anti-TNP Plaque-forming Cell Response Research Articles

Enhancement of In vitro antibody production of murine splenocytes by ascorbic acid 2-O-α-glucoside

Using an antigen-specific plaque forming cell (PFC) assay, we have studied the effect of ascorbic acid 2-O-α-glucoside (AA-2G), a new stable derivative of ascorbic acid (AsA), on antibody production in cultured murine splenocytes in comparison with that of AsA. A single addition of AA-2G (0.0625–1.0 mM) remarkably stimulated both anti-SRBC (sheep red blood cell, TD antigen) and anti-TNP (trinitrophenyl, TI antigen) PFC responses in a dose-dependent manner, although AsA failed to stimulate their responses. However, repeated additions of AsA at 12-h intervals during the cultivation resulted in enhancement of the anti-SRBC PFC response, and the magnitude of stimulation was comparable to that obtained by a single addition of AA-2G. AA-2G's effect was abrogated in the presence of castanospermine (α-glucosidase inhibitor) in the medium, indicating that the immunostimulation brought about by AA-2G is attributed to AsA released from the glucoside by α-glucosidase of cultured cells. In fact, the cells maintained a certain amount of AsA for a relatively long time after exposure to AA-2G. In contrast, AsA levels in the cells treated with AsA quickly decreased. AA-2G markedly enhanced lipopolysaccharide(LPS)-induced proliferative response, and could affect the concanavalin A (Con A) response weakly. These results suggests that AsA effectively potentiates B-cell functions in the humoral immune system. Thus, we conclude that AA-2G is capable of enhancing antibody production in the cultured splenocytes via a continuous supplementation of AsA and that AA-2G is available for long-term cell cultures as a useful tool for analyzing biological function of AsA.

Priming of peripheral lymph node B cells with TNP-Ficoll: Role of lymphokines in B cell differentiation

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/ 2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP- brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP- Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFC's were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.

Reduced T-helper cell function in mice exposed to 2, 3, 7, 8-tetrachlorodibenzo- p-dioxin(TCDD)

Antibody production to the T-dependent antigen SRBC is highly sensitive to suppression by polychlo-rinated dibenzo- p-dioxins. The present study provides evidence for a defect in T-helper (T H) cells in TCDD-exposed mice. Because spleen cells from nonimmune TCDD-exposed mice did not show suppressed antibody responses when adoptively transferred to irradiated hosts, we used a hapten-carrier (TNP-SRBC) system with cell separation/reconstitution techniques to determine the effects of TCDD on carrier-specific T H cells. In vitro cultures of spleen cells from SRBC-primed TCDD-treated (5 μg/kg) mice produced fewer anti-TNP plaque-forming cells (PFC) when immunized with TNP-SRBC, as compared to cells from primed vehicle-treated controls. A reduced anti-TNP PFC response was also observed in experiments where non-immune B-cells were induced to produce anti-TNP PFC by T H-cells obtained from carrier-primed TCDD-exposed mice, as compared to carrier-primed vehicle-exposed mice. Removal of Lyt-2 + (suppressor) T-cells in these experiments did not alter the anti-TNP PFC response. These results provide direct evidence for reduced activity of T H-cells after exposure to TCDD.

Induction of antigen-specific antibody responses in primed and unprimed B cells. Functional heterogeneity among Th1 and Th2 T cell clones.

CD4+ T cells have been recently divided into two subsets. The functions of these subsets are thought to be distinct: one subset (Th1) is responsible for delayed type hypersensitivity responses and another (Th2) is primarily responsible for induction of antibody synthesis. To more precisely define the roles of both subsets in humoral immune responses, we examined the ability of a panel of nominal antigen specific Th1 and Th2 clones to induce anti-TNP specific antibody synthesis in TNP-primed or unprimed B cells. Four of nine Th1 clones induced little or no antibody synthesis with TNP-primed B cells. However, five other Th1 clones were very effective at inducing IgG anti-TNP plaque-forming cell (PFC) responses in primed B cells. One of these Th1 clones was analysed in detail and found to also provide helper function for unprimed B cells. Cognate B-T cell interaction was required for induction of both primary and secondary responses with this clone, indicating that a Th1 clone could function as a "classical" Th cell. The seven IL-4 producing Th2 clones examined were also heterogeneous in their ability to induce antibody secretion by TNP-primed B cells. Although four of the Th2 clones induced IgG and IgM anti-TNP PFC responses, two Th2 clones induced only IgM and no IgG antibody, and another clone failed to induce any anti-TNP PFC. All Th2 clones failed to induce any anti-TNP PFC. All Th2 clones produced high levels of IL-4, but "helper" Th2 clones produced significantly greater amounts of IL-5 than "non-helper" Th2 clones. These studies indicate that some IL-2- and some IL-4-producing T cell clones can induce TNP-specific antibody in cell clones can induce TNP-specific antibody in primed and unprimed B cells, and that Th1 and Th2 clones are heterogeneous in their ability to induce Ig synthesis. Therefore, although T cell clones can be classified as Th1 or Th2 types according to patterns of IL-2, IFN-gamma, or IL-4 synthesis, the functional capacity to induce antibody synthesis cannot be predicted solely by their ability to secrete these lymphokines.

A primary in Vitro antibody assay for antigen-specific T-suppressor factor: Cross-suppression of TNP-specific antibody responses by TMA-specific TsF 1

We have previously shown that phenyltrimethylammonium (TMA)-specific, first-order suppressor T cells (Ts 1) and soluble factors extracted from these cells (TsF 1) can suppress delayedtype hypersensitivity (DTH) responses. The TsF 1, as monitored in the DTH system, was characterized and found to be a single-chain, antigen-binding, I-J +, and Id + molecule. To monitor TsF 1 in an efficient manner, an in vitro antibody system was developed. The studies show that in vitro stimulation of naive A/J spleen cells with the thymic-independent antigen, Brucella abortus, to which TMA and trinitrophenol (TNP) or fluorescein (FL) are coupled (TMA-BA-TNP or TMA-BA-FL), induces significant numbers of anti-TNP or anti-FL plaque-forming cell (PFC) responses. The addition of TMA-specific TsF 1 results in the cross-suppression of 30–50% of the total anti-TNP and FL PFC responses. This activity is antigen (TMA) dependent since suppression occurs only when the TMA ligand is present in the culture media. Analysis of the TNP-specific PFC responses in nonsuppressed cultures revealed that 20–35% of the PFC bear the cross-reactive idiotype(s) (CRI) normally associated with anti-TMA antibodies. In cultures containing TMA-TsF 1, CRI +PFC are suppressed by 90–100% while the CRI −PFC are suppressed only by 10–30%. Our studies further show that an induction-phase, antigen-binding, CRI +, and I-J + single-chain factor is responsible for the observed in vitro suppression. The possibility of utilizing this assay to monitor a variety of antigen-specific suppressor factors is discussed.

Age‐dependent isotype variation during secondary immune response in MRL/lpr mice producing autoanti‐γ‐globulin antibodies

A profound inability to produce IgG anti-2,4,6-trinitrophenyl (TNP) antibodies during the secondary immune response elicited by a T-dependent antigen was observed in aged MLR/lpr mice. This unresponsiveness is associated with a significantly low indirect anti-TNP plaque-forming cell response and a weak in vitro anti-TNP response upon the culture of keyhole limpet hemocyanin-primed T cells and TNP-primed B cells in the presence of TNP. The markedly low IgG anti-TNP response observed in aged MLR/lpr mice cannot be related to the presence of rheumatoid factors which are observed during the secondary response, since MRL +/+ and 129/J mice, (non-autoimmune disease strains), also produce significant amounts of anti-gamma-globulin antibodies yet mount a strong IgG anti-TNP response.

Two Distinct Azobenzenearsonate‐Specific Helper T‐Cell Subpopulations Mediate Different Forms of T‐B Cooperation

Popliteal lymph node T cells from mice footpad-primed with azobenzenearsonate (ABA)-protein conjugates were able to help the anti-trinitrophenyl (TNP) and anti-ABA plaque-forming cell (PFC) responses of normal syngeneic spleen cells cultured in vitro with TNP-ABA-keyhole limpet haemocyanin. Enrichment in ABA-specific helper cells was obtained by positive selection of ABA-primed T cells on ABA-pulsed syngeneic macrophages. The ABA-specific T cells induced by ABA-protein priming are able to help the anti-TNP PFC response of normal B cells through recognition of the ABA determinant either unlinked to TNP (ABA and TNP separately presented to T and B cells) or linked to TNP (ABA and TNP presented as moieties of the same molecule). These two mechanisms of T-B cooperation are mediated by two different ABA-specific helper T-cell subpopulations, which can be distinguished by their different radiosensitivities: the former mechanism is mediated by radioresistant T cells, whereas the latter is mediated by radiosensitive T cells. Both helper T-cell subpopulations bind the ABA-pulsed syngeneic macrophages, demonstrating the presence of ABA-specific receptors on both cell types.

Evidence for a mature B cell subpopulation in Peyer's patches of young adult xid mice.

Peyer's patch (PP) and mesenteric lymph node (MLN) cell cultures from young adult X-linked immunodeficient (xid) CBA/N and (CBA/N X DBA/2) F1 male mice support primary anti-sheep erythrocyte (SRBC) plaque-forming cell (PFC) responses, which suggests that gut-associated lymphoreticular tissue (GALT) contains a normal B lymphocyte subpopulation. Further support for this was provided by the observation that PP cells from xid mice gave responses to both TI-1 and TI-2 antigens that were similar to the responses of PP cell cultures from normal mice. Spleen cell cultures from xid mice were unresponsive to SRBC and TI-2 antigens. Proof that GALT of xid mice contain mature B lymphocytes was provided by the demonstration of PP B cells that bear a low density of surface immunoglobulin M. When these cells were separated by flow cytometry and immunized with trinitrophenyl (TNP)-Ficoll in vitro, good anti-TNP PFC responses were observed. These results suggest that GALT of young adult xid mice contain mature B cells and may represent the origin for the mature B cell responses seen in aged xid mice.

Enhancement of murine immune responses to orally administered haptenated Streptococcus mutans.

The induction of immune responses to orally administered trinitrophenyl (TNP)-haptenated Streptococcus mutans and its enhancement with muramyldipeptide (MDP), peptidoglycan (PG), and concanavalin A (Con A) were investigated in lipopolysaccharide (LPS)-non-responsive C3H/HeJ mice and the syngeneic, LPS-responsive C3H/HeN strain. Both mouse strains manifested similar immune responses, primarily of the IgM isotype, after a single gastric intubation (GI) with TNP-S. mutans. However, when groups of animals were first carrier-primed by GI with S. mutans for 2 consecutive days, followed by a single GI with TNP-S. mutans 1 week later, C3H/HeJ mice gave a significantly higher (P less than or equal to 0.01) splenic IgA anti-TNP plaque-forming cell (PFC) response than identically treated C3H/HeN mice. Furthermore, saliva, urine and serum from these C3H/HeJ mice possessed high levels of IgA anti-TNP antibodies as determined by the enzyme-linked immunosorbent assay, whereas C3H/HeN mice exhibited low antibody levels. Oral administration of Con A (either 250 micrograms or 500 micrograms/mouse) or purified PG (1 mg/mouse) at the time of TNP-S. mutans immunization resulted in significantly (P less than or equal to 0.01) enhanced splenic IgA anti-TNP PFC responses, especially in C3H/HeJ mice. On the other hand, MDP promoted IgA anti-TNP PFC responses in LPS-responsive C3H/HeN mice but did not augment responses in C3H/HeJ animals. A similar immune response pattern was seen when antibody levels were measured in serum, saliva, and urine of both mouse strains. These results demonstrate that haptenated S. mutans is a good antigen for the induction of high IgA responses in orally immunized C3H/HeJ mice and that this high response can be enhanced with the adjuvants Con A and PG. However, MDP is ineffective in C3H/HeJ mice but enhances IgA responses in normal LPS-responsive C3H/HeN animals.

Evidence for histamine-induced auto-anti-idiotypic antibody immunoregulation in vivo

The in vivo effects of histamine injection in LAF 1 male mice on the immune reactivity to trinitrophenylated bovine γ-globulin was studied using plaque-forming cell (PFC) responses and their avidity distributions. Splenic anti-trinitrophenyl (anti-TNP) PFC responses of mice treated with histamine (5 × 10 −6 mol or 1 mg, intravenously) were significantly reduced in number and restricted in heterogeneity and characterized by a preferential loss of high-avidity IgG PFCs. The reduced PFC response in histamine-treated mice was dose and time dependent. No evidence of suppressor cell activity in the spleens from histamine-treated mice was demonstrable. Only histamine-treated mice produced a significantly high percentage of anti-idiotype-blocked, hapten-augmentable IgG PFCs, suggesting the presence of auto-anti-idiotypic activity. Immune sera taken from histamine-treated mice caused an inhibition of anti-TNP PFC in vitro. This PFC-inhibiting factor in immune sera of histamine-treated mice was an antibody of the IgG 1 and IgG 2a class, lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of LAF 1 origin. Passive hemagglutination study of this sera showed anti-(anti-TNP F(ab′) 2-IgG) titer. Thus, the results of this study suggest that histamine in combination with antigen induces auto-anti-idiotypic antibody which, in turn, is involved in the normal regulation of the immune response to trinitrophenylated bovine γ-globulin in vivo.

A general interactive model for B cell activation II. Experimental verification.

The general interactive model described in the previous paper was subjected to experimental testing. We examined the high affinity anti-TNP plaque-forming cell response of cultures of murine lymphocytes exposed to lipopolysaccharide and trinitrophenylated lipopolysaccharide (TNP--LPS). From our model we predicted the effects of the addition of free LPS on the dose response to TNP--LPS, the effect of the addition of Polymyxin B to cultures stimulated with TNP--LPS, and the effect on the response to TNP--LPS preparations with different haptenation ratios. The results obtained were consistent with the predictions of the general interactive model.

Deregulation of mouse antibody-forming cells by streptococcal pyrogenic exotoxin (SPE): IV. Fractionation of a T-cell subpopulation which generates SPE-induced deregulation of anti-TNP PFC responses

Deregulation of the immune response to T-cell-dependent immunogens is a relevant biological property of Group A streptococcal pyrogenic exotoxin (SPE). The deregulated response induced by SPE differs from a normal regulated immune response to a T-dependent immunogen, trinitrophenylated rabbit erythrocytes (TNP-RE), by the generation of a late burst of plaque-forming cells (PFC) at 10–12 days without further immunogen stimulation. An in vitro cellular complementation system has allowed us to perform reconstruction experiments using the separate subpopulations of mouse immunocytes which include the B cells, T cells, and macrophages required for a complete anti-TNP PFC response in Marbrook-type cultures. Utilizing this system, the target of SPE activity was localized to the T-cell subpopulation. In this report we have fractionated two pools of spleen T lymphocytes from macrophage-depleted NFR/N +/nu mouse splenocytes via nylon wool columns. Analysis of these pools by cellular complementation, immunofluorescence, and antibody-complement-mediated inactivation showed the cell pools to contain different phenotypes. The subpopulation of T cells which eluted first (designated Pool 1) from the nylon wool column was capable of complementing the PFC responses of congenic NFR/N nu/nu mouse splenocytes. This result is consistent with their identity as helper T cells. The population of T cells which eluted second from the nylon wool column was designated Pool 2 and was capable of inhibiting the TNP-specific PFC responses of cultures optimally complemented with Pool 1 cells. This result is consistent with the identification of Pool 2 cells as a subpopulation with a phenotype as native negative regulatory T cells. Treatment of Pool 2 cells with SPE inhibited their suppressive activity for Pool 1 cells, and thus delineated the site of SPE-generated deregulation to Pool 2 cells.

High anti-TNP plaque-forming cell potential of residual mIg+ cells in a T cell population.

In the course of experiments designed to study the immune response of purified populations of B lymphocytes to thymus-independent (TI) antigens, a variety of cell purification procedures were followed. In using anti-immunoglobulin-coated dishes to separate lymphocytes bearing membrane immunoglobulin (mIg) from mIg- lymphocytes, it was found that the nonadherent fraction, which was predominantly mIg-, complement receptor negative, and nonresponsive to the B cell mitogen lipopolysaccharide, gave very substantial anti-TNP plaque-forming cell responses to 2 TI antigens. These responses could be inhibited by incubation of such cells in the presence of anti-mu and thus appeared to be attributable to mIg+ cells. The evidence suggests the existence of a population of B lymphocytes that constitute a minor component of mIg+ cells having a high potential to make in vitro antibody responses. Users of techniques that utilize anti-Ig as a tool for separating B and T lymphocytes should carefully assess the extent to which residual B lymphocytes in the mIg- population contribute to antibody responses being studied.

Immunoglobulin-producing cells in cultured human peripheral blood lymphocytes: Modulation by accessory cells and enhancing effect of polyacrylamide beads

In a reverse hemolytic plaque assay performed with sheep red blood cells coated either with purified antibodies polyvalent for human immunoglobulins (Ig) (anti-F(ab′) 2) or monospecific for γ chains, or with protein A, we detected few reverse plaque-forming cells (PFC) among fresh human peripheral blood lymphocytes (PBL). Upon culture, the numbers of reverse PFC remained stable during the first 4 days and then increased up to 1000–10,000 PFC 10 6 cells on Days 5–7. This phenomenon required cell proliferation and was associated with some degree of B-cell differentiation as shown by immunofluorescent study of surface and cytoplasmic Ig. Trinitrophenyl-polyacrylamide beads (TNP-PAA) and unsubstituted polyacrylamide beads significantly enhanced the numbers of reverse PFC in cultured PBL. The optimal enhancement of reverse PFC required the same experimental conditions that the induction of a specific anti-TNP PFC response by TNP-PAA required: a supportive batch of fetal bovine serum, monocytes (which could be replaced by 2-mercaptoethanol), and T cells. The latter acted in the inductive phase and was apparently not required for Ig secretion by cultured cells in our assay.

CREATION AND PROPERTIES OF HISTOCOMPATIBILITY ANTIGEN-CELL CONJUGATES

Histocompatibility (HC) antigens from Lewis (RT-11) and ACI (RT-1a) rats were solubilized and their tolerogenic potential alone or when conjugated to cellular carriers was investigated. The tolerogenic carrier potential of whole cells, crude membranes, and deoxycholate (DOC)-solubilized membranes was first determined by conjugating trinitrophenyl (TNP) groups to them and measuring their ability to prevent an anti-TNP plaque-forming cell response in rats after immunogenic challenge. Since whole lymphoid cells proved to be most effective in this system, DOC-solubilized allogeneic histocompatibility antigens were coupled to syngeneic lymphocytes, using carbodiimide cross-linking. Animals pretreated with this conjugate produced no alloantibody upon challenge with allogeneic cells. These results suggest that tolerance to allogeneic histocompatibility antigen can be produced by association of alloantigens on a tolerogenic self-carrier.

Membrane defects of the tolerant B cell: III. Defective receptor replacement

When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal. After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro, Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro. Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later. Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.

Role of self carriers in the immune response and tolerance. V. Reversal of trinitrophenyl-modified self suppression of the B-cell response by blocking of H-2 antigens.

Trinitrophenylated syngeneic spleen cells (TNP-SC) are potent tolerogens of the anti-TNP plaque-forming cell (PFC) response in vivo and in vitro. This unresponsive state requires T cells for both its induction and maintenance. Because H-2K/D-restricted cytotoxic T cells are also induced by exposure to TNP-SC, we determined the role(s) of histocompatibility antigens (K, I, and D) in the suppression of the PFC response by TNP-SC. We treated syngeneic TNP-modified stimulator cells with antiserum directed at K-, I-, or D-region determinants and found that blocking of H-2K or D antigens on TNP-SC transformed these tolerogens into immunogens capable of eliciting an anti-TNP PFC response in the absence of extrinsic immunogens like TNP-polymerized flagellin. In H-2k or H-2a(k/d) mice, only H-2Kk needs to be blocked on the stimulator cells, whereas H-2K or D recognition was apparent in B10.A(4R) mice. These observations indicate that suppression of the PFC response by TNP-SC shows the same restriction in recognition as does the cytotoxic T-cell response. Furthermore, our results suggest that TNP-I-A is recognized by the helper cells in this system as the intrinsic antigen. When both TNP-K and TNP-I-A are present and available on the same stimulator cell, suppression (via modified K recognition) is dominant over help.

Production of auto-anti-idiotypic antibody during the normal immune response to TNP-ficoll. III. Absence in nu/nu mice: evidence for T-cell dependence of the anti-idiotypic-antibody response.

Although athymic mice make an excellent immune response to the thymus-independent antigen trinitrophenyl-lys-Ficoll (TNP-F), nude mice of AKR/J and BALB/c strains lack the anti-idiotypic response that occurs in euthymic mice of both of these strains within the first 1--2 wk after injection of more TNP-F. Anti-idiotypic antibody-blocked (hapten-augmentable) anti-TNP splenic plaque-forming cells (PFC) do not occur at any time and serum anti-idiotypic antibody is absent in both congenitally athymic mice, and thymectomized, irradiated, bone marrow-reconstituted mice. Nevertheless, nu/nu mice do have PFC which can be inhibited by exposure to anti-idiotypic antibody produced in +/+ mice. As a consequence of the failure to produce anti-idiotypic antibodies, the anti-TNP PFC response is athymic as compared to euthymic mice is of greater magnitude, declines less precipitously, and shows an increase rather than a decrease in affinity between days 4 and 7 after antigen injection. It is concluded that the anti-idiotypic antibody response is thymus dependent and that athymic mice lack a helper cell required for the induction of anti-idiotypic antibodies.

Ontogeny of murine B-cell responses to thymus-lndependent trinitrophenyl antigens

The ontogeny of B-cell responsiveness to three thymus-independent trinitrophenyl (TNP) antigens has been examined in BALB/c mice in vivo and in vitro. When in vivo splenic plaque-forming cell (PFC) responses to TNP-conjugated lipopolysaccharide (TNP-LPS), Ficoll (TNP-Ficoll), and Brucella abortus (TNP- Brucella) were measured in neonatal and adult mice, a defined sequence of responsiveness was observed. Newborn mice responded well to TNP-LPS, but not to TNP-Ficoll or TNP- Brucella. Neonates injected at 1 day of age responded to TNP-LPS and TNP-Ficoll and mice 5 to 14 days of age responded to TNP-LPS, TNP-Ficoll, and TNP- Brucella. Furthermore, the antigen-reactive populations increased at different rates for the three antigens in the first 2 weeks of life. In vitro experiments confirmed the results obtained in vivo although slightly earlier responsiveness to TNP- Brucella was observed in vitro. PFC inhibition assays with free TNP hapten were performed so that avidity profiles could be examined in neonatal and adult anti-TNP PFC responses. The results clearly demonstrate that once a response becomes detectable in neonatal mice immunized with any of the three TI TNP antigens, fully heterogeneous or “adult-like” responses are found. In addition, experiments comparing avidity profiles in athymic ( nu/nu) BALB/c mice and their normal ( nu/+) littermates demonstrate that T cells are not required for the generation of fully heterogeneous anti-TNP PFC responses. These results indicate that B cells responsive to different TI TNP antigens mature at different times and at different rates during ontogeny. Late maturation events of such B cells do not include the acquisition of additional V-region specificities as detected in a PFC inhibition assay.

Membrane defects of the tolerant B cell: I. Failure of antigen-induced capping

In order to study the membrane function of tolerant B antigen-binding cells, tolerance to the trinitrophenyl (TNP) determinant was induced in mice by injecting the reactive form of the hapten, trinitrobenzene sulfonic acid (TNBS). By appropriate transfer experiments, Fidler and Golub ( J. Immunol. 112, 1891, 1974) had previously shown that this form of tolerance is a B-cell property, induced and expressed in the absence of T cells. Hapten inhibition demonstrated the TNP-specificity of receptors on TNP-donkey erythrocyte(TNP-D)-binding cells in tolerant and nontolerant mice. About 88% of these cells were B cells by immunofluorescence, and the remainder were T cells. In the tolerant mice, challenge with TNP-sheep erythrocytes failed to expand the TNP-binding population, but sheep erythrocyte binders and anti-sheep plaque-forming cells expanded normally. Despite little or no change in TNP-binding cell numbers after tolerance induction, the TNP-binding cells of tolerant animals could not cap their receptors, in contrast to the sheep erythrocyte-binding cells from the same animals which capped normally. Although there is no anti-TNP plaque-forming cell response when tolerogen and immunogen are given simultaneously, capping failure is not evident until 2–4 days after tolerogen exposure. By Day 7, substantial recovery of immune responsiveness had occurred, yet even 12 months after a single dose of tolerogen there was no restoration of capping. Thus despite the association of both capping failure and unresponsiveness with tolerogen exposure, these lymphocyte functional defects appeared not to be causally related.