Stimulation of non-Hodgkin's lymphoma via HVEM: an alternate and safe way to increase Fas-induced apoptosis and improve tumor immunogenicity.

Stimulation by CD40 ligand (L) improves B-cell malignancy immunogenicity, and also induces proliferative signals. To avoid these tumorigenic effects, we studied an alternate way of tumor-cell stimulation by homologous to lymphotoxin, inducible expression, competing for GpD of herpesvirus, which binds to the herpesvirus entry mediator (HVEM), and is expressed on T-lymphocytes (LIGHT), the ligand for HVEM, a new member of the tumor necrosis factor (TNF)/TNF-receptor (-R) family. HVEM is constitutively expressed on the surface of tumor B cells. We focused our attention on mantle cell lymphoma, a subtype of B-cell malignancy of poor prognosis. Triggering by LIGHT, in contrast to CD40L stimulation, did not increase lymphoma proliferation nor decrease chemotherapy entrance. We observed an upregulation of the TNFR apoptosis-inducing ligand Fas, and in contrast to CD40L-induced protection, an enhancement of lymphoma sensitivity to Fas-induced apoptosis. LIGHT triggering increased lymphoma cell recognition in a mixed lymphocyte response. In conclusion, LIGHT-mediated triggering renders B-cell lymphomas more immunogenic and sensitive to apoptosis, without inducing proliferation. Since LIGHT triggering also enhances the functions of T-lymphocytes and dendritic cells, it could be a unique way to restore an efficient cancer control by its pleiotropic effects on immune effectors and tumor cells.

The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity.

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.

Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor.

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.

Preclinical development of keliximab, a Primatized anti-CD4 monoclonal antibody, in human CD4 transgenic mice: characterization of the model and safety studies.

The preclinical safety assessment of biopharmaceuticals necessitates that studies be conducted in species in which the products are pharmacologically active. Monoclonal antibodies are a promising class of biopharmaceuticals for many disease indications; however, by design, these agents tend to have limited species cross-reactivity and tend to only be active in primates. Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4. In order to conduct a comprehensive preclinical safety assessment of this antibody to support chronic treatment of rheumatoid arthritis in patients, a human CD4 transgenic mouse was used for chronic and reproductive toxicity studies and for genotoxic studies. In addition, immunotoxicity studies were conducted in these mice with Candida albicans, Pneumocystis carinii and B16 melanoma cells to assess the effects of keliximab on host resistance to infection and immunosurveillance to neoplasia. The results of these studies found keliximab to be well tolerated with the only effects observed being related to its pharmacologic activity on CD4+ T lymphocytes. The use of transgenic mice expressing human proteins provides a useful alternative to studies in chimpanzees with biopharmaceutical agents having limited species cross-reactivity.

Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D)

Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle.

Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.

Herpesvirus entry mediator ligand (HVEM-L), a novel ligand for HVEM/TR2, stimulates proliferation of T cells and inhibits HT29 cell growth.

Herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family, mediates herpesvirus entry into cells during infection. Upon overexpression, HVEM activates NF-kappaB and AP-1 through a TNF receptor-associated factor (TRAF)-mediated mechanism. Using an HVEM-Fc fusion protein, we screened soluble forms of novel TNF-related proteins derived from an expressed sequence tag data base. One of these, which we designated HVEM-L, specifically bound to HVEM-Fc with an affinity of 44 nM. This association was confirmed with soluble and membrane forms of both receptor and ligand. HVEM-L mRNA is expressed in spleen, lymph nodes, macrophages, and T cells and encodes a 240-amino acid protein. A soluble, secreted form of the protein stimulates proliferation of T lymphocytes during allogeneic responses, inhibits HT-29 cell growth, and weakly stimulates NF-kappaB-dependent transcription.

Antibodies to TR2 (herpesvirus entry mediator), a new member of the TNF receptor superfamily, block T cell proliferation, expression of activation markers, and production of cytokines.

TR2 (TNFR-related 2) is a recently identified member of the TNFR family with homology to TNFRII. We have demonstrated previously that TR2 mRNA is expressed in resting and activated human T cells and that TR2-Ig partially inhibits an allogeneic mixed leukocyte proliferation response. We now characterize TR2 further by the use of specific mAbs. Flow-cytometry analysis using TR2 mAbs confirmed that resting PBL express high levels of cell surface TR2, and that TR2 is widely expressed on all freshly isolated lymphocyte subpopulations. However, stimulation of purified T cells with either PHA or PHA plus PMA resulted in decreased surface expression within 48 h of activation before returning to resting levels at 72 h. TR2 mAbs inhibited CD4+ T cell proliferation in response to stimulation by immobilized CD3 or CD3 plus CD28 mAbs. Assay of culture supernatants by ELISA showed inhibition of TNF-alpha, IFN-gamma, IL-2, and IL-4 production, which, for IL-2 and TNF-alpha was also confirmed by intracellular cytokine staining. Furthermore, expression of activation markers on CD4+ T cells, including CD25, CD30, CD69, CD71, and OX40 (CD134), was inhibited. TR2 mAbs inhibited proliferation in a three-way MLR, and a response to soluble recall Ag, tetanus toxoid. In conclusion, these results suggest that TR2 is involved in the activation cascade of T cell responses and TR2 mAbs prevent optimal T cell proliferation, cytokine production, and expression of activation markers.

A newly identified member of the tumor necrosis factor receptor superfamily with a wide tissue distribution and involvement in lymphocyte activation.

The tumor necrosis factor receptor (TNFR) superfamily consists of approximately 10 characterized members of human proteins. We have identified a new member of the TNFR superfamily, TR2, from a search of an expressed sequence tag data base. cDNA cloning and Northern blot hybridization demonstrated multiple mRNA species, of which a 1.7-kilobase form was most abundant. However, TR2 is encoded by a single gene which, maps to chromosome 1p36.22-36.3, in the same region as several other members of the TNFR superfamily. The most abundant TR2 open reading frame encodes a 283-amino acid single transmembrane protein with a 36-residue signal sequence, two perfect and two imperfect TNFR-like cysteine-rich domains, and a short cytoplasmic tail with some similarity to 4-1BB and CD40. TR2 mRNA is expressed in multiple human tissues and cell lines and shows a constitutive and relatively high expression in peripheral blood T cells, B cells, and monocytes. A TR2-Fc fusion protein inhibited a mixed lymphocyte reaction-mediated proliferation suggesting that the receptor and/or its ligand play a role in T cell stimulation.

Characterization of a novel TNF-like ligand and recently described TNF ligand and TNF receptor superfamily genes and their constitutive and inducible expression in hematopoietic and non-hematopoietic cells.

A novel (TL1), a recently described (TL2) TNF-like, and three recently described TNF receptor-like (TR1, TR2, TR3) molecules were identified by searching a cDNA database. TL1 and TL2 are type-II membrane proteins. TR2 and TR3 are type-I membrane proteins whereas TR1 appears to be a secreted protein. TL1, TL2, TR2 and TR3 were expressed in hematopoietic cells, whereas TR1 was not. Northern blots hybridized with the cDNA probes revealed multiple forms of RNA as well as inducible expression of TL1, TL2, TR2 and TR3. TL2 and TR3, in particular, were highly induced in activated CD4+ T cells. Radiation hybrid mapping localized TR1 and TL2 to 8q24 and 3q26, respectively, which are not near any known superfamily members. TL1 was mapped to 9q32, near CD30L (9q33) and TR2 and TR3 mapped to the region of chromosome 1 that contains the TNFR-II, 4-1BB, OX40 and CD30 gene cluster at 1p36. Only TR3 in this cluster possesses a death domain. Southern blot analysis revealed the presence of TL and TR genes in different mammalian species. TL2, TR1, TR2 and TR3 were recently described by others as TRAIL/Apo-2L, OPG, HVEM and DR3/WSL-1/Apo-3/TRAMP/LARD, respectively.

Differential recognition by CD28 of its cognate counter receptors CD80 (B7.1) and B70 (B7.2): analysis by site directed mutagenesis.

CD28, which is a member of the immunoglobulin superfamily of molecules (IgSF), is a homodimer of two polypeptides containing a single V-like domain with short transmembrane and cytoplasmic regions. It serves as a co-signalling molecule for T cell activation through binding to its cognate counter-receptors CD80 and B70, expressed on antigen presenting cells. In the current study, we investigated the regions of CD28 which are involved in its interactions with CD80 and B70, using site directed mutagenesis, CD28 mAb epitope mapping, receptor based adhesion assays and direct binding of Ig-fusion proteins to cell surface receptors. Truncation or substitution of a stretch of a proline rich "hallmark" sequence, "MYPPPY", abrogates binding to CD80 or B70, while retaining CD28 mAb epitopes and cell surface expression. On an Ig-fold model of the CD28 V-domain, this fully conserved motif localizes to a CDR3-like region. Mutations introduced into other loops, including the CDRI-like and CDR2-like regions, had very little effect on CD80 or B70 binding. Mutations introduced within the predicted beta-strand regions caused loss of receptor expression. Conservative substitution of both the flanking tyrosine residues within the "MYPPPY" motif with phenylalanine, caused loss of binding to B70 but not to CD80. These results show that, although the same overall region on CD28 may be involved in the interactions with CD80 and B70, subtle but important differences distinguish recognition by the two molecules. These finding, along with previous observations on the differential pattern of expression and tissue distribution of CD80 and B70, support the contention that these molecules play distinct roles in the regulation of immune responses in vivo.

CD28 signal transduction pathways. A comparison of B7-1 and B7-2 regulation of the map kinases: ERK2 and Jun kinases.

The present study compares the mitogen-activated protein (MAP) kinase responses in T cells activated with the CD28 ligands B7-1 (CD80) and B7-2/B70 (CD86). Ligands B7-1 and B7-2 do not activate the Raf-1/ERK2 cascade, but share the ability to activate related Jun kinases. These natural ligands for CD28 had no stimulatory effect alone on Jun kinase activation, but the data show that B7-1 and B7-2 could both co-operate with intracellular Ca2+ increase and protein kinase C (PKC) activation to stimulate Jun kinases. The present study shows that the interaction of CD28 with its ligands B7-1 and B7-2 can induce identical signal transduction through the MAP kinase cascades.

Comparison of CD28-B7.1 and B7.2 functional interaction in resting human T cells: phosphatidylinositol 3-kinase association to CD28 and cytokine production.

CD28 is a 44kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter-receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these co-stimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3-kinase (PI3-K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28-transfected cells or pre-activated T cells. In addition, recent reports propose that CD28-B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28-B7.1 and CD28-B7.2 interactions induce the association of PI3-K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine-phosphorylated CD28 and does not require pre-activation by CD3-T cell receptor. Worthmannin, a specific inhibitor of PI3-K enzymatic activity within the nanomolar range also inhibits the interleukin-2 production induced by costimulation mediated by either the B7.1- and B7.2-transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC50 of worthmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3-K.

Signal transduction by CD28 costimulatory receptor on T cells. B7-1 and B7-2 regulation of tyrosine kinase adaptor molecules.

This study compares the biochemical responses in T cells activated with the CD28 ligands B7-1 and B7-2. The patterns of tyrosine phosphorylation induced in T cells by these two CD28 ligands are identical, but clearly different from the tyrosine phosphorylation induced by the T cell receptor (TCR). The TCR regulates protein complexes mediated by the adapter Grb2 both in vivo and in vitro. In contrast, there is no apparent regulation of in vivo Grb2 complexes in response to B7-1 or B7-2. Rather, B7-1 and B7-2 both induce tyrosine phosphorylation of a different adaptor protein, p62. The regulation of p62 is a unique CD28 response that is not shared with the TCR. These data indicate that B7-1 and B7-2 induce identical tyrosine kinase signal transduction pathways. The data show also that the TCR and CD28 couple to different adapter proteins, which could explain the divergence of TCR and CD28 signal transduction pathways during T cell activation.

Identification of residues in the V domain of CD80 (B7-1) implicated in functional interactions with CD28 and CTLA4.

The CD80 (B7-1) molecule is a 45-60-kD member of the immunoglobulin superfamily that is expressed on a variety of cell types of haematopoietic origin. CD80 can provide a critical costimulatory signal to T cells by interacting with the T cell surface molecule CD28. CD80 also binds to the CD28-related molecule CTLA4, which is expressed on activated T cells, Recently, additional ligands of CD28 and CTLA4 have been described in mice and humans. One of them, CD86 (B-70 or B7-2) was characterized at the molecular level. Although similar in predicted structure to CD80, it is distantly related in amino acid sequence. In this study, human CD80 mutants were generated and tested for their ability to maintain the interaction with CD28 leading to adhesion and enhanced IL-2 production. Two hydrophobic residues in the V-like domain of CD80 were identified as critical for binding to CD28 and are also important for the interaction with CTLA4. These residues are adjacent to the epitope of the BB1 antibody, which inhibits CD28-CD80 interactions. One of these residues, Y87, is conserved in all CD80 and CD86 cloned from various species. These results being to unravel the structural requirements for binding to CD28 and CTLA4.

Pyridinyl imidazoles inhibit the inflammatory phase of delayed type hypersensitivity reactions without affecting T-dependent immune responses.

The effects of pyridinyl imidazoles, specifically SK&F 105809 and its metabolite, on normal T-cell and B-cell mediated immune responses were examined and compared to the fungal macrolide immunosuppressives, cyclosporin A, FK506 and rapamycin and to the corticosteroid, dexamethasone. The orally active prodrug SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo [1,2-a] imidazole[ and its metabolite, SK&F 105561 [2-(4-methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2 -a] imidazole] are dual 5-lipoxygenase (5-LO) and cycloxygenase (CO) inhibitors with potent anti-inflammatory and cytokine (IL-1/TNF) suppressive activities. The anti-inflammatory activity of SK&F 105809 and its metabolite were evaluated in an antigen-specific murine model of delayed type hypersensitivity (DTH) response, where they were found to affect only the inflammatory and not the induction phase of this response. In contrast, these compounds and other pyridinyl imidazoles (SK&F 86002 and its analog, SK&F 104351) exhibited no immunosuppressive activity under conditions where the macrolide rapamycin and the corticosteroid dexamethasone abrogated both the cellular and humoral immune responses. Thus, the ability of pyridinyl imidazoles to attenuate independently the inflammatory components of the disease without causing generalized immunosuppression enhances their profile as candidates for therapy of chronic inflammatory diseases, specifically those mediated by cytokines (e.g. IL-1, TNF) and eicosanoids.

The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras.

CD28 is a 44-kD homodimer expressed on the surface of the majority of human T cells that provides an important costimulus for T cell activation. The biochemical basis of the CD28 accessory signals is poorly understood. Triggering of the T cell antigen receptor (TCR) activates the p21ras proteins. Here we show that ligation of CD28 by a monoclonal antibody (mAb) also stimulates p21ras and induces Ras-dependent events such as stimulation of the microtubule-associated protein (MAP) kinase ERK2 and hyperphosphorylation of Raf-1. One physiological ligand for CD28 is the molecule B7-1. In contrast to the effect of CD28 mAb, the present studies show that interactions between CD28 and B7-1 do not stimulate p21ras signaling pathways. Two substrates for TCR-regulated protein tyrosine kinases (PTKs) have been implicated in p21ras activation in T cells: p95vav and a 36-kD protein that associates with a complex of Grb2 and the Ras exchange protein Sos. Triggering CD28 with both antibodies and B7-1 activates cellular PTKs, and we have exploited the differences between antibodies and B7-1 for p21ras activation in an attempt to identify critical PTK-controlled events for Ras activation in T cells. The data show that antibodies against TCR or CD28 induce tyrosine phosphorylation of both Vav and p36. B7-1 also induces Vav tyrosine phosphorylation but has no apparent effect on tyrosine phosphorylation of the Grb2-associated p36 protein. The intensity of the Vav tyrosine phosphorylation is greater in B7-1 than in TCR-stimulated cells. Moreover the kinetics of Vav tyrosine phosphorylation is prolonged in the B7-1-stimulated cells. These studies show that for CD28 signaling, the activation of p21ras correlates more closely with p36 tyrosine phosphorylation than with Vav tyrosine phosphorylation. However, the experiments demonstrate that Vav is a major substrate for B7-activated PTKs and hence could be important in CD28 signal transduction pathway.

Functional expression of human CD28 in murine T cell hybridomas.

CD28 is a 44 kDa Ig superfamily cell surface molecule expressed on most mature T cells. Through its interaction with the recently identified B7/BB1 counter-receptor, it is believed to play an important role as a co-stimulator of T cells along with the TCR-CD3 complex. Activation of T cells with CD28 mAbs synergizes with TCR-CD3 and CD2 stimulation, resulting in long term T cell proliferation, differentiation of cytotoxic T cells and production of large amounts of cytokines. In order to further delineate the role of CD28 in signal transduction and T cell activation, human CD28 was transfected into CD3+ murine T cell hybridomas. High levels of cell surface CD28 expression was achieved by protoplast fusion. The transfected molecule retained all the native CD28 mAb epitopes found on human T cells. In these transfectants, CD28 mAbs, similarly to CD3 mAbs, were able to induce Ca2+ mobilization, IL-2 promoter induction (measured as beta-galactosidase activity in T cells hybridomas pre-transfected with the IL-2-lac Z reporter gene), IL-2 secretion, TNF alpha production and apoptosis (observed as growth arrest and genome fragmentation). The parental host cells, or cells transfected with vector alone, responded only to mAbs to CD3. IL-2 secretion in the transfectants was obtained using either an IgM mAb to CD28 or IgG mAbs presented on the surface of IgG-FcR+ B lymphoma cells. Optimal activation via CD28 was inhibited by suboptimal concentrations of soluble CD3 mAb, suggesting an interaction between the two pathways. The immunosuppressive drugs Cyclosporin A and FK506 completely blocked CD28 and CD3 mediated IL-2 production in these transfectants whereas rapamycin had only a partial inhibitory effect. Finally, since the transfected human CD28 molecule confers full functional responsiveness to the murine T cell hybridomas without the need for costimulators such as PMA, this model is ideal for studying the structure-function relationships of the CD28 molecule as well as the transmembrane and cytoplasmic associations implied in CD28 signaling.

Structures of an HIV and MHC binding fragment from human CD4 as refined in two crystal lattices.

BACKGROUND: The T-cell surface glycoprotein CD4 interacts with class II molecules of the major histocompatibility complex (MHC) enhancing the signal for T-cell activation. Human CD4 also interacts, at high affinity, with the HIV envelope glycoprotein, gp120, to mediate T-cell infection by HIV. Crystal structures of amino-terminal two-domain (D1D2) fragments of human CD4, which contain the residues implicated in HIV and MHC interactions, have been reported earlier. RESULTS: We have determined the crystal structure of a new D1D2 construct by molecular replacement from a previously described crystal structure of D1D2. This structure has more uniform lattice contacts than are in the first. This gives an improved image of domain D2, which in turn has permitted further refinement of the initial structure at 2.3 A resolution against a more complete data set. The structure of the second crystal form was also refined at 2.9 A resolution. In both models, all residues from 1 to 178 are now well defined, including the loop regions in D2. CONCLUSIONS: Similarities of the molecular structure in the two lattices suggest that the D1D2 fragment works as a unit, with segmental flexibility largely restricted to the junction between domains D2 and D3. Variability of conformation in loops, including those implicated in MHC and HIV binding, requires an 'induced fit' in these interactions. Well defined density for the exposed side chain of Phe43 in both crystals confirms a prominent role for this residue in gp120 binding.