Human Lymphocytes Transcribe the Cystic Fibrosis Transmembrane Conductance Regulator Gene and Exhibit CF-defective CAMP-regulated Chloride Current*

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians, primarily affecting epithelial tissues of the lung and gut. Mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR), are responsible for this disease. A it not to cell conductances induced by pathways, volume regulation, and which are equivalent to currents described in epithelial cells. B-lymphoblasts from CF-affected humans demonstrated defective C1- conductance regula- tion by cAMP but preserved regulation by calcium-mediated and volume regulation mechanisms. CFTR involvement in cAMP regulation of C1- conductance in lymphocytes is by our of the presence of appropriately spliced CFTR mRNA segments in human B and T lymphocytes as detected by an optimized reverse-transcription and polymerase chain reaction approach. The identity of the amplified products was confirmed by hybridization to CFTR- specific probes and DNA sequencing. Furthermore, the 3’-end of the gene was found in a T cell cDNA library. We conclude that CFTR mRNA is expressed in lymphocytes, primers. The slower migrating material which hybridizes with the detection oligonucleotide is presumably single-stranded DNA generated by an imbalance of primers and/or differential efficiency of the two primers, based on observations that such bands were not visible with ethidium bromide staining of gels and appeared variably in repeated experiments.

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians, primarily affecting epithelial tissues of the lung and gut. Mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR), are responsible for this disease. Whether a physiological defect exists in the immune system of CF patients has remained controversial. A chloride ion transport defect has been described in human CF-derived lymphocytes; however, it has not been possible to detect CFTR mRNA in lymphocytes. We report here that normal human B-lymphoblasts display whole cell C1-conductances induced by calcium-mediated pathways, volume regulation, and cAMP which are equivalent to currents described in epithelial cells. B-lymphoblasts from CF-affected humans demonstrated defective C1-conductance regulation by cAMP but preserved regulation by calciummediated and volume regulation mechanisms. CFTR involvement in cAMP regulation of C1-conductance in lymphocytes is further supported by our demonstration of the presence of appropriately spliced CFTR mRNA segments in human B and T lymphocytes as detected by an optimized reverse-transcription and polymerase chain reaction approach. The identity of the amplified products was confirmed by hybridization to CFTRspecific probes and DNA sequencing. Furthermore, the 3'-end of the gene was found in a T cell cDNA library. We conclude that CFTR mRNA is expressed in lymphocytes, consistent with the cAMP regulation of chloride transport present in normal lymphocytes but defective in CF-derived lymphocytes.
Cystic fibrosis (CF)' is characterized by defective electrolyte transport of epithelial cells in several organ systems, including lung, pancreas, sweat gland, and intestine. The gene responsible for CF, which encodes for a 1480-amino acid protein that has been termed the cystic fibrosis transmembrane conductance regulator (CFTR), was recently identified and sequenced (1,2). This protein consists of 12 transmembrane (TM) spanning domains, interspersed with three cytoplasmic domains, two of which contain putative nucleotide binding folds (NBF) (3). CFTR has a similar domain organization and sequence homology to a class of prokaryotic and eukaryotic transport proteins which comprise the ATP-binding cassette superfamily (4). The NBFs common to these proteins are thought to be critical to the energy-dependent transport of a particular substrate. Many of the identified mutations of CFTR, including the most common, AF508, cluster in the first NBF (5, 6).
Although the precise function of CFTR remains to be determined, studies suggest that it plays an important role in the expression of a CAMP-dependent C1-conductance, either as a C1-ion channel or a closely associated regulator of a C1channel. CF mutant epithelial cells were shown to have defective CAMP-mediated C1-conductance (7)(8)(9). Transfection of CFTR into CF epithelial cells corrected this defect (10, l l ) , whereas transfection of CFTR into nonepithelial cells conferred a new CAMP-dependent C1-conductance (12,13). Sitedirected mutagenesis of CFTR altered the anion selectivity of the CAMP-dependent C1-current, suggesting that CFTR is the C1-channel itself (14).
CFTR has been generally thought to be expressed only in epithelial cells (2). The question of tissue specificity, however, is controversial. For example, lymphocytes of CF origin have a similar defect in C1-transport. In phosphorylation studies the catalytic subunit of CAMP-dependent protein kinase activated outwardly rectifying C1-channels in patches excised from normal B (N-B) cell lines but not in patches excised from CF-derived B (CF-B) cell lines (15). Sustained depolarization, however, could activate C1-channels in both N-B and CF-B excised membrane patches. These results were completely analogous to results published for N and CF epithelial cells (7)(8)(9). It was further demonstrated by means of fluorescence digital imaging microscopy and whole cell patch clamp recordings that macroscopic C1-permeabilities in lymphocytes are defective in CF cells (16). These studies suggest that CFTR is expressed in lymphocytes as well as epithelial cells and furthermore lend credence to a longstanding but contro-

Gene
Transcription in Lymphocytes 3243 versial view that a primary immune abnormality exists in the disease (17)(18)(19). Several important questions about the expression of a CF defect in lymphocytes were raised, however. First, the phosphorylation experiments on excised patches were challenged as being unreproducible (20). Second, there was a question of the relationship of the outwardly rectifying C1-channel to CFTR, which itself is now believed to be a C1-channel with different characteristics (21,22). Third, following the cloning of the CF gene, it was reported that both RNA blots and reverse transcription PCR failed to show CFTR gene expression in lymphocytes (2, 23).
We initiated this study with two goals. First, by means of whole cell patch clamp recording, including the use of flash photolysis of 4,5-dimethoxy-2-nitrobenzyl-cAMP (caged-CAMP), we sought to compare regulatable C1-conductances in epithelial cells and N and CF lymphocytes. Second, by means of optimized reverse transcription PCR, we sought to determine whether CFTR message is present in lymphocytes. For epithelial cells, we chose the T84 colon carcinoma cell line, since there exists a complete description of C1currents regulated by CAMP, Ca2+ (21, 24)) and volume in these cells and since they are reported to have a high level of CFTR message (2, 11). We compared T84 cells with the Epstein Barr virus (EBV)-transformed N and CF B-lymphoblasts employed in the previously cited studies. The results suggest that epithelial cells and lymphoblasts have comparable regulatable C1-conductances, that CF-derived lymphoblasts are defective in CAMP-dependent C1conductance, and that the CFTR message is expressed in T and B lymphocyte cell lines.

EXPERIMENTAL PROCEDURES
Cell Culture-Jurkat T lymphocytes and the T84 colon carcinoma cell line were obtained from the American Type Culture Collection, Bethesda, MD. EBV-transformed B-lymphoblasts from normal (GM03299 and GM03714) and CF (GM07227 and GM04330) affected humans were from the Coriel Institute for Medical Research, Camden, NJ. Cells were grown as suggested by the suppliers. In some experiments lymphocytes were incubated for 24-48 h in culture media supplemented with 1 mM hydroxyurea for cell cycle synchronization as described by Bubien et al. (16).
Electrophyswlogy-T84 cells plated on glass coverslips or B lymphoblasts in suspension were transferred to the stage of an inverted microscope and bathed in an extracellular solution containing: 150 mM Tris-HC1, pH = 7.4, 2.5 mM CaC12, 1 mM MgC12, and 5 mM glucose. Patch clamp techniques were used to measure C1-currents in the whole cell configuration (25) using an Axopatch 1B amplifier (Axon Instruments, Burlingame, CA). Unless otherwise indicated, the pipette solution contained 140 mM CsC1, 4 mM M p -A T P , 10 mM HEPES, pH = 7.2, and 5.5 mM BAPTA with 0.5 mM CaC12 (estimated pCa = 8). Glass pipettes were coated with Sylgard (Dow-Corning, Midland, MI), and tip resistances in the above solutions were 2-4 megaohms. Unless otherwise indicated, osmolarity was adjusted by use of a vapor pressure osmometer (Wescor, Logan, UT) to maintain the extracellular solution 20-30 mM hypertonic to the intracellular solution in order to prevent volume-induced C1-current (24).
When volume regulation studies were performed, reduction of osmolarity was achieved by an approximate 1:3 dilution of the bath solution with distilled water. Ca2+-dependent C1-currents were measured after the addition of either 0.5 PM ionomycin or 1.0 PM A23187 to the bath solution. In this case, the pipette solution was altered to include 0.5 mM EGTA with no added Ca2+ instead of 5.5 mM BAPTA with 0.5 mM Caz+ as listed previously. Under these conditions the calculated [Ca2+li at rest was 10-50 nM and increased to 10-100 PM upon addition of ionophore. CAMP-dependent C1-currents were measured under two different protocols. In some studies, 400 PM 8-(4-chlorophenylthio) adenosine-3',5'-cyclic monophosphate (cpt-CAMP) was added to the 1-ml bath. Alternatively, cells were loaded with 4,5-dimethoxy-2-nitrobenzyl-cAMP (caged-CAMP), which is membrane-permeable, by adding it to the bath solution to achieve a final concentration of 175-375 PM. After at least 10-min equilibration, flash photolysis was performed by 1-ms flashes from a high pressure xenon arc lamp. Quartz optics were used to focus the flash lamp onto a 2 X 3-mm area of the recording chamber. A WG305 filter was used to eliminate wavelengths below 305 nm (26). The efficiency of photolysis of caged-CAMP was approximately 6% per flash assessed by high performance liquid chromatography.
During whole cell recording, membrane potential was clamped to -50 mV and stepped to levels between -100 and +lo0 mV using a Tecmar 16-bit AD/DA converter (Scientific Solutions) and a 80386base PC. Current signals were recorded on FM analog tape and digitized for later analysis on floppy disk. For determination of current-voltage (I-V) relations, C1-current amplitudes were measured as the average current during each voltage step beginning 10 ms after the start of each step. All patch clamp recordings were carried out at Reverse Transcri~tion-PCR-Sout~rn-Total RNA was isolated from approximately 1 X 10' cells using guanidinium thiocyanate lysis followed by CsCl centrifugation (27). One microgram of total RNA served as template for cDNA synthesis by avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) for 60 min at 42 "C in the presence of 25 pmol of a specific single-stranded 25-mer oligonucleotide as primer in a total volume of 20 ~1 (28). The reverse transcription primer was the same as the antisense primer to be used in the subsequent PCR reaction. One microliter of this reaction was then used as template in 40 cycles of amplification (30 s at 94 "C, 30 s at 55 "C, 60 s at 72 "C) with 25 pmol of each primer, 1 unit of Perfect Match (Stratagene), and 1 unit of Thermalase (IBI) in a Perkin-Elmer-Cetus thermal cycler. PCR products were separated on gels of 5% acrylamide or 1.2% agarose, stained with ethidium bromide, and photographed. The gels were denatured, neutralized, and the DNA was transferred by electrophoresis for acrylamide gels or capillary action for agarose gels onto Nytran nylon membranes (Schleicher and Schuell). Filters were UV-cross-linked (UV Stratalinker, Stratagene), prehybridized, and then hybridized at 37 "C for 6-18 h in the presence of a 32P-labeled oligonucleotide probe according to standard procedures (29). All oligomer probes were 25-mers designed to hybridize to a section of the target DNA not overlapping with either amplification primer.
Contamination Precautions and Controls-All primer pairs were designed to span exon boundaries to eliminate detection of any genomic DNA contamination. PCR primers were dispensed with a separate set of pipettors and were returned to storage before cDNA reaction mixtures were added. The positive control cDNA was removed from storage only after samples, and negative control tubes were complete and sealed. Lane spillover problems in loading gels were avoided by using a negative control (PCR reaction with H20 as template) between the positive control and samples.
Control RNA-Total RNA from a neuroblastoma cell line, SH-SY5Y (30), was the gift of Dr. Ralph Aarons (Stanford Department of Neurobiology).
cDNA Library-A Jurkat T cell cDNA library was the gift of Kyoko Yokota (DNAX Research Institute). Jurkat T cells had been stimulated by phorbol 12-myristate 13-acetate plus ionomycin prior to RNA harvest. The cDNA synthesis was primed with oligo(dT) and ligated into the pcD-SRa vector (31).
PCR Primer Sequences-Primer sequences were selected for maximum uniformity in order for a single optimized set of conditions to be used in all cases. Oligonucleotide sequences of 25 bases with approximately 50-60% G-C content, and at least one G and or C at the 3' end were selected. Putative primer sequences were then screened using the Intelligenetics Seq Program (Mountain View, CA) for dyad symmetries and hybridization to the other member(s) of the primer pair. Synthetic oligonucleotides selected as amplification primers and subsequently found to amplify CFTR from lymphocyte cDNA were as follows (identified by exon number/corresponding putative region of the gene and nucleotide position in the published CFTR sequence (2) used for each sequencing reaction. PCR products were acrylamide gel-purified, electroeluted, phenol/chloroform-extracted, precipitated with ethanol, and dried. The DNA was resuspended in 9 pl of water, and 1 pl of 2 M NaOH/20 mM EDTA was added. After 5 min at room temperature for denaturation, the sequencing primer was added and the mix was neutralized with 3 rl of 3 M sodium acetate, pH 5.2.
After ethanol precipitation, sequencing reactions were carried out from the labeling step using the Sequenase (U. S. Biochemical Corp.) kit. tonicity, only T84 cells and N-B cells responded to cAMP with an increase in C1-current. CF-B cells were defective in this response. We used two methods to evaluate CAMPdependent Cl-currents. In the first method, flash photolysis of caged-CAMP was employed. Typical results are shown in Fig. 2. T84 cells and N independent and linear to slightly outwardly rectifying, as described previously (15,20). The cumulative results for the two methods of elevating intracellular cAMP are presented in Fig. 3B. Both  Lymphocytes Express CFTR mRNA-RNA blot analysis was unsuccessful in detecting CFTR message in lymphocytes even using up to 10 pg of poly(A+) mRNA, whereas T84 colon carcinoma CFTR mRNA was readily detected on blots with only 1-5 pg of total cellular RNA. Lymphocyte RNA was then + CF-6 + tested using RT-PCR. When oligo(dT) was used to prime the reverse transcription, no CFTR signals could be detected in lymphocytes. When specific oligonucleotides were used as cDNA synthesis primers, signals were generated from several different regions of the CFTR gene, including all exons which encode the putative transmembrane regions of CFTR (Fig.  4).

C1-Conductance Regulation in T84, N-B, and CF-B Cells-
The most common CF mutation, AF508, is located in exon 10 within the putative first nucleotide binding fold (2). An initial set of primers failed to amplify this region from lymphocyte message. Because of its presumed significance in the function of CFTR, effort was focused on detecting this region. Ten more primers were synthesized from the region of exons 9-11, and they were tested in 12 combinations. Of these, six combinations amplified T84 cDNA as judged by an ethidium bromide-stained PCR product of the predicted molecular weight. Of these six primer pairs, a single pair which.spanned from exon 10 to 11, corresponding to NBF1, generated the specific fragment from both B and T lymphocytes (Fig. 4).
Similar difficulties were encountered in amplifying exons 5-6 (transmembrane regions [3][4]. In this region four primer combinations failed to amplify the lymphocyte message, whereas three of the four worked for T84 cDNA. Subsequently, the presence of exons 5-6 was demonstrated using primers spanning exons 3-7 and detecting the predicted product with an exon 6-specific probe. This finding confirmed that for this region, as for exons 10-11, the difficulties were a function of primer inefficiency and not of the absence of this sequence in lymphocytes. Exons 16-17, corresponding to transmembrane regions 11-12, were amplified from two separate plasmid DNA preparations of a cDNA library constructed from oligo(dT)-primed Jurkat mRNA (Fig. 5). Using primer pairs known to be efficient in detecting lymphocyte cDNA, regions upstream (5') of exon 16 could not be amplified from the library, presumably because the clone was truncated.
PCR products generated from two segments of the T lymphocyte CFTR message were sequenced and found to be   (T84). At the left, exons 6-7 were amplified, corresponding to the fifth and sixth transmembrane regions. In the center, exons 10-11 correspond to the first nucleotide binding fold, including the region of AF508. At the right, exons 14-17 correspond to the C-terminal six predicted transmembrane regions of the protein. All primer pairs spanned exon boundaries to rule out the possibility of genomic DNA contamination. Probes were 32P-endlabeled 25-mers corresponding to regions between and not overlapping with the amplification primers. The slower migrating material which hybridizes with the detection oligonucleotide is presumably single-stranded DNA generated by an imbalance of primers and/or differential efficiency of the two primers, based on observations that such bands were not visible with ethidium bromide staining of gels and appeared variably in repeated experiments.
identical to that of the CFTR message in epithelial cells (2) (Fig. 6).
Human RNA from a neuroblastoma cell line was tested as a possible negative control for CFTR expression, since neural tissue is believed to be unaffected in CF. Using the most reliable and efficient primer pair, exon 16 sense to exon 17 antisense, no CFTR mRNA could be detected from the neuroblastoma cell line (Fig. 5). A positive control for a ubiquitously expressed gene, actin, was readily amplified from the neuroblastoma RNA sample.  (7' cell library). The exon 16-17 primer pair was chosen in this case, because it was empirically found to be the most efficient pair of CFTR primers, reliably amplifying low level CFTR from several tissues. The slower migrating material which hybridizes with the detection oligonucleotide in the B-cell lane is presumably single-stranded DNA (see legend to Fig. 4).

DISCUSSION
The studies reported here have addressed the controversial area of functional defects in lymphocytes of CF patients. We have demonstrated the following results. First, N-B uersus CF-B cells appeared to recapitulate the phenotypes of normal uersus CF airway epithelial cells (33), with respect to inducible C1-currents. Specifically, CAMP-dependent C1-currents were absent in CF-derived cells, whereas Ca2+-and volume-dependent C1-currents were preserved. Second, CFTR was expressed in lymphocytes at levels detectable by reverse transcription PCR analysis. DNA sequencing of two of the amplified products through regions of exon boundaries in addition to hybridization of all products to CFTR-specific oligonucleotide probes confirmed their identity. Careful precautions against contamination, inclusion of negative controls in every experiment, and detection of a portion of the gene in a T cell cDNA library strongly suggest that the CFTR gene is transcribed in lymphocytes. The inability of this optimized mRNA detection protocol to identify CFTR in a neuroblastoma cell line suggests that low level expression in lymphocytes may represent tissue-specific transcription. The overall conclusion is that CFTR is present in lymphocyte cell lines and that it confers a CAMP-dependent C1-conductance that is defective in CF. Cell cycle synchronization by hydroxyurea for 24 h prior to RNA harvest showed no differences in CFTR message by the nonquantitative technique of RT-PCR and no differences in base-line or CAMP-inducible C1-current.
The electrophysiological results reported herein are in general agreement with Bubien et al. (16). Both studies document Ca2+-and CAMP-dependent C1-currents in normal lymphocytes by the whole cell patch clamp technique and find a defect in CAMP-dependent C1-currents in CF-derived B cells. There are two major differences in the findings of the two studies, however. First, the study of Bubien et al. (16) documented cell cycle-dependent differences in C1-permeabilities that were not seen in the present study. The two studies were not directly comparable on this point, however. Bubien et al. FIG. 6. Schematic diagram of lymphocyte CFTR message detection. Dark lines below the schematic of the predicted CFTR gene product indicate PCR products the identities of which were verified by hybridization to probes between and not overlapping with amplification primers. The number at the end of each segment detected indicates the exon in which the primer used is located. Two different sense primers were used for exon 16 (labeled 16 and 16'). Hatched bores indicate regions through which lymphocyte PCR products were sequenced and found to be identical to the predicted structure. The double-headed arrow indicates the segment detected in each of two separate plasmid DNA preparations from a T cell cDNA library. Abbreviations: NBF, nucleotide binding fold; R, regulatory region; A, AF508, the most common CF mutation (exon 10). droxyurea treatment eliminated the heterogeneity of PC, characteristic of unsynchronized populations. GI-S phase CF lymphocytes were impermeable to C1-. In the present study we examined synchronized and unsynchronized normal B cells by whole cell patch clamp rather than FDIM and observed no difference in base-line or CAMP-inducible C1-currents. It is possible that the cell cycle-dependent macroscopic PC, documented by Bubien et al. (16) is attributable to other C1transport systems which are measured in the aggregate by FDIM. Alternatively, the differences in the two studies may be attributable to differences in the technique of cell cycle synchronization.
A second difference between the study by Bubien et al. (16) and the current study occurred in respect to Ca2+-dependent C1-conductances. In their study Ca2+-dependent C1-conductances were defective in CF-derived B cells, whereas in our study the Ca2+-dependent C1-conductances were preserved. The result was reliably (10/11 cells tested) obtained with either CsCl or NMDG-C1 as the principal intracellular salt. Ca2+-dependent C1-permeability is known to be preserved in CF-derived airway and sweat duct epithelial cells, whereas it is reportedly defective in CF-derived colonic epithelial cells (33)(34)(35)(36). In both epithelial cells and lymphocytes, Ca2+-dependent C1-secretion is mediated by Ca2+/ca1modulin-dependent protein kinase (32,33,37), which phosphorylates a C1-channel that is apparently distinct from CFTR (21). Based on this data, it does not necessarily follow that Ca2+-dependent C1-secretion would be directly affected by CF. Tissue-specific indirect effects cannot be ruled out, however.
Our results also contrast with the published data concerning the expression of CFTR message in lymphocytes. The most likely explanation for difficulty in detecting this message in lymphocytes is the low amount of CFTR mRNA, which we estimated by RNA blots and cDNA dilution experiments to be at least 100-fold lower than that in the positive control, T84. This low level of expression coupled with the large size of the message necessitated careful optimization of cDNA synthesis including the use of specific antisense primers instead of oligo(dT). PCR primer efficiency appeared to be the major determinant of success or failure in detecting regions of lymphocyte CFTR, as some primer pairs which amplified regions of T84 cDNA failed for lymphocytes, whereas other sets of primers successfully detected the same region in lymphocytes. Primer inefficiency appeared not to be due to dyad symmetry, primer-dimer formation, variable length, or G-C content as attempts were made to eliminate these possible inconsistencies (see "Experimental Procedures").
The significance of primer pair efficacy was especially pronounced in the region of exons 9-11 (NBF), in which amplification of CFTR was more difficult in both T84 cells and lymphocytes. Of 12 primer pair combinations in the exon 9-11 region, it was surprising that only six worked well even on T84 cDNA. Interestingly, all but two of the primers worked in at least one combination with another of the primers, suggesting that the quality of individual primers was not the problem. However, no primer in this region amplified T84 cDNA in all possible pairings, and only one pair was efficient enough to detect the NBFl region in lymphocyte cDNA. Problems with the quality of the RNA isolated from the tissues or of reagents such as enzymes or nucleotides were ruled out as these experiments were done in parallel with analysis of other regions of CFTR mRNA which were easily detected in lymphocytes. The difficulty in detecting this region may reflect some sequence feature which inhibits efficient polymerase activity.
Although the CFTR message appears to be present at very low levels, its functional significance is supported by the demonstration of alterations in C1-conductances in CFaffected lymphocytes. The number of molecules of CFTR protein is regulated not only by steady-state message levels but also by the half-life of the protein; furthermore, there may be post-transcriptional steps regulating CFTR expression in the lymphocyte which have not yet been discovered. Although it is not known how many CFTR molecules per cell might be required for functional significance, our data clearly demonstrate that the functional defect is present in the CF-affected lymphocyte. Other systems, such as cytokines and hormones, exist in which very low levels of message and protein show strong biological effects. Even an abundant cytokine message such as IL-2 can only be detected in normal cells by extensive amplification, even though this message can be seen by RNA blot analysis in cell lines which have been selected for high IL-2 expression (36). Yet IL-2 is certainly functional in normal lymphocytes. And rare cytokines such as IL-7 are difficult to detect after extensive amplification?
Although our study demonstrates transcription of CFTR in lymphocytes, the question remains whether the message in lymphocytes is identical over its entire length to that of epithelial cells. Alternative splicing of the CFTR gene has been reported in epithelial tissues (38). We found no evidence, however, of an alternative splicing pattern within the approximately 2 kilobases of the message which we detected (exons 3-7, 10-11, or 14-17), although we cannot rule out the possibility of alternative splicing elsewhere in the gene. Complete determination of the structure of the lymphocyte CFTR message awaits the isolation of a full-length clone.
Mechanisms by which a chloride permeability defect could lead to an altered immune response have been suggested by studies on the role of chloride ion regulation in lymphocytes. Studies on C1-conductance regulation have shown that it plays an important role in diverse processes including cytolysis by CD8+ T cells (39,40), lymphocyte volume regulation (41, 42), and the Ca2+ influx associated with T cell activation (43). Consistent with these studies and the findings reported here, clinical studies have shown altered immune responses in CF. CF patients are reported to have significantly lower helper and cytotoxic T cell function (18, 19) and hypogammaglobulinemia (17). It has not been possible, however, to determine whether these immunologic abnormalities predispose the patient to the severe infections characteristic of CF or are secondary to such infections. Further study of CFderived lymphocytes will likely be necessary in order to define a functionally significant primary immune defect.
In summary, we show that normal B-lymphoblasts have chloride conductances similar to those that can be stimulated in epithelial cells. cAMP does not regulate a C1-current in CF-derived lymphoblasts, although other methods of C1-current stimulation are intact. In addition, we show that the CFTR gene is transcribed in normal B and T lymphocytes at levels below those of epithelial tissues, but above an undetectable level in a neuroblastoma cell line. This low level of CFTR transcription is apparently sufficient to confer cAMP regulation of C1-current in lymphocytes. These results reconcile conflicting reports in the literature and are consistent with the demonstration of a chloride permeability defect in CF-derived lymphocytes.