T-cell receptor-CD3 Research Articles

Trypanosoma cruzi-induced immunosuppression: selective triggering of CD4+ T-cell death by the T-cell receptor-CD3 pathway and not by the CD69 or Ly-6 activation pathway.

In a model of experimental Chagas' disease induced with metacyclic forms of Trypanosoma cruzi, CD4+ but not CD8+ T cells undergo T-cell receptor (TCR)-CD3-mediated activation-induced cell death (AICD) in vitro. CD4+ T cells from T. cruzi-infected mice also developed unresponsiveness in proliferative responses to TCR-CD3-mediated stimulation. A linear correlation was found between extent of proliferative unresponsiveness and loss of CD4+ T-cell viability. CD4+ T-cell activation through the CD69 or Ly-6 A/E pathway, on the other hand, did not result in proliferative unresponsiveness compared with controls. Lack of suppression in proliferation assays correlated with lack of AICD by cells stimulated through the CD69 or Ly-6 A/E pathway. Concomitant stimulation through CD69, however, did not rescue CD4+ T cells from CD3-induced death. Flow cytometry study of cells stimulated in vitro showed no defect in interleukin-2 receptor expression by CD4+ T cells from infected donors, which escaped TCR-mediated AICD. In vivo injection of anti-CD3 into acutely infected mice, but not into control mice, led to splenocyte DNA fragmentation and failed to increase splenic CD4+ T-cell numbers. These results show that TCR-CD3-mediated AICD is involved in CD4+ T-cell unresponsiveness in vitro following infection with T. cruzi. In addition, successful activation of these cells through the CD69 and Ly-6 pathways is due to differences in the inability of these stimuli to trigger AICD. Since TCR-CD3-mediated AICD can be induced in vivo in infected mice, these findings may be relevant for the onset of immunological disturbances in the host.

Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody.

Elevated levels of immunoglobulin (Ig) E are associated with bronchial asthma, a disease characterized by eosinophilic inflammation of the airways. Activation of antigen-specific T helper (Th) 2 cells in the lung with the subsequent release of interleukin (IL) 4 and IL-5 is believed to play an important role in the pathogenesis of this disease. In this study, we have used a non-anaphylactogenic anti-mouse-IgE antibody to investigate the relationship between IgE, airway eosinophil infiltration, and the production of Th2 cytokines. Immunization of mice with house dust mite antigen increased serum levels of IgE and IgG. Antigen challenge of immunized but not control mice induced an infiltration of eosinophils in the bronchoalveolar lavage associated with the production of IL-4 and IL-5 from lung purified Thy1.2+ cells activated through the CD3-T cell receptor complex. Administration of the anti-IgE monoclonal antibody (mAb) 6h before antigen challenge neutralized serum IgE but not IgG and inhibited the recruitment of eosinophils into the lungs and the production of IL-4 and IL-5 but not interferon gamma. Studies performed using an anti-CD23 mAb, CD23 deficient and mast cell deficient mice suggest that anti-IgE mAb suppresses eosinophil infiltration and Th2 cytokine production by inhibiting IgE-CD23-facilitated antigen presentation to T cells. Our results demonstrate that IgE-dependent mechanisms are important in the induction of a Th2 immune response and the subsequent infiltration of eosinophils into the airways. Neutralization of IgE, for example, non-anaphylactogenic anti-IgE mAbs may provide a novel therapeutic approach to the treatment of allergic airway disease.

Function of the pre-T-cell receptor alpha chain in T-cell development and allelic exclusion at the T-cell receptor beta locus.

The pre-T-cell receptor, composed of the T-cell receptor (TCR) beta chain (TCRbeta), pre-Talpha (pTalpha) chain, and CD3 molecules, has been postulated to be a transducer of signals during the early stages of T-cell development. To examine the function of the transmembrane pTalpha chain during tbymocyte development, we generated pTalpha-/- embryonic stem cells and assayed their ability to differentiate into lymphoid cells in vivo after injection into recombination-activating gene (RAG)-2-deficient blastocysts. Thymocytes representing all stages of T-cell differentiation were detected in the thymus of pTalpha-/- chimeric mice, indicating that thymocyte development can occur without pTalpha. However, greatly reduced thymocyte numbers and substantially increased percentages of both CD4-CD8- thymocytes and TCRgammadelta+ thymocytes suggest that pTalpha plays a critical role in thymocyte expansion. To investigate the role of the pTalpha chain in allelic exclusion at the TCRbeta locus, a functionally rearranged TCRbeta minigene was introduced into pTalpha-/- and pTalpha+/- embryonic stem cells, which were subsequently assayed by RAG-2-deficient blastocyst complementation. In the absence of pTalpha, expression of the transgenic TCRbeta inhibited rearrangement of the endogenous TCRbeta locus to an extent similar to that seen in normal TCRbeta transgenic mice, suggesting that pTalpha may not be required for signaling allelic exclusion at the TCRbeta locus.

Induction of cell death via Fas (CD95, Apo-1) may be associated with but is not dependent on Fas-induced tyrosine phosphorylation

Cross-linking of the Fas-antigen (CD95, Apo-1) triggers apoptosis in activated T cells and transformed T cell lines. Fas-induced apoptosis has been previously reported to require Fas-triggered tyrosine phosphorylation of various proteins. In the present study, we have compared the protein tyrosine phosphorylation pattern and the apoptosis sensitivity in a set of Jurkat variants selected for the absence or presence of T cell receptor (TCR)/CD3 expression and resistance or sensitivity to Fas-mediated apoptosis. While tyrosine phosphorylation upon Fas-ligation was readily apparent in wild-type Jurkat cells (which are sensitive to anti-Fas-induced apoptosis), drastically reduced tyrosine phosphorylation was observed in Fas-resistant Jurkat subclones (which still express CD95 on their surface). More importantly, TCR CD3 -negative Jurkat variants which expressed normal levels of CD95 and were fully susceptible to Fas-triggered cell death, did not show any protein tyrosine phosphorylation upon Fas-ligation. Taken together, our data demonstrate that Fas-induced cell death can be associated with but is not dependent on protein tyrosine phosphorylation.

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.

Human lung mast cell IL-5 gene and protein expression: temporal analysis of upregulation following IgE-mediated activation.

The late-phase of allergic asthma is characterized by infiltration of the airway with eosinophils within 6 h of mast cell activation. Pro-eosinophilic/pro-allergic (TH2) cytokines, originally described as T-lymphocyte products, have recently been ascribed to mast cells as well. To date, however, it is unknown if TH2 cytokine gene expression by the human mast cells is subject to receptor-mediated regulation analogous to that of T-cells, and if messenger RNA (mRNA) expression results in protein secretion occurring in a temporal context consistent with the late-phase response. We examined interleukin-4 (IL-4), IL-5, and IL-6 mRNA expression induced by anti-IgE activation of human lung explants as assessed using reverse transcription/polymerase chain reaction (RT-PCR). Anti-IgE stimulation resulted in rapid and sustained upregulation of IL-5 message, but did not have analogous effects on IL-4 or IL-6. Using quantitative-competitive PCR, we demonstrated that 100 ng of total cellular RNA from human lung contained 1 fg of IL-5 mRNA; this increased to 100 fg 4 h after anti-IgE activation. The source of the anti-IgE-enhanced IL-5 mRNA is likely the mast cell itself, as anti-CD3 activation of lung led to a dissimilar array of cytokine expression. In addition, human lung mast cells purified to near homogeneity expressed IL-5 mRNA after activation, as shown by both RT-PCR and in situ hybridization. In both lung fragments and purified human lung mast cells, the modulation of IL-5 mRNA expression preceded the secretion of IL-5 protein, detected as early as 4 h after activation. Neither isolated purified mast cells nor purified peripheral blood T cells could be induced to secrete detectable amounts of IL-5 protein when activated only with antibodies against IgE or CD3-T cell receptor complex, respectively. However, mast cells (n = 4) and T cells (n = 6) cultured at comparable concentrations (4 x 10(6)/ml) activated through their respective antigen receptors in the presence of phorbol ester yielded comparable IL-5 production (253 +/- 126 pg/ml versus 183 +/- 75 pg/ml, mean +/- SE). We conclude that mast cells are analogous to T cells in the requirement of co-stimuli for the production of IL-5 protein. Moreover, the rapid kinetics of IgE-mediated IL-5 transcription and protein elaboration are consistent with a primary role for mast cell activation directly leading to late-phase airway eosinophilia.

Differential expression of the T cell receptor/CD3 genes and their lymphoid-specific transcription factor genes in murine T cell x fibroblast and T cell x B cell hybrids.

We generated cell hybrids between mouse T cell lymphoma EL4 cells and mouse fibroblast B82 cells (BELIII and BELIV) to examine the expression of T cell receptor (TcR)/CD3 genes and their lymphoid-specific transcription factor genes, which are normally detected in EL4 cells. In BELIII and BELIV, expression of the TcR alpha, TcR beta and CD3 delta genes was extinguished, whereas expression of the CD3 epsilon gene was still detected. Expression of the (lymphoid enhancer binding factor 1) LEF-1 gene was extinguished and that of the GATA-3 gene was hardly detected in BELIII and BELIV. Ets-1 gene expression, observed not only in EL4 cells but also in B82 cells, was considerably reduced in BELIII and BELIV. A much higher level of PEBP2 alpha A gene expression was observed in B82 cells than in EL4 cells and was preserved in BELIII and BELIV. To examine whether reduced expression of these genes is also found in T cell x B cell hybrids, we generated an additional cell hybrid between EL4 cells and mouse plasmacytoma S194 cells (SELIII). Marked differences were observed in the expression of the TcR alpha, CD3 delta, LEF-1 and PEBP2 alpha A genes in BEL and SEL hybrids. Expression of the TcR alpha, CD3 delta and LEF-1 genes, which was extinguished in BELIII and BELIV, was detected in SELIII. PEBP2 alpha A gene expression, not detected in S194 cells, was considerably reduced in SELIII. Almost the sum of the chromosomes from the parental cells were retained by, and the presence of every gene was proven, in each cell hybrid. These results suggest that suppression of the expression of lymphoid-specific transcription factor genes may precede that of the TcR/CD3 genes in the cell hybrids, and that the presence of a different trans-acting negative regulatory mechanism(s) suppresses the expression of T cell specific genes in fibroblasts and B cells.

Ternary complex therapy for autoimmune disease.

Induction of an immune response is remarkably simple, involving the interaction of three components: processed antigen, an MHC class II molecule, and a CD4 T-cell receptor. Each part of this ternary complex represents a novel target for interventions that may block subsequent effector phenomena of autoimmune disease. Such specific treatments will be less toxic than those currently available.

Plasma-membrane-bound macromolecules are dynamically aggregated to form non-random codistribution patterns of selected functional elements. Do pattern recognition processes govern antigen presentation and intercellular interactions?

Molecular recognition processes between cell surface elements are discussed with special reference to cell surface pattern formation of membrane-bound integral proteins. The existence, as detected by flow cytometric resonance energy transfer (Appendix), and significance of cell surface patterns involving the interleukin-2 receptor, the T-cell receptor-CD3 system, the intercellular adhesion molecule ICAM-1, and the major histocompatibility complex class I and class II molecules in the plasma membrane of lymphocytes are described. The modulation of antigen presentation by transmembrane potential changes is discussed, and a general role of transmembrane potential changes, and therefore of ion channel activities, adduced as one of the major regulatory mechanisms of cell-cell communication. A general role in the mediation and regulation of intercellular interactions is suggested for cell-surface macromolecular patterns. The dynamic pattern of protein and lipid molecules in the plasma membrane is generated by the genetic code, but has a remarkable flexibility and may be one of the major instruments of accommodation and recognition processes at the cellular level.

Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex.

Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%).

Toxoplasma gondii‐specific CD4+ T cell clones from healthy, latently infected humans display a ThO profile of cytokine secretion

Human Toxoplasma gondii (Tg)-specific T cell clones were raised by infecting peripheral blood mononuclear cells (MNC) from two healthy, latently infected individuals with Tg trophozoites. All of the clones had a CD4+ immunophenotype and produced simultaneously interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-5 upon mitogen or antigen stimulation. Tg-specific T cell clones were classified as T helper of type 0 (Th0) since most of them released roughly comparable amounts of IFN-gamma and IL-4. In some clones, a trend to an increased production of IFN-gamma following antigen-specific as compared to non-specific stimulation was observed. The Th0 phenotype was also expressed by T cell clones that had been raised from bulk cultures performed in the presence of IL-4 or IFN-gamma. All of the Tg-specific T cell clones were cytolytic in a non-specific assay which involves the triggering of the CD3-T cell receptor (TcR) complex. Some clones specifically lysed an autologous lymphoblastoid cell line (LCL) that had been infected with Tg trophozoites. Finally, most of the Tg-specific T cell clones produced IL-10, irrespective of whether they had been raised from bulk cultures incubated in the presence or absence of IL-4 or IFN-gamma. Taken together, these findings suggest that Tg-specific Th0 helper cell clones from healthy, latently infected individuals, beside activating toxoplasmacidal mechanisms through IFN-gamma release, might limit the magnitude of the immune response of the parasite by killing Tg-infected antigen-presenting cells and by releasing IL-10.

Decreased CD3-mediated interferon-gamma production in relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that has been postulated to be T-cell mediated. We examined the proliferation and cytokine secretion of mononuclear cells after stimulation with OKT3 (anti-CD3) monoclonal antibody concanavalin A, or ionomycin plus myristic acid palmityl ester in subjects with stable relapsing-remitting MS. Control subjects demonstrated good proliferation to anti-CD3 monoclonal antibody whereas subjects with relapsing-remitting MS showed a significantly decreased anti-CD3 monoclonal antibody-mediated response. There was no difference in concanavalin or ionomycin plus myristic acid palmityl ester stimulation between control subjects and MS subjects. Secretion of interferon-gamma was significantly decreased and transforming growth factor-beta was significantly increased from cultures stimulated with anti-CD3 monoclonal antibody, but not ionomycin plus myristic acid palmityl ester or concanavalin A, in MS patients compared to control subjects. Secretion of interleukin-10 and tumor necrosis factor-alpha was not different between control subjects and MS patients following stimulation with anti-CD3 monoclonal antibody, concanavalin A, or ionomycin plus myristic acid palmityl ester, or of interleukin-2 and interleukin-4 following stimulation with anti-CD3 monoclonal antibody or concanavalin A. An abnormality of signal transduction and secretion of the immunomodulatory molecule interferon-gamma may exist in MS via the CD3 T-cell receptor complex.

Crossrecognition by CD8 T cell receptor alpha beta cytotoxic T lymphocytes of peptides in the self and the mycobacterial hsp60 which share intermediate sequence homology.

Immunization of C57BL/6 mice with the mycobacterial heat shock protein (hsp) 60 in immunostimulating complexes caused the in vivo activation of autoreactive major histocompatibility complex class I (H-2Db)-restricted CD8 T cell receptor (TcR) alpha/beta cells. A CD8 TcR alpha/beta clone with specificity for the mycobacterial hsp60 peptide499-508 was derived from this immunization, which, in addition, recognized syngeneic macrophages which had been stressed by interferon-gamma (IFN-gamma) stimulation. The stress-induced, self peptide could be extracted from IFN-gamma-stressed macrophages by acid elution, suggesting that the IFN-gamma-induced self peptide is derived from an endogenous protein. Based on our observation that lysis of stressed target cells by this cytotoxic T lymphocyte (CTL) clone was specifically inhibited by hsp60-specific antisense oligonucleotides, we used synthetic peptides representing amino acid (aa) sequences of the murine hsp60 for target cell sensitization and identification of the relevant self peptide. Synthetic peptides representing 9-mer to 11-mer aa sequences of the murine hsp60 with asparagine in anchor position 4 or 5 as the minimal requirement for H-2Db binding were tested in CTL assays. The overlapping murine hsp60 peptides162-170/171 were stimulatory at a concentration as low as 10-100 pM. Seven other peptides of the murine hsp60 required intermediate peptide concentrations of 10-100 nM for recognition by the CTL clone. Although the murine and mycobacterial hsp60 peptides recognized by this CTL clone showed only intermediate homology (3 identical and 3 similar aa), our data suggest that endogenous hsp60 itself is the source of self peptide(s) presented by IFN-gamma-stressed macrophages to the cross-reactive CTL clone with promiscuous specificity. This notion is consistent with the idea of hsp as a link between infection and autoimmunity.

Susceptibility to actively-induced murine experimental allergic encephalomyelitis is not linked to genes of the T cell receptor or CD3 complexes.

The role of the T cell receptor (TCR) in the genetic control of susceptibility to autoimmune demyelinating diseases remains shrouded in controversy. We have used the CXD2 series of recombinant inbred lines (RIL) and a (B10.S/DvTe x SJL/J) x B10.S/DvTe backcross (BC1) population to test for linkage between susceptibility to actively-induced EAE and the different TCR and CD3 loci. The two populations were inoculated for induction of EAE, phenotyped for both clinical and histological parameters of disease, and genotyped using markers flanking the loci of interest in the CXD2 RIL and an SJL/J allele-specific TCR V beta assay in the BC1 mice. Comparisons between the CXD2 strain distribution pattern (SDP) for disease and the SDPs for the chromosomal regions containing the TCR alpha, beta, gamma, delta, and CD3 delta, epsilon, gamma and zeta loci showed no linkage to these loci. Additional tests between EAE susceptibility and several other immunologically important loci for which the SDPs were known also showed no linkage to the minor lymphocyte-stimulating antigen gene Mlsl, Hc, the gene encoding complement component C5, Cd8a, or Cd5. Furthermore, our data from the BC1 mice demonstrate that the Tcrb locus segregates independent of disease and does not modulate disease severity. We conclude that while autoreactive TCRs are undoubtedly necessary for disease pathogenesis, the principle non-MHC-linked loci controlling susceptibility to murine EAE in BALB/c mice are not linked to any of the individual TCR-CD3 complex genes. Similarly, the major disease genes in the SJL/J mouse are not linked to TCR V beta. Our data cannot, however, preclude the possibility that TCR/CD3 alleles are involved in epigenetic phenomena or susceptibility in other mouse strains or animal systems.

Immediate effects of reversible HTLV-ITax function: T-cell activation and apoptosis

The tax protein of Human T-cell leukemia virus type 1 (HTLV-1) is important for the transforming properties of this virus in vitro and is considered to be responsible for the early stages of leukemogenesis in infected hosts. To address the early consequences of HTLV-1 tax function, we have constructed fusion proteins containing tax sequence either aminoterminal (taxER) or carboxy-terminal (ERtax) of the hormone binding domain of the human estrogen receptor (ER). Addition of estrogen or the antagonist hydroxytamoxifen to Jurkat T-cells expressing these constructs led to the trans-activation or responsive promoters and upregulation of cell surface markers CD28, CD69 and CD5 but not CD25 (IL2R-alpha subunit) or B7 (ligand for CD28). Additional stimulation of the T-cell receptor CD3 complex, led to the upregulation of CD25. B7 was upregulated by concomittent activation of ERtax and CD3 or CD28 pathways. These events were in part reversible upon withdrawal of hormone and inactivation of ERtax. Severe inhibition of proliferation, and apoptosis was observed with cells which had been subjected to short term (3 days) activation of the tax fusion proteins and the CD3 complex. Induction of ERtax activity for longer than 3 days promoted cell death independently of CD3 stimulation. Co-stimulation through the CD28 cell surface molecule did not suppress induction of apoptosis.

Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

Advances in heterologous gene expression by Streptomyces

During the past year, noteworthy advances have been made in our ability to express foreign genes in Streptomyces. These advances were due, in part, to a detailed examination of the critical parameters that limit expression by Streptomyces of soluble forms of the human T-cell receptor CD4. Significant progress has also been made in our understanding of transcriptional regulation and protease gene expression. Application of this knowledge to expression vector design and the construction of alternative expression hosts should improve our ability to easily and routinely express foreign genes in Streptomyces.

Differences in responsiveness to CD3 stimulation between naive and memory CD4+ T cells cannot be overcome by CD28 costimulation.

Activation of naive CD4+ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4+ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen-presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7-dependent system we show that even in the presence of optimal CD28 costimulation, CD4+ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4+ cells a higher level of cross-linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4+ memory and naive cells, but that for activation of naive cells higher cross-linking of both CD3 and CD28 molecules is necessary.

Assays to detect and characterize human immunodeficiency virus type 1 (HIV-1) receptor antagonists, compounds that inhibit binding of the HIV-1 surface glycoprotein, gp120, to the CD4 receptor on human T lymphocytes

Human immunodeficiency virus type 1 infects human helper T lymphocytes by an interaction between gp120, the viral coat protein, and the T-cell receptor CD4. Two microtiter-based immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a particle concentration fluorescence assay, were developed to measure gp120-CD4 binding and were then used to screen a variety of compounds for the inhibition of this interaction. Additional protocols, called "consumption assays," were defined to distinguish inhibitors which functioned by sequestering either gp120 or CD4 to prevent the final effective bimolecular interaction. Monoclonal antibodies of defined specificity and compounds known from other published studies to inhibit gp120-CD4 binding were tested in an attempt to validate the assays used in the study. Once the capacity of these assays to detect known gp120-CD4 inhibitors was confirmed, they were used to screen synthetic agents and fermentation broths for novel compounds that might be used as human immunodeficiency virus receptor antagonists. A 2,4-diaminoquinazoline, CP-101,816-1, was found to inhibit this interaction (50% inhibitory concentration in ELISA, 32.5 micrograms/ml) and to interact more strongly with CD4 than with gp120 in the consumption assays. The identification of a novel inhibitor, a 2,4-diaminoquinazoline, confirmed that such assays are useful for the detection of human immunodeficiency virus type 1 receptor antagonists.

Expression of the ζ Protein Subunit in CD3 - NK Effectors Derived from Human Thymus

The origin, lineage derivation, and sites of human natural killer (NK) cell differentiation are presently unresolved. The vast majority of NK cells found in peripheral blood have surface membrane expression of CD2 and CD16. Both antigens trigger activation pathways which require the ζ protein, a signal-transducing subunit of the CD3-T cell receptor (CD3-TCR) complex which is found as an isolated homodimer (ζ-ζ or heterodimer (ζ-FccRIγ) in human NK cells. Unlike NK cells found in adult peripheral blood, NK cells derived in vitro from human CD34 + hematopoietic progenitor cells lack CD2 and CD16, and those found in fetal liver constitutively express CD3 and δ proteins. However, NK effectors derived in vitro from immature human CD3 - thymocytes show striking phenotypic and functional similarities to adult human NK cells. In this report, we characterize ζ protein expression in CD3 - thymocytes following short-term culture in recombinant (r)IL-2. CD3 -CD56 + thymocyte NK effectors express the ζ protein as a disulphide-linked homodimer of 32 kDa, yet lack other protein components of the CD3-TCR complex. Both CD16 + and CD16 populations were found to express ζ and within the CD16 + fraction, ζ is physically associated with CD16. These data provide evidence of additional similarities between adult peripheral blood NK cells and CD3 CD56 + NK effectors derived from human thymocytes, and suggest that under these experimental conditions, human NK cells can arise from early thymic precursors.