Secondary anti-DNP Response Research Articles

Effect of cationization on anti‐hapten antibody responses in sheep and mice

The effect on anti-hapten immune responses of altering the isoelectric point of hapten-carrier conjugates by cationization was examined in sheep and mice. No adjuvants were employed in these studies. In sheep the cationization of ovalbumin-dinitrophenyl (OVA-DNP) resulted in an enhanced primary anti-dinitrophenyl (DNP) response. The secondary anti-DNP responses were not consistently greater for animals primed with cationized (CAT) OVA-DNP than in animals primed with OVA-DNP. Similar results were obtained with mice immunized with bovine serum albumin (BSA)-DNP or CAT BSA-DNP. Mice and sheep primed with OVA-DNP failed to mount an anamnestic response following secondary immunization with CAT OVA-DNP. Failure of a cationized hapten-carrier to stimulate a secondary anti-DNP response following priming with uncationized hapten-carrier was also observed in mice using BSA-DNP as the model antigen.

Secondary response of rainbow trout ( Salmo gairdneri Richardson) to DNP-haemocyanin and Yersinia ruckeri

Yearling rainbow trout were immunized by intraperitoneal injection of 1 mg dinitrophenol-keyhole limpet haemocyanin (DNP-KLH) or 10 9 formalin-inactivated Yersinia ruckeri. Fish were regularly bled to establish individual kinetics by passive haemagglutination and agglutination, respectively. Groups of 20 to 30 fish were used. A control group was also provided which received its first injection simultaneously with the booster injection for the experimental group. The secondary anti-DNP response was never found to be enhanced when compared with the primary one but, in contrast, a noticeable secondary response was observed with the bacterial antigen.

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice : II. Both H-2-linked and -nonlinked control of induction of suppressor macrophages

The suppressive effect of Toxoplasma infection on initiation of memory cells to dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) was drastically different among inbred strains of mice. C57BL/6 (B6), C57BL/10 (B10), and SJL mice showed markedly suppressed secondary anti-DNP responses when infected. In contrast, the suppression did not occur in BALB/c mice. The infected DBA/2 and C3H/He mice produced moderately suppressed responses. In B6 mice, an injection with 1 × 10 2 organisms of T. gondii induced a suppressed elicitation of the memory cells to DNP-KLH. However, in BALB/c mice, the responses were not affected even by inoculation with 1 × 10 4 organisms. The difference in the suppressive effect of infection between B6 and BALB/c mice was also observed in the primary anti-DNP antibody responses to DNP-KLH. Both H-2-linked and -nonlinked genes appeared to be responsible for the regulation of the immunosuppression, since the suppressive effect of infection in B10.D2 mice, which have the B10 background and the same H-2 haplotype as BALB/c, was weaker than that of B10 mice, but stronger than in BALB/c mice. In vitro studies using a primary antisheep erythrocytes (SRBC) antibody response system demonstrated that the activation of plastic-adherent suppressor cells by Toxoplasma infection, in which suppressor macrophages have been proved to be the responsible cells for the suppressive activity, was controlled by both H-2-linked and -nonlinked genes.

Analysis of the negative allogeneic effect using B cells and helper T cell lines as an indicator system: B cells identified as target cells for suppression.

The target cells for H-2b T lymphocytes mediating a negative allogeneic effect in vitro were analyzed by using carrier-specific helper T cell lines of H-2b, H-2d, or F1 origin and hapten-primed T-depleted spleen cells also expressing one or both of these haplotypes. The helper T cell lines were shown to be carrier specific and H-2b or H-2d restricted. Most importantly, the lines derived from H-2b homozygous mice were devoid of alloreactivity against H-2d and vice versa. Titration of naive H-2b T lymphocytes to the indicator cultures resulted in suppression of the secondary anti-DNP response of the indicator cells whenever the B cells expressed H-2d antigens. The lack of suppression observed in mixtures in which only the helper T cell lines expressed H-2d antigens was not reversed by the increased addition of naive H-2bxd cells, indicating that an insufficient amount of H-2d antigens present on the low number of helper T cells used did not account for this finding. Moreover, the polyclonal plaque-forming cell responses of F1 spleen cells to LPS were also suppressed by naive parental T cells. From these findings it is concluded that the suppressor T cells directly recognize and inhibit allogeneic B cells without the involvement of helper T cells. In addition, it was shown that the suppression of secondary anti-hapten responses by naive allogeneic T cells is blocked by monoclonal anti-Lyt-2 antibody added at the onset of culture. Addition late in culture had no effect, pointing to a functional role of the Lyt-2-bearing structure at an early stage of the suppressive events resulting in the negative allogeneic effect.

The relationship between surface immunoglobulin isotype and the immune function of murine B lymphocytes. V. High affinity secondary antibody responses are transferred by both IgD-positive and IgD-negative memory B cells.

We examined the adoptive secondary anti-DNP responses restored by surface IgD+ and IgD- memory B cells. Several parameters that might affect the affinity and magnitude of the adoptive responses were studied: 1) time after priming of cell donors, 2) source of anti-IgD antibodies used for immunofluorescent cell staining, 3) adjuvant used for priming, 4) carrier protein used for priming, 5) amount of antigen used for the challenge of adoptive hosts, and 6) the strain of ice used as donors and recipients. In contrast to previous reports, the present results demonstrate that the selection of cells with high affinity antigen receptors can occur to the same extent in both the delta + and delta - memory cell pools. This suggests that the loss of surface IgD is not a necessary intermediate stage in the maturation of memory B cells.

Dissociation of the anti-hapten and anti-carrier responses of mice injected with dinitrophenylated lipopolysaccharide.

The quantitative and qualitative nature of the antibody responses of euthymic (normal, RML) and athymic (nude) mice injected with dinitrophenylated (DNP) lipopolysaccharide (LPS) was evaluated. Antibody responses to both the haptenic (DNP) and carrier (LPS) determinants were measured. On a quantitative basis, RML and nude mice stimulated with DNP-LPS produced only primary anti-DNP responses, whereas both primary and secondary anti-LPS responses were elicited by this conjugate. The failure of DNP-LPS to trigger secondary anti-DNP responses was not dependent on the amount of DNP-LPS given in the primary or secondary doses and could not be overcome by repeated injections of DNP-LPS. Also, the anti-DNP responses of RML mice injected repeatedly with DNP-LPS were restricted to immunoglobulin M antibodies whereas both immunoglobulin M and G anti-LPS responses were elicited. Nude mice also produced immunoglobulin G antibodies to the LPS determinants. These data showed a dissociation of the anti-hapten and anti-carrier antibody responses and suggested that different immunological signals were functioning in the respective anti-DNP and anti-LPS responses.

Hapten antibody production and the relevance of allogeneic reactions to elimination of the carrier effect.

Lewis (LEW) rats immunized 3 weeks before by injection of DNP-KLH together with Bordetella pertussis showed high levels of DNP antibody as judged by serum binding of 10(-7) M 3H-DNP-lysine 10 days after secondary immunization with DNP-KLH. Sera obtained from LEW rats following secondary immunization with DNP-BGG showed reduced DNP hapten binding. However, injection of 10(8) F1 hybrid Lewis X Brown Norway spleen cells into DNP-primed LEW rats 2 days before secondary immunization with DNP-BGG significantly increased the level of serum binding of 3H-DNP-lysine. These results provide evidence that the allogeneic cellular reaction associated with a host-versus-graft response induced by injection of F1 hybrid lymphoid cells into DNP-primed parental strain recipients partially obviates the requirement for carrier-specific T cells in the secondary anti-DNP response thereby providing a stimulus for triggering primed host B cells to produce antibody.

Activation of helper T cells by immune complexes

Spleen cells from mice primed with dinitrophenylated human γ-globulin (DNP-HGG) did not mount a secondary anti-DNP response in diffusion chamber cultures upon stimulation with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH). The same cells, however, responded to stimulation with DNP-KLH complexed with anti-KLH antibody of rabbit or mouse origin. There is an optimal antigen:antibody ratio at which the immune complexes (IC) must be formed for maximal activity. T cells are required for the immunogenic activity of IC, since T-cell-depleted cultures did not respond. It was found that IC made with carrier and anticarrier antibody stimulated the development of carrier-specific helper T cells in cultures of spleen cells, thymocytes, and nylon wool nonadherent spleen cells from nonimmune mice. In contrast, free carrier did not elicit helper T cells. IC made with carrier and the F(ab′) 2 fragment of anticarrier antibody were immunogenic, but those made with carrier and the Fab′ fragment of anticarrier antibody were not, suggesting that helper T-cell activation is triggered by crosslinking of antigen-specific surface receptors.

Adaptive Differentiation of Murine Lymphocytes

Abstract Responses to the synthetic terpolymer L-glutamic acid, L-lysine, L-phenylalanine (GLΦ) and hapten derivatives thereof are controlled by two complementing H-2-linked Ir genes in the mouse. F1 hybrids derived from two different nonresponder strains (one of which possesses the α and the other β Ir-GLΦ gene) are phenotypic responders to GLΦ and 2,4-dinitrophenyl (DNP)-GLΦ. Moreover, spleen cells from DNP-GLΦ-primed F1 mice can adoptively transfer secondary anti-DNP antibody responses to irradiated F1 recipients that have been challenged with DNP-GLΦ. When, however, GLΦ-primed F1 helper T cells are transferred together with the DNP-specific F1 B cells that had been primed in separate mice altogether by DNP coupled to an unrelated protein carrier, such mixtures failed to develop adequate adoptive secondary anti-DNP responses to DNP-GLΦ. This contrasted with the ability of the same GLΦ-primed F1 T cells to provide helper activity for DNP-primed B cells from responder recombinant B10.A (5R) mice. More important, the apparent defect of GLΦ-primed F1 T cells in providing help for DNP-primed F1 B cells (primed to a DNP-protein conjugate) could be readily overcome by using DNP-primed B cells from donor F1 mice primed with DNP-GLΦ. As discussed herein, these results suggest that interacting T and B lymphocytes pair off into partner cell sets, any pair of which interact optimally when a “best fit” reciprocal self-recognition occurs between them.

Induction and Mechanism of Tolerance to Bovine Serum Albumin in Mice Given Total Lymphoid Irradiation (TLI)

Abstract BALB/c mice given total lymphoid irradiation (TLI) were injected i.p. with bovine serum albumin (BSA) in saline, and challenged with DNP-BSA in complete Freund's adjuvant 6 weeks later. The latter animals made no anti-DNP antibody response as measured by a modified Farr assay, but made a normal anti-DNP response after challenge with DNP-BGG in adjuvant. Normal mice or mice given whole body irradiation were not tolerized by the i.p. injection of BSA in saline. Spleen cells from unresponsive mice (TLI + BSA in saline) suppressed the adoptive secondary anti-DNP response of sublethally irradiated syngeneic hosts given BSA-primed T cells, DNP-BSA-primed B cells, and DNP-BSA in saline. The suppressor cells were antigen specific, and were inactivated by in vitro treatment with anti-Thy 1.2 antiserum and complement. The findings suggest that soluble antigens administered to mice after TLI evoke a state of tolerance that is maintained by antigen-specific suppressor T cells. A similar mechanism may be involved in the maintenance of tolerance to allografts. These findings may have important clinical implications for patients treated with TLI for lymphoid malignancies.

Studies on the Immune Response in Chickens II. Functions of Carrier-Reactive Cells in Anti-Hapten Responses

Abstract Roles of T cells in the responses of B cells and B memory cells in chickens were studied using hapten-conjugated carrier antigens. The secondary anti-DNP antibody response to DNP-BSA was generated not only in chickens primed with DNP-BSA but also in those primed with the same hapten on a heterologous carrier such as DNP-BGG and DNP-KLH. Similarly, the secondary anti-DNP response to DNP-BGG occurred in chickens primed with DNP-BSA as well as in those primed with DNP-BGG. Passive administration of anti-DNP antibody did not enhance anti-DNP response to DNP on a heterologous carrier. It is likely, therefore, that carrier-primed cells are not necessary for elicitation of the secondary anti-DNP response, indicating that the carrier effect is not seen in chickens. When chickens primed with DNP-BGG were challenged by DNP-BSA, it was found that DNP-specific memory was generated within a week after priming and maintained longer than for 4 weeks. Moreover, the primary and secondary anti-DNP antibody responses to DNP-BSA of chickens were suppressed markedly by injection of BSA alone. The carrier-induced suppression of anti-hapten response was carrier-specific. The suppressive effect of BSA could be seen in the wide range of doses injected (0.1 to 1 000 mg) and was found to have no relation to immunological tolerance to BSA. The suppressive effect of BSA was exhibited when BSA was injected in the period of from 3 weeks before to 2 days after the challenge by DNP-BSA. Passive administration of anti-BSA antibody could not substitute for the stimulation by BSA in suppressing anti-DNP response to DNP-BSA. It is suggested therefore that the formation of the carrier-specific suppressor cells (T cell) can be activated by injection of carrier alone and results in suppression of anti-hapten response to the hapten on the homologous carrier. From the present study, it has been concluded that helper T memory cells do not seem to play significant roles in generation of the secondary anti-hapten response, and that stimulation by carrier alone is capable of generating the carrier-specific suppressor T cells which act to suppress antihapten response to the hapten on the homologous carrier.

Maturation of B Lymphocytes in Rats

Abstract We examined the ability of large and small thoracic duct cells obtained from Lewis rats primed to DNP to restore the adoptive secondary anti-DNP response in irradiated syngeneic hosts given an excess of T helper cells. The large cells were four to five times more active on a per cell basis than were the small cells. However, the large cells constitute only 7 to 8% of the thoracic duct cells and, therefore, make a minor contribution to the restorative activity of the unfractionated cells. Pretreatment of thoracic duct cell donors with high specific activity 3H-TdR for 48 hr before cannulation markedly reduced the memory B cell activity of the large cells, but had little effect on that of small cells. In addition, the activity of the large cells diminished with time after primary immunization, but that of the small cells remained stable. Immunofluorescent staining of the large and small cells for surface IgM and IgG, and subsequent sorting on the fluorescence-activated cell sorter (FA CS), showed that surface IgM was present on large memory B cells, and that IgG was present on small memory B cells. The experimental results suggest that two subpopulations of memory B cells in the thoracic duct lymph differ by size, rate of turnover, persistence after primary immunization, and class of surface IgG.

The lymphocyte surface. II. Separation of Fc receptor, C'3 receptor and surface immunoglobulin-bearing lymphocytes.

A one-step separation procedure is described for both depleting and obtaining in pure form Fc receptor (FcRL), C'3 receptor (CRL) and surface immunoglobulin bearing (IgL) lymphocytes from rat lymphoid populations. The method is a modification of the Bӧyum (1968) technique for separating lymphocytes from whole blood by sedimentation on Ficoll/Isopaque, and is based on the fact that when a lymphocyte forms a rosette with sensitized erythrocytes it will sediment with the red cells rather than float with the non-rosetting lymphocytes. The technique is > 99.5% efficient at depleting thoracic duct lymphocytes (TDL) of FcRL, CRL and IgL and these subpopulations can be recovered 93-98% pure. The total recovery of lymphocytes applied is usually > 90% and the separated lymphocytes are > 95% viable. This technique allowed the cellular distribution of Fc receptors, C'3 receptors and surface Ig to be determined. It was found that (a) Almost all CRL carry surface Ig, although a very small sub-population of CRL (0.2-0.8%) which lacks surface Ig could regularly be detected. (b) A substantial proportion of IgL (12-25%) lacks C'3 receptors. (c) IgL and CRL which lack Fc receptors are more frequent in spleen and lymph nodes than in TDL. The proportion of this subpopulation increases in TDL after prolonged thoracic duct drainage. (d) Some FcRL exist which lack both C'3 receptors and surface Ig. These cells are more evident in TDL after prolonged thoracic duct drainage and in lymph nodes (20-30% of FcRL) than in early TDL or spleen (5-10% of FcRL). (e) The thymus contains very few FcRL, CRL or IgL. (f) A large population of lymphocytes exists in B rats (32-42% of TDL) which is killed by an anti-B serum but which lacks surface Ig. These cells are much less frequent in normal TDL ( < 5%) and probably also lack Fc and C'3 receptors. (g) Large lymphocytes probably shed their Fc and C'3 receptors, but retain their surface Ig, during S-phase. (h) Studies on a secondary anti-DNP response showed that a substantial proportion of direct and indirect plaque forming cells (PFC) in the spleen express Fc receptors, whereas only indirect PFC carry C'3 receptors. Virtually all PFC ( > 98%) possess surface Ig.

Cellular Site of Action of Various Adjuvants in Antibody Responses to Hapten-Carrier Conjugates

Abstract The adjuvant action of concanavalin A (Con A), Escherichia coli lipopolysaccharide (LPS), beryllium sulphate (Be salt) and Bordetella pertussis vaccine have been analyzed in a system measuring humoral immune responses to hapten-carrier conjugates in mice. For the definition of the cellular locus of their action, the effect of these adjuvants on adoptive secondary anti-hapten antibody responses was studied. Spleen cells from 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH) primed donors, which normally fail to develop adoptive secondary anti-DNP responses to a heterologous conjugate such as DNP-bovine γ-globulin (BGG), can be stimulated to do so when an appropriate dose of adjuvant is administered with DNP-BGG. The capacity for the adjuvants employed to exert this effect requires the presence of thymus-derived (T) lymphocytes, since depletion of such cells by treatment of the donor cell inoculum with anti-θ serum and complement in vitro before adoptive transfer abrogates the response to DNP-BGG plus adjuvant. Moreover, evidence is presented which demonstrates that these substances exert their adjuvant action through the small number of unimmunized BGG-specific T lymphocytes in the donor cell inoculum. This conclusion derives from the failure of adjuvants to augment adoptive secondary anti-DNP responses to the DNP derivative of a non-immunogenic copolymer of d-glutamic acid and d-lysine for which there are few or no specific, functional T cells.

Mechanism of adjuvant activity of poly A:U on antibody responses to hapten-carrier conjugates

The adjuvant action of poly A:U has been analyzed in a system measuring humoral immune responses to hapten-carrier conjugates in mice. Administration of poly A:U at the time of primary immunization with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH) shortens the induction period for, and heightens the magnitude of peak anti-DNP antibody and specific memory cell production. In order to define the cellular locus of poly A:U action, the effect of this adjuvant on adoptive secondary anti-DNP antibody responses was studied. Spleen cells from DNP-KLH-primed donors, which normally fail to develop adoptive secondary anti-DNP responses to a heterologous conjugate such as DNP-bovine gamma globulin (BGG), can be stimulated to do so when an appropriate dose of poly A:U is administered with DNP-BGG. The capacity for poly A:U to exert this effect requires the presence of T lymphocytes, since depletion of such cells by treatment of the donor cell inoculum with anti-θ serum and complement in vitro prior to adoptive transfer abrogates the response to DNP-BGG plus poly A:U. Moreover, evidence is presented that demonstrates that poly A:U exerts its adjuvant action on the small number of unimmunized BGG-specific T lymphocytes in the donor cell inoculum. This conclusion derives from the failure of poly A:U to augment adoptive secondary anti-DNP responses to the DNP derivative of a nonimmunogenic copolymer of d-glutamic acid and d-lysine ( d-GL) for which there are few or no specific, functional T cells.

The allogeneic effect in inbred mice. II. Establishment of the cellular interactions required for enhancement of antibody production by the graft-versus-host reaction.

Experimental conditions have been established for the elicitation of an allogeneic effect on the adoptive transfer secondary anti-2,4-dinitrophenyl (DNP) antibody response in mice. Thus, spleen cells from DNP-keyhole limpet hemocyanin (KLH)-primed mice manifest good secondary anti-DNP responses to a challenge with DNP-KLH, but not with DNP-bovine gamma globulin (BGG), after adoptive transfer to irradiated syngeneic recipients. However, a good adoptive transfer secondary anti-DNP response of such cells can be elicited with DNP-BGG when a second transfer of allogeneic lymphoid cells, in appropriate numbers, is carried out 24 hr before secondary challenge. The advantage to this system is that the DNP-primed cell population as well as the population of allogeneic lymphoid cells are accessible to experimental manipulation such that the T lymphocytes of one or the other can be removed. Utilizing this model, we have established that the allogeneic effect on antibody production can operate on a population of primed B lymphocytes which have been depleted of their isologous T lymphocytes by in vitro incubation with anti-theta serum plus complement. The potential cellular interactions involved in the mechanism of this phenomenon are considered and discussed in detail.

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.

Histocompatibility-Linked Genetic Control of the Immune Response to Hapten Guinea Pig Albumin Conjugates in Inbred Guinea Pigs

Abstract The immune response of inbred guinea pigs to low doses of hapten-guinea pig albumin conjugates is controlled by a specific immune response gene closely linked to the gene complex controlling strain 13 histocompatibility antigens. Thus, strain 13 guinea pigs make significant anti-dinitrophenyl (anti-DNP) antibody responses to 1 μg of DNP-guinea pig albumin (GPA) while strain 2 guinea pigs make little or no response and are not prepared for secondary anti-DNP responses. Progeny of (2 × 13) F1 × 2 matings which possess strain 13 histocompatibility antigens are good responders to a low dose of DNP-GPA while offspring lacking strain 13 antigens are poor responders. The differential response of strain 2 and 13 animals to DNP-GPA is also observed with p-iodophenylsulfonyl-GPA and a 3-(2,4-dinitrophenylamino)-propylamide derivative of GPA. These observations indicate that this immune response gene is involved in carrier recognition and suggest that it operates in thymus-derived lymphocytes.

Carrier function in anti-hapten antibody responses. 3. Stimulation of antibody synthesis and facilitation of hapten-specific secondary antibody responses by graft-versus-host reactions.

The studies reported here demonstrate that immunocompetent lymphoid cells from allogeneic donor guinea pigs stimulate the synthesis of anti-DNP and anti-OVA antibodies by recipients previously primed with DNP-OVA. This allogeneic effect occurs spontaneously in the absence of any further anti-genic challenge. Furthermore, the transfer of allogeneic cells prepares DNP-OVA-primed recipients for a striking secondary anti-DNP response to DNP-BGG; this occurs in equal degree whether or not the cells are derived from BGG-primed donors. We suggest that the allogeneic cells function by virtue of a specific immunologic attack of grafted cells on host cells. This conclusion is made on the basis of the following evidence: (a) The failure of observing the phenomenon with L(2)C leukemia cells and irradiated strain 2 lymph node and spleen cells which, although capable of initiating a host-versus-graft response, are incapable of mediating graft-versus-host reactions; and (b) the inability of (strain 2 x strain 13) F(1) hybrids to mediate the allogeneic effect in strain 13 recipients. The analysis of this phenomenon may offer a key to the delineation of mechanisms involved in the activation of precursors of antibody-forming cells.