The role of B lymphocytes in cell-mediated immunity II. Delayed hypersensitivity induced by dinitrophenyl-ficoll in dinitrophenyl-keyhole limpet hemocyanin-immunized guinea pigs.

Dinitrophenyl (DNP)-Ficoll will elicit typical delayed hypersensitivity skin reactions in guinea pigs immunized with DNP-keyhole limpet hemocyanin (KLH). We observed that lymph node cells (LNC) from these animals produced the lymphokine, monocyte chemotactic factor (MNL CTX) when stimulated by DNP-Ficoll in vitro. This response was antigen and hapten specific since LNC from nonimmune guinea pigs or those immunized with nonDNP containing antigens were not stimulated by DNP-Ficoll. Lymph node cells were fractionated into T- and B-cell-enriched populations to determine the nature of the DNP-Ficoll-responsive cell. Only the B-lymphocyte-enriched population produced MNL CTX in response to DNP-Ficoll. The purity of the B-cell population was demonstrated by its failure to respond to PHA and by the fact that B cells derived from DNP-although they could no longer respond without T-cell help to the T-dependent antigen, DNP-OVA. These findings suggest that the hapten-specific response of guinea pigs to DNP-Ficoll may be a form of B-cell-mediated delayed hypersensitivity.     

The immunologic properties of Legionella cytolysin

Immunogenic properties of cytolysin were studied in experiments on guinea pigs. Preliminary immunization with cytolysin led to the suppression of response to ConA in lymphocytes not adhering to nylon wool and to the stimulation of response to Legionella antigens in lymphocytes adhering to nylon wool. For a month after infection with L. pneumophila the suppression of the proliferative activity of lymphocytes in the spleen of the immunized animals in response to ConA and Legionella antigens was observed, while in the lungs transitory suppression of response to ConA and Legionella antigens was followed by the restoration and then stimulation of proliferation in response to T-cell mitogen and specific antigens. The data obtained in these experiments indicate the capacity of cytolysin for modulating the development of immune response.     

Determinant specific suppression of antigen-induced T cell proliferation in the guinea pig. II. Determinant specific suppression of in vitro T cell responsiveness parallels a selective suppression of anti-hapten but not anti-carrier antibody responses

Using an antigen of defined physical structure with precisely mapped immunogenic sites, we asked whether those molecular sites previously shown to be critical for immune response gene-mediated initiation of T cell proliferation and T help correspond to the same molecular regions capable of inducing antigen-specific suppression of T cell proliferation and antibody production. Inbred strain 2, 13, and 2 x 13 F1 hybrid guinea pigs were immunized with various species variants or fragments of insulin adjuvant before subsequent immunization with antigen in complete Freund's adjuvant. Analysis of the patterns of depressed T cell responsiveness showed a striking correspondence to the Ir gene-dependent mechanism that controls the recognition of discrete regions within the insulin molecule observed in T cell help in antibody production. In addition, suppression of carrier-specific T cells parallels suppression of anti-hapten antibody responses when hapten is presented on the suppressed carrier without a concomitant suppression of the anti-carrier antibody response.     

Characterization and genetic control of the immune response to synthetic polypeptide antigens of defined geometry.

Both humoral and cell-mediated immune responses to the synthetic helical hapten-carrier conjugate poly-Glu-Tyr-Lys(TNP)-(Glu-Tyr-Ala)5 were found to be linked to the major histocompatibility locus in mice and guinea pigs. The responder mouse strains (H-2d haplotype) showed a primary IgM response with an IgG component appearing after the secondary immunization. The antibody response was accompanied by a positive DTH reaction in responder strains. Nonresponder mice (H-2b or H-2k haplotypes) showed neither IgM nor IgG antibodies and the DTH reaction was negative. Administration of the antigen as a complex with an immunogenic carrier was not effective in inducing a response in nonresponder mice. In guinea pig studies, it was found that strain 2 animals were able to mount an antibody response against the TNP-hapten and a DTH response against the polypeptide backbone. Strain 13 animals gave no anti-TNP antibodies at the lower dose levels and DTH activity was entirely negative for all doses of immunizing antigen. Replacement of the TNP hapten by the arsanilazo dipeptide derivative, BOC-gly-ARA-tyrosine, converted the nonresponder strain 13 guinea pigs into complete responders showing antibody and DTH reactions to both the hapten and the polypeptide backbone.     

In vitro correlates of hapten-specific delayed hypersensitivity

Azobenzenearsonate conjugates of l and d-amino acid polymers were studied in respect to their ability to stimulate lymphocytes from guinea pigs sensitized to the hapten. Using the two in vitro techniques of thymidine incorporation and inhibition of macrophage migration, it was shown that only conjugates of the l-amino acid polymers were active.The results confirm previous in vivo studies and suggest as one possibility that a preliminary processing event by macrophages may be obligatory for all associated phenomena of hapten-specific delayed hypersensitivity.     

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.     

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.     

Immune responses to myelin basic protein in mycobacterial-induced suppression of experimental allergic encephalomyelitis

Pretreatment of guinea pigs with complete Freund's adjuvant (CFA) 21 days prior to injection with myelin basic protein (BP) in CFA resulted in marked attenuation of clinical and pathologic manifestations of experimental allergic encephalomyelitis (EAE). Delayed hypersensitivity skin tests to homologous BP were likewise depressed in protected animals. The protected guinea pigs also demonstrated diminished in vitro reactivity to BP as assessed by BP-induced proliferative response of peritoneal exudate cells (PEC) and BP-induced inhibition of macrophage migration. Broad-based suppression of immunologic reactivity did not occur in these animals, as manifested by larger skin tests to PPD, a greater proliferative response to old tuberculin (OT), by PEC and peripheral blood lymphocytes (PBL), as well as marked PPD-induced inhibition of macrophage migration. Diminution of the degree of cell-mediated reactivity to BP may be one of the mechanisms by which prior treatment with CFA suppresses subsequent development of EAE.     

The delayed-type hypersensitivity response to (4-hydroxy-3-nitrophenyl) acetyl- (NP) coupled proteins is carrier-specific: in vivo and in vitro demonstrations

In vivo and in vitro approaches for measuring DTH to NP and the cross-reactive hapten, NIP, were taken. Mice were immunized subcutaneously with NP-OVA, NP-BGG or NP-CGG in CFA or NP-spleen cells, challenged intradermally with NP or NIP-coupled to a heterologous carrier, and footpad or ear swelling determined 4, 24, and 48 h later. Alternatively, draining LNC were removed and challenged in vitro with either haptenated protein or haptenated, irradiated, syngeneic spleen cells to induce lymphotoxin (LT) production or proliferation. Our results show that although carrier-specific DTH responses are easily elicited both in vivo and in vitro, NP-specific DTH effector cells cannot be evoked by conventional immunization regimens. This failure to induce hapten-specific DTH is not due to suppressor mechanisms. Attempts to induce LT production and T cell proliferation by re-exposure to NP were unsuccessful. Immunization with NP-coupled protein in CFA does elicit an intense Arthus reaction when mice are challenged with the hapten 8 days later. The antibody-mediated nature of this hapten-specific response is indicated by the kinetics of the reaction, which peaks 4 hr after challenge, intensely positive ELISA of circulating anti-NP antibodies, sensitivity to pretreatment with a high dose of cyclophosphamide, and the ability to transfer the reaction to naive recipients with serum. This early response is highly cross-reactive with NIP and is not restricted to mice of the igh-1b allotype.     

Comparison of antigen-specific T cell responses in autoimmune MRL/Mp-lpr/lpr and MRL/Mp-+/+ mice

The MRL-1 mouse develops severe autoimmune disease characterized by high titers of autoantibodies at an early age (3 to 5 mo). The congeneic MRL-n mouse, which differs only in the lymphoproliferative (lpr) gene, exhibits no such pathologic or serologic abnormalities at the same age. We examined antigen-specific T cell responses in the MRL-1 mouse and compared them to age- and sex-matched MRL-n controls. We found broad defects in these responses in the MRL-1 mouse; an inability to generate primary allospecific and hapten-specific cytolytic T lymphocytes (CTL), secondary hapten- and virus-specific CTL, as well as a deficient proliferative response to hapten and natural antigens and a weak delayed-type hypersensitivity response were demonstrated. Our data furthermore suggest a lack of interleukin 2 (IL 2) acceptor sites in the proliferating T cell, while suggesting no such lack on CTL precursors. In fact, the deficient CTL responses in MRL-1 mice can be restored to levels seen in MRL-n by the in vitro addition of IL 2. The implications of these findings and the possible explanations for the relative deficit in helper function in the MRL-1 mouse are discussed.     

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.     

Carrier-induced epitopic suppression is initiated through clonal dominance

Injection of mice with an immunogenic dose of carrier followed by immunization with hapten-carrier conjugate selectively suppresses anti-hapten antibody response. Previous studies have proposed that this epitopic suppression is related to the induction of carrier-specific Ts cells which in turn could inhibit selectively anti-hapten response. In the present study, we propose that the epitopic suppression is in fact due to clonal dominance. Immunization with a carrier such as tetanus toxoid induces a clonal expansion of carrier-specific B cells, thus decreasing the probability of hapten-specific B cells to react with the Ag. Increasing the density of the TNP-hapten on the conjugate has totally prevented the induction of the epitopic suppression. Moreover, using low hapten-carrier concentrations to challenge carrier-primed mice has enhanced the induction of the suppression. Finally, priming hapten-specific B cells before carrier/hapten-carrier immunization has also abrogated the suppression. The results of these experiments support the view that epitopic suppression is induced through the expansion of the clones specific for the carrier epitopes and resulted from intra-molecular antigenic competition between hapten and carrier epitopes. Based on these findings a regulatory role is proposed for B cells, where through their capacity to process and present antigen, they would exercise a strong influence on the selection of immune responses.     

Specific absorption of T lymphocytes committed to soluble protein antigens by incubation on antigen-pulsed macrophage monolayers.

Monolayers of macrophages (Mphi) pulsed with antigen were used as immunosorbents for T lymphocytes from guinea pigs primed to soluble protein antigens. T lymphocytes were cultured on the Mphi monolayers for 4 hr, then aspirated and reincubated on a fresh monolayer pulsed with the same antigen for a second and a third step. T lymphocytes so treated were selectively deprived of cells responding in assay for antigen-dependent proliferation against the antigen used for pulsing the absorbing monolayer, but maintained their response to other antigens. The lymphocytes adhering to the Mphi of the absorbing monolayer were capable of giving a full response to the antigen used for pulsing the Mphi of the monolyers. The proliferative response of F1 T lymphocytes to antigen in association with Mphi of either parental strain could be absorbed leaving the response to antigen in association with Mphi of the other parental strain. The absorption of the proliferative response was not inhibited by addition of excess soluble antigen to the medium of the absorption culture. Our results indicate that specific guinea pig T lymphocytes responding by proliferation to soluble protein antigens recognize and bind specifically to a complex of Ia antigen and protein antigen at the surface of the Mphi.     

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. I. Induction and suppression of delayed-type hypersensitivity and in vitro proliferative responses

Pretreatment of Lewis rats with a single i.p. injection of ABA-N-acetyl-tyrosine in incomplete Freund's adjuvant induced an unresponsiveness for delayed-type hypersensitivity to subsequent immunization with the same antigen in complete Freund's adjuvant. Complete suppression of in vitro antigen-induced proliferative responses required repeated pretreatment. Passive transfer of lymphoid cells from spleen and lymph nodes but not sera from suppressed rats induced unresponsiveness of hapten-specific T cell functions. Nylon wool-nonadherent cells and cells panned on F(ab')2 of rabbit anti-Lewis rat Ig plates suppressed the induction of DTH and in vitro antigen-stimulated proliferation. Adult thymectomy increased DTH and failed to abolish the induction of suppression.     

T cell-activating monokines in guinea pigs: comparison of high and low molecular weight factors

Guinea pig monokines produced by lipopolysaccharide-stimulated peritoneal macrophages were found in high (50,000-80,000) and low (10,000-30,000) molecular weight (m.w.) fractions by gel filtration. Both showed enhancing activity on the proliferative response of guinea pig and mouse thymocytes to PHA, but the high m.w. (65K) monokine was much more efficient than the low m.w. (15K) monokine in enhancing the response of lymph node T cells to PHA, suggesting its importance in the activation of peripheral T cells. The 65K monokine was coeluted with BSA present in the culture medium by DEAE-cellulose chromatography, but was clearly separated from it by hydroxylapatite chromatography. The immunoadsorption experiment with anti-BSA-coupled gel also indicated that 65K monokine is not a complex of low m.w. monokine with BSA. Our series of studies showed that most monokine activities were always found in the 65K fraction in guinea pigs. Thus, in guinea pigs, the 65K component appears to constitute a major class of T cell-activating monokines.     

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.     

Chronic experimental allergic encephalomyelitis in guinea pigs: immunologic studies on the two major myelin proteins

Humoral and cell-mediated immunity to the two major myelin proteins, basic protein (MBP) and proteolipid protein, have been investigated during the course of chronic experimental allergic encephalomyelitis (EAE) induced in guinea pigs with whole neural tissue. A positive proliferative response to MBP was observed at 10 and 13 days postimmunization, but was not detectable at subsequent stages. Serum antibodies to MBP first appeared during the chronic stages of the disease. A proliferative response to proteolipid apoprotein was not detected during any stage of chronic EAE. Guinea pigs immunized with proteolipid alone, however, showed a proliferative response. The data suggest that MBP is one of the antigens involved in the induction of the acute episode of chronic EAE, but its role in later stages and that of proteolipid protein remain unknown.     

IL-1 receptor signaling is required at multiple stages of sensitization and elicitation of the contact hypersensitivity response

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. The points at which IL-1R signaling is required during this complex, multistep immune response have not been clearly delineated. The role of IL-1R signaling during 2, 4 dinitro-1-fluorobenezene (DNFB) sensitization to induce hapten-specific CD8 effector T cells and in the trafficking of the effector T cells to the DNFB challenge site to elicit the response were investigated using IL-1R deficient mice. DNFB-sensitized IL-1R(-/-) mice had low CHS responses to hapten challenge that were caused in part by marked decreases in hapten-specific CD8 T cell development to IL-17- and IFN-γ-producing cells during sensitization. Hapten-primed wild type CD8 T cell transfer to naive IL-1R(-/-) mice did not result in T cell activation in response to hapten challenge, indicating a need for IL-1R signaling for the localization or activation, or both, of the CD8 T cells at the challenge site. Decreased CD8 T cell priming in sensitized IL-1R(-/-) mice was associated with marked decreases in hapten-presenting dendritic cell migration from the sensitized skin to draining lymph nodes. Transfer of hapten-presenting dendritic cells from wild type donors to naive IL-1R(-/-) mice resulted in decreased numbers of the dendritic cells in the draining lymph nodes and decreased priming of hapten-specific CD8 T cells compared with dendritic cell transfer to naive wild type recipients. These results indicate that IL-1R signaling is required at multiple steps during the course of sensitization and challenge to elicit CHS.     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.