Initiation of antibody responses by different classes of lymphocytes. V. Fundamental changes in the physiological characteristics of virgin thymus-independent ("B") lymphocytes and "B" memory cells

The life-span and migratory characteristics of rat thoracic duct cells which initiate the adoptive primary and secondary antibody response to diphtheria toxoid (DT) and horse spleen ferritin (HSF) were investigated. The experimental results show that thoracic duct lymphocytes from normal (unimmunized) donors are able to restore the adoptive response of irradiated hosts to HSF. Thoracic duct cells passaged through an intermediate host (intravenous injection and subsequent collection in the thoracic duct lymph) showed a marked reduction in their restorative action as compared with unpassaged cells. In addition, the restorative action of cells from donors treated with thymidine-³H for 48 hr before cannulation of the thoracic duct was markedly decreased. This indicates that a population of lymphocytes involved in the adoptive primary response is unable to recirculate from the blood to the lymph and is turning over rapidly (short lived). The nonrecirculating, short-lived lymphocytes are proably "B" cells, since a combination of spleen cells from neonatally thymectomized rats and passaged or thymidine-³H-treated cells restores a vigorous response to HSF. On the other hand, passaged or thymidine-³H-treated thoracic duct cells from donors immunized to DT or HSF are able to restore a vigorous adoptive secondary antibody response. Experiments with the hapten-protein conjugate, DNP-DT, show that the majority of both helper ("T") and precursor ("B") cells are able to recirculate and are slowly turning over (long lived). The findings suggest that T lymphocytes involved in both the primary and secondary antibody response are recirculating, long-lived cells. However, B lymphocytes involved in the primary response are nonrecirculating, short-lived cells ("B₁" cells) which undergo a fundamental physiological change to recirculating, long-lived cells ("B₂" cells) involved in the secondary antibody response.     

Carrier function in anti-hapten immune responses. I. Enhancement of primary and secondary anti-hapten antibody responses by carrier preimmunization

Preimmunization of either guinea pigs or rabbits to bovine gamma globulin (BGG) prepares the animals for markedly enhanced antibody responses to 2,4-dinitrophenyl-BGG (DNP-BGG). This phenomenon is observed both in the primary anti-DNP antibody response to DNP-BGG and in the secondary anti-DNP antibody response to DNP-BGG in animals primed with DNP-ovalbumin (DNP-OVA). The BGG preimmunization is most effective if the antigen is administered as a complete Freund''s adjuvant emulsion; in rabbits, a dose of 1 µg of BGG is more effective than a dose of 50 µg, whereas the reverse is true in guinea pigs. Transfusion of homologous anti-BGG sera fails to replace active immunization with BGG in the preparation of animals for these enhanced anti-DNP antibody responses. Both the immunoglobulin class and the average association constant for ε-DNP-L-lysine of the anti-DNP antibody produced in these enhanced responses is determined by the mode and time of immunization with haptenic conjugates and is not appreciably influenced by the nature of the carrier preimmunization. These studies indicate that the carrier specificity of hapten-specific anamnestic antibody responses is largely due to the interaction of two independent cell associated recognition units, one specialized for carrier and the other specific for haptenic determinants.     

PAG stimulation does not affect primary antibody responses in rats

Adult male rats, which had electrodes chronically implanted in the periaqueductal gray (PAG), were immunized with sheep red blood cells (SRBCs). The number of direct and indirect plaque-forming cells (PFCs) in the group receiving PAG stimulation after immunization did not differ significantly from that in the unstimulated group. Thus, the results indicate that short-term PAG stimulation does not suppress antibody-producing activity in the rat.     

Selective roles of thymus-derived lymphocytes in the antibody response. I. Differential suppressive effect of carrier-primed T cells on hapten-specific IgM and IgG antibody responses

Passively transferred thymocytes and spleen cells from donors primed with keyhole limpet hemocyanin (KLH) exerted differential suppressive effect on IgM and IgG antibody responses of syngeneic recipients immunized with DNP-KLH depending primarily on the time when KLH-primed cells were transferred. This was demonstrated by the decrease in the numbers of DNP-specific direct and indirect PFC in the spleen of the recipients given KLH-primed cells at different times during primary and secondary immunization. Whereas the cell transfer simultaneously with or 2 days after the primary immunization produced only slight suppression of the peak IgM antibody response, it caused profound suppression of late IgM and IgG antibody responses. By contrast, the cell transfer 3 days after the immunization produced immediate suppression of the ongoing IgM antibody response resulting in its earlier termination, while being unable to prevent the induction of IgG antibody response. KLH-primed cells could moderately suppress the secondary anti-DNP antibody response, in which IgG antibody response was found to be slightly more sensitive than IgM antibody response to the suppressive influence of KLH-primed cells. The suppressive effect of the KLH-primed spleen cells was completely eliminated by the in vitro treatment of the cells with anti-θ and C before cell transfer, indicating that cells responsible for the suppression are, in fact, T cells. The suppression of DNP-specific antibody response by KLH-primed T cells was achieved only if the recipients were immunized with DNP-KLH but not with DNP-heterologous carrier, suggesting that direct interaction between T and B cells is necessary for the suppression of the antibody response. It is concluded that susceptibility of B cells to the specific suppressive influence of T cells is inherently different depending on the differentiation stage of B cells and on the immunoglobulin class they are destined to produce.     

Type I interferon inhibits antibody responses induced by a chimpanzee adenovirus vector.

Recent studies have indicated that type I interferon (IFN) enhances antibody responses and promotes isotype switching. In this study, we analyzed the role of type I IFN signaling during the generation of transgene product-specific antibody responses elicited by recombinant adenovirus (Ad) vectors. A vector derived from a human Ad serotype (AdHu5) induced low levels of type I IFN following infection of dendritic cells (DCs) and stimulated normal transgene product-specific antibody responses in mice that have a defective type I IFN receptor (IFNAR(-/-)). A vector derived from a chimpanzee Ad serotype (AdC68) induced very high levels of type I IFN following infection of DCs, and surprisingly, primed stronger transgene product-specific antibody responses in IFNAR(-/-) mice compared to wild-type mice. The increased antibody response in IFNAR(-/-) mice vaccinated with the AdC68 vector was mainly due to the generation of IgG1 antibodies that were not elicited in wild-type mice. The induction of IgG1 antibodies correlated with an increase in transgene product expression in IFNAR(-/-) mice and was not associated with an increase in T helper 2 responses. We conclude that type I IFN, when induced at high levels, can downregulate transgene product expression of Ad vectors and inhibit the formation of optimal antibody responses.     

Clustering of dendritic cells, helper T lymphocytes, and histocompatible B cells during primary antibody responses in vitro

Mouse spleen suspensions generate discrete cell clusters within 1-2 d of culture. We have isolated these clusters by velocity sedimentation to study their contribution to primary antibody responses. Clusters represent approximately 5% of the starting spleen cells and consist of 20-50% B cells, 20-50% T cells, and 10-20% dendritic cells (DC). When the cultures are stimulated with thymus-dependent antigens, like heterologous red cells or dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), the clusters are the principal site for the development of plaque-forming cells (PFC). Noncluster fractions form few PFC and only when supplemented with fresh DC. PFC responses in all cases are antigen specific. B cells cluster only in the presence of T cells and DC (1 DC/200 B-T cell mixtures) and only after encountering specific antigen. The elimination of either DC or Lyt-1+2- T cells, with monoclonal antibody and complement, ablates B cell development into PFC. PFC responses are restored with antigen-nonspecific helper factors formed in the syngeneic mixed leukocyte reaction between DC and T cells. Since PFC to DNP-KLH do not develop de novo when B cells are exposed to antigen and helper factors, anti-DNP PFC precursors must be stimulated within clusters to become responsive to helper factors. PFC development within clusters is restricted by the major histocompatibility complex (MHC). When DC and T cells are from strain P1, then P1 but not P2 B cells develop into PFC; when DC are from strain P2 and T cells from strain P1, strain P2 B cells are selected to become PFC in clusters. The entry of B cells into clusters is itself MHC restricted, since P1 DC/T cells aggregate six times as many B cells from strain P1 as strain P2. Thus, clusters are the site in which DC, B, and T cells interact to generate PFC. One can use clusters to retrieve B cells that have been selected in an antigen-dependent, MHC-restricted fashion and to show that clustering B cells become responsive to soluble, polyclonal helper factors.     

Systemic responses to different enteral feeding schedules in rats.

The purpose of this study was to describe selected temporal systemic responses to different enteral feeding schedules using an animal model, as one prerequisite for determining optimal delivery times for human enteral feedings. The effect of feeding schedules and time of day were examined relative to body weight and plasma glucose, insulin, glucagon, and corticosterone levels. Adult male Sprague-Dawley rats (N = 80) were used in a 4 x 4 randomized block design. Rats started the feeding schedules on Day 11 post-gastrostomy tube placement. The following feeding schedules, often used by humans, were tested: 24-hour enteral; 24-hour oral; 12-hour (rest time) enteral; and 12-hour (rest time) oral. Plasma was collected on Day 20. Despite very different feeding schedules, there were no statistical differences in weight gain, or plasma glucose and glucagon levels. Levels of insulin and corticosterone were significantly influenced by time of day and/or feeding group. Temporal pattern disruption was greatest in the rest-time fed enteral group. Both route and timing of enteral delivery modified metabolic hormone patterns.     

Epitope-specific regulation. I. Carrier-specific induction of suppression for IgG anti-hapten antibody responses

The epitope-specific regulatory system selectively controls IgG antibody production to the individual (haptenic) determinants on a complex antigen. This system can be specifically induced to suppress primary and secondary IgG antibody responses to dinitrophenyl hapten (DNP) without interfering with antibody responses to epitopes on the carrier molecule on which the DNP is presented. Furthermore, once induced, it will specifically suppress responses to DNP presented on unrelated carrier molecules. Results summarized here obtained using widely different immunization conditions, and a variety of haptens and carrier molecules indicate that this regulatory system controls antibody production in most T-dependent antibody responses. Carrier-specific suppressor T cells (CTs) that arise shortly after priming with a carrier molecule such as keyhole limpet hemocyaninin (KLH) induce the epitope-specific system to suppress in situ and adoptive antibody responses to epitopes (e.g., DNP) presented subsequently on the priming carrier. These well-known regulatory T cells are commonly believed to regulate antibody production by interfering with carrier-specific help; however, by repeating the original CTs transfer experiments with additional controls that define the specificity of the mechanism mediating suppression in CTs recipients, we show that KLH-specific CTs regulate responses by inducing typical isotope- specific suppression for anti-DNP responses when the recipients are immunized with DNP-KLH. Thus, whether KLH-primed animals are immunized directly with DNP-KLH (KLH/DNP-KLH immunization sequence) or whether T cells from these animals are challenged with DNP-KLH in (nonirradiated)recipients, anti-DNP responses are persistently suppressed while anti-carrier responses proceed normally. The aqueous KLH-priming protocols usually used to generate CTs are marginally more effective in priming for in situ suppression-induction than the alum KLH-priming protocols commonly used to generate KLH-specific helper T cells and used here in KLH/DNP-KLH immunizations. Thus, studies presented show that priming with an antigenic (carrier) molecule simultaneously prepares the animal for the production of typical secondary (anamnestic) antibody responses to epitopes on the priming antigen and for the induction of epitope-specific suppression for antibody production to determinants presented subsequently on the same antigenic molecule. We discuss the mechanism(s) responsible for this duality and its significance for antibody responses in an accompanying publication that describes the bistable regulatory capabilities of the epitope-specific system.     

Polyclonal activation of human B lymphocytes by Fc fragments. I. Characterization of the cellular requirements for Fc fragment-mediated polyclonal antibody secretion by human peripheral blood B lymphocytes

Fc fragments derived from human immunoglobulin were found to be capable of inducing both a proliferative and polyclonal antibody response in human peripheral blood lymphocyte cultures. The cell population proliferating in response to Fc fragments belongs to the B cell lineage. Expression of polyclonal antibody formation requires the presence of both adherent monocytes and T cells. The role of the monocyte is to enzymatically cleave the Fc fragment into 19,000 mol wt Fc subfragments that are then able to induce polyclonal antibody secretion. Stimulation of polyclonal antibody production by Fc subfragments occurs in the absence of adherent monocytes but still requires the presence of T cells.     

Cell interactions between histoincompatible T and B lymphocytes. VII. Cooperative responses between lymphocytes are controlled by genes in the I region of the H-2 complex

The results of this study provide compelling evidence for the existence of the gene or genes controlling optimal T-B-cell cooperative interactions in the designated I region of the H-2 gene complex. Previously, we have speculated that the relevant gene(s) involved may well be located in this region based on several observations from our earlier work in this area (3, 5, 6). Thus, in the preceding paper, we showed that T and B cells from B10.BR and A strain mice developed effective cooperative interactions in vitro to DNP-KLH in a system identical to the one reported herein. Since these mice differ for genes in the S and D regions of H-2 but are identical for K and I region genes, we were able to localize the critical genes to the K-end of H-2.     

Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy

We have investigated the level of TCR occupancy required to elicit different biological responses in human CTL clones specific for an influenza matrix peptide. Specific cytotoxicity could be detected at extremely low peptide concentrations (10(-12) to 10(-15) M). However, IFN-gamma production, responsiveness to IL-2 and Ca++ fluxes were observed only at peptide concentrations > 10(-9) M, while autonomous proliferation required even higher peptide concentrations. In parallel experiments we measured TCR downregulation to estimate the number of TCRs triggered. We observed that at low peptide concentrations, where only cytotoxicity is triggered, TCR downregulation was hardly detectable. Conversely, induction of IFN-gamma production and proliferation required triggering of at least 20-50% of TCRs. Taken together these results indicate that a single CTL can graduate different biological responses as a function of antigen concentration and that killing of the specific target does not necessarily result in full activation.     

Temporal correlation of antibody responses to different epitopes of the human La autoantigen.

To investigate the temporal relationship of antibody responses to different La epitopes, sequential sera from nine patients with systemic lupus erythematosus and Sjogren's syndrome were tested by enzyme-linked immunosorbent assay for antibody binding to a series of recombinant fusion proteins containing different regions of the La molecule. The results of this analysis indicate that antibody responses to four different La fragments vary in parallel over time. This finding is supported by a statistical analysis indicating that the changes in antibody levels between the six pairs of responses were highly correlated (P less than 0.001). Furthermore, we show by immunoaffinity purification that antibodies to the three nonoverlapping La protein fragments do not cross-react with other fragments and, hence, represent independent populations. These results suggest that anti-La antibodies are coordinately produced to different epitopes on the La molecule, possibly reflecting an antigen-driven mechanism.     

Epidural opioid analgesia in infant rats I: mechanical and heat responses.

The aim of this study was to investigate the analgesic effects of epidural opioids in neonatal rat pups. The contribution of individual opioid receptor subtypes in the spinal cord to analgesia at different developmental stages was investigated using epidural mu (morphine sulphate), delta (DPDPE) and kappa (U69593) opioid receptor agonists in neonatal rats aged postnatal day (P) 3, 10 and 21. Thresholds for flexion withdrawal reflexes to mechanical stimuli (von Frey hairs) and to noxious heating of the hind paw were low in neonates and increased with postnatal age. The analgesic action of each opioid receptor agonist followed an individual developmental pattern. In mechanical tests, all three opioid agonists were considerably more efficacious analgesics in younger animals and ED50s at P3 were always lower than at P21. In heat tests, the pattern differed. The efficacy of the kappa opioid agonist decreased with postnatal age, morphine efficacy increased over the same period and the effects of the delta agonist remained relatively unchanged. The distribution and concentration of tritiated morphine in the spinal cord following epidural administration did not alter significantly with postnatal age, suggesting that opioid access is not a major determinant of the effects reported here. It is concluded that whereas heat pain is particularly sensitive to spinal kappa opioids in neonates, mechanical sensory thresholds are generally sensitive to all spinal opioids in the newborn. The differing epidural opioid requirements compared to older subjects is likely to be due to developmental changes in spinal cord opioid receptor distribution or pharmacology.     

Viruses disrupt functions of human lymphocytes. II. Measles virus suppresses antibody production by acting on B lymphocytes

Measles virus infection is associated with suppression of immune functions both in vivo and in vitro. The virus infects T lymphocytes, B lymphocytes, and monocytes, but does not produce cytolysis. One consequence of infection in vitro is the failure of T and B lymphocyte mixtures to cooperate in secreting Ig in a PWM-driven system. Here we report that this defect in Ig secretion resides in the infected B lymphocyte, but not in the T lymphocyte or monocyte. Further, NK cells are not involved, since neither their depletion nor reconstitution abrogates suppression of B cell function. Proliferation of B cells in the early culture period is suppressed, suggesting that measles virus suppresses B cell development at the activation or proliferation stages, but does not affect terminal differentiation into Ig secreting cells.     

Modulation of cellular-immune responses in vivo and in vitro by histamine receptor-bearing lymphocytes.

Histamine, one of the mediators involved in the IgE-mediated reaction, was demonstrated to influence in vivo and in vitro components of cellular-immune reactions in orthochlorbenzoyl-bovine gamma globulin-immune guinea pigs. 10(-3) M histamine reduced by half the size of a delayed hypersensitivity skin test at 24 h. Inhibition of skin reactivity by histamine could be partially reversed by H-1 receptor antagonists such as chlorpheniramine and completely prevented by H-2 receptor antagonists such as burimamide. The histamine suppression of cutaneous delayed hypersensitivity could be accounted for in part by its inhibitory effect on certain lymphocyte responses including antigen-induced migration inhibitory factor (MIF) production and proliferation. At concentrations of 10(-3)-10(-5) M histamine reversibly inhibited MIF production and its action could be blocked by H-2 antagonists but not H-1 antagonists. Thus, lymphocytes bearing H-2 receptors modulate MIF production and probably lymphocyte proliferation as well. Histamine did not interfere with the macrophage response to preformed MIF. These studies indicate that immediate hypersensitivity reactions involving histamine release might influence the subsequent expression of cellular-immune reactions.     

Nature of the antigenic complex recognized by T lymphocytes. I. Analysis with an in vitro primary response to soluble protein antigens

In order to analyze the genetic factors involved in the regulation of macrophage-T-cells interaction we have developed an in vitro primary response to soluble protein antigens in which nonimmune guinea pig T cells can be sensitized and subsequently challenged in tissue culture with antigen-pulsed macrophages. Antigen-specific T-cell activation, as measured by increased DNA synthesis, occurred when syngeneic antigen-pulsed macrophages were used for both initial sensitization and secondary challenge. No T-cell activation occurred when allogeneic antigen-pulsed macrophages were used for secondary challenge of cells primed when syngeneic macrophages. When allogeneic antigen-pulsed macrophages were used in both primary and secondary cultures it was difficult to assess antigen-specific stimulation due to the substantial mixed leukocyte reaction. However, when T cells from F1 animals were primed with parental antigen-pulsed macrophages they responded only to the parental macrophages used for initial sensitization but not to those of the other parent. These results are discussed with respect to T-cell recognition of a complex antigenic determinant which may include I-region gene products.     

Regulation of reaginic antibody production in mice. I. Suppression by antigen of IgE antibody production in vitro

Regulation of IgE production by antigen in a primed murine splenic lymphocyte culture system was described. Maximum IgE antibody production was found to occur when cells were cultured in the absence of exogenously added antigen. A cells and T lymphocytes did not affect the production of anti-DNP IgE antibody. By using a hapten-carrier antigen system (DNP-EA) for priming mice in vivo, it was found that the production of anti-DNP IgE by spleen cells in vitro was inhibited by hapten when coupled to homologous (EA) or heterologous (BGG) carrier, and was not enhanced or inhibited by homologous carrier. Anti-DNP IgE antibody production by cultures depleted of macrophages or T lymphocytes was found to be as sensitive to the suppressive effects of hapten as was the IgE production by whole spleen cell cultures. Both IgM and IgG secondary anti-DNP PFC responses in vitro were enhanced by the presence of the homologous hapten-carrier or carrier alone. DNP-BGG had no effect on the anti-DNP IgM or IgG PFC responses of the cultures. These data suggest that endogenous production of antibody (IgM or IgG) was not responsible for the observed suppression of the IgE response in vitrol The experimental results presented indicate that the regulation of the IgE production by antigen in the primed mouse splenic lymphocyte cultures was a consequence of the direct interaction of hapten with IgE B cells.     

The allogeneic effect in inbred mice. I. Experimental conditions for the enhancement of hapten-specific secondary antibody responses by the graft-versus-host reaction

The administration of allogeneic lymphoid cells to 2,4-dinitrophenyl keyhole limpet hemocyanin (DNP-KLH)-primed mice prepares such recipients for markedly enhanced secondary anti-DNP antibody responses to a DNP conjugate of a heterologous carrier. This allogeneic effect phenomenon, reflecting the development of a graft-versus-host (GVH) reaction, was first described in guinea pigs and has been extended in the present studies to inbred mice. The expression of the allogeneic effect in mice is dependent upon critical factors such as the number and route of administration of allogeneic cells, the time interval between cell transfer and secondary challenge, and the strength of histocompatibility differences between the donor and the host. The transient GVH reaction established by the transfer of allogeneic cells obviates the requirement for carrier-specific helper T cells in secondary anti-DNP responses, as evidenced by the ability to elicit such responses with DNP-D-GL, a substance which presumably does not stimulate effective T cell helper function. These studies also demonstrate that primed B cells which are not an integral part of the active GVH reaction fail to produce enhanced levels of antibody.     

Haplotype-specific suppression of antibody responses in vitro. I. Generation of genetically restricted suppressor T cells by neonatal treatment with semiallogeneic spleen cells

C57BL/10 mice were injected with semiallogeneic (B10.D2 X C57BL/10)F(1) spleen cells via the anterior facial vein within 24 h of birth to induce tolerance to B10.D2 (H-2(d)) alloantigens. Spleen cells from these mice as adults developed reduced, but significant, mixed lymphocyte and cytotoxic lymphocyte responses in vitro to H-2(d) stimulator cells and these treated mice rejected first-set B10.D2 skin grafts within a normal time-course, indicating that at best only a state of partial tolerance had been induced. Spleen cells from these mice failed to develop antibody responses to a variety of antigens in vitro when H-2(d) macrophages were in the cultures. Partially purified T cells from these neonatally treated mice suppressed primary antibody responses by normal syngeneic spleen cells in the presence of H-2(d) but not other allogeneic macrophages. These radiosensitive, haplotype-specific suppressor T (Ts) cells inhibited primary antibody responses by blocking initiation of the response, but failed to suppress secondary antibody responses and mixed lymphocyte or cytotoxic lymphocyte responses by appropriate responding spleen cells. To activate H-2(d) haplotype-specific Ts cells, stimulation with IA(d) subregion antigen(s) was necessary and sufficient; syngenicity at the I-A subregion of H-2 between the activated Ts cells and target responding spleen cell populations was also necessary and sufficient to achieve suppression. Comparable results have been obtained with spleen cells from BALB/c mice injected as neonates with (B10.D2 × C57BL/10)F(1) spleen cells where IA(b) antigens activate the haplotype-specific Ts cells. Implications for the significance of this population of haplotype-specific Ts cells in immune regulation are discussed and the properties of these Ts cells are compared and contrasted with other antigen-specific and nonspecific Ts cells whose activity is restricted by I- region products.