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.

Immunoproteomic discovery of novel T cell antigens from the obligate intracellular pathogen Chlamydia.

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.

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.

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.

Prediction of imminent complications in HIV-1-infected patients by markers of lymphocyte apoptosis.

OBJECTIVE: The aim of the study was to compare accepted surrogate markers of HIV disease progression with markers of lymphocyte apoptosis in their ability to predict short-term disease progression. METHODS: In all, 40 HIV-positive patients were studied prospectively and observed during follow-up for HIV-related adverse clinical events. Ex vivo apoptosis was measured with the markers CD95 expression, annexin V binding, and Apostain dye uptake by flow cytometry at baseline. Established markers of disease progression (CD4 count, HIV-RNA level, and CD8/38 count), CD8, B-cell, and natural killer (NK) cell counts were determined by standard procedures at baseline and after 6 months. RESULTS: In HIV-infected patients, CD95 expression and annexin V binding showed significantly elevated apoptosis in peripheral blood lymphocytes and all lymphocyte subsets at baseline compared with HIV-negative, healthy controls. Apostain failed to differentiate between HIV-infected patients and healthy controls. HIV-related complications could be predicted by CD4 and CD8/38 counts, but not HIV viral load as assessed by relative operating characteristic (ROC) analysis (CD4, p = .003; CD8/38, p = .031). A similar or even better diagnostic accuracy was found for CD95 expression in total lymphocytes (p<.001), the CD4+ (p = .003) and CD8+ (p = .005) T-cell subsets and for annexin V binding in CD4+ T cells (p = .005). When patients with CD4 counts <200 cells/microl were analyzed separately, only annexin V binding in CD4+ T cells, but none of the other prognostic markers could predict complications (p = .001). CONCLUSION: Determination of annexin V binding on CD4+ T cells may be a useful tool to monitor HIV-infected patients with low (<200 cells/microl) CD4 counts, as it can reliably assess the risk for imminent complications in such patients.

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.

Characterization of prethymic progenitors within the chicken embryo.

The thymic primordium in both birds and mammals is first colonized by cells emerging from the intra-embryonic mesenchyme but the nature of these precursors is poorly understood. We demonstrate here an early embryonic day 7 prethymic population with T lymphoid potential. Our work is a phenotypic analysis of, to date, the earliest embryonic prethymic progenitors arising in the avian para-aortic area during ontogeny. The phenotype of these cells, expressing the cell surface molecules alpha2beta1 integrin, c-kit, thrombomucin/MEP21, HEMCAM and chL12, reflects functional properties required for cell adhesion, migration and growth factor responsiveness. Importantly, the presence of these antigens was found to correlate with the recolonization of the recipient thymus following intrathymic cell transfers. These intra-embryonic cells were also found to express the Ikaros transcription factor, the molecular function of which is considered to be prerequisite for embryonic lymphoid development.

Oxidized low-density lipoproteins affect natural killer cell activity by impairing cytoskeleton function and altering the cytokine network.

Several lines of evidence indicate that oxidative imbalance can play an important role in determining an impairment of natural killer (NK) cell activity in a variety of human diseases. Because a specific role for oxidized low-density lipoproteins (LDL) as pro-oxidizing agents has been envisaged, we tested the activity of oxidized LDL (ox-LDL) on NK cell-mediated cytotoxicity, cytokine release, and membrane molecule modulation. Native LDL served as control. Treatment with ox-LDL at noncytotoxic concentrations (0.2 mg/ml) during the NK/target cell (TC) interaction markedly reduced NK cytotoxic activity against U937 tumor cells. This inhibitory activity was also noticed when NK cells were pretreated with ox-LDL. Scanning electron microscopy examination of NK-target cell conjugates failed to reveal any morphological cell damage. In addition, the number of conjugates and the expression of some adhesion molecules (CD11a, CD11b, CD18, CD2, and CD62L) were not modified by ox-LDL. These observations argued against a possible interference of ox-LDL with the binding process leading to the formation of NK/TC conjugates. By contrast, immunocytochemical analyses of cytoskeleton components of NK cells exposed to ox-LDL showed a partial depolymerization and a derangement of the microtubular apparatus. These alterations were accompanied by an evident decrease in their intracellular reduced glutathione content. Owing to the important role played by the microtubular network during the killing process, it is possible to infer that a cytoskeleton alteration underlies the inhibitory activity of ox-LDL on NK cell function. In addition, exposure of mitogen-stimulated peripheral blood mononuclear cells to ox-LDL markedly reduced specific mRNA transcription and release of cytokines relevant for NK cell activity (such as tumor necrosis factor-alpha, interferon gamma, and interleukin 12). These data suggest that the impairment of NK cell activity by ox-LDL likely reflects the concomitant dysregulation of some essential mechanisms of NK cell function.

Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/CD29).

Anti-alpha 4 integrin mAb coprecipitated CD81 (TAPA-1), a 25-kDa cell surface protein, from various alpha 4 beta1 -positive hemopoietic cell lines, including Molt4, Jurkat, Ramos, and alpha 4-transfected K562 (KX4C4) cells. In reciprocal experiments, the integrin alpha 4 beta 1 (VLA4, CD49d/CD29) could be reprecipitated from CD81 immunoprecipitates. Anti-alpha 4 integrin mAb also coprecipitated CD81 from the alpha 4 beta 7-positive B cell line RPMI 8866. In contrast, no CD81 was identified in alpha 2 beta 1, alpha 5 beta 1, or alpha L beta 2 immunoprecipitates. Abs to other members of the transmembrane-4 superfamily, including CD53, CD63, and CD82, also coprecipitated alpha 4 beta 1. As shown by confocal microscopy, CD81 and CD82 colocalized with alpha 4 beta 1 in cell surface clusters. The cytoplasmic domain of the alpha 4 integrin was not necessary for alpha 4 beta 1/CD81 association, nor was the association influenced by divalent cations, EDTA, integrin-activating mAb, or alpha 4 subunit cleavage. Notably, two independent alpha 4 adhesion-deficient mutants (D346E and D408E) were deficient in their ability to associate with CD81. Thus, CD81 and other transmembrane-4 superfamily members may participate in functionally relevant interactions with alpha 4 beta 1 and other integrins.

The amino-terminal immunoglobulin-like domain of activated leukocyte cell adhesion molecule binds specifically to the membrane-proximal scavenger receptor cysteine-rich domain of CD6 with a 1:1 stoichiometry.

Activated leukocyte cell adhesion molecule (ALCAM) was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain.

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.

Identification of a mouse protein homologous to the human CD6 T cell surface protein and sequence of the corresponding cDNA.

CD6 is a 105/130 kDa monomeric T cell surface glycoprotein that has been shown to play a role in human T cell activation. Recently a partial mouse CD6 cDNA sequence was described. We have isolated full-length cDNA clones including the initiation codon and sequence encoding the full signal peptide, as well as an additional 39 amino acids within the cytoplasmic domain as compared to the previously reported clone. The predicted full-length mouse CD6 protein contains 665 amino acids and has the features of a type I integral membrane protein. The extracellular domain of mouse CD6 is composed of three repeated cysteine-rich domains similar to those in human CD6, mouse and human CD5, and other members of a family of proteins whose prototype is the type I macrophage scavenger receptor. In marked contrast to the previously published human CD6 sequence, the mouse sequence predicts a long cytoplasmic tail that is not closely related to other proteins and possesses two proline-rich motifs containing the SH3-domain binding consensus sequence, three protein kinase C phosphorylation site motifs, nine casein kinase-2 phosphorylation site motifs, and a serine-threonine-rich motif repeated three times. Northern blot analysis revealed that mouse CD6 mRNA is expressed predominantly in thymus, lymph node, and spleen. A polyclonal antiserum was raised against mouse CD6 by gene gun plasmid DNA immunization of rabbits with the mouse CD6 cDNA in an expression vector. In immunofluorescence analysis this polyclonal antiserum positively stained the surface of cells transfected with the mouse CD6 cDNA in an expression vector, as well as most normal mouse thymocytes and peripheral T cells. CD6 protein is expressed on most CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes, and is expressed at highest levels on mature CD3high thymocytes. The expression of mouse CD6 in thymocytes and peripheral T cells correlates closely with the expression of the related CD5 molecule. The polyclonal rabbit anti-mouse CD6 Abs immunoprecipitated a major polypeptide of 128 kDa from resting and 130 kDa from PMA- and FCS-activated mouse thymocytes and lymph node cells; it is likely that this increase in size upon activation is due to phosphorylation of mouse CD6 as has been described for human CD6. These data demonstrate that mouse thymocytes and T cells express a 130-kDa cell surface protein homologous to human CD6.

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis.

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. with Cryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyte- and granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogeneously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.

CD 69 antigen of human lymphocytes is a calcium-dependent carbohydrate-binding protein.

CD69 is a signal transducing molecule of hematopoietic cells. Previous molecular cloning of CD69 has revealed a type II transmembrane orientation and the presence of an extracellular domain related to the Ca(2+)-dependent (C-type) animal lectins. As the predicted amino acid sequence for the lectin-like domain is highly divergent from those of other C-type lectin-like proteins - a feature shared with NKR-P1 of natural killer cells - CD69 and NKR-P1 are among proteins assigned to a separate group, group V. To initiate ligand identification studies, we have prepared soluble forms of CD69 protein by bacterial expression of its extracellular portion. We show that cysteine 68 located in the short membrane-proximal neck region of CD69 which adjoins the C-terminal lectin-like domain is a critical element for dimerization. We have evidence that the soluble dimeric CD69 has a tight association with calcium, a feature shared with NKR-P1, and that it is a carbohydrate-binding protein with N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as the best inhibitors: 4-8 x 10(-5) M giving 50% inhibition of binding to N-acetyl-D-glucosamine neoglycoprotein. Thus, the tight association with calcium and high affinities for carbohydrate binding appear to be features of at least two members of the C-type lectin group V.

Cellular expression of a GPI-linked T cell activation protein.

A T cell activation protein was identified by generating a monoclonal antibody (anti-D2) against a gamma delta T cell receptor bearing gibbon ape T cell line (MLA144). Immunoprecipitation studies revealed three polypeptides of 180, 150, and 120 kDa. The antigen was also found to be expressed on endothelial cells in vivo and in vitro and on tumor cell lines from a variety of tissues. Studies performed using a variety of antibodies reveal this protein to be identical to an endothelial cell protein previously identified by several antibodies to T cell activation proteins (CDw109). We demonstrate that this protein is anchored in the membrane via a glycosylphosphatidylinositol (GPI) tail in T cells, tumor cells, and endothelial cells. An analysis of tissue sections reveals this protein to be normally highly expressed on vascular endothelial cells.

Evidence for a structural difference in the CD7 polypeptide on human thymocytes and intraepithelial lymphocytes defined by a new monoclonal antibody, 3D9.

Intestinal intraepithelial lymphocytes (IEL) represent a distinct subpopulation of lymphocytes located on or above the basement membrane and adjacent to the basolateral membrane of enterocytes. Thus IEL are strategically positioned to mediate a response to the uptake of foreign antigens or to the alteration of enterocytes by injury or infection. Because of their unique location, we hypothesized that IEL might selectively express specialized cell surface proteins important in their site-specific localization or function. To identify such proteins, we immunized mice with purified human IEL and identified one monoclonal antibody, 3D9, which was found to react with a majority of IEL but with very few lamina propria lymphocytes (LPL) and weakly with most peripheral blood lymphocytes (PBL). Evaluation of this antibody with two-dimensional gels demonstrated that it reacts with CD7, previously known as an early thymocyte differentiation antigen. Interestingly, unlike all other anti-CD7 monoclonal antibodies, 3D9 identified only occasional thymocytes by immunohistochemistry suggesting that CD7 is structurally different on IEL and thymocytes. However, the CD7 polypeptide immunoprecipitated from IEL and thymocytes appeared identical in SDS-PAGE mobility and in charge by two-dimensional gels, despite being recognized differently by monoclonal antibodies. These studies emphasize the expression of CD7 by IEL T cells and reveal the existence of an undefined structural difference between CD7 molecules on IEL compared to thymocytes.

Association of four antigens of the tetraspans family (CD37, CD53, TAPA-1, and R2/C33) with MHC class II glycoproteins.

Four of the tetraspans family antigens expressed in B cells, CD37, CD53, TAPA-1, and R2/C33, as well as at least two other molecules, CD19 and CD21, coprecipitate with DR antigens from mild detergent lysates of human B-cell lines and tonsillar B cells. Coprecipitation and preclearing experiments indicate the existence of large multicomponent complexes containing jointly the seven components, although some "incomplete" complexes lacking some of the components may also exist. The complexes contain only a relatively small fraction of the total cellular pool of relevant molecules. The existence of these "tetraspans-DR complexes" may be related to the previously reported antiproliferative and signaling effects of antibodies against most of their components.

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.