Separable portions of the CD2 cytoplasmic domain involved in signaling and ligand avidity regulation.

Effective T cell immune responses require the molecular interplay between adhesive and signaling events mediated by the T cell receptor for antigen (TCR) and other cell surface coreceptor molecules. In this report, we have distinguished between the role of regulated adhesion and transmembrane signaling in coreceptor function using the T cell glycoprotein CD2. By binding its ligands on antigen-presenting cell (APC), CD2 serves both to initiate signal transduction events and to promote cellular adhesion. Furthermore, the avidity of CD2 for one ligand, CD58 (LFA-3), is regulated by TCR signaling. We have expressed wild type CD2 and a series of mutated CD2 molecules in an antigen-specific murine T cell hybridoma. Structure-function studies using these stably transfected cell lines identify two structurally and functionally distinct regions of the 116 amino acid (aa) cytoplasmic domain. One region is required for CD2-mediated signal transduction, and a separate COOH-terminal 21 aa portion is required for CD2 activity regulation. Cell lines expressing CD2 molecules lacking the cytoplasmic segment required for CD2-initiated IL-2 production retain the ability to upregulate CD2 avidity. Conversely, cell lines expressing CD2 mutants lacking the cytoplasmic segment required for avidity regulation retain the ability to initiate CD2-specific signaling. In antigen-specific T cell responses, basal binding of CD2 to its ligands enhances antigen responsiveness only minimally, whereas regulated avidity and transmembrane signaling are both required for optimal coreceptor function. Taken together, these studies demonstrate the independent contributions of regulated adhesion and intracellular signaling in CD2 coreceptor function.

Synergistic T cell activation via the physiological ligands for CD2 and the T cell receptor.

T cells may be activated either by the antigen-specific T cell receptor (TCR)-CD3 complex or the cell surface receptor CD2. A natural ligand for CD2 has been found to be lymphocyte function-associated antigen 3 (LFA-3), a widely distributed cell surface glycoprotein. To investigate the interaction of these two pathways, we have expressed the cDNA encoding the human CD2 molecule in a murine T cell hybridoma that produces IL-2 in response to HLA-DR antigens. Expression of the CD2 molecule markedly enhances IL-2 production in response to LFA-3+ antigen-bearing stimulator cells, and this stimulation is inhibited by anti-CD2 and anti-LFA-3 mAb. To further define the role of LFA-3 in antigen-dependent T cell activation, we have studied the ability of the purified ligands of CD2 and the TCR to stimulate the hybridoma. Neither liposomes containing purified HLA-DR antigens nor liposomes containing purified LFA-3 were able to stimulate the parent or the CD2+ hybridoma. However, liposomes containing both purified LFA-3 and HLA-DR, the physiological ligands for CD2 and the TCR, respectively, stimulate IL-2 production by the CD2+ but not the parent hybridoma, suggesting that complementary interactions between the TCR-CD3 complex and the CD2 pathway may regulate lymphocyte activation. To determine whether the CD2/LFA-3 interaction participates in cell-cell adhesion and provides an activation signal, we have constructed a cytoplasmic deletion mutant of CD2, CD2 delta B, in which the COOH-terminal 100 amino acids of CD2 have been replaced with a serine. Hybridomas expressing the CD2 delta B molecule were examined. Deletion of the cytoplasmic domain of CD2 did not alter binding of LFA-3 but eliminated the ability of CD2 to increase the response of the hybridoma to liposomes containing both HLA-DR and LFA-3, demonstrating that adhesion of LFA-3 to CD2 alone was insufficient for activation, and that the cytoplasmic domain was required for LFA-3 stimulation through the CD2 molecule. T cells may be activated by purified LFA-3 binding to CD2 and the TCR interacting with its ligand, and these signals appear to be synergistic for the T cell. These results suggest that the CD2/LFA-3 interaction not only plays a role in cell-cell adhesion but provides a stimulatory signal for T cell activation.

Sialophorin, a surface sialoglycoprotein defective in the Wiskott-Aldrich syndrome, is involved in human T lymphocyte proliferation.

The mAb L10 was used to determine the distribution and the function of sialophorin, the heavily glycosylated surface molecule that is deficient/defective in lymphocytes of patients with the X-linked immunodeficiency Wiskott-Aldrich syndrome. Dual-parameter FACS analysis indicated that sialophorin is expressed on CD4+ and CD8+ lymphocytes, on a subpopulation of peripheral blood B lymphocytes, on all thymocytes, and on a subpopulation of bone marrow cells. Functional studies demonstrated that L10 mAb stimulates the proliferation of peripheral blood T lymphocytes as measured by stimulation of [3H]thymidine incorporation. The time course and magnitude of increased [3H]thymidine incorporation by T lymphocytes in response to L10 mAb paralleled that induced by anti-CD3 mAb. Effective stimulation was dependent on the presence of monocytes and the Fc portion of L10 mAb. Stimulation of lymphocytes by L10, like stimulation by anti-CD3 mAb, involves increased expression of 4F2, HLA-DR, and IL-2-R. These observations suggest that sialophorin functions in T cell activation.

Ethical Challenges in Clinical Research During the COVID-19 Pandemic.

The sudden emergence of the COVID-19 pandemic brought global disruption to every aspect of society including healthcare, supply chain, the economy, and social interaction. Among the many emergent considerations were the safety and public health of the public, patients, essential workers, and healthcare professionals. In certain locations, clinical research was halted-or terminated-in deference to the immediate needs of patient care, and clinical trials focusing on the treatment and prevention of coronavirus infection were prioritized over studies focusing on other diseases. Difficult decisions were made rapidly; flexibility and reconsideration were necessary not only because the intensity and severity of infection varied over time and by locale but also because knowledge of the disease and understanding of its treatment (and prevention) grew. Here we discuss the ethical challenges in decision-making and competing ethical tensions during the pandemic in an effort to advance future preparedness.

Yeast Bim1p promotes the G1-specific dynamics of microtubules.

Microtubule dynamics vary during the cell cycle, and microtubules appear to be more dynamic in vivo than in vitro. Proteins that promote dynamic instability are therefore central to microtubule behavior in living cells. Here, we report that a yeast protein of the highly conserved EB1 family, Bim1p, promotes cytoplasmic microtubule dynamics specifically during G1. During G1, microtubules in cells lacking BIM1 showed reduced dynamicity due to a slower shrinkage rate, fewer rescues and catastrophes, and more time spent in an attenuated/paused state. Human EB1 was identified as an interacting partner for the adenomatous polyposis coli (APC) tumor suppressor protein. Like human EB1, Bim1p localizes to dots at the distal ends of cytoplasmic microtubules. This localization, together with data from electron microscopy and a synthetic interaction with the gene encoding the kinesin Kar3p, suggests that Bim1p acts at the microtubule plus end. Our in vivo data provide evidence of a cell cycle-specific microtubule-binding protein that promotes microtubule dynamicity.

Lateral mobility of class I histocompatibility antigens in B lymphoblastoid cell membranes: modulation by cross-linking and effect of cell density.

We have studied the lateral mobility of class 1 major histocompatibility complex (MHC) proteins in the membranes of human Epstein-Barr virus-transformed B cells using fluorescence photobleaching recovery. Class I MHC antigens were labeled with either W6/32 monoclonal antibody or its Fab fragment directly conjugated to fluorescein isothiocyanate. The diffusion coefficient of class I antigens labeled with Fab fragments of W6/32 was identical to that of a lipid analogue, fluorescein phosphatidylethanolamine, and was 10-fold greater than that of antigens labeled with intact W6/32. Furthermore, antigens labeled with Fab fragments but not with intact W6/32 had fractional mobilities identical to that of the lipid probe. The lateral mobility of class I antigens was dependent on the time of incubation with fluorescent antibody and on the presence of antibody microaggregates. Finally, class I MHC proteins labeled with intact W6/32 but not with Fab fragments were immobilized in the membranes of most cells grown in suspension at high cell density. These results suggest that, in the unperturbed state, class I MHC antigens diffuse as rapidly as membrane lipid, i.e., without cytoskeletal constraint. Cross-linking with bivalent ligand and growth to high cell density may trigger membrane events leading to slowing and immobilization of these proteins.

Overlapping but nonidentical binding sites on CD2 for CD58 and a second ligand CD59.

The interaction of the T cell glycoprotein CD2 with one ligand, CD58, contributes to T cell function. We have identified CD59, a glycoprotein with complement-inhibitory function, as a second physiological ligand for CD2. Antibodies to CD59 inhibit CD2-dependent T cell activation in murine T cell hybridomas expressing human CD2. In an in vitro binding assay with purified CD58 and CD59, CD2+ cells bind not only immobilized CD58 but also CD59. With two complementary approaches, it was demonstrated that the binding sites on CD2 for CD58 and CD59 are overlapping but nonidentical. These observations suggest that direct interactions between CD2 and both CD58 and CD59 contribute to T cell activation and adhesion.

Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase.

The immunosuppressant rapamycin inhibited proliferation of the H4IIEC hepatoma cell line. Rapamycin, but not its structural analog FK506, also inhibited the basal and insulin-stimulated activity of the p70 ribosomal protein S6 kinase. By contrast, insulin stimulation of the p85 Rsk S6 kinase and mitogen-activated protein (MAP) kinase activity were unaffected by drug. Rapamycin treatment of COS cells transfected with recombinant p70 S6 kinase completely inhibited the appearance of the hyperphosphorylated form of p70 S6 kinase concomitant with the inhibition of enzyme activity toward 40S subunits. Thus, rapamycin inhibits a signal transduction element that is necessary for the activation of p70 S6 kinase and mitogenesis but unnecessary for activation of p85 Rsk S6 kinase or MAP kinase.

Probing immunosuppressant action with a nonnatural immunophilin ligand.

The immunosuppressants FK506 and rapamycin bind to the same immunophilin, FK506 binding protein (FKBP), and inhibit distinct signal transduction pathways in T lymphocytes. A nonnatural immunophilin ligand, 506BD, which contains only the common structural elements of FK506 and rapamycin, was synthesized and found to be a high-affinity ligand of FKBP and a potent inhibitor of FKBP rotamase activity. Whereas 506BD does not interfere with T cell activation, it does block the immunosuppressive effects of both FK506 and rapamycin. Thus, the common immunophilin binding element of these immunosuppressants, which is responsible for rotamase inhibition, is fused to different effector elements, resulting in the inhibition of different signaling pathways. Inhibition of rotamase activity is an insufficient requirement for mediating these effects.

The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain.

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

Phenotypic and functional characterization of human cytolytic T cells lacking expression of CD5.

Although the CD5 (T1) antigen was initially described as a pan-T cell membrane glycoprotein, we report that 14 of 40 normal individuals were found to have 5% or greater of their blood mononuclear cells characterized as CD3 (T3)+ but CD5- by dual immunofluorescence flow cytometry. These cells expressed normal quantities of surface CD3 and CD2 but low levels of CD7, were CD8+ and CD4-, and CD16-. In order to determine whether cells of this phenotype were functional, six CD5- cytolytic T lymphocyte (CTL) clones isolated from normal individuals were studied. The CD5- CTL clones all demonstrated normal cytolytic activity against appropriate target cells. Monoclonal antibodies (MAbs) directed against CD3, CD8, CD2, and lymphocyte function-associated antigen 3, but not against CD5, inhibited cytolytic activity. Changes in intracellular calcium [( Ca2+]i) in response to anti-CD5 and anti-CD3 MAbs were measured. Stimulation by anti-CD5 MAb alone did not give rise to a change in [Ca2+]i. However, under conditions of limiting concentrations of anti-CD3 MAb, preincubation of normal CD5+, but not CD5-, clones with anti-CD5 MAb led to a dramatic enhancement in the ability of anti-CD3 MAb to elicit a rise in [Ca2+]i. We conclude that CD5- T lymphocytes represent a normal lymphoid phenotype. Although CD5 may be involved in T cell activation when present, these CD5- CTL clones appear to express normal cytolytic activity.

Humoral hypercalcemia of malignancy. Release of a prostaglandin-stimulating bone-resorbing factor in vitro by human transitional-cell carcinoma cells.

Secretion by tumor cells of circulating bone-resorbing factors may frequently underlie the hypercalcemia that occurs in patients with malignancy. Efforts to identify the responsible mediators have been hampered by a lack of available human tumor cell systems suitable for study of the pathogenesis of the humoral hypercalcemia syndrome. We have established a transitional-cell carcinoma (TCC) line in vitro from a patient with humoral hypercalcemia. These cells are tumorigenic and cause hypercalcemia in athymic nude mice. Culture medium conditioned by TCC cells contains potent bone-resorbing activity in vitro, the physical and biological properties of which are similar to those of bone-resorbing activity present in the original patient's urine. The bone-resorbing activity of the TCC factor is accompanied by increased prostaglandin release from bone and is blocked by indomethacin and calcitonin. The TCC-derived bone-resorbing activity coelutes with prostaglandin-stimulating activity during gel filtration with an approximate molecular weight of 15,000. This activity is nondialyzable, stable to concentrated urea and reducing agents, and destroyed by boiling. The TCC factor does not increase cyclic AMP production in bone or kidney bioassays and does not exhibit transforming growth factor activity. We conclude that a unique macromolecular factor released by TCC cells causes bone resorption by a mechanism dependent upon stimulation of bone cell cyclooxygenase, and that this factor is the probable cause of the hypercalcemia in vivo. The TCC cell line provides a new model for study of the human humoral hypercalcemia syndrome.

Abnormal cytolytic activity of lymphocyte function-associated antigen-1-deficient human cytolytic T lymphocyte clones.

The involvement of the lymphocyte function-associated antigen-1 (LFA-1) membrane molecule in cytolytic T lymphocyte (CTL) interactions with lymphoid target cells was investigated using CTL clones derived from two patients with a heritable deficiency of LFA-1. LFA-1 surface expression on the CTL clones was 1% of the normal level of LFA-1, unchanged with prolonged culture, and identical on 14 different CTL clones. The function of the LFA-1 molecule was addressed using the LFA-1-deficient CTL clones and LFA-1-deficient lymphoid target cells. The lytic activity of the LFA-1-deficient CTL clones was 43% of control when tested against a target cell line expressing normal levels of LFA-1 and less than 10% of control when tested against an LFA-1-deficient target cell line. These results demonstrate a direct involvement of LFA-1 in CTL-mediated cytolysis and suggest a more general dependence on LFA-1 in lymphoid cell-cell interactions.

Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology.

The microbial products cyclosporin A (CsA), FK506 and rapamycin are potent immunosuppressive agents. The introduction of CsA in the early 1970's significantly improved the outcome of organ and bone marrow allograft transplantation and advanced therapeutic options in autoimmune diseases. FK506 appears to have a higher therapeutic index than CsA, and has been used with encouraging results in clinical transplantation trials. FK506 and CsA, although structurally unrelated, appear to target similar signal transduction pathways in hematopoietic cells by inhibiting the action of calcineurin, a serine/threonine phosphatase. A structural analog of FK506, rapamycin, inhibits cellular function by a different molecular mechanism. These agents have advanced our understanding of signal transmission pathways in lymphocyte activation.

Adhesion receptors in lymphocyte activation.

The past several years have seen significant progress in understanding the role of T lymphocyte coreceptors in adhesion and activation. New insights have been gained in several areas: the avidity regulation of beta 1 and beta 2 integrins and their role in signal transduction; the regulation of CD8 avidity; the role of Lck in CD4 coreceptor activity; and the novel role for CD2 adhesion in the T cell antigen response.

Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.

Identification of a proline-rich sequence in the CD2 cytoplasmic domain critical for regulation of integrin-mediated adhesion and activation of phosphoinositide 3-kinase.

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in beta1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of beta1 integrin activity. CD2-induced increases in beta1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of beta1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.

Prognostic factors for treatment outcome in autotransplantation of intermediate-grade and high-grade non-Hodgkin's lymphoma with cyclophosphamide, carmustine, and etoposide.

PURPOSE: We examined a consecutive series of 78 patients with non-Hodgkin's lymphoma treated on prospective protocols with high-dose cyclophosphamide, carmustine (BCNU), and etoposide (CBV) plus autotransplantation to determine prognostic factors for time to treatment failure. PATIENTS AND METHODS: Patients with relapsed, refractory, or poor-risk intermediate- and high-grade non-Hodgkin's lymphoma were treated with CBV with autologous marrow or peripheral-blood progenitor cell support. Patient characteristics before transplantation were examined in univariate analyses by the log-rank test and simultaneously in a Cox proportional hazards regression analysis. A best-predictive model was determined from those variables significant (P < .10) in the univariate test. RESULTS: In univariate analysis, intermediate-grade and immunoblastic lymphoma, responsiveness to pretransplant salvage chemotherapy, and transplantation after primary therapy (first complete response [CR] or partial response [PR]) were associated with prolonged time to treatment failure. In proportional hazards multiple regression analysis, intermediate-grade and immunoblastic histology, responsive disease, and autotransplantation in first CR or PR were positive prognostic factors, and these characteristics are the basis of the best-predictive model for prolonged time to failure. Actuarial 3-year failure-free survival of patients with stable or responding disease at autotransplant was 54%. CONCLUSION: CBV is an effective conditioning regimen in intermediate-grade and immunoblastic non-Hodgkin's lymphoma. Patients with these histologies transplanted while responding to primary therapy, or those with stable disease or disease responding to salvage therapy at the time of autotransplant, are most likely to benefit. Patients with lymphoblastic lymphoma or diffuse undifferentiated lymphoma did poorly with CBV and should be offered alternative therapy.

The complete sequences of plasmids pFNeo and pMH-Neo: convenient expression vectors for high-level expression of eukaryotic genes in hematopoietic cell lines.

The eukaryotic expression vector, pFNeo, confers resistance to the antibiotic G418 and directs hematopoietic-specific expression of proteins under the control of the long terminal repeat from the Friend spleen focus-forming virus. Here, we report the entire nucleotide sequence of pFNeo. We have also constructed and sequenced a pFNeo-based expression vector (pMH-Neo) that carries an improved multiple cloning site region for easier subcloning. The utility of both vectors was demonstrated by transfection of murine T-cell hybridomas by electroporation. Transfection with either pFNeo or pMH-Neo yielded a high frequency (1 in 2 x 10(4)) of G418-resistant cell lines.

Structural organization of the genes encoding human and murine FK506-binding protein (FKBP) 13 and comparison to FKBP1.

FK506-binding protein (FKBP)12 and FKBP13 are members of a family of proteins which bind the immunosuppressant drugs, FK506 and rapamycin. FKBP12 and FKBP13 are encoded by distinct genes, designated FKBP1 and FKBP2, respectively. The structure of human FKBP1 was previously characterized. We now report the genomic structure of the human and murine FKBP2 genes. Comparison of FKBP1 and FKBP2 reveals significant homology and correlation of intron positions in the C-terminal region, suggesting that these genes may have evolved from a common ancestral gene.