Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

Effect of cytotoxic lymphocytes obtained by in vitro immunization with syngeneic lymphoma cells on pluripotent hemopoietic cells

The possibility of the presence of leukemia-associated antigens on pluripotent hemopoietic cells was studied with the aid of immune lymphocytes, cytotoxic against mouse syngeneic lymphoma cells. Cytotoxic lymphocytes were obtained during immunization in vitro of C57BL/6 mouse splenocytes by syngeneic T lymphoma EL-4 cells in the presence of interleukin-2. Specific cytotoxic activity of immune lymphocytes as regards EL-4 cells was not blocked by addition of normal bone marrow cells. Incubation of the bone marrow with immune killers did not lead to a decrease in the number of colony-forming units in the spleen. It was shown that using cytotoxic lymphocytes the total killing of lymphoma cells might be achieved in a mixture of bone marrow and lymphoma cells, whereas pluripotent precursor cells might be retained.     

Immune sensitization to syngeneic organ-specific intestinal antigens in the Lewis rat

In human chronic inflammatory bowel disease involving mucosal epithelium, sera and lamina propria mononuclear cells are reactive with cell surface components isolated from gut epithelial cells. To define a model system in which the disease-inducing potential of such immune factors could be rigorously evaluated, we sought to immunologically sensitize inbred murine strains to syngeneic colonic epithelial cell-associated components (ECAC-C), to define precise in vivo and in vitro conditions to optimize ECAC-C reactivity, and to initially explore whether such cells could elicit tissue injury in epithelium after adoptive transfer to naive animals. Following footpad immunization, Day 42 lymph node cells but not splenocytes were reactive with syngeneic ECAC-C, as shown by a linear increase in [3H]thymidine incorporation over a wide range of antigen concentration (0.5 to 100 micrograms/ml). A subsequent 48-hr exposure to ECAC-C and/or interleukin 2 resulted in a more restricted responsiveness, proliferation occurring only in the presence of ECAC-C or mitogen and not to a coimmunogen (PPD). Further evidence that lymph node cells from ECAC-C/CFA immunized animals were indeed sensitized to syngeneic ECAC-C included ability of donor animals to mount highly significant earlobe DTH responses to ECAC-C, indicating the presence of antigen-specific T-DTH cells, and the failure of polymyxin B, in doses sufficient to inhibit LPS-induced mitogenesis, to reduce lymph node cell responsiveness to ECAC-C, known to be contaminated with LPS. ECAC-C-specific circulating antibody and T-cytotoxic cells were not detected. Adoptive transfer of Day 42 lymph node cells, sensitized in vivo and conditioned in vitro, was not associated with tissue injury in syngeneic recipients in preliminary experiments. This model system, with antigen-specific cells analogous to those present in diseased mucosa of human chronic inflammatory bowel disease, may be an important means to determine the pathophysiologic significance of anti-epithelial cell immune responses in these disorders.     

Macrophage activation to kill Leishmania tropica: characterization of a T cell-derived factor that suppresses lymphokine-induced intracellular destruction of amastigotes

Factors obtained from phorbol myristate acetate (PMA)-stimulated EL-4 thymoma cells, a continuous T cell line, suppressed lymphokine-induced macrophage activation to kill intracellular Leishmania tropica amastigotes. Suppression of this macrophage effector activity was dependent upon concentration of EL-4 fluids admixed with lymphokines in infected macrophage cultures, and was not due to residual PMA or factors released from unstimulated EL-4 cells. Fluids from PMA-stimulated EL-4 cells did not affect the expression of microbicidal activity by macrophages activated in vivo as a consequence of infections with Mycobacterium bovis strain BCG, nor did they abrogate intracellular killing activities by C3H/HeJ macrophages primed by BCG infection and triggered by lymphokines in vitro. That the action of this EL-4 suppressor activity was at the priming stage of macrophage activation was confirmed by kinetic studies: EL-4 fluids added to lymphokine-treated cells in the first 4 hr of treatment completely suppressed intracellular killing of L. tropica; fluids added after 4 hr were not effective. The effects of these EL-4 factors appeared to be selective: of three effector activities of activated macrophages tested, induction of resistance to infection, tumor cytotoxicity, and intracellular destruction of L. tropica, only intracellular killing by lymphokine-treated macrophages was significantly suppressed. These T cell-derived soluble suppressor factor(s) may provide insight into mechanisms of immunosuppression during leishmanial disease and perhaps other intracellular parasitic infections.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Selective activation of antigen-specific human B cells in recently immunized individuals by nonspecific factors in the absence of antigen

The in vitro effect of nonspecific factors (derived from mixed lymphocyte culture [MLC] supernatants) on human B cell responses was studied in individuals recently immunized in vivo to keyhole limpet hemocyanin, tetanus toxoid, and/or diphtheria toxin. In T cell-depleted fractions of peripheral blood mononuclear cells, nonspecific factors alone, without antigen, selectively induced a specific antibody response to the antigen to which the individual had been recently immunized, at dilutions that did not generate a significant polyclonal response in the remainder of the B cell repertoire. The source of these factors, with respect to MLC donors, did not affect the antibody response. Supernatants of MLC from nonimmunized individuals induced a specific antibody response as effectively as supernatants of MLC from immunized individuals, when added to B cells plus monocytes from recently immunized individuals. Studies in which the same individuals were followed over time showed that these factor-sensitive B cells are seen in the peripheral blood of recently immunized individuals for only a finite period of time. Thus, in vivo immunization with a specific antigen results in the transient appearance in the peripheral blood of B cells that are specific for the antigen in question. These B cells are probably preactivated in that nonspecific factors selectively induce in vitro their further differentiation into antibody-secreting cells, in the absence of added antigen or mitogen. These studies may add further insight into our understanding of the sequential steps involved in the activation and differentiation of human B lymphocytes and provide a model for the combined in vivo and in vitro study of human B cell physiology.     

Activation of cytotoxic T lymphocytes requires at least two spleen cell-derived helper factors besides interleukin 2

The dependency of induction of T cell cytotoxicity on lymphokines was studied. 1 X 10(5) nylon wool-purified thymic lymphocytes or 10(4) spleen cells were cultured with TNP-haptenated syngeneic UV-irradiated spleen cells in the presence of a variety of lymphokine preparations. Concanavalin A-induced spleen cell supernatants mediated strong cytotoxic responses in this system. Three other preparations, namely, a partially purified IL 2 preparation from PMA-stimulated EL-4 thymoma cells, a Con A-induced spleen cell supernatant that was absorbed with an IL 2-dependent cell line, and a Con A-induced supernatant that was dialyzed at pH 2 were all ineffective in mediating a cytotoxic response. In reconstitution experiments, cytotoxic responses were only obtained when either the absorbed preparation or the pH 2-treated preparation was mixed with the IL 2 preparation from EL-4 cells. No reconstitution occurred after mixing of the absorbed with the pH 2-treated preparation. pH 2 treatment of the absorbed preparation did not abolish its synergistic effect when added to the IL 2 preparation from EL-4 cells. These results led to the conclusion that activation of cytotoxic lymphocyte precursors requires at least two other lymphokines in addition to IL 2. One T cell cytotoxicity-inducing factor (TCF1) remained in Con A-induced supernatants after absorption with IL 2 receptor-bearing T cell line cells. It was pH 2-resistant and was not found in EL-4 supernatants. A second T cell cytotoxicity-inducing factor (TCF2) was pH 2-sensitive and was found in Con A-induced spleen cell supernatants as well as in interferon-free supernatants of PMA-stimulated EL-4 cells. This activity co-purified with IL 2. It was absorbed by the IL 2-dependent T cell line together with IL 2. IL 2 differs from TCF2 since it is pH 2-resistant.     

Regulation of the anti-alpha (1,3) dextran response: two populations of dextran-reactive B cells that differ in their T cell requirements for induction to antibody synthesis

The in vitro antibody response to dextran B1355S, a thymus-independent Type 2 antigen, requires T cell-derived lymphokines but is not thought to require an activation signal from an antigen-specific T helper cell. The present study demonstrates that there are two dextran-reactive B cell populations in BALB/c mice with respect to the T cell requirements for the generation of antibody-forming cells. One population found among dextran-reactive spleen B cells from 12- to 14-mo-old BALB/c mice generated anti-dextran PFC in the presence of B cell growth factor (BCGF II) and IL 2 or the combination of BCGF II, IL 2, and IFN-gamma. A second population of dextran-reactive B cells found in spleen and Peyer's patches of 2-mo-old unprimed mice did not respond to these same lymphokines, but did generate anti-dextran plaque-forming cells in the presence of Thy-1.2+, L3T4+ T cells from Peyer's patches. However, splenic B cells obtained from 2-mo-old mice that had been primed with dextran 2 to 3 days after birth were shown to be responsive to the same lymphokines as dextran-reactive B cells from 12- to 14-mo-old mice. These results suggest that previous priming with dextran B1355S induces a dextran-specific B cell population that can be activated to antibody-forming cells in the presence of antigen and T cell-derived lymphokines, whereas a second, unprimed population requires an additional activation signal from L3T4+ T cells.     

Immunoregulation in the rat: cellular and molecular requirements for B cell responses to types 1, 2, and T-dependent antigens

The requirements for primary in vitro plaque-forming cell (PFC) development in cultures of purified rat splenic B cells have been examined. Rat B cells were directly responsive to the type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA), but both T cells and adherent accessory cells were required for B cell responses to the type 2 antigen TNP-Ficoll and the T cell-dependent (TD) antigen sheep erythrocytes (SRBC). However, the cellfree supernatants from concanavalin A-induced spleen cells of rat or mouse origin replaced the requirement for T cells and macrophages, and resulted in PFC development in response to TNP-Ficoll and SRBC and augmented PFC numbers in response to TNP-BA. Culture supernatants from induced murine T cell and macrophage cell lines were used to partially deduce the molecular requirements for the support of PFC development by rat B cells to these three antigens. Supernatants from the EL-4 (EL-4 sup) and B151 K12 (B15 sup) T cell lines augmented TNP-BA responses, suggesting that B cell growth factor II (BCGF-II) mediated this effect. An admixture of purified interleukin 2 (IL 2) and B15 sup supported PFC development to SRBC; indicating that IL 2, BCGF-II, and the T cell-replacing factor in B15 sup (B15-TRF) were sufficient to support this response. In addition, the IL 2 plus B15 sup-supported anti-SRBC PFC response was increased by the addition of an interleukin 1-containing fraction from the supernatant of the macrophage line P388D1. PFC development in response to TNP-Ficoll had the most stringent requirements and only occurred in the presence of EL-4 sup and B15 sup (IL 2, BCGF-I, BCGF-II, EL-TRF, B15-TRF). These data indicate that different cellular and molecular requirements exist for PFC development in response to types 1, 2, and TD antigens by rat B cells.     

Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction

In this report we show that three mutagenized sublines of (murine) EL-4 thymoma cells can constitutively activate human and/or murine B cells via an MHC-nonrestricted cell-cell interaction. The activation signal is not by itself mitogenic but renders B cells capable of proliferating in response to interleukin 2 (IL 2). In addition, one of the mutant EL-4 sublines can constitutively respond by release of IL 2 in the presence of IL 1-containing macrophage (P388D1) supernatant. The exact relationships between these functional properties of the mutant EL-4 thymoma cells and those associated with activated normal T helper-cells remain to be established. However, the EL-4 cells provide a unique system to study in parallel murine and human B cell responses. In particular, the following observations were made during the present study. First, anti-Ig antibodies (anti-Ig) were required for B cell activation in conjunction with two EL-4 sublines acting only on murine B cells, whereas with a third subline acting on both murine and human B cells, anti-Ig was not required. Anti-Ig by itself did not lead to significant B cell activation in the absence of mutant EL-4 (or normal T) cells. Second, the growth factor-stimulated proliferation of EL-4-activated B cells, following separation of the B cells from the EL-4 cells, lasted only 2 days. These results, thus, indicate that the requirement for a surface Ig-mediated B cell activation signal depends on the quality/intensity of a direct T cell signal and that cell-cell interactions may exert a more stringent control over the growth factor responsiveness of B cells as compared with T cells.     

Activation of cation transport by lymphokines in B cells without induction of DNA synthesis or immunoglobulin gene transcription

We report on an experimental model that permitted us to evaluate the biologic relevance of membrane-associated biochemical events with respect to cell proliferation and maturation, each induced by distinct sets of signals. Antigen-affinity-enriched murine B cells cultured in the presence of a proliferative signal induced by LPS showed activation of Na+/K+ ATPase and enhanced the uptake of proline, followed by RNA, protein, and DNA synthesis, without the generation of antibody. Stimulation with both the proliferative signal(s) and the maturation signal(s) derived from lymphokines of an EL-4 thymoma induced B cells to proliferate and synthesize mRNA encoding mu-chain of IgM and to mature into IgM-secreting cells. Most important, the secretory product of EL-4, in the absence of LPS, activated Na+/K+ ATPase but failed to stimulate uptake of proline and synthesis of DNA or mu-specific mRNA. A similar response was observed in splenocytes depleted of T cells and in unfractionated spleen cells. Thus a component secreted by EL-4 can induce some of the early molecular events characteristic of the proliferative response but lacks the ability to initiate blast transformation and DNA synthesis.     

Antigen-reactive cloned helper T cells. II. Exposure of murine cloned helper T cells to IL 2-containing supernatant induces unresponsiveness to antigenic restimulation and inhibits lymphokine production after antigenic stimulation

Cloned murine helper T lymphocytes (HTL) reactive to alloantigen or to ovalbumin (OVA) become unresponsive to antigenic restimulation after exposure to antigen or to culture supernatant fluids (SF) containing multiple lymphokine activities. Unresponsiveness is manifest by a failure of antigen-stimulated cells to incorporate thymidine or to produce lymphokines after antigenic challenge. Antigen-unresponsive HTL, however, will incorporate thymidine when exposed to an exogenous source of interleukin 2 (IL 2). The duration of unresponsiveness to antigen is correlated with the concentration of IL 2 in SF to which the cloned HTL had been exposed. Chromatographic fractionation of IL 2-containing supernatant from EL-4 thymoma cells (EL-4 SF) yielded a pool of SF that was enriched for IL 2 activity. Exposure of HTL to lymphokines contained in this pool induced unresponsiveness to antigen that was comparable to that observed when HTL were exposed to unfractionated EL-4 SF. Unresponsiveness to antigen also developed after cloned HTL were stimulated with concanavalin A (Con A) or with OVA and syngeneic splenic filler cells. We have used monoclonal antibody (mAb) GK1.5 (anti-L3T4) to investigate the role of lymphokine production in the induction of unresponsiveness. This antibody did not inhibit IL 2-induced thymidine incorporation by cloned HTL, and did not inhibit the induction of unresponsiveness after exposure of cloned HTL to EL-4 SF. In the presence of mAb GK1.5, however, HTL that were stimulated with Con A or OVA did not become unresponsive to antigenic restimulation, an effect that was overcome by the addition of EL-4 SF. These results suggest that HTL become unresponsive to antigen after exposure to IL 2-containing SF, and that stimulation by antigen or Con A can induce the unresponsive state by virtue of stimulating lymphokine production.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

Immune dysfunction associated with graft-vs-host reaction in mice transplanted across minor histocompatibility barriers. II. Reversible defect in T-dependent antibody responses

B cell and Th cell functions were assessed in mice undergoing a graft-vs-host reaction (GvHR) in response to minor histocompatibility Ag by using the plaque-forming cell (PFC) response to the T-independent Ag TNP-Brucella abortus and the T-dependent Ag TNP-SRBC. Bone marrow plus spleen cells from B10.D2 mice were transplanted into lethally irradiated B10.D2 (syngeneic recipient) or H-2d-compatible BALB/c (allogeneic recipient) to produce a chronic form of GvHR. BALB/c recipients of an allogeneic transplant demonstrated a marked and proportional lymphoid depletion of the spleen with normal percentages of B cells, T cells, and CD4+ and CD8+ T cell subsets. Mice with GvHR made normal numbers of PFC/10(5) spleen cells in response to the T-independent Ag, but a significantly depressed number of PFC/10(5) spleen cells to the T-dependent Ag compared with normal B10.D2 mice and with irradiated B10.D2 recipients of syngeneic B10.D2 marrow plus spleen cells. Mice undergoing the minor Ag GvHR made significantly larger numbers of PFC/10(5) spleen cells after secondary immunization with TNP-SRBC compared with controls. In vitro assays demonstrated that B cells from mice with GvHR responded to T help from normal B10.D2 mice and that T cells from mice with GvHR provided help to normal B cells after in vivo immunization. These data demonstrate that radiation chimeras with GvHR in response to minor histocompatibility Ag have relatively normal B cell function and an apparent defect in T helper cell function that is reversible by immunization with appropriate Ag.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Further studies on the regulation of lymphokine biosynthesis in contact sensitivity

Contact sensitivity (CS) results from a series of cellular interactions involving CD4+ T cells and macrophages. We have studied the production of lymphokines by T cells from mice immunized by contact sensitization and by a variety of stimuli leading to CS or tolerance to CS. Primed T cells from mice immunized for CS are predominantly of the TH1 type. Conversely, a failure in the activation of these antigen-specific TH1 cells is a key step in the induction of tolerance to CS. Spleen cells from tolerant mice can suppress the production of lymphokines from primed cells, both in an adoptive transfer system and when mixed with effector cells in vitro. The relative importance of lymphokine competition, prostaglandin production by adherent cells, and other mechanisms underlying this suppression is discussed.     

Experimental allergic encephalomyelitis: clinical disease and enhanced cellular transfer in the absence of lymphocyte proliferative responses against syngeneic MBP

Experimental allergic encephalomyelitis (EAE) was induced in Lewis rats using several different immunization protocols, and draining lymph node cells from these animals were assayed for proliferation against heterologous, homologous, and syngeneic MBP, and syngeneic spinal cord. Proliferative responses were largely stimulated by nonsyngeneic antigenic determinants and correlated better with the antigen used to induce EAE than with signs of autoimmune disease. Lymph node cells from rats immunized with either guinea pig spinal cord or syngeneic MBP did not proliferate measurably when restimulated in vitro with syngeneic MBP, yet lymphoid cells from these animals were enhanced in their capacity to transfer EAE following in vitro stimulation with syngeneic MBP.     

Idiotope antigens (Ab2 alpha and Ab2 beta) can induce in vitro B cell proliferation and antibody production

We previously showed that immunization of various strains of mice with three types of antigen--PC-Hy (nominal antigen), F6-Hy (Ab2 alpha-Hy, and 4C11-Hy (Ab2 beta-Hy)--induces a differential PC-specific, T15-Id+ antibody response. In this report, the in vitro phosphorylcholine (PC)-specific B cell responses induced by these three antigens were studied. A hemocyanin-specific long-term T helper cell line was used to provide help for primary and secondary in vitro T cell-dependent B cell responses. At low doses (0.005 to 0.5 micrograms/ml) of antigen, a significant increase in the proliferation of PC-OVA-primed BALB/c B cells was observed with Ab2-Hy or PC-Hy conjugate, but not unconjugate, antigens. Similar low doses of antigen could stimulate naive B cells to secrete IgM and stimulate PC-OVA- or 4C11-Hy-primed B cells to secret IgM and IgG1 anti-PC antibodies. The percentage of T15-Id of the PC-specific antibodies produced in the in vitro T-B culture was found to be less dominant than that produced by in vivo immunization, suggesting that certain regulatory mechanisms occur in the in vivo environment that may help to maintain the T15-Id dominance. Taken together, our in vivo and in vitro results indicate that idiotope antigens can function like nominal antigens to induce antigen-specific B cell responses. The mechanisms of thymic-dependent B cell activation induced by idiotope and nominal antigen are similar in that the T-B interaction is MHC-restricted and requires cognate recognition.     

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.