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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.

The secretion rate of anti-TNP IgM antibodies has been assessed on spleen cells from unprimed or HRBC-primed mice following immunization with TNP-HRBC. Measurement of the antibodies secretion rate was performed by the hemolytic plaque inhibition assay. HRBC-priming induced greater numbers of anti-TNP PFC/spleen but had no effect on the antibody secretion rate and on its heterogeneity. It is noteworthy that both secretion rate and its heterogeneity were found to oscillate with time after immunization.

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.

The effect of varying the temperature over a wide range (-3 dergees -67 degrees) on the binding of xi-DNP-L-Lysine to bovine colostral anti-DNA IgG1, and also rabbit anti-DNP IgG revealed non-linear van't Hoff plots. The extent of the curvatures were found to be indicative of large positive heat capacity changes; and the thermodynamic parameters, calculated using a non-linear least-squares computer procedure for these anti-DNP antibody preparations, revealed an enthalpy-entropy compensation mechanism for hapten-antibody binding. The enthalpy factor was found to be the primary contributor for the binding process at low temperatures, but at increasing temperatures the entropy factor assumed greater importance. At physiological temperature (37 degrees), the entropy factor was the major contributor to the free energy of reaction for rabbit anti-DNP IgG, while for bovine colostral anti-DNP IgG it was predominant at temperatures higher than 37 degrees.

The immediate effects and mechanisms of desensitization of contact sensitivity to dinitrofluorobenzene (DNFB) were investigated. Intravenous injection of dinitrophenol (DNP)-labeled syngeneic spleen cells caused significant antigen-specific inhibition (greater than 40%) of the contact response within 24 hr in mice that had been sensitized 2 weeks prior to desensitization. With low concentrations of the hapten used for labeling, allogeneic spleen cells were found to be more efficient than syngeneic ones in inducing the down-regulation of the contact response. The most efficient desensitization was produced by DNP-cells that differed from the recipient at the MLS locus. Haptenated spleen cells induced the production of suppressor mechanisms, as spleen cells from animals desensitized with DNP-cells were able to down-regulate recipients in adoptive transfer and could block the passive transfer of contact sensitivity. Procedures that interfere with the development of suppressor cells, e.g., cyclophosphamide and adult thymectomy, interfered with desensitization by DNP-cells. These results are in contrast to desensitization with soluble dinitrobenzenesulfonic acid (DNBS), where suppressor mechanisms have not been shown. The mechanisms for desensitization depend upon the form of the desensitizing antigen.

Evidence is presented in humans that the dinitrophenylated (DNP) antigen, previously shown to be restricted in cell-mediated cytotoxic assays by the HLA-A and -B antigens, is restricted in the proliferative response by HLA-D antigens. A gene dose effect was observed. Some influence of DNP conjugation on the mixed lymphocyte culture stimulatory capacity is shown, but the restriction phenomenon is obvious with the degree of conjugation chosen. The functional part of HLA-D is more likely to follow DR than D antigens. Unspecific conditioning of alloreactivity in secondary cultures is shown by negative as well as by positive selection procedures to be different from the major part of the DNP-specific clones.

The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.

An improved adoptive transfer procedure has been developed for the thymus-independent conjugate TNP-LPS. This technique involving delayed immunization results in increased sensivity and makes possible studies of various cell types, the response of which is normally very low or which are difficult to manipulate experimentally in situ. The standard adoptive immune response of adult spleen cells to TNP-LPS was low in comparison to the primary AFC response of intact mice. Adult bone marrow cells gave a 30-fold lower and neonatal spleen cells an 8-fold lower adoptive response than an equivalent number of adult spleen cells. If the administration of antigen was delayed past the normal time of immunization (1 hr after cell transfer), the resulting AFC response of adult spleen, adult bone marrow, and neonatal cells was enhanced. The peak response occurred with a delay interval of 4 days, and the magnitude decreased with greater delay. Part of the reason for the decline was an acceleration in the attainment of a peak response in mice given antigen past the 4-day optimum. The enhancement of the adult splenic response was not restricted to the antigen TNP-LPS, and was a transferrable phenomenon which did not require antigen to persist past the 4-day optimum delay period. The ineffectiveness of host pre-irradiation indicated that host recovery alone was not involved. Although the results are more compatible with B cell proliferation causing the enhancement, a combination of effects may be involved.

A microculture and radioimmunoassay is described which is capable of generating and detecting very small levels of human anti-DNP antibody. The antibody produced is hapten specific and the response is T-cell dependent. The microculture system described avoids the need for high serum supplements, mitogens, in vivo immunisation or large cell numbers. As this system is simple, sensitive and reproducible it has considerable potential in the study of human immune response regulation and especially the role of lymphokines in human antibody responses.

The primiary structure of the 83 residues of the NH(2)-terminus of the V(II), region was determined for each of three different antibodies to hapten which were produced in inbred guinea pigs. Each antibody had a different and distinctive primary structure within each of the two "hypervariable" regions (Hv1 and Hv2) included in the analyzed part of the variable region of the heavy chain. The sequences of Hvl and Hv2 in the three antibodies were either unique or of restricted variability compared with those of "normnal" immunoglobulin G2. Further implication of Hv1 and Hv2 in contributing to ligand-binding specificity of antibodies came from the placement of residues modified by affinity labeling reagents in these hypervariable regions.

Pig antibodies to the dinitrophenyl group and fragments derived from them by limited proteolysis were studied by temperature-perturbation and solvent-perturbation spectroscopy with particular attention to differences between the number of perturbed chromophores in free antibodies and in antibody-hapten complexes. The position of the maxima in the difference spectra show that solvent-perturbed chromophores are exposed to water, but thermally perturbed chromophores are located in a microenvironment the polarity of which corresponds to 25-50% ethylene glycol. A significant fraction of tyrosine residues (65-90%) and tryptophan residues (20-45%) is perturbed by temperature. Much lower fractions, i.e. 35-45% of tyrosine residues and less than 15% of tryptophan residues, are perturbed by 20% glycerol. The numbers of perturbed chromophores in fragments constituting the molecule are lower than or equal to the numbers in the original molecule. The effect of hapten binding is significant only with one of the antibody types, the precipitating antibody. The number of thermally perturbed tyrosine residues is by about 17% lower in the liganded antibody. The absence of an analogous effect in the Fab fragment suggests that the fine conformational mechanism of signal transfer from the binding sites operates only in intact antibody molecules.

Pre-resonance Raman spectroscopy was used to study the interactions of the nitro groups of dinitrophenyl haptens with three dinitrophenyl-binding antibody fragments: M315 Fv, M460 Fab' and X25 Fab'. The observed changes in frequency of modes associated with the nitro moieties are compared with solvent-induced changes for the model hapten 2,4-dinitroaniline. These comparisons demonstrate a specific interaction via the H2N--C--C--2-NO2 and 4-NO2 groups with the protein. The interaction with the 4-NO2 group appears to be absent for epsilon-N-2,4-dinitrophenyl-L-lysine bound to M315 Fv fragment in contrast with either 2,4-dinitrophenylaspartate or 2,4-dinitrophenylglycine bound to M315 Fv fragment, despite the much tighter binding of the lysine derivative. The implications of this for M315 Fv fragment in terms of the antibody specificity are discussed. Comparisons of the effect of binding to M460 Fab' and X25 Fab' fragments also revealed significant differences in the shifts of the nitro group vibrations of 2,4-dinitrophenyl-lysine and 2,4-dinitroaniline.

A new method for the induction of cellular immune response to commonly used haptens in the absence of detectable antibody response is described. Different haptens were convalently coupled to Mycobacteria and they were injected into guinea pigs in incomplete Freund's adjuvant. Humoral and cellular immune response to haptens were examined at weekly intervals for 5 weeks. Our results show that a significant anti-hapten cellular response was induced and subsequently elicited by both in vivo (skin test) and in vitro (Lymphocyte transformation and macrophage migration inhibition) assays.

Carp anti-DNP antibodies were raised by various DNP-carrier conjugates. Their intrinsic affinity (K0) to monovalent E-DNP-L-lysine and functional affinity (DF) for binding to the multivalent DNP-T4 bacteriophages were determined. The functional affinity of antibodies elicited by T-cell-dependent DMPn-HSA (n = 3, 15, 33) is relatively high (KF):1010-1012 M-1). These KF values increase more than K0 during the immune response. The functional affinity is dependent on the molar KNP: HSA ratio. The mediate coupled DMP15-HSA elicits antibodies with the highest functional affinity. Carps immunized with T-cell-independent DNP-conjugates synthesize antibodies which have similar K0 as the antibodies elicited with DNP-HSA. However, the KF-values are in the range from 107 to 1010 M-1 only. The KF of antibodies raised with DNP-BA are 103-104 fold, those of DNP-S III and DNP-Ficoll elicited are only 4 . 101 to 4,7 . 102 fold higher than their corresponding K0-values. This means that these antibodies are not very effective in binding the multivalent DNP-T4. Specifically purified antibodies have also such low functional affinities. These strong differences in the functional affinity of carp DNP-antibodies elicited by T-cell dependent and independent DNP-conjugates are discussed with regard to stimulation of different B-cell subpopulations.

Methods are described for the efficient induction of primary anti-hapten antibody responses to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) in vitro. Mouse leukocytes stimulated in vitro with particulate (bentonite-adsorbed) TNP-KLH demonstrated 10-fold more IgM-secreting, plaque-forming cells (PFC) as measured by a hemolytic plaque assay than did leukocytes stimulated with soluble TNP-KLH. The T dependency of the enhanced antibody response was confirmed using highly enriched B and T lymphocyte populations. These methods should greatly facilitate studies regarding primary in vitro antibody responsiveness to T-dependent antigens.

The co-operation of spleen cells of carrier- and hapten--carrier-primed mice in antibody formation against the hapten part of complexes was studied in 550 rad whole body irradiated mice. Hapten--carrier complexes were prepared with the 2,4-dinitrophenyl group (DNP) as a hapten and heterologous bovine serum albumin (BSA) and isologous mouse immunoglobulin (MIg) as carriers. Priming of donor mice with carrier alone did not prepare for a secondary (IgG) response in the recipients of hapten--carrier. Priming of donors and challenge of recipients with the same hapten--carrier complex resulted in high IgG responses. Whereas donor and recipient immunization with complexes differing in the carrier did not give a secondary response, addition of cells of donors immunized with the carrier of the complex used for challenge, resulted in a secondary response. This was only possible when at least one of the complexes had an intermediate hapten:carrier ratio. Only an IgM or a low IgG response was obtained if both complexes had a high hapten:carrier ratio. Three determinants, namely hapten and carrier groups and new antigenic determinant (NAD), are suggested for antibody formation against hapten--protein complexes. In vivo treatment of donor cells with anti-thymocyte serum (ATS) or anti-plasma cell serum (APCS) and complement (C) suggested that: (1) T-cell epitopes are present on the carrier; (2) DNP groups are B-cell epitopes; (3) NAD and possibly DNP are T-cell epitopes; (4) synergism exists in the collaborative antibody response of B cells recongnizing DNP, T cell recognizing carrier and T cells recognizing NAD. Mitomycin treatment of donor cells was used to test whether cell division was mandatory. While the B cells were sensitive to mitomycin treatment, no effect of this drug was found on the helper activity of T cells.

The ability of the chemically reactive hapten 2-4-6-trinitrobenzene sulphonic acid (TNBSA) to inhibit anti-TNP IgM antibody plaque-forming cells (APFC) was investigated. TNBSA at a concentration of 1 mg (3 X 10(-3)M) to 0.3 mg (10(-3] completely inhibited IgM anti-TNP APFC after a 10-min incubation period at 37 degrees C. This inhibition was specific and did not alter the antifluorescein IgM antibody response. After a 24-h culture period in tissue culture medium without antigen, the non-dividing APFC remained inhibited, whereas the dividing APFC escaped this inhibition.

Bovine serum albumin (BSA) was derivatized, under mild conditions, with trifluoromethyl-dinitrophenyl (CF3-DNP), a haptenic group cross-reacting with trinitrophenyl (TNP). High-field nuclear magnetic resonance of fluorine (19F-NMR) permitted to calculate the number of covalently and noncovalently bound haptenic groups per BSA molecule. Further dialysis against paratoluene sulfonic acid permitted to obtain CF3-DNP-BSA conjugates from which noncovalently bound hapten had been removed. Soluble conjugates containing 4 covalently bound plus 1 noncovalently bound hapten groups, or only 4 covalently bound groups, were added to splenocytes in culture. These splenocytes, after such treatments, were added to effector lymphocytes in a 5-day culture aimed at the generation of cytotoxic T lymphocytes (CTL) against the CF3-DNP-induced cell surface modification antigens. It was found that only the BSA conjugates that contained noncovalently bound haptens were able to generate CTL against target cells whose surface had been directly modified with CF3-DNP, whereas BSA bearing only covalently bound hapten groups were not. Replacing BSA by human serum albumin, or CF3-DNP by TNP, gave comparable results. Thus, under the conditions used, haptenic groups covalently bound to their soluble carrier protein did not generate hapten-dependent CTL, but noncovalently bound or free haptenic groups at very low concentration were able to do so.

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.

The ability of haptenated subpopulations of epidermal cells (EC) to induce contact hypersensitivity (CH) in Syrian hamsters was investigated. Crude haptenated EC and haptenated EC enriched for Langerhans cells (LC) inoculated i.v. into hamsters induced CH that was equivalent in intensity to that induced by epicutaneous application of hapten. By contrast, haptenated EC, relatively depleted of LC, failed to induce CH hypersensitivity responses. Upon subsequent reimmunization of all animals with epicutaneously applied hapten, hamsters that had first received haptenated EC depleted of LC failed to respond in CH assays, indicating that these animals had been rendered unresponsive. These data suggest that haptenated LC are capable of inducing CH regardless of the route of inoculation, whereas haptenated EC, when depleted of LC, deliver a down-regulating signal via the i.v. route.

The immunological competence of foetal (85 days of gestation), neonatal and older pigs has been assessed by their ability to make heterogeneous IgG antibody to dinitrophenyl (DNP) hapten. Antibody heterogeneity was resolved by isoelectric focusing (IEF) followed by overlay of labelled antigen. Choice of protein carrier (bovine serum albumin, keyhole limpet haemocyanin or ovalbumin) had little effect on anti-DNP responses. Pigs of all three ages were capable of producing isoelectrically heterogeneous antibody which did not become more diverse, either with repeated immunization or with increasing age. Foetal pigs did not all respond equally strongly as assessed by IEF, but those with clearly heterogeneous antibody showed IEF patterns similar to 'adult' type anti-DNP. This demonstrates that by 85 days of gestation the pig foetus is capable of a mature response to an antigen which is T-dependent in rodents.

The hapten binding characteristics of the covalent light chain dimer, derived from the murine IgA secreted by plasmacytoma MOPC-315, to two nitroaromatic compounds, epsilon-N-(2,4-dinitrophenyl)-L-lysine and 4-(alpha-N-alanine)-m-nitrobenz-2-oxa-1,3-diazole, were investigated by differential spectroscopic titrations. The binding curves for both haptens were found to display sigmoidity similar to that reported earlier for the reduced and alkylated dimer held together by noncovalent bonds only. However, the presence of the interchain disulfide bond in the covalent dimer was found to cause marked changes in its binding properties. The data, like those obtained for the noncovalent dimer, fit the allosteric model of Monod, Wyman, and Changeux in which binding of the first hapten to the dimer causes a conversion of both sites of the protein molecule from a lower to a higher affinity conformation. However, the binding parameters show that both the affinity and the positive cooperativity in the interaction between haptens and the covalent dimer are significantly enhanced. The differences in the parameters of the binding and of the allosteric transition caused by the presence of the interchain disulfide bond demonstrate the existence of longitudinal interactions in immunoglobulin derivatives. These properties of the light chain dimer make it a potential model for the receptors present on thymus-derived lymphocytes.

Several anti-H-2Kk but not anti-H-2Dd monoclonal antibodies (mAb) exhibited enhanced binding to B10.A murine spleen cells after modification of the cells with trinitrobenzene sulfonate (TNBS). The number of antibody molecules bound to TNP-modified B10.A spleen cells increased by a factor of two or more. The same anti-2Kk mAb that exhibited enhanced binding to modified B10.A cells did not bind to unmodified C57BL/10 spleen cells, as expected, but did bind to TNP-modified C57BL/10 spleen cells. This TNP-dependent binding was not a result of cross-reactions with cell surface TNP groups nor with Fc receptors. TNP modification of a variant cell line that does not express class I H-2 products did not result in enhanced binding by these mAb. These findings can account for preferential recognition of TNP-Kk by B10.A and B10.BR CTL, and also for cross-reactive lysis by C57BL/10 CTL stimulated by C57BL/10-TNP against unmodified H-2Kk targets.

Reaginic antibodies to DNP and ovalbumin were indcued readily in B6D2F1 mice by a single intraperitoneal injection of 1 mug of DNP-ovalbumin suspended with 1 mg aluminum hydroxide in 0.5 ml of saline. The formation of anti-DNP reaginic antibody was completely suppressed by treatment of mice with a conjugate consisting of the hapten coupled to an isolgous nonimmunogenic carrier, viz., murine phi-globulins. However, this treatment did not affect the level of antibody formation to the carrier of the immunizing antigen. The induction of unresponsiveness was dose dependent, complete suppression being achieved with 1 mg of the tolerogen. The state of unresponsiveness could be maintained for prolonged periods (these observations were made over a period of at least 8 months) by repeated injections of the tolerogen at intervals of 2 months. More importantly, the state of unresponsiveness could be induced readily not only in normal, but also in sensitized mice, i.e., this treatment was capable of abrogating an ongoing reaginic response, and the suppression was immunologically specific. Hence this system appears to have a great potential for adaptation to the treatment of allergic diseases in man.

Suppressor T cells acting on the efferent phase (Ts-eff) of dinitrofluorobenzene-contact sensitivity (DNFB-CS) were induced in BALB/c mice by injection of dinitrobenzenesulphonate (DNBSO3) and subsequent painting (sensitization) with DNFB. Ts-eff cells released a suppressor factor, antigen and strain specific, which was active in vivo in sensitized recipients. Administration of DNBSO3 alone generated suppressor T cells, which acted only on the afferent phase (Ts-aff). Suppressor T cells could not be detected in mice painted with DNFB and desensitized later by injection of DNBSO3.

The conjugation of drugs to autologous proteins is thought to be a key step in the hapten mechanism of drug hypersensitivity. We have studied the mild arylating agent dinitrofluorobenzene (DNP-F) as a model compound with which to investigate the relationship between the disposition and immunogenicity of drug haptens in two species, the rat and rabbit. Intramuscular administration of DNP-F (0.027-27 mumol/kg/day) for 4 days to male Wistar rats produced a dose-dependent (ED50 2.7 mumol/kg) IgG anti-DNP antibody response, measured by enzyme-linked immunosorbent assay. Subsequent monthly administrations (for 4 days) increased both the frequency and titre of antibody response. Intravenous administration of [3H]DNP-F (0.27 or 2.7 mumol/kg) for 9 days to male New Zealand White rabbits produced an IgG and IgM anti-DNP response in all animals from day 9 onwards. Formation of circulating (serum) DNP-protein conjugates was determined by radiometric analysis, and found to reach steady state (0.12-0.17% dose/ml) between days 6 and 8 and decline with a half-life of 7.4 days. The immunogenicity of fully characterized haptenated, autologous proteins was investigated in further experiments in which dinitrophenylated serum protein conjugates (DNP-RSP) and albumin conjugates (DNP-RSA) were prepared ex vivo and then administered (50 mg/kg; i.v.) to the rabbit from which the protein had been obtained. The plasma clearance and immunogenicity of DNP-RSA conjugates was dependent on epitope density. Anti-DNP antibodies were detected after administration of an RSA-DNP15 conjugate but not after either RSA-DNP0.5 or RSA-DNP5. Plasma concentrations of RSA-DNP15 conjugate declined slowly initially, but then fell rapidly between days 8 and 10. The plasma clearance of DNP-RSP conjugates showed a dependence on epitope density from day 1 onwards and anti-DNP antibodies were detectable after administration of all conjugates investigated (range of epitope densities 0.5-30 DNP/albumin equivalent). Thus conjugates derived from proteins other than albumin are likely to be the effective immunogens, for the model hapten DNP. These studies show that DNP-F is a useful model compound in studies of the disposition and immunogenicity of drugs acting as haptens, and may therefore be used as a positive control in experiments designed to assess the potential immunogenicity of drugs and other xenobiotics.

The capacity to respond to haptenated Ficoll, though thymus-independent in mammals, is lost in Xenopus laevis following thymectomy at any stage of development. This capacity can be restored to thymectomized Xenopus by whole allogeneic thymus implants, by thymus implants from animals treated with N-methyl-N-nitrosourea (which have an active thymus medulla but no cortex, and which lack certain thymus-dependent immunological functions), and Concanavalin A. These results are discussed in terms of the cell populations and/or factors which may be involved.

Injection of mice with the chemically reactive hapten 2,4,6-trinitrobenzene sulphonic acid (TNBSA) induces a specific unresponsiveness as judged by reduction or abolition of the anti-TNP response to TNP-KLH. The normal response to a nonrelated hapten, oxazolone, bound to the same carrier (OX-KLH) is unaffected. Reduction of the anti-TNP response was also observed after TNBSA treatment, in nude mice (nu/nu) and their littermates (nu/+) challenged with TNP-POL, an antigen to which the response is thymus independent. Injection of the chemically non-reactive hapten TNP-glycyl-glycine did not induce unresponsiveness. A similar failure was observed with TNP autologous red cells or serum proteins from mice previously injected with TNBSA. The specific unresponsiveness of spleen cells of TNBSA injected mice was maintained after their transfer into lethally irradiated syngeneic host mice. Finally tolerant cells do not inhibit specifically an adoptive anti-TNP secondary response.

The immunogenic properties of the dihapten and monohapten derivatives of polyethylene glycol with different nitroaromatic groupings were studied. 2,4-dinitrophenyl, 2, 4, 6-trinitrophenyl and trinitrophenyl-ethyl groupings were used as hapten groups. The injection of monohapten compounds was found to induce the accumulation of antibody-forming cells secreting antibodies to trinitrophenyl in the spleen of normal and athymic nude mice. As early as on day 3 the number of antibody-forming cells considerably exceeded their background level, the process of B-cell activation being, to a certain extent, thymus-independent. Dihapten compounds were not immunologically active. The effect rendered by the nitroaromatic derivatives of polyethylene glycol, revealed in this study, is linked with the known capacity of polyethylene glycol to adsorb on the surface of cell membranes.

Techniques are reported for the induction and assay of cytotoxic effector cells capable of specifically lysing hapten-coupled EL4 leukemia targets. It is shown that EL4 cells survive coupling with TNP-sulfonic acid and retain the hapten on their cell surface for a prolonged period of time. Although TNP-EL4 cells are readily lysed by anti-TNP serum in a complement-mediated reaction, they are inefficiently killed in an antibody-dependent cell-mediated reaction. Cytotoxic effector cells, able to lyse TNP-EL4 targets, are induced when C57BL/6 spleen H-2-b cells are cultured with the following cell types which have been coupled with TNP: 1) ALLOGENEIC P815 tumor cells (H-2-d), 2) syngeneic EL4 tumor cells, 3) allogeneic BALB/c spleen cells (H-2-d), 4) syngeneic C57BL/6 spleen cells. Further experiments show that TNP-coupled xenogeneic chicken erythrocytes, which by themselves are unable to induce cytotoxic effectors, are capable of doing so if uncoupled P815 cells are present simultaneously. On the basis of these findings, it can be hypothesized that two stimuli are required for induction of these cytotoxic effector cells--one provided by the hapten, and the other by the P815 cell. Treatment of cytotoxic spleen cells induced by hapten-coupled allogeneic tumor cells with anti-Thy-1 serum and complement abrogates their cytotoxicity, indicating that T cells play a central role in the cytotoxic reaction. TNP-coupled erythrocytes do not serve as targets for these cytotoxic T cells, but do cause competitive inhibition of TNP-EL4 when added to the reaction mixture at high ratios. However, because the inhibition is relatively low, and because no such inhibition can be demonstrated with TNP-lysine, it is concluded that the receptor on the cytotoxic effector cell has a low affinity for hapten. This low affinity could be due to the receptor recognizing an antigen comprising mouse cell surface antigen in addition to the TNP moiety. Supporting this interpretation is the finding that TNP-EL4 cells competitively inhibit cytotoxicity much more efficiently than TNP-CRBC and that even uncoupled EL4 cells inhibit to some extent.

The immune response of mouse spleen cells to hapten-conjugated polymer of flagellin (DNP-POL, NIP-POL) was studied using a microculture system. When increasing numbers of spleen cells were added to a 'filler' cell system, negative feedback effects became apparent and resulted in the generation of progressively lower numbers of plaque-forming cells (PFC) per input cell. This feedback inhibition was shown to be antigen-specific and mediated by factors released into the culture medium. The effect precludes calculation of the frequency of PFC precursors in cultures containing spleen cells alone and complicates the analysis of tolerance using in vitro assay systems. The addition of small numbers of spleen cells to a constant number of thymocytes provided a system in which Poisson analysis could be used to determine the frequency of PFC precursors capable of being activated by hapten-POL conjugates. This system was used to estimate the frequency of anti-NIP-PFC precursors in CBA spleen cells.

Attempts were made to induce antibody response of hapten-primed cells by stimulation with anti-immunoglobulin (Ig) antibody and cellfree supernatant (CFS) which contained nonspecific enhancing factor. Mesenteric lymph node cells were obtained from rabbits which were primed with dinitrophenylated ragweed antigen (DNP-Rag), and the primed cells were incubated in vitro with either anti-rabbit gamma-chain or anti-Fab antibody for 24 hr. After washing, the cells were cultured in CFS which was obtained from the culture of DNP-Ascaris (DNP-Asc)-primed lymph node cells with free carrier (Asc). The results showed that hapten-primed cells were triggered for IgG anti-DNP antibody response by the two reagents, i.e., anti-Ig antibody and CFS, both which did not share any antigenic specificity with the priming antigen (DNP-Rag). Neither anti-Ig alone nor CFS alone induced anti-hapten antibody response. Anti-Ig and CFS triggered not only hapten-primed B cells but also the other IgG-bearing (B) cells for IgG synthesis. Lymph node cells of umprimed animals were activated for IgG synthesis by the stimulation with anti-Ig followed by culture in CFS. Evidence was obtained that anti-Ig antibody has to be divalent for the activation of B cells. Stimulation of DNP-primed cells with the F(ab')2 fragment of the antibody and CFS-induced anti-hapten antibody response, whereas the Fag' fragment of the anti-Ig plus CFS failed to do so. The results suggested that bridging of cell-surface Ig receptors by multivalent ligand is the initial step of B cell activation. Another evidence for the activation of hapten-primed B cells by anti-Ig was obtained by supplemental immunization of DNP-Asc-primed animals with the Fc fragment of goat IgG. Stimulation of mesenteric lymph node cells from these animals with goat anti-rabbit-Ig alone (no CFS) resulted in the formation of anti-DNP IgG antibody. The results indicated that lymphocytes primed for goat IgG collaborated with DNP-primed IgG-B cells when the lymph node cells were stimulated with goat anti-Ig.

The in virto immunogenicity of the solid-phase hapten, dinitrophenyl-ornithine-Bio-Gel (DNP-O-Bio-Gel), was investigated in cultures of mouse spleen cells. Appropriate combinations of cells and immobilized hapten were determined. Large numbers of direct anti-hapten plaque-forming cells (PFC) were generated when 1 times 10-7 C57BL/6 or C57BL/10 spleen cells were cultured with 4 times 10-3 DNP-O-Bio-Gel beads. Specificity studies of the responses of cultured spleen cells to DNP-O-Bio-Gel yielded the following results: soluble DNP-ornithine or DNP-bovine gamma-globulin inhibited the induction of anti-hapten PFC by DNP-O-Bio-Gel; neither dinitrophenyl-Bio-gel (DNP-Bio-gel) nor ornithine-Bio-Gel (O-Bio-Gel) induced anti-hapten responsiveness; furthermore, neither DNP-Bio-Gel nor O-Bio-Gel inhibited the induction of PFC by DNP-O-Bio-Gel. It was concluded, from the results of these specificity experiments, that a spacer, ornithine, is required for immunogenicity of immobilized DNP; and that the Bio-Gel bead, itself, acts solely as a physical carrier for the hapten.

A dinitrophenyl hapten capable of protein SH-group modification was synthesized and the specificity of its reaction with SH-groups of the E. coli ribosomal proteins was studied. The possibility of incorporation of the Dnp-modified protein into ribosomal subunits by in vitro reconstitution was demonstrated with the ribosomal protein S12. The Dnp-hapten attached to the protein S12 was found to be accessible for interaction with the Dnp-specific antibodies and therefore to be exposed on the surface of the reconstituted 30S subunit. Thus, the approach for incorporation of the antigenic groups into the protein components of supramolecular structures was proposed.