Viral superantigen drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection: this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Immunoglobulin switch transcript production in vivo related to the site and time of antigen-specific B cell activation.

Immunoglobulin (Ig) class switch recombination is associated with the production and splicing of germline IgCH messenger RNA transcripts. Levels of gamma 1 transcripts in mouse spleen sections were assessed by semiquantitative analysis of reverse transcriptase polymerase chain reaction (PCR) products during primary and secondary antibody responses to chicken gamma globulin (CGG). This was correlated with the appearance of CGG-specific B cells and their growth and differentiation to plasma cells. After primary immunization with CGG, gamma 1 switch transcripts appeared after 4 d, peaked at a median of six times starting levels between 10 and 18 d after immunization, and returned to background levels before secondary immunization at 5 wk. By contrast, after secondary challenge with CGG, a sevenfold increase in transcripts occurs during the first d. The level again doubles by day 3, when it is six times that which is seen at the peak of the primary response. After day 4, there was a gradual decline over the next 2-3 wk. Within 12 h of secondary immunization, antigen-specific memory B cells appeared in the outer I zone and by 24 h entered S phase, presumably as a result of cognate interaction with primed T cells. Over the next few hours, they migrated to the edge of the red pulp, where they grew exponentially until the fourth day, when they synchronously differentiated to become plasma cells. The same pattern was seen for the migration, growth, and differentiation of virgin hapten-specific B cells when CGG-primed mice were challenged with hapten protein. The continued production of transcripts after day 3 indicates that switching also occurs in germinal centers, but in a relatively small proportion of their B cells. The impressive early production of switch transcripts during T cell-dependent antibody responses occurs in cells that are about to undergo massive clonal expansion. It is argued that Ig class switching at this time, which is associated with cognate T cell-B cell interaction in the T zone, has a major impact on the class and subclasses of Ig produced during the response.

Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool.

The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool.

Compromised OX40 function in CD28-deficient mice is linked with failure to develop CXC chemokine receptor 5-positive CD4 cells and germinal centers.

Mice rendered deficient in CD28 signaling by the soluble competitor, cytotoxic T lymphocyte-associated molecule 4-immunoglobulin G1 fusion protein (CTLA4-Ig), fail to upregulate OX40 expression in vivo or form germinal centers after immunization. This is associated with impaired interleukin 4 production and a lack of CXC chemokine receptor (CXCR)5 on CD4 T cells, a chemokine receptor linked with migration into B follicles. Germinal center formation is restored in CTLA4-Ig transgenic mice by coinjection of an agonistic monoclonal antibody to CD28, but this is substantially inhibited if OX40 interactions are interrupted by simultaneous injection of an OX40-Ig fusion protein. These data suggest that CD28-dependent OX40 ligation of CD4 T cells at the time of priming is linked with upregulation of CXCR5 expression, and migration of T cells into B cell areas to support germinal center formation.

Gamma delta T cell help of B cells is induced by repeated parasitic infection, in the absence of other T cells.

BACKGROUND: gamma delta T cells, like alpha beta T cells, are components of all well-studied vertebrate immune systems. Yet, the contribution of gamma delta T cells to immune responses is poorly characterized. In particular, it has not been resolved whether gamma delta cells, independent of any other T cells, can help B cells produce immunoglobulin and form germinal centers, anatomical foci of specialized T cell-B cell collaboration. RESULTS: TCR beta-/- mice, which lack all T cells except gamma delta T cells, routinely displayed higher levels of antibody than fully T cell-deficient mice. Repeated parasitic infection of TCR beta-/- mice, but not of T cell-deficient mice, increased antibody levels and induced germinal centers that contained B cells and monoclonal gamma delta cells in close juxtaposition. However, antibody specificities were more commonly against self than against the challenging pathogen. gamma delta T cell-B cell help was not induced by repeated inoculation of TCR beta-/- mice with mycobacterial antigens. CONCLUSIONS: In the absence of any other T cells, gamma delta T cell-B cell collaboration can be significantly enhanced by repeated infection. However, the lack of obvious enrichment for antibodies against the challenging pathogen distinguishes gamma delta T cell help from alpha beta T cell help induced under analogous circumstances. The increased production of generalized antibodies may be particularly relevant to the development of autoimmunity, which commonly occurs in patients suffering from alpha beta T cell deficiencies, such as AIDS.

Sequential antigen-specific growth of T cells in the T zones and follicles in response to pigeon cytochrome c.

The sites of accumulation and growth of antigen-specific T cells was assessed during the V alpha 11/V beta 3 T cell receptor-restricted response of IEk+ mice to pigeon cytochrome c. In the T zone, the response was early but transient; V alpha 11/V beta 3+ T cell numbers fell to background levels as germinal centers developed. The follicles were a major site of specific T cell growth, but V alpha 11/V beta 3+ T cells were not obvious in foci of extrafollicular B cell growth in the red pulp. As germinal centers waned, V alpha 11/V beta 3+ T cells in the T zones again rose above background levels and some persisted in the follicles. After the initial stage of T cell priming, specific T cell growth seems to occur where specific interaction can occur with B cells that are presenting processed antigen.

Memory B-cell clones and the diversity of their members.

Memory B-cell clones develop from virgin B cells that take up processed antigen, make cognate interaction with primed T cells and then grow in germinal centres. Within the germinal centre the proliferating B cells undergo Ig variable-region mutation and are subsequently selected on their ability to bind antigen held on follicular dendritic cells and then to make cognate interaction with germinal centre T cells. The selected cells emerge as memory B cells or plasmablasts. Although many of the memory B cells and most of the plasma cells emerging from follicles have undergone Ig class switch recombination a substantial minority of the memory B cells have not switched. These non-switched memory cells can be induced to switch on re-exposure to antigen. Affinity maturation following a single immunization ceases as germinal centres wane some 3-4 weeks after immunization - memory cells and antibody production, on the other hand, persist for months and even years.

Co-stimulation and selection for T-cell help for germinal centres: the role of CD28 and OX40.

Given the importance of responding to infections with the right defensive strategy, much interest has focused on cytokine differentiation in CD4+ T cells. However, relatively little is known of the logistics of T-cell help for B cells. Here, Lucy Walker and colleagues propose key roles for CD28 and OX40 in coordinating the selection, expansion and migration of CD4+ T cells to B-cell follicles.

CD4 T cell traffic control: in vivo evidence that ligation of OX40 on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the accumulation of CD4 T cells in B follicles.

We report here that CD40- but not lipopolysaccharide (LPS)-activated murine dendritic cells (DC) express OX40-ligand (OX40L) as has been reported in humans. To understand how OX40 ligation affects differentiation of CD4 T cells at the time of priming, we constitutively expressed OX40L on DC using the DC-specific promoter of CD11c. Transgenic mice showed greatly increased numbers of CD4 but not CD8 T cells in their B cell areas. This effect was to a great extent immunization dependent, as spleen and lymphoid tissue with no germinal center reactions from mice which had not been deliberately immunized did not show marked CD4 T cell accumulation. The increased numbers of CD4+ CD62low cells in transgenic mice suggest that it is activated CD4 T cells that accumulate within B cell follicles. These data are consistent with the notion that physiological engagement of OX40 (CD134) on activated CD4 T cells either initiates their migration into or causes them to be retained in B follicles. In contrast, LPS-treated CD did not up-regulate OX40L expression. This dichotomy provides a molecular explanation of how DC might integrate environmental and accessory signals to control cytokine differentiation and migration in CD4 effector cells.

Germinal center formation in mice lacking alpha beta T cells.

T cells are essential for inducing clonal B cell expansion in germinal centers during T cell-dependent antibody responses. However, class-switched antibodies are readily detectable in TCR alpha-deficient mice that congenitally lack alpha beta T cells, including those such as IgG1 that are considered to be dependent on collaboration between B cells and alpha beta T cells. This observation suggests that a novel form of B:T collaboration may be evident in TCR alpha-/- mice. We report that germinal centers develop spontaneously in mice lacking T cell receptor alpha genes (TCR alpha-/-), despite the absence of alpha beta T cells. They are not seen in TCR beta-/- mice kept in similar conditions. Both strains of mice have gamma delta T cells, but it is a subset of T cells expressing TCR beta and CD4 that is dominant in the germinal centers of TCR alpha-/- mice. Exceptionally, germinal centers were associated with CD4+ gamma delta T cells. The expression of CD4 seems to be important, for few extrafollicular T cells have CD4 and CD4 is largely absent from TCR beta-/- T cells. The CD4+ TCR beta cells may help B cells produce autoantibodies that have been identified in TCR alpha-/- mice.

Defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

Vav, a guanine nucleotide exchange factor for members of the Rho family of small GTPases, is activated through engagement of B and T lymphocyte antigen receptors. It is important for establishing the signaling threshold of the TCR, as mice lacking Vav display defective thymocyte selection. Here, conventional B cells are shown to develop normally in Vav-deficient mice but these mice have few B-1 B cells. The threshold for inducing B cell proliferation through BCR engagement in vitro is greater in Vav-deficient B cells. Nevertheless, in vivo the mutant mice have normal antibody responses to haptenated Ficoll. In contrast, Vav-/- mice show defective class switching to IgG and germinal center formation when immunized with haptenated protein. Interestingly, this defect is reversed in chimeras where normal T cells are present. Antigen-specific proliferation of T cells in the T zone was found to be similar in wild-type and Vav-/- mice but the induction of IL-4 mRNA and switch transcripts was specifically impaired. These results suggest that defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

Dendritic cells associated with plasmablast survival.

A subset of myeloid dendritic cells is described which is associated with the ability of splenic and lymph node plasmablasts to survive and differentiate into plasma cells. Plasmablast-associated dendritic cells (PDC) are CD11c(high), DEC-205(-) and unlike conventional dendritic cells do not associate with T cells. The following findings suggest a requirement for PDC if plasmablasts are to differentiate to plasma cells. First, when large numbers of B cells are recruited into antibody responses and plasmablasts outgrow the PDC stroma, only those associated with PDC survive and differentiate into plasma cells. Conversely, if the number of PDC is increased by ligating their CD40, more plasmablasts survive on the expanded PDC stroma and differentiate into plasma cells. Finally, in T cell-deficient mice, the plasma cells that develop atypically in the T zones in response to thymus-independent antigens are associated with ectopic PDC.