Normal recombination substrate VH to DJH rearrangements in pre-B cell lines from scid mice.

To further analyze the VDJ recombination defect in lymphoid pre-B cells from mice with severe combined immune deficiency (scid mice), we have assayed the ability of Abelson murine leukemia virus (A-MuLV) transformed pre-B cells from scid mice to rearrange a recombination substrate in which inverted VH to DJH joins activate a selectable (gpt) gene. In unselected populations, substrate rearrangements occurred frequently, but were aberrant and probably analogous to the aberrant rearrangements observed at endogenous scid Ig gene loci. In contrast, populations of scid pre-B lines selected for gpt activity within the substrate contained mostly "normal" VH to DJH joins within the introduced substrate. These findings demonstrate that scid pre-B cells can make normal joins at low efficiency and are discussed with respect to the potential mechanism of the scid defect and the occurrence of Igs in leaky scid mice.

Non-antigen signals for B-cell growth and differentiation to antibody secretion.

Recently, significant progress had been made in understanding the T-B lymphocyte interactions that control humoral immunity. This review highlights experiments that demonstrate a central role for interactions between T-cell-B-cell-activating molecule (CD40 ligand) expressed on T cells and CD40 on B cells in B-cell activation and immunoglobulin isotype switching, both in vitro and in vivo.

The effect of the scid mutation on mechanism and control of immunoglobulin heavy and light chain gene rearrangement.

Most Abelson murine leukemia virus (A-MuLV)-transformed cell lines derived from scid (severe combined immune deficient) mice actively rearrange their endogenous immunoglobulin (Ig) heavy (H), but not light (L) chain variable region genes. Such cell lines express germline VH segments and other RNA transcripts that are characteristically produced by early precursor (pre)-B lymphocytes, but do not express high levels of transcripts from the germline kappa (k) constant region (C kappa) locus. However, we have derived scid A-MuLV transformants that express germline C kappa transcripts and attempt kappa gene assembly. In one case kappa gene expression and rearrangement occurred in the absence of mu H chain expression, and in another was not induced efficiently by introduction of a mu-expression vector. Although the vast majority of scid H and L chain coding sequence joins are grossly aberrant, scid A-MuLV transformants can form normal coding joints at a very low frequency. In contrast, these cells form generally normal signal sequence joins at an approximately normal efficiency. Thus, these findings mechanistically distinguish coding and signal join formation. Subcloning analyses suggest that scid A-MuLV transformants that do not attempt chromosomal coding sequence joining may have a relative survival advantage, and therefore that these events may often result in unrepaired chromosomal breakage and cell death.

Isolation of cDNAs encoding T-BAM, a surface glycoprotein on CD4+ T cells mediating contact-dependent helper function for B cells: identity with the CD40-ligand.

"T-cell B-cell Activating Molecule" (T-BAM) is an activation-induced surface protein on CD4+ T cells that mediates a contact-dependent signal for B cell differentiation and immunoglobulin (Ig) secretion. The T-BAM protein on a helper clone of Jurkat (D1.1) was affinity purified using the anti-T-BAM mAb, 5c8. The NH2-terminal amino acid sequence of purified T-BAM was determined and found to be highly homologous to the predicted NH2-terminal sequence of a T cell ligand to the B cell CD40 molecule (CD40-L). From a D1.1 cDNA library, a clone was isolated that encodes CD40-L by sequence and drives expression of T-BAM protein on transfected cells, demonstrating that the T-BAM and CD40-L genes and proteins are identical. Moreover, transfection of T-BAM was shown to confer to non-lymphoid cells, the ability to induce B cells to upregulate the expression of surface CD23 molecules. In previous studies we showed that T-BAM was expressed predominantly on activated CD4+ and on few if any CD8+ cells. Although the current work confirms that T-BAM is largely restricted to activated CD4+ T cells, we now provide definitive evidence that T-BAM can be expressed by a small population of CD8+ T cells after activation. Importantly, a subset of CD8+ T cells do not express T-BAM after activation and this T-BAM- phenotype is maintained on certain CD8+ T cell clones. Taken together, these data unify the biology and structure of T-BAM and CD40-L and this synthesis has implications for understanding the T cell regulation of the humoral immune response.

CD40:CD40L interactions in X-linked and non-X-linked hyper-IgM syndromes.

Hyper-IgM (HIM) syndrome is a rare immunodeficiency characterized by low or absent IgG, IgA, and IgE with normal or elevated levels of IgM. This disorder can be acquired or familial with either X-linked or autosomal patterns of inheritance. The X-linked form of the disease is a consequence of mutations in the CD40 ligand (CD40L) gene that encodes a protein expressed primarily on activated CD4+ T cells. The cognate interaction between CD40L on T cells and CD40 on antigen-stimulated B cells, macrophage, and dendritic cells is critical for the development of a comprehensive immune response. The non-X-linked form of HIM syndrome is heterogeneous and appears in some cases to be a consequence of mutations in the AlD gene which encodes a B cell specific protein required for class switch recombination, somatic mutation, and germinal center formation. However, mutations in other unidentified genes are clearly the basis of the disease in a subset of patients. In this article, we review the essential features of the X-linked and non-X-linked forms of HIM syndrome and discuss the critical role the CD40:CD40L receptor-ligand pair plays in the pathogenesis of these immune deficiencies.

CREB/ATF proteins enhance the basal and CD154- and IL-4-induced transcriptional activity of the human Igamma1 proximal promoter.

To understand the underlying basis for the strong IL-4- and CD154-mediated Igamma1 promoter activity in Ramos 2G6 B cells, we carried out transient transfection assays with luciferase-based constructs containing approximately 2.2 kb and 500 bp of the human Igamma1 proximal promoter region. As a comparison, the corresponding regions of the human Igamma3 promoter were tested under identical conditions. We found that both Igamma1 and Igamma3 promoter constructs were activated upon transfection into Ramos B cells and that activity was significantly up-regulated by CD154 and IL-4 signals. However, the Igamma1 promoter was measurably stronger than the Igamma3 promoter with respect to both basal and induced responses. Sequence comparison revealed a divergent 36-bp region containing multiple putative transcription factor binding sites in the Igamma1 but not the Igamma3 promoter. A mutational "swap" of this sequence resulted in a marked decrease and increase in Igamma1 and Igamma3 basal and induced promoter activity, respectively. Gel retardation assays with Igamma1-specific probes revealed CREB-containing complexes that were not observed with the corresponding Igamma3 probes. Mutation of a single nucleotide in overlapping CREB sites in the Igamma1 sequence resulted in a significant decrease in basal activity with a corresponding reduction in the level of IL-4- and CD154-mediated transcription.

Growth response to insulin in mouse melanoma cells and fibroblast X melanoma hybrids.

PG19 mouse melanoma cells arrest growth when they become confluent in medium containing low concentrations of serum. Under these conditions, insulin does not stimulate DNA synthesis in the mouse melanoma cells, whereas it does in mouse embryo fibroblasts and fibroblast X melanoma hybrids. A detailed examination of the binding of insulin to the melanoma cells and fibroblast X melanoma hybrids in the presence of bacitracin has shown that they have approximately equal numbers of insulin receptors, and that these receptors have similar affinities for insulin. These results indicate that the unresponsiveness of the melanoma cells to the mitogenic action of insulin is not attributable to a lack of insulin receptors. Although insulin does not stimulate incorporation of 3H-thymidine into DNA in confluent cultures of the melanoma cells, it does stimulate uptake of alpha-aminoisobutyrate, indicating that the insulin receptors are functional and that insulin elicits an acute response in these cells. In hormone-supplemented serum-free medium, insulin does not stimulate growth of the melanoma cells, while it does stimulate growth of th fibroblast X melanoma hybrid. This suggests that an acute response to insulin is not sufficient for stimulation of growth either in confluent growth-arrested cells or in exponentially growing cells in serum-free medium.

VH to VHDJH rearrangement is mediated by the internal VH heptamer.

To elucidate the mechanism of VH to VHDJH joining (VH replacement), we have assayed a pre-B cell line for ability to perform V(D)J recombinase-mediated rearrangement events between an unrearranged VH gene segment and a VHDJH rearrangement within a retroviral recombination substrate. V(D)J recombinase-mediated inversional joins between the VH gene segment and the VHDJH rearrangement within this substrate activate a drug-resistance gene allowing selection of cells that performed the event. We have used this method to identify and isolate VH to VHDJH joins within the introduced substrate. Analyses of the resultant signal and coding joins of such rearrangements demonstrated that VH replacement recombination is mediated by a heptamer-like sequence embedded in the 3' region of the VH segment in the assembled VHDJH join and the heptamer-spacer-nonamer recognition signal of the unrearranged VH segment.

A polymorphic CD40 ligand (CD154) molecule mediates CD40-dependent signalling but interferes with the ability of soluble CD40 to functionally block CD154:CD40 interactions.

We report the characterization of a naturally occurring polymorphism in CD40 ligand (CD40L, CD154) expressed by activated T cells from a young female patient. This polymorphism encodes a nonconservative Gly --> Arg substitution in amino acid 219 in the extracellular, CD40 binding domain of the molecule. Studies carried out with 293 epithelial cells ectopically expressing the polymorphic protein (CD154/G219R) revealed reduced levels of binding to different anti-CD154 monoclonal antibodies (mAb) and CD40-immunoglobulin (CD40-Ig). However, recognition of the polymorphic and wild-type CD154 molecules by a polyclonal antiserum was comparable, suggesting that the polymorphism affects the ability of the protein to interact with CD40 but does not significantly alter its surface expression. To determine if reduced cross-linking of CD40 mediated decreased functional effects, three CD40-dependent properties were measured. We found that pathways leading to the induction of surface CD23, CD80, and Igamma transcription were activated in response to CD154/G219R signalling. However, the decrease in affinity for CD40 by the mutated CD154 affected the ability of CD40-Ig to efficiently interfere with the binding and effectively block induced CD80 expression. In contrast, we found that the 5c8 mAb, which recognized the polymorphic molecule to a similar extent as wild-type CD154, effectively blocked the interaction between CD154/G219R and CD40 as measured by CD80 expression. These findings suggest that naturally occurring polymorphisms in the CD154 molecule may affect the ability of CD40-mediated functions to be blocked by soluble CD40 or anti-CD154 mAb in the therapeutic treatment of disease and graft rejection.

A transcriptional defect underlies B lymphocyte dysfunction in a patient diagnosed with non-X-linked hyper-IgM syndrome.

To establish the underlying cause of hyper-IgM syndrome in one female patient, B cell function was examined in response to CD40- and IL-4-mediated pathways. When CD40-induced functional responses were measured in unfractionated B cells, CD80 up-regulation, de novo Cmu-Cgamma recombination, and Igamma transcription were all found to be relatively unaffected. However, CD40- and IL-4-mediated CD23 up-regulation and VDJ-Cgamma transcription were clearly diminished compared to control cells. IL-4-induced CD23 expression was measurably reduced in the CD20- population as well. These results suggested that the patient's defect is positioned downstream of CD40 contact and affects both CD40- and IL-4 signal transduction pathways. Further analysis of B cell function in CD19+ B cells revealed a clear B cell defect with respect to Igamma and mature VDJ-Cgamma transcription and IgG expression. However, under the same conditions Iepsilon transcription was relatively normal. Partial restoration of B cell function occurred if PBMC or CD19+ B cells were cultured in vitro in the presence of CD154 plus IL-4. Because addition of IL-4 to cocultures containing activated T cells failed to induce B cells to undergo differentiation, the ability of the patient's B cells to acquire a responsive phenotype correlated with receiving a sustained signal through CD40. These findings support a model in which the patient expresses an intrinsic defect that is manifested in the failure of specific genes to become transcriptionally active in response to either CD154 or IL-4 and results in a functionally unresponsive B cell phenotype.

T cell receptor DJ but not VDJ rearrangement within a recombination substrate introduced into a pre-B cell line.

To elucidate mechanisms that regulate ordered and tissue-specific assembly of Ig and TCR variable region gene segments, we have introduced a recombination substrate comprised of germline TCR beta V, D, and J gene segments into an Abelson murine leukemia virus-transformed pre-B cell line that actively rearranges endogenous Ig H chain variable region gene segments but does not rearrange endogenous light chain or TCR variable region gene segments. We find that these cells efficiently join D beta segments to J beta segments within the mini-locus, but that they do not make any detectable site-specific rearrangements of the introduced V beta segment even though it is closely linked in the same construct to the D beta. These findings suggest that factors necessary for V beta to (D beta)J beta joining may be absent in these pre-B cells and also imply that the order in which TCR V beta, D beta, and J beta segments are rearranged can be influenced by factors other than the 12/23 recombination rule. Furthermore, in agreement with the an accessibility model of VDJ recombinase control, the D beta region of the construct was found to be relatively more sensitive to DNAase I digestion in isolated nuclei when compared to the unrearranged V beta region.

CD40 ligand exerts differential effects on the expression of I gamma transcripts in subclones of an IgM+ human B cell lymphoma line.

The CD40:CD40 ligand (CD40L) interaction plays a critical role in T cell-dependent isotype switching. To elucidate the role of CD40 signaling in the activation of gamma germline transcription and as an extension, in targeting Cgamma regions for isotype switching, an IgM+ Burkitt lymphoma cell line (Ramos 2G6) was assayed for the up-regulation of germline gamma transcripts after CD40L stimulation. Independent Ramos 2G6 subclones that either expressed (Igamma+) or did not express (Igamma-) basal levels of Igamma transcripts were assessed for their transcriptional response to CD40L signaling by contact with either a Jurkat T cell line (D1.1) or a transfected CD40L-expressing epithelial cell line (293/CD40L) in the presence or absence of IL-4. Both Igamma- and Igamma+ Ramos 2G6 subclones cultured with IL-4 and CD40L markedly up-regulated germline transcription predominantly from the gamma1, gamma2, and gamma3 subclasses over levels obtained with IL-4 alone. In addition, these two signals were required to obtain de novo switch recombination. However, incubation with CD40L alone resulted in a substantial increase in germline transcription only in the Igamma+ and not the Igamma- subclones. Observed basal transcription at the gamma1 locus also correlated with the ability of not only the gamma1 locus, but also the gamma2 and gamma3 loci, to up-regulate germline transcripts in response to CD40 signaling. These data are consistent with CD40:CD40L contact up-regulating germline transcription only after the B cell has received a signal that alters the transcriptional state of the heavy chain locus.

Regulation of CD154 (CD40 ligand) mRNA stability during T cell activation.

The CD154 protein (CD40 ligand), which is critical to the regulation of both humoral and cellular immune responses, is expressed transiently on the surface of activated CD4+ T cells. To determine whether control of mRNA stability contributes to the highly regulated expression of CD154 during T cell activation, CD4+ T cells were isolated from human peripheral blood and stimulated for various lengths of time with plate-bound anti-CD3 mAb. At early times after anti-CD3 activation, the CD154 message was found to be very unstable, however, the stability measurably increased after 24-48 h of activation. Similar analyses of TNF-alpha and c-myc mRNA decay throughout a time course of T cell activation revealed patterns of regulation that were distinct from CD154. Similar to the effect on TNF-alpha mRNA, stimulation of T cells with PMA + ionomycin greatly increased the stability of CD154 message. However, CD154 message stability was only modestly increased in T cells coactivated with anti-CD3 and anti-CD28 at 5 h and not increased by costimulation at 24 h. Finally, an analysis of both mRNA and surface protein expression over a time course of T cell activation with anti-CD3 revealed a rapid induction of expression early after activation. This induction was followed by a more gradual decrease in expression over the next 48 h. Together, these data support a role for posttranscriptional regulation in the control and overall expression of CD154 in activated T cells.

Identification of a complex that binds to the CD154 3' untranslated region: implications for a role in message stability during T cell activation.

CD154 expression is regulated throughout a time course of CD3-dependent T cell activation by differential mRNA decay. To understand the molecular basis of the "stability" phase of this pathway, experiments were conducted to identify sequences and specific complexes important in this regulation. Gel retardation assays using extracts from both Jurkat T cells and CD3-activated CD4(+) T cells revealed a major complex (complex I) that bound a 65-bp highly CU-rich region of the CD154 3' untranslated region. The specificity of the CU-rich element for complex-I formation was confirmed by disruption of this complex by oligo(dCT) competition. Formation of complex I strongly correlated with CD154 mRNA stability across a time course of T cell activation. UV cross-linking identified a major oligo(dCT)-sensitive species at approximately 90 kDa that showed induced and increased expression in extracts from 24- and 48-hr anti-CD3-activated T cells, respectively. This protein was absent in equivalent extracts from resting or 2-h-activated T cells. Using an in vitro decay assay, we found that a CD154-specific transcript was more rapidly degraded in 2-h-activated extract and stabilized in the 24- and 48-h extracts compared to extracts from resting T cells. Disruption of complex I resulted in the rapid decay of a CD154-specific transcript demonstrating a functional role for complex I in mRNA stabilization in vitro. These studies support a model of posttranscriptional regulation of CD154 expression being controlled in part by the interaction of a poly(CU)-binding complex with a specific sequence in the 3' untranslated region.

CD40 molecules induce down-modulation and endocytosis of T cell surface T cell-B cell activating molecule/CD40-L. Potential role in regulating helper effector function.

The T-BAM/CD40-L molecule on CD4+ T cells interacts with B cell CD40 molecules to deliver contact-dependent signals that drive B cell activation and Ig secretion. Cell surface T-BAM/CD40-L expression is transient and may be closely regulated in order to limit the activation and clonal selection of noncognate B cells. We demonstrate that B cells, but not non-B cells, rapidly and specifically down-modulate surface T-BAM/CD40-L expression in a contact-dependent and temperature-sensitive manner that renders T cells unable to activate resting bystander B cells. Because the ability to down-modulate T-BAM/CD40-L correlated with CD40 expression, the role of CD40 molecules in down-modulating its ligand was directly assessed. Anti-CD40 mAb, but not control mAb, block B cell-induced T-BAM/CD40-L down-modulation. Furthermore, CD40+ nonlymphoid transfectants specifically down-modulate surface T-BAM/CD40-L expression. B cells induce T-BAM/CD40-L internalization into cytoplasmic compartments in a process that is inhibited by cytochalasin B. Pretreatment of activated T cells with lysosomotropic agents does not affect CD40-induced down-modulation of surface T-BAM/CD40-L but results in a marked accumulation of T-BAM/CD40-L in cytoplasmic vesicles. Together, these studies strongly suggest that CD40 induced T-BAM/CD40-L down-modulation occurs, in part, by receptor-mediated endocytosis followed by lysosomal degradation and may represent a mechanism to regulate CD4+ T cell helper effector functions.

T-BAM/CD40-L on helper T lymphocytes augments lymphokine-induced B cell Ig isotype switch recombination and rescues B cells from programmed cell death.

An important component of T cell help for B lymphocyte differentiation is the contact-dependent signaling mediated by the T cell-B cell activating molecule (T-BAM/CD40-L), an activation-induced surface membrane protein on CD4+ T helper cells in lymphoid follicles that interacts with the B cell surface molecule, CD40. The present study dissects the roles of T-BAM/CD40-L in helper function by means of a neutralizing anti-T-BAM/CD40-L mAb (5c8), a T-BAM/CD40-L-expressing T cell tumor subclone (Jurkat D1.1), and a T-BAM/CD40-L-responsive IgM+ B cell tumor of germinal center origin (RAMOS 266). Like activated T cells, D1.1 cells induce B cells to synthesize IgG, IgA, and IgE in a process that is specifically inhibited by the mAb 5c8. Although rIL-4 alone, but not Jurkat D1.1, induces IgH C gamma mRNA transcripts in RAMOS 266, the T-BAM/CD40-L molecule on D1.1 acts on rIL-4-primed RAMOS B cells to augment expression of C gamma transcripts. In addition, IgG+ RAMOS 266 clones were expanded from D1.1- and rIL-4-stimulated cultures that had undergone deletional IgH isotype switch recombination events. Furthermore, T-BAM/CD40-L signals delivered by the D1.1 clone dramatically rescue RAMOS 266 from mAb anti-IgM-induced apoptosis. Taken together, these data support the idea that T-BAM/CD40-L plays important roles in inducing Ig isotype switch recombination and the clonal selection of isotype-switched B cells.

T lymphocyte T cell-B cell-activating molecule/CD40-L molecules induce normal B cells or chronic lymphocytic leukemia B cells to express CD80 (B7/BB-1) and enhance their costimulatory activity.

Activation-induced cell surface molecules are involved in mediating bidirectional T-B lymphocyte signaling that is important in the induction of T or B lymphocyte effector functions. In this regard, T-BAM/CD40-L is an activation-induced CD4+ T cell surface molecule known to be important in inducing B cell effector functions. This report demonstrates that T-BAM/CD40-L molecules on a Jurkat T cell leukemia subclone (D1.1) or nonlymphoid 293 kidney cell transfectants induce B cells or B-CLL cells to express CD80 (B7/BB-1) in a manner that is specifically inhibited by anti-T-BAM/CD40-L mAb 5C8. Because activation-induced B cell surface molecules, such as CD80, deliver costimulatory signals to T cells that augment T cell proliferation, the functional costimulatory capacity of T-BAM/CD40-L-primed B cells and B-CLL cells was studied. T-BAM/CD40-L-primed B cells or B-CLL cells augment the proliferative responses of allogenic T cells. Furthermore, T-BAM/CD40-L priming is specifically inhibited by mAb 5C8. Together, these studies demonstrate that T-BAM/CD40-L induces CD80 expression on resting B cells or B-CLL cells. Moreover, T-BAM/CD40-L signaling enhances B cell costimulatory capacity. These studies suggest that T-BAM/CD40-L molecules not only induce B cell differentiative processes that result in Ab secretion, but also enable B cells to prime Ag-specific T cells for subsequent clonal expansion.

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre-) B cell differentiation, we find that most Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A-MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre-B cell lines from normal mice, attempted V kappa-to-J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back-to-back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site-specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.