Human immune responses to hapten-conjugated cells. I. Primary and secondary proliferative responses in vitro.

An in vitro model was developed to study both primary and secondary proliferative responses of human lymphocytes to hapten-conjugated peripheral blood mononuclear cells. Coculture of human lymphocytes with autologous trinitrophenyl (TNP)-conjugated stimulator cells resulted in primary proliferative responses. Subjects segregated into high and low primary responders with mean stimulation indices of 11 and 2.1, respectively. Restimulation of primed cells from high responder subjects 3 wk after initial sensitization generated secondary proliferative responses. To investigate the antigenic requirements for secondary stimulation, autologous TNP-conjugate primed responders were restimulated with both autologous and allogeneic TNP-conjugated stimulators. In all experiments restimulation with autologous conjugated cells yielded substantially greater proliferative responses than with allogeneic conjugates. Experiments were then performed to ascertain whether HLA determinant homology between primed responder and stimulator cells influenced the level of secondary responsiveness. Homology for HLA-A and B locus serologic determinants was not associated with enhanced responsiveness. In contrast, D region determinant homology, detected by B-cell antigen typing, showed a highly significant positive correlation with the magnitude of secondary responses. The data thus strongly suggest that for secondary proliferative responses to TNP, human T cells recognize hapten in association with HLA-D region determinants.

Long-term growth of lines of murine dinitrophenyl-specific B lymphocytes in vitro.

Studies of cellular events associated with antigen-induced triggering and differentiation of B cells would be greatly facilitated by the availability of homogeneous cell lines of antigen-specific lymphocytes that can be maintained in long-term culture. By combining the techniques of enrichment of lymphocytes for antigen-specific cells, cloning in soft agar, and long-term propagation of B cells we have been able to isolate, propagate, and maintain two lines of dinitrophenyl (DNP) -specific B lymphocytes. These cell lines are B lymphocytes that have 70% and greater than 80% DNP-specific rosette-forming cells, respectively. Both cell lines secrete small amounts of antibody spontaneously but can be stimulated by antigen in vitro in the presence of either supernatants from phytohemagglutinin-stimulated spleen cells or irradiated normal filler cells. Thus far these lines have been maintained in vitro for greater than 9 mo. They will be useful in studying factors associated with B cell response.

Regulation of T-cell-mediated lympholysis by the murine major histocompatibility complex. II. Control of cytotoxic responses to trinitrophenyl-K and -D self products by H-2K- and H-2D-Region genes.

An H-2K/IA recombinant mouse strain was used to map the genes within the H-2 complex which determine the ability to respond in cell-mediated lympholysis (CML) to low doses of trinitrophenyl-self (TNP-self). It was found that gene(s) which map to the K region are involved in regulation of CML response to low-dose TNP-self. It was also found that CML response to TNP recognized in association with H-2Dq was not detectable in this recombinant. These findings are discussed with respect to the involvement of the H-2K and H-2D regions by structural and/or regulator gene functions.

Ir gene controlled carrier effects in the induction and elicitation of hapten-specific delayed-type hypersensitivity responses.

The genetic requirements of carrier recognition were examined in the priming and elicitation of hapten specific, T-cell mediated, delayed-type hypersensitivity (DTH) responses. It was shown that nitrophenyl acetyl-poly-(L-glu56-L-lys35-L-phe9) (NP-GLO) could prime for NP responses only in strains of mice which are Ir gene responders to GLO. In contrast to this requirement, NO-GLO could elicit an NP-specific response in NP-bovine gamma globulin primed mice, even in GLO nonresponder strains. Furthermore, the nonimmunogenic molecule, NP-GL, could elicit an NP-specific DTH response in animals primed with NP on an immunogenic carrier.

The immune response of allophenic mice to 2,4-dinitrophenyl (DNP)-bovine gamma globulin. I. Allotype analysis of anti-DNP antibody.

The question of whether or not lymphoid cells can cooperate across a histocompatibility difference barrier has been studied in several laboratories. Using an adoptive transfer system, Katz et al. (1) first showed that T cells from (low responder x high responder) F(1) mice, primed to the terpolymer L-glutamic acid, L-lysine, L-tyrosine (GLT), could collaborate with 2,4-dinitrophenyl (DNP)-primed B cells from a high responder, but not a low responder strain, in response to DNP-GLT. The response to GLT is under H- 2-1inked Ir gene control. In contrast, studies with mouse bone marrow chimeras have shown that T cells can interact with H-2-histoincompatible B cells in response to antigens not under Ir gene control (2-4). Another type of chimera, the allophenic mouse, has been used to study possible histoincompatible cell interactions to a number of antigens, including DNP-L- glutamic acid, L-lysine, L-alanine; L-glutamic acid, L-alanine, L-tyrosine; L-glutamic acid, L-lysine, L-phenylalanine; and poly-L (Tyr, Glu)-poly D,L- Ala-poly-L-Lys[T,G)-A-L] (5-9). The response to each of these antigens is under H-2-1inked Ir gene control. It was initially reported (8, 9) that in allophenic mice containing both high and low responder cells, the antibody to (T,G)-A-L was of both the high and low responder allotype. This was interpreted to mean that high responder T cells had cooperated with low responder B cells across a histocompatibility difference barrier in the environment of the allophenic mice. However, Press and McDevitt (10) have recently reported that additional and more accurate analyses of these allophenic mouse sera failed to detect any anti-(T,G)-A-L antibody of the low responder allotype. Moreover, in an experiment using bone marrow chimeras, there was no low responder allotype antibody produced in response to (T,G)-A- L(10). The present study was undertaken to test the immune response of allophonic mice to an antigen, DNP-bovine gamma globulin (DNP(56)BGG), known to be controlled by genes both inside and outside the H-2 complex (11, 12).(1) When high and low responder cells to DNP(56)BGG are present in allophenic mice, only antibody of the high responder allotype is produced. The results suggest that cell cooperation in allophenic mice cannot occur across a histocompatibility difference barrier in response to an antigen whose genetic control is at least partially within the H-2 complex.

Variation and control of specific antigen-binding cell populations in individual fetal mice.

To determine the extent and nature of individual variation in the development of specific antigen-binding cells, the numbers of cells specific for each of two antigens in the spleens of individual random-bred Swiss-L and inbred CBA/J and BALB/c fetal mice were measured as a function of spleen size. For Swiss-L fetuses, the ratio of antigen-binding cells to nucleateated cells varied more than would arise from sampling fluctuation. For each inbred strain, however, the number of cells specific for a given antigen was a constant proportion of the total number of nucleated cells within sampling error. These proportions varied from antigen to antigen, and from strain to strain. The ratio of the proportions of cells specific for the two antigens, however, differed no more from CBA/J to BALB/c mice than would be expected in repeated samples of cells from the spleen of a single fetus. These results confirm at the level of the individual fetus the uniform pattern of development seen for populations of fetuses. They reveal a surprising precision in the proliferation of specific antigen-binding cell populations and suggest that the development of these cells may be subject to strong genetic controls.

Antigen structural requirements for immunoglobulin isotype switching in mice.

L-Tyrosine-p-azobenzene-p-arsonate (RAT) is immunogenic and serves as a carrier for anti-hapten antibody responses in guinea pigs, rats, and mice. However, the murine anti-N-2,4-dinitrophenyl (DNP) plaque-forming cell (PFC) response to the bifunctional antigen 2,4-dinitrophenyl-6-amino-caproyl-L- tyrosine-p-azobenzene-p-arsonate (DNP-SAC-RAT; or BI-1) is extremely weak (2,000-4,000 PFC/spleen) and exclusively IgM in both primary and secondary responses. The 6-amino-caproyl group serves as a spacer in this antigen between the DNP haptenic and RAT carrier epitopes. In view of recent evidence indicating that different T helper cells synergize for optimal antibody responses, a trifunctional antigen, N-2,4- dinitrophenyl-6-amino-caproyl-L-tyrosine-p-azobenze-p-arsonate-(proline)9-L- tyrosine-p-azobenzene-p-arsonate (DNP-SAC-RAT-PRO(9)-RAT; or TRI), was prepared to investigate the effect of adding a second RAT epitope to BI-1. The nonaproline spacer between the two RAT epitopes in TRI is assumed to be a rigid rod of approximately 28 A. TRI induced about twice as many PFC as BI-1 in primary responses of A/J mice, and induced both IgM and IgG PFC in secondary responses. Furthermore, TRI induced IgG PFC responses in mice primed with p-azobenzene-p-arsonate-keyhole limpet hemocyanin, BI-1, or RAT, whereas boosting with BI-1 failed to induce IgG PFC, even in mice primed with TRI. These findings indicate that the minimum antigen structural requirements for inducing IgG PFC in mice are two carrier epitopes and one haptenic epitope. In addition, priming with a mono-epitope carrier (RAT) is sufficient preparation for IgG responses to a trifunctional immunogen. Because TRI differs from BI-1 by the (proline)(9) spacer as well as the additional RAT epitope, two other compounds, N-2,4-dinitrophenyl-6-amino- caproyl-(proline)(9)-L-tyrosine-p-azobenzene-p-arsonate (DNP-SAC-PRO(9)-RAT; or BI-2) and N-2,4-dinitrophenyl-6-amino-caproyl-(proline)(9)-L-tyrosine-p- azobenzene-arsonate (DNP-SAC-RAT-PRO(10); or BI-3), were prepared to evaluate the possible role of the spacer in the observed responses. BI-2, but not BI-3, induced IgG as well as IgM PFC in TRI-primed mice. However, BI-2 failed to induce IgG responses in RAT-primed mice, indicating that TRI and BI-2 were not equivalent immunogens. Because anti-prolyl antibodies had been found in guinea pigs immunized with N-2,4-dinitrophenyl-(proline)10-L-tyrosine-p- azobenzene-p-arsonate (DNP-PRO(10)-RAT), it seemed possible that priming with TRI might induce anti-prolyl antibodies, which, in turn, could cross-link BI-2 molecules into aggregates containing at least two carrier epitopes. To help resolve this question, mice were immunized with acetyl-(proline)10-L- tyrosine-p-azobenzene-p-arsonate and boosted with BI-2. IgG PFC responses were detected, suggesting that anti-prolyl antibodies were indeed responsible, because priming with RAT and boosting with BI-2 did not induce IgG formation. Accordingly, the observations that IgG responses in RAT-primed mice were induced only by TRI and not by any of the bifunctional antigens indicate that two carrier epitopes per antigen molecule are indeed required for IgG induction. They also provide indirect evidence for synergistic help in the switching of immunoglobulin isotypes.

Human cytotoxic T cell responses to trinitrophenyl hapten and influenza virus. Diversity of restriction antigens and specificity of HLA-linked genetic regulation.

This report compares both the HLA restriction patterns and Ir gene regulation of human in vitro T cell-mediated cytotoxic responses to the trinitrophenyl (TNP) hapten and the type A and B influenza viruses. Comparison of the restriction patterns of these cytotoxic responses indicates that A/HK and B/HK are recognized in conjunction with polymorphic HLA-A and -B self determinants, whereas TNP is recognized in association with a more complex spectrum of self determinants. These self determinants include polymorphic HLA-A and -B determinants, polymorphic non-HLA-A and -B determinants that probably include DR antigens, and non-polymorphic determinants that appear to be species specific. Analysis of the self determinants recognized by human T cells in conjunction with influenza virus demonstrates that (a) the antigens recognized by virus-immune T cells can be distinguished from the serologically defined HLA-A and -B antigenic determinants, and (b) there may be multiple self determinants on individual HLA-A molecules that T cells can recognize in conjunction with virus. The results of family studies indicate that donors' T cells often preferentially respond to virus (and to a lesser extent TNP) in conjunction with products of one parental HLA haplotype (haplotype preference). In the family study, three HLA-identical siblings preferentially recognize paternal HLA antigens in conjunction with A/HK, and maternal HLA antigens in conjunction with B/HK and TNP, which indicates antigen-specific HLA-lined genetic control. Population studies demonstrate virus-specific differences in the ability of donors to respond to selected self HLA-A and -B antigens in conjunction with virus. These differences may be controlled by Ir genes that are distinct from HLA-A and -B, because differences are observed in the response patterns of HLA-A- and -B-matched individuals.

Studies of the antibody-dependent killing of schistosomula of Schistosoma mansoni employing haptenic target antigens. I. Evidence that the loss in susceptibility to immune damage undergone by developing schistosomula involves a change unrelated to the masking of parasite antigens by host molecules.

A method was developed for coupling a hapten, trinitrophenyl (TNP), to the surface of schistosomula of Schistosoma mansoni which results in a minimal loss in their viability as judged by morphological examination in vitro and survival after injection in vivo. Skin-stage (3-h-old) and lung-stage (5-d-old) schistosomula surface labeled in this manner were then compared for their susceptibility to killing by anti-TNP antibody-dependent effector mechanisms both in vivo and in vitro. TNP skin-stage larvae were readily rejected in mice actively immunized against TNP bovine gamma globulin and were highly susceptible to anti-TNP-dependent killing mediated either by complement or purified human eosinophils in vitro. In contrast, TNP-lung-stage schistosomula, which were shown by microfluorimetry to bind anti-TNP antibody to approximately the same extent as skin-stage schistosomula, were found to be resistant to killing by the same in vivo and in vitro mechanisms. These findings suggest that the insusceptibility of postskin-stage schistosomula to antibody-dependent killing must result at least in part from an intrinsic structural change in the integument of the parasite and cannot be caused solely by the masking of parasite antigens by acquired host molecules, a mechanism of immune evasion previously proposed for schistosomes.

Induction of tolerance to 1-fluoro-2,4-dinitrobenzene contact sensitivity with hapten-modified lymphoid cells. II. Selective tolerance in F1 mice of T cell subsets recognizing 1-fluoro-2,4-dinitrobenzene associated with parental major histocompatibility complex antigens.

F1 animals were tolerized to 1-fluoro-2,4-dinitrobenzene (DNFB) contact sensitivity with parentally derived, in vitro hapten-modified spleen cells. This tolerant state was found, upon adoptive transfer to naive parental strain recipients, to affect only that T cell subpopulation that recognized the parental haplotype of the cell used as the tolerogen, and did not inhibit the ability of the remaining T cell subset to confer immunity. This demonstrates that this tolerant state involves the inactivation of a cell required for the expression of contact sensitivity by recognizing DNFB in association with self major histocompatibility complex gene products.

Demonstration of an antibody-mediated tolerance state and its effect on antibody affinity.

We have described a model of immunological tolerance induced, in adult mice, by a single injection of a moderate dose of a hapten-protein conjugate. The data suggest that the mechanism of this tolerance state is the production of small amounts of high affinity antibody in response to the tolerance-inducing antigen injection. This antibody acts to inhibit the response to a subsequent challenge with antigen in complete Freund's adjuvant by a mechanism comparable to that of passive antibody-medicated immune suppression. It was shown that a small but high affinity. Tolerance was not terminated by transfer of normal syngeneic spleen or peritoneal cells into tolerant animals. Spleen cells from tolerant mice, when transferred into lethally irradiated, syngeneic animals, produced a PFC response which is greater in magnitude and tolerance state had a significant degree of carrier specificity which was shown to be comparable to the carrier specificity of antibody-mediated immune suppression. hus, evidence was presented to show that one mechanism of tolerance in adult animals in the suppressive effect of small amounts of high affinity antibody formed in response to the tolerizing injection of antigen.

Haptens can serve as surrogate transplantation antigens in a manner that demonstrates H-2 restriction of graft rejection.

Hapten-immune mice are capable of rejecting syngeneic skin grafts that are derivatized with the relevant hapten, but only if the hapten is applied while the graft is "healing in." This model system was used to demonstrate that the hapten-specific immune effectors responsible for rejection are restricted by H-2 determinants of the recipient. Thus, haptens can be used in vivo as surrogate transplantation antigens for the study of immunopathogenic mechanisms in transplantation immunity.

Soluble factors in tolerance and contact sensitivity to 2,4-dinitrofluorobenzene in mice. III. Histocompatibility antigens associated with the hapten dinitrophenol serve as target molecules on 2,4-dinitrofluorobenzene-immune T cells for soluble suppressor factor.

Previous studies have shown that suppression of 2,4-dinitrofluorobenzene (DNFB) contact sensitivity by soluble suppressor factor (SSF) requires that the donor of immune lymph node (LN) cells and of SSF share either the H-2K and/or H-2D region of the major histocompatibility complex. Thus, target or acceptor molecules for SSF appear to be coded for by genes within the H-2K and H-2D loci. Experiments were done to investigate the nature of these target molecules and to determine what cell types expressed them. It was found that purified lymph node T cells are suppressed by SSF indicating that T cells express the acceptor molecules. Adsorption experiments showed that the only cells capable of adsorbing the suppressor factor are DNFB-immune T cells from donors which share with the factor-producing strain either the H-2K or H-2D locus. This adsorption can be specifically blocked by pretreating the immune LN cells with antibodies directed against H-2K and/or H-2D determinants or against the hapten DNP but not by antibodies against Ia or theta-antigens. Collectively, these results indicate that the target molecules are expressed only by DNFB-immune T cells and are comprised of histocompatibility antigens associated with DNP.

Suppressor T-cell mechanisms in contact sensitivity. III. Apparent non-major histocompatibility complex restriction is a result of multiple sets of major histocompatibility complex-specific suppressor T cells induced by syngeneic 2,4-dinitrophenyl-modified lymphoid cells.

This report has examined the mechanisms by which major histocompatibility complex (MHC) non-restricted suppressor T cells (Ts), induced by the i.v. injection of 2,4-dinitropheny (DNP)-modified, syngeneic lymphoid cells (DNP-LC), suppress the passive transfer of contact sensitivity mediated by syngeneic and allogeneic immune delayed hypersensitivity T cells (TDH). In terms of suppression of syngeneic TDH, it was found that the suppressive action of the Ts was only blocked by pretreatment with soluble syngeneic DNP-LC membrane preparations. Monomeric DNP-lysine, polymeric DNP-protein conjugates, and syngeneic TNP-LC membranes did not inhibit Ts function. Further experiments showed that inhibition of syngeneic suppression could be achieved by DNP-modified-membrane preparations that were only H-2D-region compatible with the Ts donor. Thus, Ts antigen receptors in this system specifically recognize DNP-modified H-2D-region determinants. In contrast, it was found that pretreatment os syninduced Ts with syngeneic DNP-LC membranes did not inhibit the ability to suppress allogeneic TDH. However, pretreatment of Ts with DNP-allogeneic membranes which were H-2D-end compatible to the allogeneic target TDH eliminated their ability to suppress the specific allogeneic TDH, leaving intact suppression of syngeneic or third party TDH. It is proposed that perturbation of the immune system by i.v. injection of syngeneic NDP-LC leads to the induction of a polyclonal wave of DNP-specific Ts activity. Some members of this set of Ts recognize DNP-self MHC determinants with moderate affinity and are thus specifically inhibited after pretreatment with those DNP-self determinants. Other members of this set display receptors which cross-react with high affinity with DNP-allogeneic determinants and thus suppress allogeneic TDH cells. These allosuppressive clones can thus be specifically inhibited only by pretreatment with DNP-LC membranes, MHC-compatible with the target TDH. The data are discussed in terms of current models of T-cell cross-reactivity and T-cell-receptor recognition.

Cellular basis of regulation of expression of idiotype. II. Immunity to anti-MOPC-460 idiotype antibodies increases the level of anti-trinitrophenyl antibodies bearing 460 idiotypes.

The antibody response of BALB/c mice to trinitrophenyl (TNP)-levan or TNP-Nocardia water-soluble mitogen (NWSM) includes a small but significant fraction of antibodies which share idiotypes (Id) with the dinitrophenyl (DNP)- and TNP-binding myeloma protein MOPC-460. Active immunization of BALB/c mice with MOPC-460 or passive administration of anti-460-Id antibodies suppresses the 460-Id+ component of the anti-TNP response. By contrast, active immunization of BALB/c with anti-460-Id antibodies or passive administration of BALB/c anti-[anti-460-Id] antibodies leads to an enhanced 460-Id+ component in the anti-TNP antibodies produced in response to TNP-levan or TNP-NWSM. This enhanced 460-Id+ response appears to be a result of the elimination of suppressor T lymphocytes specific for the 460-Id as T lymphocytes from such mice are unable to suppress the in vitro 460-Id+ response to TNP-NWSM whereas normal T cells are suppressive. These results indicate that suppressor cells specific for 460-Id normally regulate the activation of precursors of cells capable of secreting 460-Id+ anti-TNP antibodies.

Antigen requirements for induction of B-memory cells: studies with dinitrophenyl coupled to T-dependent and T-independent carriers.

Mice were primed with dinitrophenyl (DNP) (trinitrophenyl, TNP) coupled to thymus-independent (TI) or thymus-dependent (TD) carriers. B cells from these mice were transferred to irradiated recipients with T cells from keyhole limpet hemocyanin (KLH)-primed mice. After secondary immunization with DNP-KLH a significant DNP-specific IgG memory response was produced only by mice which received B cells which had been primed with TD antigens. TI antigens were unable to induce differentiation of B-cell precursors to IgG producing memory B cells but they did not suppress the induction of B-memory cells by TD antigens. The results indicate that TI antigens fail to activate a cell type which is required for the induction of memory B cells.

Genetic restrictions for the induction of suppressor T cells by hapten-modified lymphoid cells in tolerance to 1-fluoro-2,4-dinitrobenzene contact sensitivity. Role of the H-2D region of the major histocompatibility complex.

Genetic restrictions governing the induction and expression of suppressor T cells (Ts) in tolerance to 1-fluoro-2,4-dinitrogenzene (DNFB) contract sensitivity were studied. Tolerance was induced by using 2,4-dinitrophenyl (DNP)-modified lymphoid cells (DNP-LC) as tolerogen. Two kinds of Ts were found-those produced by DNP-LC syngeneic to the donor of the Ts (syninduced Ts), and those produced by DNP-LC allogeneic to the donor of Ts (alloinduced Ts). Studies employing congenic resistant mouse strains indicated that recognition of DNP-modified-major histocompatibility region determinants on the tolerogenic DNP-LC was essential for the induction of both types of Ts. Non-H-2 genetic background was irrelevant to Ts induction. Mapping studies indicated that induction of both syninduced and alloinduced Ts was associated with recognition of DNP-modified-MHC region determinants which map to the right of the H-2G region (i.e., H-2D gene products). Tolerization of donor mice with DNP-LC which were H-2D region compatible, but not with H-2K or I region compatible DNP-LC, was both sufficient and required for the induction of hapten-specific syninduced Ts. Tolerization of donor mice with DNP-LC which were incompatible only at the H-2D region was sufficient for the induction of alloinduced Ts. These Ts were capable of suppressing recipient mice only if the recipients shared the H-2D region with the strain providing the DNP-LC tolerogen, and were not capable of suppressing recipients sharing all but the H-2D region with the tolerogen.

H-2-restricted cytotoxic effectors generated in vitro by the addition of trinitrophenyl-conjugated soluble proteins.

Murine spleen cells from normal donors were cultured in vitro with trinitrobenzene sulfonate (TNBS)-conjugated soluble proteins, i.e., bovine gamma globulin (TNP-BGG) or bovine serum albumin (TNP-BSA). Addition of 100 mug of any of these TNP-proteins to the spleen cell cultures led to the generation of cytotoxic T-cell effectors which were H-2-restricted and TNP- specific. The lytic potential of such effectors was comparable to that generated by sensitization with TNBS-modified syngeneic cells, and was restricted to haplotypes shared at the K or K plus I-A, or the D regions of the H-2 complex. Greater effecter cell activity was generated by addition of TNP-BGG against TNBS-modified targets which shared K plus I-A than against modified targets which shared the D region with the responding cells, which suggests that the same immune response genes are involved when the response is generated by the addition of TNP-conjugated soluble proteins or of TNBS- modified cells. H-2-restricted, TNP-specific effecter cells were generated by culturing mouse spleen cells with syngeneic cells which had been preincubated with TNP- BGG or TNP-BSA for 1.5 h. The addition of unconjugated soluble proteins to the cultures did not result in cytotoxic effectors detectable on H-2-matched targets, whether the targets were prepared by modification with TNBS, or by incubation with either the unconjugated or TNP-conjugated proteins. Depletion of phagocytic cells in the tumor preparation by Sephadex G-10 column fractionation before incubation with TNP-BSA had no effect on their lysis by the relevant effector cells. Immunofluorescent staining of tumor target cells with anti-TNP antibodies indicated that TNP could be detected on the tumor cells within 10 rain of incubation with TNP-BSA. The cytotoxic response generated by addition of the TNP-proteins to spleen cell cultures was found to be T-cell dependent at the effector phase, as shown by the sensitivity of the lytic phase to absorbed RAMB and complement. Furthermore, the response did not appear to be attributable to antibody-dependent cellular cytotoxicity. Three mechanisms were considered which could account for the generation of H-2-restricted, TNP-specific, cytotoxic T-cell effectors by the addition of soluble TNP-proteins. These include covalent linkage of activated TNP groups from the soluble proteins to cell surface components, macrophage processing of the soluble conjugates and presentation to the responding lymphocytes in association with H-2-coded self structures, or hydrophobic interaction of the TNP-proteins to cell surfaces. Results obtained from sodium dodecyl sulfate gel patterns indicating that cell-bound TNP was still linked to BSA, and the observation that phagocytic-depleted cells could interact with the soluble TNP-proteins and function as H-2-restricted targets, appear not to favor the first two proposed mechanisms.

Expression of an idiotype (Id-460) during in vivo anti-dinitrophenyl antibody responses. II. Transient idiotypic dominance.

After immunization of mice with 2,4-dinitrophenyl-ovalbumin (DNP-OVA), it was shown previously that strains having Igh-Va genes and able to express light chains of the Vk1 group produce high levels of anti-DNP antibody bearing an idiotype (Id-460) associated with the combining site of the BALB/c DNP-binding myeloma protein MOPC 460. Expression of Id-460 in serum is transient; Id-460 levels peak early in the response and are regulated independently of total anti-DNP antibody. In this paper, the transient dominance of Id-460 expression has been confirmed at the cellular level by inhibition of splenic anti-DNP plaque-forming cells (PFC) with rabbit anti-Id-460 antiserum. Id-460+ PFC can account for 52-91% of anti-DNP PFC early after secondary challenge with DNP-OVA. Furthermore, Id-460 is represented at these high levels in IgM, IgG, and IgG1, and IgG2a, the three isotypes tested in the PFC assay, as well as in IgE, as tested by passive cutaneous anaphylaxis. Thus, there is no preferential association of Id-460 with a given isotype. We conclude from these studies that Id-460 is a dominant idiotype in the anti-DNP antibody response of BALB/c mice to DNP-OVA. This dominance is expressed transiently and is independent of isotype. A further conclusion from these studies is that regulation of isotype expression is independent of the regulation of idiotype expression in this system. We would suggest that regulation of Id-460 expression involves Ig-dependent helper T cells specific for Id-460 that induce Id-460+ B cells and also activate suppressor T cells, both events occurring via idiotype-anti-idiotype interactions.

Major histocompatibility complex-restricted self-recognition in responses to trinitrophenyl-ficoll. Adaptive differentiation and self-recognition by B cells.

The present study has examined the possibility of TNP-Ficoll-responsive B cells recognize the MHC determinants expressed by the accessory cells with which they interact for the generation of T cell-independent responses to "high" concentrations (10(-2) micrograms/ml) of TNP-Ficoll. In experiments with B cells from normal mice, it was found that MHC homology between the TNP-Ficoll-responsive B cells and accessory cells was not required. Nevertheless, TNP-Ficoll-responsive B cells from both fully allogeneic (A leads to B) and F1 leads to parent radiation bone marrow chimeras were triggered by accessory cells expressing host-type, but not uniquely donor-type, MHC determinants. The MHC gene products responsible for this apparent B cell-accessory restriction were encoded in the left side, i.e., the K and/or I-A region, of H-2. Such genetic restrictions were shown not to be imposed by the residual T cells contaminating the chimeric B cell populations because T cell reconstitution experiments using "unrestricted" F1 T cells from normal mice did not fully overcome the marked preference of the chimeric B cells for accessory cells expressing appropriate (host-type) MHC determinants. To directly determine whether TNP-Ficoll-responsive B cells from fully allogeneic chimeras are unable to recognize and cooperate with syngeneic strain A accessory cells, unfractionated spleen cells from A leads to B chimeras are co-cultured with unfractionated spleen cells from essentially syngeneic normal strain A mice. In such co-cultures, all the accessory cells express strain A MHC determinants, and all T cell requirements would be fulfilled by the T cells present in the normal strain A spleen cell population. After stimulation of the co-cultures with TNP-Ficoll, it was found that virtually all the PFC that had been generated in the co-cultures were derived from the normal B cell population, and essentially none were derived from the chimeric A leads to B B cell population. The failure of the chimeric B cells to be activated in such co-cultures was specifically due to their maturation in a fully allogeneic host environment because TNP-Ficoll-responsive B cells from A leads to (A X B) F1 chimeric mice were successfully triggered in co-cultures with normal spleen cells. These experiments demonstrated that the co-culture conditions did fulfill the MHC restriction requirements for activating TNP-Ficoll-responsive strain A B cells that had matured in a syngeneic or semi-syngeneic differentiation environment, but did not fulfill the MHC restriction requirements for activating TNP-Ficoll-responsive strain A B cells that had matured in a fully allogeneic differentiation environment. Taken together, these results demonstrate that (a) TNP-Ficoll-responsive B cells recognize the MHC determinants expressed by accessory cells, and (b) their MHC specificity is influenced by the MHC haplotype of the host environment in which the B cells had differentiated.