Selection of human anti-CD28 scFvs from a T-NHL related scFv library using ribosome display.

Engineered antibodies have become an invaluable source of biopharmaceuticals against a wide range of diseases. About 200 antibody-based biologicals have been tested in clinical trials. Single chain variable fragments of antibodies (scFvs) provide binding specificity and offer an increased ease of in vitro display selection. Here, we present the generation of a human scFv library from peripheral blood lymphocyte RNA of a patient with relapsed T-cell non-Hodgkin lymphoma (T-NHL) who experienced a rare case of "spontaneous" remission. Antibodies against human T-cell antigen CD28, a co-stimulatory protein that influences the immune response by amplification of the transcriptional effects of T-cell receptors, might have contributed to the patient's remission. The scFv library was panned against CD28 using ribosome display and further subjected to affinity maturation. Isolated scFv were assessed for binding specificity and affinity and may provide the basis for the development of novel immunotherapeutic strategies. This work demonstrates the selection of a fully human antibody fragment from a patient-derived gene pool by in vitro ribosome display technology.

Markers of cell death-activation in lymphocytes of vertically HIV-infected children naive to highly active antiretroviral therapy: the role of age.

BACKGROUND: Apoptosis plays a major role in depleting CD4(+) lymphocytes during infection with HIV-1. Few data exist on its role during HIV infection of children. Sensitivity of peripheral blood lymphocytes (PBLs) to apoptotic stimuli and the importance of the patient's age remain unclear. OBJECTIVES: We sought to analyze the following: (1) markers of cell death-activation (CD95, CD45 isoforms, and CD28) in PBLs from vertically HIV-infected children of different ages before highly active antiretroviral therapy; (2) changes in other PBL populations; (3) PBL sensitivity to cell death and mitochondrial damages; and (4) role of age during progression of infection. METHODS: Cell culture techniques and flow cytometry were used to analyze surface antigens, PBL susceptibility to apoptosis, or PBL susceptibility to change of mitochondrial membrane potential. RESULTS: Donor age had a strong negative correlation with numbers of CD4(+) and CD8(+) T cells. Virgin T lymphocyte (CD45RA(+), CD95(-)) levels and those of CD95(+) cells showed no correlation with the children's clinical status but did show a correlation with patient age. CD28(-) T lymphocytes were markedly augmented in HIV-infected children but were unrelated to stage of infection or age. A relevant decrease in B lymphocytes and an increase in natural killer cells were also found. Finally, PBLs from HIV-positive children had a marked tendency to undergo apoptosis and mitochondrial damage. CONCLUSION: Changes in PBL phenotype, increased expression of CD95, and high sensitivity to apoptosis suggest that a precocious aging of the immune system occurs in HIV-infected children.

Expression of CD28 and CD86 by human eosinophils and role in the secretion of type 1 cytokines (interleukin 2 and interferon gamma): inhibition by immunoglobulin a complexes.

Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-gamma secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-gamma were biologically active, as supernatants from anti-CD28-treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A-anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-gamma release. These results suggest that the release of type 1 (IFN-gamma and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines.

Novel phenotype associated with in vivo activated CTL precursors.

A previously undefined phenotype of CD8(+) cells that appears to represent in vivo activated CTL precursors (CTLP*) has been identified in the spleens of C57Bl/6 mice responding to a P815 tumor allograft. This population was first evident by the transient expression of very high levels of CD28 and CD44 on day 5 of the allograft response and reached maximal levels on days 7 and 8 before declining on day 9. A transient increase in CD69 expression was also observed on these cells on day 5. In contrast, CTL effectors (CTLE), identified by their CD8(+)CD44(hi)CD62LloCD45RBlo phenotype, were not appreciably detected in the spleen until day 8 and reached maximal levels on day 10. Further characterization of CTLP* on day 7 revealed that they represented blasting cells by increased light scatter and also expressed very high levels of CD54 but not CD122, CD152, or CD154. In addition, the cells had already up-regulated CD49d, asialo GM1, CD11a, and CD95L, and down-regulated their expression of CD62L. A small percentage of these cells also expressed CD25. Day 7 CTLP* sorted on the basis of their CD44(xhi) and CD54(xhi) phenotype did not exhibit cytolytic activity in a standard chromium release assay but became cytotoxic when they were cultured in the presence of exogenous murine IL-2 for 5 days. Granzyme B activity, however, was detected in CTLP* on day 7 at levels equivalent to CTLE on day 10. In order to establish a potential precursor relationship between CTLP* and CTLE, mice were treated with various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a chemical that has been shown to dose-dependently suppress the in vivo generation of CTLE to P815 tumor cells by altering an early stage of CTLP activation. Results indicated that CTLP* were suppressed by TCDD on day 7 to the same degree that CTLE were suppressed on day 10. Importantly, for controls and for all doses of TCDD, there were approximately 12.5 CTLE on day 10 for every CTLP* detected on day 7. These results suggested that TCDD acted identically across all doses to inhibit the early stages of activation of CTLP but did not affect the final stages of differentiation and expansion to CTLE. This interpretation supports the previous observation that TCDD exposure had to occur within the first 3 days of the allograft response in order to induce suppression of CTLE activity. Taken together, these results support the conclusion that in vivo activated CTLP can be identified by their unique expression of very high levels of CD44, CD28, and/or CD54 prior to their full maturation and clonal expansion to functional CTLE.

Coordinate transactivation of the interleukin-2 CD28 response element by c-Rel and ATF-1/CREB2.

The interleukin-2 CD28 response element (CD28RE) acts as a composite enhancer, in conjunction with a 3'-12-O-tetradecanoylphorbol-13-acetate response element (TRE)-like element, to confer CD28 receptor-dependent inducibility to the interleukin-2 promoter in T-cells. When inserted as a single copy upstream of a basal promoter, this composite enhancer, termed the CD28RE-TRE, is both highly active and CD28-inducible in transactivation assays. A multicomponent nuclear protein complex that binds the CD28RE-TRE was isolated by DNA affinity chromatography from nuclear extracts of mitogen- and CD28 receptor-costimulated human T-cells. Immunological and biochemical analyses of this complex reveal the presence of c-Rel, ATF-1, and CREB2 as major DNA-binding components. Coexpression of c-Rel in combination with ATF-1, CREB2, or ATF-1/CREB2 leads to synergistic transactivation of a CD28RE-TRE reporter plasmid in quiescent Jurkat T-cells. Furthermore, CD28-dependent transactivation of the CD28RE-TRE is specifically inhibited by cAMP response element-binding protein (CREB) dominant-negative expression vectors. Moreover, mutant promoter constructs in which the internal 5'-CD28RE and 3'-TRE-like sequences have been topologically positioned 180 degrees out of phase with one another show loss of mitogen- and CD28-dependent inducibility. Finally, the addition of the CREB-binding transcriptional coactivator p300 leads to a dramatic CREB-dependent increase in both mitogen- and CD28-mediated transactivation of the CD28RE-TRE. These findings demonstrate that full physiological responsiveness to CD28 receptor stimulation in T-cells is dependent on topologically linked sequences within the CD28RE-TRE composite enhancer and provide strong support of a direct role for the CREB family of transcription factors and p300/CREB-binding protein coactivator proteins in cytokine gene induction during T-cell activation.

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.

Stimulation of CD28 triggers an association between CD28 and phosphatidylinositol 3-kinase in Jurkat T cells.

The T cell surface molecule CD28 can provide costimulatory signals that permit the full activation of T cells. Here we demonstrate that stimulation of CD28, either by B7, its natural ligand, or by the anti-CD28 monoclonal antibody 9.3, induces an association between CD28 and phosphatidylinositol 3-kinase (PI3-K) in Jurkat T cells, raising the possibility that an interaction with PI3-K contributes to CD28-mediated signaling. To examine the mechanism of the association, we synthesized tyrosine-phosphorylated oligopeptides corresponding to each of the four tyrosines in the CD28 cytoplasmic domain. When added to lysates of B7-stimulated Jurkat cells, the oligopeptide corresponding to Tyr 173 inhibits the coimmunoprecipitation of PI3-K with CD28; the other oligopeptides have no effect. Tyr 173 is contained within the sequence YMNM, a motif that is also found in the platelet-derived growth factor receptor and that, when phosphorylated, forms a high affinity binding site for the p85 subunit of PI3-K. These observations suggest that phosphorylation of Tyr 173 may mediate the interaction between CD28 and PI3-K. However, because CD28 is not known to be phosphorylated, it remains possible that CD28 interacts with PI3-K through a mechanism independent of tyrosine phosphorylation.

Activation of src family kinase lck following CD28 crosslinking in the Jurkat leukemic cell line.

T lymphocytes require a signal via their antigen specific receptors (the T cell receptors) and an antigen independent costimulatory signal. Signals through CD28 can costimulate T cells in the presence of limiting amounts of T cell receptor signal, or in the presence of PMA, providing this second signal. CD28 signaling is known to involve the activation of protein tyrosine kinases. Using the Jurkat leukemic cell line as a model, we have tested CD28 crosslinking for its effects on the protein tyrosine kinases p56lck. We report that following crosslinking of CD28, p56lck kinase activity is increased. Crosslinking CD28 causes a shift in the relative mobility of p56lck from 56 to 60 kD similar to that seen after crosslinking of CD2 and CD4, cell surface receptors known to be associate with and activate p56lck. Finally, lck could be found in anti-CD28 immunoprecipitates in exponentially growing Jurkat and in "activated" CD28 (i.e., cross linked), but not in CD28 in resting Jurkat cells. These findings suggest an important role for p56lck in CD28 signal transduction.