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A Transcriptional Defect Underlies B Lymphocyte Dysfunction in a Patient Diagnosed with Non-X-Linked Hyper-IgM Syndrome¹

Ameesha Bhushan^*, Bryan Barnhart^*, Scott Shone^*, Charles Song and Lori R. Covey^2,*

^* Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, NJ 08854; and Harbor General Hospital, University of California at Los Angeles, Torrance, CA 90502

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 Cµ-C recombination, and I transcription were all found to be relatively unaffected. However, CD40- and IL-4-mediated CD23 up-regulation and VDJ-C 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 I and mature VDJ-C transcription and IgG expression. However, under the same conditions I 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.

	Introduction

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Hyper-IgM (HIM)³ syndrome is a heterogeneous group of disorders defined by an increase in IgM and severely reduced levels of other circulating isotypes. This group of disorders includes defects in both CD40 signaling and other distinct molecular pathways that have not been clearly elucidated. The defect in the production of most isotypes renders affected individuals extremely susceptible to recurrent bacterial infections, autoimmune diseases, frequent neutropenia, and lymphoproliferative disease (reviewed in Ref. 1). Opportunistic infections, such as cryptosporidium and Pnuemocystis carinii, are also seen in affected individuals at an abnormally high frequency, indicating that cell-mediated immune functions are jeopardized in these individuals as well (1, 2).

Although most reported cases of HIM syndrome are related to mutations in CD40 ligand (CD154, gp39, CD40L) (3, 4, 5, 6, 7, 8), a percentage of cases involve females displaying a non-X-linked form that arises either sporadically or involves autosomal recessive (9, 10, 11) or autosomal dominant (12, 13) transmission. An analysis of a subset of patients presenting symptoms consistent with autosomal recessive transmission revealed a reduction in CD154 levels on activated T cells compared with those in unaffected controls. In addition, this patient subset displayed B cell defects relating to the secretion of IgG and IgA after in vitro stimulation (14). In a separate study a subset of patients with HIM and normal CD154 expression failed to activate phosphatidylinositol 3-kinase and induce NF-B and AP-1, transcription factors up-regulated by CD40 signaling (15). Further studies with a subset of non-X-linked HIM patients confirmed that one underlying basis of immune dysfunction is associated with downstream defects in the CD40 signaling pathway. Specifically, B cells from a subset of CD154⁺ HIM patients were found to be deficient in distinct CD40-mediated functions, including proliferation, up-regulation of CD23, and, in combination with IL-4, production of IgE (16). The complexity of HIM is further underscored in studies demonstrating the defective activation and proliferation of B cells isolated from X-linked HIM patients (17).

A second primary immunodeficiency, termed common variable immunodeficiency (CVI), is characterized by hypogammaglobulinemia and impaired functional Ab responses (18, 19, 20). An analysis of a subset of patients with CVI revealed that B cells exhibited anergic properties that were altered in vitro in response to stimulation with anti-CD40 mAb and IL-10 (21). Specifically, CVI B cells displayed a hierarchy of Ig class-specific responsiveness: IgM responses were the least affected, IgG responses were affected to an intermediate degree, and IgA responses were the most affected (21, 22). In vitro recovery of Ab secretion was interpreted as the acquired ability of B cells to differentiate in culture through a process of switch recombination and expression of differentiated isotypes.

In this report we have characterized the lymphocytes from a young female patient (GP) with a diagnosis of non-X-linked HIM. At an early age GP displayed very high serum IgM levels with little to no circulating IgG, IgA, or IgE. To determine the cellular basis of immune unresponsiveness we analyzed CD154 expression and CD40-mediated functions in lymphocyte subsets from both unfractionated and purified populations. Analysis of CD23 and CD80 expression in unfractionated B cells revealed defective CD40- and IL-4-mediated induction of CD23, but normal CD80 up-regulation. Also, when this population was cocultured under conditions of exogenous CD154 and IL-4 stimulation, B cells acquired partial functional responsiveness, as revealed by induction of germline transcripts, switch recombination, and expression of IgG and IgA. Using purified B and T cells we clearly show that the patient’s immunodeficiency resides primarily in the B cell compartment and results from a failure to properly respond to T cell signaling. This defect is manifested in an inability to express I transcripts in response to activated T cells in the presence or the absence of IL-4. However, under the same conditions of coculture the patient’s B cells were able to express I transcripts. These results strongly suggest that the basis of immune dysfunction in this individual is associated with a defect in B cell responsiveness that can be partially restored by in vitro and sustained stimulation with CD154. Furthermore, the failure of the patient’s B cells to transcribe I transcripts or express normal levels of CD23 in response to either CD40 or IL-4 signaling suggests that defective regulation or expression of a signaling molecule or transcription factor may underlie this immunodeficiency.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patient

The female patient presented at the age of 2 years with a near absence of IgG, IgA, and IgE and highly elevated levels of IgM (986 mg/dl). T cell subsets and B cell number were normal. A skin test for cell-mediated immune response showed anergy to purified protein derivative, mumps, and Candida. After 4 years of age the patient’s clinical condition began to deteriorate, with recurrent otitis media, pneumonia, and urinary tract infections. Lymph nodes, liver, and spleen all increased in size, and serum IgM levels increased progressively beyond 3000 mg/dl. Fine needle aspirates of a left cervical node at 4.5 years demonstrated caseating necrosis and multiple Pneumocystis carinii organisms. The patient was placed on i.v. Ig replacement therapy (1 g/kg) at a frequency of once per 3-wk period. There is no reported history of immunodeficiency in the patient’s family, and recently her mother has given birth to a healthy boy.

Cell lines and Abs

293 cells are derived from a primary human embryonal kidney cell transformed by adenovirus 5 and are available from American Type Culture Collection (Manassas, VA). The CD154-expressing 293 cell line (293/CD154) was constructed by stable transfection of pCT-BAM into 293 cells as previously described (23). FITC-labeled mouse anti-human CD23 was purchased from Sigma (St. Louis, MO). FITC-labeled mouse anti-human CD80 and Cy-Chrome-labeled mouse anti-human CD3 mAb were purchased from PharMingen (San Diego, CA). FITC-labeled anti-CD154, PE-labeled anti-CD69 mAbs, and biotin-conjugated mouse anti-human CD20 were purchased from Ancell (Bayport, MN).

Surface CD154 expression analysis

CD4⁺ T cells were removed from 50-cc human leukocyte preparations provided by the New Brunswick Affiliated Hospitals blood center (New Brunswick, NJ). Briefly, the leukocyte preparation was spun through Histopaque (Sigma) to isolate PBMC. Cells (10⁶) were activated with PMA (20 ng/ml) and ionomycin (1 µg/ml) (Sigma) in 3 ml of culture medium for 5 h. Activated and nonactivated cultures were incubated either with saturating amounts of FITC-conjugated mouse anti-human CD154 mAb, PE-labeled mouse anti-human CD69 mAb, and Cy-Chrome-labeled mouse anti-human CD3 mAb or with the appropriate labeled isotype control Abs. Analysis of CD3⁺/CD154⁺/CD69⁺ cells was conducted using an EPICS Profile II flow cytometer (Coulter, Hialeah, FL). To analyze CD154 expression in response to anti-CD3 and anti-CD28 mAb stimulation, a multiwell plate was coated with 15 µg/µl anti-CD3 mAb clone OKT3 (American Type Culture Collection), and 4 µg/µl anti-CD28 mAb clone 9.3 (a gift from Dr. Carl June, Uniformed Armed Services University, Bethesda, MD) for 1 h at 37°C. Wells were washed twice with 1x PBS, and 5 x 10⁵ CD4⁺ cells were added to coated wells for 8 h at 37°C. CD154 expression was determined by FACS analysis of 1.5 x 10⁵ cells using FITC-conjugated anti-CD154 mAb or isotype-matched control (Ancell).

Up-regulation of CD23 and CD80

PBMC (5 x 10⁵) were cultured with 2 x 10⁵ 293 cells, 2 x 10⁵ CD154-transfected 293 cells, or 200 U/ml IL-4 (PeproTech, Rocky Hill, NJ). Cocultures were established in 1.0 ml of RPMI/10% FBS in 24-well plates at 37°C in 5% CO₂. After 24 h cells were harvested and assayed for CD23 and CD80 up-regulation by incubating them with saturating amounts of biotin-labeled mouse anti-human CD20 mAb followed by FITC-labeled anti-CD23, FITC-labeled anti-CD80 mAbs, or matching isotype controls (Ancell) and streptavidin-PE. Cells were analyzed for two-color expression by FACS.

Isolation of B and T cells

CD4⁺ T cells were removed from total PBMC by biomagnetic separation using anti-CD4-coated magnetic beads following protocols provided by Dynal (Lake Success, NY). B cells were isolated by positive selection over anti-CD19-conjugated superparamagnetic beads (Dynal). CD19⁺ cells were washed and resuspended in RPMI (minus bicarbonate)/10% FBS/20 mM HEPES at a concentration of 2 x 10⁸ cells/ml. IgG⁺ cells were removed by two rounds of plating onto anti-IgG-coated plates for 30 min at 25°C. After the second round of negative selection, cells were washed in 1x PBS and collected by centrifugation.

Cocultures established with PBMC or purified B cells

PBMC were cultured for 6 days with either 5 x 10⁵ mitomycin C (mitoC)-treated CD154-transfected (293/CD154) or untransfected 293 cells in the presence or the absence of 200 U/ml of IL-4. CD19⁺IgG^- purified B cells were cultured with 5 x 10⁵ mitoC-treated 293 cells alone or with 10 µg/ml pokeweed mitogen (PWM; Sigma), and mitoC-treated 293/CD154 cells were cultured in the presence of 10 µg/ml PWM, 200 U/ml IL-4, or 200 ng/ml IL-10 (PeproTech). In cocultures in which CD19⁺ B cells were incubated with activated T cells, CD4⁺ T cells from the patient and the control were activated by incubation on 96-well plates that had been precoated with anti-CD3 and anti-CD28 mAbs according to the protocols described above. After initial activation, 5 x 10⁴ CD4⁺ T cells were cultured in Ab-coated wells for 6 days with 5 x 10⁴ B cells from the patient or the control in the presence or the absence of 200 U/ml IL-4. RNA was extracted from all samples. In duplicate PBMC cocultures, DNA was extracted for switch circle product analysis, and in all samples supernatants were assessed for Ig expression.

In vitro production of Ig

ELISA for Ig isotype quantitation was performed according to previously published protocols (24). Goat anti-human IgM, IgG, or IgA was purchased from Southern Biotechnology Associates (Birmingham, AL). For detection of IgE, the ELISA Amplification System (Life Technologies, Gaithersburg, MD) was used according to protocols provided by the manufacturer. Absorbance was read at 490 nm on a Microplate Autoreader (Bio-Tek, Burlington, VT). The limit of sensitivity was for IgM, IgG, and IgA was typically 0.8 ng/ml. The limit of sensitivity of IgE was 62 pg/ml.

Identification of I, I, and VDJ-C transcripts

RNA isolation, synthesis of cDNA, PCR amplification, and identification of germline and mature transcripts have been previously described (25, 26). Briefly, a 5' I primer (5'-gccctcctctcagccaggacc-3') and a 3' C_H2C primer (5'-tccttgggttttggggggaa-3') were used to amplify I transcripts from all four subclasses. For amplification of the V_HDJ_H-C transcripts the 5'J_H primer (5'-acc(c/a)tggtcaccgyctcctca-3') was used with the 3' C_H2C primer. Reactions to amplify I transcripts were established with 5 µl of cDNA in a 100-µl reaction volume containing 10 mM Tris (pH 8.3), 75 mM KCl, 1.5 mM MgCl₂, 200 µM dNTP, and 4 ng of each primer. V_HDJ_H-C transcripts were amplified in a reaction containing 5 µl of cDNA, 10 mM Tris (pH 8.8), 25 mM KCl, 1.5 mM MgCl₂, 200 µM dNTPs, and 4 ng of each primer. Amplification of all reactions was conducted for 30 cycles at 1.5 min at 94°C, 1 min at 55°C, and 1 min at 72°C. PCR products were identified using hinge region-specific oligonucleotides as previously described (25). For amplification of the I transcripts, reactions were conducted essentially as described above, except the concentration of KCl was 50 mM and the I-specific primers were 5'IgE (5'-gacgggccacaccatcc-3') and 3'IgE (5'-cggaggtggcattggagg-3'). Semiquantitative PCR was performed using 5, 0.5, and 0.05 µl of cDNA in the reaction conditions described above. GAPDH transcripts were amplified with the 5'GAP primer (5'-gtcttcaccaccatggagaaggct-3') and the 3'GAP primer (5'-catgccagtgagcttcccgttca-3') and were probed with an internal oligonucleotide (27). Quantitation of VDJ-C transcripts was performed by dividing the VDJ-C signal at each point by the GADPH signal using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Identification of switch circle products

PBMC were cocultured with 293 cells or 293/CD154 cells with or without IL-4 (200 U/ml) for 6 days. Preparation of DNA was conducted as previously described with minor modifications (26). Briefly, 0.5 µl of digested circular DNA was amplified in a 50-µl reaction containing 50 mM KCl, 15 mM Tris-HCl (pH 9.0), 1.5 mM MgCl₂, 200 µM dNTP, 5% DMSO, 0.5 U Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA), and 100 nM of each primer. Samples were initially incubated at 95°C for 10 min, 60°C for 10 min (at which time the enzyme was added), and 72°C for 10 min. These steps were followed by 40 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 2 min. The primers used to amplify Sµ-S switch circle products were as follows: M1, 5'-ggtgagtgtgatggggaacgcagtgta-3', corresponding to nucleotides 3810–3785 of Sµ (GenBank accession no. X56795); and G1, 5'-gggcttccaagccaacagggcaggaca-3', corresponding to nucleotides 1859–1885 in the S4 region (GenBank accession no. X56796) (28). The segment between nucleotides 1280 and 1546 of the S1 region (266 bp) (28) was amplified by PCR for use as a switch region-specific probe.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD154 expression in activated T cells from a female patient with HIM

A female patient (GP) presented at 2 years of age with frequent urinary tract infections and otitis media. Early examination of blood Igs revealed elevated serum IgM levels (>800 mg/dl) with trace levels of serum IgG, IgA, and IgE. Fine needle aspirates of a left cervical node at 4.5 years demonstrated caseating necrosis and multiple Pneumocystis carinii organisms. Analysis of blood lymphocytes revealed normal numbers of CD20⁺ and CD3⁺ cells as well as a normal distribution of CD4⁺ and CD8⁺ T cells. While the patient clearly fell within the non-X-linked category of HIM based on family history and gender, evaluation of CD154 expression was conducted to establish whether defective expression was an underlying cause of the Ab deficiency. PBMC were isolated from GP and the control, activated with PMA/ion (Fig. 1A, left panels) or immobilized anti-CD3 plus anti-CD28 mAb (Fig. 1B), and analyzed for the expression of CD154. After PMA/ion activation we detected a level of CD154 surface expression on GP T cells that was consistently reduced compared with that on control T cells. However, the expression of CD154 after anti-CD3/anti-CD28 activation was similar to the expression of control cells, although the absolute numbers of cells were far fewer (Fig. 1B). Because the expression of CD154 is dependent on CD4⁺ T cell activation, we analyzed the expression of a separate activation marker, CD69, to determine whether depressed CD154 expression using PMA/ion was a consequence of a general defect in T cell activation. Analysis of CD69 up-regulation on CD3⁺ cells revealed no significant difference between patient and control samples, suggesting the presence of normal activation pathways in GP’s T cells (Fig. 1A, right panels, shown is the single-color parameter). Together, these findings suggested that the patient’s T cells were activated upon stimulation, and CD154 expression appeared to be relatively normal. Therefore, we turned our focus to a potential B cell defect as the underlying basis of GP’s immune dysfunction.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Analysis of CD154 expression on activated PBMCs from the patient and the control. A, PBMCs were purified from whole blood and activated with PMA/ion for 5 h. T cells were gated with Cy-Chrome-labeled anti-CD3 mAb and stained with either FITC-labeled anti-CD154 mAb (anti-TRAP; left panels) or PE-labeled mouse anti-human CD69 mAb (right panels). Shown are the single-color histograms for CD154 and CD69 expression, where solid line peaks represent the cells staining positively with the indicated Ab, and the dotted line represents the level of background staining with a matched isotype control. Numbers in the upper right corner of each histogram indicate the percentage of CD3+ cells that are also positive for either CD154 or CD69 over the mean fluorescence of the positive population. These data are representative of two independent experiments. B, PBMC were stimulated for 8 h in 12-well plates with immobilized anti-CD3 mAb (15 µg/µl) and anti-CD28 mAb (4 µg/µl). Cells were incubated with either a FITC-conjugated anti-human CD154 mAb (solid line) or a FITC-conjugated mouse IgG1 isotype control mAb (dotted line) and were analyzed by FACS for CD154 expression. Numbers in the upper right corner indicate the percentages of positive cells and the mean fluorescence levels of the positive population.

To examine the responsiveness of the patient’s B cells to CD40 and cytokine signaling, CD23 expression was analyzed on CD20⁺ cells stimulated with 293 cells, 293 cells expressing CD154 (293/CD154), or 200 U/ml IL-4 (Fig. 2A). After 24 h we found that GP’s CD20⁺ B cells up-regulated CD23 in response to both IL-4 and CD154, although the degree of expression was measurably depressed compared with that of control cells (compare middle and right panels). Whereas the patient’s CD20⁺ B cells did increase CD23 after activation with both stimuli, the increases of 22% (CD154) and 18% (IL-4) over unstimulated CD20⁺ cells represented approximately half the increase observed for the control cells under identical conditions of stimulation. In addition, the change in CD23 expression on CD20^- cells in response to IL-4 was much lower on the patient’s cells than on control cells (0.8 vs 6.4%; see right panels, lower right quadrant). Another difference in the CD23 profile between patient and control was that there was no observable CD20⁺/CD23^lo population in GP’s unstimulated PBMC, although there were significantly more B cells as a percentage of the total cells (compare, left panels, upper right quadrants). These results suggested that although a fraction of the patient’s B cells were competent to up-regulate CD23 in response to IL-4 or CD154, the majority of the unstimulated CD20⁺ population appeared significantly more refractory than the control population. These data also suggest that GP’s defect affects both IL-4 and CD40 signaling pathways.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. Expression of CD23 and CD80 on PBMC following stimulation with CD154 or IL-4. A, Shown are fluorescence-activated flow cytometric analysis of PBMC from the patient and the control before and after overnight incubation with 2 x 105 293 cells (left panels), 293/CD154 cells (middle panels), or IL-4 (right panels). CD20+ cells were identified by staining with anti-CD20-PE, and the expression levels of CD23 were determined by staining with anti-CD23-FITC. B, PBMC from the patient and the control were stimulated for 24 h with 2 x 105 293 cells (left panels) or 293/CD154 cells (right panels). CD80 up-regulation on B cells was determined by incubation with PE-labeled mouse anti-human CD20 mAb and FITC-conjugated mouse anti-human CD80 mAb. Numbers in the upper right quadrant indicate the percentages of CD20+ B cells that are also positive for CD23 or CD80.

Secretion of Igs by in vitro stimulation of PBMC

To examine the ability of B cells from the patient to express "switched" classes of Ig in response to in vitro stimulation, PBMC were cultured with either 293 cells or 293/CD154 cells in the presence or the absence of IL-4 for 6 days (Table I). Supernatants were collected, and concentrations of IgM, IgG, and IgA were assayed by ELISA. As expected, the patient’s B cells expressed IgM in the absence of stimulation, and this expression increased in response to stimulation with 293/CD154 cells with or without IL-4. Because of passive Ab therapy given to the patient we could not accurately measure the expression of IgG in PBMC. However, when we compared the IgG response in the separate samples under different conditions of coculture, we observed no increase in IgG in the patient’s samples in response to all conditions of stimulation. This result was significantly different from our results with control samples, which also displayed a very high level of IgG in the unstimulated cultures. Here, we observed a slight increase in IgG after stimulation with CD154 alone (from 332 to 419 ng/ml IgG) and a significant increase in IgG after stimulation with CD154 plus IL-4 (1231 ng/ml IgG). Unstimulated control cultures also showed considerable expression of IgA, and this expression increased in response to both CD154 alone and CD154 plus IL-4. In accordance with the patient’s phenotype we detected no IgA in unstimulated supernatants or in cultures stimulated with IL-4. Surprisingly, IgA expression was significantly induced in patient cocultures established with both 293/CD154 cells alone or 293/CD154 cells plus IL-4 (265 and 412 ng/ml, respectively). Although the patient’s B cells appeared to be refractory to IgA expression in vivo, our ELISA results suggested that under specific in vitro conditions the intrinsic defect could be reversed, and B cells could be induced to express Ig at a relatively high level.

Patient’s B cells can be induced to express I transcripts and undergo switch recombination

Because a failure to express switched isotypes in vivo may be explained by an impairment in mechanisms that lead to the expression of I and mature transcripts or the induction of Cµ-C switch recombination, we examined whether these processes were intact in GP’s B cells. To assay germline transcription, PBMC from the patient and the unaffected control were incubated with either 293 cells or 293/CD154 cells in the presence or the absence of IL-4, RNA isolated, and RT-PCR employed to amplify I transcripts (Fig. 3A). Using probes to the hinge regions to differentiate subclass expression, we found a similar level of I2 transcripts in both unstimulated patient and control cultures (lanes 1 and 5) and relatively equal induction of 1, 2, and 4 transcripts in cultures stimulated with either IL-4 or CD154 (lanes 2, 3, 6, and 7). One surprising finding was the complete down-regulation of I transcription in both patient and control cultures in response to 293/CD154 plus IL-4 stimulation (lanes 4 and 8). The one significant difference in the I profiles of stimulated cultures was a much higher level of IL-4-induced 3 transcription in patient vs control B cells.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. A, Patient B cells can be induced to express germline I transcripts and undergo de novo switch recombination. PBMC (1 x 106) from the patient (lanes 1–4) and the control (lanes 5–8) were incubated with 5 x 105 293 cells (lanes 1 and 5), 5 x 105 293 cells plus 200 U/ml IL-4 (lanes 2 and 6), 5 x 105 293/CD154 cells (lanes 3 and 7), or 5 x 105 293/CD154 cells plus 200 U/ml IL-4 (lanes 4 and 8). RNA was isolated, reverse transcribed, and amplified using primers homologous to sequences in all four subclasses. Identification of subclass-specific transcription was conducted using replica gels and 32P end-labeled hinge region probes specific for each subclass. All reactions included primers for the amplification of GAPDH as a control for RNA and cDNA integrity, and the GAPDH product is shown on a separate blot below the I blots. B, Detection of circular reciprocal recombination products (switch circles) following stimulation with CD154 and IL-4. PBMC from patient and control blood were isolated and incubated under various coculture conditions for 6 days. DNA was isolated and analyzed by PCR for the presence of reciprocal products from recombination events between Sµ and S. Shown are the products isolated from PBMC of the patient (lanes 1–4) and the control (lanes 5–8) cocultured with 293 cells (lanes 1 and 5), 293 cells and 200 U/ml IL-4 (lanes 2 and 6), 293/CD154 cells (lanes 3 and 7), and 293/CD154 cells plus 200 U/ml IL-4 (lanes 4 and 8).

Mature C expression is reduced in patient B cells

When mature transcripts were assayed in stimulated PBMC by RT-PCR, we observed a decreased level in GP samples, especially for the 2, 3, and 4 subclasses. However, we found that 1, 2, and 4 mature transcripts in the patient’s B cells were all modulated in response to IL-4 alone or IL-4 plus CD154 (Fig. 4A). As an extension of our switch circle data, these results suggested that the patient’s B cells were being induced in culture to undergo heavy chain gene rearrangement to all four C loci. The absolute level of transcripts from all subclasses in GP cultures was significantly lower than the level observed in control cultures; however, much of this difference is accounted for by the fact that there was significant expression of C transcripts in unstimulated control cultures (compare lanes 1 and 5). Nevertheless, there did appear to be a significant difference in both the absolute level of VDJ-C1 transcription and the degree of inducibility of this locus in GP vs control B cells.

View larger version (46K): [in this window] [in a new window]   FIGURE 4. Transcription of mature C transcripts in stimulated PBMC. A, Analysis of mature C transcription was conducted using RNA isolated from PBMC and RT-PCR to amplify germline transcripts from all four subclasses. Six-day cocultures were established under the following conditions: 293 cells alone (lanes 1 and 5), with 293 cells and 200 U/ml IL-4 (lanes 2 and 6), with 293/CD154 cells (lanes 3 and 7), with 293/CD154 cells and 200 U/ml IL-4 (lanes 4 and 8). Replica blots were hybridized with 32P end-labeled hinge region probes corresponding to the different C subclasses. An equal number of counts per minute was added to each blot. GAPDH primers were included in the PCR reactions as a control for integrity and quantity of cDNA. B, Semiquantitative PCR of mature VDJ-C1 transcripts from PBMC stimulated with 293 cells, 293 cells plus IL-4, 293/CD154 cells, or 293/CD154 cells plus IL-4 was conducted with decreasing amounts of cDNA (5, 0.5, and 0.05 µl). Duplicate blots of amplified products were hybridized with probes specific for C1 and GAPDH transcripts. Signals were quantitated by PhosphorImager analysis, and fold induction was determined by dividing the C1 signal by the signal for GAPDH at the 1/10 dilution.

A B cell transcriptional defect underlies GP’s immunodeficiency

Our results with in vitro stimulation of PBMC suggested that the patient’s B cells were fully capable of responding to a majority of signal transduction pathways via CD154 and IL-4 contact. Although quantitative differences were observed between patient and control B cells with respect to C expression, these differences failed to adequately explain the complete absence of non-IgM and non-IgD isotypes in the patient. These observations suggested that our in vitro assay conditions for B cell stimulation were not accurately reflecting in vivo conditions of activation. Also, these results did not reveal whether the defect was restricted to a particular lymphocyte subset. Therefore, to assay B cell responses under more physiological conditions and to locate GP’s defect to either the T cell or B cell subset a series of cocultures was established using different combinations of control and patient lymphocytes. CD4⁺ T cells and CD19⁺ B cells were isolated from patient and control PBMC, T cells were activated on immobilized anti-CD3 and anti-CD28 mAb plates, and cocultures were established with equal concentrations of patient and control T cells and B cells in the presence or the absence of IL-4. After 6 days, RNA was isolated, reverse transcribed into cDNA, and amplified using primers specific for germline and mature transcripts.

Surprisingly, under these conditions we observed a remarkably different pattern of I transcription in patient vs control B cells in response to either IL-4 and/or autologous or control T cells (Fig. 5A). Here, patient B cells were almost completely unresponsive to activated T cell signals (either patient or control) in the presence or the absence of IL-4 (see lanes 3, 4, 9, and 10). In contrast, control B cells expressed I transcripts in response to either patient or control T cells with or without IL-4 (lanes 5, 6, 11, and 12). We did observe a very low, but detectable, level of inducible I2 and I3 transcripts in patient B cells stimulated with IL-4 (lane 2). However, the IL-4-induced pattern of I1 and I4 transcription from patient B cells was severely repressed (compare lanes 2 and 8). One difference that we did observe between patient and control T cells was in the ability to induce I4 transcripts in control B cells in the presence of IL-4 (compare lanes 6 and 12). This result suggested that GP’s defect may also extend to specific expression of T cell factors as well.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Analysis of I and VDJ-C transcription in purified GP B cells stimulated with patient and control activated CD4+T cells. A, CD19+ B cells from patient (lanes 1–4, 9, and 10) and control (lanes 5–8, 11, and 12) were cultured alone (lanes 1 and 7), with 200 U/ml IL-4 (lanes 2 and 8) or with patient CD4+ T cells (lanes 3–6) or control CD4+ T cells (lanes 9–12) in the presence (lanes 4, 6, 10, and 12) or the absence (lanes 3, 5, 9, and 11) of IL-4. After 6 days RNA was isolated from all samples and reverse transcribed, and I transcripts were amplified using PCR. PCR products were probed with hinge-region oligonucleotides specific for the four subclasses designated on the left. Primers to specifically amplify GAPDH were included in the reactions to control for cDNA concentration and integrity of the reactions. The higher GAPDH signal in lanes 3–6 and lanes 9–12 reflects the total increased number of cells (B and T cells) in these samples. C and P designate the lymphocyte population as being from either the control or the patient, respectively. B, The same samples as those described in A were amplified using primers specific for mature C transcripts. Identification of specific products was conducted using the hinge region-specific oligonucleotides to the different subclasses as described above.

Culturing with 293/CD154 cells overrides defective I and V_HDJ_H-C transcription in patient B cells

The difference in I and V_HDJ_H-C expression in unfractionated (Figs. 3 and 4) vs purified B cells (Fig. 5) may be explained either by the presence of additional cells and factors in the PBMC and/or by the different stimulator cells used in the cocultures (293/CD154 vs activated T cells). To elucidate the basis for this difference CD19⁺IgG^- cells were purified and cultured with 293/CD154 cells in the presence of PWM, IL-4, or IL-10 for 2 wk (Fig. 6). Like unfractionated B cells we did observe germline transcription from I1, I2, and I3 subclasses in response to all conditions of stimulation (Fig. 6A). We also observed a low level of I4 transcription in response to 293/CD154 cells plus IL-4 (lane 4). What was surprising was that at this time there was a greater I response from the patient’s cells compared with the control B cells. However, when we examined V_HDJ_H-C transcription from all four subclasses we found a higher amount of mature transcripts from the control vs the patient B cells (Fig. 6B). This suggested that the patient B cells were less differentiated at this time point and that a greater percentage of control B cells had already undergone switch recombination. This interpretation is consistent with the idea that the GP B cells are initially refractory to differentiation signals but can be induced to respond to signals under specific conditions of activation over a set time period.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Purified B cells can be induced to express I and VHDJH-C transcripts after stimulation with CD154 and IL-4. CD19+IgG- B cells from the patient and the control were cultured for 14 days in the presence of 293 cells alone (lanes 1 and 6), with PWM (lanes 2 and 7), or in the presence of 293/CD154 cells plus PWM (lanes 3 and 8), IL-4 (lanes 4 and 9), or IL-10 (lanes 5 and 10). Total cellular RNA was isolated, and RT-PCR was performed. Equal amounts of product were loaded on four separate gels, and germline I (A) or mature (VHDJHC) transcription (B) was analyzed for each subclass as described in Materials and Methods.

Patient B cells can up-regulate I transcripts in response to IL-4

We also assessed whether the putative transcriptional defect extended to the expression of other classes of germline transcripts. Using RNA from T cell:B cell cocultures, we showed that GP and control B cells up-regulate I transcripts in response to either IL-4 alone (lanes 2 and 8) or to activated T cells plus IL-4 (lanes 4, 6, 10, and 12). However, the I response is not enhanced in GP B cells after incubation with activated T cells and IL-4 compared with stimulation with IL-4 alone (compare lanes 2 and 4 (patient) to lanes 8 and 6 (control)). Also we found a complete absence of I expression in GP B cells after incubation with control or patient T cells (compare lanes 3 and 9 to lane 11). Together these results suggest that the patient’s defect does not extend to IL-4-inducible expression of I transcripts; however, the patient’s B cells again fail to respond to CD40-mediated signals in the absence of IL-4. Also, T cell function again appears to be slightly compromised, as shown by the inability of activated patient T cells to induce I transcripts in control B cells in the absence of exogenous IL-4.

	Discussion

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In this report we have made several novel observations with respect to the functional characteristics of lymphocytes isolated from a female patient diagnosed with HIM syndrome. Specifically, we have determined that GP has relatively normal T helper function, but displays deficits in subsets of CD40-and cytokine-mediated pathways that underlie B cell differentiation. The nature of the defect is especially apparent in the altered expression of germline transcripts in B cells after coculturing with activated T cells and in CD23 surface expression in B cells and non-B cells after stimulation with CD154 and IL-4. However, we have also observed that the expressed defect can be reversed, and the patient’s B cells induced to become functionally responsive if unfractionated or purified B cells are stimulated in vitro with 293/CD154 cells and/or IL-4. Our conclusion that germline transcription from different classes is not equally affected by the defect is based on the observation that under conditions of little or no I expression, the patient’s B cells express normal levels of I transcripts in response to IL-4 and IL-4 plus activated T cells. Finally, we have detected a subtle difference in the ability of the patient’s T cells to induce control B cells to express I4 and I transcripts.

Although our findings present a complex picture of this particular immunodeficiency, they also reveal two salient characteristics of the defect that allow us to hypothesize as to its underlying nature. Because the defect affects the expression of a number of immune-related genes, it most likely resides in a protein, such as a transcription factor or signaling molecule, common to their expression. Second, because the defect can either be by-passed or reversed by in vitro culture with CD154 and IL-4, it appears that this environment may allow other molecules to substitute for the defective molecule, and/or the sustained stimulus in the in vitro environment allows the defect to be overcome. The second model is supported by results showing low levels of secreted IgG and IgA and expression of both germline and mature transcripts in purified B cells after incubation with 293/CD154 cells plus IL-4, but not with activated T cells plus IL-4. That activated T cells rapidly down-regulate CD154 expression upon contact with CD40 by receptor-mediated endocytosis (29) and 293/CD154 cells do not (L. Covey, unpublished observations) would support this model.

This second model does not necessarily exclude the possibility that signals from other cells, such as macrophage, may also influence and/or accelerate the release from functional unresponsiveness. In fact, our observation that B cells in unfractionated PBMC rapidly become transcriptionally competent, undergo switch recombination, and express a level of inducible V_HDJ_H-C transcripts would support this model. Independent of the signal source, our data point toward the importance of a threshold of signaling that drives the in vitro differentiation of GP B cells under conditions that clearly do not exist in vivo. Other undefined interactions or signals in the unfractionated cultures may explain a number of intriguing observations seen with the PBMC but not with the CD19⁺IgG^- cultures. These observations include the expression of I transcripts by the patient’s B cells in response to IL-4 alone, the expression of I2 in the patient’s unstimulated B cells, and the complete down-regulation of I transcription in both patient and control cultures after stimulation with 293/CD154 plus IL-4. These results suggest that a number of signaling pathways in addition to CD154 and IL-4 may influence germline transcription with both positive and negative results.

Previously, it was demonstrated that B cells from a group of CVI patients were effectively anergized; however, defective B cell function could be rescued in vitro over a period of extended IL-10 and CD40 stimulation (21). Although, GP’s B cells show many characteristics of being functionally anergic, the fact that CD40-directed CD80 expression is indistinguishable from that in control cells at a time that the CD23 expression is highly compromised suggests that the cell is differentially unresponsive depending on the specific signaling pathway. Thus, these B cells appear to be different in nature than a truly anergized B cell.

The exact correspondence between the molecular defect in GP’s B cells and CD40 and IL-4 signaling is still undefined. Theoretically, it could be in any molecule that is part of the IL-4/CD154 signaling cascade or a transcription factor that is common to both pathways. The cytoplasmic tail of CD40 interacts with a number of molecules of the TNF receptor-associated factor (TRAF) family of proteins (30, 31). Recently, it has been shown that different TRAF molecules are involved in signal transduction pathways that lead to distinct CD40-mediated functions. For example, it was found that binding of TRAF2 and TRAF3 to CD40 was not required for Ab secretion, although binding of these molecules appeared to modulate the in vitro responses (32). CD40 signaling also induces multiple transcription factors, including NF-B (33, 34, 35), NF-AT (35), and AP-1 (35, 36). To address the question of the role of NF-B in different CD40-mediated B cell responses Hsing and Bishop determined that NF-B activation was required for Ab production and B7-1 up-regulation (37). In light of these findings, we would surmise that either GP’s defect is not related to NF-B activation, because we observed normal CD80 induction in response to CD40 signaling, or that a different NF-B dimeric complex is responsible for CD80 vs CD23 and I expression. One recent report revealed that the p50-RelA and p50-RelB dimers were the major complexes binding to NF-B sites in the murine germline 1 promoter (38). Thus, one possibility is that the defect resides in a particular NF-B/Rel subunit and that sustained signaling allows for a less efficient NF-B dimeric complex to initiate transcription. We are currently carrying out experiments to test these and other potential hypotheses.

Because we only assayed IgE secretion in 14-day cocultures, we were initially unable to determine whether the molecular defect by-passed the expression of this particular class or whether IgE expression was assayed after the patient’s B cells had been released from functional unresponsiveness. This question was answered in part by measuring I transcription in 6-day lymphocyte cocultures. Here, we clearly established that the transcriptional defect differentially affected I and I transcription. In fact, at this time we observed normal induction of I transcripts in the patient’s B cells after stimulation with CD154 and IL-4. These data indicate that the molecular pathways and/or transcription factors responsible for mediating I region transcription are different between the and classes. It is interesting to note that our results showing normal expression of IgE by GP’s B cells concur with those of another study showing that B cells from CVI patients express IgE, but not necessarily IgG or IgA, after extended in vitro culture (22).

The subtle T cell defect that was revealed with regard to decreased I4 and I transcription in control B cells is very interesting because of the dependence of I4 and I promoter activity on IL-4. One possible explanation for these observations is that GP’s defect extends to the expression of IL-4 in activated T cells. Thus, only under our experimental protocol, where activated T cells were added to B cell cultures, did we observe a difference with the activities of these promoters. Experiments to understand the basis for this defect in GP T cells and how it is related to CD40 and IL-4 signaling pathways in her B cells are currently underway.

View larger version (29K): [in this window] [in a new window]   FIGURE 7. Patient B cells express I transcripts in response to IL-4 and IL-4 plus activated T cells. To detect I transcripts in CD19+B cells derived from the control and from patient GP, RT-PCR was conducted on RNA isolated from 6-day cocultures. CD19+ B cells from the patient (lanes 1–4, 9, and 10) and the control (lanes 5–8, 11, and 12) were cultured alone (lanes 1 and 7), with 200 U/ml IL-4 (lanes 2 and 8), or with patient CD4+ T cells (lanes 3–6) or control CD4+ T cells (lanes 9–12) in the presence (lanes 4, 6, 10, and 12) or the absence (lanes 3, 5, 9, and 11) of IL-4.

	Acknowledgments

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI37081 (to L.R.C.).

² Address correspondence and reprint requests to Dr. Lori R. Covey, Department of Cell Biology and Neuroscience, Nelson Biological Laboratories. Rutgers, The State University of New Jersey, 604 Allison Road, Piscataway, NJ 08854. E-mail address:

³ Abbreviations used in this paper: HIM, hyper-IgM syndrome; CVI, common variable immunodeficiency; mitoC, mitomycin C; PWM, pokeweed mitogen; TRAF, TNF receptor-associated factor.

Received for publication September 23, 1999. Accepted for publication December 29, 1999.

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