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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.

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., (Published by Elsevier Inc.)

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.

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.

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.

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.

Recently the platelet and T-cell activation antigen 1 (PTA1) was assigned as CD226 at the 7th Conference and Workshop on Human Leukocyte Differentiation antigens (HLDA). PTA1 is mainly expressed on activated T cells, natural killer (NK) cells, platelets and stimulated endotheliocytes, and involved in the differentiation of cytotoxic T lymphocytes (CTL) and NK, as well as platelet activation and aggregation. We raised hybridomas secreting monoclonal antibodies (MAbs) to PTA1 by using the natural PTA1 as immunogen, which was purified from platelets via affinity chromatography. These MAbs, designated FMU1, FMU2, FMU3, FMU4, FMU5, FMU6 and FMU7, could recognize PTA1 cDNA transfected COS7 cells detected by flow cytometry (FCM), and also react with both natural PTA1 and PTA1/Ig fusion protein in indirect enzyme-linked immunoadsorbent assay (ELISA). The biosensor epitope mapping assay showed that the seven MAbs, together with previous PTA1-specific MAbs Leo A1 and New E1, could bind seven distinct epitopes of PTA1, respectively. The panel of MAbs might be new powerful tools to study the structure-function relationship of PTA1 molecule, and to search for the ligand of PTA1.

Understanding the basic immunology of an infectious disease requires insight into the pattern of T cell reactivity and specificity. Although lymphatic filariasis is a major tropical disease, the predominant T cell Ags of filarial species such as Brugia malayi are still undefined. We have now identified a prominent T cell Ag from B. malayi microfilariae (Mf) as Bm-SPN-2, a serpin secreted exclusively by this stage. Mf-infected mice mounted strong, but short-lived, Bm-SPN-2-specific Th1 responses, measured by in vitro production of IFN-gamma, but not IL-4 or IL-5, 14 days postinfection. By day 35, responsiveness to Bm-SPN-2 was lost despite enhanced reactivity to whole Mf extract. Single immunization with Mf extract also stimulated typical Th1 reactions to Bm-SPN-2, but IgG1 Ab responses dominated after repeated immunizations. Human patients displayed potent humoral responses to Bm-SPN-2 in both IgG1 and IgG4 subclasses. Thus, 100% (20 of 20) of the microfilaremic (MF(+)) patients bore IgG4 responses to Bm-SPN-2, while only 30% of endemic normal subjects were similarly positive. Following chemotherapy, Bm-SPN-2-specific Abs disappeared in 12 of 13 MF(+) patients, although the majority remained seropositive for whole parasite extract. PBMC from most, but not all, endemic subjects were induced to secrete IFN-gamma when stimulated with Bm-SPN-2. These findings demonstrate that Bm-SPN-2 is recognized by both murine and human T and B cells and indicate that their responses are under relatively stringent temporal control. This study also provides the first example of a stage-specific secreted molecule that acts as a major T cell Ag from filarial parasites and is a prime candidate for a serodiagnostic probe.

Whether immunity against opportunistic pathogens can be fully restored by control of HIV-1 replication remains open to question. This longitudinal study was conducted to measure anti-tuberculosis (TB) cellular immunity in 13 HIV-1/TB-coinfected patients effectively treated by highly active antiretroviral therapy (HAART) in a period of 12 months. In this study, anti-TB cellular immunity was assessed by determining the frequencies of CD 69 expression on CD4+ and CD8+ T cells in response to purified protein derivative (PPD) stimulation (abbreviated as %CD4+CD69 to PPD and %CD8+CD69 to PPD). Here, we show that %CD4+CD69 to PPD correlated with the results of tuberculin skin tests and interferon-gamma (IFN-gamma) production from PPD-stimulated CD4+ T cells, and %CD8+CD69 to PPD also correlated with CD8+ T cell-mediated PPD-specific cytolysis. In overall analysis for these 13 patients, both %CD4+CD69 to PPD and %CD8+CD69 to PPD increased significantly during the 12 months (p =. 003 and p <.001, respectively). However, we found %CD4+CD69 to PPD or %CD8+CD69 to PPD failed to increase substantially in some patients (i.e., immunologic nonresponders). A significantly higher proportion of patients whose baseline CD4+ count was <50 cells/mm3 were considered to be CD4+ nonresponders compared with those whose baseline CD4+ count was >50 cells/mm3. Furthermore, baseline CD4+ cell count in nonresponders is significantly lower than that in responders, although the effectiveness of HAART did not differ between them. Our results indicate that PPD-specific frequencies of CD69 expression may be used as surrogate markers of anti-TB cellular immunity. By this method, we show that full reconstitution of anti-TB cellular immunity in HIV-1/TB coinfected patients may not necessarily be achieved by "successful" HAART and may be influenced by the baseline immune status when HAART is started. These data suggest that the decision to discontinue secondary prophylaxis for opportunistic infections should be cautiously made, even when the CD4+ cell count has significantly increased.

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.

We reevaluate here an earlier report of monosaccharide binding by the C-type lectin-like, leukocyte surface protein CD69 in the form of a recombinant soluble dimer, and we examine polysaccharide binding by the protein. We have expressed in Escherichia coli a new construct of the extracellular part (Q(65)-K(199)) of human CD69. We describe the folding in vitro to produce, in good yield, the protein in a soluble, disulphide-linked, dimeric form, and the results of binding experiments with monosaccharides: glucose, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine, linked to bovine serum albumin. Monosaccharide-binding signals are not detectable. Among the polysaccharides, heparin, chondroitin sulphates A, B, and C, fucoidan, and dextran sulphate, CD69 dimer gives a weak binding signal with fucoidan., (Copyright 1999 Academic Press.)

We investigated a role of chemokines in thymocyte trafficking. Genes encoding stromal cell-derived factor-1 and its receptor CXCR4 were detected in the cortex by in situ hybridization. Early immigrant cells did not express CXCR4, whereas their descendant CD44+CD25+CD4-CD8- cells did. CXCR4 expression was down-modulated when CD4+CD8+ double-positive cells became CD4+CD8- or CD4-CD8+ single-positive (SP) cells. Positively selected CD69+CD3intermediate cells gained CCR4, of which ligand, thymus activation-regulated chemokine, was expressed in the medulla. At the next developmental stage, CD69-CD3high cells lost CCR4 but gained CCR7. These results suggest that thymocytes use different chemokines along with their development. Blockade of chemokine receptor-mediated signaling by pertussis toxin perturbed the normal distribution of SP cells and resulted in the accumulation of SP cells in the cortex. Thus, a pertussis toxin-sensitive event controls the trafficking of SP cells across the corticomedullary junction.

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., (Copyright 1999 Academic Press.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

We have previously shown that myelinating Schwann cells associated with motor, but not sensory, axons in peripheral nerves of adult mice express the L2/HNK-1 carbohydrate epitope. This carbohydrate structure carried by glycolipids and neural cell adhesion molecules has been suggested to specifically foster regrowth of motor as opposed to sensory axons after infliction of a lesion. To determine which molecular components may be the carriers of the L2 carbohydrate in motor axon-associated myelinating Schwann cells, we have isolated the purely sensory, cutaneous branch and the mixed sensory and motor muscle branch of the femoral nerve of adult mice, isolated the myelin fraction thereof and analysed the molecules expressing the L2 carbohydrate by several immunochemical methods. L2 immunoreactivity in myelin of the muscle branch was four to five times higher than that of the cutaneous branch. The 110 kDa L2-immunoreactive glycoprotein in myelin of the muscle branch, which is not L2-immunoreactive in the cutaneous branch, was identified as the myelin-associated glycoprotein by a combination of immunoprecipitation and Western blot analysis. Myelin extraction with organic solvents additionally revealed the two L2-carrying glycolipids, which amounted to approximately 40 ng glycolipid/mg dry weight in myelin of the muscle branch, whereas no significant amounts of the L2 glycolipids were found in myelin of the cutaneous branch. These observations suggest an astonishing degree of differential regulation of carbohydratesynthesizing activities in myelinating Schwann cells.

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.

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.

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.

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.

CD69 is a very early activation Ag of T lymphocytes. It is a cell surface glycoprotein that can only be detected after stimulation of lymphocytes. Despite extensive studies on its biologic activities, little is known about its molecular function. To investigate the latter in more detail, we have cloned a cDNA encoding CD69 on the basis of its expression in COS cells. The nucleotide sequence of clone CD69.13 is 1676 bp in length and contains a single open reading frame of 600 bp encoding a protein of 199 amino acids. The predicted molecular mass of 22,559 Da could be confirmed by in vitro translation. The protein contains a hydrophobic transmembrane region between amino acids 41 and 61 but no N-terminal signal peptide, which suggests that it is a type II membrane protein. It has one potential N-glycosylation site at amino acid 166. Two glycosylated forms of 26 to 28 kDa and 32 to 34 kDa were detected both in transfected COS cells and in in vitro translation in the presence of canine microsomes. Proteinase K degradation of the N-terminal part after in vitro protein synthesis supports the view of CD69 being a type II integral membrane protein with the N-terminal 40 amino acids in the cytoplasm, a transmembrane domain of 21 amino acids, and C-terminal 138 amino acids as the extracellular domain. Homology searches revealed sequence similarity with members of a supergene family of type II integral membrane proteins with a C-type lectin domain, indicating that CD69 is involved in signal transduction.

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.

CD28, initially detected on human T lymphocytes with the help of antibodies, and CTLA-4, obtained by reverse genetics through its preferential expression in mouse activated T cells, are both single-V domain members of the Ig superfamily. Early work showed a relationship between these two molecules, which we wished to further document, in particular because of the growing realization of the functional importance of CD28 in some T cell activation pathways. Isolation and analysis of the mouse CTLA-4 gene and further analysis of the human CTLA-4 gene showed that both of these and the human CD28 gene share the same overall intron/exon organization. The nucleic acid sequence homology of the exons was found to extend across both molecules and species, whereas the 5' and 3' flanking regions exhibited homology across species but not between molecules. Message expression of human CTLA-4 was only detected in activated T cells and, thus, shares with that of mouse CTLA-4 and of mouse and human CD28 a lymphoid tissue distribution, although apparently broader for the latter. Two main human CTLA-4 transcripts of about 1.8 and 0.8 kb were detected, the smaller of which may derive, as reported for human CD28, from the use of an alternate degenerated polyadenylation signal sequence. The nucleic acid sequence data allowed a direct comparison of the four putative complete protein sequences of CD28 and CTLA-4 in the mouse and the human, showing striking homologies, especially in some stretches (such as a MYPPPY hexamer in the hinge region) conserved across molecules and across species. The mouse CD28 gene was localized to chromosome 1 band C by in situ hybridization with three different radioactive probes, indicating, together with previous data, that the CD28 and CTLA-4 genes map to the same chromosomal region in both the mouse and the human. Thus, CD28 and CTLA-4 were found to be strikingly similar in most respects, in terms of structure, sequence, expression, and gene location, furthermore in two species, strongly suggesting that their genes are the direct products of a duplication event and raising the possibility of functional homologies between the corresponding proteins.

CD6 (T12) is a 130-kDa glycoprotein present on the surface of human T cells. Previously, we demonstrated that the anti-T12 and anti-2H1 monoclonal antibodies recognized different epitopes on CD6, and both were capable of transducing activation signals to T cells. Anti-T12 augmented suboptimal signaling via the TCR/CD3 complex and directly activated separated CD4+ but not CD8+ cells. Structural characterization of CD6 revealed that it contained intrachain disulfide bonds, was N-glycosylated, and in activated cells was phosphorylated on serine. Given the functional significance of CD6 and its involvement in signaling via CD3 and CD2 pathways, we examined in detail the biosynthesis, structural characteristics, and phosphorylation properties of this receptor-like molecule. These studies demonstrate that the nascent CD6 polypeptide on both T cells and thymocytes in 88 kDa, and the immature N-glycosylated form is 110 kDa. After maturation of N-linked glycan and addition of sulfated O-linked oligosaccharide, CD6 appears on the cell surface as a molecule of 130 kDa. CD6 is phosphorylated in resting cells and can be hyperphosphorylated when stimulated by phorbol 12-myristate 13-acetate, indicating that it may participate in the major common signaling pathway mediated through protein kinase C. Concanavalin A-activated cells are phosphorylated at an additional site(s) on the molecule and cannot be hyperphosphorylated with phorbol 12-myristate 13-acetate. These physical features reveal additional clues about the physiological role of CD6 and its mechanism of signal transduction and strongly suggest that CD6 represents a physiologically important membrane receptor involved in T cell activation.

Using a polyclonal heterologous anti-soluble E-receptor serum, we identified molecules of molecular weight circa 58,000 and 150,000. The soluble receptor molecule with molecular weight of approximately 58,000 (Rs1) was initially purified from supernatant of heated lymphocytes through chromatography on Sephadex G-200 and/or DEAE-cellulose. The soluble receptor molecule with molecular weight of approximately 150,000 (Rs2) is detected at high levels in the serum of patients with cancer and uremia. Rs1 and Rs2 present in serum from cancer patients were purified by chromatography on Sephadex G-200 and by affinity chromatography using anti-Rs1 IgG. 131I-labelled supernatant of heated lymphocytes binds to sheep erythrocytes and the elution and analysis of the molecules adsorbed showed bands of molecular weights approximately 58,000 and 150,000, confirming the receptor activity of these molecules.

Using a specific serum anti-soluble T lymphocytes receptor for sheep erythrocytes (E) and SDS-PAGE, we detected radioactive bands of molecular weight 58,000 in immunoprecipitates of supernatant of heated human lymphocytes (SHL), in the supernatant of PHA stimulated lymphocyte cultures (SLC), normal human serum (NHS), and serum from cancer and uremia patients, labelled with 131I. By Sephadex G-200 chromatography, in addition to this fraction, we detected molecules of molecular weight higher than 150,000 which interact with the anti-soluble receptor serum (anti-RS), in serum from cancer and uremia patients. These molecules were detected in NHS or SHL after concentration or by prolonged exposure of SDS-PAGE with some labelled and immunoprecipitated SHL samples. The soluble receptors of molecular weights 58,000 (RS1) and more than 150,000 (RS2) were fully identical when analyzed by immunodiffusion with anti-RS serum. When submitted to immunoelectrophoresis, RS1 showed electrophoretic migration similar to that of albumin, while RS2 showed a pattern close to that of alpha 2-globulin. However, RS2 did not show antigenic relationship with IgM and was not an immune complex with IgG. Even though the presence of RS in human saliva has not yet been reported, molecules that interact with anti-RS serum have been detected in human saliva and are fully identical to molecules found in supernatant of heated human T lymphocytes and NHS. The RS molecules present in human saliva have a molecular weight and electrophoretic migration similar to those of RS1 from SLC and from human serum and have no antigenic relationship with human albumin.

A new surface molecule has been discovered on mouse intestinal intraepithelial lymphocytes (IEL) using a rat anti-mouse IEL monoclonal antibody, M290. It was expressed at high levels on nearly all IEL and on a majority of T cells in the gut lamina propria. M290 stained, with lower intensity, a small minority of T cells in other lymphoid tissues. Expression was biased towards the CD8+ subset. Stimulation of peripheral T cells with mitogens did not induce expression of the new antigen but addition of transforming growth factor beta to stimulated T cells had a marked inductive effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IEL surface components precipitated with M290 showed principal bands at 135, 120, 28 and 24 kDa (reduced) and 135, 100, 24 and 21 kDa (nonreduced). Precipitation with antibodies to integrin subunits showed that the new molecular complex was not a member of the beta 1, beta 2, or beta 3 integrin families although all of these were represented on IEL. A 13-amino acid N-terminal sequence obtained from the 120-kDa beta subunit of the antigen prepared from an M290+ T hybridoma (MTC-1) did not show homology with integrins. Pulse-chase studies using MTC-1 cells showed that the 135-kDa alpha subunit was derived from a 147-kDa precursor. The function of this new molecular complex is not yet known.

In a T cell antigen receptor complex (TCR), the clonotypic disulfide-linked Ti heterodimer is noncovalently associated with the invariant CD3 polypeptides. The latter are composed of three monomeric subunits (gamma, delta, epsilon) and either a disulfide-linked homodimer (zeta zeta) or a disulfide-linked heterodimer (zeta eta). The exact stoichiometry of the Ti-CD3 subunits in a given complex is still largely unknown. Here, we report the presence of a CD3 epsilon dimer in a fraction of the TCR. When TCRs from both human and murine T lymphocytes were immunoprecipitated with monoclonal antibodies against either CD3 epsilon or Ti, a 40-kDa disulfide-linked dimer was coprecipitated with the other TCR subunits from digitonin lysates. Amino acid sequence analysis of peptides obtained by in situ CNBr cleavage of the 20-kDa product blotted to polyvinyl difluoride membranes from reducing/nonreducing two-dimensional gels identified human CD3 epsilon. Assuming this CD3 epsilon to derive from a homodimer, then either some TCRs contain more than one CD3 epsilon chain or several TCRs are covalently associated with one another via their CD3 epsilon subunits. Although it has been suggested that a putative TCR association with CD2 exists under similar conditions to those utilized to detect CD3 epsilon dimers, the CD2 molecule was not coimmunoprecipitated with the TCR by any of a series of anti-CD3 epsilon monoclonal antibodies. In conjunction with the fact that CD2 and the TCR do not colocalize during conjugate formation between T cells and antigen-presenting cells (Koyasu, S., Lawton, T., Novick, D., Recny, M. A., Siliciano, R. F., Wallner, B. P., and Reinherz, E. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2603-2607), we conclude that CD2 and the TCR are not physically associated on the T cell surface.

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.

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.

Sequential effects on cellular protein expression following human immunodeficiency virus (type 1) infection of a CD4+ T-cell line in vitro were investigated. Events in the human interleukin 2-dependent helper T-cell line WE17/10 are similar in several respects to the clinical progression in acquired immunodeficiency syndrome. WE17/10 cell infection is characterized by an extended period during which viral replication occurs without accompanying cytotoxicity and with a maximum 30% decrease in surface CD4. Cellular protein expression generally remains unaffected during this first phase of infection. However, after 2-3 months, a severe defect in the expression of the T-cell receptor/CD3 complex both on the cell surface and inside the cell becomes apparent. Other cell membrane markers, such as CD2 and CD25, remain constant throughout the course of infection; after its initial decrease, CD4 remains at 70% of control values. Lack of surface expression of the TCR/CD3 complex is correlated with a specific defect in transcription of the CD3 gamma-chain gene.

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.

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.

A monoclonal antibody (mAb 12-15) reactive with the mouse CD2 was found to co-precipitate a high-molecular-weight glycoprotein from mouse thymocyte, splenic lymphocyte, Con A blast, and T cell tumor detergent lysates which was identified as the leukocyte common T 200 glycoprotein (CD45). The reactivity was specific for CD2 since antibodies to CD3 did not co-precipitate the T200 glycoprotein. mAb 12-15 did not react with immunoaffinity-purified T200 glycoprotein, ruling out the possibility that the antibody detected a cross-reactive epitope. Biochemical data indicated that the association of CD2 with T200 was not generated during lysis of the cell and that the molecular complex was non-covalently linked since it could be destroyed by high salt washing or boiling in SDS. Distribution analysis in Triton X114-H2O revealed that, in contrast to free T200 molecules, the complexed T200 was enriched in the detergents phase. To investigate the CD2-T200 association in more detail at the cell surface, modulation of CD2 and T200 was studied. Modulation could be induced on Con A blasts by monoclonal antibodies followed by cross-linking with a FITC-conjugated second antibody. Within 24 h the expression of CD2 or T200 was reduced to approximately 10-20% of the initial value on the majority of cells. However, two-color fluorescence showed that modulation of CD2 did not lead to co-modulation of CD3 or T200. A possible physiological role of CD2-T200 complexes is discussed.

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 CD8 Ag is a cell surface heterodimer which demarcates predominantly cytotoxic T cells which are restricted by class I MHC Ag. The disulfide bonds within the murine structure were assigned in this study and the alpha-beta-interchain bond involves one or more cysteine residues located in each chain proximal to the plasma membrane or included within it. The location of the intrachain disulfide loop within the CD8 beta-chain confirms its proposed structural homology to an IgV domain but no corresponding disulfide loop is present within the alpha-chain. The invariant IgV disulfide loop has been replaced by a unique, short loop involving an unusual cysteine which is conserved in the CD8 alpha-chains of man, mouse, and rat. Despite its lack of precedent in other Ig-related structures, this unusual disulfide loop can be parsimoniously accommodated into a modified domain which has retained the major features of the Ig structural motif.

The recognition of foreign antigen by T lymphocytes requires direct contact with cells expressing the antigen. It has recently become clear that T lymphocytes can form conjugates with other cells in the absence of foreign antigen expression. Studies using monoclonal antibodies (mAbs) to inhibit conjugate formation have suggested that a portion of the antigen-dependent adhesion is mediated by T lymphocytes interacting with cells expressing lymphocyte-function-associated antigen 3 (LFA-3), a widely distributed cell surface protein. We have investigated antigen-independent adhesion by incorporating affinity-purified LFA-3 into the lipid membrane of an artificial target cell (ATC; a nylon-matrix vesicle with a lipid membrane). These vesicles are similar in size and density to intact cells, so that conjugates between cells and ATCs may be seen by light microscopy. ATCs expressing a density of LFA-3 similar to that on intact cells were found to form conjugates with T cells, but only if the T cells expressed the sheep erythrocyte receptor, CD2 (T11; LFA-2). Previous studies using mAbs have implicated the CD2 molecule in both adhesion and T-cell activation. ATCs prepared without surface protein or with purified HLA class I protein failed to interact with the CD2-positive T cells, indicating that the adhesion found was mediated by the LFA-3 molecule. Furthermore, mAb against LFA-3 or CD2 was able to block the LFA-3-mediated vesicle-cell interaction, whereas mAb against LFA-1 or HLA failed to inhibit the interaction. These results provide direct evidence that LFA-3 functions as an adhesion molecule by serving as a ligand for the CD2 molecule on T cells.

YE1/48 is a murine cell surface disulphide-linked dimeric Ag consisting of two 45,000-50,000 Mr subunits. It is expressed on some T lymphoma lines at high levels but its expression on normal lymphocytes is very low. The functional significance of this Ag is currently unknown. We have now cloned a cDNA encoding the YE1/48. Sequence analysis revealed that it encodes a Type II membrane protein of 262 amino acids (30,500 MW), with 44 amino acids in the N-terminal cytoplasmic domain, 22 amino acids in the transmembrane domain and 196 amino acids in the C-terminal extracellular domain. There are three potential N-linked glycosylation sites in the extracellular domain all of which are probably used in the mature protein. No significant homology can be identified with other known protein sequences in the data base or with human CD28(T44), a human T cell activation antigen consisting of two 44,000 Mr subunits. The protein sequence includes in its extracellular domain the arginine-glycine-aspartic acid tripeptide, a potential cell-adhesive binding site, and a sequence similar to the consensus domain of any metal-binding proteins. However, whether these sequences are functional is unknown. Genomic Southern analysis of C57BL/6, BALB/c and C3H mice has demonstrated a restriction fragment length polymorphism. The analysis has also strongly suggested the existence of some other genes with sequences highly homologous to the YE1/48 gene. The YE1/48 gene appears to be expressed at very low levels in a wide range of lymphoid cells with no restriction to their differentiation stages. Interestingly, YE1/48 expression appears to be induced in pre-B cells after transformation by Abelson virus, suggesting an association of YE1/48 expression with the transformation of T and pre-B Cells.

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.

The T-cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. We have examined the glycosylation of CD4 and asked whether carbohydrate addition is essential for proper expression of the glycoprotein on the cell membrane. Under conditions where treatment of CD4+ human acute lymphoblastic leukemia cells (CEM-CM3 cells) with the glycosylation inhibitor tunicamycin decreased surface expression of CD4 in a time- and concentration-dependent manner, the surface expression of several other glycoproteins was unaffected. Incubation with tunicamycin for 48 h inhibited mannose incorporation by 98%, caused a 76% decrease in CD4 surface expression as judged by flow cytometry, and had little effect on methionine incorporation. Scatchard analysis showed a decrease in the total number of CD4 molecules on the cell surface from 17,000 to 8,900 after 24 h of tunicamycin treatment. Immunoprecipitation of metabolically labeled CD4 revealed the presence of an unglycosylated precursor in tunicamycin-treated cells. The observed difference between the Mr of the glycoprotein and its precursor is consistent with glycosylation at two potential N-linked sites. However, this precursor could not be detected by measuring steady state levels by immunoblotting. Also, no intracellular accumulation of CD4 in tunicamycin-treated cells was detectable using immunofluorescence microscopy. We conclude that surface expression of CD4 depends on glycosylation of the protein and that the unglycosylated precursor is preferentially degraded.