Production and characterization of recombinant 9 and 15 kDa granulysin by fed-batch fermentation in Pichia pastoris.

Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.

Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system.

Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.

CD69 is expressed on platelets and mediates platelet activation and aggregation.

CD69, a surface dimer so far considered an early activation antigen restricted to lymphocytes, was found constitutively expressed on human platelets. Biochemical analysis revealed that platelet CD69 appears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad 55-65-kD band, in which three 55-, 60-, and 65-kD components were detectable when nonreduced, and as two 28- and 32-kD bands when reduced, corresponding to the two disulfide-linked chains of the dimer. It therefore closely resembles lymphoid CD69, although the resolution of the three bands under nonreducing conditions is not usually seen in lymphoid cells. Moreover, as CD69 expressed on activated lymphocytes and CD3bright thymocytes, both chains are constitutively phosphorylated. CD69 stimulation by anti-Leu-23 monoclonal antibodies induced platelet aggregation in a dose-dependent fashion. This effect was associated with Ca2+ influx and platelet degranulation, as revealed by adenosine triphosphate release. In addition, CD69 stimulation in platelets induced production of thromboxane B2 and PGE2, suggesting activation of arachidonic acid metabolism by cycloxygenase. As observed for CD69-mediated T cell activation, platelet activation through CD69 requires molecular crosslinking. These results suggest that CD69 may function as an activating molecule on platelets, as on lymphocytes, and point toward a more general role of this surface dimer in signal transduction.

Definition of a specific interaction between the early T lymphocyte activation 1 (Eta-1) protein and murine macrophages in vitro and its effect upon macrophages in vivo.

The Eta-1 gene specifies a secreted product of activated T cells and is associated with genetic resistance to infection by an obligate intracellular bacterium. Previous studies have suggested that eta-1 might affect the ability of macrophages to migrate to the site of bacterial infection and/or to inhibit intracellular bacterial growth. We therefore examined the interaction of eta-1 with macrophages in vitro and in vivo. We find that macrophages express approximately 10(4) eta-1 receptors/cell and each receptor has a Kd of approximately 5 x 10(-10) M. The subsequence of eta-1 containing an RGD motif is required for binding because a synthetic peptide containing the eta-1 RGD domain inhibited protein attachment to macrophages. We also found that subcutaneous inoculation of mice with eta-1 resulted in a cellular infiltrate comprised primarily of macrophages. We propose that the interaction between eta-1 and its receptor on macrophages results in a change in macrophage physiology resulting in accumulation of these cells at extravascular sites.

Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule.

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.

T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages.

To identify the maturational stage(s) during which T cell receptor (TCR)-mediated positive and negative selection occurs, we followed the development of CD4+8- and CD4-8+ T cells from TCRlo CD4+8+ thymic blasts in the presence of different positive and negative selecting (major histocompatibility complex or Mls) elements. We describe novel lineage-committed transitional intermediates that are TCRmed CD4+8lo or TCRmed CD4lo8+, and that show evidence of having been positively selected. Furthermore, negative selection is not evident until after cells have attained one of the TCRmed transitional phenotypes. Accordingly, we propose that negative selection in normal mice occurs only after TCRlo CD4+8+ precursors have been positively selected into either the CD4 or CD8 lineage.

Dissection of the human CD2 intracellular domain. Identification of a segment required for signal transduction and interleukin 2 production.

To evaluate those residues in the 117 amino acids of the CD2 cytoplasmic domain required for transduction of T lymphocyte activation signals, a full-length human CD2 cDNA and a series of deletion and substitution mutants were inserted into the ovalbumin-specific, I-Ad-restricted murine T cell hybridoma 3DO54.8 using a retroviral system. The resulting cells express surface CD2 protein and unlike the parental murine line, are reactive with murine anti-human CD2 antibodies. Anti-T11(2) plus anti-T11(3) antibody stimulation of cells expressing a full-length CD2 cDNA results in a characteristic rise in cytosolic-free calcium [( Ca2+]i), and subsequent IL-2 secretion that accompany CD2 stimulation in human T lymphocytes. Transfectants expressing CD2 delta C98 and CD2 delta C77, partially deleted CD2 molecules containing the entire extracellular and transmembrane CD2 segments but only 98 and 77 amino acids of the cytoplasmic domain, respectively, are also activated by anti-CD2 mAbs. In contrast, clones expressing more severely truncated CD2 structures, CD2 delta C43 and CD2 delta C18, are not stimulated. These data show that the cytoplasmic domain plays an essential role in transduction of activation signals via CD2, and that the segment between amino acid residues 253 and 278 is necessary for activation. This region contains two tandem repeats of the sequence PPPGHR, thought to form part of a putative cationic site. Disruption of the latter by site-directed mutagenesis does not affect IL-2 gene induction, suggesting that only one of the repeats is required for activating this function of the CD2 molecule.

The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors.

The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.

Synthetic CD4 peptide derivatives that inhibit HIV infection and cytopathicity.

Synthetic peptide segments of the CD4 molecule were tested for their ability to inhibit infection of CD4+ cells by the human immunodeficiency virus (HIV) and to inhibit HIV-induced cell fusion. A peptide mixture composed of CD4(76-94), and synthesis side products, blocked HIV-induced cell fusion at a nominal concentration of 125 micromolar. Upon high-performance liquid chromatography, the antisyncytial activity of the peptide mixture was found not in the fraction containing the peptide CD4(76-94) itself, but in a side fraction containing derivatized peptide products generated in the automated synthesis. Derivatized deletion and substitution peptides in the region CD4(76-94) were used to demonstrate sequence specificity, a requirement for benzyl derivatization, and a core seven-residue fragment required for antisyncytial activity. A partially purified S-benzyl-CD4(83-94) peptide mixture inhibited HIV-induced cell fusion at a nominal concentration of less than or equal to 32 micromolar. Derivatized CD4 peptides blocked cell fusion induced by several HIV isolates and by the simian immunodeficiency virus, SIV, and blocked infection in vitro by four HIV-1 isolates with widely variant envelope gene sequences. Purified CD4(83-94) dibenzylated at cysteine 86 and glutamate 87 possessed antisyncytial activity at 125 micromolar. Derivatization may specifically alter the conformation of CD4 holoreceptor peptide fragments, increasing their antiviral efficacy.

Adult T cell leukemia cell lines that originated from primary leukemic clones also had a defect of expression of CD3-T cell receptor complex.

Surface phenotypes and genotypes of six T cell lines established from four patients with adult T cell leukemia (ATL) were compared with those of fresh ATL cells. The surface antigen densities of CD3 and T cell receptor (TCR) complex examined by OKT3 and WT-31 mAbs on fresh ATL cells were low. But three IL-2-dependent cell lines, SKT-2, SKT-5, and ATL-17-2, established from three patients expressed a high density of CD3-TCR complex on their surfaces. On the contrary, three other cell lines (SKT-1A, SKT-1B, and MMT-2), established from two other patients, expressed a low density of CD3-TCR complex. Genotypic analysis of TCR beta chain (T beta) gene and integration sites of the proviral genome of human T cell lymphotropic virus type 1 demonstrated that SKT-1A, SKT-1B, and MMT-2 were derived from the original leukemic clones. These apparent associations between a defect of expression of CD3-TCR complex and identical genotypes of fresh and cultured cells suggest that the defect of expression of CD3-TCR complex may play a key role for development of ATL.

Purification and identification by amino acid sequence analysis of the subunits of the CD3 antigen of human tonsil T-lymphocytes.

The CD3 antigen was purified from human tonsils by immunoaffinity chromatography and preparative SDS-PAGE; the overall yield was 10%. Amino acid sequence analysis of the separated gamma, delta and epsilon polypeptides revealed that the gamma and epsilon polypeptides were blocked at the N-terminus, whereas the partial N-terminal amino acid sequence for the delta chain was identical to that described by Borst et al. [Nature 312, 455-485 (1984)]. The gamma and epsilon chains were cleaved with formic acid and cyanogen bromide respectively in order to obtain amino acid sequence data. The sequences obtained corresponded exactly to the amino acid sequences deduced from the nucleotide sequences of putative gamma and epsilon cDNA clones.

Molecular characterization of mouse T-cell ecto-ADP-ribosyltransferase Rt6: cloning of a second functional gene and identification of the Rt6 gene products.

RT6 is an enzymatically active GPI-anchored membrane protein that was originally discovered in the rat as a peripheral T cell alloantigen. It has attracted interest as an activation antigen and because defective RT6-expression coincides with increased susceptibility for autoimmune type I diabetes in the BB rat. Southern blot analyses indicate that the rat carries a single copy RT6 gene whereas the mouse carries a duplication of the homologous locus. We had previously cloned and sequenced a RT6-homologous cDNA from BALB/c mouse spleen. We now report the cloning and characterization of a second RT6-homologue from BALB/c and 129/Sv mice. The two mouse Rt6 genes (designated Rt6-1 and Rt6-2) encode similar open reading frames that are disrupted by conserved introns. The nucleotide sequences of the Rt6-1 and Rt6-2 coding regions show 87% sequence identity, the deduced amino acid sequences 79% identity. The amino acid sequences reveal significant similarity to recently cloned ADP-ribosylating ectoenzymes from rabbit and human skeletal muscle as well as chicken bone marrow cells. RT-PCR analyses reveal that the two Rt6 genes are differentially expressed in distinct inbred mouse strains and that their transcripts are properly processed. Western blot analyses demonstrate that the respective gene products are released from cells by treatment with PI-PLC. The results further show that both mouse Rt6 genes are translated into GPI-anchored cell surface molecules and that Rt6 gene expression is restricted to peripheral lymphoid tissues.

Development of a procedure for the direct cloning of T-cell epitopes using bacterial expression systems.

Although single bacterial recombinant antigens have been used successfully to stimulate individual T-cell clones and elicit recall responses in peripheral lymphocytes, the broader use of molecular cloning systems for the identification of autoantigens recognised by the cellular arm of the immune system has met with only limited success. In a systematic approach to address this problem, a series of bacterial expression vectors were examined for their potential use as cloning vectors to elicit a proliferative response in vitro from a non-obese diabetic (NOD) mouse T-cell clone which recognises the immunodominant ovalbumin epitope (aa 323-339). The use of the vector pRSET, which produces a hexa-histidine tagged fusion protein, was confounded by non-specific responses to bacterial protein contaminants. pGEX, which generates a glutathione-S-transferase hybrid, avoided this problem but suffered from the disadvantage that a universally applicable purification procedure for the hybrid antigen could not be easily developed. A practical screening protocol was developed using the pUEX expression system (beta-galactosidase hybrid) and purification based upon electroelution of the hybrid protein from purified inclusion bodies subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This system can be used to screen expression libraries for the detection of T-cell epitopes provided that the T-cell clones give low background responses to irrelevant pUEX recombinant proteins. Low abundance antigens may be obtained using this system in combination with subtractive hybridisation to construct cDNA libraries enriched in the target antigen.

Plasmacytoma with aberrant expression of myeloid markers, T-cell markers, and cytokeratin.

Plasmacytomas are localized neoplastic proliferations of monoclonal plasma cells. When multifocal, the process is referred to as multiple myeloma. These lesions exhibit a pattern of antigen expression and cytomorphology that usually leads to a ready diagnosis. However, potentially troublesome variations in immunophenotype occur. We describe a case of a plasmacytoma from a patient who presented with sudden onset of pain and a lytic lesion of the left proximal humerus. Hematoxylin and eosin-stained sections showed a lymphoproliferative lesion composed of large lymphoid cells, some with plasmacytoid and immunoblastic features. The lesion also showed significant mitotic activity. Immunohistochemical staining was positive for CD45 (LCA), CD56 (N-CAM), CD43 (MT1), and cytokeratin CAM5.2. There was also clonal staining for lambda light chains. In addition, flow cytometric analysis showed positivity for myeloid markers such as CD13, CD33, CD38, and CD138. Significant negative markers include CD20 (L26), CD45RO (UCHL-1), and CD79alpha. The unusual phenotypic features of this plasmacytoma illustrate potential diagnostic pitfalls. It is important to fully study such lesions to correctly classify them, because this has significant impact on prognosis and management.

A single-step purification procedure and partial amino acid sequence analysis of picomole amounts of the rat T cell alloantigen RT6.2.

A single-step immunoaffinity purification procedure for the rat T cell marker RT6.2 is described which permits the isolation of microgram quantities of protein from the RT6.2+ T-T hybridoma EpD3. The amino terminus was sequenced directly from a polyvinylidene (PVDF) membrane blot prepared after SDS-PAGE. Further internal sequence data were obtained from peptides generated from purified RT6.2 digested with different endoproteases and separated by reverse-phase micro-HPLC. A computer search in data banks did not reveal any significant homology to other proteins.

Serological identification of thymocyte differentiation antigens.

We have examined subfractions of human thymocytes for the expression of novel differentiation antigens. Non-HLA alloantisera procured from multiparous women served as antibody probes. Thymocytes from five individuals were sequentially separated by discontinuous Percoll density gradient centrifugation and a peanut agglutinin (PNA) panning technique. Subfractions were selected and examined for their relative intensity of HLA class I and CD1 antigens as determined by cytofluorometric analysis. Two subfractions were characterized as follows: an immature population (Fr6 PNA-) expressed a high level of CD1 (OKT6 binding) antigen and a low level of class I HLA antigen; and a more mature fraction (Fr3 PNA-) expressed minimal amounts of CD1 antigen and relatively high levels of HLA class I molecules. Fr6 PNA+ and Fr3 PNA- thymocytes were tested for their reactivity with a panel of non-HLA alloantibodies as determined by cytofluorometric analysis. We observed that three alloantibodies demonstrated strong fluorescence staining with Fr6 PNA+ thymocytes only, whereas three other alloantibodies reacted with both the Fr6 PNA+ and the Fr3 PNA- subfractions. All six alloantibodies failed to react with peripheral T cells. However, the six antibodies did react with a panel of cultured T lymphoblastoid leukemic cells and fresh leukemic T cells. Blocking studies demonstrated that these alloantibodies do not bind beta 2-microglobulin-associated determinants. These results suggest that the alloantibodies detect thymocyte differentiation antigens (TDA) that are shared by or are cross-reactive with antigens expressed on certain leukemia T cells. The non-beta 2m-associated TDA antigens are not expressed on normal resting T cells.

A novel 26 kilodalton antigen expressed on the surface membrane of activated T cells.

We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+. 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb 1B10 recognizes a novel <> appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.

Characterization of new monoclonal antibodies identifying avian T lymphocyte antigens.

Six monoclonal antibodies (mAb) were produced to identify and characterize surface antigens of chicken T cells. Determination of their reactivity with different lymphatic cells using immunofluorescence analysis demonstrates that mAb KH8, NA6, PD4 and TH8 stained 32-43% blood lymphocytes, 72-77% thymocytes and 19-27% spleen cells, mAb OC5 approximately 99% thymocytes and 55% blood and spleen lymphocytes each, and mAb OC2 36% blood lymphocytes, 79% thymocytes and 62% spleen cells. The KH8, NA6, PD4 and TH8 antibodies immunoprecipitated from lysates of surface-labeled chicken thymocytes a polypeptide of M(r) 60,000 under non-reducing conditions and the OC5 antibody a glycoprotein of M(r) 68,000 under reducing conditions. MAb OC2 precipitated a single polypeptide of M(r) 40,000 under both conditions. The mAb KH8, NA6, PD4, TH8 and OC2 inhibited ConA-induced proliferative responses of blood T cells in vitro. However, sepharose-bound or soluble OC5 antibody was able to increase DNA synthesis significantly. These results indicate that (a) the mAb KH8, NA6, PD4 and TH8 identify the avian homologue of the mammalian CD4 molecule, (b) the mAb OC2 detects the avian CD2 antigen, and (c) the mAb OC5 recognizes the putative avian CD5 homologue.

Antibody binding profile of purified and cell-bound CD26. Designation of BT5/9 and TA5.9 to the CD26 cluster.

The CD26 activation antigen (Ag) which is expressed on a subpopulation of human T cells has been characterized as dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5). In this paper, we describe the antibody binding profile of CD26/DPP IV, purified from human peripheral blood lymphocytes. The purified molecule binds to the anti-Ta1, anti-1F7 and anti-134-2C2 monoclonal antibodies (mAb), reported to react with cell-bound CD26 Ag. Among unclustered mAb recognizing T cell antigens, two, anti-BT5/9 and anti-TA5.9 were found to react with purified and cell-bound CD26 Ag. The classification of the BT5/9 Ag, the functional properties of the BT5/9+ T cell subset, as well as the in vivo effect of anti-BT5/9 mAb administration, are re-interpreted in the light of its specificity. Applying the anti-TA5.9 mAb in three color FACS analyses, we demonstrated that CD26+bright cells co-express CD45RO but not HLA-DR and CD38.

Expression of recombinant human ICOS and in vitro characterization of its bioactivity on B lymphocytes.

Inducible costimulator (ICOS) is a novel costimulatory molecule expressed in activated T cell and has critical regulation effect on special immune response. In this study, the cDNA encoding human ICOS was cloned from activated tonsil cells via RT-PCR, and was expressed in E. coli on pET28 expression vector. The recombinant ICOS protein expressed from E. coli showed a molecular weight of 14 kD on SDS-polyacrylamide gel electrophoresis and was further confirmed by Western blot. In presence of IL-10, the purified rhICOS significantly increased in vitro B cell growth stimulated by pokeweed mitogen (PWM), and enhanced the secretion of IgG from B cells.