Genetically restricted immune responses in guinea pigs primed in vivo with antigen-bearing macrophages.

Guinea pigs injected intradermally with antigen pulsed macrophages generate a population of immune T cells that proliferate in vitro on second exposure to antigen. T cells from F1 (2 X 13) guinea pigs immunized with DNP-OVA on one parental macrophage respond in vitro only to DNP-OVA on macrophages identical to those used for immunization and not to DNP-OVA associated with the other parental macrophages. These results demonstrate that the immunogenicity of antigen is dependent upon the macrophages used for priming in that, with this approach, strain 2 or 13 guinea pigs immunized with allogeneic macrophages pulsed with antigen do not respond to either allogeneic or syngeneic antigen-bearing macrophages. However, lysates of antigen-pulsed macrophages can still immunize either allogeneic or syngeneic recipient via their own macrophages. F1 (2 X 13) guinea pigs are immunized by insulin B chain pulsed strain 13 macrophages (responder) but not by strain 2 macrophages (nonresponder) suggesting that whether a F1 (nonresponder X responder) guinea pig recognizes antigen bound to a parental macrophage is genetically restricted before immunization to the same extent as the donor parental macrophages used for immunization.     

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.     

Enhanced transfer of experimental allergic encephalomyelitis with strain 13 guinea pig lymph node cells: requirement for culture with specific antigen and allogeneic peritoneal exudate cells

Previously, we reported that transfer of experimental allergic encephalomyelitis (EAE) with sensitized peritoneal exudate cells (PEC) in strain 13 guinea pigs is markedly enhanced if the cells are first cultured with specific antigen, myelin basic protein (BP). These cells also undergo considerable antigen-specific proliferation. In contrast, the data reported here show that lymph node cells (LNC) from sensitized animals display neither enhanced transfer nor antigen-specific proliferation after culture with BP. Enhanced transfer is obtained, however, if a second nonspecific signal is available. This second signal is provided by the presence of normal allogeneic strain 2 PEC in culture. After culture with BP and strain 2 PEC, 2.5 to 5 x 10(7) strain 13 LNC transfer disease reproducibly, in contrast with approximately 1 x 10(9) previously required for successful transfer. Addition of allogeneic or syngeneic PEC without antigen does not lead to enhanced transfer by LNC. Culture with normal syngeneic PEC plus BP oly infrequently enhances transfer by LNC. The intense mixed lymphocyte reaction (MLR) induced by addition of strain 2 PEC to strain 13 LNC precludes the use of 3H-TdR incorporation for detection of proliferation by EAE effector cells. However, inhibition of transfer with low doses of mitomycin C (2 to 5 micrograms/ml) pluse the fact that EAE effector cells are found almost exclusively in the light fraction of BSA gradients after (but not before) culture suggests that the latter are induced to proliferate in culture.     

Separation of lymphocyte mitogen from lymphotoxin and experiments on the production of lymphotoxin by lymphoid cells stimulated with the partially purified mitogen: a possible amplification mechanism of cellular immunity and allergy.

A mitogenic factor (MF) from guinea pig lymph node cells (LNC), which was produced by stimulating immune LNC with the specific antigen, ovalbumin, was partially purified by the sequential use of Sephadex G-200 gel filtration. CM-cellulose ion exchange chromatography, DEAE-cellulose ion exchange chromatography, and polyacrylamide disc gel electrophoresis. The product was purified at least 100-fold with regard to protein content. In addition, the purified MF was functionally pure with respect to the absence fo lymphotoxin (LT), another guinea pig lymphokine.     

5-HT(3) receptors mediate inflammation-induced unmasking of functional tachykinin responses in vitro.

Exogenously applied tachykinins produce no measurable electrophysiological responses in the somata of vagal afferent neurons [nodose ganglion neurons (NGNs)] isolated from naive guinea pigs. By contrast, after in vitro antigen challenge of nodose ganglia from guinea pigs immunized with chick ovalbumin, approximately 60% (53 of 89) of NGNs were depolarized an average of 13 +/- 1.2 mV by substance P (SP; 100 nM; n = 53). Receptor antagonists and enzyme inhibitors were utilized to screen a number of mast cell-derived mediators for their role in the uncovering or "unmasking" of functional tachykinin receptors after antigen challenge. Two chemically distinct 5-hydroxytryptamine-3-receptor antagonists significantly reduced the percentage of NGNs displaying depolarizing SP responses. Treatment with Y-25130 (1 or 10 microM) or tropisetron (1 microM) 15 min before and during antigen challenge reduced the percentage of SP-responsive neurons to approximately 20 and approximately 15% respectively. These results suggest that activation of 5-hydroxytryptamine-3 receptors plays an integral role in the unmasking of functional tachykinin receptors after specific antigen challenge of nodose ganglia. The mediator(s) underlying tachykinin-receptor unmasking in the remainder of the NGNs has yet to be characterized. However, it does not appear to be histamine, prostanoids, or peptidoleukotrienes.     

The role of myelin lipids in experimental allergic encephalomyelitis. III. Transfer of suppression from guinea pigs recovering from EAE, induced by myelin basic protein--galactocerebroside complexes

Juvenile strain 13 guinea pigs were immunized with myelin basic protein (MBP) combined with galactocerebrosides (MBP + GC) or with total myelin lipids without GC [MBP + (TL-GC)] in CFA. Control animals received dinitrophenylated-ovalbumin (DNP-OA) in CFA, CFA or IFA alone. The animals injected with MBP + GC showed a higher rate of recovery from the first EAE episode (83%) than those treated with MBP + (TL-GC) (50%). With the exception of the group treated with IFA alone, all animals were refractory to EAE following rechallenge with MBP in CFA 90 days after the first exposure. The in vitro proliferative response to MBP, of peripheral blood lymphocytes (PBLs) derived from guinea pigs freshly sensitized to MBP in CFA, was drastically suppressed in the presence of PBLs from animals injected with MBP + GC. Upon transfer to normal syngeneic recipients, spleen cells from MBP + GC-treated animals completely suppressed the clinical and histological manifestations of EAE following recipient challenge with MBP in CFA. Cell-free supernatants from PBLs and spleen cells of strain 13 guinea pigs treated with MBP + GC inhibited lymphocyte proliferation to MBP, of allogeneic responder cells, and spleen cell supernatants completely suppressed the induction of EAE upon transfer to allogeneic recipients. Suppression could not be transferred with cells from other treated groups. These results suggest that animals immunized with MBP + galactocerebrosides in CFA develop suppressor cells that may be in part responsible for the recovery from the first EAE episode and for protection against rechallenge with MBP in CFA. Their cell-free supernatants act in an MHC-nonrestricted fashion. These results do not rule out an additional protective mechanism since all animals exposed to CFA were refractory to rechallenge despite lack of demonstrable suppressor cell activity.     

Reduced anaphylactic responsiveness of strain 2 guinea pigs.

Strain 2 guinea pigs have been shown to have diminished anaphylactic responsiveness. In the present study, experiments were conducted comparing various characteristics of the anaphylaxis-resistant Strain 2 guinea pigs to those of an outbred anaphylaxis-prone Dunkin-Hartley strain. To bypass the possibility that differences in antibody titers accounted for the difference in anaphylactic reactivity, both strains of guinea pig were passively sensitized with the same amount of IgG antibody to ovalbumin. Measures of anaphylactic responsiveness to subsequent antigen challenge with ovalbumin included (i) systemically induced respiratory responses; (ii) isolated cardiac responses; and (iii) cutaneous responses. In all cases, using an amount of antibody sufficient to sensitize Dunkin-Hartley guinea pigs, the anaphylactic responses of the Strain 2 guinea pigs were either nonexistent or significantly less than those of the Dunkin-Hartley strain. To further determine which factors might be responsible for this difference, tissue histamine content, histamine releasability, and histamine responsiveness of the two strains were measured. The results of these studies indicated that the respiratory hyporesponsiveness of the Strain 2 guinea pigs may be due to a low pulmonary histamine content combined with reduced pulmonary responsiveness to histamine. However, since the cardiac histamine content and the responsiveness of the Strain 2 guinea pigs were not different from those of the Dunkin-Hartley strain, these factors cannot contribute to the reduced Strain 2 cardiac anaphylactic responsiveness. Compound 48/80 released equal quantities of histamine from the isolated hearts of the Strain 2 and the Dunkin-Hartley animals, but antigen challenge evoked histamine release only from the isolated Dunkin-Hartley hearts. We conclude that the cardiac anaphylactic hyporesponsiveness of the Strain 2 guinea pigs may be due to an inability of antigen to evoke release of anaphylactic mediators such as histamine.     

Susceptibility and resistance to experimental autoimmune encephalomyelitis and neuritis in the guinea pig correlate with the induction of procoagulant and anticoagulant activities

Activation of macrophage procoagulant activity (MPCA) is involved in the manifestation of EAE and EAN in susceptible guinea pigs and provides a mechanism for the deposition of fibrin, which is a feature of histologic lesions of EAE. Peritoneal exudate cells (PEC) from susceptible (strain 13) guinea pigs immunized with either central or peripheral nervous tissue antigens produce procoagulant activity when incubated with the immunogen in vitro. The production of the procoagulant is quantitative and antigen-specific and is maximal at the time of clinical signs of the disease. After recovery, the production of procoagulant activity decreased. The MPCA test was able to discriminate the biochemical differences existing between chicken and mammalian peripheral nerve proteins, thus providing a quantitative and sensitive indicator of cell-mediated immunity in EAE and EAN. The autoimmune response to brain and nerve antigens in nonsusceptible (strain 2) guinea pigs was coincident with the antigen-specific production of a cell-bound anticoagulant activity by stimulated mononuclear cells. The production of anticoagulant activity followed the same sequence of time changes after immunization as that of the MPCA in susceptible guinea pigs, and high immunizing doses of nerve antigens induced high levels of anticoagulant activity. The same cells produced high levels of procoagulant when incubated with tuberculin or lipopolysaccharide. The recalcification time of normal plasma was prolonged by the anticoagulant, and the decreased clotting time of plasma induced by the procoagulant activity obtained by incubating sensitized strain 13 PEC with myelin basic protein was suppressed by the anticoagulant produced by culturing sensitized strain 2 PEC with myelin basic protein. Preliminary evidence indicates that the anticoagulant has properties similar to antithrombin III. The anticoagulant could play a role in the control of effector cell function, and therefore in recovery from clinical features of EAE and EAN in susceptible guinea pigs.     

In vitro studies on transplantation immunity. I. MIF production by sensitive lymphocytes in mice

Sensitized lymphocytes from mice immunized with skin homografts produce migration inhibitory factor upon incubation with lymphocytes (antigen) from the sensitizing strain. The MIF is produced within 14 hr following incubation of sensitized lymphocytes and antigen. In this reaction, antigenic specificity is a prerequisite for MIF production; however, the action of MIF transcends the strain barrier. Also, MIF produced in homograft reactions in mice inhibited the migration of peritoneal cells from normal guinea pigs. Finally, lymphocytes from mice bearing skin homografts do not develop the capacity to produce MIF prior to the rejection of the sensitizing skin grafts.     

Effect of anti-lymphotoxin on cell-mediated cytotoxicity. Evidence for two pathways, one involving lymphotoxin and the other requiring intimate contact between the plasma membranes of killer and target cells.

A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro. The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro, one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells.     

Immunogenicity of Nanogram to Milligram Quantities of Inactivated Foot-and-Mouth Disease Virus. I. Relative Virus-neutralizing Potency of Guinea Pig Sera

Quantitative antigen dose-neutralizing antibody response curves were established in guinea pigs for purified foot-and-mouth disease virus (FMDV), type A, strain 119, inactivated for 48 hr with N-acetylethyleneimine (AEI). Inactivation of FMDV by 0.05% AEI at 25 C occurred without virus degradation and followed first-order kinetics over a 10(8)-fold decrease in plaque-forming units (PFU) extrapolating to 10(-5) PFU/ml at 48 hr. The AEI-treated virus was administered in doses ranging from 10 ng to 2.62 mg, alone or emulsified in oil adjuvant. Sigmoidal dose-response curves were obtained with 160 ng as the minimum effective dose. The maximum effective dose was 163 mug and 2.62 mg or more at 6 and 28 through 84 days postinoculation, respectively. Oil adjuvant had little effect at 6 days postinoculation, but its use markedly increased the amount of neutralizing antibody obtained at the later testing periods.     

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.     

Effect of brucellosis protective antigen on the phagocytic activity of the cells]

The authors present the results of study of the protective function of cells of the local inflammatory focus caused by the administration of live brucella culture to guinea pigs immunized with a protective antigen in the optimal (0.6) and increased (2 mg) doses. Macrophages of guinea pigs vaccinated with the optimal immunized dose of the protective antigen phagocytized brucellae actively; an increased dose of the antigen suppressed the ingestive and the digestive functions of macrophages, this apparently being caused by the manifestation of the immunodepressive activity of the residual amount of lipopolysaccharide (endotoxin) in the protective antigen.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity. II. Suppression of cutaneous delayed-type hypersensitivity by macrophage disappearance reaction

Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 micrograms of EA was injected into these animals and the animals were divided into two groups. One group of animals was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine gamma-globulin (DNP-BGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGG-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supernatants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.     

Alloantiserum-mediated supperssion of Ir gene controlled antigen-induced lymphocyte activation: effect on uridine and leucine incorporation.

Antisera to guinea pig histocompatibility antigens specifically suppress Ir gene-controlled antigen-stimulated, DNA-synthetic responses in vitro. To define further the mechanisms of alloantiserum-mediated suppression and to utilize this suppression as a probe of the cellular events occurring during lymphocyte activation, we have examined the effects of alloantisera on 14C-leucine and 3H-uridine incorporation, earlier events after antigen stimulation. The RNA and protein synthetic responses of peritoneal exudate lymphocytes from immunized guinea pigs to DNP-GL (controlled by a strain 2-linked Ir gene) are 40 to 50% of maximum by 24 hr in culture and at or near maximum by 48 hr. DNA synthesis was only 2% of maximum at 24 hr and maximum at 72 hr. Comparisons of the degree of stimulation of the incorporation of all three precursors reveals an excellent correlation between leucine and uridine but poor correlation between leucine and thymidine. Despite the observed differences in time course of incorporation and magnitude of response, the susceptibility to suppression by alloantisera of all three responses was virtually identical. Anti-2 sera, added at the initiation of culture, completely suppress all three responses. If addition was delayed 8 hr, 50 to 60% suppression was still demonstrated, whereas only minimal suppression was evidenced if addition was delayed 18 hr. These results suggest that by 8 to 10 hr of incubation with antigen, the responding cells have undergone the necessary biochemical and structural changes to eventuate in an immune response; and, furthermore, that alloantisera do not suppress by blocking antigen access to, or release from, its lymphocyte membrane receptor, but rather by a dynamic alteration of the lymphocyte membrane surface which interferes with the stimulus being generated by the antigen-receptor complex.     

Quantitation of the Antigenicity and Immunogenicity of Purified Foot-and-Mouth Disease Virus Vaccine for Swine and Steers

The antigenicity and immunogenicity of a purified preparation of foot-and-mouth disease virus [type A(12), strain 119 (FMDV A-119)] inactivated with 6.0 mmN-acetylethylenimine at 37 C were compared in swine and steers. Three antigen doses were tested, 640, 160, and 40 ng. In accordance with findings for guinea pigs, as previously determined by dose-response curves, as little as fourfold changes in antigen in the region of the minimum effective dose produced marked differences in the serological and immune responses of swine. The minimum effective dose of antigen for antibody formation in swine and guinea pigs, as determined by mouse median protective dose (PD(50)) values, was 160 ng. The minimum immunogenic dose for swine was also 160 ng. The vaccinated swine were challenged with either FMDV A-119 or with heterologous subtype A(24) strain Cruzeiro or type A strain A-CANEFA-1. Those immunized with 640 ng of antigen were about equally immune to the three challenge viruses; most swine having a mouse PD(50) value of 2.0 or greater were immune regardless of which strain was used for challenge. In steers, the smallest dose tested, 40 ng, was satisfactory in eliciting circulating antibodies and immunity. Physical and biological tests indicated that the antigen used in the vaccine is stable for at least 9 months at 4 C.     

Macrophage-T cell interaction mediated by immunogenic and non-immunogenic forms of a monofunctional antigen

Summary As an approach to the elucidation of the essential steps in the immune pathway, the uptake and retention of immunogenic and non-immunogenic analogs of a monofunctional antigen by guinea pig macrophages and the efficiency of macrophages pulsed with the compounds to present antigen to sensitized T lymphocytes were compared. L-Tyrosine-azobenzene-p-arsonate (RAT) and its non-immunogenic analog, 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate (RAN), react similarly with antiarsonate antibody, but RAN, unlike RAT, is unable to induce cellular immunity in guinea pigs. The uptake and retention patterns of the two compounds by macrophages differed in that, at a given time, more RAN than RAT was retained and detectable on cell surfaces by anti-arsonate antibody. Equivalent numbers of T lymphocytes from guinea pigs sensitized to RAT formed antigen-dependent clusters with macrophages pulsed with either RAT or RAN after 24 hr in culture, but not with macrophages pulsed with an azobenzenoid compound of unrelated specificity. On the other hand, T lymphocytes from guinea pigs immunized with RAN showed no significant capacity to bind to macrophages which had been pulsed with any of the compounds. The number of lymphocytes from RAT-sensitized animals which bound to RAT-pulsed macrophages remained relatively stable over a 48 hr period, whereas clusters of the same lymphocytes with RAN-pulsed macrophages dissocitated to background levels within that time. Early cluster formation mediated by RAN, as well as its ability to induce transient specific T cell unresponsiveness to RAT in vivo, indicate that T cells are capable of recognizing (binding) the non-immunogen. However, such early, and perhaps weak, interaction with RAN-pulsed macrophages did not induce DNA synthesis by T cells. Anti-Ia serum completely blocked cluster formation mediated by either RAT or RAN. Thus, the only significant distinction disclosed by these studies between the immunogenic and non-immunogenic compounds was the stability of macrophage-T cell interaction as determined by the persistence of antigen mediated cell clusters in culture, suggesting that this may be a factor in immunogenic discrimination.Abbreviations ABA azobenzenearsonate - BSA bovine serum albumin - CFA complete Freund's adjuvant - IFA incomplete Freund's adjuvant - KLH keyhole limpet hemocyanin - LNC Lymph node cells - MHC major histocompatibility complex - PEC peritoneal exudate cells - PEL peritoneal exudate lymphocytes - RAN 4-hydroxyphenyl-n-propane-3-azobenzene-p-arsonate - RAT L-tyrosine-azobenzene-p-arsonate - TAT L-tyrosine-azobenzene-p-trimethylammonium chloride Aided by USPHS Grant AI 05664.     

Alveolar macrophages in pulmonary immune responses. I. Role in the initiation of primary immune responses and in the selective recruitment of T lymphocytes to the lung

Antigen inoculated intratracheally (IT) into animals can induce primary immune responses and selectively recruit specific T cells to the lung. In the current study, the role of alveolar macrophages (AM) in these two responses was investigated. Antigen-pulsed bronchoalveolar cells (BAC) inoculated IT into guinea pigs generated a population of immune T cells that proliferated in vitro on reexposure to antigen-pulsed macrophages (M?). The possibility that antigen-pulsed donor BAC shed antigen that was subsequently processed and presented by host M? was ruled out by genetic experiments. Thus, peritoneal exudate lymphocytes (PEL) from (2 X 13)F1 guinea pigs primed with antigen-pulsed BAC from strain 2 animals responded preferentially to antigen-pulsed strain 2 M? rather than to antigen-pulsed strain 13 M?. In a second set of studies, antigen-pulsed BAC inoculated IT into guinea pigs selectively recruited antigen-specific T cells to the lung. Genetic experiments verified that inoculated BAC were the source of the antigen-presenting cells responsible for selective recruitment. Thus, antigen-pulsed strain 2 BAC inoculated IT recruited a greater proportion of (2 X 13)F1 T cells that recognized antigen in the context of strain 2 M? than F1 T cells that recognized antigen on strain 13 M?. Taken together, these studies suggest that AM contribute to the regulation of pulmonary immunity by both inducing T lymphocyte immunity and selectively recruiting specific T cells to the lung.     

Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

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.