, 1, 3, 10, 30 mg/kg or placebo. METHOD: This was a randomized, double-blind, placebo-controlled, dose-escalation Phase I study. The trial had a duration of 90 days, with dosing on Day 1 and follow-up visits on Days 2, 4, 7, 14, 21, 30, 45 and 90. Serum was collected for PK, binding activity and immunogenicity analysis at screening and up to Day 90. Safety was recorded throughout the study by performing laboratory tests, recording vital signs and electrocardiograms (ECGs), by monitoring the occurrence of adverse events (AEs). The use of concomitant medications was also recorded. RESULTS: All 40 subjects received CR002 or placebo, and completed the trial. No dose-limiting toxicities (DLTs) occurred, the maximum tolerated dose (MTD) was not reached and was estimated as > 30 mg/kg. There were no deaths during this study and no SAEs or other significant AEs reported. The most frequent drug-related treatment-emergent AE (TEAE) was headache in 4 of 30 subjects (13.3%) in the CR002 group vs. 0 of 10 subjects in the placebo group. CR002 exhibited linear PK parameters, had a long half-life (t1/2 in the range 15.5 â 48.1 days) and a volume of distribution at steady state in the range 4.7 â 6.5. Free PDGF-D in the serum bound to CR002 in a reversible manner, as shown in the lowest dose cohort. However, levels of total circulating PDGF-D remained constant throughout the study. There were no anti-CR002 antibodies detected in subjects dosed with CR002. CONCLUSIONS: CR002 was safe and well-tolerated at all doses tested as a single i.v. administration. The MTD was estimated to be above 30 mg/kg, the highest dose tested. CR002 had a long half-life, low clearance and a limited tissue distribution. Although total levels of PDGF-D at all dose levels remained relatively constant, there was no detectable circulating free PDGF-D after CR002 administration. At the lowest CR002 dose tested (0.3 mg/kg), PDGF-D was detectable again by Day 21 and the levels increased near to pre-infusion levels by Day 90. In this study, CR002 was not immunogenic during the 90-day study period.

Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development.

Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.

Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation.

The most potent antigen presenting cell present in peripheral blood, lymphoid and non-lymphoid tissue is the dendritic cell (DC). The study of human DC has been restricted by their low frequency in the tissues and the lack of a truly DC specific surface marker to assist in identification and isolation. Standard techniques for the isolation of blood DC generally employ a period of in vitro culture followed by flotation on dense albumin gradients, or more recently, discontinuous gradients of metrizamide. Dense albumin gradients are time consuming to prepare, giving low and variable yields of DC. Metrizamide is more convenient, although exposure of monocytes to metrizamide can decrease the expression of CD14 and alter the accessory cell properties of antigen presenting cells. Here we demonstrate that Nycodenz gradient centrifugation of 16 h cultured, T lymphocyte depleted, peripheral blood mononuclear cells (PBMC) reliably yields a population of low density cells that is highly enriched for DC. Most B and residual T lymphocytes are depleted and NK cell numbers are reduced two-fold from the interface cell population. The high density pellet fraction exhibits very little allostimulatory activity, indicating that few DC pass into the pellet. The low density fraction contains a significant population (20 +/- 5 (SD)%, n = 8) of cells which fail to stain for the lineage markers CD3, CD11b, CD14, CD16, CD19 and CD57. Nycodenz exhibits low toxicity, does not alter the allostimulatory activity of antigen presenting cells, and is therefore ideal for the isolation of cultured DC.

Selection of an unrelated donor for marrow transplantation facilitated by the molecular characterization of a novel HLA-A allele.

Precise HLA typing is crucial in the selection of marrow donors for the treatment of patients with hematologic malignancy. This study was undertaken to characterize an unusual variant of HLA-A30, designated HLA-A30JS, identified in a patient with leukemia who was a candidate for unrelated donor marrow transplantation. IEF and cDNA-sequencing analyses revealed that A30JS is a novel variant differing from the IEF-defined subtype A30.1 (encoded by the A*3002 allele) by a single amino acid substitution. An unrelated marrow donor was identified who was matched with the patient for HLA-A3, B7, B18, DR2, and DR3, but mismatched within the A30 antigen family for the two distinct alleles A*3002 versus A30JS. These two alleles encode a single amino acid substitution, Arg versus Gly, at position 56 in the alpha 1 domain. Position 56 is located outside the antigen-binding cleft of the class I molecule, suggesting that this substitution may not be functionally significant. Transplantation from this donor was performed and the patient is surviving free of leukemia for more than 700 days after transplant. The maximum acute GVHD observed was scored as grade II, but immunosuppressive therapy is still required for control of chronic GVHD. This study demonstrates how the molecular characterization of a novel HLA-A allele in a patient could facilitate the selection of an unrelated donor. Lacking this information, it would not have been possible to select a donor for this patient, and thus apparently successful marrow transplant could not have occurred.

A novel HLA-A*8001 allele identified in an African-American population.

HLA polymorphism varies among different racial origins. Certain antigens are restricted to a particular ethnic group, suggesting that genetic events might have generated further unique polymorphism following racial diversification. In this study, we have identified and characterized a novel HLA-A allele, officially designated A*8001. The class I molecule, HLA-AXBG, encoded by this allele could not be defined by commonly available HLA antisera. We have identified two alloantisera that appear to be monospecific for this new antigen. The observed frequency of AXBG antigen specificity is 2% in African Americans among the total of 254 tested, but it has not been found in 305 Caucasians tested. IEF and cDNA sequencing analyses on multiple individuals revealed that AXBG antigen defined by the two antisera are encoded by an identical HLA-A*8001 allele and it has a number of unique amino acid residues that have not been observed among other HLA-A alleles but are known to occur in certain human nonclassic class I genes and nonhuman primate class I genes. The ability to identify the new HLA-AXBG antigen will improve the precision of HLA-A typing in black populations and enable us to match a patient with a donor for this otherwise undefined antigen in transplantation.

Role of tumour necrosis factor-alpha in dendritic cell-mediated primary mixed leucocyte reactions.

Tumour necrosis factor (TNF alpha) is a major inflammatory cytokine with potentiating effects on specific immune responses, including graft-versus-host disease. This study examined the contribution of TNF alpha to dendritic cell (DC)-mediated primary allogeneic T lymphocyte responses. Purified blood DC were shown to produce minimal amounts of TNF alpha mRNA but no significant TNF biological activity or secreted TNF alpha as measured by ELISA. Amplification of DC mRNA by PCR using oligonucleotide primers to CD120a (TNFRI, p55) and CD120b (TNFRII, p75) and probing with specific internal oligonucleotides, suggested that DC express the CD120b but little if any CD120a. These results were confirmed using monoclonal antibodies to the TNF receptors. Polyclonal antiserum specific for TNF alpha blocked the blood DC-stimulated allogeneic mixed leucocyte reaction (MLR). The addition of TNF alpha to suboptimal MLRs (limited DC stimulators), increased the proliferation of responding T lymphocytes. Having confirmed that T lymphocytes produce TNF alpha and express CD120b after stimulation, we sought to clarify whether the contributing effect of TNF alpha to the allogeneic MLR resulted from a TNF alpha-mediated signal stimulating DC activity, or as a result of autocrine stimulation of T lymphocytes. Pre-incubation of DC with TNF alpha did not increase DC stimulatory capacity and late addition of anti-TNF serum (up to 72 h) still had a significant inhibitory effect on the MLR. We conclude that TNF alpha is probably not involved in the initial DC-T lymphocyte interaction, but acts as an autocrine growth factor for DC induced T lymphocyte proliferation.

Identification of a circulating soluble form of CD80: levels in patients with hematological malignancies.

The release of soluble forms of CD80 provides a potentially powerful mechanism for the modulation of anti-tumor responses. In this report we investigated whether a soluble form of CD80 (sCD80) circulates in vivo and whether levels are altered in patients with hematological malignancies. Circulating sCD80 was detected by ELISA in all normal donor (0.024-0.318 ng/ml) and patient (0.02-3.75 ng/ml) blood analyzed. The majority of acute myeloid leukemia (13/17) and multiple myeloma (11/12) patients had normal sCD80 levels. Significantly elevated levels were detected in chronic lymphocytic leukemia (CLL, P = 0.0001) and mantle cell lymphoma (MCL, P = 0.0002) patients. MCL patients had the highest levels with 8/9 having levels > 0.318 ng/ml. Increased sCD80 levels in CLL were significantly associated with poor prognosis markers such as low platelet (P = 0.01) and hemoglobin (P = 0.002) levels, elevated WBC counts (P = 0.03) and expression of CD38 (P = 0.048). The immunoreactivity of the sCD80 in both normal and patient plasma was inhibited by the presence of CTLA-4-Ig, suggesting sCD80 is functional. Comparison of sCD80 and soluble CD86 levels demonstrated that these molecules were independently elevated in 39% of patients. The finding that a proportion of CLL and the majority of MCL patients contain elevated levels of sCD80 and the demonstration that sCD80 can interact with CTLA-4-Ig suggests a potential role for sCD80 in modulating anti-tumor responses during the malignant process.

Hairy cell leukemia cells are relatively NK-insensitive targets.

The hypothesis that interferon alpha (IFN alpha) has its beneficial effects in hairy cell leukemia by activating natural killer cells against hairy cells was examined. Leukemic cells from patients with hairy cell leukemia were tested for their susceptibility to lysis by fresh and IFN alpha activated peripheral blood mononuclear (PBMC) cells from normal donors. All hairy cells tested were relatively insensitive to cytolysis by PBMC and IFN alpha activated PBMC. The low levels of 51Cr release obtained with a few donors was due to lysis of leukemic cells, not residual normal cells, and was mediated by a natural killer cell (T cell receptor independent) mechanism. Chronic lymphatic leukemic cells before and after treatment with phorbol ester were also resistant to cytolysis. Hairy cells were not susceptible to lymphokine activated killer (LAK) cells but were sensitive to lysis by antibody and complement. The insensitivity to cell mediated cytolysis against hairy cells was shown by cold target inhibition to be a lack of target recognition by NK cells.

CD45 molecule cross-linking inhibits natural killer cell-mediated lysis independently of lytic triggering.

The fact that certain CD45 [anti-leucocyte common antigen (LCA)] monoclonal antibodies (mAb) inhibit natural killer (NK) cell non-major histocompatibility complex (MHC)-restricted cytolysis led to the suggestion that these mAb block a 'trigger' for NK cell lytic activity. However, the discovery that the intracytoplasmic portion of the leucocyte common molecule has protein tyrosine phosphatase activity raises the possibility that the mAb initiate a direct inhibitory signal, independent of the triggering apparatus. To clarify this, we have tested the ability of CD45 antibodies to trigger NK cells and redirect cytotoxicity against mAb-producing hybridoma cells and autologous monocytes, an approach which has identified other cytotoxic trigger molecules. Peripheral blood NK cells failed to kill the CD45 antibody-producing hybridomas, although a CD3 antibody expressing hybridoma was susceptible to cytotoxic T-cell lysis. Furthermore, the CD45 mAb CMRF-12 + 26, 13.3 and HuLyM4 did not redirect lysis of autologous monocytes by NK cells, whereas the isotype-matched CD16 mAb did so. Bivalent CD45 antibody was necessary to block NK lysis of K562, as F(ab')2 but not F(ab') fragments of CMRF-12 + 26 antibody inhibited killing. Capping of the LCA appeared to correlate with the ability of the CD45 mAb to block killing, suggesting that cross-linking of LCA molecular isoforms on the NK cell surface is required for CD45 mAb to inhibit non-MHC-restricted cytolysis.

Promotion of activated human B cell apoptosis and inhibition of Ig production by soluble CD95 ligand: CD95-based downregulation of Ig production need not culminate in activated B cell death.

CD95/CD95L interactions are vital to normal lymphoid homeostasis and in the protection against autoimmunity. To directly assess the effects of CD95L on activated B cell survival and Ig responses, purified human peripheral blood B cells, activated in vitro with SAC + rIL2, were incubated with a soluble CD95L fusion protein (fp) and assayed for apoptosis and IgG/IgM production. CD95L fp reproducibly increased apoptosis of these activated B cells and inhibited their Ig production. However, CD95L fp-mediated effects on activated B cell survival could be uncoupled from those on Ig production in that a soluble CD40L fp was incapable of reversing CD95L fp-mediated downregulation of Ig responses despite inhibiting CD95L fp-mediated apoptosis. Moreover, despite the specific caspase-8 inhibitor z-IETD-fmk substantially protecting transformed CL-01 B cells from CD95L fp-mediated apoptosis and permitting their ongoing proliferation, caspase-8 inhibition had no protective effects on CD95L fp-mediated inhibition of constitutive IgM production by CL-01 B cells. Collectively, these results point to a CD95-based downregulatory pathway in activated B cells that need not necessarily culminate in their death.

Inhibition of natural killer-cell mediated cytolysis with monoclonal antibodies to restricted and non-restricted epitopes of the leucocyte common antigen.

Three monoclonal antibodies recognizing different epitopes of the leucocyte common molecule, CMRF-11 (against the restricted or B-220 leucocyte common molecule), CMRF-12 and CMRF-26 [each against a different epitope on the non-restricted or T200 leucocyte common (CD45) molecule], were tested for their effects on lymphocyte cytotoxicity. The individual monoclonal antibodies inhibited human natural killer cell-mediated cytolysis (NK-CMC) weakly, but a mixture of CMRF-11 + 12 + 26 antibodies inhibited cytolysis more consistently and to a greater extent. This mixture did not inhibit cytotoxic T lymphocytes derived from secondary mixed lymphocyte cultures. The CMRF-11 + 12 + 26 mix was shown to inhibit a post-conjugate formation stage of lysis at the effector cell level. Inhibition of NK-CMC of a wide range of target cells, including the T-cell lines Jurkat, HSB2 and Molt 4, was demonstrated.

B7/BB-1 is a leucocyte differentiation antigen on human dendritic cells induced by activation.

Activation of a primary T-lymphocyte response requires additional signals apart from interaction of the T-cell receptor (TcR)/CD3 complex with major histocompatibility complex (MHC) antigens on the antigen-presenting cell. The CD28 antigen on T lymphocytes provides an important co-stimulatory signal to T lymphocytes and we therefore searched for the presence of its ligand, the B7/BB-1 antigen, on blood and tonsil dendritic cells (DC). Blood DC, prepared from peripheral blood mononuclear cells with a minimal period of in vitro culture, did not stain with the monoclonal antibody BB-1 using flow cytometry analysis. In contrast, tonsil DC stained weakly for B7/BB-1 compared to positive control cell lines. Polymerase chain reaction (PCR) was used to amplify a 605 base pair (bp) fragment from human B7/BB-1 mRNA and demonstrated significant amounts of B7/BB-1 mRNA in tonsil DC but no specific product was obtained from blood DC, confirming the surface-staining results. Weak expression of B7/BB-1 antigen was detected by immunofluorescence analysis following culture of blood DC with either interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF). These data support the concept that blood DC give rise to tissue and/or lymphoid DC, which acquire co-stimulatory ligands as a result of activation and/or differentiation.

The Ly-1.1 and Ly-1.2 epitopes of murine CD5 map to the membrane distal scavenger receptor cysteine-rich domain.

CD5 is a member of a superfamily of proteins which contain one or more extracellular domains homologous to the type I macrophage Scavenger Receptor cysteine-rich (SRCR) domain. The extracellular region of CD5 is composed of three SRCR domains (D1, D2, D3). Murine CD5 (mCD5) is polymorphic (Ly-1.1 and Ly-1.2 alleles), however, the only murine CD5 gene characterized to date encodes the Ly-1.2 allele (mCD5.2). Likewise, the domain specificity of many of the available anti-mCD5 mAb recognizing either Ly-1.1 or Ly-1.2 or both has not been examined. Herein we describe the isolation and characterization of cDNA encoding the Ly1.1 allele (mCD5.1) and map the location and molecular nature of the mCD5 allelic variation. We also determined which SRCR domain of mCD5 is recognized by a panel of anti-mCD5 mAb. The mCD5.1 protein differs from mCD5.2 in only three amino acids, all of which map to the most amino terminal SRCR domain (D1) of mCD5. An additional seven silent substitutions were observed in the nucleotide sequence encoding mCD5 D1, D2 and transmembrane domains. Immunoglobulin (Ig) fusion proteins consisting of various combinations of mCD5.1 or mCD5.2 SRCR domains were produced and used to determine that allele specific mAb bound to D1, confirming sequence data. MAb against monomorphic determinants on mCD5 bound to each mCD5D11g.

Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) involved in the initiation of primary T-lymphocyte responses. However, despite their importance, no DC-specific surface marker has been identified in humans and many aspects of their ontogeny and the mechanisms underlying their potent functional activity remain unknown. In this report we describe a novel monoclonal antibody (mAb), CMRF-44, which recognizes an early activation antigen expressed by human DC and acts as a marker of allostimulatory activity within preparations of peripheral blood mononuclear cells (PBMC). The CMRF-44 antigen was expressed strongly on DC isolated from blood and tonsil by standard techniques, but was not detectable on Langerhans' cells within skin or on DC isolated directly from blood using a cell-sorting method which involves minimal DC manipulation/activation. Normal resting peripheral blood leucocytes did not label with CMRF-44, although weak staining of a small subpopulation (15%) of blood B lymphocytes was identified by double labelling. However, following overnight culture at 37 degrees, moderate staining of a subpopulation of PBMC was detected. Confirmation that CMRF-44 recognized an early marker of activated DC and hence allostimulatory activity was obtained by sorting cultured cell preparations on the basis of CMRF-44 reactivity. A marked enrichment of allostimulatory activity was observed in the CMRF-44-positive cellular population, whereas the CMRF-44-negative cells showed only minimal stimulatory activity. Activation studies established that the CMRF-44 antigen was an early activation marker, expressed constitutively on the majority of tonsil B lymphocytes, which can be induced on peripheral blood B lymphocytes and subpopulations of monocytes. Expression of the CMRF-44 antigen on cell lines was similarly restricted, CMRF-44 antigen being detected only on Hodgkin's disease-derived and B-lymphoid lines. The cellular distribution, expression kinetics and biochemical characteristics of the CMRF-44 antigen identify it as a new early marker of activated allostimulatory (DC) populations.

Superantigen-driven, CD8+ T cell-mediated down-regulation: CD95 (Fas)-dependent down-regulation of human Ig responses despite CD95-independent killing of activated B cells.

Staphylococcal superantigens, including staphylococcal enterotoxin B (SEB), promote vigorous T cell-dependent Ig responses at low dose (0.01 ng/ml). In contrast, more mitogenic high dose SEB (100 ng/ml) profoundly inhibits the Ig responses. To assess the contribution of CD8+ T cells to this inhibition, high dose SEB-dependent killing of activated B cells and down-regulation of Ig responses were determined. Rapid killing (4 h) of activated B cells was effected by high dose SEB-activated CD8+ T cells (CD8*), but not by high-dose SEB-activated CD4+ T cells (CD4*), and required the presence of high dose SEB during the cytotoxicity assay. This killing was abrogated by chelation of extracellular calcium or by treatment with concanamycin A but was only modestly affected by treatment with brefeldin A, suggesting a perforin-based pathway of killing. Despite their widely disparate abilities to rapidly kill activated B cells, CD8* and CD4* demonstrated similar quantitative abilities to effect high dose SEB-dependent down-regulation of Ig responses. Antagonist anti-CD95 mAb substantially reversed high dose SEB-dependent downregulation effected by CD8* but had no appreciable effects on high dose SEB-dependent killing of activated B cells. These observations strongly suggest that the small fraction of activated B cells that secrete Ig are selectively sensitive to CD95-based killing but resistant to CD95-independent killing. This finding may help explain why clinical autoimmunity associated with increased titers of autoantibodies is a predominant feature of defects in CD95 or CD95 ligand.

Analysis of the ligand binding site in Fas (CD95) by site-directed mutagenesis and comparison with TNFR and CD40.

Fas and its ligand (FasL) are members of the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamilies, respectively. Fas-FasL interactions trigger controlled cell death (apoptosis) in the immune system and thus play a key role in the regulation of immune responses. Structural details of the Fas-Fas ligand interaction are currently unknown. Previously, six Fas residues were identified by mutagenesis as important for ligand binding. We have now extended our mutagenesis analysis and identified additional residues which contribute to the Fas-FasL interaction. Candidate and control residues were selected based on a molecular model of the Fas extracellular region. Although residues in all three extracellular domains were identified to contribute to binding, the Fas-FasL interaction is centered on the second TNFR-like domain. Important residues were compared to critical positions in TNFR and CD40, another member of the TNFR family.

Expression of the CMRF-35 antigen, a new member of the immunoglobulin gene superfamily, is differentially regulated on leucocytes.

A new monoclonal antibody, CMRF-35, has been generated that recognized a 224 amino acid cell surface protein which is a novel member of the immunoglobulin gene superfamily. The antibody, raised against large granular lymphocytes (LGL), stains LGL, monocytes, macrophages and granulocytes but not platelets or erythrocytes. In addition, a subset of peripheral blood T lymphocytes (26.6 +/- 13.4% CD5+ cells) and B lymphocytes (13.7 +/- 6.8% CD20+ cells) stained with CMRF-35 but tonsil T and B cells were essentially negative. Expression of the CMRF-35 antigen (Ag) on different leucocyte populations was markedly influenced by stimulation of the cells with mitogens and cytokines. Activation of peripheral blood T cells with phytohaemagglutinin (PHA), or phorbol myristate acetate (PMA) and calcium ionophore (CaI) led to a decrease in the proportion of CMRF-35+ T lymphocytes. In contrast, PHA activation of tonsil T lymphocytes resulted in an increase in CMRF-35 Ag expression (47.1 +/- 1.5% CD5 cells at 6 days). An increase in CMRF-35 Ag was also seen on phorbol ester and CaI-activated tonsil B cells. No change in CMRF-35 expression on natural killer (NK) cells occurred following activation with interleukin-2 (IL-2) but the CMRF-35 Ag was down-regulated following Fc receptor stimulation. A moderate increase in CMRF-35 expression occurred during monocyte-macrophage differentiation and the expression of the Ag on monocytes was differentially regulated by interferon-gamma (IFN-gamma). This regulation of the CMRF-35 Ag on the leucocyte surface suggests that the molecule has an important function common to diverse leucocyte types.