T helper cell lines that augment in vivo cytotoxic T-cell responses to minor alloantigens

We have investigated the ability of long-term cultured T helper (Th) cell lines to help an in vivo cytotoxic T lymphocyte (CTL) response to non-H-2 alloantigens (minor antigens). Th cell lines specific for various single or undefined minor antigens were selected by regular restimulation with antigen in vitro. They were antigen specific and H-2 restricted in proliferation assays and were found to be able to help primary CTL responses to multiple minor antigens and secondary CTL responses to single minor antigens. Although the Th were antigen specific they did not determine the specificity of the CTL. Th cells were both necessary and limiting for an effective CTL response indicating that "helper-independent" CTL are not in themselves sufficient to generate a strong in vivo response. Under conditions where a CTL response was clearly H-2 restricted, Th cells were not. Thus, the Th cells appeared to be activated by reprocessed antigen rather than antigen on the surface of the injected antigenic cells even though the CTL themselves reacted directly to the injected antigen.     

Idiotope antigens (Ab2 alpha and Ab2 beta) can induce in vitro B cell proliferation and antibody production

We previously showed that immunization of various strains of mice with three types of antigen--PC-Hy (nominal antigen), F6-Hy (Ab2 alpha-Hy, and 4C11-Hy (Ab2 beta-Hy)--induces a differential PC-specific, T15-Id+ antibody response. In this report, the in vitro phosphorylcholine (PC)-specific B cell responses induced by these three antigens were studied. A hemocyanin-specific long-term T helper cell line was used to provide help for primary and secondary in vitro T cell-dependent B cell responses. At low doses (0.005 to 0.5 micrograms/ml) of antigen, a significant increase in the proliferation of PC-OVA-primed BALB/c B cells was observed with Ab2-Hy or PC-Hy conjugate, but not unconjugate, antigens. Similar low doses of antigen could stimulate naive B cells to secrete IgM and stimulate PC-OVA- or 4C11-Hy-primed B cells to secret IgM and IgG1 anti-PC antibodies. The percentage of T15-Id of the PC-specific antibodies produced in the in vitro T-B culture was found to be less dominant than that produced by in vivo immunization, suggesting that certain regulatory mechanisms occur in the in vivo environment that may help to maintain the T15-Id dominance. Taken together, our in vivo and in vitro results indicate that idiotope antigens can function like nominal antigens to induce antigen-specific B cell responses. The mechanisms of thymic-dependent B cell activation induced by idiotope and nominal antigen are similar in that the T-B interaction is MHC-restricted and requires cognate recognition.     

Antigen-induced T suppressor cells regulate the autoreactive T helper-B cell interaction

The T suppressor (Ts) cell population that functions to regulate antigen-specific MHC-restricted T helper (Th)-B cell interactions also regulates the activation of B cells by cloned autoreactive Th cells. Activated Ts cells were generated by in vivo priming and restimulation in vitro with high concentrations of the specific priming antigen. Once generated, this Ts population inhibits the Th-dependent activation of primed B cells by both antigen-specific and autoreactive T cells in an antigen-nonspecific manner. This suppression requires the participation of both Lyt-1+2- and Lyt-1-2+ T cells. It was also demonstrated that accessory cells were required for the induction of Ts cells. Moreover, the generation of suppression was MHC-restricted and required the recognition by T cells of Ia antigens on accessory cells. These studies demonstrate that the same or a very similar Ts cell population can function to inhibit the activation of B cells by antigen-specific as well as autoreactive T cells.     

Th1 and Th2 CD4+ T cells provide help for B cell clonal expansion and antibody synthesis in a similar manner in vivo

The relative ability of Th1 and Th2 T cells to help B cells remains controversial as do the mechanisms by which both T cell subsets provide help in vivo. Whether this help affects the clonal expansion and/or differentiation of B cells has been difficult to assess due to the low frequency of Ag-specific T and B lymphocytes. We have employed a novel technique to directly monitor the clonal expansion of Ag-specific T and B lymphocytes in vivo. OVA-specific TCR transgenic T lymphocytes were polarized toward a Th1 or Th2 phenotype in vitro. These cells were then transferred into syngeneic recipients, along with B cell receptor transgenic hen egg lysozyme-specific B lymphocytes. Our results indicate that Th1 and Th2 cells support B cell responses to a similar extent in vivo and that they achieve this in the same manner by migrating into B cell follicles to promote CD154-dependent B cell clonal expansion and Ab production.     

B cell presentation of a tolerogenic signal to Th clones.

Lightly irradiated (950 R) splenic B cells were inefficient, in comparison to unseparated spleen cells, in stimulating antigen-specific proliferation of Th1 clones specific for human gamma globulin (HGG). This inefficiency was due to antigen-specific inactivation: Th1 clones preincubated with HGG and lightly irradiated B cells or mitomycin C-treated B cells were unable to proliferate to HGG in secondary cultures. In contrast to Th1 clones, Th2 clones proliferated well in response to B cell APC, and showed no decrease in their subsequent antigen-induced proliferative capacity after exposure to lightly irradiated B cells and HGG. However, preincubation of Th2 with lightly irradiated B cells and HGG did inactivate the capacity of Th2 to provide help for antibody production in secondary cultures. These results suggest that under certain conditions B cells may present antigen to Th1 and Th2 cells in a tolerogenic rather than an immunogenic manner.     

Collaboration of Th1 and Th2 T cell clones in specific antibody responses: regulation of the IgM response to phosphorylcholine

Carrier (KLH)-specific type 1 T cell clones (Th1), which are defined by secretion of IL-2 and IFN-gamma but not IL-4, and type 2 (Th2) clones, which secrete IL-4, but not IL-2 or IFN-gamma, have been isolated and analyzed for their ability to collaborate in providing help for B cells to secrete phosphorylcholine-specific IgM antibodies. The resulting antibody responses exhibited a characteristic pattern suggesting two distinct regulatory interactions among the Th1, Th2, and B cells. At low doses of antigen, Th1 cells enhanced the helper function of the Th2 cells, an effect due primarily to IL-2. At high doses of antigen, Th1 cells or IFN-gamma inhibited Th2-dependent antibody responses. The inhibitory effect of Th1 or IFN-gamma affected primarily the hapten-carrier-linked portion of the response. The overall effect was a modulation of the antigen dose-response curve for antibody production, eliminating the sharp increases in dose response mediated by isolated T cell clones. The data suggest that collaborative interactions of Th1 and Th2 cells in antibody production may have important physiological consequences.     

Synergistic effects of the xid gene in X chromosome congenic mice. I. Inability of C3.CBA/N mice to respond to thymus-dependent antigens in adoptive transfer assays

The xid gene, which causes a B lymphocyte immune defect in CBA/N mice, has been bred onto the C3H/HeN background. The resulting X chromosome congenic mice (C3.CBA/N) exhibit immunologic defects that are much more profound than the defect exhibited by CBA/N mice; thus, the B cells from C3.CBA/N mice not only fail to respond to thymus-independent (TI) type 2 antigens such as TNP-Ficoll, but they fail to respond in vitro to TI-type 1 antigens such as TNP-Brucella abortus (BA) and B cell mitogens such as LPS and Nocardia water-soluble mitogen. In this paper we show that the synergistic defect seen in C3.CBA/N B cells is also elicited in adoptive transfer assays to thymus-dependent (TD) antigens such as TNP-KLH and PC-KLH, antigens to which both parental strains respond. Thus, the secondary adoptive transfer response of C3.CBA/N spleen cells is generally less than 5% of the immune response produced by CBA/N or C3H/HeN spleen cells. This synergistic defect is restricted to the C3.CBA/N B cells, since C3.CBA/N T cells can provide help to CBA/N B cells that is equivalent to the help obtained with CBA/N T cells. The low responsiveness of C3.CBA/N spleen cells to TD antigens, which is elicited in adoptive transfer assays, is not seen when the intact animal is immunized with antigen in CFA; this, intact C3.CBA/N mice produce anti-PC-KLH and anti-TNP-KLH responses only slightly lower than the responses of CBA/N mice to these same antigens. In contrast, when these mice are immunized with phenol-extracted LPS, a TI-type 1 antigen, their antibody responses are severely depressed. These data suggest that under conditions in which T cell help may be limiting or in which the intact physiology of the T and B cells has been disrupted, C3.CBA/N B cells demonstrate profound immunologic impairment; however, when adequate T cell help is available and the splenic architecture is not disrupted, their immune responses appear to progress in a normal fashion.     

Immunoregulation in the rat: cellular and molecular requirements for B cell responses to types 1, 2, and T-dependent antigens

The requirements for primary in vitro plaque-forming cell (PFC) development in cultures of purified rat splenic B cells have been examined. Rat B cells were directly responsive to the type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA), but both T cells and adherent accessory cells were required for B cell responses to the type 2 antigen TNP-Ficoll and the T cell-dependent (TD) antigen sheep erythrocytes (SRBC). However, the cellfree supernatants from concanavalin A-induced spleen cells of rat or mouse origin replaced the requirement for T cells and macrophages, and resulted in PFC development in response to TNP-Ficoll and SRBC and augmented PFC numbers in response to TNP-BA. Culture supernatants from induced murine T cell and macrophage cell lines were used to partially deduce the molecular requirements for the support of PFC development by rat B cells to these three antigens. Supernatants from the EL-4 (EL-4 sup) and B151 K12 (B15 sup) T cell lines augmented TNP-BA responses, suggesting that B cell growth factor II (BCGF-II) mediated this effect. An admixture of purified interleukin 2 (IL 2) and B15 sup supported PFC development to SRBC; indicating that IL 2, BCGF-II, and the T cell-replacing factor in B15 sup (B15-TRF) were sufficient to support this response. In addition, the IL 2 plus B15 sup-supported anti-SRBC PFC response was increased by the addition of an interleukin 1-containing fraction from the supernatant of the macrophage line P388D1. PFC development in response to TNP-Ficoll had the most stringent requirements and only occurred in the presence of EL-4 sup and B15 sup (IL 2, BCGF-I, BCGF-II, EL-TRF, B15-TRF). These data indicate that different cellular and molecular requirements exist for PFC development in response to types 1, 2, and TD antigens by rat B cells.     

Characterization of the effector mechanism of help for antigen-presenting and bystander resting B cell growth mediated by Ia-restricted Th2 helper T cell lines

The mechanism of help for resting B cell growth in MHC-restricted T-B collaboration was investigated using an in vitro polyclonal model for these T cell-B cell interactions. In the presence of rabbit anti-mouse Ig, small, size-selected B cells elicit help from syngeneic Ia-restricted Th2 cell lines specific for F(ab')2 rabbit globulin. Both Ag-presenting and bystander B cells receive signals from stimulated Th cells that lead to B cell proliferation. The results suggest that the direct activation of resting Ag-presenting and bystander B cells by Th2 cells is mediated by a similar effector mechanism. Although proliferative responses by Ag-presenting B cells are of greater magnitude, help for both Ag-presenting and bystander B cell populations is characterized by the lack of a requirement for membrane Ig cross-linking, by identical kinetics, and by the necessity for direct cell contact or close proximity with Th cells. B cell proliferation is not induced by exposure to the sequence of diffusable mediators released from a synchronized Ag-specific T-B interaction. The T cell-dependent proliferation by both B cell populations can be inhibited by excess mitomycin C-treated syngeneic "cold target" B cells, demonstrating a requirement for a short-range T cell-B cell interaction. mAb inhibition experiments fail to identify a role for class II, LFA-1, or CD4 membrane molecules in the delivery of help to bystander B cells. Antibody against H2d bystander class II molecules has no effect on bystander B cell proliferation at concentrations that completely block Ag presentation by H2d B cells to an H2d-restricted Th cell line. Antibodies against the cell adhesion molecule LFA-1 or the Th cell molecule CD4 do inhibit bystander B cell proliferation, but only to the extent that they block T cell activation and the induction of help. The inductive stimulus leading to resting B cell growth results from an early, short-range interaction with Th cells. B cell proliferation is supported by T cell soluble mediators as a consequence of this interaction, which is required for at least 8 hr after T cell recognition of Ag/Ia on the surface of Ag-presenting B cells.     

Functions of accessory cells in B cell responses to thymus-independent antigens

The functions of adherent accessory (A) cells in thymus-independent (TI) B cell activation were investigated using homogeneous A cell lines with distinct cell surface and functional characteristics, as well as inhibitors of antigen processing and interleukin 1 (IL 1) secretion. B cell responses to both type 1 and type 2 TI antigens were found to be strictly A cell dependent. Only A cells capable of IL 1 secretion could restore responsiveness in A cell-depleted spleen cells, regardless of Ia expression or antigen-processing capability. Moreover, recombinant IL 1 completely replaced A cell function in B cell responses to both TI 1 and TI 2 antigens. Finally, T cell depletion did not diminish the reconstitution by IL 1. Thus in contrast to T cell activation, IL 1 secretion is the only A cell function required in TI B cell activation, and the data are consistent with a direct role for IL 1 in B cell activation.     

Cloned allospecific human helper T cell lines induce an MHC-restricted proliferative response by resting B cells

To analyze helper T (Th) cell-induced B cell proliferation in man, we have cloned allospecific Th cells, grown them as long-term IL 2-dependent T cell lines (TCL), and analyzed their phenotypic and functional properties. The two TCL described in this report, A-7 and A-57, are both composed exclusively of T3+, T4+, T8- T cells blasts. In proliferative assays, with a panel of x-irradiated allogeneic stimulator cells, A-7 was found to proliferate in response to DR3-bearing cells, whereas A-57 responds to DR2-positive stimulators. Both TCL are capable of providing MHC-restricted polyclonal help for allogeneic B cells, as measured in the reverse hemolytic plaque assay. Of greater interest, x-irradiated A-7 and A-57 cells are capable of inducing a proliferative response by allogeneic B cells that is absolutely MHC restricted at the inductive (Th-APC) level. Thus, x-irradiated A-7 cells only trigger proliferation by DR3+ B cells, whereas A-57 cells selectively activate DR2+ B cells. In contrast, after antigen-specific activation, x-irradiated A-7 and A-57 cells can recruit a significant proliferative response by allogeneic B cells bearing "irrelevant" DR antigens. The possibility that Th-induced B cell proliferation may be restricted at the effector (Th-B cell) level was addressed by fractionating B cell populations into "activated" and "resting" subsets by discontinuous Percoll density gradient centrifugation and further purification by employing a monoclonal antibody directed against an antigen expressed on activated B cells (4F2). These studies demonstrate that activated B cells are readily and nonspecifically recruited to proliferate by activated Th cells, whereas optimal proliferative responses by resting B cells require MHC restricted Th-B cell interaction.     

Functional characterization of cloned intrahepatic, hepatitis B virus nucleoprotein-specific helper T cell lines

We have previously reported the establishment and preliminary characterization of polyclonal hepatitis B virus (HBV) nucleoprotein (HBcAg)-specific CD4+ and CD8+ T cell lines derived from the hepatic lymphomononuclear cell infiltrate of several patients with chronic active hepatitis B. The isolated subsets from these lines were specifically activated by HBcAg and displayed antigen-specific help and suppression with respect to proliferation of the alternate subset. One of these lines was recently cloned by limiting dilution, and four HBcAg-specific CD3+ CD4+ CD8-DR+ T cell lines were produced that had a 95.3% likelihood of monoclonality. Antigenic specificity was confirmed by dose-dependent, HLA class II (DR)-restricted proliferation in response to recombinant and human serum-derived HBcAg and the lack of proliferation to HBV envelope antigens (HBsAg and pre-S(2)Ag). All cloned lines were interleukin 2 dependent, produced interferon-gamma in an antigen-specific manner, and provided antigen-specific help to autologous B cells with respect to anti-HBc production. We conclude that HBcAg-specific, HLA-class II restricted helper T cells capable of inducing antigen-specific functional responses by autologous B lymphocytes and T lymphocytes are present at the site of viral antigen synthesis and hepatocellular injury in HBV infection.     

The use of marsupial x eutherian somatic cell hybrids to study marsupial cell surface antigens

Buck and Bodmer (1976) have developed a technique for identifying an antigen on the surface of human x mouse somatic cell hybrids, specified by a gene on a particular human chromosome. We have successfully adapted this technique to a study of marsupial cell surface antigens. Somatic cell hybrids between Macropus rufus (Marsupialia) lymphocytes and the mouse cell lines PG19 and 1R were injected intraperitoneally into mice of the same inbred strain from which the above cell lines were derived (C57B16J and C3H, respectively). The only identified M. rufus chromosome present in the hybrid cells was the X chromosome. The antisera, after adsorption with PG19 or 1R, were tested using indirect immunofluorescence, against the hybrid cells, and also against sub-clones (derived from hybrids) which had apparently lost the M. rufus X chromosome, or at least its long arm. The results of these tests showed that the absorbed antisera contained reactivity against an M. rufus cell surface antigen (or antigens). The reactions of one of the antisera were most simply interpreted by supposing that it was detecting an M. rufus X-lined antigen(s).     

B7, a B-cell-restricted antigen that identifies preactivated B cells

After activation with antigen or mitogen, a number of cell surface proteins appear that are not expressed on resting B cells. To date, a number of B lineage restricted and associated activation antigens have been reported that appear at distinct intervals after in vitro activation. In this report, we describe a new B lineage restricted activation antigen (B7) that appears within 24 hr of in vitro stimulation. The expression of B7 antigen, which is detected on a minor subpopulation of B cells isolated from peripheral blood and lymphoid tissues, is strongly induced following stimulation with either anti-immunoglobulin or Epstein-Barr virus. In contrast, B7 was not detected on resting or activated T cells or monocytes. The B7 antigen was expressed on a subset of B cell lines and B cell neoplasms, but was not detected on leukemias and lymphomas of T cell or myeloid origin. B7 was distinguished from other B cell restricted and associated activation antigens by its unique pattern of expression on a variety of hemopoietic cell lines. The biochemical characterization of B7, that it is a single chain protein of 60 kDa, further distinguishes it from other B cell activation antigens. The functional importance of the B7 antigen was demonstrated when splenic B cells were fractionated into the B7+ and B7- populations. The peak of proliferation in response to anti-Ig, appeared earlier within the B7+ population. These studies suggest that B7 antigen identifies a subpopulation of B cells that are preactivated or primed in vivo, and have an accelerated response to subsequent activation via cross-linking of surface Ig.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Inability of IL-2 and IL-10 to counteract B cell clonal deletion.

The B cell antigen receptor (BCR) delivers inhibitory signals in nascent B cells leading to the establishment of tolerance via clonal deletion or clonal anergy depending upon the type of antigen to which the B cells are exposed. In previous work, it has been demonstrated that activated Th2 cells, as well as some recombinant lymphokines, prevent the inhibition of growth and subsequent cell death induced through the BCR in model B cell lymphomas. Herein, we extend this work to another Th2 lymphokine, IL-10, that in contrast to IL-4 does not interfere with the deletion promoted by IgM crosslinking. The effect of individual lymphokines has also begun to be analyzed in a transgenic model of B cell clonal deletion. To this end, we have administered a recombinant vaccinia virus producing human IL-2 to mice expressing an autoreactive H-2Kk,b-specific transgenic IgMk and found that IL-2 does not abrogate B cell deletion in vivo.     

Immunoregulation in the rat: ontogeny of B cell responses to types 1, 2, and T-dependent antigens

The development of rat B cells has been examined in neonatal and adult Fischer rats through the use of type 1 (TNP-Brucella abortus), type 2 (TNP-LPS(Ph), TNP-Ficoll) and T cell-dependent (TD) (SRBC) antigens. In vivo splenic PFC responses to TNP-Brucella abortus could be induced in newborn rats and by 12 days of age had reached adult levels. In contrast, the responses to the type 2 and TD antigens were 30% and 70%, respectively, of the adult levels at 30 days of age. Adoptive transfer of the B cells from neonatal and young rats into irradiated adult hosts demonstrated that the kinetics in the development of responses to these antigens (early for type 1, intermediate for TD, and late for type 2) were not due to limiting accessory cell or T cell help in immature rats. In vitro cultures of purified B cells from neonatal and adult rats were responsive to TNP-BA and TNP-LPS(Ph) but not to TNP-Ficoll and SRBC. However, the addition of spleen cell-derived Con A supernatant to the B cell cultures resulted in responses to all four antigens, which arose as a function of B cell age, with kinetics that were identical to those observed in vivo. Fluorescent staining of B cells from rats of various ages for cell surface IgM and analysis on the fluorescence-activated cell sorter (FACS) revealed that all splenic B cells from rats 4 days of age expressed a relatively high level of sIgM, and that a subpopulation that expressed a relatively low level of sIgM increased with age until it represented approximately 50% of the adult splenic B cells. Challenging Con A supernatant-supplemented cultures of FACS-prepared low sIgM+ and high sIgM+ cells revealed that B cells responsive to TNP-Ficoll were confined to the ontogenically late-arising low sIgM+ subpopulation but that B cells responsive to TNP-BA, TNP-LPS(Ph), and SRBC were present in both subpopulations.     

B cell sensitivity to natural killing: correlation with target cell stage of differentiation and state of activation

When 13 B cell lines were phenotyped with a panel of B cell, stage-specific monoclonal antibodies and ordered with respect to differentiation state; their sensitivity to natural killer (NK) cell-mediated conjugate formation and cytolysis was found to be stage dependent. Target cell lines expressing an early B cell phenotype (B1+B2-CALLA+DU-ALL1 +/-) or an intermediate phenotype (B1+B2+CALLA-DU-ALL1+) were NK resistant. Late phenotypic B cells (B1+B2-CALLA-DU-ALL1-) were highly susceptible to NK cytolysis. Individual B cell lines when induced with sodium butyrate or retinoic acid expressed altered B cell phenotype and NK susceptibility. For example, SB, an intermediate B cell line and initially NK resistant, when induced to express a later B cell phenotype became NK sensitive. BJA.B, a late B cell line and initially NK sensitive, when induced to differentiate lost most of its sensitivity to natural killing. Since loss of the B2 antigen is associated with B cell activation, we further phenotyped the B cell lines and induced B cell lines with the 4F2 and 5E9 (anti-transferrin receptor) monoclonal reagents. All cell lines tested expressed each of these antigens, but with varying intensities. While the intensity of 4F2 expression appeared to correlate well with NK sensitivity on both resting and differentiated B cell lines, the intensity of 5E9 expression did not. Peripheral blood B cells express a similar pattern of reactivity to natural killing when stimulated with pokeweed mitogen (PWM) in vitro. B cell sensitivity to NK-mediated events peaked at day 4 of incubation and correlated with the loss of the B2 antigen and acquisition of the 4F2 and 5E9 antigens; sensitivity to natural killing was diminished in the presence of the PCA-1 antigen. The expression of the NK-susceptible phenotype among B cells appears to be differentiation stage- and activation state-dependent. It is during this period of ontogeny that the NK cell may cytolytically regulate the B cell transition to a plasma cell.     

Two stages of b cell memory development expressing differential sensitivity to stimulation with thymus-dependent and thymus-independent antigenic forms

We have shown that spleen cells specifically primed to the decapeptide determinant (a.a. 103-112) of the thymus-dependent (TD) antigen, tobacco mosaic virus protein (TMVP), can be secondarily stimulated to antibody synthesis by either TD or TI forms of the decapeptide. Further, TD- and TI-responsive memory B cells consisted of overlapping populations which were indistinguishable by the specificity and isotype composition of the antibodies which they synthesized. In the present study, two functionally distinct but developmentally related memory B cell subsets were identified by their differential sensitivity to repeated in vivo stimulation with various combinations of TD and TI decapeptide antigens. One subset of memory B cells responded only to TMVP or decapeptide conjugated to succinylated human gamma-globulin (SHGG) with a strict requirement for carrier-primed theta-positive cells. Secondary challenge with these two TD antigens elicited antidecapeptide antibodies and specific memory regeneration. This TD-responsive memory B cell subset contained the precursors of a second memory B cell subset which was activated by decapeptide-Brucella abortus (TI.1 prototype), as well as by TMVP or decapeptide-SHGG. Stimulation of the second memory B cell subpopulation by decapeptide-BA (previously shown not to be dependent on conventional T cell help) was typified by differentiation into antibody-forming cells without significant memory regeneration.