Antibodies engineered with IgD specificity efficiently deliver integrated T-cell epitopes for antigen presentation by B cells.

We have developed a strategy for improving the stimulation of T cells during immune responses by constructing recombinant antibodies that enhance the delivery of antigen to antigen-presenting cells, such as B cells. These antibodies have variable regions specific for surface molecules on B cells, and a constant region with an inserted antigen. In vitro, such antibodies make B cells approximately 1000-fold more efficient at presenting antigen and stimulating specific T cells. In vivo, the antibodies turn B cells of the spleen into potent stimulators of T cells. This approach may be useful for the generation of new vaccines.     

High efficiency presentation of TNP-conjugated islet antigen to islet-specific T cells by a hybrid B cell line expressing TNP-specific surface Ig.

There is compelling evidence from animal models that type I diabetes is a consequence of T cell-mediated destruction of islet beta-cells. The recent isolation of islet-specific T cell clones from nonobese diabetic mice provides a means of identification of the Ag on islet cells that are responsible for stimulation of autoreactive T cells. We describe an APC line constructed by fusion of spleen B cells obtained from nonobese diabetic mice to a B lymphoma that was transfected with the H and L chains of an IgM specific to the hapten TNP. Using this hybrid APC we have observed a dramatic increase in the efficiency of presentation of TNP-conjugated islet cell protein preparations compared to that seen with conventional APC. Our results illustrate the potential use of this APC line for isolation and characterization of islet Ag relevant to the T cell response.     

Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells

B cells are well documented as APC; however, their role in supporting and programming the T cell response in vivo is still unclear. Studies using B cell-deficient mice have given rise to contradictory results. We have used mixed BM chimeric mice to define the contribution that B cells make as APC. When the B cell compartment is deficient in MHC class II, while other APC are largely normal, T cell clonal expansion is significantly reduced and the differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells). The development of the memory T cell populations is also decreased. Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin alpha (influencing lymphoid tissue architecture) had any effect on the T cell response. We conclude that in vivo B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells, optimizing expansion and allowing the generation of memory and effector T cells.     

Autoreactive B cells escape clonal deletion by expressing multiple antigen receptors

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2-/-) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2-/- knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.     

Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells

We have shown previously that specific Ag presentation is prevented by the inhibition of protein synthesis but nonspecific presentation is not. In the present paper, Ag presentation by Ag-specific B cells was examined for sensitivity to brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum. A20-HL B lymphoma expressing surface receptors specific for TNP was used as a B cell, and TNP-OVA was used as a specific Ag. The presence of BFA during pulsing of A20-HL cells with TNP-OVA inhibited the ability of the pulsed cells to stimulate 42-6A T cell clone, specific for OVA323-339 and Iad. The inhibition was not due to nonspecific toxicity of BFA, because the presence of BFA during pulsing of A20-HL cells with OVA323-339 did not affect their APC function. Ag binding to the receptor on A20-HL cells and internalization by the cells were observed in the presence of BFA. Thus, BFA might inhibit intracellular processing of specific Ag or intracellular complex formation of antigenic peptide from specific Ag with MHC class II molecules. Nonspecific Ag presentation by A20-HL cells, however, was resistant to BFA. A20-HL cells pulsed with OVA in the presence of BFA, even after fixation, could stimulate 42-6A cells to produce IL-2, although the IL-2 production was lower than that induced by A20-HL cells pulsed in the absence of BFA. These results suggest that the processing pathways for specific Ag and nonspecific Ag are different from each other, at least partly, in A20-HL cells.     

Efficiency of antigen presentation after antigen targeting to surface IgD, IgM, MHC, Fc gamma RII, and B220 molecules on murine splenic B cells

Bispecific heteroconjugate antibodies can bind soluble protein Ag to APC and thereby enhance Ag presentation. We used such antibodies to bind hen egg lysozyme (HEL) to various structures on the surface of normal splenic B cells to determine which structures would provide the best targets for enhanced presentation. We found that HEL was presented efficiently to hybridoma T cells if bound to sIgD, sIgM, or class I or II MHC molecules, but not at all if bound to Fc gamma RII, or B220 molecules on B cells. The efficiency of presentation of HEL was measured as a function of the amount of 125I-HEL bound per cell. HEL was presented with 5 to 10 times greater efficiency when bound to sIg, than when bound to MHC molecules. When compared on the basis of the amount of HEL bound, sIgD and sIgM functioned equally as target structures, as did class I and class II MHC molecules. Large amounts of HEL bound to B220, but no presentation resulted, indicating that focusing HEL to the APC surface was not sufficient for presentation to occur. HEL was internalized rapidly and in large amounts when bound to sIgD or sIgM, but slowly and in small amounts, when bound to class I or class II MHC molecules. Thus, a rapid rate of internalization may in part explain the high efficiency of Ag presentation after binding to sIg. However, the small amount of HEL internalized via MHC molecules was utilized efficiently for presentation. These results indicate that sIgM and sIgD serve equally on normal B cells to focus and internalize Ag and enhance Ag presentation, but that class I or class II MHC molecules can also be used to internalize Ag and enhance Ag presentation, perhaps by a separate intracellular processing pathway.     

Memory CD8+ T cell responses expand when antigen presentation overcomes T cell self-regulation

Antimicrobial memory CD8+ T cell responses are not readily expanded by either repeated infections or immunizations. This is a major obstacle to the development of T cell vaccines. Prime-boost immunization with heterologous microbes sharing the same CD8+ epitope can induce a large expansion of the CD8+ response; however, different vectors vary greatly in their ability to boost for reasons that are poorly understood. To investigate how efficient memory T cell expansion can occur, we evaluated immune regulatory events and Ag presentation after secondary immunization with strong and weak boosting vectors. We found that dendritic cells were essential for T cell boosting and that Ag presentation by these cells was regulated by cognate memory CD8+ T cells. When weak boosting vectors were used for secondary immunization, pre-established CD8+ T cells were able to effectively curtail Ag presentation, resulting in limited CD8+ T cell expansion. In contrast, a strong boosting vector, vaccinia virus, induced highly efficient Ag presentation that overcame regulation by cognate T cells and induced large numbers of memory CD8+ T cells to expand. Thus, efficient targeting of Ag to dendritic cells in the face of cognate immunity is an important requirement for T cell expansion.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. I. Relevance of antigen presentation persistence

Various mechanisms of peripheral T cell tolerization have evolved to avoid responses mediated by autoreactive T cells that have not been eliminated in the thymus. In this study, we investigated the peripheral conditions of Ag presentation required to induce T cell tolerance when the predominant APCs are B cells. We show that transient Ag presentation, in absence of inflammation and in a self-context, induces CD4(+) T cell activation and memory formation. In contrast, chronic Ag presentation leads to CD4(+) T cell tolerance. The importance of long-lasting Ag presentation in inducing tolerance was also confirmed in the herpes stromal keratitis autoimmune disease model. Keratogenic T cells could be activated or tolerized depending on the APC short or long persistence. Thus, when APCs are B cells, the persistence of the Ag presentation itself is one of the main conditions to have peripheral T cell tolerance.     

Analysis of H2-O influence on antigen presentation by B cells

HLA-DM (DM; in mouse H2-DM) promotes the exchange of MHC class II-associated peptides, resulting in the accumulation of stable MHC class II-peptide complexes. In naive (but not germinal center) B cells, a large part of DM is tightly associated with HLA-DO (DO; in mouse H2-O), but the functional consequence of this association for Ag presentation is debated. Here, we have extended previous studies by examining the presentation of multiple epitopes after Ag internalization by fluid phase endocytosis or receptor-mediated uptake by membrane Ig (mIg) receptors. We find that the effects of H2-O are more complex than previously appreciated; thus, while only minor influences on Ag presentation could be detected after fluid phase uptake, many epitopes were substantially affected after mIg-mediated uptake. Unexpectedly, the presentation of different epitopes was found to be enhanced, diminished, or unaffected in the absence of H2-O, depending on the specificity of the mIg used for Ag internalization. Interestingly, epitopes from the same Ag did not necessarily show the same H2-O dependency. This finding suggests that H2-O may control the repertoire of peptides presented by B cells depending on the mIg-Ag interaction. The absence of DO/H2-O from germinal center B cells suggests that this control may be released during B cell maturation.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. II. Chronic antigen presentation overrules antigen-presenting B cell activation

Ag presentation in the absence of danger signals and Ag persistence are the inductive processes of peripheral T cell tolerization proposed so far. Nevertheless, it has never been definitively shown that chronic Ag presentation per se can induce T cell tolerance independent of the state of activation of APCs. In the present work, we investigated whether chronic Ag presentation by either resting or activated B cells can induce tolerance of peripheral Ag-specific T cells. We show that CD4(+) T cells that re-encounter the Ag for a prolonged period, presented either by resting or activated Ag-presenting B cells, become nonfunctional and lose any autoimmune reactivity. Thus, when the main APCs are B cells, the major mechanism responsible for peripheral T cell tolerization is persistent Ag exposure, independent of the B cell activation state.     

B cells expressing Ig transgenes respond to a T-dependent antigen only in the presence of Ia-compatible T cells

We sought a quicker and easier method for the isolation of B cells enriched for a given Ag specificity. Here, we report our results using transgenic mice bearing mu- and kappa-transgenes that encode an IgR against the hapten phosphorylcholine. Splenic B cells from such mice had a high percentage of phosphorylcholine-binding cells and differentiated in vitro in response to T cell hybridomas in an Ag-specific, Ia-restricted manner. Supernatants from such in vitro cultures could not transfer helper activity, nor could the T cell be replaced in other ways tried when using a classical T-dependent Ag. These results support the model of a cognate interaction between T and B cell, and we present evidence that the interaction may consist of two or more steps.     

Microbial and T cell-derived stimuli regulate antigen presentation by dendritic cells in vivo

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.     

Functional clonal deletion of class I-specific cytotoxic T lymphocytes by veto cells that express antigen

Intravenous injection of class I incompatible spleen cells into mice results in a drastic reduction of the recipient's cytotoxic T lymphocyte (CTL) response against the injected, but not against third party, class I antigens when measured in bulk cultures initiated 5 to 6 days after the injection. This specific suppressive effect is partly due to T cells but can also be seen when high numbers of anti-Thy-1 and complement-treated spleen cells of nude mice are injected. Such cells suppressing CTL responses against self histocompatibility antigens are called "veto cells." The precursor frequency of CTL specific for the injected class I antigen is found to be reduced greater than 200-fold at days 5 to 6 after the injection, whereas the frequencies of CTL specific for third party class I antigens are not significantly changed. These results indicate that there is a functional clonal deletion of the CTL recognizing class I incompatible veto cells in vivo. The role of such a veto phenomenon in the induction and maintenance of self tolerance and allograft tolerance is discussed.     

IgD receptor-mediated signal transduction in T cells

Upregulation of immunoglobulin D-specific receptors (IgD-R) on CD4+ T cells may facilitate their interaction with specific carbohydrate moieties uniquely associated with membrane IgD on B cells. Previous studies have shown that upregulation of IgD-R facilitates cognate T-B cell interactions by mediating bidirectional signaling resulting in increased antibody responses and clonal expansion of antigen-specific T cells. Murine T hybridoma cells, 7C5, constitutively express IgD-R, as has been confirmed by staining with biotinylated IgD. Earlier studies have shown that inhibitors of protein tyrosine kinase (PTK) completely prevented upregulation of IgD-R in response to oligomeric IgD, suggesting that cross-linking of IgD-R may induce signal transduction and functional consequences through one or more PTK activation pathways, leading to upregulation of IgD-R. In the present study we show that cross-linking of IgD-R by oligomeric IgD indeed results in (a) T cell activation as seen by tyrosine phosphorylation of several intracellular proteins, (b) tyrosine phosphorylation of p56 Lck and PLC-gamma in 7C5 T hybridoma cells, and (c) phosphorylation of an approximately 29-kDa band that exhibits strong affinity for IgD. We analyzed tyrosine phosphorylation of p56 Lck and PLC-gamma in BALB/c splenic T cells that were exposed to oligomeric IgD both in vivo and in vitro. In vitro cross-linking as well as in vivo followed by in vitro cross-linking of IgD-R resulted in enhanced phosphorylation of p56 Lck and moderate tyrosine phosphorylation of PLC-gamma. These results suggest that interactions between IgD-R and IgD mediate signal transduction and support our previous findings that IgD-R+ T cells enhance cognate T cell-B cell interactions and antibody production.     

Tumor antigen presentation by murine epidermal cells

The ability of epidermal Langerhans cells to present Ag for CD4-dependent immunity is well documented, and it has been hypothesized that Langerhans cells participate in the generation of immunity against incipient epidermal neoplasms by presentation of tumor-associated Ag in situ. This study examined the ability of murine epidermal cells (EC) to present tumor-associated Ag for the induction of in vivo antitumor immunity. Murine epidermal cells were deleted of Thy-1-bearing cells, cultured in 50 U/ml granulocyte-macrophage-CSF for 14 to 18 h, and pulsed with tumor fragments (TF) derived from S1509a-fibrosarcoma cells. These TF-pulsed EC were injected s.c. into syngeneic recipients at weekly intervals for a total of three immunizations and challenged with viable S1509a tumor cells 1 wk after the last immunization. Control animals received TF-pulsed allogeneic EC or EC treated identically but not pulsed with TF. EC that were pulsed with tumor cell fragments were able to induce protective immunity to tumor growth in vivo and to immunize for a significant delayed-type hypersensitivity response to injected tumor cells. The induction of antitumor immunity with TF-pulsed EC was genetically restricted, and culture of EC in granulocyte-macrophage-CSF was required for development of significant immunity. Furthermore, deletion of I-A+ cells by antibody and complement-mediated lysis eliminated the generation of immunity. Thus, I-A+ epidermal cells are capable of presenting S1509a tumor Ag for the generation of protective antitumor immunity in vivo.     

Antigen presentation by human B cells: T cell proliferation induced by Epstein Barr virus B lymphoblastoid cells

T cell proliferation in response to antigen requires the presence of an antigen-presenting accessory cell. The accessory cell most commonly associated with antigen presentation has been the macrophage (Mo). This study demonstrates that B cells in the form of Epstein Barr virus-transformed B lymphoblasts (EBV-B) are able to present tetanus toxoid (TT) to human T cells and induce proliferation of these cells. T cells freshly isolated from peripheral blood were shown to undergo blast transformation and proliferation in response to TT and irradiated EBV-B cells. Furthermore, TT-reactive T cell blasts were shown to proliferate in the presence of EBV-B cells and TT, but not with other antigens. There was a progressive increase to a plateau in T blast proliferation with increasing numbers of EBV-B cells added to the culture. The concentration of TT required for optimal antigen presentation was similar for EBV-B cells and for Mo. TT-specific cloned T cells also proliferated in response to TT and EBV-B cells and could be continuously grown in culture with TT, interleukin 2-containing supernatant, and EBV-B cells in place of autologous Mo. EBV-B cells pulsed with TT could also act as antigen-presenting cells. The proliferative response of T cell clones to TT in the presence of EBV-B cells was inhibited by antiserum to human p29,34 glycoprotein but not by anti-beta 2-microglobulin. This inhibition was shown to result from interaction with Ia-like determinants on EBV-B cells. These results indicate that B lymphoblastoid cells in man are able, like Mo, to present antigen to T cells in the context of Ia-like determinants.     

Murine T lymphoma cells express a novel membrane-associated antigen with unique features

A novel membrane-associated antigen expressed on various murine T lymphoma cells has been detected by a rat monoclonal antibody. The antibody YE6/6 initially produced against Moloney leukemia virus-transformed T lymphoma line MBL-2, reacted with several other lymphoma lines including non-T lymphoma lines as well as thymocytes from leukemic AKR mice, but it did not show significant reactivities with resting or mitogen-activated normal lymphocytes by flow cytometric analysis. The antibody did not bind to some Abelson leukemia-transformed cells, which express Moloney virus antigens, suggesting that the antigen is unlikely to be encoded by Moloney virus genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the antigen molecules immunoprecipitated by the antibody revealed three major polypeptides. Two of the polypeptides, with approximate m.w. of 95,000 and 35,000, can be labeled by the cell surface iodination and, therefore, seem to be exposed on the cell surface. The third polypeptide, with approximate m.w. of 65,000, is not labeled by the surface iodination but it is readily detected by [35S]methionine labeling. The third polypeptide was labeled with [32P]orthophosphate indicating that it is a phosphoprotein. Western blot analysis showed that YE6/6 antibody primarily reacts with 35,000 m.w. polypeptide. Furthermore, the same 35,000 m.w. protein was also detected in concanavalin A-activated spleen cells at a low level by Western blot, but normal resting lymphocytes were negative. These results suggest that the antigen detected by YE6/6 antibody may be a cell proliferation-associated antigen and its expression is highly elevated on transformed lymphoma cells as compared to normal mitogen-activated lymphocytes.     

Electron microscopy of hepatitis B virus core antigen expressing yeast cells by freeze-substitution fixation

We have used the freeze-substitution fixation technique for electron microscopy of yeast cells that express the hepatitis B virus core antigen (HBcAg) following transformation with the cloned gene. Abundant spherical particles were found within the transformed cells. These particles had a uniform size and shape, measured about 21 nm in diameter, had electron-lucent centers, and consisted of many subunits. They were localized in both the cytoplasm and the nucleus. None of these particles was found in the cells of the parent strain. Comparison of the HBcAg particles isolated from the yeast cells and the particles within the yeast cells demonstrated that the 21-nm particles were in fact ultrastructurally superimposable on HBcAg. Thus, the HBcAg particles within the yeast cells were similar to the HBcAg particles in human liver tissues infected with hepatitis B virus, not only in their size and appearance, but also in their intracellular localization. These results suggest that the yeast cell has the same machinery for synthesis and intracellular translocation of the HBcAg polypeptides as the human cell.     

Physiology of IgD. VII. Induction of receptors for IgD on cloned T cells by IgD and interleukin 2

The role of IgD in the immune response has remained elusive, although the predominance of IgD on the B cell surface and the paucity of IgD in serum have suggested a receptor function. In support of this hypothesis, it has recently been shown that receptors for IgD on helper T cells can be induced by exposure to IgD in vivo and in vitro. Such IgD receptor-positive T cells (i.e., T delta cells), detectable as RFC using IgD-coated SRBC, augment antibody responses. In this report, we demonstrate that cloned, antigen-specific T cells of helper phenotype show only very low percentages of IgD-RFC, if allowed to rest in vitro after antigen exposure in the absence of IL 2. Exposure to IgD or to IL 2 for 24 hr causes the IgD-specific RFC to increase as much as 25-fold to nearly 80%. Clones that have recently been stimulated with antigen, or T cell hybridomas prepared from such clones, exhibit 40 to 50% IgD-RFC before exposure and twofold higher levels after exposure to IgD. IL 2 also causes a dose-dependent induction of OgD-RFC in normal splenic T cells. Thus, antigen stimulation, IL 2 and IgD can all induce these receptors for IgD which presumably enable helper T cells to interact more effectively with IgD+ B cells.     

Colitis immunoregulation by CD8+ T cell requires T cell cytotoxicity and B cell peptide antigen presentation

Deficient immunoregulation by CD4+ T cells is an important susceptibility trait for inflammatory bowel disease, but the role of other regulatory cell types is less understood. This study addresses the role and mechanistic interaction of B cells and CD8+ T cells in controlling immune-mediated colitis. The genetic requirements for B cells and CD8+ T cells to confer protective immunoregulation were assessed by cotransfer with colitogenic Galphai2-/- T cells into immune-deficient mice. Disease activity in Galphai2-/- T cell recipients was evaluated by CD4+ T intestinal lymphocyte abundance, cytokine production levels, and large intestine histology. B cells deficient in B7.1/B7.2, CD40, major histocompatibility complex (MHC) II (Abb), or native B cell antigen receptor (MD4) were competent for colitis protection. However, transporter-1-deficient B cells failed to protect, indicating a requirement for peptide MHC I presentation to CD8+ T cells. CD8+ T cells deficient in native T cell receptor repertoire (OT-1) or cytolysis (perforin-/-) also were nonprotective. These finding reveal an integrated role for antigen-specific perforin-dependent CD8+ T cell cytotoxicity in colitis immunoregulatory and its efficient induction by a subset of mesenteric B lymphocytes.