Bolus injection of aqueous antigen leads to a high density of T-cell-receptor ligand in the spleen, transient T-cell activation and anergy induction.

In vivo anergy can be modelled by administration of soluble peptide to T-cell receptor (TCR) transgenic mice specific for the moth cytochrome c peptide 88-103 (MCCp). Two weeks after initial peptide treatment, T cells were present in normal numbers but were unresponsive to antigen stimulation in vitro. Only bolus injections of peptide, either subcutaneous or intravenous, were effective at inducing tolerance, while slowly released antigen administered via mini-osmotic pump failed to result in anergy. Examination of T cells soon after bolus peptide administration revealed that anergy induction was preceded by a transient hyperactivation of T cells in vivo. Within 2 hr of peptide treatment, interleukin-2 was detectable in the plasma of the transgenic mice. Interestingly, only bolus injections of peptide led to high levels of T-cell activation, while adjuvant emulsified and pump-administered peptide resulted in very low stimulation in vivo. When the dose of bolus-injected peptide used for tolerization was titrated, the extent of anergy induction directly correlated with the intensity of early T-cell activation. Indirect measurements of TCR-ligand density on the surface of antigen-presenting cells following peptide administration revealed that aqueous peptide delivered via bolus injection generated a large number of major histocompatibility complex-peptide complexes, while pump-delivered and adjuvant-emulsified peptide did not. These data suggest that high levels of TCR ligand are required for in vivo T-cell hyperactivation and induction of anergy.

Experimental autoimmune encephalomyelitis induced with a combination of myelin basic protein and myelin oligodendrocyte glycoprotein is ameliorated by administration of a single myelin basic protein peptide.

Multiple sclerosis is an autoimmune disease of the central nervous system in which T cell reactivity to several myelin proteins, including myelin basic protein (MBP), proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG), has been implicated in the perpetuation of the disease state. Experimental autoimmune encephalomyelitis (EAE) is used commonly as a model in which potential therapies for multiple sclerosis are evaluated. The ability of T cell epitope-containing peptides to down-regulate the disease course is well documented for both MBP- and proteolipid protein-induced EAE, and recently has been shown for MOG-induced EAE. In this study, we describe a novel EAE model, in which development of severe disease symptoms in (PL/J x SJL)F1 mice is dependent on reactivity to two different immunizing Ags, MBP and MOG. The disease is often fatal, with a relapsing/progressive course in survivors, and is more severe than would be predicted by immunization with either Ag alone. The MOG plus MBP disease can be treated postinduction with a combination of the MOG 41-60 peptide (identified as the major therapeutic MOG epitope for this strain) and the MBP Ac1-11[4Y] peptide. A significant treatment effect can also be obtained by administration of the MBP peptide alone, but this effect is strictly dose dependent. This MBP peptide does not treat the disease induced only with MOG. These results suggest that peptide immunotherapy can provide an effective means of mitigating disease in this model, even when the treatment is targeted to only one component epitope or one component protein Ag of a diverse autoimmune response.

Peripheral tolerance in T cell receptor-transgenic mice: evidence for T cell anergy.

T cell tolerance can be induced in adult mice by injection of soluble antigenic peptide. The underlying mechanism has been difficult to establish in normal mice due to the low precursor frequency of T cells specific for any given antigen. Therefore, we examined peripheral tolerance in mice transgenic for a T cell receptor specific for a cytochrome c peptide bound to I-Ek. Antigen-specific hyporesponsiveness could be induced in the transgenic mice. We followed the transgene-bearing T cells with a clonotypic monoclonal antibody and found similar numbers of clonotypic T cells in tolerized and control mice. To prevent de novo differentiation of T cells we analyzed thymectomized mice in which antigen-specific hyporesponsiveness was induced. Our analysis of thymectomized transgenic mice showed that antigen-specific T cell hyporesponsiveness following injection of peptide intravenously is not caused by gross elimination of T cells. These data provide evidence for the role of anergy in peripheral tolerance.

Strong priming of T cells adoptively transferred into scid mice.

We have examined the requirements for activating unprimed T cells in vivo by transferring T cells into scid mice, which lack mature B and T cells. Purified adult thymocytes and a protein antigen, keyhole limpet hemocyanin (KLH), were injected into scid mice. scid mice injected with T cells and KLH developed cellular lymph nodes containing CD4+ and CD8+ T cells. Cells recovered from the lymph nodes of injected scid mice proliferated and secreted interleukin 2 in response to KLH in vitro. The results indicate that T cells can be primed to KLH in the scid mouse in the absence of B cells.

The presentation of L-tyrosine-azobenzenearsonate by different mouse Ia molecules uses a common agretope.

The T cell response to L-tyrosine-azobenzenearsonate (ABA-tyr) has been studied using T cell lines and clones derived from three different mouse strains, B10.BR, B10.A (5R) and C57B1/6. In all cases, the arsonate group in conjunction with the amino group of tyrosine formed the functional T cell epitope. Molecules without any one or both of these groups are non-stimulatory. The hydrophobic moiety consisting of the azo-linked benzene rings forms the agretope of the molecule, as is evident from competitive inhibition of T cell stimulation by non-stimulatory analogues lacking the epitope. Substitutions on the benzene ring at ortho or meta positions resulted in decreases in ability to compete, indicating the likelihood of steric inhibition of binding of the agretope with the Ia molecule. This pattern was observed for clones and lines restricted by IAk, IAb and IEb/k MHC class II molecules. Peptides from lambda repressor protein, P84-98 and P73-88, showed haplotype specificity in their ability to inhibit ABA-tyr-induced proliferation of T cell clones, BRTC-4 and B6TC, respectively. The binding constants of ABA-tyr analogues were considered to be comparable to those of lambda repressor peptides because equimolar concentrations resulted in similar levels of competition. A cluster of aromatic amino acids on the floor of most MHC class II molecule binding sites might provide strong hydrophobic interaction with azo-linked benzene rings of ABA-tyr, thus accounting for its immunogenicity in all strains of mice studied.

Role of the thymus in natural tolerance to an autologous protein antigen.

C5-deficient mice grafted with thymus from C5-sufficient donors and immunized with C5 failed to make humoral antibody to C5, suggesting that the transfer of thymus had induced tolerance. Irradiated C5-deficient hosts repopulated with lymphoid cells from thymectomized C5-deficient mice grafted with C5-sufficient thymus also failed to respond to immunization with C5, thus showing that the state of tolerance can be adoptively transferred. These results demonstrate that natural tolerance to self-protein antigen is "learned" in the thymus.

Neonatal tolerance induction to Mlsa. II. T cells induce tolerance to Mlsa.

We have investigated the ability of murine T cell lines to induce neonatal tolerance to Mlsa (minor lymphocyte stimulating). Mlsb mice were injected within 24 hr of birth with MHC (major histocompatibility complex) identical T cell lines generated by culturing responders from Mlsa strains with stimulators from Mlsb strains. Injected mice were tested at 6 to 8 weeks of age for responses in either primary mixed leukocyte reaction or IL-2 limiting dilution analysis. Mlsa specific responses by injected tolerant mice relative to noninjected controls were reduced by 92-98% in MLR and by 2- to 10-fold in IL-2 LDA. In contrast, responses against third-party MHC antigens by either the injected or the noninjected mice were identical. Fifty percent of all mice injected with the T cell lines were tolerant to Mlsa. These results strongly suggest that murine T cells express the Mlsa gene product.

Neonatal tolerance induction to Mlsa. I. Tolerance to Mlsa is restricted by shared MHC determinants.

In this paper we have examined the influence of MHC (major histocompatibility complex) on neonatal tolerance to Mlsa (minor lymphocyte stimulating). By employing a novel approach we have shown that tolerance to Mlsa is restricted by shared MHC determinants. Thus, neonatal Mlsb mice, injected at birth with spleen cells from Mlsa mice, were tested as adults for Mlsa specific responses by interleukin-2 limiting dilution analysis, a technique which allows us to discriminate between responses to MHC + Mlsa and to MHC alone. Tolerance to Mlsa was in the context of any MHC type examined--donor, host, and third-party MHC products. These results show that tolerance to Mlsa is restricted by shared MHC determinants and extend previous studies indicating that activation of Mlsa responses is similarly restricted.

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes.

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.

Comparative accessory cell function of Langerhans cells isolated from mouse skin.

We have isolated Fc receptor bearing cells from murine epidermis and examined these cells using both structural and functional methods. From 60 to 70% of these cells are Ia positive, phagocytic and express Mac 1 and F4.80 markers. They do not have Birkbeck granules in their cytoplasm. In functional studies these cells are not active as inducer cells in allogeneic and mitogen responses. The results do not support direct homology between these Langerhans cells and the interdigitating dendritic cell. Thus within the epidermis these cells may be more closely related to a macrophage, possibly involved in antigen uptake.

Hapten-specific T-cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. IV. Rat dendritic cells present soluble and insoluble azobenzenearsonate conjugates to T cells.

We have used a fractionation procedure involving bovine serum albumin gradient floatation, adherence to glass, and rosetting with antibody-coated sheep erythrocytes to purify an accessory cell fraction from Lewis rat spleens. The fraction which is of light buoyant density, nonadherent, and FcR- is markedly Ia+, lacks phagocytic ability, is nonspecific esterase negative and under scanning electron microscopy has a heterogeneous morphology with a variety of protuberences described for rat dendritic cells. This putative dendritic cell preparation is very effective at stimulating proliferative responses with concanavalin A and allogeneic cells. When used to reconstitute the reactivity of peritoneal exudate cells of rats immunized with azobenzenearsonate-N-acetyl-L-tyrosine (ABA-Tyr) and subsequently depleted of Ia+ cells, it was shown to be highly effective with ABA conjugates of tyrosine, Ficoll, and polystyrene beads. Thus, despite the apparent absence of phagocytic or degradative abilities, this cell was very efficient at presenting large soluble and insoluble antigens. It is suggested that processing may occur at the cell surface without requiring internalization.

Antigen presentation by spleen dendritic cells.

It is now recognized that dendritic cells (DC) isolated from mouse spleen play an important role in activating T lymphocytes. These DC, which show many similarities to veiled cells found in the paracortical regions of spleen and lymph node, may be closely related to the epidermal Langerhans cells. It is known that DC are extremely effective allostimulators. We have also found that although DC lack demonstrable phagocytic ability, they are extremely potent at presenting soluble polypeptide antigens to primed T cells. Since T lymphocytes comprise several distinct subsets (particularly cytotoxic, helper, and suppressor) in our most recent studies we have asked whether DC are able to trigger all these different subsets of T cells. We examined the ability of different spleen cell types coupled with the hapten NP to induce antigen-specific T suppressors for a delayed type hypersensitivity (DTH) response. It was found that T suppressors were generated only when hapten was conjugated to a spleen-derived antigen-presenting cell. Further analysis revealed that macrophages but not DC were able to induce defined sets of suppressor cells in vivo (although DC were able to trigger a very powerful DTH response). We also examined the ability of DC to activate T cells which are required to cooperate with B cells in the production of antibody. Even though DC were able to trigger T lymphocytes to produce lymphokines, these activated T cells did not act as helper cells in a standard hapten-carrier system. Possible mechanisms for this dichotomy of DC function are discussed.

Evaluation of accessory cell heterogeneity. III. Role of dendritic cells in the in vitro activation of the antibody response to soluble antigens.

Dendritic cells and macrophages obtained from spleen and peritoneal exudate were tested as accessory cells for the activation of lymphokine production by T cells, for supporting T-B cooperation and for the induction of antigen-specific T helper cells. Dendritic cells as well as macrophages were able to activate T cells for interleukin-2 secretion and functioned as accessory cells in T-B cooperation, but only macrophages induced T helper cells, which cooperate with B cells by a linked recognition interaction, to soluble antigens. Dendritic cell- and antigen-activated T cells also did not help B cells in the presence of Con A supernatants which contained various T cell- and B cell-stimulatory factors. The failure of dendritic cells to differentiate memory into functional T helper cells, but their efficient accessory cell function in T-B cooperation, where functional T helper cells are already present, can be best explained by a differential accessory cell requirement for T helper cell activation dependent on the differentiation stage of the T helper cell.

Augmentation of cell-mediated responses in vitro by a monoclonal anti-helper factor antibody.

Previous studies have shown that monoclonal antibody AF3.44.4 has specificity for a constant region determinant on mouse antigen-specific helper factors and that it also binds to cultured T cells with functional helper cell characteristics. The antibody synergizes with antigen to enhance in vitro antibody responses; here we demonstrate that it will also enhance cell-mediated responses in vitro such as in the generation of proliferating cells in mixed lymphocyte responses and in the generation of specific killer cells in cytotoxic T lymphocyte cultures. The mechanism of AF3.44.4-generated enhancement was investigated. Increased levels of the lymphokines IL-2 and BCDF were detected in supernatants of AF3.44.4-treated cultures but the antibody itself could not replace interleukin-2 (IL-2), and would not stimulate primed cells in the absence of antigen. This type of monoclonal antibody which augments immunological responses in an antigen-dependent fashion may provide a new class of immunostimulant and a new approach to augmenting the responses of weak immunogens.

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities.

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.

Evaluation of accessory cell heterogeneity. II. Failure of dendritic cells to activate antigen-specific T helper cells to soluble antigens.

The activation of antigen-specific T cells requires Ia+, antigen-presenting accessory cells (AC). Dendritic cells (DC) and macrophages (M phi) isolated from spleen an peritoneal exudate were tested as AC for the activation of the activation of T helper cells and the induction of T cell proliferation. The cell separations to obtain DC and splenic M phi were performed by discontinuous bovine serum albumin gradients, adherence on petri dishes and rosetting with opsonized sheep erythrocytes. DC as well as the M phi were able to induce antigen-specific T cell proliferation, but only the M phi and not the DC activated antigen-specific T helper cells which help B cells for antibody production to soluble antigens. Keyhole limpet hemocyanin-specific T cells repeatedly stimulated with DC and antigen also did not express helper activity. The failure of DC to induce T helper cells was not due to the activation of a suppressor pathway. Thus, dendritic cells, although very efficient as AC in the induction of various T cell functions, are not able to activate T helper cells required for carrier-specific T-B cooperation and therefore cannot be the sole accessory cells. Based on these results and on previous data using Ia+ tumor cell lines as AC, we confirm the existence of functional AC heterogeneity.

Characterization of stimulator cells for alloreactive cytotoxic-T-lymphocyte responses in vivo.

Mouse spleen cells were fractionated and tested for their ability to induce alloreactive cytotoxic-T-lymphocyte responses in vivo. The cells with allostimulatory potential are enriched maximally in a population of low density, Ig-negative, Thy 1-negative cells. This fraction has the cytochemical and ultrastructural characteristics of cells of the early myeloid series and not those of typical dendritic cells or macrophages. These myeloid cells represent a new subset of accessory cells which could be a potential source of allostimulation in organ grafts. They should be considered along with other accessory cell types as potential elements which induce transplantation responses.

Requirements for suppressor T cell activation.

Third-order (Ts3) suppressor cells are generated after conventional immunization. These cells, however, will not mediate suppressor cell function unless specifically triggered by an activating signal, termed TsF2. This report analyzes the mechanism of this TsF2-mediated triggering event. TsF2-mediated suppression is genetically restricted by genes in the I-J and Igh-V regions. The target of the I-J restrictions is a firmly adherent accessory cell, which appears to express I-J-related determinants. These accessory cells are sensitive to cyclophosphamide treatment and 500 R irradiation. In contrast, the target of the Igh-V restriction of TsF2 appears to be the Ts3 cell, which carries antigen-specific, idiotype-related receptors. The mechanism of suppressor cell activation appears to involve two stages. Presentation of I-J-restricted TsF2 by I-J-compatible presenting cells and a second step involving idiotype-anti-idiotype interactions between TsF2 and the Ts3 cell. I-J compatibility is not required with the accessory cell for Ts3 activation. Finally, we hypothesize that the anti-idiotypic determinants expressed on TsF2 can serve as an internal image of antigen, thereby permitting specific targeting of the factor.

A role for macrophages in suppressor cell induction.

A mechanism responsible for the induction of NP-specific first order (inducer) suppressor cells (TS1) is described. TS1 cells are induced by i.v. administration of hapten-coupled splenic cells. Their activity is assessed by the adoptive transfer of NP-specific suppression during the afferent phase of the contact sensitivity response. NP-coupled firmly adherent, FcR+, I-A-bearing macrophages induce TS1. The antigen-presenting cells required for TS1 induction lack the Thy-1 and Lyt-1 markers, and are resistant to 500 R irradiation and to cyclophosphamide treatment. NP-coupled dendritic cells fail to induce TS1 activity. The induction of TS1 cells is genetically restricted by genes that map in the I-J region of the H-2 complex. The NP-coupled antigen-presenting cells must share at least one I-J allele with the TS1 donor for effective induction of TS1 activity. To minimize allogeneic effects in these studies, the activity of the TS1 population was assessed by adoptive transfer into syngeneic recipients. The present results are compared with the mechanisms required for the induction of second and third order suppressor cells.