Abstract: OX40 ligand (OX40L), a member of the TNF family, was shown to be capable of signaling both the cells on which it is expressed and those expressing OX40, its cognate receptor. Here we show that OX40L is expressed on dendritic cells (DC), the most efficient APC to prime naive T cells. The expression and the functional activity of OX40L were examined by means of mAbs used to stain or cross-link OX40L on 1) freshly isolated human blood DC (bDC) and 2) monocyte-derived DC at different stages of differentiation. These were derived from monocytes cultured either with IL-4 and granulocyte-macrophage CSF (IL-4-Mo-DC) or with IL-4 and granulocyte-macrophage CSF plus TNF-alpha. Both types of Mo-DC expressed OX40L after stimulation through CD40; ligation of OX40L on activated IL-4-Mo-DC enhanced by 4- to 35-fold their cytokine production (TNF-alpha, IL-12 p40, IL-1 beta, and IL-6) and increased CD80, CD86, CD54, and CD40 expression. Stimulation of activated IL-4-Mo-DC through OX40L strikingly enhanced their maturation as evidenced by CD83 up-regulation, CD115 (CSF-1R) down-regulation, and typical morphologic changes. OX40L was constitutively expressed on a subset of bDC, and its ligation slightly enhanced CD40L-stimulated IL-12 production. OX40L was down-regulated after overnight culture and spontaneously reexpressed on a subset of mature bDC (CD83high, CD33high, CD11chigh, CD5+). Thus, the expression of OX40L on DC suggests a physiologic role of this molecule during T cell priming by virtue of its ability to costimulate both T cell and DC activation and differentiation.

Abstract: Nucleic acid immunization is a novel vaccination technique to induce antigen-specific immune responses. We have developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules that play an important role in the induction of T cell-mediated immune responses. Coimmunization of these expression plasmids, along with plasmid DNA encoding for HIV-1 antigens, did not result in any significant change in the humoral response; however, we observed a dramatic increase in cytotoxic T-lymphocyte (CTL) induction as well as T-helper cell proliferation after the coadministration of CD86 genes, in contrast, coimmunization with a CD80 expression cassette resulted in a minor, but positive increase in T-helper cell or CTL responses. This strategy may be of value for the generation of rationally designed vaccines and immune therapeutics.

Abstract: T cell activation is brought about by recognition of peptide/MHC complexes on an antigen-presenting cell (APC) by the T cell receptor (TCR). However, in general this appears to be insufficient for the full development of T cell responses and therefore additional signals are required, provided by ligation of counter-receptors on the T cell by APC accessory molecules. Although many studies have suggested that B7 molecules (CD80/CD86) binding to CD28 induce this second signal, it is now evident that any one of a number of molecules may provide accessory function and that efficient response is only generated following multiple interactions. It has also become clear that T cells exist in varying states of activation or differentiation, and that requirements for accessory molecules and costimuli are not always equivalent. This review covers much of the recent data regarding accessory molecule regulation of T cell responses. A modified version of the two signal model is presented, suggesting that the major function of accessory molecules during the initial stages of activation is to augment the ability to signal through the TCR, and that the primary role of costimulatory signals is to allow IL-2 secretion and growth. The requirement for multiple accessory molecules interactions is discussed in relation to activation of naive T cells and how such interactions are less critical at the memory and effector stages. Finally, this new information is related to how T cells interact with varying APC and how these interactions may modulate T cell response.

Abstract: Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunately, also very toxic. Here we present a powerful new approach to separate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells. The CTA1 was genetically linked at its C-terminal end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli. The highly purified, monomeric CTA1-DD fusion protein, with a molecular mass of 37 kDa, was found to exhibit strong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses--IgE, IgA, and IgM. After i.v. injection of the fusion protein, FACS analysis revealed binding of CTA1-DD to splenic IgM+ B cells, but not CD3+ T cells, indicating cell-specific targeting in vivo. Strikingly, we found that the adjuvant ability of CTA1-DD to enhance systemic IgG as well as mucosal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocyanin, administered i.v or intranasally, was comparable to that of intact CT. In addition, the enhancing effect on specific IgG1, IgG2a, and IgG2b responses mimicked that of CT and suggested involvement of both Th1 and Th2 CD4+ T cell activity. The CTA1-DD, as well as CT, up-regulated expression of the CD80 and CD86 molecules on the targeted B cells, indicating that enhanced T cell costimulation may be responsible for the adjuvant effect. Contrary to CT, however, CTA1-DD was completely nontoxic. Thus, the CTA1-DD adjuvant should find general applicability in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation.

Abstract: Immune surveillance of skin cancer involves the stimulation of effector T cells by tumor-derived antigens and antigen-presenting cells (APCs). An effective APC must not only display processed antigen in the context of MHC molecules but also express co-stimulatory molecules that are required to fully activate T cells. One of the most common cutaneous neoplasms is basal cell carcinoma. To investigate expression of the co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) on tumor-associated dendritic cells (TADCs), cryosections from basal cell carcinomas were immunostained. In basal cell carcinomas, only 1 to 2% of intratumor and 5 to 10% of peritumor APCs expressed CD80 or CD86. In contrast, biopsies of immunological/inflammatory dermatoses revealed that 38 to 73% of APCs expressed CD80 and CD86. To further evaluate their phenotype and function, TADCs were isolated from tissue samples of basal cell carcinomas; they were non-adherent to plastic, displayed a typical dendritic morphology, and expressed high levels of major histocompatibility class II molecules on their surface. When TADCs were compared with dendritic cells from blood for presentation of superantigens (staphylococcal enterotoxins A and B) to resting autologous T cells, TADCs were consistently weaker stimulators of T cell proliferation than blood dendritic cells. When analyzed by flow cytometry, TADCs expressed high levels of HLA-DR, but only 5 to 10% co-expressed CD80 or CD86. A 3-day culture in granulocyte/macrophage colony-stimulating factor-containing medium partially reconstituted the TADC expression of CD80 and CD86 as well as their immunostimulatory capacity. Thus, in this common skin cancer, although there are prominent collections of HLA-DR-positive APCs in and around tumor cells, the TADCs are deficient in important co-stimulatory molecules as well as being weak stimulators of T cell proliferation. The paucity of co-stimulatory molecule expression and functional activity of TADCs may explain why the local T lymphocytic infiltrate fails to become fully activated to eradicate adjacent tumor cells. From a clinical perspective, these findings suggest a novel immunotherapeutic strategy targeting T cell co-stimulatory molecules on professional APCs in cutaneous oncology.

Abstract: alpha-Melanocyte-stimulating hormone (alpha-MSH) is released by immunocompetent cells as well as the pituitary gland and functions as a potent inhibitor of immune and inflammatory reactions Therefore, it was investigated whether normal human monocytes express melanocortin (MC) receptors specific for alpha-MSH Upon FACS analysis using biotin-labeled alpha-MSH, a low number of alpha-MSH binding sites was detectable on unstimulated monocytes alpha-MSH receptor expression was up-regulated when monocytes were treated with endotoxin (LPS) or mitogen (PHA) for 3 to 5 days and was further augmented by the addition of cytokines such as IL-2, IFN-gamma, IL-4, and IL-10 Adrenocorticotropin, a precursor of alpha-MSH, but not the structurally unrelated beta-MSH, competitively inhibited alpha-MSH binding, suggesting that the receptor expressed on monocytes is specific for alpha-MSH This was further confirmed by reverse transcription-PCR, which demonstrated that monocytes express mRNA specific for the MC receptor MC-1, which binds alpha-MSH and adrenocorticotropin, whereas mRNA specific for other known melanocortin receptors was not detectable To investigate whether the immunosuppressing capacity of alpha-MSH is associated with the up-regulation of MC-1, its effect on the expression of costimulatory molecules (CD86 and CD80) on human monocytes was investigated alpha-MSH significantly inhibited the expression of CD86 on LPS-treated monocytes, which exhibited a high density of MC-1, whereas CD80 expression was not altered These findings indicate that human monocytes, depending on their activation and maturation state, are able to express MC-1, and up-regulation of MC-1 seems to be required to enable alpha-MSH to modulate immune responses in which costimulatory molecules play a decisive role

Abstract: Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.

Abstract: We compared the immunological functions of interferon-gamma (IFN-gamma)-induced, classically activated macrophages (caM phi) and of interleukin-4 (IL-4)- and glucocorticoid-induced, alternatively activated macrophages (aaM phi) in a human co-culture system in vitro. Proliferation of peripheral blood leucocytes (PBL) or CD4+ T cells mediated by optimal doses of phytohaemagglutinin (PHA) or concanavalin A (Con A) was only marginally influenced by caM phi, but was strongly inhibited by aaM phi. The degree of lymphocyte proliferation sustained in the presence of caM phi was gradually reduced in a dose-dependent fashion by the addition of aaM phi. Flow cytometric analysis revealed that expression of costimulatory molecules such as CD11a, CD40, CD54, CD58, CD80 and CD86 did not vary significantly between caM phi and aaM phi and was low for CD58, CD80 and CD86. As shown by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, IL-10 was expressed in caM phi, aaM phi and control macrophages; the level of expression of IL-10 was slightly enhanced in aaM phi. Neither neutralizing anti-IL-10 antibodies, indomethacin nor NG-monomethyl-L-arginine (NMMLA) was able to reverse aaM phi-mediated inhibition of lymphocyte proliferation. Of several agents interfering with various second messenger pathways, cAMP and the Ca(2+)-ionophore A23187 inhibited differentiation of cultured human monocytes into phenotypically mature aaM phi expressing MS-1 high molecular weight protein (MS-1-HMWP) and RM 3/1 antigen, and prevented the suppressive action of aaM phi on lymphocyte proliferation. In conclusion, these results who that aaM phi actively inhibit mitogen-mediated proliferation of PBL and CD4+ T cells independently of the expression of costimulatory molecules and of IL-10, NO or prostaglandin synthesis, and that inhibition of phenotypic differentiation of aaM phi is paralleled by a lack of functional maturation. Thus, fully matured aaM phi may be functional in down-regulating CD4+ T-cell-mediated immune reactions by an as yet unknown mechanism.

Abstract: Treatment of mice with the recently cloned hemopoietic growth factor Flt3 ligand (FL; 10 microg/day for 10 days) resulted in a large increase in myeloid lineage cells within the liver. While the number of nonparenchymal cells (NPC) harvested from liver increased about 9-fold, a 90-fold increase was observed in the proportion of CD11c+ dendritic cells (DC) recovered from NPC following overnight (18-h) culture in granulocyte-macrophage CSF. In contrast, only a 50% increase was seen in CD11c+ cells within heart single cell suspensions and in the number of DC obtained from hearts after 18-h culture. Liver NPC and heart cell suspensions freshly isolated from 10-day FL-treated animals exhibited increased T cell allostimulatory capacity compared with controls. Overnight cultured DC from livers of FL-treated animals expressed both higher levels of costimulatory molecules (CD80 and CD86) and allostimulatory activity than those from controls. Heart-derived DC also displayed enhanced stimulatory capacity. Pretreatment of organ donors with FL for either 5 or 10 days before transplant of organs to normal recipients abrogated the spontaneous liver allograft acceptance normally observed and resulted in delayed or acute graft rejection (median survival times, 40 and 12 days, respectively). Heart rejection was significantly accelerated by pretreatment of donors with FL for 5 or 10 days (median survival times, 8 and 7 days, respectively, vs 12 days in controls). These novel findings reveal the potent immunologic adjuvant properties of FL in vivo. They also show that substantial augmentation of the number of potential allostimulatory cells in donor organs before transplantation favors rejection rather than tolerance induction.

Abstract: Bacillus Calmette-Guerin (BCG) mycobacteria have been used as adjuvant in the active immunotherapy of various human cancers. In addition, dendritic cells, which are the most potent antigen-presenting cells, have been shown to be capable of initiating anti-tumor immune responses. Here we investigated the effects of BCG on dendritic cells cultured from human blood. Addition of BCG resulted in rapid homotypic adhesion of dendritic cells. Moreover, BCG concentrations ranging from 10(4) to 10(6) bacteria/ml enhanced expression of the dendritic-cell-maturation antigen CD83 and of the T-cell co-stimulator CD86 (B7-2) in a dose-dependent manner. Concomitant with the increase of CD83 and CD86 expression, the cells lost the ability to capture soluble antigens, as determined by the exclusion of fluoresceinated Dextran molecules. Strikingly, the same dosages of BCG-bacteria stimulated TNF-alpha-gene transcription and TNF-alpha-protein release from dendritic cells in a dose-dependent fashion. BCG infection of dendritic cells in the presence of a neutralizing antibody directed against TNF-alpha inhibited CD83 expression by more than 50% indicating that the BCG-induced maturation of dendritic cells was at least partially mediated by dendritic-cell-derived TNF-alpha. The finding that BCG activates the most potent antigen-presenting cells reveals a plausible immunological mechanism of the occasionally observed anti-tumor activity of BCG.

Abstract: The molecules B7.1 and B7.2 deliver costimulatory signals of critical importance to naive T cells, and may thus be involved in abrogation of oral tolerance in IBD. Functional disparity apparently exists among antigen-presenting cells in vivo. We wanted to examine if differential B7 expression occurs on mucosal macrophage subsets. Cryosections of bowel specimens from patients with IBD and normal controls were subjected to immunofluorescence and immunoperoxidase staining. In normal mucosa, selective subepithelial accumulation of B7.2+ cells was found. In inflamed IBD mucosa, however, subsets appeared consisting of both B7.2(hi) and B7.1(hi) cells as well as CD14(hi) macrophages. Notably, outside lymphoid aggregates the prominent fraction of recently recruited CD14(hi) macrophages comprised most (approximately 80%) of the B7.1(hi) cells, whereas most (approximately 70%) B7.2(hi) cells were identified as resident mucosal macrophages (CD14(lo) or CD14-). Differential expression of B7.1 and B7.2 on two functionally different subsets of intestinal macrophages implies separate immunoregulatory roles for the two molecules. This finding is in keeping with recent experimental data demonstrating that monocyte-derived cells are crucial for immune responses at mucosal surfaces. Preferential B7.1 up-regulation might be critical in breaking the immunological tolerance to luminal antigens in IBD, but it cannot be excluded that it is a secondary pathogenic event.

Abstract: Several costimulatory molecules play a key role in the differentiation of B lymphocytes and in T-B-cell interactions. In this study, we addressed the question of whether different receptors and counter-receptors may be expressed on malignant B lymphocytes from chronic B-cell malignancies. Using flow cytometry and reverse transcription PCR analyses, the expression of molecules belonging to the tumor necrosis factor receptor (TNFR) and tumor necrosis factor ligand (TNFL) families, as well as the expression of CD80 and CD86 molecules, was analyzed in normal B cells and in different chronic lymphoproliferative disorders of B-cell type, including B-cell chronic lymphocytic leukemia (CLL), mantle cell lymphoma, hairy cell leukemia (HCL), and HCL variant. Different patterns of expression of TNFR and TNFL superfamily molecules were demonstrated among B-cell malignancies. In particular, CD40 was commonly observed on all B cells (both tumor and normal), whereas its ligand (CD40L), which is usually undetectable on resting normal B lymphocytes, was expressed in CLL and HCL but not in other chronic lymphoproliferative disorders. CD27 was not shown in normal B cells, although it was present in all malignancies and with particularly high density in mantle cell lymphoma. CD70 was widely distributed on tumor B lymphocytes, but not on the CD5+ normal counterpart. CD30 was strongly expressed in HCL variant and weakly in B-cell CLL, whereas its ligand showed a wide pattern of expression, including all neoplastic and normal B cells. TNFR II (CD120b) and CD80 were distributed on neoplastic B cells from all groups, usually at an intermediate to high degree of intensity, whereas the CD86 molecule was present at lower intensity than CD80. Finally, reverse transcription PCR analysis confirmed the presence of CD40L, CD30, and CD30L mRNAs in those B cells expressing the corresponding membrane-bound proteins at low density. Our data indicate that TNFR and TNFL molecules are of use clinically both in differentiating B-cell malignancies from the normal counterpart (i.e., CD27, CD70, CD40L, CD30, and CD80) and in defining different chronic B-cell disorders (i.e., CD40L, CD27, and CD30). Interestingly, the observation that several receptors and their ligands (i.e., CD40/CD40L, CD30/CD30L, and CD27/CD70) can be expressed on the same cell suggests that these molecules play a role in initiating and maintaining the neoplastic process by mediating B-T and B-B interactions.

Abstract: Dendritic cells with potent Ag-presenting function can be propagated from peripheral blood using recombinant cytokines, and these cells have potential usefulness as immunotherapeutic agents in the treatment of cancer and other disease states. However, it is not known if these in vitro differentiated dendritic cells have the capacity to migrate in vivo, especially to T cell areas of lymphoid tissue. We have used a fluorescent marker system to track the migration of dendritic cells, propagated in vitro from chimpanzee peripheral blood, following s.c. injection. We report that injected dendritic cells migrate spontaneously and rapidly to draining lymph nodes, where they remain for at least 5 days. The injected cells interdigitate with T cells in the parafollicular and paracortical zones and retain high level expression of CD86, CD40, and MHC class II molecules, reflecting a phenotype of potent APC. We conclude that dendritic cells differentiated in vitro from peripheral blood and administered s.c. behave in a manner very similar to endogenous Langerhans cells. These data provide strong experimental support, in a highly relevant large animal model, for the use of in vitro differentiated dendritic cells as vehicles for immunotherapy. More importantly, they show that the s.c. route of injection delivers these APC to sites of T cell activation, a prerequisite for the generation of an effective immune response.

Abstract: Human interdigitating dendritic cells (IDC) were isolated from tonsils based on their CD40+ lineage-negative expression in situ. Isolated IDC displayed a phenotypic profile similar to that of IDC in tonsils and spleen in situ, characterized by high-level expression of major histocompatibility complex class II, the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), expression of the late DC maturation marker CD83, and no expression of CD1a, CD13, or CD33. IDC also showed weak nonspecific esterase staining and had the ability to induce an allogeneic mixed lymphocyte reaction. In this study, we further show that in the presence of surrogate activated T cells in the form of CD40 ligation and IL-2, IDC enhance the proliferation of naive B cells and induce their differentiation into plasma cells producing IgM. Evidence for the anatomical co-localization of naive B cells and IDC in the T cell area together with the data obtained in vitro implies a role for IDC in the initiation of the extrafollicular reaction.

Abstract: The B7 family of costimulatory molecules provides the second signal necessary for activation of T cells In the absence of the second signal, responding T cells become anergic Although predominantly expressed on professional APCs, recent evidence shows that the B7 molecules are also expressed on T cells To study the functions of B7 molecules on T cells, we transfected murine B71 (CD80) and B72 (CD86) cDNAs into the EL4 T cell thymoma cell line and examined the transfectants for their ability to costimulate T cell proliferation in vitro and to induce antitumor immunity in vivo Here we show that although EL4-B71 cells costimulate T cells and induce tumor regression, EL4-B72 transfectants failed to costimulate T cell proliferation or induce tumor regression To understand the cellular basis for this difference, we examined the binding of EL4-B71 and EL4-B72 to CTLA4 and CD28 Whereas EL4-B71 cells bound both CTLA4-Ig and CD28-Ig, EL4-B72 transfectants preferentially bound CTLA4-Ig, but not CD28-Ig Similar binding data were obtained with freshly isolated murine T cells, which have been shown to constitutively express B72 Our data suggest, therefore, that B72 expressed on T cells may not costimulate but instead inhibit the T cell response by preferential binding to CTLA4

Abstract: T cells require at least two signals for activation and clonal expansion. The first signal conferring specificity is initiated by interaction of the T cell receptor with antigenic peptides in the context of MHC molecules. The second, costimulatory signal can be provided by cell surface molecules on APCs such as B7-1 (CD80) and B7-2 (CD86), which interact with their counter-receptors on T cells. The absence of costimulatory signals presents one possible mechanism for tumor cells to escape immune surveillance. In experimental models transfection of B7 genes into tumor cells can result in T cell-dependent tumor rejection. We have developed a novel approach to direct the costimulatory B7-2 molecule to the surface of target cells. Our approach is based on a chimeric fusion protein that consists of the extracellular domain of human B7-2 fused to a single-chain Ab domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas. This B7-2(225)-scFv(FRP5) molecule expressed in the yeast Pichia pastoris and purified from culture supernatants is functionally active and binds to B7 counter-receptors and to ErbB2. B7-2(225)-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal that results in enhanced proliferation of syngeneic T cells. Our results suggest that effective tumor vaccines for cancer immunotherapy could be created by targeting such chimeric ligands to the surface of tumor cells.

Abstract: The study of liver dendritic cells (DC) and their progenitors is restricted by the small numbers that can be isolated or propagated from normal hepatic tissue. We examined the ex vivo growth, phenotype, and function of these cells after the administration to mice of the recently cloned hemopoietic growth factor flt3 ligand (FL), which is highly effective in mobilizing stem/progenitor cells. FL treatment (10 microg/day for 10 days) resulted in a mean 14-fold increase in the absolute number of nonparenchymal cells recovered from collagenase-digested livers compared with the control value. Culture of these nonparenchymal cells in granulocyte-macrophage CSF (GM-CSF; 1000 U/ml) resulted in the early formation of proliferating cell clusters and maximal release (within 4-5 days) of markedly increased numbers of nonadherent, low buoyant density cells per liver. Maximal release of low buoyant density cells propagated from control livers was at the later time of 6 to 8 days. Cells from both sources were DEC-205+, CD11c+, MHC class II+, CD80(low) (i.e., low level of CD80), CD86(low) and CD40(low). This immature phenotype was linked to poor T cell allostimulatory activity, indicative of DC progenitors. Propagation of cells from livers of FL-treated mice in GM-CSF and IL-4 resulted in a more mature DC phenotype and function. Maturational changes were also observed following exposure of the GM-CSF-stimulated progenitors to type 1 collagen for 3 additional days. The ability of FL to boost production of large numbers of liver DC progenitors provides opportunities for the further study of these important APC in normal liver immunobiology and in immune-mediated hepatic disorders.

Abstract: Optimal T cell activation requires signals delivered via both the TCR and the costimulatory receptors. Considerable experimental data now suggest that this costimulatory signal is generated predominantly by CD28 when engaged by its ligands CD80 (B7-1) and/or CD86 (B7-2). Whether T cell activation is controlled in part by regulated CD80 and/or CD86 expression has been incompletely explored. Here, we report that CD80 can be expressed constitutively by murine fibroblasts and up-regulated after treatment with IFN-gamma plus TNF-alpha. CD80 expression and function was confirmed by 1) Northern analysis, 2) specific immunoprecipitation of a approximately 69-kDa surface protein that comigrated with CD80 precipitated from CD80-transfected CHO cells, and 3) two independent assays for costimulation of Ag-specific T cell activation. Taken together, these observations suggest that CD28/CTLA-4 ligands are expressed on a wider variety of tissues than previously suspected and that their expression is dynamically regulated. Consequently, these results might explain previous observations that inflammatory cytokines can result in autoimmune responses.

Abstract: T cell anergy refers to a functional state in which the cells are alive but unable to produce IL-2 after appropriate triggering. Lack of CD28 costimulation through CD80 and CD86 molecules on APC might play a causative role in anergy induction, as previously shown with T cell clones. We now developed a model of anergy induction in cultures of freshly isolated memory T cells. Addition of either CTLA-4Ig or blocking anti-CD80 and anti-CD86 mAbs, in combination with cyclosporin A, to cultures of PBMC with soluble Ag consistently resulted in Ag-specific unresponsiveness, as evidenced upon antigenic rechallenge. In most experiments, the presence of cyclosporin A was not required, and blocking the B7-CD28 interaction during antigenic stimulation was sufficient to induce unresponsiveness. Unresponsiveness was apparent at the level of T cell proliferation as well as at the level of IL-2 and IFN-gamma production, and T cell responses to unrelated Ags were intact. Induction of unresponsiveness correlated with lack of T cell proliferation in the induction culture and could largely be prevented by supplementing the induction cultures with rIL-2, indicating that lack of IL-2 was responsible for this altered functional state. Unresponsive T cells did not suppress the proliferation of autologous T cells in response to original or third-party Ags. On the other hand, culture with IL-2 and Ag could reverse established T cell unresponsiveness, pointing to anergy rather than deletion as the underlying mechanism. Anergy induction in freshly isolated memory T cells opens perspectives for treatment of autoimmune and allergic diseases.

Abstract: Previous studies have shown that HIV-1 exploits dendritic cells (DCs) to replicate and spread among CD4+ T cells. The DCs within mucosal surfaces may be especially important, but these are more difficult to access. To study more extensively the properties of DCs and other leukocytes from skin and different mucosae, DCs were isolated from uninfected macaques and their sensitivity assessed to infection with SIV in vitro. Dendritic cells and T cells readily emigrated from organ cultures of macaque skin, as described previously for humans. In addition, characteristic cells emigrated from expiants of mucosae, both nasopharyngeal (adenoid and tonsil) and genital (vagina and cervix). The macaque DCs reacted with the monoclonals that are used to study human DCs, such as MAbs to CD40, CD86, CD83, and the p55 protein. When SIV was added to the DC-T cell mixtures from these different organs, extensive replication was observed in all but the cervical leukocytes. SIV replication occurred without the use mitogens, and ...

Abstract: CD80 (B7-1) and CD86 (B7-2) on APC provide a major costimulatory signal through interactions with CD28 on T cells. Absent from resting human T cells, CD86 is up-regulated early upon T cell activation, whereas CD80 expression appears later. Whereas T cell expression of CD80 has been implicated in costimulation, the functional significance of CD86 expression on T cells is unclear. We now demonstrate that CD86 expressed on human CD4+ T cell clones does not provide a costimulatory signal for other CD4+ T cell clones. Binding studies using CD28-Ig and CTLA-4-Ig fusion proteins demonstrate that CD86 expressed on T cells has significantly reduced binding affinity for CTLA-4 and no detectable binding to CD28. Biochemical analysis demonstrates that post-translational modifications of CD86 in human T cells are different from those of CD86-transfected Chinese hamster ovary cells or EBV-transformed B cells, in that T cells express a hypoglycosylated form of CD86 on the surface membrane. Thus, our results suggest that while CD86 is expressed on a number of different cell types, its costimulatory function and affinity for its ligands may be regulated by cell type-specific post-translational modifications.

Abstract: Dendritic cells (DC) are potent APC that, in mature form, can be distinguished from other mononuclear cells on the basis of their distinct morphology, absence of lineage markers, and dense expression of MHC and costimulatory molecules. While comparing different DC preparation methods, we observed that DC derived from cultured PBMC that had been depleted of CD2+ cells before culture were functionally distinct from DC derived from PBMC that had not been depleted of CD2+ cells. Thus, both types of DC stimulated allogeneic T cells to proliferate in the MLR, but only DC derived from CD2+ precursors could sensitize naive T cells to soluble Ags such as keyhole limpet hemocyanin and HIV gp160 glycoprotein. Subsequent studies confirmed the existence of CD2+ and CD2- DC precursor populations among HLA-DRbright, lineage-negative PBMC. Immediately after their isolation, these populations were morphologically similar to one another by light and electron microscopy, and neither had substantial Ag-presenting activity. After culture for 24 to 48 h with supernatant from PHA-activated PBMC, both populations developed dendrites, formed clusters with T cells, and stimulated allogeneic T cell responses in the MLR as well as autologous T cell responses to tetanus toxoid, a recall Ag. However, CD2+ DC precursors alone gave rise to APC that presented soluble Ags to naive CD4+ T cells, a property that could be inhibited by Abs to CD4, CD11a, and CD28 on T cells or CD86 on DC. The expression of CD54 and CD86 on CD2+ DC precursors was increased markedly after their culture and differentiation, while the expression of these molecules on CD2- DC precursors was not remarkably changed. These findings reveal the existence of two functionally distinct populations of DC, each derived from a phenotypically distinct precursor present in monocyte-depleted peripheral blood.

Abstract: The origin and function of decidual stromal cells (DSC), the main cellular component of the decidua, are uncertain. Although the general consensus is that they are fibroblastic cells involved in fetal trophoblast nutrition, several authors have demonstrated that these cells can carry out immunological functions and that at least a subpopulation of them may be of hematopoietic origin. Human DSC precursors or predecidual cells (preDSC) purified by expansion in culture express a surface phenotype recalling that of dendritic cells. In the present study, we show by flow cytometry that these cells also express B7-1 (CD80) and B7-2 (CD86), two antigens involved in the costimulation of T cells by antigen-presenting cells. Cultured DSC were also able to stimulate allogenic T cells in vitro. Using an immunohistochemical technique, we found that in cryostatic sections of early human decidua, CD80 and CD86 were expressed mainly by DSC located around the decidual vessels, a location compatible with preDSC rather than fully differentiated DSC. Our results suggest that preDSC are involved in antigen presentation.