The asymmetry in idiotype-isotype expression in the response to phosphocholine is due to divergence in the expressed repertoires of Lyb-5+ and Lyb-5- B cells

The X-linked CBA/N immune defect was used to investigate the roles of Lyb-5- and Lyb-5+ B cells in the memory response to PC-KLH (phosphocholine-conjugated keyhole limpet hemocyanin). (CBA/N X BALB/c)F1 (CB) male mice express the xid mutation and thereby lack the Lyb-5+ B cell subset, whereas their female littermates are normal and express both Lyb-5+ and Lyb-5- B cells. After priming with PC-conjugated hemocyanin (PC-Hy) in complete Freund's adjuvant, female B cells produce three phenotypic sets of PC-KLH-specific antibody. The first set (group I) is dominated by T15+, IgM, IgA, and IgG3, PC-specific antibodies. The second subset (group II) is specific for phenylphosphocholine (PPC), and is dominated by T15-, IgG1, and IgG2 antibodies. The third set (group III) recognizes an epitope(s) composed of both the PPC hapten and carrier determinants. PC-Hy-primed B cells from immune defective CB male mice produce the same number of IgG1 and IgG2 plaque-forming cells (PFC) as do PC-Hy-primed normal female cells, and these PFC are also predominantly T15- and PPC specific (group II). In addition, a significant amount of group III IgG1 and IgG2 antibody is observed in the immune defective male response. In contrast to female B cells, immune defective male B cells produce a low IgM, IgA, and IgG3 memory response that is not composed of PC-specific (group I) antibodies; in fact, most of these antibodies arise from group III precursors and are not inhibited by either PC or PPC. PC-specific antibodies usually represent less than 25% of the anti-PC-KLH response in immune defective mice; however, these PC-specific antibodies are predominantly T15-. These data suggest that the Lyb-5-B cells in both normal and immune defective mice produce the T15-, IgG1, and IgG2 antibodies that dominate the secondary immune response to PC-KLH, and that the Lyb-5+ B cells produce the T15+, IgM, IgA, and IgG3 portion of the secondary response in normal mice. This hypothesis was confirmed by priming normal mice with the R36a strain of Streptococcus pneumoniae or with PC-Hy in saline. These forms of PC-antigen prime only the Lyb-5+ B cell subset. The adoptive transfer of these two B cell sources results in an anti-PC-KLH response that is T15 dominant and totally PC inhibitable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of T15 idiotype dominance. I. Mice expressing the xid immune defect provide normal help to T15+ B cell precursors

The immune response to phosphocholine (PC) in many strains of mice is dominated by the T15 idiotype family of anti-PC antibodies. By introducing the CBA/N X-linked immune defect (xid gene) into these mice, one profoundly alters their ability to make a T15-predominant, IgM anti-PC response. This loss of T15 dominance in mice expressing the xid gene is not due to the presence of suppressor T cells or the lack of T15 idiotype-specific helper cells in these mice. Thus, one can reconstitute a T15 idiotype-dominant response in immune defective mice with B cells from normal mice, and in adoptive transfer assays the primed T helper cells from immune-defective mice provide qualitatively the same help to normal B cells as the T helper cells from normal mice. T15 idiotype dominance appears to be controlled by the expression and activation of Lyb-5+ PC-specific B cells. Thus, the majority of T15+ B cell precursors are restricted to this B cell subset, whereas the Lyb-5- B cell subset contains predominantly T15-, anti-PC B cell precursors, which produce mainly IgG antibodies after activation by PC-containing antigens.     

Regulation of T15 idiotype dominance. II. Genes unlinked to the Igh locus regulate T15 dominance of the secondary adoptive transfer response to phosphocholine

We have studied the idiotype and fine specificity of the secondary immune response to phosphocholine (PC) in C57BL (B10, B10.D2, and B.C8) and BALB (BALB/c, BAB-14, and C.B20) congenic strains of mice. In vivo IgM responses of mice from these two genetic backgrounds differed in their T15 idiotypic representation. BALB strains expressed the T15 idiotype on greater than 90% of their IgM, PC-specific plaque-forming cells (PFC), whereas C57BL strains expressed the T15 idiotype on approximately 50% of their IgM PFC. All strains examined expressed greater than 75% PC-inhibitable, VHPC idiotype-positive, IgM PFC. The IgG3 and IgA memory responses were similar to the IgM memory response; BALB strains produced a higher proportion of T15+ PFC than C57BL strains; however, the majority of IgG3 and IgA PFC in all strains were VHPC+, and PC-inhibitable. In contrast, the IgG1 memory response was not dominated by T15+, VHPC+, PC-inhibitable PFC in any of the strains tested. The IgG1 PFC required nitrophenylphosphocholine (NPPC) for efficient inhibition. The IgG2 memory response generally mimicked the IgG1 response with respect to idiotype and specificity. These data demonstrate that the representation of the T15 idiotype in the anti-PC immune response is determined by genes outside both the MHC and Igh genetic loci. Control of T15 expression in secondary IgM, IgG3, and IgA anti-PC responses was examined by using a cell-mixing protocol with primed T and B cells from BALB/c and B10.D2 mice. T15 representation in these responses was determined by the genotype of the B cell, not by the genotype of the helper T cell. Similarly, the B cell genotype was responsible for the idiotypic profile of a primary, in vitro, T-dependent, anti-PC response.     

Contribution of Lyb 5+ and Lyb 5- B cells to the primary and secondary phosphocholine-specific antibody response

Due to a mutation on their X-chromosome, CBA/N mice lack the Lyb-5+ subset of B cells. The loss of this B cell subset results in a profound alteration in the immune response of these mice to the hapten phosphocholine (PC). Thus, when these mice are immunized with high doses of PC-KLH (200 micrograms) in CFA, they: 1) fail to produce IgM anti-PC antibodies; 2) produce little or no anti-PC antibody bearing the normally predominant T15-idiotype; and 3) produce IgG anti-PC antibodies only late in the primary response. In order to more fully delineate this defect in responsiveness to PC, the splenic focus assay was used to analyze Lyb-5- B cell precursors from both normal and immune defective mice. Lyb-5- cells were obtained from normal (CBA/N x DBA/2)F1 (CD) female spleens by treatment with anti-Lyb-5 serum and complement. These normal Lyb-5- cells and Lyb-5- cells from immune defective CD male mice were stimulated in vitro with either PC-Hy or TNP-Hy in the presence of Hy-primed T helper cells. The results demonstrate that primary Lyb-5- PC-specific B cells fail to respond in the splenic focus assay, while secondary Lyb-5- PC-specific precursors respond normally, and that both primary and secondary Lyb-5- TNP-specific precursors respond in the splenic focus assay. These data suggest that Lyb-5- PC-specific precursors must differentiate into memory cells before they can be activated to secrete antibody, and they also indicate that the Lyb-5- B cell subset may be composed of two subsets with different activation requirements.     

Lyb-5+ B cells can be activated by major histocompatibility complex-restricted as well as unrestricted activation pathways

It has previously been demonstrated that B cells can be activated through two distinct T helper (Th) cell-dependent pathways, one requiring both carrier-hapten linkage and MHC-restricted T-B interaction and the other requiring neither. In addition, it has been shown that different B cell subpopulations exist and that these subpopulations differ in their activation requirements. Previous studies demonstrated that resting B cells containing an Lyb-5+ subpopulation were activated by MHC-unrestricted T cell signals, whereas resting Lyb-5- B cells were activated only through MHC-restricted T-B interaction. It was suggested that this difference resulted from the ability of Lyb-5+ but not Lyb-5-B cells to respond to soluble MHC-unrestricted Th signals. Because Lyb-5+ B cells were responsive in these previous experiments to MHC-unrestricted Th signals, it could not be determined whether Lyb-5+ B cells were also responsive to MHC-restricted Th signals. Consequently, the present study was undertaken to directly address the question of whether Lyb-5+ B cells can be activated under appropriate conditions by MHC-restricted as well as unrestricted T cell-B cell interactions. It was found that unprimed normal B cells (containing Lyb-5+ and Lyb-5-B cells) but not unprimed xid-defective populations (Lyb-5- only) can be activated by cloned KLH-specific and MHC-restricted Th cells in response to either high or low concentrations of TNP-KLH. The IgM response of Lyb-5+-containing B cells to a high concentration of antigen (10 micrograms/ml) was MHC unrestricted, whereas the IgM response of unprimed Lyb-5+ B cells to a low concentration of antigen (0.001 micrograms/ml) was MHC restricted. Thus, unprimed Lyb-5+ B cells can be activated through both MHC-restricted and unrestricted pathways. It was further demonstrated that the activation requirements of Lyb-5+ and Lyb-5- B cells differed even for MHC-restricted B cell activation.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Human helper-T-cell function does not require T4 antigen expression

The relationship between immunoregulatory T-cell function and the expression of T-cell subset-specific differentiation antigens was examined using a phenotypically anomalous human T-cell line (TCL), termed H-1. H-1 cells were found to express T11, extremely high levels of T3, but no T4 nor T8 antigen. Despite their lack of T4 antigen expression, H-1 cells could be activated by coculture with pokeweed mitogen (PWM), anti-T3 antibody, or autologous B cells to provide potent help for B-cell differentiation into plaque-forming cells (PFC). In contrast, H-1 cells did not suppress the PFC response triggered by PWM-activated T4+ cells. These results demonstrate that the expression of the T-cell subclass-specific differentiation antigen, T4, is not required for a T cell to become activated and to implement the program for helper function. In addition, enhanced expression of T3 on the T4-, T8-, H-1 cell surface may reflect a compensatory upregulation of the T3/Ti receptor complex on T cells which are deficient in these nonpolymorphic associative recognition structures.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

Helper T lymphocytes from xid and normal mice support anti-phosphocholine antibody responses with equivalent T15, 511, and 603 idiotypic composition

We have examined the idiotypic composition of secondary adoptive transfer antibody responses to phosphocholine (PC) supported by KLH-primed helper T cells derived from normal mice or xid mice. CBA/N x BALB/c F1 male xid mice have diminished anti-PC responses and virtually undetectable levels of the T15 idiotype; xid mice do express the 511 and 603 idiotypes. Nonetheless, we find helper T cells derived from such mice are indistinguishable from T cells primed in a normal environment in their ability to cooperate with B cells producing anti-PC antibody bearing the T15, 511, or 603 idiotype markers. This result is in contrast to a previously published report from this laboratory. T cells from xid mice did support more IgG PFC than normal T cells, but serum IgG anti-PC antibody levels were similar in both groups. The IgM anti-PC response was predominantly of the T15 idiotype, whereas the 511 idiotype was associated with a minor fraction of IgG1 antibodies. The majority of the secondary IgG "anti-PC" antibody response bore none of the idiotypic markers associated with PC-binding myeloma or hybridoma antibodies, and was directed against phenyl-PC rather than PC. The phenomenon of T15 clonal dominance in the anti-PC response therefore is largely confined to the IgM response. We would conclude that the idiotype levels in the T cell priming environment do not influence the subsequent ability of such primed T cells to support anti-PC antibody responses.     

Regulation of the secondary in vitro antibody response by endogenous natural killer cells: kinetics, isotype preference, and non-identity with T suppressor cells

Our previous studies had demonstrated that depletion of endogenous natural killer (NK) cells resulted in an augmented primary antibody response in vivo and in vitro. We have now examined the effect of NK cell depletion on the in vitro secondary response to antigen. Treatment of primed murine spleen cells with anti-NK-1.1 allo-antibody and complement before culture resulted in a significant increase in the magnitude of the antigen-specific plaque-forming cell (PFC) response. This treatment did not affect the proportions of Lyt-2+, L3T4+, or sIg+ cells in the population, however, indicating that the augmentation in PFC was not due to changes in the ratio of T to B cells. Removal of endogenous NK cells had a greater effect on the IgG (indirect) PFC response (100 to 200% increase) than on the IgM (direct) PFC response (25 to 50% increase). In contrast, removal of Lyt-2+ cells before culture affected the IgM and IgG responses similarly. Moreover, the kinetics of augmentation differed between cultures depleted of Lyt-2+ cells and those depleted of NK-1.1+ cells. NK cells appeared to act earlier in the response than did T suppressor cells. The NK-1.1+ cells involved in antibody regulation were not involved in the generation of the in vitro derived T suppressor cells. The conclusion that the regulation of the antibody response by NK-1.1+ cells is distinct from that involving T suppressor cells was confirmed in experiments in which removal of both regulatory cell populations resulted in an increase in PFC that was greater than in cultures depleted of either NK or T suppressor cells.     

Role of self carriers in the immune response and tolerance. X. A lymphoid dendritic-like tumor, P388AD.2, acts as a novel immunogenic carrier for hapten

Hapten-modified spleen cells, peritoneal exudate cells, and certain lymphoid tumors preferentially induce specific tolerance after i.v. administration. In contrast to these tolerogenic carrier cells, we found that a haptenated lymphoid dendritic-like tumor, P388AD.2, acts as a potent immunogen after i.v. injection. The immunogenicity of P388AD.2 was analyzed by measuring the specific augmentation of plaque-forming cell (PFC) responses when spleen cells from mice previously injected with haptenated tumor cells were challenged in vitro with thymus-independent antigens. Optimal immunization was found to be dependent on cell dose and hapten concentrations. Further studies indicated that P388AD.2 elicited a response which was T cell-dependent and which involved both the so-called Lyb-3,5,7- and Lyb-3,5,7+ B cell populations. Injection of haptenated tumor into different mouse strains suggested that H-2 compatibility was required to prime B cells in vivo, although significant augmentation could also be achieved in allogeneic C57B1/6J mice. The enhanced PFC responses elicited in H-2b mice could not be explained by allo-recognition of class I or II MHC determinants. In toto, these results suggest that P388AD.2 acts as a unique accessory cell for the presentation of hapten-modified self.     

Keyhole limpet hemocyanin-propagated Peyer's patch T cell clones that help IgA responses

Four T cell clones, isolated from Peyer's patches of keyhole limpet hemocyanin (KLH)-primed BALB/c mice, were selected on the basis of their ability to help IgA responses by TNP-KLH-primed BALB/c mouse B cells. Two were KLH-dependent both in terms of their own proliferative response and in terms of their help for that of B cells. The other two were autoreactive and helped B cells proliferate independently of the presence of Ag. Both primed and unprimed B cells proliferated to some extent when helped by the KLH-reactive clones in the presence of high concentrations of either KLH or TNP-KLH. Much higher proliferation was, however, induced when primed, but not unprimed, B cells were exposed to the T cells in the presence of low concentrations of TNP-KLH but not KLH, i.e., under conditions favoring direct, cognate interaction between the T and B cells. Only modest IgM, and no IgG or IgA plaque-forming cell (PFC) responses were generated by TNP-primed B cells upon interaction with either autoreactive T cells in the absence of Ag or KLH-reactive T cells in the presence of high concentrations of KLH. For high IgM responses as well as for the appearance of IgG and IgA PFC responses, TNP-KLH was required whatever the source of the T cell help. The isotype ratios depended on the TNP-KLH concentration; IgA responses were highest and IgM responses lowest at the lowest TNP-KLH concentrations suggesting that the precursors of the IgA PFC have higher average affinity for TNP than the precursors of IgM PFC. Overall, the results are compatible with the idea that the precursors of IgA and IgG PFC and many of the precursors of IgM PFC in the long term primed B cell populations used in these experiments require engagement of their Ag-receptors before they express sufficient class II Ag and/or receptors for "switch" and differentiation factors for cognate interaction with T cells leading to PFC responses.     

Human peripheral blood B lymphocyte subpopulations: functional and phenotypic analysis of surface IgD positive and negative subsets

Highly purified human peripheral blood B cells stimulated with Cowan I Staphylococcus aureus (SA) and mitogen-activated T cell supernatants (T supt) generated large numbers of immunoglobulin (Ig)-secreting cells (ISC), whereas fewer ISC developed in cultures containing T supt in the absence of SA. To determine whether surface Ig isotype expression defined responsive B cell subsets, IgD+ and IgD- B cells were prepared with the fluorescence-activated cell sorter. Whereas both the IgD+ and IgD- B cells responded to SA + T supt, only the IgD- subset generated substantial numbers of ISC in response to T supt alone. Analysis of secreted Ig revealed that IgG and IgA were the predominant isotypes secreted by IgD- B cells in response to T supt or SA + T supt. By contrast, the IgD+ cells secreted predominantly IgM in response to SA + T supt but not to T supt alone. When responsiveness to pokeweed mitogen (PWM) was examined in the presence of supplemental T cells, the IgD- subset was found to be greatly enriched for responsive cells, and again, IgG and IgA were the predominant isotypes secreted, although these cells were also capable of secreting some IgM. The magnitude of the response induced by PWM from IgD- B cells was usually greater than that induced by SA + T supt. Although IgD+ B cells responded poorly to PWM, the differentiation of a small number of IgM-secreting cells was routinely stimulated by this polyclonal activator in the presence of T cells. The magnitude of the PWM response by IgD+ B cells was always greatly diminished compared with that stimulated by SA + T supt. Cell cycle analysis after acridine orange staining, cell volume measurement, and staining for expression of activation antigens (transferrin receptor and 4F2) indicated that the IgD- B cells were largely resting, but did contain a population of activated cells. Removal of activated 4F2+ cells from the IgD- subset diminished but did not abolish their capacity to generate ISC in response to SA + T supt or PWM in the presence of T cells. These results suggest that the IgD- population contains both an activated 4F2+ and a resting 4F2- subset. The data emphasize that multiple subpopulations of peripheral blood B cells contain precursors of ISC. Moreover, the responsiveness of the subsets to various stimuli and the Ig isotype subsequently secreted appear to be intrinsic features of each subset.     

The Lyb-3+5+ subset of B cells is not required for lupus-like autoantibody formation caused by graft-vs-host reaction

We asked the question whether or not the Lyb-3+5+ B cell subset, which is lacking in CBA/N immune defective mice, is required for the lupus-like autoantibody formation caused by graft-vs-host reaction (GVHR). (CBA/N X DBA/2)F1 male defective mice injected with DBA/2 T cells produced IgG autoantibodies to the same extent as did nondefective F1 mice suffering from GVHR. Although a very small number of DBA/2 B cells might have contaminated the T cell inocula, it was shown that these were B cells of the defective F1 mice that produced autoantibodies during the GVHR. This was demonstrated by detecting autoantibodies carrying an immunoglobulin allotype of the F1 recipient. Furthermore, the defective F1 male mice injected with CBA/N lymphoid cells, which were lacking Lyb-3+5+ B cells, also produced autoantibodies. Isotype analysis of antinuclear antibodies revealed that some of them belonged to IgG3 isotype. It was concluded that the ontogenically late-appearing B cell subset is not required for GVH autoimmunity.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

Primed lymphoid cell tolerance. II. In vivo tolerization of highly tolerogen-sensitive hapten-primed, potentially IgG-producing B cells

The relative ease of tolerizing IgM-bearing versus IgG-bearing B cells was investigated. Previous work had shown that IgG-bearing trinitrophenyl (TNP)-specific B cells from mice primed and boosted with TNP-keyhole limpet hemocyanin (TNP-KLH) are highly susceptible to tolerization in vitro by TNP presented on an unrelated carrier. TNP-OVA was used as tolerogen, as it may represent a more general class of tolerogens than those which are nonmetabolizable or immunoglobulin containing. This study showed that highly primed B cells are tolerizable in vivo using TNP-OVA, with the IgG response to TNP-KLH easier to tolerize than the IgM response. To determine if the ease of tolerization of the IgG response in vivo was due to intrinsic differences in B-cell precursors of the IgM and IgG responses, tolerance was performed in vitro with B cells of defined surface isotypes. A T-independent antigen, TNP-endotoxin, was employed to minimize T-cell effects. At least 10 times as much TNP-OVA was required to tolerize B cells bearing the IgM surface isotype than those with the IgG surface isotype. Thus, the ease of inhibition of the IgG response as compared to the IgM response in vivo by preexposure to TNP-OVA may be at least partially explained by inherent differences in IgM and IgG B-cell precursors.     

IgG recruiting component (GRC): B cell-derived signal for IgG antibody synthesis.

The B cell-derived soluble factor that has been described as an IgG-recruiting component (GRC) was investigated to: a) ascertain whether it is governed by genetic constraints, b) determine what triggers its synthesis, and c) identify its cellular target. GRC has been shown to be unrestricted by histocompatibility barriers since it enhanced IgG antibody production in mice of diverse genetic backgrounds. Further, we report that eliminating IgG-bearing cells from B cells to be immunized in vitro allows T cells-replacing factor (TRF) to increase the number of IgM but not IgG PFC. Thus, TRF appears to act on IgM-bearing cells by expanding the IgM PFC number. Adding GRC 48 hr after the addition of TRF to such IgG-depleted cells caused expression of IgG PFC. Hence, B cells lacking IgG but possessing IgM surface immunoglobulins appear to be those that are acted upon by GRC. These data indicate that in whole splenic cell populations, GRC is derived from IgG-bearing B cells that are stimulated by antigen and a component in TRF.     

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.     

Regression of Epstein-Barr virus-induced B-cell transformation in vitro involves virus-specific CD8+ T cells as the principal effectors and a novel CD4+ T-cell reactivity

T-cell memory to Epstein-Barr virus (EBV) was first demonstrated through regression of EBV-induced B-cell transformation to lymphoblastoid cell lines (LCLs) in virus-infected peripheral blood mononuclear cell (PBMC) cultures. Here, using donors with virus-specific T-cell memory to well-defined CD4 and CD8 epitopes, we reexamine recent reports that the effector cells mediating regression are EBV latent antigen-specific CD4+ and not, as previously assumed, CD8+ T cells. In regressing cultures, we find that the reversal of CD23+ B-cell proliferation was always coincident with an expansion of latent epitope-specific CD8+, but not CD4+, T cells; furthermore CD8+ T-cell clones derived from regressing cultures were epitope specific and reproduced regression when cocultivated with EBV-infected autologous B cells. In cultures of CD4-depleted PBMCs, there was less efficient expansion of these epitope-specific CD8+ T cells and correspondingly weaker regression. The data are consistent with an effector role for epitope-specific CD8+ T cells in regression and an auxiliary role for CD4+ T cells in expanding the CD8 response. However, we also occasionally observed late regression in CD8-depleted PBMC cultures, though again without any detectable expansion of preexisting epitope-specific CD4+ T-cell memory. CD4+ T-cell clones derived from such cultures were LCL specific in gamma interferon release assays but did not recognize any known EBV latent cycle protein or derived peptide. A subset of these clones was also cytolytic and could block LCL outgrowth. These novel effectors, whose antigen specificity remains to be determined, may also play a role in limiting virus-induced B-cell proliferation in vitro and in vivo.     

B- and T-lymphocyte responses to an immunodominant epitope of human immunodeficiency virus.

Using synthetic peptides, we characterized the B-lymphocyte (antibody) and T-lymphocyte (proliferation) responses to an immunodominant epitope of human immunodeficiency virus type 1 (HIV-1) located near the amino-terminal end of the transmembrane glycoprotein (env amino acids 598 to 609). Both immunoglobulin M (IgM) and IgG antibodies against this epitope appeared early after primary infection with HIV-1. In an animal model, the IgG response to a synthetic peptide derived from this sequence was T-helper-cell dependent, whereas the IgM response was T-cell independent. In addition, antibody generated by immunization with this peptide had HIV-1-neutralizing activity. Greater than 99% (201 of 203) of patients infected with HIV-1 generated antibody to this peptide in vivo; however, only 24% (7 of 29) had T cells that proliferated in response to this peptide in vitro. These observations suggest that different HIV-1 gp41 epitopes elicit B-cell and T-cell immune responses.