Antigen presentation by hapten-specific B lymphocytes. III. Analysis of the immunoglobulin-dependent pathway of antigen presentation to interleukin 1-dependent T lymphocytes

The ability of antigen-specific murine B lymphocytes to present antigen to a normal (nontransformed) antigen-specific, interleukin (IL)1-dependent T cell clone and to heterogeneous T lymphocytes was investigated. Hapten-specific lymphocytes present protein antigens to both the D10G.4.1 T cell clone and heterogeneous immune T cells. When the antigen bears an epitope recognized by the specific B cell, the subsequent presentation of this antigen is 10(2)-10(4)-fold more efficient, as compared to the same, but nonhaptenated protein. The requisite B lymphocyte binds hapten, is radiosensitive and is nonadherent to plastic. The hapten-specific antigen presentation is blocked by antibodies to the surface Ig receptor, while the presentation of unmodified protein is unaffected. These observations are identical to our findings in studies of B cell antigen presentation to T-T hybridomas and therefore demonstrate the generality of the immunoglobulin-dependent pathway of presentation. However, in contrast to the results with T-T hybridomas, the specific B lymphocytes are necessary, but not sufficient, to activate IL 1-dependent T cells. A third cell is required for both B lymphocyte immunoglobulin-dependent and nonspecific antigen-presentation. This cell is radioresistant, plastic adherent and of low density. The requirement for this third cell can be circumvented by supplementing cultures with highly purified IL 1. These results demonstrate that the remarkably efficient ability of B lymphocytes to present antigen is operative with factor-dependent T lymphocytes. Furthermore, conventional accessory cells also appear to play a role in this process, which is consistant with their known requirement in antigen-specific T-B interactions in the generation of antibody responses.     

In vivo antigen presentation

Monoclonal antibodies specific for defined peptide-MHC complexes are now being used to physically detect T-cell receptor ligands. These reagents have resulted in the identification of the cells that present antigen in lymphoid and non-lymphoid tissues after various forms of antigen administration. In addition, recent advances in real-time imaging technology have begun to measure the rate and directionality of T-cell movement relative to antigen-presenting cells in lymph nodes, shedding light on the earliest events in T-cell activation in a physiological setting.     

Requirement of the T cell receptor for antigen presentation by T lymphocytes. Effect of envelope glycoproteins of HIV-1 on antigen presentation by T cells.

We have developed CD4+, tetanus antigen-specific T cell clones that proliferate in the presence of tetanus antigen and autologous irradiated peripheral blood leucocytes (PBL) as antigen-presenting cells (APC). There have been several reports that T cells can present antigen themselves. We have used tetanus antigen-specific T cell clones to examine the effects of envelope glycoproteins of HIV-1 on processing and presentation of antigen to T cells. Cloned T cells were pre-incubated with soluble crude preparation of tetanus antigen for 4 h at 37 degrees C, irradiated, and used as APC (T-APC). These cells could present antigen, as assessed by the ability of the autologous cloned T cells to proliferate. Resting T cells and phytohaemagglutinin-activated T cell blasts from autologous PBL could not present tetanus antigen to the responder cloned T cells. Antigen presentation by T-APC was abrogated by treating cells with anti-HLA-DR but not by anti-HLA-DQ monoclonal antibodies; treatment of tetanus antigen-pulsed T-APC with anti-tetanus antibody also blocked the ability of these cells to induce proliferation in responder T cells. Antigen presentation by cloned T cells was by a chloroquine-resistant pathway. Pretreatment of T-APC with envelope glycoprotein of HIV-1, gp120, did not affect the proliferative responses of the responder T cells. These data suggest that gp120 does not inhibit the antigen-presenting function while suppressing antigen-specific responses.     

Antigen presentation by B lymphocytes to CD4+ T lymphocytes in vivo: importance for B lymphocyte and T lymphocyte activation.

B lymphocytes, like macrophages and dendritic cells, can present antigen to CD4+ T cells. Antigen presentation by B cells is essential for the generation of an in vivo T cell dependent antibody response, and repeated antigen presentation by B cells to T cells is necessary to induce B cell clonal expansion. Presentation of antigen by resting B cells to unprimed T cells tolerizes T cells, while anti-IgD antibody activates B cells and allows B cell antigen presentation that productively activates T cells. However, activation is not all that is required for B cells to productively present antigen to T cells.     

In vivo retroviral marking of antigen-specific B lymphocytes.

A major obstacle in the study of B lymphocyte development subsequent to immunization has been the paucity of distinctive phenotypic markers which might permit the separation and scrutiny of a mature antigen-experienced or memory cell population. To address this issue, we have introduced a recombinant non-replicating retrovirus into antigen-reactive lymph nodes in vivo using the expression of a reporter gene to monitor the trafficking, phenotype and persistence of retrovirus-marked B cells. Marked cells are first observed in the medullary cords where they proliferate for approximately 10 days prior to migration to the spleen. Up to half of the marked cells bear antigen-specific immunoglobulin receptors, but otherwise phenotypically resemble conventional B cells. The abundance and persistence of the marked cells suggest an association with the memory compartment. These experiments demonstrate a feasible approach for the identification and lineage analysis of antigen-responsive B cells, and suggest that the lymph node medulla supports a transient phase of post-antigenic B cell development associated with a long-lived antigen-specific B cell subset.     

Recruitment of effector lymphocytes by initiator lymphocytes. In vivo migration of in vitro sensitized initiator T lymphocytes.

We previously found that mouse T lymphocytes sensitized in vitro against allo- or syngeneic fibroblasts, upon injection into syngeneic recipients, do not themselves differentiate into effector cells, but recruit effector T lymphocytes within the draining lymph nodes. As a result of sensitization, these initiator lymphocytes acquire a trypsin-sensitive membrane property which is necessary for recruitment. We now report studies on the in vivo migratory behavior of initiator lymphocytes following sensitization. We injected 51Cr-labeled initiator lymphocytes into recipient footpads and found significantly increased migration of sensitized cells to the draining popliteal lymph node (PLN) during the first day. By amputation of the foot at various times, we showed that migration during the first 12-24 hours was critical for subsequent recruitment. Trypsin treatment of initiator lymphocytes abolished this accelerated migration. Lymphocytes triggered nonspecifically by Con A migrated to the PLN like antigen-sensitized cells. We also compared the migration of injected lymphocytes from the footpad to the PLN in graft-versus-host and host-versus-graft reactions, and found these reactions to differ both from each other and from recruitment in terms of lymphocyte migration. These findings are discussed in terms of the physiology of the cell-mediated immune response and the notion of peripheral sensitization.     

Recruitment of effector lymphocytes by initiator lymphocytes. In vivo migration of in vitro sensitized initiator T lymphocytes

We previously found that mouse T lymphocytes sensitized in vitro against allo- or syngeneic fibroblasts, upon injection into syngeneic recipients, do not themselves differentiate into effector cells, but recruit effector T lymphocytes within the draining lymph nodes. Asaresult of sensitization, these initiator lymphocytes acquire a trypsin-sensitive membrane property which is necessary for recruitment. We now report studies on the in vivo migratory behavior of initiator lymphocytes following sensitization. We injected ⁵¹Cr-labeled initiator lymphocytes into recipient footpads and found significantly increased migration of sensitized cells to the draining popliteal lymph node (PLN) during the first day. By amputation ofthe foot at various times, we showed that migration during the first 12—24 hours was critical for subsequent recruitment. Trypsin treatment of initiator lymphocytes abolished this accelerated migration. Lymphocytes triggered nonspecifically by Con A migrated to the PLN like antigen-sensitized cells. We also compared the migration of injected lymphocytes from the footpad to the PLN in graftversus-host and host-versus-graft reactions, and found these reactions to differ both from each other and from recruitment in terms of lymphocyte migration. These findings are discussed in terms of the physiology of the cell-mediated immune response and the notion of peripheral sensitization.     

In vitro activation of human T and B lymphocytes by pokeweed mitogen.

In previous in vitro studies the DNA synthesis in human blood lymphocytes induced by low concentration of PWM correlated with the percentage of bone marrow-derived (B) lymphocytes in cell suspensions. No correlation with lymphocyte subpopulations was noted when lymphocytes were activated by high concentrations of PWM. In this paper the hypothesis that low concentration of PWM mainly activates B lymphocytes was tested by measuring the [14C]thymidine incorporation into purified T or B lymphocytes from healthy donors. B lymphocytes were purified to 90--95% by buoyant density centrifugation of T lymphocytes rosetted with sheep red blood cells. T lymphocytes were enriched by passage of lymphocytes through an IgG-anti-IgG-coated column. Low concentration of PWM-stimulated B lymphocytes but not T lymphocytes, while high concentrations of the stimulant activated both cell types. It was also noted that the B- but not the T-lymphocyte fraction contained cells which synthesized DNA in the absence of PWM.     

Downmodulation of antigen presentation by H2-O in B cell lines and primary B lymphocytes

Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory molecule H2-M, which helps to exchange the invariant chain-derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2-O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2-M and selectively expressed in B cells. Using stable H2-O ribozyme-antisense transfectants, H2-O overexpressing murine B cell lines, and H2-O-transgenic mice, we investigated the effects of H2-O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levels of H2-O in the antigen-presenting B cells. Thus, increased H2-O expression downmodulated, whereas reduced H2-O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2-O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2-O levels. The intracellular location of H2-O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2-O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2-O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.     

Human Thy-1 antigen: cell surface expression on early T and B lymphocytes.

Human Thy-1 is present on the surface of only a small proportion of haemopoietic cells (thymus--0.2-10%; bone marrow--0.1-0.5%). We have analysed these Thy-1+ cells in more detail using immunofluorescence on a wide range of human haemopoietic cell lines and fresh leukaemic blasts; both situations where rare cells can become clonally expanded. The data demonstrate that Thy-1 is confined to the early stages of T- and B-lymphocyte development, and is absent from all myeloid cells. The Thy-1+ B cells represent late pre-B (c mu+)/early sIgM+ B cells. The Thy-1+ T cells are situated in the outer thymic cortex. Dual immunofluorescence analysis of FACS separated Thy-1+ thymocytes shows them to have the antigenic phenotype and morphology of prothymocytes/early thymocytes (some overlap seen with CALLA, TdT, OKT6 and OKT1).     

Obligatory role of macrophages in dengue virus antigen presentation to B lymphocytes.

The study was undertaken to investigate the role of dengue type 2 virus (DV)-infected mouse peritoneal macrophages (M phi) in presentation of the DV antigen to B lymphocytes as shown by counting virus-specific IgM antibody plaque-forming cells (PFC). It was observed that heat-killed or glutaraldehyde-fixed M phi did not present the antigen. Pretreatment of M phi with the lysosomotropic compounds ammonium chloride and chloroquine inhibited the antigen presentation. Depletion of M phi from the spleen cell cultures abrogated the immune response to DV. The tryptic-digested DV antigen could stimulate immune responses in B-lymphocyte enriched (depleted of M phi and T cells) spleen cell cultures, and the digested antigen could be presented by glutaraldehyde-fixed M phi. Pretreatment of M phi with a trypsin inhibitor abrogated antigen presentation. The findings thus show that even for presentation to B cells the DV antigen must be processed by M phi by a trypsin-like protease.     

In vivo efficacy of virus-derived peptides and virus-specific cytotoxic T lymphocytes.

The in vivo efficacy of virus- and tumor-specific cytotoxic T lymphocytes (CTL) is discussed as well as ways to activate these cells in vivo with lymphokines, monoclonal antibodies and immunization. In vivo and in vitro peptide immunization can induce such virus- and tumor-specific CTL but several questions have to be answered before peptide vaccination can be implemented as a novel immunotherapeutic or preventive approach in man.     

Capacity of antigen uptake by B cells, fibroblasts or macrophages determines efficiency of presentation of a soluble self antigen (C5) to T lymphocytes.

Self antigens in the body fluids must be taken up, processed and presented by antigen-presenting cells (APC) in order to induce T cell tolerance. For self antigens like the fifth component of complement (C5) which is not picked up by APC via antigen-specific receptors, presentation has to rely on uptake by nonspecific means. C5 was used as a model soluble self antigen to study the capacity of different APC (B lymphoma cells, fibroblasts and macrophages) of taking up, processing and presenting low concentrations of soluble C5 to C5 specific T cell hybrids. Under conditions of limiting antigen amounts macrophages and fibroblasts exhibited similar presentation capacity for soluble C5 while B cells did not. C5 presentation by macrophages was enhanced in the presence of C5-specific antibody and augmented further if antigen was added in the form of particulate latex-antigen-antibody complexes indicating enhanced uptake via Fc receptor-mediated endocytosis or phagocytosis. B cells presented soluble C5 only in the presence of C5-specific antibody. Uptake of C5 under these conditions occurred via Fc receptors type II. This pathway of antigen uptake did not operate with other antigens which were presented efficiently after nonspecific endocytosis. In light of these findings it seems reasonable to propose that nonspecific endocytosis of serum proteins like C5 by B cells is normally limited in order to avoid interference with their critical role in antigen receptor-mediated uptake and presentation for the initiation of an antibody response. It seems likely that presentation of soluble self antigens present in the circulation may normally be the task of dendritic cells and macrophages depending on the physical shape of the antigen.     

T cell-mediated antigen presentation

Differentiation of the T cell repertoire and the physiology of T cell-mediated antigen presentation are reviewed in relation to mechanisms of self-tolerance. Recent research has indicated that T cell development is a continual process that optimizes partial recognition of self as a homeostatic set-point. Specific T cell antigen recognition of partial agonists is intrinsically linked to expression of class II MHC glycoproteins on T cells. Even ligands that act as TCR antagonists in IL-2 production assays have sufficient agonistic strength to induce expression of class II MHC glycoproteins on T cells. Thus, the intrinsic self-reactivity of the T cell repertoire may promote T-APC activity in vivo and may explain why thymic and peripheral T cells express low but significant levels of class II MHC glycoproteins. T-APC activity induces extensive apoptosis among responder T cells, causes desensitization among surviving responders, and has been implicated in the adoptive transfer of tolerance in the Lewis rat model of experimental autoimmune encephalomyelitis. Overall, these findings support a relationship between the partial recognition of self MHC ligands, expression of class II MHC glycoproteins on mature peripheral T cells, tolerogenic T cell-mediated antigen presentation, and desensitization of pathogenic self-reactive T cells.     

Identical forms of the CD2 antigen expressed by mouse T and B lymphocytes

A monoclonal antibody (12-15) reactive with the mouse CD2 was used to study the expression of the antigen in different lymphoid cell subsets. By two-color immunofluorescence using B or T cell-specific reagents and cell sorting in combination with biochemical analysis we provide evidence that the CD2 antigen is present on mouse B and T cells. The antigen is expressed by both subsets at similar density and appears to be biochemically indistinguishable.     

Induction of cytotoxic T lymphocytes specific for paraneoplastic cerebellar degeneration-associated antigen in vivo by DNA immunization.

Paraneoplastic cerebellar degeneration associated with gynecological and breast malignancies (PCD) is known to develop autoantibodies and autoreactive cytotoxic T lymphocytes (CTLs) specific for a cytoplasmic protein of Purkinje cells PCD17/cdr2, in the blood of patients. The functional roles of these antibodies and CTLs in the pathogenesis of PCD are unknown. Induction of immune response to this antigen in experimental animals should be useful to clarify the immune mechanisms in PCD patients. We immunized Balb/c mice with naked DNA, which is encoded human PCD17 neural protein in an eukaryotic expression plasmid under control of CMV promoter, and explored whether or not humoral and cell-mediated immune responses against PCD17 could be induced in vivo. We show that DNA immunization with naked pcd17 cDNA could induce autoantibodies against the cytoplasmic protein of Purkinje cells and CTLs could lyse syngenic myeloma cells pulsed with H-2K-restricted PCD17 peptide. In spite of the generation of anti-Purkinje cell antibodies and PCD17-specific CTLs in vivo, neither clinical nor pathological changes consistent with significant cerebellar degeneration have been detected.     

The role of lymphocyte function-associated antigen 1 (LFA-1) in the adherence of T lymphocytes to B lymphocytes

The functional role of the LFA-1 molecule in the interaction between helper T lymphocytes and B lymphocytes was investigated using lymphocytes from patients with leukocyte adhesion deficiency, an inherited immunodeficiency characterized by a defective leukocyte expression of the LFA-1, Mac-1 (CR3) and p150,95 molecules. The ability of LFA-1- T lymphocytes to provide antigen-specific help for HLA-identical LFA-1+ B lymphocytes was reduced while their antigen-specific activation was normal. Antigen-independent conjugate formation between resting, nonactivated LFA-1- T lymphocytes and LFA-1+ B lymphocytes was impaired while LFA-1- B lymphocytes bound LFA-1+ T lymphocytes normally. Conjugate formation of activated LFA-1- T lymphocytes was mostly mediated by the CD2-LFA-3 adhesion pathway while the ICAM-1 molecule, a ligand of LFA-1, had no function. These results demonstrate that LFA-1 plays a major role in the cognate interaction between helper T lymphocytes and B lymphocytes that cannot be mediated instead by CD2 or other molecules on resting T lymphocytes.