T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.

Exposure of T94, a CD4+ V beta 8-expressing murine Th cell clone, or immediately ex vivo CD4+ T cells to deaggregated, bivalent antibodies specific for either the TCR or CD3 failed to induce an increase in [Ca2+]i, or activation of phosphatidylinositol hydrolysis unless cross-linked with a secondary anti-Ig antibody. In contrast, we show that a combination of two mAb directed against different components of the TCR/CD3 complex (145.2C11, anti-CD3 epsilon and F23.1, anti-V beta 8) successfully induce second messenger formation, that is, without any requirement for a secondary antibody. This requirement for either a secondary antibody or two independent bivalent antibodies to activate second messenger production in T cells suggested that the signal transduction apparatus may be activated by multiple TCR/CD3 complexes being brought together on the T cell surface. This was supported by the observation that conditions inducing increased T cell [Ca2+]i through the TCR/CD3 complex also resulted in aggregation of the TCR/CD3 complex on the T cell surface. Conversely, binding of anti-TCR/CD3 antibodies to the T cell under conditions that did not induce increased [Ca2+]i also failed to induce surface TCR/CD3 redistribution. Cross-linking of the CD4 accessory molecule on T94 also resulted in increased [Ca2+]i, with kinetics similar to those observed after TCR/CD3 oligomerization. CD4 is involved in the recognition of invariant regions of MHC class II during Ag presentation and has been proposed to be associated with TCR/CD3 in the absence of Ag. Aggregation of TCR/CD3 and subsequent second messenger formation was achieved by combinations of mAb to distinct determinants within the complex due to the stable association of these determinants within the T cell membrane. We therefore assessed the functional association of CD4 with the TCR/CD3 complex by examining whether a combination of mAb directed against CD4 and CD3 or TCR induced second messenger formation. We found that anti-CD4 in combination with F23.1 or with 145.2C11 failed to induce increases in [Ca2+]i. Furthermore, mAb to CD4 failed to inhibit the increase in [Ca2+]i observed with the combination of 145.2C11 and F23.1. We therefore conclude that CD4 is not stably associated with TCR or CD3 in the absence of Ag/MHC class II composites.

Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen.

Effector T cells are restricted to recognizing antigens associated with major histocompatibility complex (MHC) molecules. Specific recognition is mediated by the alpha beta heterodimer of the T-cell receptor (TCR)/CD3 complex, although other membrane components are involved in T-cell antigen recognition and functions. There has been much controversy in this regard over the part played by the CD4 glycoprotein. It is known that expression of CD4 correlates closely with the cell's ability to recognize antigens bound to class II MHC molecules and that CD4 can bind to class II molecules. Also monoclonal antibodies to CD4 can modify signals generated through the TCR/CD3 complex. It has therefore been proposed that CD4 binds to class II molecules, coaggregates with the TCR-CD3 complex and aids the activation of T cells. But given that TCR can itself impart restriction on the cell, it remains unclear whether the contribution of CD4-derived signals to those generated through the TCR alpha beta-CD3 complex is central to this activation. Here we report that when preceded by ligation of CD4, signalling through TCR alpha beta results in T cell unresponsiveness due to the induction of activation dependent cell death by apoptosis. These results imply that CD4 is critically involved in determining the outcome of signals generated through TCR, and could explain why the induction of effector T cells needs to be MHC-restricted.

Immune status of a mu, kappa transgenic mouse line. Deficient response to bacterially related antigens.

We have examined the immune repertoire and immune response of a mouse that carries transgenes for a mu heavy chain and kappa light chain. The expression of these genes is under the regulation of their own controlling elements. The transgenes are expressed early in ontogeny and are easily detectable from day 13 of gestation onwards. The pre-B cells seem to function normally as they generate IgM-secreting colonies at normal frequencies. Colonies show predominantly the transgenic specificity. Expression of the transgenes is not limited to B cells since around 10%-20% of peripheral T cells and 50% of thymocytes express the mu transgene as an intracellular protein. Ectopic expression of kappa was not seen. The spleen size of the transgenic mouse is decreased by around 20%; this reduction is largely caused by a reduction of the B cell pool. Almost all B cells express the transgenes, only 30% co-express endogenous heavy chain genes and all co-express endogenous light chain genes. Serum Ig levels for IgM and IgA were normal, 20% of the IgM consist of the transgenic product. Serum IgG levels were decreased. T cell functions (helper and cytotoxic) were normal. Immune responses to conventional antigens were impaired, especially in the early phases of the immune response, but after boosting they were virtually normal, except for IgG3 which remained low. Primary antibody responses to T cell-independent antigens of the class II type (bacterially related antigens) were absent, although precursor frequencies for these antigens were within the expected range. The significance of this finding, as it relates to allelic exclusion of Ig genes, is discussed.

T helper cell-dependent induction of resting B cell differentiation need not require cognate cell interactions.

We have analyzed the role of cognate interaction with helper T cells (Th) in support of resting B cell differentiation to plaque formation. Co-culture of histoincompatible resting B cells and resting Th cells resulted in the induction of plaque-forming cells when dimeric but not monomeric fragments of anti-T cell receptor (TcR) antibody were added to culture. The efficiency of B cell activation was comparable to that supported by lipopolysaccharide and lectin-mediated Th-B cell conjugate formation. Further, if resting Th cells were preactivated with antigen and histocompatible antigen-presenting cells, the requirement for addition of anti-TcR to mixtures of histoincompatible Th and B cells was obviated. These results demonstrate that TcR-mediated Th recognition of major histoincompatibility complex class II/antigen composites on the resting B cell membrane does not provide obligate signals for B cell differentiation to plaque formation. We are left with two possibilities. Either the entire process of Th cell-dependent induction of resting B cell differentiation is mediated by soluble lymphokines or if Th-B cell contact is mandatory, it is mediated through nonpolymorphic cell surface determinants.

The molecular interactions with helper T cells which limit antigen-specific B cell differentiation.

Helper T (Th) cell-dependent activation requirements for 2,4,6-trinitrophenyl (TNP)-specific resting B cells obtained from mice transgenic for Sp-6 mu, kappa genes were analyzed. Carrier-specific T cell help required linked recognition of TNP carrier and was functionally restricted by the B cell major histocompatibility complex. However, histoincompatible T cell-B cell conjugates formed by bridging surface immunoglobulin and Th cell receptor for antigen (TcR) through TNP-conjugated anti-TcR antibodies resulted in the efficient differentiation of TNP-specific B cells. Thus, Th cell-dependent cognate recognition of B cells is not obligatory. Specific conjugate formation could be obviated by using unconjugated fragments of anti-TcR antibodies. If dimeric, these fragments supported the Th cell-dependent differentiation of co-cultured histoincompatible resting B cells. Unconjugated monomeric fragments were ineffective, demonstrating the necessity for TcR cross-linking. Resting B cells from Sp-6+ mice rendered TNP-conjugated monomeric fragments of anti-TcR antibodies effectively multivalent, thereby satisfying conditions for the activation of co-cultured Th cells. The results demonstrate that Th cells do not transduce activation signals through TcR recognition of B cell membrane-associated ligand which limit the induction of B cell differentiation. Cross-linking of TcR on Th cells is required, sufficient and can be induced through interaction with the antigen-specific B cell surface.

A colostral protein that induces the growth and differentiation of resting B lymphocytes.

We describe the first protein of mammalian origin that induces the growth and differentiation of resting B lymphocytes. A proline-rich protein has been isolated from sheep colostrum. A purified proline-rich protein preparation (PRPP) induced resting mouse B cells into and supported their progression through the cell cycle at frequencies comparable with those seen for LPS. Differentiation of resting B cells to plaque formation was also supported as efficiently by PRPP as it was by LPS. However, PRPP was distinct from LPS in that it supported the growth and differentiation of resting B cells derived from either C3H/Tif or C3H/HeJ mice. Splenocytes from neonatal mice responded robustly to PRPP with the growth and differentiation of contained B cells to plaque formation. Unlike LPS, PRPP did not induce detectable Ig isotype switching.

Early changes in quiescent B cell physiology subsequent to cognate and bystander interaction with helper T cells.

We have assessed early changes in quiescent B cells following cognate and bystander interaction with cloned helper T cells. Variables monitored include Ia expression, blastogenesis, G0 to G1 transition, and progression through cycle. We have also assessed the antigen specificity, Ia restriction, and dependence on membrane immunoglobulin crosslinking of both generation and delivery of effectors that mediate B cell responses. The results demonstrate that antigen presentation by quiescent B cells to T cells resulting in the generation of effectors that activate B cells is Ia-restricted and dependent on antigen and an (mIgM and mIgD) crosslinking signal. However, once generated, T cell effector functions act independently of Ia haplotype to promote Ia expression, blastogenesis, and G0 to G1 transition by most small B cells. Although these responses can be mediated by T cell supernatants, further progression of B cells through S, G2 and M is only efficient when Th cells are present in cultures. Thus, results suggest that one or more Ia unrestricted, labile and/or cell-associated factors, not active in most conventional T cell supernatants, are necessary to stimulate proliferation of small B cells.

Targeting cytotoxic T cells to antigen-specific B lymphocytes.

A recent development in immunomanipulation involves the targeting of cytotoxic T lymphocytes (CTL) to cell-bound antigens using bispecific antibodies. These antibodies have been engineered such that specificity is directed against the T cell receptor (TCR) or TCR-associated T3 molecules, as well as against the chosen antigen. The present study was aimed to force interactions between T and B cells by bridging their receptors. F23.1 antibodies, which are specific for gene products of the TCR V beta 8 gene family, were conjugated with TNP (2,4,6-trinitrophenyl) and this construct was used to bridge the receptors of V beta 8+ T cells with the receptors of TNP-specific B cells. The bridging was demonstrated by direct killing of both a TNP-specific B hybridoma and of blast cells from mice transgenic for mu, kappa of the TNP-specific antibody Sp6. Further, F23.1-TNP constructs in conjunction with V beta 8+ CTL were shown to specifically deplete Ig-secreting B cells from Sp6 transgenic mice. Conjugates of TCR-specific antibodies and antigen are theoretically useful in vivo to either deplete or expand B cells of a given specificity by coupling their receptors to the TCR of CTL or T helper cells, respectively.

Neither interleukin 2 nor gamma interferon directly promote growth or differentiation of mouse B cells.

The roles of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) as direct mediators of B-cell growth and differentiation were analysed. Products of cloned genes were used in both cases. The use of flow cytometric assays coupled with density fractionation of responding splenic B-cell populations enabled both the characterization of B cells responding to various stimuli and the estimation of their frequency. B cells responding to non-IL-2 related lymphokines promoting growth and differentiation were restricted to low buoyant density fractions. In addition, these cells expressed densities of IL-2 receptor determinants comparable to those found on T cells, although, IL-2 did not support their growth or differentiation. The inability to demonstrate any direct effect of either IL-2 or IFN-gamma on B cells in any state of activation suggests that their physiological roles are mediated through additional cell types.

Autoreactive or self restricted?

Putative major histocompatibility complex (MHC) class II-specific T cell clones were isolated from nominal antigen-primed T cell populations propagated in vitro under antigen selection. However, unlike the proliferative responses of nominal antigen-specific, MHC class II-restricted T cell clones derived from the same population, the former's MHC-specific proliferative responses required that the culture medium be supplemented with serum. The role of xenogeneic antigens in the generation of this category of "autoreactive" T cells is discussed.

Abortive activation of B lymphocytes by monoclonal anti-immunoglobulin antibodies.

Toward a better understanding of the signaling role of antigen-mIg binding in the generation of humoral immune responses, we have assessed the effects of soluble, monoclonal anti-Ig antibodies on various cell physiologic parameters known to change during B cell activation. These parameters include membrane potential, I-A antigen expression, narrow angle light scattering properties (size), and cell cycle state. Results indicate that all monoclonal antibodies that bind cell to surface IgM or IgD, or both, induce virtually all small B cells to undergo membrane depolarization and increased I-A expression. Only a small subset of these antibodies, exemplified by b-7-6 anti-mu, induce all small B cells to enter G1. An increasingly smaller proportion of these cells traverse each subsequent cell cycle phase, with 10% of cells reaching G2 or M phases by 60 hr of culture. The kinetics of this response to b-7-6 are considerably slower than those of the response induced by LPS. Finally, analysis of Percoll density-fractionated cells revealed that although B blasts made by b-7-6 stimulation of small cells remain b-7-6 responsive, natural B blasts isolated from the spleen are refractory to monoclonal anti-Ig stimulation as indicated by membrane depolarization, increased IA expression, blastogenesis, and [3H]thymidine uptake.

Mouse B cells do not proliferate in human interleukin 2.

By pre-activation with a monoclonal anti-IgM antibody coupled to Sepharose, either in the absence or presence of low doses of lipopolysaccharide, mouse B cell populations were rendered responsive to recombinant DNA derived human interleukin 2 (IL 2). However, if the B cell populations were subjected to separation based on their buoyant density before pre-activation, only low but not high buoyant density cells became responsive to IL 2. Both cell populations subsequent to anti-IgM pre-activation were equally responsive to a Sephadex G-100 fraction of supernatants from rat spleen cells stimulated with concanavalin A. Furthermore, the differences in the IL 2 titration curves on T and B cell populations indicate that only a subpopulation of cells are responding to IL 2 in the B cell populations. In B cell populations that did respond to IL 2, a population of Thy-1.2-positive cells appeared. The proportion of IL 2-responsive T cells needed to give a significant signal when admixed with nonresponding B cell populations was quantitated to be less than 1%.

Supernatant from a cloned helper T cell stimulates most small resting B cells to undergo increased I-A expression, blastogenesis, and progression through cell cycle.

Helper T cell clone 52.3 supernatant (52.3 SN) was previously shown to be able to stimulate gradient-purified murine resting B cells in the absence of any additional stimulus. However, the proportion of cells that were accounting for the thymidine uptake and the Ig production was unknown. In this paper, we have studied induced changes that can be measured at the single cell level, and have thus determined the frequency of resting B cells that respond to 52.3 SN. Results indicate that 52.3 SN induces an increased I-A expression and a cell size enlargement on virtually all resting B cells. A significant proportion (30%) of these cells later becomes large blasts. Acridine orange staining revealed that in the presence of 52.3 SN a large fraction of the resting B cells undergoes the G0 to G1 transition. Furthermore, 52.3 SN is able to induce at least 20% of the cells to continue through the cell cycle into S phase as indicated by propidium iodide staining of DNA. Finally, a fraction of the 52.3 SN-stimulated cells differentiate to Ig-producing cells. Our present results suggest that resting B cells express functional receptors for some lymphokines and that these lymphokines can act in the absence of membrane Ig occupancy. Our findings further support the existence of a B cell-activating factor acting in a MHC-unrestricted manner and responsible for the entry of resting B cells into cell cycle. The relationship between this factor and other lymphokines is discussed.

Trans-stimulation of T cells: characterization of targets and involvement in loss of alloreactivity.

The rapid loss of alloreactivity within populations of antigen-primed, in vitro propagated T cells cannot be explained by the appearance of suppressor cells nor by the dilution effect of the proliferative antigen-specific T cells alone. The involvement of trans-stimulation in loss of alloreactivity, i.e. the recruitment of non-antigen-specific T cells into proliferation in an antigen-dependent and specific fashion, was assessed. Susceptibility to trans-stimulation was found to correlate directly with state of activation at the outset of assay. Large T cells (low buoyant density) but not small T cells (high buoyant density) are susceptible to trans-stimulation. Moreover, in vitro pre-activation of small T cells by mitogen confers susceptibility to trans-stimulation. Analysis of the alloreactivity in Percoll fractions of antigen-primed lymph node T cells revealed activity in both large- and small-T-cell fractions with some enrichment in the latter. The small T cells, refractive to trans-stimulation, are diluted out of the population within the early weeks of antigen-mediated in vitro propagation, accounting for a rapid loss of considerable alloreactivity. The loss of all detectable alloreactivity within antigen-selected populations suggests that the state of activation conferring sensitivity to trans-stimulation must be maintained, and that neither the antigen nor the culture conditions employed met this requirement.

Heterogeneity in the response of T cells to antigens presented by B lymphoma cells.

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstituted the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.

Induction of resting B cells to DNA synthesis by soluble monoclonal anti-immunoglobulin.

A novel monoclonal anti-immunoglobulin is described which in soluble form is comparable to lipopolysaccharide in its ability to induce DNA synthesis in populations of resting B cells. It inhibits lipopolysaccharide-induced B cell maturation to immunoglobulin secretion while not suppressing mitogen-induced proliferative responses. B cells from C3H/HeJ mice are stimulated as well as those from C3H/Tif mice demonstrating that the induction capacity of this monoclonal antibody does not involve functional lipopolysaccharide receptor molecules.