Effects of cyclosporine on responses of murine B cells to T cell-derived lymphokines

Cyclosporine (CS) inhibits the stimulation of both T and B lymphocytes by certain agents, but not by others. Here we have studied the effects of the drug on the responses of murine B cells to T cell-derived B cell growth and differentiation factors. We show that activation of resting B cells by B cell-stimulatory factor-1 (BSF-1) is resistant to CS, whereas stimulation by anti-Ig antibodies is not, which is in agreement with earlier findings. Furthermore, B cell proliferation elicited by co-stimulation with anti-Ig plus BSF-1 remains drug susceptible. In contrast, the stimulation of large (presumably preactivated) B cells by B cell growth factor II to synthesize DNA or to secrete Ig is inhibited by low concentrations of CS. These results therefore contrast with earlier findings with human B cells, and with those using T cells from various species, which showed that the responses of preactivated cells to growth factors are resistant to the drug. It thus appears that in the mouse CS can affect all stages of B cell stimulation.     

Requirement for BSF-1 in the induction of antigen-specific B cell proliferation by a thymus-dependent antigen and carrier-reactive T cell line

We report here a role of B cell stimulatory factor 1 (BSF-1) in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes by a thymus-dependent antigen and a carrier-reactive T cell line. By using an ovalbumin-reactive T cell line (designated Hen-1), which does not produce BSF-1 following activation, it was possible to demonstrate that the antigen-specific proliferative response of trinitrophenyl (TNP)-binding B cells to TNP-ovalbumin required exogenous BSF-1 in addition to direct interaction with irradiated Hen-1 T cells. The activation obtained under these conditions was highly efficient, being sensitive to antigen doses as low as 0.001 microgram/ml. The addition of saturating amounts of BSF-1 did not alter the antigen-specificity or the requirements for hapten-carrier linkage or major histocompatibility complex-restricted T-B interaction in this system. The involvement of BSF-1 was confirmed by the ability of 11B11 anti-BSF-1 antibody to specifically suppress the response of TNP-binding B cells to TNP-ovalbumin, BSF-1, and irradiated Hen-1 T cells. Finally, this response was augmented by addition of the monokine interleukin 1. These data indicate that the proliferative response of small B cells to the thymus-dependent antigen and carrier-reactive T cell line used in our experiments can be regulated by the same factors that govern B cell proliferation induced by thymus-independent type 2 antigens or anti-IgM antibodies.     

Lyb-5- B cells of CBA/N mice can be induced to synthesize DNA by culture with insolubilized but not soluble anti-Ig

The use of anti-immunoglobulin (anti-Ig) antibodies to stimulate B cell proliferation (1-4), and to stimulate B cell differentiation in the presence of T cell derived-lymphokines (5-8), has simplified investigations into the mechanisms of B cell growth and maturation that are dependent on the cross-linking of surface Ig (sIg). It is only the ontogenetically late appearing Lyb-5+ murine splenic B cells, however, that proliferate in response to anti-Ig antibodies, whereas B cells of the Lyb-5- phenotype obtained from neonatal mice or from mice with the xid immune defect cannot be induced to proliferate in response to this stimulus (1, 9, 10). Thus, the analysis of B lymphocyte physiology of the Lyb-5- B cell population has been hampered by the unavailability of B cell stimulants that mimic an antigen-induced sIg cross-linking event that leads to B cell activation. The inability of soluble anti-Ig antibodies to induce the proliferation of Lyb-5- cells has been particularly difficult to explain because these cells can be induced to increase in size (11) and to show an increase in their expression of surface Ia (sIa) after exposure to anti-Ig (12). Apparently, therefore, these cells are not entirely refractory to this stimulus but are simply unable to progress to the latter stages of cell activation. In view of our observations that the cells of CBA/N mice cannot respond to soluble trinitrophenyl-(TNP) dextran or TNP-polyacrylamide (13) but can respond to insolubilized forms of these antigens, we evaluated their ability to respond to insolubilized anti-Ig. In this paper we report that B cells from CBA/N mice can be stimulated to proliferate in response to anti-Ig conjugated to Sepharose beads, but in contrast to normal B cells they need to be stimulated with beads expressing a high-epitope density of anti-Ig antibodies.     

B-cell activation stimulated by monoclonal anti-Lyb2.1 antibody is mediated through a receptor distinct from the BSF-1 receptor

The experiments in this paper demonstrate that monoclonal anti-Lyb2.1 antibody enhances the proliferative response of anti-immunoglobulin (anti-Ig)-stimulated but not of dextran sulfate-stimulated B cells. The magnitude of this enhanced B-cell proliferation is comparable to that induced by BSF-1 on anti-Ig-stimulated cells. The ability of this antibody to enhance B-cell proliferation does not result from its ability to neutralize the suppressive effects on B-cell activation that is mediated by the Fc fragment of anti-Ig antibody as it is equally as effective in enhancing B-cell proliferative responses stimulated by F(ab')2 fragments of anti-Ig. BSF-1 and Anti-Lyb2.1 appear to stimulate nonoverlapping pathways leading to B-cell activation since the enhanced responses induced by the combination of BSF-1 and anti-Lyb2.1 on anti-Ig-stimulated cells are additive even when maximum quantities of these activators are employed. There is also a marked difference in their activity on T cells; while BSF-1 can enhance T-cell proliferation in synergy with phorbol ester, anti-Lyb2.1 is ineffective in this regard. These data, while consistent with the suggestion that the Lyb2 surface determinant on B cells may be involved in B-cell activation, indicate that it is distinct from the receptors for BSF-1 or BCGF-II.     

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.     

T cell regulation of polyclonal B cell responsiveness. II. Evidence for a deficit in T cell function in mice with an X-linked B lymphocyte defect.

In addition to the x-linked B cell maturation deficit previously reported in CBA/N mice, a functional T cell defect has now been observed. T lymphocyte regulation of the polyclonal PFC response was studied within the context of this x-linked immunodeficiency model. The ability of 1) B cells from (CBA X CBA/CaJ)F1, male mice to respond to nonspecific T cell helper signals and 2) T cells from NCF1 male mice to provide such signals was investigated under in vitro conditions by using bacterial lipopolysaccharide (LPS) as the polyclonal activator. B lymphocytes from both male and female NCF1 mice were receptive to T cell help rendered by NCF1 female T cells. Male T cells. however, were unable to augment polyclonal B cell responses of either NCF1 male or female B cells to LPS. Treatment with ATS + C reduced the polyclonal response of female but not male spleen cells to LPS. This deficit could not be overcome by the use of greater numbers of NCF1 male T cells. The observation that this deficiency in T cell regulation is not due to active suppression suggests that the results may be attributable to an intrinsic T cell defect.     

Role of T cell-replacing factor (TRF) in the murine B cell differentiation: induction of increased levels of expression of secreted type IgM mRNA

T cell-replacing factor (TRF) is known to play a critical role in the regulation of B cell growth and differentiation. In this study, the role of TRF in the expression of mRNA for both IgM and IgG1 class was investigated. The TRF was purified from cellfree supernatants from a T cell hybridoma, B151K12. RNA was isolated from chronic B cell leukemia (BCL1) cells, DNP-KLH-primed B cells, or normal B cells cultured with or without LPS, and LPS plus TRF or LPS plus BSF-1. The steady state level of isotype-specific mRNA was assessed by Northern blot analysis with a mu-specific or a gamma 1-specific probe. It was demonstrated that BCL1 and purified B cells cocultured with TRF expresses increased levels (twofold and fourfold, respectively) of secreted forms of mu mRNA. Purified B cells from DNP-KLH-primed mice also expressed increased levels (twofold to fourfold) of mu as well as gamma 1 mRNA for secreted form by stimulation with TRF. Total expression of mu mRNA, however, was approximately threefold higher than that of gamma 1 mRNA. The stimulation of normal B cells with LPS plus TRF induced an increase in the levels of mu mRNA and gamma 1 mRNA expression, fourfold and threefold, respectively. However, the levels of gamma 1 mRNA expression was one-third of that induced in B cells stimulated with LPS plus BSF-1. These results indicate that TRF preferentially induces increased levels of secreted type of mu mRNA and induces less gamma 1 mRNA than BSF-1. The differential role of TRF from BSF-1 in the expression of Ig mRNA will be discussed.     

B cell stimulatory factor 1 (IL-4) enhances the development of cytotoxic T cells from Lyt-2+ resting murine T lymphocytes

B cell stimulatory factor 1 (BSF-1) (IL-4) was shown to synergize with phorbol esters or with monoclonal anti-TCR antibody in stimulation of the development of CTL from small resting murine T cells. IL-2 also synergized with PMA in such differentiation but was less effective than BSF-1. The combination of these two lymphokines with PMA had the most potent effect on the development of CTL. BSF-1 plus PMA stimulated a significant increase in the intracellular content of N-benzyloxycarbonyl-L-lysine thiobenzylester esterase, a granule-associated biochemical marker, whereas IL-2 plus PMA was only marginally effective. Depletion of L3T4+ cells did not result in the abrogation of these effects. Lyt-2+ T cells that were incubated for 72 h with BSF-1 plus PMA accumulated N-benzyloxycarbonyl-L-lysine thiobenzylester esterase and secreted this intragranular marker after interaction with immobilized anti-T cell receptor mAb. These BSF-1/PMA-stimulated Lyt-2+, L3T4- T cells were also able to kill FcR positive target cells in a retargeting assay with a mAb to murine T3 Ag, providing evidence that BSF-1 plus PMA acted directly on precursors of cytotoxic T cells.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Delayed clearance of filarial infection and enhanced Th1 immunity due to modulation of macrophage APC functions in xid mice.

Bruton's tyrosine kinase (Btk) mutant CBA/N mice show delayed clearance of injected microfilaria (mf) compared with wild-type CBA/J mice. Anti-mf T cells from CBA/N mice make relatively more IFN-gamma than those from CBA/J mice. The anti-mf T cell proliferative responses are also greater in CBA/N mice. This CBA/N immune phenotype is not restricted to filarial Ags, because immunization with pure proteins also yields T cell responses of greater proliferative magnitude skewed away from Th2 cytokines in CBA/N compared with CBA/J mice. The increased magnitude of CBA/N T cell proliferative responses is reflected in increases in both precursor frequencies and clonal burst sizes of responding Ag-specific T cells, and is independent of the source of re-stimulating APCs. Transfer of CBA/J peritoneal resident cells (PRCs) into CBA/N mice before pure protein immunization leads to a wild-type immune phenotype in the recipient CBA/N mice, with a reduction in the proliferative response and a relative decrease in the IFN-gamma produced. When wild-type PRC subpopulations are similarly transferred, the wild-type immune phenotype is transferred by macrophages rather than by B cells. Transfer of wild-type PRCs into CBA/N mice before injection of mf also causes similar changes in the anti-mf T cell responses and enhances the clearance of mf. Thus, Btk is involved in critical macrophage APC functions regulating priming of T cells, and can modulate these responses in pathophysiologically relevant fashion in vivo.     

Detection of receptors for murine B cell stimulatory factor 1 (BSF1): presence of functional receptors on CBA/N splenic B cells

The presence of receptors specific for murine B cell stimulatory factor 1 (BSF1) was demonstrated by utilizing an internally radiolabeled recombinant BSF1. Radiolabeled BSF1 was efficiently produced in Xenopus laevis oocytes injected with a cloned mRNA for BSF1 and 35S-methionine. The labeled BSF1 specifically bound to splenic B cells. A Scatchard analysis indicated the existence of one class of receptor sites. BSF1 receptors were found to be distributed on a wide range of hematopoietic lineage cells, including B cells, T cells, macrophages, and mast cells. B cells from CBA/N mice with the xid gene defect had a similar level of BSF1 binding capacity compared with BALB/c strain B cells, and responded well to insoluble anti-Ig and BSF1 in proliferation assays, indicating that CBA/N B cells express functional BSF1 receptors at normal levels. Pre-B cell lines showed low levels of BSF1 binding, suggesting that cells in the B cell lineage acquire BSF1 responsiveness early in development.     

Induction of antigen-specific proliferation in affinity-purified small B lymphocytes: requirement for BSF-1 by type 2 but not type 1 thymus-independent antigens

We report here the role of B cell stimulatory factors in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes. TI-1 antigens such as TNP-LPS and TNP-BA induced proliferation of hapten-binding B cells in the absence of exogenous B cell stimulatory factors. TI-2 antigens such as TNP-Ficoll required the co-stimulator BSF-1 to induce antigen-specific proliferation, and this response could be augmented by IL 1. TD antigens such as TNP-OVA were unable to induce antigen-specific proliferation either in the absence or presence of B cell stimulatory factors, and showed an absolute activation requirement for carrier-specific helper T cells. No role for IL 2 or BCGF II could be found in the factor-dependent proliferative response of hapten-binding B cells to TI-2 antigens, either as primary co-stimulators or as modulators of the response obtained with TNP-Ficoll, BSF-1, and IL 1. In contrast, concentrations of IFN-gamma that were nontoxic for normal B cells and B cell hybrids effectively abrogated the proliferative response of affinity-purified cells to TNP-Ficoll, BSF-1, and IL 1. By all of these criteria, the B cell activation requirements of TI-2 antigens appear to be identical to those previously published for soluble anti-IgM antibodies.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: I. Study of proliferative response

Culture of normal CBA lymphocytes on monolayers of syngeneic thyroid epithelial cells leads to significant thymidine incorporation. The specificity of this model was demonstrated by depletion of the CBA lymphocytes binding to syngeneic thyroid cells and the increase of thymidine uptake after secondary exposure on syngeneic thyroid monolayers. Removal of B cells (by treatment with anti-Ig serum plus complement) or of adherent cells does not modify the proliferative response whereas T-cell depletion strongly diminishes the response. Thus T cells are stimulated to undergo DNA synthesis and are sensitized when exposed to syngeneic thyroid epitelial cells. The nature of the antigens recognized by T cells (native autoantigen, enzyme, or virus-modified autoantigen) is not yet determined. Whether such autoreactive T cells play a role in the onset of experimental autoimmune thyroiditis as regulatory T-cells or cytotoxic effector cells is discussed.     

Activity of a partially purified human BCGF on murine assays for B-cell stimulatory factors. I. BCGF II-like activity of human BCGF

To further characterize a human B-cell growth factor (BCGF) produced by phytohemagglutinin (PHA) P-stimulated peripheral blood T cells, a partially purified preparation of this material was tested in a number of murine assays for B-cell stimulatory factors (BSF). Human BCGF lacked murine BSF-1 activity as assessed via the induction of polyclonal proliferation of anti-IgM-stimulated murine B cells; however, this material consistently augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1. Human BCGF also induced proliferation in unstimulated murine B cells, and augmented the proliferative response of dextran sulfate activated murine B cells. Human BCGF is therefore capable of causing proliferation of unstimulated and activated murine B cells, and by these criteria closely resembles murine BCGF II. In contrast to murine BCGF II, however, human BCGF failed to stimulate proliferation or immunoglobulin (Ig) secretion by murine BCL1 B lymphoma cells. A murine analog of this human BCGF showing the same pattern of biological responses was found in concanavalin A-stimulated supernatants of the murine MB2.1 T-cell line and D9-Cl T-cell hybridoma. The active component of the human BCGF preparation was not due to contaminating PHA, interleukin 1, interleukin 2; interferon-gamma, or endotoxin. Comparison between the above human BCGF and a commonly used source of murine BCGF II, i.e., supernatant from antigen-stimulated D10.G4.1 T cells, provided information suggestive of BCGF II heterogeneity. Both human BCGF and D10.G4.1 supernatant caused proliferation of unstimulated and dextran sulfate-stimulated murine B cells; however, only the human BCGF preparation augmented the proliferative response of murine B cells to anti-IgM and a saturating dose of murine BSF-1, and only the D10.G4.1 supernatant stimulated BCL1 cell proliferation and immunoglobulin secretion. The data therefore indicate that the different assays for BCGF II used in this study respond to different factors, and suggest the existence of two BCGF II-like activities.     

Enhancement by monoclonal anti-Lyb-2 antibody of antigen-specific B lymphocyte expansion stimulated by TNP-Ficoll and T lymphocyte-derived factors

By using antigen-specific populations of B cells (TNP-ABC) we have demonstrated that the type-2 antigen TNP-Ficoll was capable of initiating B cell proliferation only in the presence of T cell-derived factors. Monoclonal-anti-Lyb-2.1 antibody acted synergistically with a T cell-derived supernatant, as well as with B cell-stimulating factor (BSF-1) to enhance the level of B cell expansion obtained in this in vitro system. This effect of anti-Lyb-2.1 mAB was observed at each day of the antigen-driven B cell expansion and was seen only with B cells purified from strains expressing the Lyb-2.1 allele. The epitope density of hapten on the Ficoll plays a critical role in this process, because Ficoll that is haptenated with low density of hapten was not found to be stimulatory. These results suggest that the Lyb-2 surface molecule influences the antigen-driven B cell growth that is stimulated by type 2 antigens and BSF-1.     

Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1alpha (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.     

Regulation of murine T cell proliferation by B cell stimulatory factor-1

The proliferation of mitogen-activated primary T cells, antigen-activated memory T cells from mixed leukocyte culture, and antigen-dependent alloreactive T cell clones in response to purified murine recombinant B cell stimulatory factor-1 (also known as interleukin 4) was examined. We found that B cell stimulatory factor-1 (BSF-1) stimulated optimal proliferation of these T cells only after their recent activation by antigen or mitogen. Analysis of cell surface BSF-1 receptor expression indicated that although T cell activation is accompanied by a small increase in BSF-1 receptor expression, the cells also express BSF-1 receptors prior to activation at a time when they do not proliferate in response to BSF-1. BSF-1 was as effective a stimulus as interleukin 2 for inducing proliferation of the Lyt-2+ subpopulation of concanavalin A-activated murine spleen cells and an alloreactive cytolytic T cell clone. However, the L3T4+ subpopulation of concanavalin A-activated spleen and an alloreactive helper T cell clone were less responsive to BSF-1 than to interleukin 2. Taken together, the data indicate an important role for BSF-1 in the regulation of normal T cell proliferation.     

A reappraisal of "T-independent" antigens. II. Studies on single, hapten-specific B cells from neonatal CBA/H or CBA/N mice fail to support classification into TI-1 and TI-2 categories

Spleen cells from adult CBA/H or CBA/N mice, or from neonatal CBA/H mice, were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B lymphocytes. A single cell, or small numbers ranging from 1 to 10, were cultured in 10-microliter microcultures together with various antigens and mitogens. The results were compared with those of bulk culture or limiting dilution cultures supported by thymus filler cells. B cell growth and differentiation-promoting conditioned media (BGDA) were added to some cultures. The CBA/N results gave no support to the commonly used classification of T cell-independent (TI) antigens into TI-1 and TI-2 categories. A typical supposed TI-1 antigen, FLU-LPS, strongly stimulated normal adult single FLU-specific B cells to proliferate and form antibody, but virtually failed to trigger CBA/N B cells of comparable antigen-binding avidity. The same was true of LPS or LPS plus dextran sulfate acting as mitogens. The allegedly TI-2 antigen FLU-Ficoll, although still triggering comparatively poor responses, was actually marginally more active than FLU-LPS. FLU-Brucella abortus (FLU-BA) + BGDA gave the best results with single CBA/N B cells, but still induced only 1.27% of cells to develop into antibody-forming clones vs 12.2% with CBA/H cells. The results obtained with single neonatal B cells also lent no support to the distinction between TI-1 and TI-2. Both "TI-1" and "TI-2" stimuli caused adequate proliferation, one "TI-2" antigen stimulating 23.2% of the cells. None of the antigens caused good antibody formation, however, probably because multivalent antigens can deliver signals impeding the differentiation of immature B cells. It is therefore suggested that the classification of TI-1 antigens into two subcategories be abandoned, at least for the time being.