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NC/Nga mice have pathological and behavioral features similar to those seen in human atopic dermatitis. There are two known dermatitis models in NC/Nga mice, one being spontaneous-induced dermatitis under conventional conditions and the other 2,4,6-trinitrochlorobenzene (TNCB)-induced allergic contact dermatitis. However, there are significant differences in time course on development of dermatitis. We studied the role of scratching behavior (sign of itch) on the development of dermatitis on spontaneous- and TNCB-induced dermatitis. We measured scratching counts, transepidermal water loss (TEWL), and skin inflammation score, under conventional conditions or by applying 5% TNCB once a week for 6 weeks in NC/Nga mice. In spontaneous-induced dermatitis, scratching counts increased with the passage of time. The scratching counts were significantly increased only 1 week after housing the mice under conventional conditions, but no changes were observed in cases of TNCB-induced dermatitis. In spontaneous-induced dermatitis, TEWL and skin-inflammation score were gradually increased, time-dependently. On the other hand, in TNCB-induced dermatitis, these dependent values rapidly increased and reached a maximum only after 24 h TNCB application. These data suggest that pathogenesis of spontaneous- and allergic contact-induced dermatitis was clearly different. It will be of major interest to identify the pruritic mediators causing profound scratching behavior and scratching-induced aggravation of inflammation in the spontaneous-induced dermatitis, as opposed to the inflammatory mediators that cause contact allergic dermatitis without major scratching.

Spleen and bone marrow (BM) are the major sites of antibody production and anamnestic response in systemically immunized mice. We examined the VDJ segment repertoire of antibody plaque-forming cells (APFC) in those two sites in the course of antibody responses to the hapten nitrophenyl (NP). Individual IgG APFC expressed any one of 10 V(H) segments of the V186.2/V3 (J558) gene family: 186.2, 102, 23, C1H4, 165.l, CH10, 3, 593.3, 24.8 and 671.5. The majority of cells in both spleen and BM expressed the V186.2 gene joined to a D segment with Tyr95. During a 2-month period after a single immunization, the V186.2(+) APFC in BM accumulated 3 times as many somatic mutations than splenic APFC (average 8.5 versus 3 mutations/V(H)); this process was T(h) dependent as shown by in vivo depletion of CD4(+) lymphocytes. However, the V186.2(+) APFC in both spleen and BM shared a recurrent W33L replacement, indicating their common origin from germinal centers. The APFC expressing the other (analogue) V(H) segments were evenly represented in the spleen and BM, but they accumulated few, if any, mutations. The anamnestic V186.2(+) APFC were highly mutated both in the spleen and BM; they represented a new and unexpected clonotype. The V/D segments were joined by Gly95 instead of Tyr95, the W33L was absent and a new shared K58R replacement appeared. The APFC expressing the 'analogue' V(H) genes comprised approximately 20% of the anamnestic response and did not accumulate more mutations, but their affinities were in the range of the memory V186.2(+) cells. These data suggest that the late primary and secondary responses to a hapten may be born by different B cell lineages, and that some clonotypes may reach the memory pool without an extensive mutation and expansion.

Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the alpha- and beta-forms of p-nitrophenyl-labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.

After exposure to antigen, Langerhans cells (LC) migrate from the epidermis to lymph nodes, where they initiate primary immune responses in T cells. The adhesion molecule E-cadherin mediates adhesion of LC to keratinocytes in vitro and may be responsible for localization of LC in epidermis. To determine if levels of LC E-cadherin are modulated during LC emigration from epidermis, we utilized flow cytometry to evaluate E-cadherin expression on BALB/c LC exposed in situ to the contact allergen 2,4,6-trinitrochlorobenzene (TNCB). TNCB induced increased I-A/E antigen and decreased E-cadherin expression on a subpopulation of LC as early as 12 h, and as late as 48 h, after application. At 24 h, approximately 30% of LC in TNCB-treated skin expressed increased I-A/E antigens; of these activated LC, approximately 40% expressed decreased levels of E-cadherin. E-cadherin levels on this latter subset were approximately 15% of those expressed by LC in normal skin, and were similar to levels on cultured LC and LC that migrated from skin explants. The effect was specific for allergens; no changes occurred in LC following treatment with several contact irritants or the tolerogen dinitrothiocyanobenzene. To determine if cytokines modulated LC E-cadherin expression, we introduced various cytokines into BALB/c ear skin and assayed I-A/E antigen and E-cadherin levels. Of the cytokines tested, only interleukin-1 and tumor necrosis factor alpha reproduced the effects of TNCB. We propose that downmodulation of E-cadherin expression occurs as a consequence of local cytokine production during antigen-induced LC activation, facilitating LC emigration and the initiation of immune responses against antigens encountered in epidermis.

In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens. TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nu/nu, and CBA/N mice. In contrast, N-2,4-dinitrophenyl-beta-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice. These results indicate that LPS from B. abortus acts as a TI-1 carrier in generating antibody responses. In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin G1 (IgG1), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC. Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold). This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B. abortus and E. coli to stimulate C3H/HeJ B cells. The ability of LPS from B. abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B. abortus organisms in vaccine development.

The relative complement-mediated lytic capability of the IgG subclass isotypes was studied using a matched set of mouse-human chimeric anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies. The subclass pattern was shown to be highly dependent on variations in antigen concentration and to lesser extent on variation in epitope patchiness, antibody binding affinity and complement concentration. In general, the IgG3 subclass was most effective in inducing cytolysis at the different conditions used and only at high antigen concentration did the IgG1 subclass mediated more efficient cytolysis than IgG3. The IgG2 isotype required a relative high antigen concentration to be cytolytic while the IgG4 isotype was not cytolytic at any of the conditions tested. These individual characters of each of the IgG subclasses makes it conceivable that a subtle system of immunoregulation exists among the subclasses.

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.

Prenatal tolerization with trinitrobenzenesulphonic acid (TNBS) leads to expansion of trinitrophenyl (TNP)-specific B cells, the majority of which become refractory to stimulation during postnatal development. One possible explanation could be that they belong to the repertoire of naturally activated B cells which are limited in expansion after antigenic stimulation due to a high degree of idiotypic connectivity. To evaluate this hypothesis, 59 thymus- and 490 spleen-derived B-cell hybridomas from 6-day-old prenatally untreated and prenatally TNBS-treated mice were tested for reactivity against 33 arbitrarily chosen clones derived from the same fusions, 17 being derived from control and 16 from tolerized litters. Two major points could be deduced: (1) Idiotypic connectivity, including connectivity of TNP- and anti-TNP-reactive monoclonal antibodies (MoAb), was maintained after prenatal tolerization. This accounted for thymus- and spleen-derived MoAb. (2) Only TNP- and anti-TNP-reactive MoAb derived from prenatally untreated and prenatally tolerized mice displayed significantly distinct idiotypic profiles. Differences were pronounced, especially with thymus-derived MoAb. Thus, TNP-specific B cells in prenatally tolerized newborns do not behave like B cells of adult mice stimulated by external antigen, but rather like a part of the naturally activated, idiotypically connected B-cell repertoire of the newborn. This could explain B-cell unresponsiveness at older age as a consequence--at least partly--of their high idiotypic connectivity.

Myosin contains reactive lysine residues which are trinitrophenylated by 2,4,6-trinitrobenzene sulfonate much faster than the rest of the lysines. Here we find the location of these residues in the primary and spatial structure of myosin with the help of an anti-trinitrophenyl antibody. This antibody was raised against trinitrophenyl hemocyanin in rabbits. It reacted with trinitrophenylated myosin, and with some of the tryptic fragments of trinitrophenylated myosin. By analyzing the reaction with Western blots, it was found that the antibody preferentially reacts with the 27 kDa N-terminal fragment of the myosin head, and more weakly with the light meromyosin region of the myosin rod. The 27 kDa fragment contains the most reactive lysine residue, while the intermediate lysine residue is located in the light meromyosin region. The locations of the epitopes of the antibody were visualized on electron microscope images of rotary-shadowed trinitrophenylated myosin-antibody complexes. The distances of the epitopes to the head-rod junction of myosin were measured as 13 and 113 nm for the epitope on the head (reactive lysine residue) and for that on the rod (intermediary reactive lysine residue), respectively.

It is still a matter of debate, whether tolerance toward self-non-MHC antigens is due to intrathymic deletion or to regulatory processes in the periphery. To further pursue this question, responsiveness toward TNP and an anti-TNP monoclonal antibody (Sp6) carrying a recurrent idiotype was evaluated in prenatally trinitrobenzenesulfonic acid (TNBS)-treated mice. In prenatally untreated as well as in TNBS-treated mice, thymocytes proliferating in the absence of nominal antigen were double negative (L3T4-/Lyt2-), but antigen-specific thymocytes were single positive (L3T4+/Lyt2- or L3T4-/Lyt2+). TNBS-treated mice differed from controls inasmuch as in their first week of life T cells proliferating in response to TNP were found in the thymus and detected at increased frequencies in the spleen. The frequency of TNP-specific thymocytes and spleen cells declined rapidly, finally reaching in the spleen a level of 20-30% of controls. Furthermore, after antigenic stimulation, the frequency of thymocytes and spleen cells proliferating in response to TNP was found to be increased in control mice, but TNP-specific T cell were no more recovered in the thymus or the spleen of tolerized mice. The same accounted for thymic and splenic T cells proliferating in response to Sp6. They were expanded in control mice after antigenic stimulation, but were undetectable in TNBS-treated mice. Thus, T cells with specificity for an internal (Sp6) and an external (TNP) antigen, provided the latter was present during ontogeny, were detected in the thymus of control and, transiently, in the thymus of tolerized mice. But, the fate of antigen-specific thymocytes was different in prenatally untreated and TNBS-treated mice. The data are interpreted in the sense that tolerance toward non-MHC antigens may be acquired subsequently to tolerance toward self-MHC antigens and possibly after imprinting of antigen specificity.

Tripalmitoyl-S-glycerylcysteinyl lipopeptides are B-cell and macrophage activating and may be used as low molecular weight immunogens of considerable potency and even as vaccines when conjugated with suitable epitopic structures. Selected lipopeptides carrying single Dnp haptens were found to evoke mild passive cutaneous anaphylaxis in guinea pigs sensitized against Dnp. The reactions were observed after intravenous injection whereas intradermally applied antigen was negative. The anaphylactogenicity seems unrelated to micelle or aggregate formation of the insoluble peptides which require lecithin additions as well as sonication to become solubilized. The dinitrophenylated lipopeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-lysine produced toxic reactions which were not observed with the lipopeptide devoid of Dnp. Dinitrophenylated tripalmitoyl-S-glycerylcysteiny-1,6-diaminohexane and tripalmitoyl-S-glyceryl-cysteinyl-lysine did not show these toxic reactions.

The role of metabolic detoxication (glutathione conjugation) in the humoral response to three model haptens, dinitrofluorobenzene (DNFB), dinitrochlorobenzene (DNCB) and dinitrobenzenesulphonate (DNBS), was investigated in male Wistar rats. All three haptens produced a measurable anti-dinitrophenyl IgG antibody response over a wide dose range (2.7 nmol/kg to 0.27 mmol/kg) given for 4 days. A significant difference in antibody titre was only observed at the highest dose (DNFB greater than DNCB = DNBS), despite a marked difference in reactivity towards a protein carrier (albumin) and N-acetyl-lysine in vitro. These observations can be partly explained by the fact that the most reactive hapten (DNFB) conjugates most rapidly with glutathione in vitro and in vivo.

T cell-containing lymphoid populations produce a nonantigen-specific mediator(s) (NSM) which can replace T cell helper function in vitro in the response of B cells to sheep red blood cells (SRBC), but not to the hapten-protein conjugate, trinitrophenyl-keyhole limpet hemocyanin, (TNP-KLH). NSM produced under three conditions: 1) stimulation of KLH-primed cells with KLH; 2) allogeneic stimulation of normal spleen cells; and 3) stimulation of normal spleen cells with Con A (but not PHA) are indistinguishable on the basis of their biologic activity and m.w., estimated as 30 to 40,000 daltons by G-200 chromatography. Production of NSM is dependent on the presence of T cells. The action of NSM on B cells responding to SRBC in the presence of 2-mercaptoethanol is unaffected by severe macrophage depletion. Extensive absorption of NSM with SRBC failed to remove its activity, confirming its nonantigen-specific nature.

The activity of a complement-dependent cytotoxic antibody in the sera of 21 melanoma patients was investigated using a microcytotoxicity assay. Heat-inactivated sera were caused to react against mechanically dispersed fresh tumor cells in the presence of exogenous blood group AB complement. Cytotoxicity was evaluated relative to pooled normal sera as a control. Sera were cytotoxic against autochthonous tumor cells in 9 of 10 patients with localized or regional melanoma and in 1 of 11 patients with disseminated metastases. Cytotoxicity of sera was unrelated to size of tumor burden. Six of 7 antibody-positive sera (autochthonous system) were noncytotoxic to between 2 and 7 different allogeneic melanoma tumor cell preparations. Immunological reactivity of the cytotoxic antibody-positive and -negative groups was similar with respect to their capacity to be sensitized to dinitrochlorobenzene, produce positive skin tests to microbial antigens, and produce antibodies to typhoid vaccination; serum immunoglobulins were comparable. These results support the reported findings of the presence of cytotoxic antibody in the sera of melanoma patients without disseminated metastases.

Trinitrophenyl (TNP)-couple epidermal cells (EC) injected subcutaneously (s.c.) were more capable of inducing contact sensitivity (CS) to 2, 4, 6-trinitro-1-chlorobenzene (TNCB) than similarly substituted spleen cells (TNP-SC). Furthermore, the intravenous (i.v.) or intraperitoneal (I.P) injection of TNP-EC also induced CS response, whereas the i.v. or i.p. injection of TNP-SC failed to induce them. Treatment of mice with cyclophosphamide (Cy; 50 mg/kg) or anti- I-J serum allowed animals injected with TNP-SC i.v. to develop significant CS responses, suggesting that Cy-sensitive and I-J positive regulatory cells were involved in the induction of unresponsiveness by the I.V. injection of TNP-SC. Mapping studies o the major histocompatibility gene complex (MHC) region demonstrated that identity at the I-A subregion alone between EC donor and recipient mice was sufficient for the induction of CS by TNP-EC given i.v. Blocking experiments using antisera in the absence of complement indicated that I-A subregion-encoded antigens on the surface of TNP-EC apparently are involved in the induction of CS, and are not simply phenotypic markers on the surface of accessory cells.

Tolerance to major histocompatibility antigens as well as the ability to mount a cytotoxic response to hapten-modified cells of bone marrow donor and host origin was studied in allogeneic radiation chimeras. Lethally irradiated (C57BL/6 X DBA/2)F1 hosts reconstituted with anti-Thy 1.2 + C-treated bone marrow from (C57BL/6 X CBA)F1 mice showed tolerance to the MHC antigens of the three parental strains as measured by MLC and CML assay. The chimeras responded normally to unrelated allogeneic cells. Chimeric animals generated a cytotoxic response to hapten-modified cells of both donor (CBA) and host (DBA/2) haplotypes, as well as to C57BL/6, demonstrating that tolerance to the hapten-presenting host haplotype is sufficient to allow a cytotoxic antihapten response, and that processing through a semiallogeneic host environment does not affect the ability to generate a response to hapten in conjunction with self-determinants. Chimeras failed to mount a cytotoxic response to hapten presented on nontolerated allogeneic spleen cells.

Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectiviely a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be medicated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-D surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.

Secondary cell-mediated lympholysis (CML) and mixed lymphocyte reactions (MLR) were generated in a tissue culture system against trinitrophenyl (TNP)-modified murine syngeneic spleen cells. H-2 homology between primary and secondary TNP-modified stimulating cells was required in order to restimulate in the secondary CML. Strong proliferative responses (MLR) were detected only in the secondary cultures, for which H-2 homology was also required between TNP-modified primary and secondary immunogens. Intra-H-2 mapping for the secondary MLR indicated that the relevant regions of homology were I, D, and K and/or I-A. Homology throughout the entire major histocompatibility complex or at K plus I-A gave stronger MLR than did cultures in which there was homology between the primary and secondary phases at I or D only.

Dinitrophenylated-type III pneumococcus has been used to induce biliary IgA anti-DNP antibody responses in rats. A solid phase radioimmunoassay was employed to quantitate the biliary IgA anti-DNP antibodies after subcutaneous, i.v., or intragastric immunization. All routes were shown to effectively elicit biliary IgA antibody responses. The microscale sucrose isoelectric focusing technique was employed to assess molecular diversity of the secretory IgA anti-DNP antibodies in bile. Subcutaneous and intragastric immunization resulted in complex antibody spectrotypes whereas i.v. immunization yielded restricted antibody spectrotypes, demonstrating that the route of immunization influenced the molecular diversity of the biliary secretory IgA antibodies. These findings are discussed in terms of mechanisms governing the induction of secretory antibodies.

After injection of TNP-KLH in mice and TNP-BGG-PEN in rabbits, anti-TNP antibody-forming cells were observed in the spleen. When cryostat sections of the stimulated spleen were incubated with TNP-HRP conjugate, and then treated for HRP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained red. When similar sections were incubated with TNP-AP conjugate, and then treated for AP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained blue. After simultaneous incubation with TNP-AP conjugate and PEN-HSA-HRP conjugate, followed by treatment for HRP cytochemistry and AP cytochemistry, anti-TNP-forming cells with a blue cytoplasm and anti-PEN-forming cells with a red cytoplasm could be distinguished in the same spleen section.

Two samples of murine monoclonal antibodies to dinitrophenyl groups were studied by difference thermal perturbation spectroscopy with particular attention to changes in the amount of perturbed chromophores induced in antibodies as a result of hapten binding (epsilon-2,4-dinitrophenyl-L-lysine). Despite the fact that both antibody samples belong to immunoglobulin G1 and have the same type of light chain, kappa, they were found to differ significantly in the number of the chromophores perturbed by temperature. The binding of hapten decreases the perturbation of chromophores only in the sample with the less rigid structure, as regards thermal perturbation. These data provide evidence that differences in the rigidity of the structure of variable domains affect the extent of conformational changes induced in the antibodies due to the interaction with an antigen.

This report defines a methodology for the production and characterization of an antigen-specific, monoclonal T cell hybrid-derived suppressor T cell factor (TsF) that suppresses the passive transfer of 2,4-dinitrofluorobenzene (DNFB) contact hypersensitivity. Fusion of T cells from BALB/c (H-2d) mice tolerized with syngeneic DNP-spleen cells to BW 5147 thymoma cells resulted in several hybrids that constitutively produce a soluble regulatory molecule. One of these hybrids, 26.10.2, was subsequently cloned, and its soluble factor was characterized with respect to its antigen specificity, biochemical nature, MHC restriction pattern, and identity of its target cell. 26.10.2 TsF suppresses the passive transfer of delayed-type hypersensitivity (DTH) mediated by DNP- but not trinitrochlorobenzene- or oxazalone-primed DTH T cells (TDH) after a 1 hr incubation at 37 degrees C. In contrast, 26.10.2 TsF had no suppressive effect on secondary in vitro DNP-specific T cell proliferative responses. 26.10.2 TsF therefore represents an antigen-specific factor with effector (efferent-acting) function. The monoclonal TsF was shown to consist of a two-chain, disulfide-bonded molecule, and to bear a receptor(s) specific for DNP and determinants encoded by the I region of the H-2 complex. Effector suppressive activity of 26.10.2 TsF was restricted by Class I H-2Dd determinants. One cellular target of this monoclonal factor was shown to be the DNP-specific TDH cell, because DNFB-primed lymph node cells from cyclophosphamide-pretreated donors (lacking Ts-auxiliary (Ts-aux) cells) were efficiently suppressed. The TsF appears to focus on passively bound, TDH receptor-associated, DNP-Class I determinants, as suggested by the observation that freshly prepared, but not overnight cultured, DNP-specific TDH cells were susceptible to suppression.

A primary in vitro antibody response to DNP-KLH by peripheral blood lymphocytes in culture was developed. Optimal anti-DNP antibody production, as measured by solid phase radioimmunoassay occurred at a cell density of 1 X 10(6), and an antigen concentration of 5 to 10 micrograms per culture when PBL was incubated with the antigen for 5 days and then cultured in the absence of DNP-KLH for 4 additional days. The antibody produced was shown to be primarily of the IgM isotype and was specific for DNP. In the absence of T lymphocytes, no antibody was generated. The regulatory effects of T4+ and T5+/T8+ subsets on antigen-specific antibody production were determined and it was found that T4+ subset alone provided help for anti-DNP antibody production. In contrast, the T8+ subset did not provide help and more importantly, could suppress anti-DNP antibody production in the presence of T4+ inducer T cells. This in vitro antibody forming system should be of considerable use in the analysis of the cellular requirements for antibody production and genetic control of the immune response in man.

Dinitrochlorobenzene (DNCB) test was applied to 22 patients with chronic nephritis. Only 22% of the patients gave positive results. This was concluded to be due to the high NPN levels. It was demonstrated that this inhibition is transitory and the normal responses appear 12 hours after the return of the NPN to normal levels. When it was considered that some patients with high NPN levels showed normal DNCB reactivity and delayed rejection time, the chronic nephritis patiens reacted to organ transplants despite their normal NPN levels. It was suggested that it is one of the components of NPN which should be responsible for the above mentioned inhibition.

The primary and secondary immune responses to a thymus-independent antigen, TNP-LPS, were investigated in C57BL/6, DBA/2 and (C57BL/6 x DBA/2)F1 mice. While there is no evidence that TNP-LPS induces immunological memory in DBA/2 mice, a definite priming effect has been observed in C57BL/6 mice. Memory is revealed by the appearrance of antibody-forming cells of the IgG isotype. The differentiation of B micron precursors into B gamma memory cells is a dominant phenotype, since it is also found in the F1 C57BL/6 x DBA/2 hybrids.

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.

A bivalent affinity label, bis(alpha-bromoacetyl-epsilon-2,4-dinitrophenyllysylproline)ethylenediamine, has been synthesized. Treatment of anti-2,4-dinitrophenyl antibodies with this compound produces a mixture of covalently and noncovalently cross-linked material. Only specific antibodies are covalently cross-linked, suggesting that covalent attachment occurs in the variable regions. Covalently cross-linked dimers and trimers have been isolated from the reaction mixture in a high state of purity, in yields of about 12 and 4%, respectively. The complexes are stable in solutions containing 10(-4) M hapten and can therefore be used as sensitive probes of immune effector functions.

The question of whether TH cells are required for the priming of CTL precursors (CTLp) in vivo was studied by using Txbm mice (Thymectomized, irradiated, and stem cell-reconstituted mice). In these mice, TNP-specific CTL could be induced in vitro with TNP-coupled spleen cells only if the cultures were supplemented with an IL 2-containing supernatant (ConAsup). In contrast to normal mice, TNP-specific Lyt-2-TH cells could not be induced by skin painting with trinitrochlorobenzene (TNCB) (as tested by the ability to help CTL formation from thymocyte or normal spleen precursors). These data confirm previous findings that Txbm mice possess CTLp but that their TH compartment is deficient. TNCB skin painting had, however, a clear priming effect on the CTLp population: spleen cells from TNCB-painted mice could give rise to specific CTL with a lower amount of ConAsup than spleen cells from unprimed mice. In addition to this, priming changed the CTLp so that stimulation with lightly coupled cells (0.1 mM trinitrobenzene sulfonic acid [TNBS] instead of 10 mM TNBS) became effective. These changes took place without a significant increase in the frequency of TNP-specific CTL precursors. The data obtained are consistent with the concept that at least with some antigens, CTLp proliferation (clonal expansion), which is probably caused by activated TH cells, is not required for the induction of immunologic memory in vivo.

The effect of specific and nonspecific stimuli on the cycle status of subsets of primary B lymphocytes was assessed by preinjecting donor CBA mice 1 to 2 days previously with various substances, and then incubating the isolated spleen cells with high specific activity 3H-TdR before assay. AFC-progenitor activity was assessed as a response to NIP-POL antigen, either by adoptive transfer to irradiated recipients or by cell culture. Previous studies showed these assays reflected the activity of different subsets of B cells, termed "pre-progenitors" (adoptive assay) and "direct progenitors" (culture assay). Most functional primary B cells, whether assayed in culture or by adoptive transfer, were not initially in rapid cell cycle in normal adult mice. However, nonspecific stimulation for 1 day caused NIP-specific adoptive transfer IgM AFC-progenitors to enter rapid cell cycle. This effect was independent of T cells and not related to the antigenicity of the stimulus: particulate peritoneal irritants were the most effective stimulants. In contrast to adoptive transfer results. AFC-progenitors assayed in cell culture were unaffected by nonspecific stimuli, but were activated into cell cycle by specific antigen.

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.

A man, with a dermatitis acquired while working in a factory producing a weed killer, showed sensitivity to 4-methylsulfonyl 2,6-dinitro-N,N-dipropylaniline (DNA-nitralin) and its precursor, 4-chloro 3,-5-dinitrophenylmethyl sulfone (DNC), and cross-sensitivity to dinitrochlorobenzene (DNCB). Sensitization capacities of DNA-nitralin and DNC compared with DNCB, and cross-sensitizations among 11 dinitrobenzene derivatives, including DNA-nitralin, DNC, and DNCB, were studied in guinea pigs. We found that the order of potency was DNCB, DNC, and DNA-nitralin for the sensitization capacity, and that cross-sensitizations may occur among DNCB, DNC, DNA-nitralin, and dinitrofluorobenzene, in comparatively high incidence.

TNBSA injected at tolerogenic doses with a trace label of 14C showed an immediate fixation on serum proteins, red cells, lymphoid cells and, as far as could be determined, all organs in the body. The fixation on lymphoid cells was non-specific, at least initially. These results are discussed in relation to the mechanism of tolerance induction by TNBSA.

The importance of cell interaction for thymic independent antigen responses has not been widely appreciated. The present report demonstrates, however, that macrophage-B cell interaction may be an important feature of B ce-l activation for the response to at least one polysaccharide thymic independent antigen, TNP-Ficoll. Experiments were performed demonstrating that a strict accessory cell requirement exists for the thymic independent response to soluble TNP-Ficoll, and that such accessory cells are both adherent and phagocytic, that is, macrophages. It was further demonstrated that macrophages could be pulsed with TNP-Ficoll and that these pulsed macrophages could activate B cells to respond, but only if the pulsed macrophages were viable. Thus, one function that macrophages can fulfill in responses to TNP-Ficoll is the specific function of antigen presentation. Such presentation of TNP-Ficoll by macrophages to B cells suggests that the antigen may not be activating B cells directly, and raises the possibility that the interaction of B cells and macrophages might be genetically restricted.

We have used fluorescence photobleaching recovery (FPR) to study the lateral diffusion of antigen-receptor complexes during stimulation of DNP-specific mouse B cells by the T-independent antigens DNP-polymerized flagellin (DNP-POL). Depending on epitope density and dose, these antigens behave either as immunogens or tolerogens. Lymphocyte DNP receptors binding DNP0.5 flagellin monomer show a diffusion constant D of 2.2 X 10(-10) cm2 sec-1 and ca 50% fluorescence recovery after bleaching. For DNP-POL bound to DNP-specific lymphocytes, the observed diffusion constants decrease monotonically with increased antigen dose and epitope density. Under optimally immunogenic conditions of DNP2.3-POL at 1 micrograms/ml, D = 1.5 X 10(-11) cm2 sec-1, some 14-fold less than for a single DNP receptor. Under tolerogenic conditions lower diffusion constants approaching 0.8 X 10(-11) cm-2 sec1 are observed. The fraction of aggregates mobile on the time scale of the experiment remains constant at about 50 to 60% in all immunogenic situations, but falls abruptly to about 24 to 32% in precisely those situations where the antigen/dose combination is tolerogenic. This might support the hypotheses that there exist critical epitope densities above which antigens and receptors form rigidly cross-linked aggregates that bring about B cell tolerance. The mobility of DNP0.5 flagellin monomer bound to receptors left unoccupied after treatment with various doses and batches of DNP-POL is independent of DNP-POL presence. Receptor aggregate diffusion is unaffected by treatment with colchicine or cytochalasin B.

Cultured RDM4 cells were modified with 1 mM trinitrobenzene sulphonate (TNBS) and assayed for lysis by either in vitro generated cytotoxic T-lymphocytes (CTL) or complement-mediated humoral immunity. It was observed that cells harvested from the exponentially growing phase culture (as assessed by an important incorporation of thymidine) were more sensitive to CTL, but less sensitive to humoral immunity, than cells harvested from resting phase culture. Radiolabelled [14C]TNBS permitted to show that exponentially growing cells bound about 5 times less TNBS than cells at the resting phase. It is therefore concluded that the efficiency of the CTL on TNP-modified cells is not proportional to the density of TNP-groups on the targets, and that CTL on the one hand, and complement-mediated humoral immunity on the other hand, must have very different cell surface requirements to be able to lyze the targets.