anti-TNP Response Research Articles

Functional Characteristics of Peyer's Patch Lymphoid Cells

Abstract The frequency of B cells in Peyer's patches from normal BDF1 and sheep red blood cell (SRBC)-fed BDF1 mice that could respond to antigenic determinants on SRBC and trinitrophenyl (TNP) was determined using an in vitro system of limiting dilution analysis. In normal mice, one B cell in 1.9 × 104 Peyer's patch cells could be induced to an anti-SRBC response and one B cell in 3.6 × 104 Peyer's patch cells could be induced to an anti-TNP response. The frequency of B cells capable of responding to SRBC in normal mice was similar in Peyer's patches and spleen. However, after feeding mice SRBC for 3 weeks, there was a 6-fold reduction in the frequency of B cells in Peyer's patches capable of responding to SRBC but no change in the frequency of B cells capable of responding to TNP. The average clone size of Peyer's patch B cells responding to SRBC was similar in normal and SRBC-fed mice. Although antigen-feeding does not stimulate Peyer's patch B cells in situ to humoral antibody synthesis, antigen-feeding can markedly alter the reactivity of the antigen-sensitive cell population in Peyer's patches. We previously demonstrated that T cells in Peyer's patches could be specifically carrier primed for helper function by SRBC feeding. We have now demonstrated that antigen-feeding reduced significantly the frequency of B cells in Peyer's patches capable of responding to the fed antigen. Peyer's patches appear to serve an important function as a sampling site for intestinal antigens.

Primary in Vitro Antibody Response from Human Peripheral Blood Lymphocytes

Abstract A method for the induction of a primary in vitro antibody response from human peripheral blood lymphocytes is presented. Upon cultivation with trinitrophenyl conjugated polyacrylamide beads (TNP-PAA), an anti-TNP response can be obtained as indicated by the appearance of direct plaque-forming cells from day 5 of culture, with a reproducible peak on day 8. These plaques correspond to cells actively producing antibody of the IgM type, as shown by their inhibition by cycloheximide and by anti-human IgM serum, but not by anti-human Fc γ serum. Their specificity for the TNP hapten can be demonstrated by the effector cell blockade phenomenon, with highly substituted TNP-human IgG. Although the anti-TNP response induced by TNP-PAA in mouse spleen cell cultures appears T independent the same response in human PBL may involve in addition the participation of T cells, since E-RFC depletion before culture led to a markedly decreased number of plaque-forming cells. A significant response could be obtained from the PBL of all of the 30 normal individuals tested. Importantly, the response was reproducible in its magnitude in the six individuals tested in at least three different experiments. Thus, the in vitro stimulation of human PBL by TNP-PAA can be proposed as a reliable test for the study of human B cell function in a specific primary antibody response.

Carrier-specific unresponsiveness in reaginic antibody formation: I. Induction of helper cell tolerance in rats

Rats pretreated with 10 mg soluble sheep gamma globulin (soluble SGG or SGGSo) showed a marked reduction in IgE anti-TNP antibody upon challenge with TNP-SGG in alum. This effect was found to be carrier-specific since SGGSo tolerant rats gave normal IgE anti-TNP responses when challenged with TNP-KLH. Carrier tolerance persisted when a secondary challenge with TNP-SGG was given. The degree of tolerance induction was shown to be tolerogen dose-dependent. As expected, IgE anti-SGG responses were also reduced in this system by pretreatment with SGGSo. These results indicate that carrier-specific helper cells for IgE antibody responses can be rendered functionally unresponsive. However, in contrast to the long duration of carrier-specific tolerance for IgM and IgG responses, IgE tolerance in this system appeared to be only short-lived. This difference suggests that IgE helper and/or suppressor T cells may represent a separate subclass from those involved in IgM and IgG responses.

The affinity and spectrum of cross reactivity of anti-body production in senescent mice: The IgM response

Experiments were performed to determine whether the IgM component of the primary humoral immune response of senescent mice differs from that of young-adult mice in its affinity, specificity or heterogeneity. For this purpose, mice were immunized with the T-dependent antigen, TNP-KLH, or the T-independent antigen, TNP-LPS. Affinity and specificity of the anti-TNP response were studied at the cellular level by the plaque inhibition technique using the cross-reacting haptens TNP-ϵ-aminocaproic acid (TNP-EACA), DNP-EACA and ONP-EACA. It was found that when the immunogen was TNP-KLH, the peak IgM response was delayed in senescent animals by several days as compared to the response in young adults. However, the number of PFC on the day of peak response was comparable. No age related delay in the peak of response was detected when the antigen was TNP-LPS although the magnitude of response was reduced in old age. The average affinity of the plaque forming cell responses to both immunogens is comparable in young and old mice.

Regulation of the Immune Response in Neonatal Piglets by Maternal Antibody

The ability of maternal antibody to regulate the humoral immune response to sheep erythrocytes and the hapten-carrier conjugate trinitrophenylated sheep erythrocytes (TNP-SRBC) was investigated in neonatal piglets. It was found that in this system, passively acquired maternal antibody would completely inhibit the in vitro primary immune response to SRBC while leaving the response the TNP intact. Data suggest that maternal antibody is regulating the in vitro response at the B-cell level since T-cell helper function to SRBC must not have been inhibited in order for an anti-TNP response to have occurred to TNP-SRBC. The regulation of immune responses by maternal antibody is temporary and disappears before 3 months of age.

Autoimmunity and Aging: The Age-Related Response of Mice of a Long-Lived Strain to Trinitrophenylated Syngeneic Mouse Red Blood Cells

Mice of 1.5, 9, 22, and 31 to 32 months of age were injected with the thymus-dependent antigen, TNP-SRC, or the thymus-independent antigen, TNP-SRC, TNP-MRC. The anti-SRC and TNP immune responses to TNP-SRC were markedly reduced in older mice, whereas the anti-TNP response to the TNP-MRC showed no substantial decline. Young mice produced higher anti-TNP plaque-forming cell responses after injection of TNP-SRC than after TNP-MRC, whereas in older mice the reverse obtained. Old mice but not young mice displayed a high anti-SRC cross-reactive response after injection of TNP-MRC. The avidity of anti-TNP antibody of young mice immunized with TNP-SRC was higher than that following immunization with TNP-MRC, whereas the avidities of anti-TNP antibodies from old mice injected with these two reagents were the same. Those individual mice which showed a poorly regulated immune response also displayed an autologous anti-MRC plaque-forming cell response after injection of either TNP-SRC or TNP-MRC. It is suggested that mechanisms mediated by suppressor T cells may be responsible for regulating the autoimmune response to modified self antigens, and that these are severely impaired in age individuals.

Immune response of New Zealand mice to trinitrophenylated syngeneic mouse red cells.

NZB and NZB/W mice have reduced anti-sheep red cell (SRC) and 2,4,6-trinitrophenyl-plaque-froming cell (TNP-PFC) responses with age after injection of either the thymus-dependent antigen TNP-SRC or the thymus-independent antigen TNP-mouse red cells (MRC). However, the thymus-dependent response diminished much faster than the thymus-independent response. As a consequence, young New Zealand mice have a higher anti-TNP response after injection of TNP-SRC than after injection of TNP-MRC, while old New Zealand mice have a higher anti-TNP response after injection of TNP-MRC than after injection of TNP-SRC. The PFC avidity of NZB/W mice injected with TNP-SRC diminished with age, while the PFC avidity of mice injected with TNP-MRC did not change with agrc or TNP-SRC. Old NZB/W mice had few spontaneous anti-MRC-PFC. The number of anti-MRC PFC in old mice was increased 4 to 10 times after injection with either TNP-SRC or TNP-MRC. It is suggested that surveillance mechanisms are responsible for suppressing the autoimmune response to modified self-antigens. The unregulated immune system of NZB and NZB/W mice appears to be an expression of impairment of such a hypothetical surveillance mechanism.

In Vitro Responses of CBA/N Mice: Spleen Cells of Mice with an X-Linked Defect that Precludes Immune Responses to Several Thymus-Independent Antigens Can Respond to TNP-Lipopolysaccharide

Spleen cells from CBA/N mice with an X-linked B cell defect were examined for their ability to form antibody in vitro after stimulation with the T-independent antigen TNP-LPS. In contradistinction to their failure to respond to some conventional T-independent antigens such as type III pneumococcal polysaccharide or DNP-AECM-Ficoll, spleen cells from (CBA/N X DBA/2)F1 male mice were able to make a specific anti-TNP PFC response after culture with TNP-LPS. Their response differed from that of phenotypically normal (CBA/N X DBA/2)F1 female littermate spleen cells in that more TNP-LPS was required to elicit the peak anti-TNP response and the anti-TNP antibody secreted by F1 male cells was of lower avidity than that of F1 female cells. The polyclonal antibody response to unsubstituted LPS did not differ substantially between normal and defective B cells. Tnymus-derived cells were not required for the TNP-LPS response by either F1 male or female cells. We conclude that CBA/NB cells can respond to certain T-independent antigens that are able either to induce a very strong activating signal upon ligand-surface receptor interaction and/or to stimulate immature B cells (with a characteristic high surfact immunoglobulin profile) which fail to respond to antigens like DNP-AECM-Ficoll.

Modulation of Mouse Anti-Trinitrophenyl Plaque-Forming Cell Affinity by Adjuvants or Lectins

The efffects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.

Modulation of Help and Suppression in a Hapten-Carrier System

Provision of beta-galactosidase (GZ) under defined conditions of dose and time can either help or suppress a subsequent response to trinitrophenyl (TNP)-GZ in CBA/J mice. The optimal helper effect occurs when 10(7) spleen cells from mice primed 9 or more days previously with 10 mug GZ are adoptively transferred to irradiated recipients which are than challenged with 10 mug TNP50GZ. Optimum suppression results from the transfer of spleen cells from mice primed 3 days previously with 100 mug GZ and challenge of recipients with TMP150GZ. Both help and suppression are carrier-specific and mediated by T cells. In experiments where helper or suppressor cells were mixed with normal cells, the anti-TNP response was proportional to the number of primed cells transferred. The results point to a wave of suppression as the initial event after immunization, which is succeeded by period in which the helper effect dominates.

In Vitro Immune Response to the 2,4,6-Trinitrophenyl Determinant in Aged C57BL/6J Mice: Changes in the Humoral Immune Response to, Avidity for the TNP Determinant and Responsiveness to LPS Effect with Aging

An in vitro anti-TNP response of the spleen cells from aged C57BL/6J mice showed approximately 4-fold less PFC than did that from young adult mice. Anti-theta serum-treated young spleen cells gave an anti-TNP response that was definitely greater than the response of the anti-theta serum-treated aged spleen cells in the presence of the exogenous activated thymus cells as helper cells. These results suggest that the deficits in B cells may be partly responsible for the imparied anti-TNP response of the aged spleen cells. To examine further the capacity of stem cells in the bone marrow to generate B cells responsible for anti-TNP response in the spleen, we injected i.v. 1.5 to 2.0 times 10(7) bone marrow cells from young or aged mice into lethally irradiated syngeneic recipients that had previously been thymectomized. Four to 6 weeks later, 10(7) spleen cells from the two groups of these recipient mice were immunized with TNP-SRBC in the presence of the exogenous activated thymus cells and assayed for anti-TNP PFC. The response of the aged marrow-derived B cells was approximately one-half of that of the young marrow-derived B cells. The avidity for TNP determinant of the antibodies produced by the PFC was determined by the plaque-inhibition technique. The avidity of the antibodies produced by the aged mice was approximately 33 times lower than that by the young mice. Anti-TNP response of the young spleen cells were markedly enhanced by the addition of LPS to the cultures, whereas no or little enhancement of the response was induced in the aged spleen cells even in the presence of high concentration of LPS. In contrast, DNA synthesis of both the young and aged spleen cells was comparably stimulated by 1 mug/ml and 10 mug/ml of LPS, however, it was rather less in the aged spleen cells at a concentration of 100 mug/ml. Mechanisms responsible for the changes in avidity and responsiveness to LPS with aging are discussed.

In Vitro Immunogenicity of Trinitrophenylated Bacteriophage T4

Abstract A primary immune response was obtained in vitro to trinitrophenylated bacteriophate T4 (TNP-T4) by using normal BALB/c spleen cells challenged in vitro. The primary response was limited to anti-TNP; we failed to find evidence of a primary anti-T4 response in spleen cell cultures challenged with either TNP-T4 or native T4. In vivo priming with the carrier T4 failed to enhance the subsequent in vitro response to TNP-T4 although such priming sometimes caused suppression of the anti-hapten response. The addition of excess free carrier to spleen cell cultures challenged with TNP-T4 failed to suppress the anti-TNP response. Spleen cells treated with anti-θ plus complement were unaffected in their ability to respond to TNP-T4. We conclude that TNP-T4 should be classified among the thymus independent antigens.

Induction of an immune response through interaction of helper cells with an immune complex bound to the surface of B cells.

Adoptive anti-trinitrophenyl (Tnp) responses were elicited from Tnp-hemocyanin (Tnp-KLH)-primed cells by challenge with an immune complex of KLH and a Tnp conjugate of the Fab fragment of rabbit anti-KLH. Removal of T cells by treatment with anti-Thy 1.2 (phi C3H) + complement abolished this effect. Tnp-KLH primed cells that had been incubated with a Tnp conjugate of pneumococcal polysaccharide type III (Tnp-SIII), and which were then unable to respond to Tnp-KLH, made an anti-Tnp response upon incubation with rabbit anti-Dnp when transferred together with cells primed with rabbit gamma globulin. Since Tnp-KLH was not added in these experiments, it would appear that the cells were triggered by immune complexes on their surfaces consisting of Tnp-SIII and rabbit anti-Dnp with the help of T cells primed with rabbit gamma globulin; the presence of free antigen was evidently not required. Therefore, antigen-antibody complexes, when bound to the surface of B cells, may mediate T-B cell cooperation in the humoral immune response.

Lack of requirement for thymocytes for efficient antibody formation to trinitrophenylated mouse red cells in mice: role for thymocytes in suppression of the immune response.

The requirement of thymus-derived cells for the immune response to trinitrophenylated syngeneic mouse red cells (TNP-MRC) was investigated. In three sets of experiments the following results were obtained: a) irradiated mice which were reconstituted with bone marrow cells alone showed a better anti-TNP response after injection with TNP-MRC than those reconstituted with both bone marrow cells and thymocytes. b) Anti-thymocyte serum augmented the mouse anti-TNP response to TNP-MRC. c) Nude thymusless mice showed a better anti-TNP response to TNP-MRC than their normal littermate controls. These results indicate that the anti-TNP response of mice to TNP-MRC does not require thymus-derived helper cells. Moreover, thymus-derived cells have a suppressive effect on the anti-TNP response.

Induction of primary and inhibition of secondary antibody response to hapten by hapten conjugates of type III pneumococcal polysaccharide

Tri- or dinitrophenylated pneumococcal polysaccharide type III (TNP- or DNP SIII)) induced a primary 19S anti-TNP response without generating immunological memory to the hapten in LAF 1 mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. abortus organisms (DNP-BA) induced both 19S and 7S primary responses and memory to the hapten. Spleen cells from mice immunized with TNP-KLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SIII and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLH-primed spleen cells for as short as 5 min at 0 °C with 10 μg of TNP-SIII per milliliter virtually abolished their ability to give 19S and 7S memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help.

Carrier-Determined Tolerance in Vitro

A primary immune response to normal BDF1 spleen cells was obtained in vitro to the T-independent antigen TNP-T4 coliphage. This anti-TNP response was suppressed by exposing the spleen cells for 6 hr to TNP bound either to isologous or heterologous gamma-globulin. The suppression was hapten specific. In contrast, TNP-albumin conjugates did not induce tolerance in vitro.

Carrier-and hapten-antibody producing cells, in vitro II. Effect of purine antimetabolites

To determine the effects of purine antimetabolites on the various developmental stages of cells involved in in vitro carrier and hapten responses, purine antimetabolites were added on day 0, day 1, day 2 or day 3 of the in vitro immune response. Anti-HRBC and anti-TNP responses were assessed on day 2, day 3 and day 4 of culture. Our results indicated that purine antimetabolites were: (1) most effective on early and intermediate precursor cell types, (2) most effective when added early, during the in vitro immune response and (3) more effective in inhibiting immunogen-stimulated immune response than nonspecific enhancement of the in vitro immune response.

Carrier and hapten antibody-producing cells, in vitro. I. Cytokinetics.

SummaryNormal spleen cells or HRBC-primed spleen cells were cultured with HRBC, TNP-HRBC or without an in vitro immunogen. Anti-HRBC and anti-TNP responses were assessed on Days 2, 3, and 4. Antibody-producing cells detected on Day 2 arose mainly from late, on Day 3 from intermediate and on Day 4 from early precursor cell types. We envision APC development proceeding through the following maturation sequence: early precursor cells → intermediate precursor cells → late precursor cells → APC. The relative proportion of these precursor cell types at any given time is dependent on the immunological status of the animal. Later precursor cell types can be triggered non-specifically to develop into APC.This work was supported by GM 15956 and AEC Contract No. C00-1632-30. T.L.P. was supported by NIH 1 F02 GM 50743-01. We thank Ahmed Elashkar for his skillful technical assistance.

Induction of Tolerance to a Hapten

Abstract A single injection of trinitrobenzenesulfonic acid (TNBS) induces a state of immunologic tolerance to the trinitrophenyl (TNP) hapten, which is specific and dose dependent. multiple injections of TNBS induce a longer lasting state of unresponsiveness than single injections. Escape from tolerance is characterized by the production of only 2-mercaptoethanol (ME)-sensitive (19S) anti-TNP antibody whether tolerance is induced by single or multiple injections. multiple injections of hapten-carrier conjugate (TNP-sheep erythrocytes) extend the duration of the tolerance induced by multiple injections of TNBS. The anti-TNP response of animals escaping from tolerance induced by multiple TNBS injections and extended by multiple hapten-carrier challenges consisted entirely of ME-sensitive (19S) antibody. Substitution of carrier for hapten-carrier conjugate in the initial challenge injection resulted in a normal anti-TNP response pattern in terms of class and temporal sequence of antibody produced during escape from tolerance. TNBS treatment had no effect upon the “accelerated” response to carrier induced by low-dose carrier priming, and TNBS treatment induced tolerance to TNP in low-dose carrier-primed mice.

Development of Primary γM, γG, and γA Anti-TNP Plaque-Forming Cell Responses in Vitro

Abstract Trinitrophenylated bacteriophage φX174 (TNP-φX) is a potent in vitro immunogen capable of stimulating specific anti-TNP plaque-forming cell (PFC) responses in cultures of mouse spleen cells. Under optimal conditions, 10 × 106 spleen cells and 3 × 109 TNP-φX particles in 1 ml of culture medium, TNP-specific PFC responses representing the major mouse immunoglobulin classes, γM, γ1, γ2, and γA were detected. A study of kinetics of appearance of these immunoglobulin class-specific PFC revealed that an increase in γM PFC was detected within 24 hr of initiation of the cultures; the maximum γM PFC responses were on days 4 and 5. γ1 and γ2 PFC were not detectable until day 3 of culture and reached maxima on days 5 and 6. The anti-TNP PFC response to TNP-φX was almost completely suppressed by the addition of 100 µg TNP guinea pig albumin; the addition of free φX at a 10-fold excess over the TNP-φX immunogen suppressed the anti-TNP response about 70%.