B70 antigen is a second ligand for CTLA-4 and CD28

The membrane antigen B7/BB1 (refs 1, 2) is expressed on activated B cells, macrophages and dendritic cells, and binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. Discrepancies between results with anti-CD28 and anti-B7 antibodies have suggested the existence of a second ligand for CD28 and CTLA-4 (refs 3, 6-8). We have generated a monoclonal antibody, IT2, that reacts with a 70K glycoprotein (B70). B70 complementary DNA was cloned from a B-lymphoblastoid cell line library and encodes a new protein of the immunoglobulin superfamily with limited homology to B7. B70 is expressed on resting monocytes and dendritic cells and on activated, but not resting, T, NK and B lymphocytes. IT2 substantially inhibited the binding of a CTLA4-immunoglobulin fusion protein to human B-lymphoblastoid cell lines and, together with anti-B7 antibody, completely blocked CTLA-4 binding. Further IT2 efficiently inhibited primary allogeneic mixed lymphocyte responses. These findings indicate that B70 is a second ligand for CD28 and CTLA-4 and may play an important role for costimulation of T cells in a primary immune response.     

Developmental and tissue-specific expression of human CD4 in transgenic rabbits

A major obstacle to understanding AIDS is the lack of a suitable small animal model for studying HIV-1 infection and the subsequent development of AIDS, and for testing diagnostic, therapeutic, and preventive modalities. Our goal is to produce a rabbit model for the study of AIDS. Here we report on the generation of transgenic rabbits that express the human CD4 (hCD4) gene. The transgene, which contains the coding region for hCD4 and approximately 23 kb of sequence upstream of the translation start site, was used previously to direct hcD4 expression on the surface of CD4+ T cells of transgenic mice (Gillespie et al., 1993: Mol Cell Biol 13:2952-2958). The hCD4 transgene was detected in five males and two females derived from the microinjection in five males and two females derived from the microinjection of 271 rabbit embryos. Both hCD4 RNA and protein were expressed in peripheral blood lymphocytes (PBLs) from all five males but neither of the females. Human CD4 was expressed on PBLs from F1 offspring of all founder males. T-cell subset analysis revealed that hCD4 expression was restricted to rabbit CD4 (rCD4) expressing lymphocytes; mature rCD4- rCD8+ lymphocytes did not express hCD4. In preliminary studies, PBLs from hCD4 transgenic rabbits produced greater amounts of HIV-1 p24 core protein following HIV-1 infection in vitro than HIV-1 p24 antigen in nontransgenic rabbit infected cultures. These results extend to rabbits our previous observation that this transgene contains the sequence elements required for high-level expression in the appropriate cells of transgenic mice. Furthermore, these and previous studies demonstrating that expression of hCD4 protein enhances HIV-1 infection of rabbit T cells in vitro, coupled with reports that normal, nontransgenic rabbits are susceptible to HIV-1 infection, suggests that the hCD4 transgenic rabbits described herein will have an increased susceptibility to HIV-1 infection. In vivo HIV-1 infection studies with these rabbits are under way.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus, bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4+ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25+ and CD69+ lymphocytes were higher. Seventy-four percent of the CD25+ PBMC but only 55% of the CD69+ cells were CD3+ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69+ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

Monoclonal anti thy 1.2 antibodies from hybridoma HO13-4 do not react with mouse natural killer cells

The susceptibility of NK cells and immune cytotoxic T-cells to treatment with (a) monoclonal anti thy 1.2 antibodies from hybridoma HO13-4, (b) rabbit anti-mouse T-cell antiserum and (c) gamma globulins prepared from AKR/J anti C3H/HeJ antiserum was studied in the presence of rabbit complement. Monoclonal anti thy 1.2 antibody treatment completely abolished the cytotoxic activity of immune T-cells derived from C57BL/6J mice (H-2b) immunized with (C57BL/6J x DBA/2)F1 spleen cells (H-2bd) against P815 (H-2d) target cells. The same treatment had no significant effect on the NK activity of spleen cells from unimmunized mice against YAC target cells. Rabbit anti-mouse T-cell and mouse anti theta antisera also abrogated completely the immune T cell activity of spleen cells. This treatment however also resulted in a partial loss of NK activity. These results indicate that conventional anti theta antisera contain antibodies which recognize antigenic specificities on T-cells as well as on a population of NK cells. The cross reactivity is not a result of thy 1.2 antigen expression on NK cells and T-cells as recognized by the monoclonal antibodies. The specificity recognized by the monoclonal antibody (HO13-4) is only expressed on T-cells.     

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.     

BCMAp: an integral membrane protein in the Golgi apparatus of human mature B lymphocytes

BCMA is a human gene expressed preferentially in mature B lymphocytes as a 1.2 kb mRNA, which encodes a 184 amino acid peptide (BCMAp). The study of BCMA mRNA expression, using human malignant B cell lines characteristic of different stages of B lymphocyte differentiation, demonstrated that the BCMA mRNA is absent in the pro-B lymphocyte stage. It is expressed faintly at the pre-B cell stage and its expression increases with B lymphocyte maturation. Polyclonal antibodies were used to show, by cellular fractionation and immunoprecipitation, that BCMAp is a non-glycosylated integral membrane protein. Furthermore, BCMAp inserts, in vitro, into canine microsomes, as a type I integral membrane protein. Cell surface labeling showed that BCMAp is not expressed in the plasma membrane of mature B lymphocytes. Immunofluorescence studies revealed that BCMAp lies in a cap-like structure near the nucleus, that was identified as the Golgi apparatus by co-localization of BCMAp with CTR433, a marker of the medial cisternae of the Golgi apparatus. Confocal scanning laser microscopy of U266 plasma cells labeled with markers of various Golgi apparatus subcompartments strongly suggests that BCMAp is located in the cis part of the Golgi apparatus. Thus, BCMAp is the first Golgi resident protein with a tissue specificity and whose expression is linked to the stage of differentiation of B lymphocytes. The location of BCMAp in the Golgi apparatus and its high expression in plasmocytes (secreting large amounts of Ig) suggest that BCMAp is implicated in the intracellular traffic of Ig.     

Cloning of BY55, a novel Ig superfamily member expressed on NK cells, CTL, and intestinal intraepithelial lymphocytes

Expression of the BY55 protein has been shown to be tightly associated with NK and CD8+ T lymphocytes with cytolytic effector activity. To determine the function of this protein, we molecularly cloned BY55 cDNA. The cDNA sequence predicts a cysteine-rich, glycosylphosphatidylinositol-anchored protein of 181 amino acids with a single Ig-like domain weakly homologous to killer inhibitory receptors. Reduction and carboxyamidomethylation of immunoprecipitated BY55 gave a band of 27 kDa, whereas reduction alone led to an 80-kDa species, suggesting that BY55 is a tightly disulfide-linked multimer. RNA blot analysis revealed BY55 mRNAs of 1.5 and 1.6 kb whose expression was highly restricted to NK and T cells. BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity, but was not expressed and was not induced on the CD56bright, CD16-subset of NK cells, a subset with high proliferative, but low cytolytic, capacity. In human tissues, BY55 mRNA was expressed only in spleen, PBL, and small intestine (in gut lymphocytes). BY55 was expressed on all intestinal intraepithelial lymphocytes, which were predominantly CD3+TCRalpha/beta+CD4-CD8+CD11b+CD28-CD45RO+C D56-CD101+CD103+ (alphaEbeta7 integrin). In addition, BY55 was expressed on most CD8+CD28- peripheral blood T cells. These phenotypic relationships suggest that CD8+CD28+ precursor CTL may terminally differentiate into CD8+CD28-BY55+ effector CTL and that some of the peripheral blood CD8+CD28- subset may represent recirculation from mucosal epithelial immune sites.     

Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts

Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.     

Tumour radiosensitization by high-oxygen-content gases: influence of the carbon dioxide content of the inspired gas on PO2, microcirculatory function and radiosensitivity

The rat testis is considered to be an immunologically privileged site because of its reduced capacity to support antigen-specific immune responses. To understand this phenomenon, it is essential to characterize both the lymphocyte subpopulations normally present in the testis and their regulation by testicular cytokines. Peripheral blood was obtained from adult male Dark Agouti or Sprague-Dawley rats, and testicular interstitial tissue was collected after perfusion of the testes to remove blood. Blood and testis lymphocytes were isolated using discontinuous Percoll density gradients, and the testicular lymphocytes were further purified by selective adherence to remove mononuclear phagocytes. The isolated lymphocytes were analyzed by flow cytometry using specific monoclonal antibodies and fluorescein labeling and were enumerated as total T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells. In contrast to peripheral blood, in which the CD4+ T-cell subset was the major lymphocyte subset, rat testis T cells were predominantly of the CD8+ subset, and a large population of NK cells also were present. Subsequently, peripheral blood lymphocytes were stimulated with the polyclonal T-cell activator, phytohemagglutinin, and cultured in the presence of activin, inhibin, or transforming growth factor beta (TGFbeta) prior to flow cytometric analysis. Activin and TGFbeta suppressed T-cell proliferation without any selective effect on either T-cell subset, and inhibin had no effect. The predominance of CD8+ T cells and NK cells, and the relatively minor proportion of CD4+ T cells, are consistent with both increased cellular immune surveillance and a reduced capacity for initiating antigen-specific immune responses in the adult rat testis.     

CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.     

Heat shock cognate protein 71-associated peptides function as an epitope for Toxoplasma gondii-specific CD4+ CTL

HLA-DR-restricted CD4+ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4+ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4+ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4+ CTL.     

Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes

We are developing strategies to use naive T lymphocytes in cancer therapy. For this purpose, we are deriving T cells with specificity of recognition for defined tumor cells. To direct effector lymphocytes toward tumor cells, we have manipulated the recognition specificity of naive rat and mouse T lymphocytes and a mouse T-cell line. The cells were stably transduced with a chimeric T-cell receptor (TCR) component. The zeta chain of the TCR consists of a single transmembrane protein with a short extracellular domain and an intracellular domain for TCR signaling. We provided an extracellular tumor cell recognition domain to the zeta chain. Human heregulin beta1 (ligand to the erbB-3 and erbB-4 receptors) and three different single-chain antibodies specific for the human and rat Neu/erbB-2 receptors were used. One single-chain antibody (C11) is directed against the rat Neu protein, and one single-chain antibody (FRP5) is directed against the human erbB-2 receptor. The single-chain antibody (R-AK) directed against the Mr 14,000 fusion protein of orthopox viruses served as a control. An efficient procedure was devised to introduce the chimeric genes into primary rat and mouse T lymphocytes. Retrovirus-producing packaging cell lines were cocultured with the T cells activated by phytohemagglutinin and interleukin 2. T-cell lines were transduced by exposure to retrovirus-containing supernatants from helper cell lines. Expression of the fusion genes was determined by fluorescence-activated cell sorting analysis. More than 80% of the naive rat and mouse T cells and 85-100% of the cells from the established T-cell lines expressed the fusion genes within 48 h after infection. The expression of the fusion genes was maintained for at least 10 days after infection. Target cells expressing Neu/erbB-2, erbB-3, or erbB-4 were lysed in vitro with high specificity by T cells expressing the corresponding recognition proteins. No selection of a marker gene is necessary to confer a predetermined recognition specificity. The described experiments are important for a gene therapy approach to cancer treatment with autologous T cells.     

Idiotype immunization combined with granulocyte-macrophage colony-stimulating factor in myeloma patients induced type I, major histocompatibility complex-restricted, CD8- and CD4-specific T-cell responses

Idiotypic structures expressed on the myeloma Ig protein might be regarded as a tumor-specific antigen. Five patients with IgG myeloma were immunized with the purified serum M-component by repeated intradermal injections together with soluble granulocyte-macrophage colony-stimulating factor (GM-CSF). All patients developed an idiotype (Id)-specific T-cell immunity, defined as blood T cells predominantly secreting interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) (type I cells). Id-specific DNA synthesis was induced in one patient. Delayed-type hypersensitivity against the Id was not evoked. The specific IFN-gamma/IL-2 T-cell response was inhibited (46% to 100%) by a major histocompatibility complex (MHC) class I monoclonal antibody (MoAb) in all five patients. A 5% to 37% inhibition by an MHC class II MoAb was seen in four patients. CD4+ as well as CD8+ T cells enriched by magnetic microbeads contained Id-specific cells. The T cells recognized peptides corresponding to the complementarity-determining regions 1, 2, and 3 of the heavy chain of the Id. There was a transient rise of B cells producing IgM anti-idiotypic antibodies in all patients. The results indicate that immunization of myeloma patients using the autologous M-component and soluble GM-CSF may evoke an Id-specific predominantly MHC class I-restricted type I T-cell response.     

Clonal expansion of Vgamma3/Vdelta3-expressing gammadelta T cells in an HIV-1/2-negative patient with CD4 T-cell deficiency

Immunological investigations were carried out in an HIV-1/2//HTLV-1-negative patient with CD4 T-cell deficiency (0.357-0.6 x 10(9)/l) and expansion of gammadelta T cells which accounted for 26-42% of peripheral blood lymphocytes during an observation period of 3 years. Flow cytometry analyses with a panel of available Vgamma/Vdelta-specific monoclonal antibodies indicated that the pathologically expanded gammadelta population expressed Vgamma2 or Vgamma3 paired with Vdelta3 on the surface but lacked the expression of activation antigens such as CD38 or CD71. Cloning and sequencing of RT-PCR products obtained after amplification of cDNA with Vgamma-Cgamma and Vdelta-Cdelta specific primers confirmed the presence of a clonally expanded Vgamma3/Vdelta3 population in the peripheral blood of this patient. Cytotoxicity assays performed with purified gammadelta T cells as effectors and resting or preactivated autologous CD4 T cells as targets failed to reveal evidence for autoreactive cytotoxicity of Vgamma3/Vdelta3 cells as a possible mechanism of CD4 T-cell deficiency in this patient.     

Simultaneous cross-linking of CD6 and CD28 induces cell proliferation in resting T cells

In the present study, we showed that simultaneous ligation of the monoclonal antibodies (mAb) against CD6 and CD28 induces T-cell proliferation in purified resting T lymphocytes in the absence of T-cell receptor (TCR) occupancy. No cell proliferation was observed when the mAb were cross-linked alone or used simultaneously in the soluble form. T-cell proliferation mediated through CD6/CD28 is accompanied by the up-regulation of interleukin-2 (IL-2) mRNA and expression of IL-2 receptors on the cell surface. In the presence of IL-2-neutralizing mAb the proliferative response of the T cell induced through CD6/CD28 was inhibited dose dependently. Cross-linking mAb to CD6 and CD28 alone or together did not down-regulate the CD3/TCR complex. T-cell proliferation mediated through CD6/CD28 was only partially blocked by the immunosuppressive drug, cyclosporin A (CsA), whereas anti-CD28-induced T-cell proliferation in the presence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was unaffected. In sharp contrast T-cell proliferation mediated by anti-CD6 in the presence of TPA was efficiently blocked by CsA. In addition, two protein kinase C (PKC) inhibitors, GF 109203X and H-7 dose-dependently inhibited T-cell proliferation mediated through CD6/CD28, suggesting that PKC activation may be involved. Furthermore, there was a marked differential dose-dependent inhibitory effect of the PKC inhibitors on T-cell proliferation mediated by the co-ligation of anti-CD6 or anti-CD28 in the presence of anti-CD3, with the former being more sensitive to PKC inhibition. Taken collectively, our results suggest that T-cell activation can occur through an antigen-independent pathway by cross-linking the accessory molecules, CD6 and CD28, and that these two cell surface antigens may have distinct signalling pathways.