A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2 fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed to plastic resulted in weak T cell activation, and these T cells did not induce B cell responses. Haptenated B cell populations, although recognized by E9.D4, were not activated. Separation of T and B cells by a 0.4-micron membrane prevented T-dependent B cell activation, although Th cell-derived B cell-activating lymphokines would be assayed across these membranes. These results suggest a polyclonal noncognate B cell activation that depends on physical contact between B cells and activated T cells. The requirement for a cognate interaction of Th with B cells for the production and delivery of B help can therefore be overcome by activating Th cells with high densities of T cell receptor ligands.     

Antigen-specific major histocompatibility complex-restricted helper T cell clones are sufficient to induce unprimed B cells to switch and to secrete IgG and IgA in a primary in vitro polyclonal response

To investigate the role of helper T (Th) cells in the regulation of the production of the various immunoglobulin (Ig) classes and subclasses, we have used poly (Glu60 Ala30Tyr10) (GAT)-specific, major histocompatibility-complex-restricted Th cell clones to stimulate unprimed B cells. The T cells used in these studies were Thy-1+, Lyt-1+, Lyt-2- and lacked Fc receptor for IgM, IgG and IgA, and the unprimed splenic B cells were selected by the fluorescence-activated cell sorter for their lack of expression of surface (s)IgG and by panning for their lack of expression of sIgA. We have taken advantage of the ability of some antigen-specific major histocompatibility complex (MHC)-restricted Th cell clones to polyclonally activate unprimed B cells in vitro in the presence of high doses of antigen. We have shown that under these conditions, an antigen-specific MHC-restricted Th cell clone is sufficient to induce the switch of sIgG- sIgA- unprimed B cells to IgG and IgA, as well as the expansion of these cells and their differentiation into IgG and IgA-secreting cells. Isotype-specific Th cells thus do not seem to be an absolute requirement for the production of the various IgG subclasses and of IgA.     

A significant proportion of normal resting B cells are induced to secrete immunoglobulin through contact with anti-receptor antibody-activated helper T cells in clonal cultures

This report describes single-cell techniques to address the nature of a cellular interaction in which activated T lymphocytes stimulate small resting B cells to develop into antibody-forming cell clones in the absence of any surface immunoglobulin ligand or an antigen bridge. The cloned T helper cell line E9.D4 was stimulated with the anti-V beta 8 antibody F23.1 bound to the plastic of Terasaki 10-ul culture wells. When an excess of T helper lymphocytes was used (1,000 X-irradiated or 600 unirradiated, stimulated E9.D4 cells), 10-25% of B cells responded by antibody formation as judged by an enzyme-linked immunosorbent assay performed after 5 days of culture. When one of a very small number of B cells were present, the rate-limiting step to antibody-forming cell formation was the number of T cells present. Far fewer T cells sufficed for stimulation when culture trays were tilted to force T and B cells into proximity at the sulcus formed at the bottom edge of the culture wells. When T cell numbers were limiting, unirradiated T cells out-performed irradiated T cells. Some cell clones held for 7 days switched to IgG antibody production. E9.D4 supernatants were virtually ineffective in causing B cell stimulation, even when 3T3 filler cells were added to support cultures. The results suggest that cell contact, and perhaps conjugate formation, with a strongly activated T cell can cause changes in the adjacent resting B cells akin to those of Ig receptor cross-linking, following which a lymphokine flux (even one not involving IL 4 and 5) promotes antibody-forming cell development.     

Idiotype-specific neonatal suppression of phosphorylcholine-responsive B cells.

The effect on neonatal anti-idiotypic suppression on the expression of B cells of the T15 clonotype has been investigated at the level of individual clonal precursor cells. The results indicate that B cells of the T15 clonotype are almost completely eliminated from the repertoire for four months after neonatal injection of allogeneic anti-idiotypic serum. The degree of this suppression is dependent on the amount of anti-idiotypic antibody administered and is less profound if anti-idiotypic antibody is given after the first week of life. No suppression was observed when anti-idiotypic antisera were administered to mice 30 days of age or older, which may indicate that immature B cells are the population most susceptible to suppression. However, since suppression could be reversed by administration of T15 myeloma protein several days after injection of anti-idiotype, the inability to suppress adult BALB/c mice may have been due to the high level of T15 idiotype normally present in their serum. Finally, phosphorylcholine-responsive B cells of identifiable clonotypes other than T15, even a clonotype sharing antigen-combining site determinants with T15, appear unaffected by anti-T15 suppression.     

Priming of helper T cell-dependent antibody responses by hemagglutinin-transgenic B cells

Mice expressing the hemagglutinin (HA) gene of influenza virus PR8 (H1 subtype) under the control of x light chain promoter and enhancer have been generated. They express HA in and on B cells, and are tolerant to HA. In vitro, only lipopolysaccharide (LPS) blasts but not resting B cells of transgenic mice can stimulate HA-specific helper T cells of HA-specific α/β T cell receptor (TCR)-transgenic mice. Transfer of HA-transgenic LPS blasts into syngeneic, non-transgenic recipients primes HA-specific antibody responses. Resting, small HA-transgenic B cells, which were purified by fluorescence-activated cell sorting, prime lower antibody responses. Host B cells produce the HA-specific antibody response. The donor HA-transgenic B cells need to express major histocompatibility complex (MHC) class II molecules and need to be alive to induce the antibody response in the host. Most notably, the host antibody response never produces detectable levels of IgM, but only of switched IgG isotypes. Neither resting nor activated HA-transgenic B cells induce tolerance in antibody responses. These results suggest that HA-transgenic B cells, presenting both the intact antigen on the cell surface and peptides of the antigen on MHC class II, are effective inducers of helper T cell responses, and as judged by the Ig-isotype response pattern, which is mainly IgG1, of Th2 type.     

CD4+ helper T cells are required for resistance to a highly metastatic murine tumor

A role of CD4+ T helper cells in induction of tumor transplant rejection leading to complete regression of a highly metastatic DBA/2 mouse lymphoma was analyzed. Using an anti-CD4 monoclonal antibody (GK1.5) which eliminates T helper cells in vivo and in vitro, we found that CD4+ cells are required for tumor resistance in syngeneic DBA/2 mice or allogeneic but major histocompatibility complex-identical B10.D2 mice. In contrast, in allogeneic C57BL/6 mice tumor rejection was independent of CD4+ cells. An analogous requirement for immune CD4+ cells for in vitro induction of CD8+ tumor-specific cytotoxic T cells was found in these respective strains. The requirement for immune CD4+ cells in vitro could be replaced by recombinant interleukin 2. These results demonstrate a role of CD4+ regulatory T cells and T-T cell cooperation in the induction of anti-tumor immunity and tumor rejection, and point to possible therapeutic interventions in the afferent phase of anti-tumor immune responses.     

'Educated' dendritic cells act as messengers from memory to naive T helper cells.

Ingested antigens lead to the generation of effector T cells that secrete interleukin 4 (IL-4) rather than interferon-gamma (IFN-gamma) and are capable of influencing naive T cells in their immediate environment to do the same. Using chimeric mice generated by aggregation of two genotypically different embryos, we found that the conversion of a naive T cell occurs only if it can interact with the same antigen-presenting cell, although not necessarily the same antigen, as the effector T cell. Using a two-step culture system in vitro, we found that antigen-presenting dendritic cells can act as 'temporal bridges' to relay information from orally immunized memory CD4 T cells to naive CD4 T cells. The orally immunized T cells use IL-4 and IL-10 (but not CD40 ligand) to 'educate' dendritic cells, which in turn induce naive T cells to produce the same cytokines as those produced by the orally immunized memory T cells.     

Mini-review CD4 T cells are required for CD8 T cell memory generation

Whereas the role of CD4 T cells in B cell memory generation is well established and unequivocal, the role that CD4 T cells play in CD8 responses was until recently far more elusive and controversial. A series of recent reports, however, have re-assessed the role of CD4 help on CD8 responses and have given rise to surprisingly unambiguous conclusions. While studying very different systems, they demonstrated that CD4 T cells are absolutely required for the generation of bona fide CD8 memory cells; the reports allow, for the first time, strong analogies to be made between B and CD8 memory cell generation. These data invite us to drastically change our idea of CD4 help on CD8 responses because they show that the old dichotomy - Th-dependent versus Th-independent CD8 responses - is no longer accurate.     

Naive T-cell receptor transgenic T cells help memory B cells produce antibody

Injection of the same antigen following primary immunization induces a classic secondary response characterized by a large quantity of high-affinity antibody of an immunoglobulin G class produced more rapidly than in the initial response - the products of memory B cells are qualitatively distinct from that of the original naive B lymphocytes. Very little is known of the help provided by the CD4 T cells that stimulate memory B cells. Using antigen-specific T-cell receptor transgenic CD4 T cells (DO11.10) as a source of help, we found that naive transgenic T cells stimulated memory B cells almost as well (in terms of quantity and speed) as transgenic T cells that had been recently primed. There was a direct correlation between serum antibody levels and the number of naive transgenic T cells transferred. Using T cells from transgenic interleukin-2-deficient mice we showed that interleukin-2 was not required for a secondary response, although it was necessary for a primary response. The results suggested that the signals delivered by CD4 T cells and required by memory B cells for their activation were common to both antigen-primed and naive CD4 T cells.     

Lymphoid reservoirs of antigen-specific memory T helper cells

How vaccines control the development of antigen-specific effector and memory T helper cells is central to protective immunity but remains poorly understood. Here we found that protein vaccination selected high-affinity, CXCR5+ICOS(hi) follicular B-helper T cells (T(FH) cells) that developed in draining lymphoid tissue to regulate B cell responses. In the memory phase, reservoirs of antigen-specific CXCR5+ICOS(lo) T(FH) cells persisted with less effector activity but accelerated antigen-recall ability. This new compartment of memory T(FH) cells was retained in draining lymphoid sites with antigen-specific memory B cells, persistent complexes of peptide and major histocompatibility complex class II and continued expression of CD69. Thus, protein vaccination promotes B cell immunity by selecting high-affinity effector T(FH) cells and creating lymphoid reservoirs of antigen-specific memory T(FH) cells in vivo.     

CD8+ immunoregulatory cells in the graft-versus-host reaction: CD8 T cells activate dendritic cells to secrete interleukin-12/interleukin-18 and induce T helper 1 autoantibody

Initiation of cell-mediated immunity or autoimmunity requires secretion of interleukin (IL)-12 from dendritic cells (DC), which drives the generation of T helper 1 (Th1) effector cells in synergy with IL-18. Induction of IL-12 can be triggered by microbial stimuli but also requires signals from activated T cells. We investigated interactions between alloreactive CD4 and CD8 T cells in mixed lymphocyte reactions (MLR) in vitro and in the graft-versus-host reaction (GVHR) in vivo. In a parent-into-F1 model of GVHR, donor CD8 cells were found to suppress the hyper-immunoglobulin E (IgE) syndrome, anti-DNA immunoglobulin G1 (IgG1) autoantibodies and donor CD4-cell expansion, but were essential for Th1-dependent immunoglobulin G2a (IgG2a) autoantibody production and release of serum IL-12 p40. In vitro, addition of alloreactive CD8 cells to CD4 cells and mature DC enhanced Th1 development. CD4 and CD8 T cells induced IL-18 from DC and primed for IL-12 p70 secretion via interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha). However CD8 T cells, but not CD4 cells, released IFN-gamma/TNF-alpha after primary stimulation. The data suggest that rapid release of inflammatory cytokines from central memory-type CD8 cells early in immunity is critical for induction of Th1 cells via DC activation and IL-12 production. This pathway could provide a means for amplification of cell-mediated autoimmunity in the absence of microbial stimuli.     

T helper cells.

B-cell proliferation and differentiation is controlled by T helper cells. Recent studies have determined that the expression of a novel, 39 kD, T-cell membrane protein is responsible for inducing T-cell-dependent B-cell activation. The receptor for this protein on the resting B cell is CD40. Once activated, B cells are induced to grow and differentiate by the elaboration of interleukin-4 and interleukin-5 from activated T cells. Together, T cell-B cell contact and soluble factors provide all the signals required for B-cell growth and differentiation.     

Antigen-specific helper T cells are essential for cytotoxic T cell responses to metabolically inactivated stimulator cells.

Primed spleen cells respond well to metabolically inactivated stimulator cells while normal spleen cells do not. This observation has been interpreted as showing that cytotoxic T cell precursors are different from unprimed precursors in their antigen recognition requirements for induction. A different model is proposed here which accounts for these observations as due to enhanced helper cell levels in primed populations. Experiments are described in this study which test several predictions of this model. These experiments show that in the presence of in vitro primed helper T cells, normal cells are able to respond efficiently to glutaraldehyde-fixed stimulator cells. The helper effect is antigen-specific. Since unprimed spleen cells can be efficiently induced by metabolically active stimulators (γ-irradiated cells) and can respond to glutaraldehyde-fixed antigen (metabolically inactive cells) only in the presence of specific helper cells, it seems reasonable to propose that helper cell signals are enhanced by a nonantigenic property of γ-irradiated stimulator cells requiring metabolic activity. It is also clear that glutaraldehyde-fixed cells are anti- genically intact as helper cells, primed to antigens on γ-irradiated stimulator cells, efficiently and specifically help a response to fixed stimulators. Conversely, helper cells primed in vitro to glutaraldehyde-fixed stimulators recognize antigen on γ-irradiated stimulator cells. The level of help generated in response to glutaraldehyde- fixed stimulator cells is at least 10-fold higher in primed cells than in normal cells. In addition, primed spleen cells can be induced in vitro to yield helper function by both fixed or unfixed stimulator cells. Normal helper cell precursors are induced at least 100-fold more efficiently by γ-irradiated as compared to glutaraldehyde-fixed stimulator cells. This work supports the idea that a major effect of priming, which allows primed cells to respond to metabolically inactive stimulators, is to enhance levels of helper T cells in the primed population.     

Is the activated T helper stimulus able to induce Ig isotype switching in cultured human B cells?

It Is confirmed that large amounts of IgM, IgG, and IgA areproduced when human B cells are cultured with T cells activatedby immobilized CD3 antibody (CD3 system). IL-2 was essential;lowerlevels of Ig production with different isotype ratios were obtainedif IL-4 or IL-6 replaced IL-2. Depletion of sIgG⁺ or sIgA⁺ cellsfrom the B population to be cultured markedly reduced productionof IgG or IgA. Culturesof B cells selected with the pan-B markersCD19, CD72, or CD21 contained similar levels of Ig of all threeisotypes, whereas B cells selected for sIgM or sIgD expressionproduced IgM but very little IgG or IgA indicating that littleisotype switching was occurring. Production of IgG or IgA fromcells expressing these isotypes was more efficient than productionof IgM from IgM⁺ IgD⁺ cells. These results are considered inthe light of the demonstration by others of the production ofmultiple isotypes from single sIgM⁺-selected B cells. Clonedhuman T cells from a single donor induced production of allthree isotypes, but the proportions varied indicating that thepotent T-B cell interactions inducing B cell activation mayoverride and conceal the operation of isotype specific cellinteractions. Some T clones used at an optimal dose were aseffective untreated as X-irradiated, whereas with other clonesmaximumIg production was not achieved without irradiation.     

Stimulation of a memory B cell response does not require primed helper T cells

The use of universally immunogenic T cell epitopes, such as those identified in tetanus toxin or malaria circumsporozoite protein, could represent a major improvement in the development of synthetic vaccines. However, one limitation of this approach is the lack of T cell cross-reactivity between the vaccine and the pathogen. To determine whether the memory B cell response elicited by immunization with a synthetic peptide containing a B cell epitope linked to a T cell epitope can be restimulated by the same B cell epitope linked to different T cell epitope(s), we used a synthetic peptide which contains non-overlapping B and T cell determinants from hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV). The results of this study clearly show that primed T cells can increase the antibody response against a B cell epitope linked to the priming T cell determinant. However, the antibody response obtained was weaker than that obtained after two injections of the peptide containing both B and T cell epitopes, showing the important role played by memory B cells in secondary antibody responses. Moreover, a strong antibody response against the B cell epitope was elicited by boosting mice with the B cell epitope linked to a heterologous carrier, thus demonstrating that a strong B cell memory response can be revealed in the absence of primed T cells. These results therefore provide new important information for the design of synthetic or recombinant vaccines.     

Expansion of mycobacterium-reactive gamma delta T cells by a subset of memory helper T cells.

Human gamma delta T cells expressing the V gamma 9/V delta 2 T-cell receptor have been previously found to proliferate in response to certain microorganisms and to expand throughout life, presumably because of extrathymic activation by foreign antigens. In vitro expansion of V gamma 9/V delta 2 cells by mycobacteria has been previously shown to be dependent on accessory cells. In order to gain an insight into the mechanisms involved in the expansion of these cells, we have undertaken to identify the peripheral blood subset of cells on which proliferation of V gamma 9/V delta 2 cells in response to mycobacteria is dependent. Contrary to their role in antigen presentation to alpha beta T cells, professional antigen-presenting cells, such as monocytes, B cells, and dendritic cells, were unable to provide the cellular support for the expansion of V gamma 9/V delta 2 cells. Selective depletion of T-cell subsets, as well as the use of highly purified T-cell populations, indicated that the only subset of peripheral blood cells that could expand V gamma 9/V delta 2 cells were CD4+ CD45RO+ CD7- alpha beta T cells. These cells underwent distinct intracellular signaling events after stimulation with the mycobacterial antigen. Expansion of V gamma 9/V delta 2 cells by alpha beta T cells was dependent on cell-cell contact. This is the first evidence that a small subset of the memory helper T-cell population is exclusively responsible for the peripheral expansion of V gamma 9/V delta 2 cells. These data illustrate a unique aspect of antigen recognition by gamma delta T cells and provide new means to study their immune defense role.     

Tweaking of memory T helper 2 cells by TSLP

Thymic stromal lymphopoietin conditions dendritic cells to support homeostatic proliferation of central memory T cells. In this issue of Immunity, Wang et al. (2006) show that these dendritic cells are critical in maintaining T helper 2 central memory cells and impart them with expression of unique proallergic molecules.     

Cognate interactions between helper T cells and B cells

The mechanism by which mammals produce an antibody response after exposure to antigen has intrigued biologists for over a hundred years. Here, Randolph Noelle and Charles Snow review some of the experimental findings since the early 1970s that have advanced understanding of the mechanisms operating during B-cell activation by thymus-dependent (TD) antigens. They also propose a model for B-cell activation that emphasizes the critical role played by direct cellular interactions between B cells and helper T(TH) cells and seek to place into perspective the role played by the membrane immunoglobulin (mlg) receptor in cognate responses.