Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro.     

Discordant expression of CD28 ligands, BB-1, and B7 on keratinocytes in vitro and psoriatic cells in vivo.

The process for optimal T-cell activation requires not only engagement of the T-cell receptor/CD3 complex, but also the delivery of additional co-stimulatory signals that synergize with the primary response mediated through the T-cell receptor. Thus, the regulated expression of ligands for such co-stimulatory molecules can be critical in determining whether a cell can effectively activate T cells following the presentation of a foreign antigen. The CD28 antigen has recently been shown to mediate such co-stimulatory signals by interacting with the B7/BB-1 molecule expressed on activated B cells and monocytes. We show in this study that activated keratinocytes, both in vitro and in vivo display a discordance in expression between B7 and BB-1 based on differential monoclonal antibody (MAb) reactivity. Activated keratinocytes in vitro, as well as psoriatic keratinocytes and epithelial cells in the thymus, are reactive with the BB-1 MAb but not anti-B7 MAbs. These BB-1 positive cells fail to express detectable B7 messenger RNA by Northern blot analysis. Furthermore, keratinocytes bind specifically to CD28-transfected COS7 cells, and this binding is inhibited by anti-CD28 and anti-BB-1 but not B7 MAbs. These studies suggest: 1) that the MAb against BB-1 binds a functional epitope on a molecule distinct from B7 as detected on activated keratinocytes in vitro and in vivo and 2) that keratinocytes in skin and epithelial cells in thymus can express cell-surface molecules that might mediate T-cell co-stimulation via CD28.     

The immune response induced in vivo by dendritic cells is dependent on B7-1 or B7-2, but the inhibition of both signals does not lead to tolerance

Dendritic cells (DC) can be used as physiological adjuvant in vivo. Indeed, a single injection of DC, pulsed in vitro with antigen, induces activation of specific T and B lymphocytes in syngeneic mice. The unique capacity of DC to sensitize naive T lymphocytes correlates with elevated expression of MHC antigens as well as co-stimulatory molecules. The aim of this work was to evaluate the functional role of the individual CD28 ligands in the induction of primary humoral and cellular responses, and to characterize the nature of the immune response induced in the presence of selected co-stimulatory molecules. Our data show that the primary response is strictly B7 dependent, and that B7-1 and B7-2 mediate overlapping co-stimulatory functions, as either molecule alone is sufficient to initiate an immune reaction. Inhibition of B7-1 and B7-2, however, does not lead to tolerance as predicted by the two-signal hypothesis. Rather, recognition of antigen in the absence of B7 appears as a null event, since subsequent immunogenic stimulation results in a primary response. Blockade of B7-2 co-stimulatory molecules significantly inhibits antigen-specific IgG1 but not IgG2a production, suggesting that B7-2 may direct the development of Th2 cells. These data emphasize the critical role of the CD28/B7 pathway in the induction of the immune response by DC, which appear to be the initiating antigen-presenting cells in situ.      

B7/BB-1 antigen expression on adult human microglia studied in vitro and in situ

In this study, we have examined the expression and function of B7/BB-1 on individual glial cells, by utilizing surgically resected adult human central nervous system (CNS) tissues, tissues derived from fetal human CNS, and pathology material from cases of multiple sclerosis (MS). Immunofluorescence analysis using enriched adult human derived cultures of microglia and oligodendrocytes, and mixed microglia/astrocyte cultures, demonstrated that B7/BB-1 was expressed on microglia. Adult human-derived oligodendrocytes and astrocytes, and human fetal astrocytes were B7/BB-1 negative under all culture conditions. Flow cytometry studies demonstrated a low basal level of B7/BB-1 expression on microglia that was up-regulated following incubation with interferon-γ (IFN-γ). Co-culture of purified fresh allogeneic CD4⁺ T cells with microglia for 24 h resulted in clustering of T cells around microglia and microglial B7/BB-1 expression. Preincubation of microglia with an anti BB-1 monoclonal antibody (mAb) prior to microglia: CD4⁺ T cell co-cultures resulted in partial inhibition of the ability of microglia both to present recall antigen to autologous CD4⁺ T cells and to present antigen to allogeneic CD4⁺ T cells in primary mixed lymphocyte reaction (1°MLR). The CTLA-4 Ig fusion protein inhibited the ability of microglia to present antigen in both antigen presentation assays to an even greater extent than did the anti BB-1 mAb. The BB-1 antibody also inhibited the ability of microglia to stimulate previously activated T cells in a secondary 2° MLR. In sections of multiple sclerosis brain, B7/BB-1 expression was observed on activated microglia in select parenchymal lesions, and on perivascular cells and infiltrating monocytes. B7/BB-1 immunoreactivity was not found in normal appearing white matter from MS brain or from non-inflammatory brain specimens. Our results indicate that the B7/BB-1 molecule plays a functional role in the capacity of microglia to serve as CNS antigen-presenting cells that can both initiate and perpetuate CD4⁺ T cell activation.     

Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand-dependent pathway

The CD40: CD40 ligand (CD40L) interaction provides T lymphocyte-mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co-stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up-regulated by culture, and its expression was further enhanced by interleukin (IL)-1α, IL-1β, IL-3, tumor necrosis factor-α and granulocyte/macrophage-colony-stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross-linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up-regulation. Nevertheless, the same cross-linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA-4Ig, only minimal and variable additional inhibition of DC-stimulated allogeneic T lymphocyte proliferation and IL-2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86-dependent and -independent components of DC-T lymphocyte CD40: CD40L co-stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co-stimulatory molecules.     

Priming to Mycobacterial Antigen In Vivo Using Antigen-Pulsed Antigen Presenting Cells Generated In Vitro is Influenced by the Dose and Presence of IL-4 in APC Cultures

Antigen presenting cells (APC) similar to immature dendritic cells can be generated in vitro from bone marrow precursors. The authors have compared the yield, the phenotype and the function of murine bone marrow cells cultured for 7 or 11 days in either granulocyte macrophage colony stimulating factor alone (GM BMAPC) or in combination with interleukin-4 (GM/IL-4 BMAPC). The results showed that GM/IL-4 BMAPC expressed the highest levels of MHC Class 2 molecules, CD86 /B7-2 and CD80/B7-1 co-stimulatory molecules and the lowest levels of F4/80 macrophage marker. However, when these APC were pulsed with BCG culture filtrate antigen or PPD they were not correspondingly more effective at stimulating activated T lymphocytes in vitro or priming naive T lymphocytes in vivo . Also, in contrast to GM BMAPC, high backgrounds recorded following injections of GM/IL-4 BMAPC without antigen were not consistently reduced by lowering the dose and irradiating the cells prior to administration. The authors conclude that the degree of maturity of BMAPC varies with culture conditions and that this may be an important consideration where BMAPC are to be used in vivo in immunotherapeutic regimens.     

Expression and functional role of CD23 on T cells

We have found that approximately 10%-15% of tonsil, but not peripheral blood, T cells express the CD23 antigen following activation with 12-O-tetradecanoylphorbol 13-acetate (TPA), phytohemagglutinin (PHA) or recombinant interleukin 4. The proliferative response of tonsil T cells is significantly increased when CD23 monoclonal antibodies (mAb) are present in the cultures. In contrast, no such proliferative augmentation is seen when peripheral blood T cells are cultured in this way. Supernatant (SN) of Epstein-Barr Virus-transformed B lymphoblastoid cell lines (EBVLCL), is found to have a similar co-stimulatory effect on the proliferation of tonsil T cells to that seen with CD23 mAb. This effect is greatly diminished by preclearing SN with CD23 mAb. Similarly, SN from a CD23+ L cell transfectant augments the proliferative response of tonsil T cells to both TPA and PHA. The CD23 molecule expressed by TPA-driven T cell blasts appears identical in size to the 45-kDa glycoprotein present on EBVLCL and activated B cells. In contrast, a 42-kDa molecule is observed when CD23 is precipitated from T cells activated with PHA. The results presented here demonstrate that CD23 is expressed on activated tonsil, but not peripheral blood T cells and plays a role, via the binding of CD23 mAb and CD23+ material, present in EBVLCL and CD23+ transfectant SN, in the regulation of T cell proliferation in response to mitogens such as PHA and TPA.     

A novel pathway of human B cell activation initiated by CK226 surface antigen

In this study we analyzed the effect of CK226 monoclonal antibody (mAb) on human B cell activation and proliferation. This mAb was shown to recognize a 75-kDa surface molecule expressed on both T and B lymphocytes and to mediate T lymphocyte activation and proliferation. Flow cytometry analysis of B cell populations isolated from peripheral blood, tonsil and spleen showed that CK226 surface antigen is highly expressed on 40-80% of surface Ig+ cells. When purified B cells were cultured in the presence of CK226 mAb, up-regulation of major histocompatibility complex class II and CD23 surface structures and the de novo expression of CD25 antigen could be detected within 48 h. In addition, B cells underwent proliferation ([3H] thymidine uptake) in the absence of either T cells or exogenous lymphokines. Proliferation was potentiated by the addition of suboptimal concentrations (0.5 ng/ml) of phorbol 12-myristate 13-acetate (PMA). Cells recovered at day 5 were surface Ig+ and no CD3+ cells could be detected. CK226-induced proliferation (either in the presence or in the absence of PMA) was not inhibited by anti-CD25 mAb. Addition of exogenous interleukin 2 to CK226-stimulated B cells resulted in further increase of B cell proliferation. On the other hand, CK226 mAb did not display a co-stimulatory effect with submitogenic concentrations of either anti-Ig antibody or Staphylococcus aureus Cowan strain I bacteria. In addition proliferation induced by mitogenic concentrations of the above stimuli was inhibited in a dose-dependent fashion by CK226 mAb.     

Expression of a gp33/27,000 MW activation inducer molecule (AIM) on human lymphoid tissues. Induction of cell proliferation on thymocytes and B lymphocytes by anti-AIM antibodies.

We have recently described several monoclonal antibodies (mAb) that recognize a heterodimeric structure (gp33/27,000 MW) expressed on the surface of human peripheral blood T lymphocytes upon activation with different mitogenic stimuli. Such mAb, when used in combination with submitogenic doses of phorbol ester, were capable of triggering T-cell proliferation. The antigen has been designated as activation inducer molecule (AIM). In the present study we have investigated the expression of the AIM in different lymphoid and non-lymphoid tissues. In addition, we have analysed the ability of lymphocyte subsets derived from thymus and tonsil to proliferate in response to anti-AIM mAb. The presence of AIM on subpopulations of lymphoid cells from thymus, tonsil, lymph node and spleen has been demonstrated by immunoprecipitation, flow cytometry and immunoperoxidase staining of tissue sections. By contrast, non-lymphoid cells from tissue such as brain, kidney, liver, lung or skin did not react with anti-AIM mAb. In thymus, the AIM expression was restricted to a subset of CD3+ medullary thymocytes, whereas CD1+ CD3- cortical thymocytes did not express this antigen. Nevertheless, the majority of both purified CD1- and CD3- thymocytes expressed AIM antigen after treatment with PMA. In tonsil and lymph node, a strong staining of a subset of CD3+ T lymphocytes located in the germinal centre was observed by immunohistochemical labelling with anti-AIM mAb. Certain T cells from the paracortical zone and CD19+ B lymphocytes from mantle region were also reactive. Both purified tonsillar T and B lymphocytes strongly expressed AIM after activation with PMA. The anti-AIM mAb was able to induce a strong proliferative response on purified CD1- thymocytes as well as on both purified tonsillar T and B lymphocytes in the presence of submitogenic doses of PMA. By contrast, no proliferative response was induced through the AIM in the CD3- immature thymocyte subset.     

Human sunlight-induced basal-cell-carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells.

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.     

Interleukin-7 is a potent co-stimulus of the adhesion pathway involving CD2 and CD28 molecules.

Co-stimulation of highly purified peripheral T lymphocytes from healthy blood donors with the adhesion molecules CD2 and CD28 in association with recombinant interleukin-7 (rIL-7) induced T-cell proliferation, multiple cytokine secretion and IL-2 receptivity. We demonstrated that rIL-7 is as potent as rIL-2 in inducing the proliferation of unseparated, CD4+ and CD8+ T cells. In contrast to low or undetectable levels of IL-1 alpha, IL-6 and IL-2, high levels of tumour necrosis factor-alpha (TNF-alpha), IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were secreted. Experiments using blocking antibodies suggested a direct mechanism for rIL-7 co-stimulatory effect, although induction of the CD25/IL-2 receptor alpha-chain (CD25/IL-2R alpha) was observed. Monoclonal antibodies (mAb) against the adhesion molecules CD2 and CD28 are likely to mimic the interaction with their respective physiological ligands [lymphocyte function-associated antigen-3 (LFA-3)/CD58, CD59 and CD48 for CD2, B7/BB1 for CD28]. Taken together, these in vitro data suggest that IL-7 could participate in paracrine interactions between T lymphocytes and thymic stromal cells or dendritic cells, via its potent co-stimulatory activity with CD2 and CD28 adhesion molecules.     

Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) involved in the initiation of primary T-lymphocyte responses. However, despite their importance, no DC-specific surface marker has been identified in humans and many aspects of their ontogeny and the mechanisms underlying their potent functional activity remain unknown. In this report we describe a novel monoclonal antibody (mAb), CMRF-44, which recognizes an early activation antigen expressed by human DC and acts as a marker of allostimulatory activity within preparations of peripheral blood mononuclear cells (PBMC). The CMRF-44 antigen was expressed strongly on DC isolated from blood and tonsil by standard techniques, but was not detectable on Langerhans' cells within skin or on DC isolated directly from blood using a cell-sorting method which involves minimal DC manipulation/activation. Normal resting peripheral blood leucocytes did not label with CMRF-44, although weak staining of a small subpopulation (15%) of blood B lymphocytes was identified by double labelling. However, following overnight culture at 37 degrees, moderate staining of a subpopulation of PBMC was detected. Confirmation that CMRF-44 recognized an early marker of activated DC and hence allostimulatory activity was obtained by sorting cultured cell preparations on the basis of CMRF-44 reactivity. A marked enrichment of allostimulatory activity was observed in the CMRF-44-positive cellular population, whereas the CMRF-44-negative cells showed only minimal stimulatory activity. Activation studies established that the CMRF-44 antigen was an early activation marker, expressed constitutively on the majority of tonsil B lymphocytes, which can be induced on peripheral blood B lymphocytes and subpopulations of monocytes. Expression of the CMRF-44 antigen on cell lines was similarly restricted, CMRF-44 antigen being detected only on Hodgkin's disease-derived and B-lymphoid lines. The cellular distribution, expression kinetics and biochemical characteristics of the CMRF-44 antigen identify it as a new early marker of activated allostimulatory (DC) populations.     

Hodgkin's disease cell lines: a model for interleukin-1-independent accessory cell function.

The haemopoietic origins of the Hodgkin's disease (HD)-derived cell lines L428, KM-H2 and HDLM-2 remain controversial. Analysis of T-cell receptor (TcR) and Ig rearrangements cannot resolve this, and lineage promiscuity limits the interpretation of isolated surface antigen expression. Nonetheless the cell marker profile of L428 has similarities with human dendritic cells (DC), and L428 strongly stimulates in the mixed leucocyte reaction (MLR). We therefore undertook an extended immunophenotypic comparison of the HD lines with that recently defined for DC, prior to examining their ability to stimulate allogenic T lymphocytes, and comparing the molecular interactions involved with those of primary MLR stimulatory cells. The immunophenotype of the HD lines failed to establish either a lymphoid or monocytoid derivation. The profile of L428 appeared similar to the human DC. All three lines were potent stimulators in the primary MLR, and each expressed relevant adhesion and signal-transducing molecules important for co-stimulating T lymphocytes. Inhibition studies using monoclonal antibodies indicated similar contributions within HD line-T cell MLR to that documented in human tonsil DC-T cell MLR. The HD lines produced no detectable interleukin-1 (IL-1) by biological or immunological analysis. Moreover they stimulated allogeneic T lymphocytes in the presence of anti-IL-1 antibodies. Thus although IL-1 mRNA can be detected in both HDLM-2 and KM-H2 by polymerase chain reaction, these lines, and L428, share with DC the ability to stimulate allogeneic T lymphocytes in an IL-1-independent manner [corrected]. HD lines, particularly L428, may provide a standardized, reproducible, IL-1-independent model for dissection of the co-stimulatory requirements of the human primary MLR.     

Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells

Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APC) are used to elicit T cell responses, has become part of the search for alternative cancer and infectious disease treatments. Here, we report on the feasibility of using mRNA-electroporated CD40-activated B cells (CD40-B cells) as alternative APC for the ex vivo induction of antigen-specific CD8(+) T cell responses. The potential of CD40-B cells as APC is reflected in their phenotypic analysis, showing a polyclonal, strongly activated B cell population with high expression of MHC and co-stimulatory molecules. Flow cytometric analysis of EGFP expression 24 h after EGFP mRNA-electroporation showed that CD40-B cells can be RNA transfected with high gene transfer efficiency. No difference in transfection efficiency or postelectroporation viability was observed between CD40-B cells and monocyte-derived dendritic cells (DC). Our first series of experiments show clearly that peptide-pulsed CD40-B cells are able to (re)activate both CD8+ and CD4(+) T cells against influenza and cytomegalovirus (CMV) antigens. To demonstrate the ability of viral antigen mRNA-electroporated CD40-B cells to induce virus-specific CD8+ T cell responses, these antigen-loaded cells were co-cultured in vitro with autologous peripheral blood mononuclear cells (PBMC) for 7 days followed by analysis of T cell antigen-specificity. These experiments show that CD40-B cells electroporated with influenza M1 mRNA or with CMV pp65 mRNA are able to activate antigen-specific interferon (IFN)-gamma-producing CD8(+) T cells. These findings demonstrate that mRNA-electroporated CD40-B cells can be used as alternative APC for the induction of antigen-specific (memory) CD8(+) T cell responses, which might overcome some of the drawbacks inherent to DC immunotherapy protocols.     

Targeting antigen to CD19 on B cells efficiently activates T cells

CD19 is a B cell-surface molecule that participates as an important regulatory signaling complex for antigen bound at the surface by Ig. Triggering of CD19 through its linkage with CD21 amplifies signals transduced through the Src family kinases and modulates B cell differentiation in response to antigen. This study examines the kinetics of antigen uptake and processing of antigen directly targeted to the CD19 protein on purified B cells. We have demonstrated that the antigen internalized within minutes through CD19 forms a cap at the B cell surface and can be found within lysosomes in the cytoplasm in 90 min. B cells acquiring antigen via CD19 express elevated levels of B7-1 and B7-2 co-stimulatory molecules. Moreover, antigen-anti-CD19 complexes administered intravenously bind B cells in vivo and activate antigen-specific T cells more efficiently than non-specific uptake and in a manner similar to antigen taken up through surface IgM on B cells. This work illustrates an important and previously unrecognized mechanism for targeting proteins to B lymphocytes for antigen presentation and activation of CD4 T cells.     

Cellular distribution of a human I-A-like (DS/DC) antigen on normal and neoplastic lymphoid cells

Previous biochemical studies have shown that B cell specific, monoclonal antibody, McAb 33.1, reacts with a class II antigen that represents a human analogue of murine I-A (DS/DC) antigens (J. Exp. Med. 158:1924, 1983). McAb 33.1 recognizes a polymorphic human B lymphocyte specific antigen present on mu+, B1+ peripheral B cells, B lymphoid cell lines, activated B cells, and neoplastic B lymphoid cells. Of 100 HLA-D/DR typed EBV-transformed lymphoblastoid cell lines tested, only those from DR3,3 and DR7,7 individuals failed to react with McAb 33.1. The 33.1 antigen is present at lower concentrations on B cells from blood, tonsil, spleen, and lymph node when compared to B cell lines. By contrast, the antigen is not detectable on blood T lymphocytes, T cell lines, or mitogen activated T cells and it is absent on monocytes of some individuals or is present only on a minor subpopulation (approximately 20%). McAb 33.1 should facilitate the functional, structure, and molecular dissection of the human Ia system.