Selective suppression by auto-anti-idiotypic antibody of B-cell idiotype repertoires generated after stimulation with the same hapten on T-dependent and T-independent carriers

In the present study, we investigated whether auto-anti-idiotypic antibody in the immune sera from old mice could recognize antitrinitrophenyl (TNP) plaque-forming cells (PFC) generated after stimulation with the T-dependent and T-independent forms of the hapten, TNP. Young and old C57BL/6J male mice were immunized with a variety of T-dependent (TNP-bovine gamma-globulin, TNP-BGG; TNP-keyhole Limpet hemocyanin, TNP-KLH; ovalbumin, OVA; bovine serum albumin, BSA; BGG) and T-independent (TNP-Brucella abortus, TNP-BA; TBP-Ficoll; TNP-polyacrylamide beads, TNP-PAA) antigens either in complete Freund's adjuvant (CFA) or in soluble form. Splenic anti-TNP or antiprotein PFC responses were assayed for anti-idiotype-blocked, hapten- or protein-augmentable IgM, IgG and IgA PFC, 1-2 weeks after immunization. It was found that 8-month-old mice produced significantly a higher percentage of hapten augmentable (26-42%) IgM PFC response to T-independent antigens as compared with the 2-month-old mice (3-6% augmentation). Similarly, old mice produced a significantly higher percentage of hapten or protein augmentable (25-129%) IgG PFC response to T-dependent antigens as compared with the 2-month-old group (2-6% augmentation). The data support the view that age-related regulation of auto-anti-idiotypic antibody is a general phenomenon for immune responses to T-dependent and T-independent antigens. Hapten-reversible inhibition of plaque formation was used to determine whether anti-idiotypic antibody containing antisera from old mice could inhibit B-cell idiotype repertoires generated after stimulation with the same hapten, TNP, on T-dependent and T-independent carriers. Pools of immune sera from 8-month-old mice primed with T-dependent TNP-BGG or TNP-KLH antigens but not with T-independent TNP-PAA or TNP-BA antigens, or with the proteins OVA, BSA, or BGG selectively inhibited IgM, IgG, and IgA anti-TNP PFC from 2-month-old mice that were previously primed with either TNP-BGG or TNP-KLH. In contrast, immune sera from old mice primed with TNP on either T-dependent or T-independent carriers inhibited anti-TNP PFC from mice primed with T-independent TNP-PAA or TNP-BA antigens. Immune sera from old mice primed with OVA or BSA only inhibited the respective antiprotein PFC. The immune sera from young mice did not show any appreciable inhibition of PFC generated after stimulation by any of the antigens studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of haptenated immunoglobulins to induce B cell tolerance in vitro. The roles of hapten density and the Fc portion of the immunoglobulin carrier

The relationship between the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig), and their ability to induce tolerance was examined. It was found that adult B cells responding to a T-independent (TI) antigen were tolerized by TNP11 human gamma globulin (HGG), but not by TNP10F(ab')2 fragments of HGG. Increasing the hapten density on the F(ab')2 fragments overcame their inability to induce tolerance. Thus, a TNP17-F(ab')2 was an effective tolerogen. Murine myeloma proteins of different IgG subclasses were similarly tested. A TNP12-IgG2a and a TNP11-IgG1 induced tolerance, whereas two TNP11-12-IgG3 did not. However, a more heavily haptenated TNP18-IgG3 was tolerogenic. These results suggest that lightly haptenated immunoglobulins depend upon Fc receptor binding to induce tolerance in adult B cells. Non-Fc receptor-binding carriers are not tolerogenic unless they are more heavily haptenated. Finally, T cell and macrophage depletion experiments suggest that the tolerogens act directly on the B cells.     

C-Reactive protein (CRP)-mediated inhibition of the induction of in vitro antibody formation. I. T-cell dependence of the inhibition.

Purified human C-reactive protein (CRP) inhibited the in vitro anti-hapten antibody plaque-forming cells (PFC) response of both carrier keyhole limpet hemocyanin (KLH)-primed and unimmunized Balb/c spleen cells to TNP-KLH. The inhibitory effect was neutralized by the CRP-substrate, C-polysaccharide. The response to the T-independent antigens, TNP-T4 and DNP-lys-Ficoll, was not inhibited by CRP. A cell population that was suppressive for the in vitro PFC response was generated by incubating normal spleen cells with CRP. These cells suppressed the PFC response of syngeneic KLH-primed cells to TNP-KLH in proportion to the number of added lymphoid cells with bound CRP. Selective depletion of B cells, T cells or macrophages before incubation with CRP revealed that T cells were required for the induction of suppressive cells. Treatment of spleen cells after incubation with CRP, with T cell-specific antisera and C abolished suppressor-cell activity. Mitomycin-C treatment of the CRP-binding cells did not alter their suppressive activity. These results indicated that CRP mediates suppression of antibody induction to T-dependent antigens by interacting with T cells and generating a suppressive T-cell population.     

Inhibition of the in vitro 19S and 7S antibody response by immunoglobulin-binding factor (IBF) from alloantigen-activated T cells

A soluble factor secreted by alloantigen-activated mouse T cells which binds to the Fc fragment of IgG and inhibits complement activation by IgG (immunoglobulin-binding factor, IBF) suppressed the in vitro 19S and 7S antibody response by mouse spleen cells to T-dependent as well as T-independent antigens. IBF inhibited the 19S plaque response best when it was added late during PFC generation (between 48 and 72 hr). On the other hand, when it was left in cultures for up to 60 hr and then removed, antibody synthesis was not inhibited. However, its presence for only 2 hr starting after 72 hr of incubation was sufficient to inhibit PFC formation. The suppressive activity of IFB could be neutralized by adding aggregated mouse IgG prior to the critical stage around 72 hr. These data favour the view that IBF could be a suppressive T cell factor and point to the possibility that IBF may act on already triggered B cells during their final differentiation to active PFC.     

Effects of the MER tubercle bacillus fraction on the production of antibodies in vitro: I. Effect on the primary response

The effect of the methanol extraction residue (MER) fraction of BCG tubercle bacilli on the primary antibody response in vitro to sheep red blood cells (SRBC), TNP conjugates, and the monovalent hapten DNP-glycine was studied. Addition of MER to whole splenocyte cultures simultaneously with antigen presentation potentiated the antibody response to SRBC and TNP-SRBC, and facilitated reactivity to DNP-glycine; there was no effect on the response to the T-independent entity TNP-LPS (lipopolysaccharide from E. coli 055-B5). Immunopotentiating activity of MER for SRBC and DNP-glycine was also evident in macrophage-depleted cultures. Peritoneal exudate cells (PEC) taken from MER-treated donors were more efficient than PEC from untreated donors in reconstituting antibody formation to SRBC by macrophage-depleted spleen cell populations. The results obtained indicate that activation of both macrophages and of certain lymphocyte population(s) by MER may play a role in the potentiation of antibody responsiveness in vitro by this agent.     

Transmission of hapten-specific tolerance to an unrelated carrier

The principle of linked recognition is well defined in response and suppression. Yet, to our knowledge, it is not explored in the context of tolerance. To investigate, whether the status of tolerance toward a hapten (TNP) can be transferred to a subsequently introduced carrier, animals which were tolerized by a subimmunogenic dose of hapten (TNP) coupled to syngeneic monoclonal anti-TNP IgG, with the rationale of combining the phenomena of low zone tolerance and syngeneic IgG-induced suppression, were challenged with TNP-horse red blood cells (HRBC). Conjugates of high density (40 mM) TNP-syngeneic IgG (TNP40-IgG) were immunogenic and after challenge with TNP-HRBC, animals responded to TNP and to HRBC. Yet, spleen cells (SC) of mice injected with TNP2.5-IgG and challenged with TNP-HRBC were tolerant against TNP as well as the carrier. Limiting dilution (LD) analysis revealed that subimmunogenic doses of TNP coupled to IgG resulted in diminished activation of help, failure to activate contrasuppressor T cells (TCS), and significantly augmented activation of suppressor T cells (TS). On the other hand, after challenge with TNP-HRBC, activation/expansion of carrier-specific helper (TH), suppressor, and contrasuppressor T cells were not affected by previous immunization with subimmunogenic or immunogenic doses of TNP-IgG conjugates, but HRBC-specific TCS could not interact with TNP-specific TS. Hence, to initiate tolerance it was necessary (and sufficient) that an activated and expanded TS population was not counterregulated by TCS. In this situation, an established status of dominance of suppression for the epitope TNP could not be disrupted by an immunogene carrying a multitude of new epitopes; i.e., tolerization by subimmunogenic doses of the individual epitope TNP resulted in unresponsiveness against any immunogen carrying this epitope.     

Effects of cyclosporin A on humoral immune response and resistance against vesicular stomatitis virus in mice.

The effect of cyclosporin A (CS-A) on the antiviral humoral response was studied by using vesicular stomatitis virus (VSV); VSV provided the opportunity to simultaneously assess both T-independent and T-dependent antibody responses. The T-independent anti-VSV immunoglobulin M (IgM) response was virtually unaffected, whereas the T-dependent primary anti-VSV IgG response was suppressed by CS-A; in contrast, the secondary IgG response was highly resistant to CS-A. Moreover, once the switch from IgM to IgG had occurred, the primary response also became refractory to suppression by CS-A. We concluded that the effect of CS-A on the primary anti-VSV antibody response was mediated via impairment of a T-dependent mechanism; in contrast, memory T cells or memory B cells or both were quite resistant to the suppressive effects of CS-A. CS-A treatment rendered mice highly susceptible to VSV infection; under CS-A treatment, mortality was 100% after infection via footpads, whereas immunocompetent mice survived. Since CS-A does not impair induction of early T-independent anti-VSV IgM neutralizing antibodies, this high mortality in CS-A treated mice illustrates the crucial role of CS-A-sensitive cells in resistance against VSV.     

Immune responses to T-dependent and T-independent antigens during visceral leishmaniasis in mice: evidence for altered T-cell regulation of immune responses to non-parasite antigens

Antibody responses to T-dependent and T-"independent" antigens were studied in disease-susceptible (BALB/c and C57BL/10) and disease-resistant (A/J) mice infected with Leishmania donovani chagasi. Disease-susceptible mice but not disease-resistant mice showed a transient decrease in PFC responses to TNP on a T-dependent carrier (BGG) during the period of 4-8 weeks after infection. Infected disease-susceptible animals also showed increased responses to TNP on a type II T-independent carrier (Ficoll), which persisted until at least 14 weeks after infection. The increased responses were associated with a significant increase in anti-TNP antibody of the IgG2b subclass. When T-enriched spleen cells from infected mice and B-enriched spleen cells from uninfected mice were transferred to irradiated recipients immunized with TNP-Ficoll, increased anti-TNP PFC were observed over numbers seen in irradiated recipients which received both B and T cells from uninfected mice. Increased responses to TNP-Ficoll were also induced by prior administration of soluble leishmania extract in CFA. Infected mice immunized with TNP-LPS, a T-independent type I antigen, also had increased anti-TNP antibody responses, but had normal anti-LPS antibody responses. The elevated antibody production which occurred in response to the T-"independent" antigens could not be attributed to the relatively low polyclonal response which occurred in both disease-resistant and disease-susceptible mice infected with L. donovani chagasi. The observations are consistent with leishmania induced, transient alterations in some T-cell functions including response to haptens on T-dependent carriers, and a lack of down regulation of T-"independent" responses. Subtle lesions in immunoregulation may be important correlates of successful protozoal infection and may be responsible for some of the immunologic manifestations of the disease.     

Energy transfer distance measurements in immunoglobulins. II. Localization of the hapten binding sites and the interheavy chain disulfide bond in rabbit antibody

The distance between the hapten combining site and the single interheavy chain disulfide bond in rabbit immunoglobulin G has been determined by measuring the efficiency of energy transfer between chromophores specifically attached at these sites on the molecule. The donor chromophore, DnsLys³, was non-covalently bound in the combining sites of high-affinity antiDns antibody molecules, in one case, and in the combining site of the pepsin Fab′ fragment of antiDns in another. The acceptor chromophore, fluorescein, was covalently attached by disulfide interchange of di-FlCys with sulfhydryls generated by selective reduction of the interheavy chain disulfide bond of whole antiDns antibody and of the (Fab′)₂ pepsin fragment. The presence of acceptor decreased the donor fluorescence lifetime by about 1.0 nanosecond in both cases, i.e. for the whole antibody, from 23.6 to 22.7 nanoseconds, and for the Fab′ fragment from 23.6 to 22.5 nanoseconds. An average separation distance of 81 Å was calculated from an average observed transfer efficiency of 3.7%. This value agrees closely with the over-all length of a Fab′ fragment of a human IgG myeloma protein (Poljak et al., 1972). These results strongly suggest that the antibody combining site is at, or very close to, the tip of the Fab fragment and that the inter-heavy chain disulfide bond is at or near the edge of the C_L?C_H1 domain.     

Studies on Fc receptor function. I. IgG-mediated inhibition of B lymphocyte activation by T-dependent and T-independent antigens.

Mouse aggregated IgG, when continuously present in cultures of mouse spleen cells immunized with sheep erythrocytes, causes a dose dependent inhibition of the generation of plaque forming cells with a maximum of about 90% at 400 μg IgG/culture. Unaggregated IgG induces a similar inhibition, whereas treatment with mouse albumin or F (ab¹)₂, under the same conditions, does not affect the generation of plaque forming cells.It has been reported that unaggregated IgG binds poorly to Fc receptors of B lymphocytes and thus should not be expected to inhibit PFC generation if the effect is at the level of B lymphocyte Fc receptors. Competitive binding experiments were carried out and showed that aggregated and unaggregated IgG compete similarly with ¹²⁵I-labeled aggregated IgG for binding to Fc receptors of mouse spleen cells.The same inhibition of PFC can be induced by aggregated IgG in cultures of B lymphocytes immunized with the T-independent antigen DNP-Ficoll. When IgG is absorbed extensively with sheep erythrocytes and added to cultures immunized with sheep erythrocytes, PFC generation is inhibited to a level comparable to that of nonabsorbed IgG.These results suggest that IgG binding to Fc receptors leads to a severe inhibition of the induction of PFC by both T-dependent and T-independent antigens. This and other work from our laboratory indicate that this effect may be at the level of B lymphocyte Fc receptors.Taken together with reports from several laboratories, the data presented here suggest that Fc receptors may have a regulatory role on the activation of B lymphocytes by antigens or mitogens.     

Clonal analysis of B-lymphocyte subpopulations separated on the basis of Lyb surface antigens

The aim of these experiments was to see whether antisera of the Lyb series could be used to identify B cells capable of responding differentially in T-dependent and T-independent systems. The antisera tested were against the alloantigens Lyb 1.1,2.1,3,4.1,5.2, and LyM 1. A polyvalent sheep anti-mouse immunoglobulin (Ig) antibody acted as a positive control for the identification of B cells. As a first step, all spleen B cells were treated to remove this surface Ig by a capping procedure. They were then washed, reacted with a mouse alloantiserum, and allowed to form rosettes with sheep erythrocytes to which a sheep anti-mouse IgG had been coupled. Rosetted fractions were prepared on a Percoll density gradient. After removal of erythrocytes by osmotic shock, the cells were tested for their capacity to respond to antigenic stimulation. To allow accurate estimation of functional potential, two B-cell cloning assays were used. To enumerate T-dependent B cells, the Klinman splenic microfocus assay was employed using haptenated KLH⁴ as antigen. To study T-independent cells, a limiting-dilution liquid microculture method employing hapten-polymerized flagellin as antigen was used. The results showed that none of the Lyb antigens clearly demarcated T-dependent from T-independent B cells. Rosetted fractions consisting of Lyb 1.1-, 2.1-, 3-, or 4.1-positive cells responded well in both assays. Fractions enriched for LyM 1-positive cells behaved like unfractionated spleen cells. Only the Lyb 5.2-rosetted fraction showed any discordance between the two assays, the fraction being enriched for cells responding in the T-dependent system and slightly depleted of cells responding in the T-independent system. Taken as a whole, the results suggest that these alloantigens will not serve as useful markers to characterize T-dependent and T-independent B-cell subsets. In fact, the experiments cast further doubt on whether such a distinction is valid.     

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

Two processes for B-cell triggering by T-independent antigens as evidenced by the effect of azathioprine.

The effect of azathioprine (Az) on the in vitro antibody response induced by two T-independent antigens has been evaluated. The response to trinitrophenyl (TNP)-polyacrylamide is highly sensitive to Az whereas that to TNP-lipopolysaccharide (LPS) is Az resistant. A similar Az resistance is found for both the polyclonal B-cell activation and the B-cell proliferation induced by LPS, suggesting that Az resistance is related to the presence of a mitogenic carrier. These data suggest that at least two different processes may operate in B-cell triggering by T-independent antigens. On the basis of Az sensitivity, the response to T-independent antigens without a mitogenic moiety such as TNP-polyacrylamide is comparable to the responses to T-dependent antigens which are Az sensitive.     

Human B cell differentiation. II. Suppression by T cells of T-dependent and T-independent plasma cell maturation.

In vitro human plasma cell generation induced by both T-dependent (PWM) and T-independent (NWSM) mitogens was found to be suppressed by peripheral blood lymphocytes preincubated with human aggregated IgG. T cells, but not B lymphocytes, were able to mediate the suppressive activity; since aggregated (Fab)'2 fragments were found unable to generate suppressor cells, it was concluded that the suppressor cell was a T lymphocyte bearing Fcgamma receptors. These cells appeared to be largely radiosensitive. In most cases the proliferative responses remained unchanged. Since NWSM-induced activation is not dependent on the presence of T cells, these results show that, at least in this case, T cells exert their suppressor function directly on B lymphocytes. Whether PWM-induced B cell differentiation is suppressed by the same mechanism or/and by inactivation of T helper lymphocytes remains under investigation.     

Defective B cell tolerance induction in New Zealand black mice. I. Macrophage independence and comparison with other autoimmune strains

B cell unresponsiveness was examined in vitro by using spleen cells from autoimmune NZB, BXSB/Mp male, MRL/Mp-Ipr/Ipr (MRL/l), and control mice, and the tolerogen trinitrophenyl human gamma-globulin (TNP-HGG). The B cell subset responsive to TNP-Brucella abortus in each autoimmune and control strain that was tested was highly susceptible to tolerance induction with the use of high epitope density conjugates (TNP30HGG and TNP32HGG). When a tolerogen with a lower epitope density was used (TNP7HGG), several control strains were all rendered tolerant in a thymic-independent and hapten-specific manner. NZB B cells were resistant to all concentrations of TNP7HGG tested, whereas B cells from BXSB/Mp male and MRL/1 mice were resistant to low concentrations of this tolerogen. NZB mice were resistant in addition to tolerance induction with TNP9HGG, TNP10HGG, and TNP12.7HGG. Experiments were performed to determine whether splenic macrophages played a role in resistance to tolerance in NZB mice. The mixing of NZB and control DBA/2J T cell-depleted splenocytes revealed no modulatory effects by the accessory cells in culture. Moreover, B cells rigorously depleted of macrophages by double Sephadex G-10 column passage exhibited characteristic patterns of resistance or susceptibility in NZB and control strains, respectively. These findings support the conclusion that resistance to tolerance in NZB mice is determined at the B cell level and are consistent with the hypothesis that diverse immunoregulatory disturbances contribute in varying degrees to the development of systemic lupus erythematosus in different inbred strains of mice.     

In vivo immune response to TNP hapten coupled to thymus-independent carrier lipopolysaccharide

Lipopolysaccharide has been utilized as a carrier for the TNP hapten, producing an antigen which induces an in vivo thymus-independent antibody response to TNP as determined using athymic nude mice and their normal littermates. The immune response to TNP-LPS was investigated at both the antibody-forming cell and the serum antibody levels.The primary response to an optimal dose of TNP-LPS (1.0 μg) exhibited unusual kinetics reaching a sharp peak on day 3 of 58,000 anti-TNP PFC/spleen. Serum antibody to TNP was first detected on day 3 and reached a maximum log₂ titer of 17.5 on day 5, an uncommonly high level for hapten-carrier conjugates and most carriers. Both the anti-TNP serum antibody and PFCs were exclusively IgM. No IgG antibody was detected in the primary response through 28 days postimmunization, nor was any detected in any experiment described in this paper. The primary PFC response to 1.0 μg of TNP-LPS was specific for TNP, producing no evidence of polyclonal antibody synthesis. The relative affinities of PFC-secreted antibody were investigated using hapten inhibition. The hapten inhibition curves for TNP-LPS and TNP-SRBC were very similar, indicating that relatively high affinity antibody was elicited by TNP-LPS. The secondary response to this dose following priming with TNP-SRBC or TNP-LPS was similar to the primary response, though the peak was less sharp in both cases. The response to the homologous secondary challenge shifted somewhat, reaching a peak on days 3–4. The effect of various doses in priming or challenging for the secondary response to TNP-LPS was investigated. Using an increased PFC response as a criterion, no dose was optimal for priming or immunological memory to TNP-LPS. While the adoptive primary response to TNP-LPS reached a low level peak on day 7, the adoptive secondary attained a maximum on day 6. This shift in kinetics in intact mice and in adoptive hosts in comparing primary to secondary responses indicated that a state of B cell priming may be induced. However, its full expression may be suppressed by endogenous factors at the time of priming, such as the high level of circulating anti-TNP antibody or residual antigen. Adoptive transfer would remove the cells from these influences, allowing such B cell priming to manifest itself fully.     

Neutralizing Activities of Early and Late IgG Fragments from Rabbits Immunized with Herpes Simplex Virus

Rabbits immunized with herpes virus were bled periodically and bivalent and univalent fragments of IgG from each serum sample were prepared by enzymatic digestion. The 2-week F(ab′)₂ showed a low neutralizing activity only after addition of anti-IgG. F(ab′)₂ of the 4-week serum retained almost all of the neutralizing activity of IgG, while its univalent fragments demonstrated none even when tested with anti-IgG. In contrast to these early IgG fragments, univalent fragments of the 9-week and 20-week IgG neutralized the virus to considerable extents in the absence of anti-IgG; after addition of anti-IgG the activity equaled that of intact IgG in the cases of Fab′ and Fab-II, though the activity of Fab-I was relatively low. The three univalent fragments were all sensitive to heating at 70 C and to ultraviolet irradiation, whereas intact IgG resisted these treatments. F(ab′)₂ was resistant to the heating and less sensitive to ultraviolet irradiation than univalent fragments. Neutralization kinetic curve experiments to test blocking effects of IgG fragments against the neutralization by intact IgG suggested that the early Fab′ did combine with the virus and that the late Fab′ exerted a higher blocking effect than the early Fab′.     

The influence of epitope density on the immunological properties of hapten-protein conjugates. I. Characteristics of the immune response to hapten-coupled albumen with varying epitope density

The relationship between the epitope density of hapten-protein conjugates (DNP· BSA), and their immunogenicity in mice has been investigated. As others have found, lightly substituted protein (DNP₅BSA) elicited primary and secondary antibody responses which were mainly IgG. In contrast, DNP₅₀BSA induced a primary IgM response with relatively little IgG, and little or no immunological memory. The transition in immunogenic behaviour from “low” to “high” occurred with a hapten: protein molar ratio around 30. DNP₅₀BSA does not contain any serologically detectable native BSA determinants or neodeterminants resulting from dinitrophenylation. Although this antigen elicits a mainly IgM response as do thymus-independent antigens, antibody production to both DNP₅BSA and DNP₅₀BSA is highly thymus dependent. The possible reasons for the thymus dependence of immune responsiveness to highepitope-density hapten-protein conjugates are discussed.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Universal vaccine carrier. Liposomes that provide T-dependent help to weak antigens

The design of a thymus-dependent synthetic vaccine that will provide a universal T cell epitope for B cell epitopes is described in this study. Simultaneous incorporation into liposomes of both a peptide recognized by Th lymphocytes and a lipophilic hapten and the IgG antibody responses to this hapten were assessed in outbred mice. DNP-aminocaproyl phosphatidylethanolamine (DNP-CapPE) is a well characterized T-independent hapten Ag. HA2 peptide derived from the hemagglutinin protein of influenza virus contains amino acid sequences recognized by Th and T cytotoxic lymphocytes. In addition, HA2 contains a sequence of hydrophobic amino acids near the carboxyl terminus, allowing its incorporation into liposomes. Results of immunization show that (i), when DNP-CapPE is carried by liposomes without the HA2 peptide, an IgM antibody response is induced, (ii) liposomes carrying both HA2 and DNP-CapPE elicit an IgG antibody response to DNP in a dose-dependent fashion for both HA2 and DNP, (iii) the liposomes must be processed intracellularly in order to elicit a response, (iv) the system leads to a memory response for DNP, and (v) all of the IgG subclasses are elicited. These data suggest that liposomes containing the HA2 peptide exhibit a T-dependent carrier effect for a T-independent Ag. The significance of these findings is discussed in conjunction with the characteristics of the liposome model used.