B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

Maternal IgG stimulates B lineage cell development in the progeny

To examine the physiological role of maternal natural IgG antibodies on the development of B lineage cells of the progeny, we have bred homozygous μMT/μMT or heterozygous μMT/+ females to μMT/μMT or μMT/+ males, respectively. We could thus compare normal or B cell-deficient mice born from Ig-deprived (Ig^?) or phenotypically normal mothers (Ig⁺). B cell-deficient progeny of heterozygous mothers contain no detectable serum IgA or IgM, but IgG concentrations that peak at 2 mg/ml by 7–21 days of age, decay after weaning with a half-life of 7 days, and remain detectable for 2 months after birth. At 7 days after birth, μMT/+ progeny born of Ig⁺ mothers contain two- to threefold higher numbers of bone marrow (BM) pre-B and B cells, and of splenic B cells, compared to mice of the same age born from Ig^? mothers. In contrast, the former progeny exhibit two to four times lower numbers of Ig-secreting plasma cells in spleen and thymus, and contain sixfold lower serum IgM concentrations. A similar maternal IgG-dependent stimulation of BM B cell precursors is also observed in μMT/μMT progeny. No significant differences were detected between the groups on day 3 after birth, suggesting the requirement for a minimal IgG concentration in the serum.     

Mice with an inactivated joining chain locus have perturbed IgM secretion

A mouse with an inactivated joining chain locus was produced by gene targeting in embryonic stem cells by deleting the first exon. Heterozygote (J^{+ / −} ) and homozygote (J^{− / −} ) offspring from these mice showed normal total serum immunoglobulin levels and a normal peripheral B cell compartment when compared to wild-type littermates. The distribution of serum immunoglobulin isotypes in serum was different; IgA levels were elevated while IgM levels were reduced in J^{− / −} mice as compared to wild-type mice. High molecular weight serum IgM was reduced in J^{+ / −} and J^{− / −} mice and instead found in oligomeric form of undefined structure. Furthermore, serum IgM from J^{+ / −} and J^{− / −} mice showed a reduced ability to activate complement. The number of splenic and bone marrow IgM plaque-forming cells were reduced in unimmunized J^{+ / −} as well as in J^{− / −} mice. Furthermore, the number of plaque-forming cells was reduced in B cells from both J^{+ / −} and J^{− / −} mice after stimulation with lipopolysaccharide in vitro. The perturbation of IgM production in J^{− / −} mice appears to affect a late stage of differentiation, because cells with intracellular IgM were readily detected both in vivo and in vitro. Finally, after immunization with T-dependent or T-independent antigens the IgM component of the immune response was reduced in J^{− / −} mice while only a marginal reduction of the IgG response was detected.     

In vivo blockage of B cell maturation by splenic T cells of total lymphoid irradiated mice

The effect of fractionated total lymphoid irradiation (TLI) on the induction of cells blocking in vivo B cell maturation was examined. (BALB/c x C57BL/6)F1 mice received 200 rds lymphoid irradiation daily for 8 days. One month after termination of the irradiation splenic T cells of TLI-treated mice were transferred, together with normal bone marrow cells, to lethally irradiated hosts. In addition, splenic cells or splenic B cells of TLI mice were transferred to irradiated hosts. Two months later, we checked the reconstituted mice for their ability to produce in vivo anti-trinitrophenyl (TNP)-Ficoll antibodies and their capacity to respond in vitro to dextran sulfate and lipopolysaccharide. In parallel, stained spleen cells were analyzed on the fluorescence-activated cells sorter using monoclonal anti-mu, anti-delta and anti-B220 surface marker antibodies. The results indicate that splenic T cells originating from TLI-treated mice cause maturation arrest of normal and TLI-treated B cells, since normal bone marrow cells injected together with those cells have immature phenotypic and functional features. In addition, neither splenic cells nor splenic B cells of TLI-treated mice can mature upon transfer to normal irradiated hosts. The B cells are IgM+, IgD-; they respond with a high proliferation rate to dextran sulfate and low proliferation rate to lipopolysaccharide, and poorly to TNP-Ficoll challenge.     

Immature B cells from human and mouse bone marrow can change their surface light chain expression

The capacity of bone marrow-derived surface immunoglobulin-positive (sIg⁺) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg⁺ B cells were generated in vitro from sIg^? precursor cells from human or mouse bone marrow. The immature sIg⁺ cells expressed RAG-1. Human sIg⁺ cells expressed xfr; and λ L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse xfr;⁺sIg⁺ cells, about half of them remained xfr;⁺, a quarter became λ⁺, and another quarter became sIg^?. Between 1 and 3% expressed both xfr; and λ chains. Of the human λ⁺ cells, about two-thirds remained λ⁺, only 1 to 2% became xfr;⁺, while the other third became sIg^?. Again, between 1 and 3% expressed both xfr; and λ L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in xfr; versus λ gene rearrangements. Hence, human and mouse xfr;⁺ immature B cells can become λ⁺, but very few of the λ⁺ cells can become xfr;⁺, and both can become sIg^?. Further, human CD10⁺/sIg⁺ xfr;⁺ and λ⁺ cells and mouse B220^low/sIg^low xfr;⁺ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg⁺ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.     

Flexibility of the Thyroiditogenic T Cell Repertoire for Murine Autoimmune Thyroiditis in CD8-Deficient (B2m -/-) and T Cell Receptor VBC Congenic Mice

In murine experimental autoimmune thyroiditis (EAT), previous studies have revealed a highly adaptable thyroiditogenic T cell repertoire which involves both CD4⁺ and CD8⁺ T cells in the susceptible H2k strain. To further test this flexibility, congenic B10.K mice lacking CD8⁺ T cells (B2m -/-) or harboring 70% T cell receptor (TCR) V6 gene deletions (V6^C) were immunized with mouse thyroglobulin (MTg) and evaluated for EAT 28 days later. All 62m -/- mice developed moderate antibodies to MTg, and thyroidal inflammation was comparable to B10.K mice, averaging 35-40%. Spleen cells (SC) from MTg-immunized mice were then injected into syngeneic recipients after stimulation in vitro with MTg or with conserved, thyroxine (T4)- or thyronine (TO)- containing 12mer peptides, hT4(5), hT0(2553), or hT4(2553), derived from the primary hormonogenic sites at position 5 or 2553 of human Tg. As previously shown in another H2^k strain (CBA/J), all three peptides activated MTg-primed SC to transfer EAT in B10.K mice. hT4(5) and hT4(2553) were further tested in B10.K-V6^C and ß2m^- B10.K mice. Both peptides expanded thyroiditogenic T cells in either strain, resulting in severe thyroiditis in syngeneic recipients. That EAT can develop in the absence of CD8⁺ T cells or in the presence of a severely restricted TCR repertoire underscores the remarkable flexibility of the thyroiditogenic T cell profile in the susceptible k haplotype.     

Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies

Acute infection with Trypanosoma cruzi, the aetiological agent of Chagas’ disease, results in parasitaemia and polyclonal lymphocyte activation. It has been reported that polyclonal B‐cell activation is associated with hypergammaglobulinaemia and delayed parasite‐specific antibody response. In the present study we analysed the development of a B‐cell response within the different microenvironments of the spleen during acute T. cruzi infection. We observed massive germinal centre (GC) and extrafollicular (EF) responses at the peak of infection. However, the EF foci were evident since day 3 post‐infection (p.i.), and, early in the infection, they mainly provided IgM. The EF foci response reached its peak at 11 days p.i. and extended from the red pulp into the periarteriolar lymphatic sheath. The GCs were detected from day 8 p.i. At the peak of parasitaemia, CD138⁺ B220⁺ plasma cells in EF foci, red pulp and T‐cell zone expressed IgM and all the IgG isotypes. Instead of the substantial B‐cell response, most of the antibodies produced by splenic cells did not target the parasite, and parasite‐specific IgG isotypes could be detected in sera only after 18 days p.i. We also observed that the bone marrow of infected mice presented a strong reduction in CD138⁺ B220⁺ cells compared with that of normal mice. Hence, in acute infection with T. cruzi, the spleen appears to be the most important lymphoid organ that lodges plasma cells and the main producer of antibodies. The development of a B‐cell response during T. cruzi infection shows features that are particular to T. cruzi and other protozoan infection but different to other infections or immunization with model antigens.     

Characterization of immature B cells by a novel monoclonal antibody,by turnover and by mitogen reactivity

The transit of immature to mature sIgM⁺ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493⁺ from mature, 493⁻ B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493⁺ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10 – 20 % of the cells reach the spleen as immature 493⁺ cells. The life span of 493⁺ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493⁻ B cells where they gain a longer life span of 15 – 20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti-CD40, however, stimulates mature B cells better than immature B cells. IgM cross-linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by co-stimulation with anti-CD40 or by overexpression of bcl-2. We speculate that Ig receptor ligand-mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.     

An interleukin-6 transgene expressed in B lymphocyte lineage cells overcomes the T cell-dependent establishment of normal levels of switched immunoglobulin isotypes

Long-term proliferating, stromal cell/interleukin (IL)-7-reactive precursor B cell lines established from fetal liver and bone marrow of human IL-6-transgenic B6L^d46 mice produce and secrete human IL-6. When transplanted into severecombined immunodeficient (SCID) or Rag2 knockout (Rag2-T) mice, these pre-B cell lines establish a part of the B cell compartment but yield no T cells, as do pre-B cell lines from genetically matched non-transgenic mice. Within 2 to 3 months after transplantation, the serum of mice transplanted with pre-B cells from normal mice contains normal levels of IgM (200–600 μg/ml) but 10–100-fold lower levels of the IgG subclasses and of IgA. In contrast, the sera of mice transplanted with IL-6 transgenic pre-B cells contain not only IgM, but also IgG and IgA at nearly normal levels. The results indicate that at least a part of the plasmacytosis and elevated IgG production observed previously in the IL-6-transgenic mice appears to be due to a T cell-independent activation of IgG and IgA production by the IL-6-secreting pre-B cells and their differentiated progeny in the immunodeficient hosts.     

CD3 components at the surface of pro-T cells can mediate pre-T cell development in vivo

Developmentally arrested pro-T cells (CD4^?8^?, IL-2R⁺, HSA⁺⁺) of RAG-1-deficient mice appear to express low levels of CD3 molecules in the absence of T cell receptor (TcR) chains at their surface, while developmentally arrested pre-T cells of TcRα-deficient mice express low levels of a disulfide-linked TcRβ chain in association with CD3 molecules. Cross-linking of the CD3 modules on pro-T cells of RAG-1^?/- mice in vivo, with either of two different CD3′-specific monoclonal antibodies, induces differentiation of these pro-T cells into pre-T cells (CD4⁺8⁺, IL-2R^?, HSA⁺), concomitant with a rapid expansion of the thymic T cell compartment, up to 175-fold within 12 days. The same effects can be produced by introduction of a mutant TcRβ transgene lacking most of the variable domain (ΔV-TcRβ) into the RAG-1^?/- background. These experiments suggest that cross-linking of the CD3 modules on pro-T cells mimics the signaling function expected of the pre-TcR complex, which is found at the surface of pre-T cells prior to functional TcRa gene rearrangement. The variable domain of the TcRβ chain is apparently not essential for inducing these aspects of T cell development.     

Mature helper T cell requirement for immunoglobulin production by neonatal naive B cells injected intraperitoneally into severe combined immunodeficient (SCID) mice

It is accepted that human neonatal naive B cells produce mainly IgM in vivo as well as in vitro. Our previous work has demonstrated that i.p. injection of neonatal B cells together with adult mature T cells induces substantial levels of human IgG in the serum of SCID recipient mice. The present study was further attempted to determine the cellular components required for immunoglobulin production by neonatal B cells in SCID mice. When neonatal B and adult T cells were transferred into the SCID mice, human immunoglobulins, largely of IgG, were maximally detected in the serum around 6 weeks after a cell transfer. Depletion of CD4⁺ T cells from adult T cells resulted in undetectable levels of human immunoglobulin in the serum. By contrast, CD4⁺ T cell-enriched populations exhibited an enhancing effect on immunoglobulin production by neonatal B cells. Higher levels of immunoglobulin, including IgA and IgM, were detected in the peritoneal fluid than in the serum as early as 2 weeks after the cell transfer. Human T cells expressing activation antigens such as CD45RO and HLA-DR antigens were identified in the peritoneal lavages. These results suggest that neonatal naive B cells are able to differentiate into cells producing all classes of immunoglobulin in the presence of mature CD4⁺ T cells in a SCID mouse environment. The peritoneal cavity of SCID mice appears to provide a suitable place for immune responses by human cells, possibly in association with a certain xenogeneic reaction.     

B cell development in mice with a defective λ5 gene

The surrogate light chain encoded by the two pre-B cell-specific genes V_preB and λ₅ plays a critical role in B cell development of the mouse. It has been shown that targeted disruption of the λ ₅gene results in a depletion of B220⁺ CD43^? IgM^? pre-B cells in bone marrow, and in a delayed appearance both of CD5⁺ as well as CD5^? surface immunoglobulin (slg)⁺ B cells in the periphery. In this report we show that D_HJ_H-rearranged B220^? and B220⁺, CD43⁺, c-kit⁺, sIgM^? pro- and pre-B-I cells with long-term capacity to proliferate in vitro on stromal cells in the presence of interleukin-7 are present in normal numbers in the bone marrow of λ ₅T/λ.₅T mice at various ages. They express normal levels of V_preB mRNA but, in contrast to normal pre-B-I cells, do not express surrogate light chain on their surface. Pre-B-I cells from fetal liver and bone marrow of λ ₅T/λ ₅T mice differentiate with normal kinetics and in normal numbers to sIg⁺, mitogen-reactive B cells. These results suggest that the delayed generation of sIg⁺ B cells in the peripheral, mature compartments of CD5⁺ and CD5^? cells could be accounted for by the daily production of approximately 5 × 10⁵ sIg⁺ B cells from the pre-B-I cell pool in the absence of a normal pool of pre-B-II cells.     

Marked depletion at the late pro-B cell stage in the bone marrow of lpr mice correlates with the development of lymphadenopathy but not autoimmunity

We have found that old lpr mice exhibit a loss of mature B cells in the bone marrow. The deleted population is HSA^lo B220^hi and is generated from the peripheral pool. Abnormalities in the microenvironment could explain the absence of mature B cells. Thus, old lpr bones were grafted under the skin of normal adult Ly5.1 hosts and examined 3 weeks later for the presence of Ly5.1⁺ B220^hi cells. Our data show that the lpr medullary compartment was efficiently restored by host B cells. These results suggest that the bone marrow microenvironment of old lpr mice is able to sustain mature B cells. However, transfer of T cell-depleted bone marrow cells from old lpr mice to Rag-2^−/− mice leads to incomplete and inefficient repopulation of the host medullary compartment. Thus, a defect at an early stage of B cell differentiation was detected: using four-color flow cytometry, we found a profound depletion of the late pro-B B220⁺ CD43⁺ HSA⁺ BP-1⁺ cell population in aging lpr mice. This depletion was not observed in old autoimmune-prone MRL-+/+ mice which develop only autoantibodies but was present in B6-lpr mice which develop a lymphadenopathy and an indolent autoimmune syndrome. Altogether, our results demonstrate an age-linked defect in the progression of B cell differentiation in lpr mice independent of the presence of autoantibodies and targeted to the late pro-B cell subset.     

Transitional B cell subsets in human bone marrow

B cells originate from precursors in the bone marrow, and the first cells which migrate to the peripheral blood have been classified as ‘transitional B cells’. Transitional B cells have been characterized in human blood with stage 1 (T1) and stage 2 (T2) subsets being proposed. In the present study, 27 normal human bone marrow samples were analysed for transitional B cell markers by eight‐colour flow cytometry. T1 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD^lo) and T2 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD⁺) were identified in normal bone marrow samples at a mean frequency of 3·2 and 3·1% of total B lineage cells, respectively. A majority of the bone marrow transitional B cells were CD24^hiCD38^hi, the phenotype of blood transitional B cells. Consistent with recent peripheral blood data, T2 B cells had a significantly higher CD21 expression compared with T1 B cells (72·4 versus 40·9%) in the bone marrow. These data raise the possibility that transitional B cells are capable of differentiating from T1 to T2 B cells within the bone marrow. Furthermore, transitional cells at either stages 1 or 2 might be capable of migrating out of the bone marrow.     

The Yaa gene-mediated acceleration of murine lupus: Yaa− T cells from non-autoimmune mice collaborate with Yaa+ B cells to produce lupus autoantibodies in vivo

The BXSB Y chromosome-linked mutant gene, Yaa, promotes autoimmune responses in mice predisposed to a lupus-like autoimmune disease. We have previously shown that a cognate interaction of T cells with B cells expressing the Yaa gene appears to be responsible for the accelerated production of autoantibodies. To investigate whether T cells that provide help for autoantibody production by Yaa⁺ B cells need to express the Yaa gene, we have made radiation bone marrow chimeras containing two sets of T and B cells from mice with or without the Yaa gene and differing by the Thy-1 and Igh allotypes. We then determined autoantibody production following the selective elimination of T cells of Yaa⁺ origin by treating mice with allele-specific anti-Thy-1 monoclonal antibody. Our results demonstrated that the selective production of autoantibodies by Yaa⁺ B cells in Yaa⁺-Yaa^? double bone marrow chimeras can be mediated as efficiently by T cells from non-autoimmune mice lacking the Yaa gene as by T cells from autoimmune mice bearing the Yaa gene. This indicates that T cells from non-autoimmune Yaa^? mice are capable of providing help for autoimmune responses by collaborating with Yaa⁺ B cells. These data thus strongly suggest that the Yaa gene defect is not functionally expressed in T cells, but only in B cells, and contrast with parallel experiments in the lpr model, in which defects of the Fas antigen in both T and B cells are crucial for the lpr gene-mediated promotion of autoantibody production.     

Photodynamic Virus Inactivation of Blood Components

Staphylococcal enterotoxin B (SEB) can activate specific T cell clones bearing specific TcR Vβ domains together with MHC class II ligands on accessory cells. The release of proinflammatory cytokines is the consequence of this activation as well as the main pathological aspect involved in SEB infection. This current study looked at the active role of both T and B cells during the induction of anergy by SEB in vivo Euthymic and nude BALB/c mice were injected with SEB and over a period of 8 days, cells from the spleen and sera from the blood were collected. After a single injection with SEB (50 μg/mouse), a transient increase of CD4⁺Vβ8⁺ T cells were detected after 2 days followed by a decrease after 4 days, which persisted until day 8. These clones were rendered anergic upon restimulation in vitro with SEB. Interestingly, cells taken out 2 days after SEB injection, exhibited reduced proliferation in response to Con A. However, this response gradually recovered on days 4, 6 and 8. Furthermore, early IgM antibody production (day 2) was observed after SEB injection. SEB-induced IgM antibody production in euthymic BALB/c was found to have specificity against SEB, cardiolipin (CL) and phosphatidylethanolamine (PE). SEB-treated nude mice did not produce antibody secreting cells in response to SEB, indicating that this process is T cell dependent.     

A re-evaluation of the effects of X-linked immunodeficiency (xid) mutation on B cell differentiation and function in the mouse

CBA/N (xid) mice have a point mutation in Bruton's tyrosine kinase (btk), which results in their failure to respond to T-independent type 2 (TI-2) antigens, and to several B cell mitogens [most notably anti-immunoglobulin (Ig)] in vitro. They have reduced numbers of peripheral (B2) B cells, which are regarded as being phenotypically and functionally immature. We show here that adult CBA/N mice in fact have two distinct B cell populations: some 60% of the cells are CD23⁺ HSA^lo sIgD^hi and hence resemble recirculating, follicular (RF) B cells from normal mice, except that they are sIgM^hi. The remaining 40% of xid B cells are CD23^? HSA^hi sIgD^?/lo and resemble immature transitional (TR) B cells. TR B cells from xid mice do not synthesize DNA when cultured with lipopolysaccharide (LPS), whereas those from normal mice do so. Only the RF cells from either xid or normal mice proliferate in response to ligation of CD40. In neonatal normal mice the emergence of mitogen responsiveness followed the chronological sequence LPS → anti-CD40 → anti-Ig ≈ anti-CD38. The same developmental sequence was seen with B cells from xid mice (for LPS and anti-CD40), but it occurred at a significantly slower tempo and this correlated with the later appearance of RF-type cells. TR xid B cells express very low levels of bcl-2 and we conclude that these cells resemble very immature (bone marrow) B cells, rather than normal transitional cells. We, therefore, propose that the xid mutation imposes a multistage brake on B cell differentiation in the mouse. The available data suggest that btk is required for the positive selection of B cells throughout their differentiation in the periphery. This in turn implies that low level signaling via surface Ig is needed throughout this process in order for peripheral B cells to become functionally mature.     

Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: Dose-response relationship

Ligand-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. T cell receptor (TcR) transgenic mice offer a clear advantage for studying post-thymic tolerance mechanisms in vivo that are operational in a monoclonal T cell population with preselected antigen specificity. Yet it is unclear whether the rules defined in monoclonal T cells of genetically manipulated mice reflect those operative in clonally diverse peripheral T cells of normal mice. To analyze acute tolerance mechanisms in unselected peripheral T cells, we challenged normal mice with the superantigen staphylococcal enterotoxin B (SEB) and analyzed ligand-reactive Vβ8⁺ T cells for TcR-triggered tolerance mechanisms such as anergy, TcR down-regulation, or apoptosis. Upon challenge with graded doses of SEB (0.001–10 μg) Vβ8^? T cells become anergic within 6–16 h. Importantly, a dosage effect of SEB in regard to the level of anergy induced was observed. Anergy induced by low concentrations of SEB (0.001–0.1 μg) is transient and is overcome by clonal growth, while higher concentrations of SEB (0.1–10 μg) cause long-lasting anergy resistant to cell cycle progression. At high SEB concentrations (1–10 mg) about 50% of the anergic Vβ8⁺ T cells additionally down-regulate their TcR-CD3 complex, followed by a loss of CD2, CD4, CD8 accessory molecules. In parallel, T cell phenotypenegative but genotypically Vβ8⁺ T cells are generated. The T cell phenotypenegative cells reacquire their Vβ8⁺ T cell phenotype upon culture in vitro. In vivo, a subset of Vβ8⁺ cells, defined by an intermediate stage of TcR down-regulation, i.e. Vβ8^lowCD3⁺ cells, but not T cell phenotype-negative cells are selectively programmed for apoptosis, which occurs within 1 h. These data suggest that SEB triggers distinct tolerance pathways which operate in a hierarchical fashion in clonally diverse ligand-reactive T cells. Specifically, the results illustrate the power of exogenous superantigens to exploit these distinct tolerance pathways, thereby achieving distinct levels of immunosuppression.