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The Journal of Immunology, 2006, 176: 1978-1987.
Copyright © 2006 by The American Association of Immunologists

TLR9 Activation Is Defective in Common Variable Immune Deficiency1

Charlotte Cunningham-Rundles2, Lin Radigan, Adina K. Knight, Li Zhang, Laura Bauer and Atsushi Nakazawa

Department of Medicine and the Immunobiology Center, Mount Sinai Medical Center, New York, NY 10029


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Common variable immune deficiency (CVID) is a primary immune deficiency characterized by low levels of serum immune globulins, lack of Ab, and reduced numbers of CD27+ memory B cells. Although T, B, and dendritic cell defects have been described, for the great majority, genetic causes have not been identified. In these experiments, we investigated B cell and plasmacytoid dendritic cell activation induced via TLR9, an intracellular recognition receptor that detects DNA-containing CpG motifs from viruses and bacteria. CpG-DNA activates normal B cells by the constitutively expressed TLR9, resulting in cytokine secretion, IgG class switch, immune globulin production, and potentially, the preservation of long-lived memory B cells. We found that CpG-DNA did not up-regulate expression of CD86 on CVID B cells, even when costimulated by the BCR, or induce production of IL-6 or IL-10 as it does for normal B cells. TLR9, found intracytoplasmically and on the surface of oligodeoxynucleotide-activated normal B cells, was deficient in CVID B cells, as was TLR9 mRNA. TLR9 B cell defects were not related to proportions of CD27+ memory B cells. CpG-activated CVID plasmacytoid dendritic cells did not produce IFN-{alpha} in normal amounts, even though these cells contained abundant intracytoplasmic TLR9. No mutations or polymorphisms of TLR9 were found. These data show that there are broad TLR9 activation defects in CVID which would prevent CpG-DNA-initiated innate immune responses; these defects may lead to impaired responses of plasmacytoid dendritic cells and loss of B cell function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Common variable immunodeficiency (CVID)3 is a primary immunodeficiency disease with the hallmarks of low levels of serum IgG and IgA and/or IgM, and Ab deficiency to both T-dependent and -independent Ags (1, 2, 3, 4). Many patients have recurrent sinopulmonary infections, but some have gastrointestinal inflammation or autoimmune disease as the first symptom (1, 5, 6). CVID has been viewed as a heterogeneous group of genetic disorders, because in addition to the clinical variability and range of age at diagnosis, patients have T cell-, cytokine-, and monocyte-derived dendritic cell defects, in addition to the profound lack of Ab production (7, 8, 9, 10, 11, 12, 13, 14). There has been an intensive search for genes which, when mutated, could lead to the CVID phenotype. This search has led to the discovery that mutations of the inducible costimulatory in a related kindred (15, 16)–or more recently, TNF family members, B cell-activating factor receptor, or transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)–can lead to autosomal recessive or dominant B cell defects of this phenotype or, for TACI, also IgA deficiency (17, 18, 19). However, for the great majority of patients with CVID, no molecular basis has yet been provided.

Because the most fundamental defect in CVID is a lack of maturation of B cells into functioning plasma cells, recent studies have focused on somatic hypermutation of IgG genes, a process which normally occurs in germinal centers and leads to the selection of long-lived, high-affinity immune globulin-secreting B cells. In CVID, IgG-secreting cells often lack somatic hypermutations (20, 21, 22), which is also closely related to both reduced numbers of memory B cells (CD27+) (23) and paucity of (switched memory) CD27+IgD–IgM– peripheral blood B cells (24). One of the most potent mechanisms for triggering B cells to proliferate and mature into plasma cells, in the absence of Ag, is oligonucleotides containing selected CpG-DNA sequences (CpG oligodeoxynucleotides (ODN)) (25). Unmethylated DNA in specific sequence contexts is known for its potent immunostimulatory activity; a single nucleotide substitution or methylation of a cytosine residue within this motif blocks the stimulatory effect. CpG-DNA ODN of type B (CpG-B), which contains phosphorothioate backbones, activate human B cells which then increase surface expression of essential costimulators CD86 and MHC class II. CpG-DNA-activated B cells proliferate, produce cytokines, and become high secretors of immune globulins (26, 27, 28). CpG-DNA, especially in combination with IL-10, initiates germline C{gamma}1, C{gamma}2, and C{gamma}3 gene transcription and induces IgG class switch DNA recombination (29). Cellular recognition of CpG-DNA depends solely on the presence and function of TLR9, a member of the conserved family of TLRs, pattern recognition structures involved in mediating innate immunity (30).

TLRs are differentially expressed on B cells; while naive human B cells express TLRs at low levels, memory B cells, characterized by the presence of CD27 (31, 32), express several TLRs, especially TLR9 at constitutively higher levels (33). As a result, memory B cells have greater responses to the stimulating effects of CpG-DNA than naive B cells (33, 34). These data suggest that selected bacterial or viral DNAs, interacting with memory B cell TLR9, might be an important means whereby long-term B cell memory could be sustained and periodically reinforced, in the absence of the specific inciting Ag (34, 35).

Aside from expression on B cells, the expression of TLR9 is also a characteristic of the plasmacytoid dendritic cell (PDC), which is also highly responsive to CpG-DNA stimulation. Activation of PDCs by the constitutively expressed TLR9 results in cellular maturation and secretion of large amounts of type 1 IFN-{alpha} and IFN-beta. Although CpG-DNA sequences in the CpG-B conformation stimulate B cells, PDCs are responsive to ODNs with CpG motifs arranged on different backbone structures, designated CpG-A and CpG-C (36, 37). Once activated, PDCs become potent APCs and immune stimulators which prime T cells, direct Th1 polarization, and induce cytotoxic T cell responses and the development of CD4+CD25+ T regulatory cells (38, 39, 40). Type 1 IFNs not only sponsor and drive T cell immunity, but the secreted IFN-{alpha} also enables B cells to undergo isotype switch and mature into Ab-secreting plasma cells (41). Thus, TLR9 signaling plays both a direct role on B cells and an indirect role involving PDCs, in both initiating and supporting humoral immunity.

Our previous work showed that B cells of CVID patients had an impaired ability to proliferate or secrete immune globulins, if stimulated with an ODN-DNA corresponding to the rev gene of the HIV virus (42), an oligonucleotide now known as an early example of an CpG-DNA of type B (26, 43). In the current experiments, we hypothesized that the lack of B cell activation in response to CpG-B ODNs could to be due to the relative lack of CD27+ memory B cells. However, we found that B cells of the CVID subjects had pronounced TLR9 defects, regardless of B cell memory phenotype. CpG-B did not activate the CVID B cells even if costimulated by the BCR. TLR9 was insufficiently expressed on the B cell surface after activation, both intracytoplasmic TLR9 protein and mRNA were deficient, and CpG-DNAs did not promote CVID B cell IL-6 or IL-10 production. These defects were not restricted to B cells, as PDCs did not produce IFN-{alpha} in normal amounts after exposure to appropriate CpG-DNAs even though these cells expressed intracytoplasmic TLR9 in amounts similar to PDCs of normal controls. These results demonstrate that patients with CVID have broad TLR9 activation defects, resulting in impaired CG sponsored innate immunity, with potential consequences for both B cell and PDC function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and controls

A group of 32 patients with CVID (ages 18–71) were evaluated (Table I). All were healthy at the time of study. There were 16 males and 16 females. Subjects who had other causes of congenital or acquired hypogammaglobulinemia were excluded; none had relatives known to have CVID or IgA deficiency. All had reduced serum IgG, IgA, and/or IgM two or more confidence intervals below the normal ranges for age; Ab deficiency was verified by lack of protective levels of Ab to tetanus, diphtheria, measles, mumps, rubella, and pneumococci after vaccination. The control group included 35 healthy adult volunteers and normal blood bank donors.


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  		Table I. Immunologic profiles of patients

 
Isolation and culture of cells

Heparinized peripheral blood was obtained from controls or CVID patients using an Institutional Review Board approved informed consent. PBMCs were isolated by Ficoll-Hypaque (Pharmacia) density gradient centrifugation. In some experiments, T cells were removed from PBMC by rosetting with sheep RBC, and B cells were negatively isolated using RosettSep (Stem Cell Technologies) yielding preparations that were 86–90% B cells. Human PBMC were incubated (2 x 105/ml) and activated in cultures with various amounts (0.1, 0.3, 1.3, 3, or 10 mg/ml) of CpG-B ODN 2006 or as a control, CpG-B ODN 2117 which contains methylated cytosines (27). In other experiments, an optimum amount of ODN 2006 (0.6 mg/ml) was added to cultures with 10 µg/ml goat F (ab')2 anti-human µ-chain Ab (Calbiochem) to cotrigger the BCR. PDC were isolated using Ab on microbeads to PDC Ag (BDCA-4; Neuropilin-1) according to the manufacturer’s protocol (BDCA-4 cell isolation kit; Miltenyi Biotec). PDCs (5 x 105/ml) were cultured in 100 µl with 0.6 µg/ml ODN CpG-A 2336 or its control, 2243, or CpG-C 2395 or control 2137, for 48 h; cell culture supernatants from these and a media control, were batched for quantitation of IFN-{alpha}. All cell cultures were performed in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, 1.5 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin.

Cytokines

IL-6 and IL-10 produced by B cells stimulated with a range of concentrations of CpG-B 2006, were quantitated using BD Opt EIA kits (sensitivity 47–3000 pg/ml; BD Biosciences). IFN-{alpha} produced by PDCs was quantitated using sandwich ELISA reagents (Bender MedSystems) with a sensitivity of 30–3000 pg/ml, diluting supernatants as required.

Abs and flow cytometry

Numbers of B cells, memory and switched memory B cells, were determined for CVID subjects and controls using freshly isolated PBMCs (2.5 x 106/50 µl in RPMI 1640 medium (Invitrogen Life Technologies) plus 10% FCS) incubated at 20 min at 4°C with 10 ml of the following premixed Abs at optimal concentrations: CD27-FITC (DakoCytomation), CD19-PC5, or CD22 (Coulter-Immunotech), anti-IgD-PE (Southern Biotechnology Associates), and anti-IgM-Cy5 (Jackson ImmunoResearch Laboratories). Four-color data acquisition was performed with a FACSCalibur (BD Biosciences). Data analysis (CellQuest; BD Biosciences) was performed by forward vs side scatter, gating on viable lymphocytes, in combination with gating on CD19+ and CD22+ cells. The percentages of naive, and switched (IgM–IgD–), memory B cells were calculated. Patients were grouped on the basis of the number of switched memory B cells as previously defined; those with 0.4% or less of switched memory B cells, as a percent of PBMC, were classified in group 1, and those with more than this number were classified in group 2 (24). B cell surface expression of CD86 using FITC-conjugated Ab was determined before and after culture with ODNs, with or without goat anti-µ Ab. PDCs were examined using conjugated anti-human Abs, CD4-FITC, anti-MHC class II- PE and-CD11c-Cy5 (Coulter-Immunotech). Cell surface and intracellular expression of TLR9 was determined on both intact and permeabilized B cells and permeabilized PDCs. For intracellular staining, cells were washed, suspended in PBS containing 0.1% saponin with 5% nonfat dry milk for 30 min, and washed in PBS containing 0.1% saponin with 1% FBS. To detect TLR9, flow cytometry was performed using PE-mouse anti-human TLR9 (clone 26C593; Imgenix), gating on B cells by anti-CD19 or on PDCs using FITC-anti-CD86 using the corresponding isotype controls (BD Pharmingen).

Oligonucleotides

CpG oligonucleotides were obtained from Coley Pharmaceutical Group (25, 26, 36). CpG-B ODN 2006 has a fully phosphorothioate backbone: TCG TCG TTT TGT CGT TTT GTC GT; control ODNs 2117 or 2137 contain methylated cytosines which lead to inactivation. CpG-A oligonucleotide 2336 has the sequence G*G*G GAC GAC GTC GTC GTG G*G*G*G*G*G; the CpG-A control is ODN 2243, sequence G*G*G GGA GCA TGC TGG* G*G*G*G*G (G* indicates phosphorothioate bonds, the rest are phosphodiester bonds). CpG-C oligonucleotide 2395 has the sequence TCG TCG TTT TCG GCG CGC GCC G (fully phosphorothioate backbone); the CpG-C control is ODN 2137, sequence TGC TGC TTT TGT GCT TTT GTG CTT (fully phosphorothioate backbone).

Real-time RT-PCR

Total RNA was isolated from purified, freshly isolated and lysed B cells (on average 90% pure) using an RNeasy Mini kit (Qiagen). First-strand cDNA was synthesized from 0.5 mg of RNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies). Two microliters of cDNA was used per 20-µl reverse transcriptase reaction. The reaction was conducted using the LightCycler FastStart DNA Master SYBR Green I kit from (Roche Applied Science) according to manufacturer’s instructions using parameter- specific primer sets optimized for the LightCycler. Each sample was measured in triplicate. The RT-PCR primers used were as follows: TLR9, sense: 5'-CCTTCGTGGTCTTCGACAAAAC-3', antisense: 5'-TTGTACACCCAGTCTGCCACTG-3'; product size 52 bp (human TLR9 cDNA: GenBank code AF259262); beta-actin, same product size, sense: 5'-GGACTTCGAGCAAGAGATGG-3'; antisense: 5'-CTTCTCCAGGGAGGAGCTG-3' (GenBank code: X00351) (33). A range of primer concentrations was tested to identify optimum amplification efficiency. The amplification was determined by plotting against the threshold concentration. TLR9 mRNA copy number was normalized with beta-actin and expressed as copy number per microgram of RNA added to the reaction. The specificity of the amplification was controlled by analysis of the melting curve analysis and crossing points. No amplification of nonspecific products was observed.

TLR9 DNA sequencing

The TLR9 gene spans 5 kb with two exons, the second of which is the major coding region. Peripheral mononuclear cells were isolated from the blood of 20 CVID subjects, chosen because each had <0.4% peripheral blood switched memory B cells; CD27+CD19+ B cells in these subjects ranged between 0.2 and 40% of total B cells. Peripheral cells were lysed; genomic DNA was isolated using Puregene DNA Purification kits (Gentra Systems) and then treated with RNase. Approximately 1500 bases of exons 1 and 2 were PCR amplified from cDNA using HotStarTaq DNA Polymerase (Qiagen). TLR9 was PCR amplified for 35 cycles using primers for sequences from 502 to 2252 (forward primer, 5'-AAA ATC TTA CTT CCT CTA TTC TCT GAG CCG-3' and reverse primer, 5'-AGT GCT CGT GGT AGA GGT CCA GCT TAT TGT-3') and second sequences from 2004 to 3843 (forward primer, 5'-CAG TGG ACA CTC CCA GCT CT-3' and reverse primer, CTG CTC TGT GTC AGG TGT GG-3'). PCR products were visualized and isolated from 1% agarose gels using 0.8–4.0% ethidium bromide under UV transillumination. DNA amplicons were sequenced on an ABI PRISM 377 DNA Sequencer (Applied Biosystems). Sequences were aligned to the wild-type TLR9 sequence (44) using DNAStar software.

Statistical analysis

Data are expressed as median values, ranges, and/or 10th and 25th (interquartile) percentiles. To compare data for CVID to controls, the Mann-Whitney U test was used. For determination of correlations, the Spearman correlation test was used. Statistical differences were determined by the paired Student t test. Differences between groups were considered significant at p < 0.05. Statistical analyses were performed using StatView 4.51 software (Abacus Concepts)


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CVID patients and B cell populations

CVID subjects had variable but overall reduced numbers of CD27+ B cells, with a median percentage of 14.8% as compared with 35 normal controls who had a median of 29.9% of CD27+ B cells, which is similar to that previously described in CVID (23). Table I gives percentages of peripheral blood B cells, absolute lymphocyte counts, memory (CD27+) B cells, and switched memory B cells for patients. Using the percentage of isotype-switched memory B cells (CD27+IgD–IgM–) to categorize these subjects (24), 23 of the 32 subjects (10 males and 13 females) had <0.4% CD27+IgD–IgM– B cells (group 1). Nine other subjects (6 males and 3 females) had >0.4% switched memory B cells (group 2). Normal controls had 19–34% CD27+IgD–IgM– B cells, or 1.1–2.9% of the total PBMC population, which is similar to previously described normal values (23, 24, 31).

Defective activation of CVID B cells by ODN CpG-B

Phosphorothioate ODNs with specific CpG motifs (ODN type B) activate normal human B cells and up-regulate surface CD86 (27, 36). Testing a range of concentrations of CpG-B ODN 2006, B cells of normal subjects exhibited the expected increase in CD86 as measured by mean fluorescence intensity (MFI) but had no increase after exposure to the corresponding control CpG DNA, ODN 2117 (top panel, Fig. 1a). However, in cultures under the same conditions, B cells of CVID subjects (Fig. 1a, lower panel) expressed little or no enhancement of surface CD86 expression. Fig. 1b shows a dose response experiment for eight representative CVID subjects (of 23) in comparison to 7 (of 21) control subjects tested in parallel. CVID B cells did not attain the level of activation found for B cells of normal controls at any CpG concentration. Although CVID CD27+ B cells might be more likely to respond to this TLR9 stimulator, there was no correlation between the numbers of CD27+ B cells in peripheral blood of the CVID subjects tested and peak expression of CD86 after ODN activation (r = 0.389; p = 0.16).


Figure 1
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  		FIGURE 1. a, B cell activation with CpG: MFI for CD86+CD19+ B cells after culture with 0.6 µg/ml ODN2006 (open area) as compared with the control ODN 2117 (shaded area.) Comparing a CVID subject (lower panel) with a control (NL top panel), adding ODN 2006 does not increase surface CD86 expression. b, B cell activation with CpG: dose-response curve testing a range of concentrations of ODN2006, 0.37–10 µg/ml, shows very little activation for 8 representative CVID subjects of 23 tested, as compared with 7 controls at any concentration.

 
Lack of augmentation of activation by CpG with the BCR

Stimulation of normal B cells by type B CpG DNA coupled with triggering of the BCR, synergizes the TLR9 activation and augments the up-regulation of surface CD86 (34, 45). Although CVID B cells displayed less activation than normal B cells when cultured with anti IgM alone, CVID B cells showed much less augmentation of CD86 when stimulated by the potent-activating combination, ODN2006 with anti-IgM, than normal B cells (Fig. 2). (The median CD86 MFI for CVID B cells cultured with ODN 2006 + anti-IgM activation was 130.0; for control B cells, the median MFI was 283.2; differences were significant, p = 0.002.) A control CpG-DNA of type B, 2137, was similarly tested; as expected, it was less effective than ODN 2006 in activating normal B cells.


Figure 2
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  		FIGURE 2. Activating with CpG and BCR stimulation: MFI of CD86 on CD19+ B cells was tested after culture with 0.6 µg/ml ODN 2006 or the ODN control 2137, or with 1 µg/ml F(ab')2 of µ-chain-specific goat anti-human IgM, or with both anti µ-chain and ODN 2006, for 48 h. CVID subjects (n = 26) (left panel) are compared with 18 normal controls (right panel). The median (center line), 10% of the range (box), 25% percentile lines, and all outliers are shown for each condition.

 
Lack of CpG induced production of cytokines IL-6 or IL-10

Normal B cells cultured with type B CpGs produce both IL-6 and IL-10 (37, 46). Testing PBMCs from subjects with CVID using a range of concentrations of CpG-B ODN 2006, neither IL-6 (Fig. 3a) nor IL-10 (Fig. 3b) were produced in the amounts generated by similarly tested PBMCs from normal donors. These cytokines are largely produced by B cells, as demonstrated by the inclusion of PBMCs of one subject with X-linked agammaglobulinemia and no B cells; little or no IL-6 or IL-10 was found in these cultures. The percent of B cells in blood of the CVID subjects tested was between 5 and 24%, and 0.2–42.9% of these B cells were CD27+; however, there was no correlation between production of either cytokine (at any ODN concentration) and the percent of B cells, absolute number of B cells, CD27+ B cells, or isotype-switched CD27+ B cells. As expected, the control ODN CpG-B 2137 did not cause either normal or CVID B cells to produce amounts of IL-6 or IL-10 in the ranges found for ODN 2006 (Fig. 3, c and d).


Figure 3
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  		FIGURE 3. a, Lack of IL-6 production: measuring IL-6 in picograms per milliliter after activating PBMC of 14 CVID subjects (–{circ}–) by varying concentrations of ODN2006 (0–3.3 µg/ml) in comparison to PBMCs of 5 normal controls (–{blacksquare}–) for 24 h showed that CVID cells produced very little IL-6. The number of CD27+ B cells in the CVID cultures ranged from 0.2 to 42.9%, but there was no relationship between the number of B cells or CD27+ B cells and IL-6 produced. A PBMC sample from a patient with XLA (no B cells) is included as a control (–{blacktriangleup}–). b, Lack of IL-10 production: similar results were found for other cultures in which IL-10 was measured for stimulated cultures of 16 CVID subjects and 6 normal controls. A PBMC sample from a patient with XLA is also included as for a. c, Control type B ODN and IL-6: very little IL-6 production by PBMCs of 9 normal controls and 8 CVID subjects’ PBMCs cultured with ODN-B control 2137. d, Control type B ODN and IL-10: very little IL-10 production by PBMCs of 8 normal controls and 7 CVID subjects’ PBMCs cultured with ODN control 2137.

 
Deficient B cell surface expression of TLR9

TLR9 is primarily an endosomal protein detecting CpG-DNA ligands only as these compounds become internalized (47); however, a recent study showed that TLR9 can also appear on the cell surface of some tonsillar lymphocytes, especially after LPS activation (48). Investigating the expression of TLR9 on activated peripheral B cells, we found that a subpopulation of B cells from control subjects exposed to CpG-B (2006) for 24 h do develop detectable surface TLR9; up to 29% of B cells (median 19.6%; range 12.3–29.5%) of 10 normal controls became TLR9 surface-positive. However, after exposure to CpG-B 2006, significantly fewer CVID B cells became surface TLR9+ (median 8.1%; range 1.9–25%) (p = 0.001). Fig. 4 shows the CpG-B 2006 responsive TLR9+ B cell populations for two CVID subjects (of 22 subjects tested) in contrast to B cells of a normal subject. The top panels show results for unstimulated B cells and the lower panels, cultures of CpG-stimulated B cells. B cells of CVID subjects expressed little or no TLR9 after CpG exposure, in contrast to the controls. (For the normal control shown here, 18% of B cells became TLR9+). Because previous studies suggested that B cell TLR9 mRNA may be more abundant in CD27+ memory B cells (33, 34), CD27+ and CD27– cells were examined separately; for both CVID and control subjects, surface TLR9 was mostly a feature of CD27+ B cells and not CD27– B cells (Table II). However, for CVID subjects, the percent of CD27+ B cells in peripheral blood was not correlated to the number of TLR9+ B cells after activation with CpG-B 2006 (22 subjects: r = 222; p = 0.342).


Figure 4
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  		FIGURE 4. CD27+ B cell surface expression of TLR9: CD27+ B cells not cultured with ODN2006 (top panels) or after culture (bottom panels) with ODN2006 were examined for surface TLR9. CpG-activated B cells of two CVID subjects are shown in comparison to similarly activated B cells of a normal control. The percentage of TLR9+ B cells for each is given. CVID subjects had a median of 8.1% TLR+ B cells (interquartile range 8.8%, range, 1.95–25%) in contrast to the control subjects, with a median of 19.6% TLR+ B cells (interquartile range 9.3% and range 12.3–29.5%). For 22 CVID subjects tested, the percentage of TLR9+ B cells after activation with ODN was not correlated to the number of CD27+ B cells in peripheral blood.

 

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  		Table II. Percent CD27+ and CD27– B cells positive for surface TLR9 in the resting or CpG-activated state

 
Reduced intracellular TLR9 in peripheral B cells

Because most TLR9 is found in the endosomal compartment, freshly isolated CVID and normal B cells were examined for intracytoplasmic TLR9. These analyses demonstrated that CVID peripheral blood B cells had significantly less detectable intracellular TLR9 than B cells of normal donors. Fig. 5 shows results for six representative patients shown here because they had highly variable numbers of peripheral blood CD27+ B cells (0.2–46%); while the amounts of intracellular TLR9 were also variable, none approached the amounts found in B cells of control subjects. Because CD27+ B cells may contain greater amounts of TLR9 mRNA (34), CD27+ and CD27– B cell populations were again examined separately. For CVID subjects, the CD27+ B cells contained less stainable TLR9 than CD27+ normal B cells and the differences between CD27+ and CD27– B cell populations were smaller for CVID subjects. For controls, CD27+ B cells had a median of 1.4 times more TLR9 (MFI) as compared with CD27– B cells while CVID CD27+ B cells had a median of 1.1 times more TLR9 than CD27– B cells (data not shown).


Figure 5
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  		FIGURE 5. B cell intracytoplasmic TLR9: intracytoplasmic TLR9 in permeabilized peripheral blood B cells for CVID subjects (green) was compared with the B cells of normal controls (purple). Results for six representative CVID subjects and the controls tested at the same time are shown indicating the MFI for TLR9 (isotype controls are pink). The percentage of CD27+ B cells in peripheral blood is given for each CVID subject. Isotype controls are shown at the left of each panel. B cells of all CVID subjects contained less intracytoplasmic TLR9 than normal controls tested in parallel. There was no relationship between TLR9 and the number of CD27+ B cells.

 
Deficient TLR9 mRNA in B cells

Isolated B cells were examined for TLR9 mRNA by real-time PCR. Fig. 6 shows results for 12 CVID subjects (chosen because they had a range of CD27+ B cells, 1.4–38.6%) in comparison to 10 normal controls with CD27+ B cells in the range of 25–32%. Although one subject (Table I, patient no. 8 with only 9% B cells, and 4% CD27+ B cells) had an amount of TLR9 mRNA similar to normal controls, the B cells of CVID subjects overall had significantly less TLR9 mRNA than the control group (p = 0.004). The amount of B cell TLR9 mRNA was unrelated to the number of B cells, CD27+ B cells, or switched memory B cells in peripheral blood (Table III.)


Figure 6
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  		FIGURE 6. Real-time TLR9 mRNA CVID and control B cells: TLR9 mRNA copy number for isolated peripheral blood B cells (90% pure populations) from 11 CVID and 10 controls are given. TLR9 mRNA was standardized to actin to determine the copy number of micrograms of RNA added to the reaction. The median values are indicated. Results are significantly different (p = 0.002).

 

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  		Table III. TLR9 mRNAa and CD27+ B cell populations

 
Abundant TLR9 in PDCs but lack of IFN-{alpha} production after CpG treatment

Normal PDCs express abundant TLR9 and exposure to CpG DNA leads to activation, maturation, and production of large amounts of type 1 IFNs (49). Isolated CVID PDCs were found phenotypically similar to PDCs isolated from the blood of control subjects (CD4+, MHC class II+, CD11c–), and these contained similar amounts of intracytoplasmic TLR9 based on MFI. To illustrate, Fig. 7a shows intracytoplasmic TLR9 in PDCs from four CVID subjects in comparison to PDCs from four control subjects tested at the same time; cells of all CVID subjects tested contained substantial and similar amounts of TLR9 as compared with the controls. However, on culture with appropriate ODNs, CVID PDCs were defective in cytokine production (Fig. 7b). On exposure to an ODN of type A or C, CVID PDCs produced somewhat variable, but overall strikingly less IFN-{alpha} than PDCs of normal donors (differences for ODN type A, p = 0.0005; ODN type C, 0.0002). As a further control, PDCs of three subjects with X-linked agammaglobulinemia (XLA) were also tested; PDCs from these patients produced the same amounts of IFN-{alpha} as normal controls and thus significantly more IFN-{alpha} as compared with CVID subjects (ODN type A, p = 0.0071; ODN type C, 0.0017). (The one subject who had more normal PDC IFN-{alpha} production was patient no. 27, Table I). ODN-CpGs of type B do not have such a powerful effect on PDCs. However, as illustrated here, ODN2006 also had less stimulating capacity on CVID PDCs than on normal or XLA PDCs (comparing control or XLA PDCs, differences are p = 0.0009). Using the ODN controls for the type A or C ODN-CpGs resulted in little or no production of IFN-{alpha}.


Figure 7
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  		FIGURE 7. a, Intracellular TLR9 in plasmacytoid DCs: PDCs of four representative CVID subjects (green) contain the same amounts of intracellular TLR9 in comparison to 4 normal controls (purple) tested at the same time (isotype controls are pink). b, IFN-{alpha} from PDCs: isolated PDCs (5 x 104/100 µl) from up to 23 CVID subjects and 16 controls were cultured for 48 h with ODN-A (left panel), ODN-B (middle panel), or ODN-C (right panel). The amount of IFN-{alpha} in culture supernatants was compared (expressed as picograms per milliliter, y-axis, log scale). The median values are indicated. CVID cells produced significantly less IFN-{alpha} in response to ODN-A (p = 0.0005), ODN-B (p = 0.009). or ODN-C (p = 0.0002) as compared with PDCs of normal controls. (PDCs of 2 CVID subjects produced <10 pg/ml in each condition, and due to the log scale, are not seen). Cells of subjects with XLA produced equivalent amounts of IFN-{alpha} as controls to each ODN, but as for normal controls, significantly more IFN-{alpha} than CVID subjects (ODN-A, p = 0.0071; ODN-B = 0.69; ODN-C, 0.0017). Note that ODN-B is not an optimum stimulator for PDCs. As controls, the ODN-A control (2249) and ODN-C control (2137) resulted in negligible IFN-{alpha} production, median = 0 for each, range, 0 = 67 pg/ml).

 
TLR9 gene sequence analysis

The TLR9 gene was sequenced from genomic DNA of 20 CVID subjects who had low numbers of both CD27+ and CD27+IgM–IgD– switched memory B cells (group 1); all TLR9 DNA sequences were found identical to the standard published sequence (44). Although single nucleotide genetic polymorphisms have been identified in various populations (50), none were found in this CVID patient group.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Bacterial and viral products activate cells of the immune system by binding to pattern recognition receptors such as TLRs. These receptors are differentially expressed on many cells of the hemopoietic system, with the most extensive expression on professional APCs. All TLRs sense microbial infection and trigger an array of antimicrobial and inflammatory responses. Human B cells express a distinct TLR expression profile with high levels of TLR7 and TLR9 in particular (34, 51) and, in fact, were the first cellular target in humans found to respond to TLR9 triggering by CpG-DNA. CpG-mediated B cell activation results in enhanced surface expression of costimulatory molecules CD86, MHC class II, and CD40, rapid cell proliferation (26, 36, 45), cytokine (37, 46), and IgM production (25). CpG also induces C{gamma}1, C{gamma}2, and C{gamma}3 gene transcription and class switch to IgG1, IgG2, and IgG3, activating a pathway which includes NF-{kappa}B, Rel, STATs, and IFN-responsive factors (29). For human B cells, CpG-DNA synergizes with CD40 signals, with or without the presence of PDCs to further induce secretion of immune globulins (45), and markedly augments the generation of plasma cells from naive and memory B cells (52). In the presence of IL-4, CpG DNA exerts another effect on B cells, inhibiting IgG and IgE production, resulting in a protective effect in a setting of allergic inflammation (53). BCR cross-linking, in conjunction with CpG-DNA and IL-10 and/or T cell CD40L, may augment TLR9 mRNA expression, further increasing the responsiveness of B cells to CpG-DNA (33).

Although many TLRs are found on different tissues, TLR9 appears restricted to B cells and PDCs, allowing only these two cell types to be responsive to CpG-DNA. However, not all DNA motifs are equally stimulatory. CpG-ODNs of type B strongly stimulate B cells to proliferate and produce IL-6 and IL-10, but CpG-ODNs of type A or C induce PDC to mature and produce large amounts of IFN-{alpha} and -beta. The mature PDC becomes a potent APC which drives T cell proliferation, Th1 polarization, IL-12 production, memory cytotoxic T cell responses, and the development of CD4+CD25+ regulatory T cells (54). In addition to the effects on T cells, the secreted IFN-{alpha} exerts a direct effect on humoral immunity as it enables B cells to undergo isotype switching and stimulates the terminal differentiation into plasma cells (41).

Recent work has suggested that human memory B cells contain constitutively higher levels of TLR9 mRNA, and as a result, are more responsive to CpG-DNA than naive B cells (33, 34). B cells that have been activated by the BCR are most susceptible to TLR9 triggering, leading to an essentially T-independent amplification of Ab production (34). The relative enrichment of TLR9 mRNA in CD27+ B cells, and the heightened responsiveness of these cells to CpG, has lead to the suggestion that bacterial or viral DNA might be able to support specific serological memory in the absence of the actual Ag (34, 35). This hypothesis is based on the potent and multiple biological activities of CpG on B cells, the fact that circulating plasma cells secreting IgG Abs to recall Ags are detectable in blood of immunized donors years after immunization, and the observation that the level of specific serum Abs is proportional to the frequency of memory B cells in blood (35).

In the current work, we investigated the effects of CpG DNA in CVID because late B cell differentiation is particularly abnormal. Most patients have normal numbers of B cells but decreased numbers of circulating memory and switched memory CD27+ B cells and a lack plasma cells (23, 24, 55, 56). In fact, patients with the fewest switched memory B cells have the lowest levels of serum IgG and poorer vaccine responses (57). Abnormalities of B cell TLR9 function were suspected because in a previous study we showed that B cells of CVID subjects did not proliferate normally or in many cases, secrete immune globulin, when exposed to an ODN antisense to the rev gene of HIV, a compound now identified as a potent type B CpG-containing ODN which targets B cells via TLR9 (42). Further investigating this with an ODN reagent specifically targeted to B cells, we now show that B cells of CVID subjects do not become activated and up-regulate adhesion molecule CD86 to the same extent as controls, after exposure to the prototypic type B ODN 2006, even when costimulated by triggering through the BCR. After CpG-DNA activation, TLR9 appears on the surface of CD27+ B cells from normal donors, while CVID B cells demonstrate little or no surface TLR9. Although most TLR9 is intracellular (47), our observation of surface TLR9 on activated normal B cells is compatible with a recent report which noted surface TLR9 on a population of activated human tonsillar lymphocytes, presumably B cells (48). Compared with normal B cells, CVID B cells subjected to CpG-B stimulation produced very little IL-6 or IL-10, both of which enhance B cell growth and maturation. Although normal control B cells, especially CD27+ B cells, contained large amounts of intracytoplasmic TLR9, B cells of CVID subjects (both CD27+ and CD27–) had markedly reduced amounts of both TLR9 protein and TLR9 mRNA as compared with normal B cells. We had assumed that the defects of B cell CpG triggering were most likely related to the deficient numbers of CD27+ B cells, but this was not the case, as patients who had relatively larger numbers of memory B cells did not have more normal responses to CpG-DNA, or greater amounts of intracellular TLR9 protein or mRNA in B cells. Further, investigating the effects of TLR9 triggering on PDCs, we found that CVID cells produced strikingly less IFN-{alpha} after exposure to appropriate CpG-DNAs, even though intracytoplasmic staining of these cells demonstrated the same amounts of TLR9 as PDCs of normal subjects.

The results described demonstrate that there are broad TLR9 defects in CVID, consisting of both lack of TLR9 in B cells and defective TLR9 function in B cells and PDCs. The reason(s) for this are not known. Mechanisms which control the expression of TLR9 in either B cells or PDCs are controversial. BCR triggering has been reported to either enhance (34) or depress expression of TLR9 mRNA (45); exposure of B cells to CpG-DNA has been reported to either up-regulate (33) or down-regulate TLR9 mRNA (45). In contrast, IL-10 does appear to increase the expression of B cell TLR9 (29), thus, it is possible that environmental or other cellular factors lead to the lack of TLR9 in CVID B cells. Whatever the explanation, subjects with relatively more B cell intracytoplasmic TLR9 had no better activation responses after CPG-DNA triggering, and the results for PDCs suggest more pervasive defects of TLR9 function. Mutations of the TLR9 gene itself were not demonstrated in the subjects analyzed here. However, defects in TLR9 pathway members, leading to NF-{kappa}B translocation have not been excluded; in fact mutations in three of these are known to lead to defects of immune function: hypomorphic mutations of NF-{kappa}B essential modulator, as well as mutations of the IL receptor-associated kinase-4, and an inhibitor of NF-{kappa}B {alpha} (46, 58, 59). In particular, patients with mutations of IL receptor-associated kinase-4, which is downstream in TLR signaling, have impaired Ab responses to polysaccharide Ags, and ODN2006 does not stimulate PBMC production of IL-6 or IL-10 (46). Intriguingly, for one patient with this defect studied more extensively after multiple vaccinations, Ab production and B cell memory were severely impaired (60). To further investigate the TLR9 pathway, analysis of biologically related TLR7 and TLR8, which like TLR9, sense pathogen-derived DNA and RNA nucleic acid motifs and similarly recruit MyD88 to endosomal vesicular structures, may help to clarify this defect(s) in CVID.

Over the past decades, a number of studies have investigated immune defects in CVID. Although T cell subsets are perturbed (61, 62), T cell activation (9), proliferation (63), and cytokine production are subnormal (64, 65), apoptosis is enhanced (12, 66), and monocyte dendritic cell defects are present (13, 14); the defining hallmark of the disease is hypogammaglobulinemia and the lack of functioning B cells. Based on this characteristic, investigators have used a number of in vitro B cell functional studies to stratify patients into those with nonfunctional B cells, poorly functioning B cells or more normally responsive B cells (67, 68, 69). In contrast to these studies, we found that B cells of almost all CVID subjects studied here were unable to respond normally to CpG-DNA, suggesting that the TLR9 defect may be a central abnormality.

Although it is currently unclear whether the TR9 deficiency is primary, or secondary to other causes, the result in both cases would be markedly impaired B cell and PDC immunity. B cell surface CD86, normally up-regulated 16 to 20 h after activation, supplies an important link between innate and adaptive immunity because the microbial recognition phase accelerates the growth and effector functions of Ag-specific T and B cells. CVID B cells thus lack the link to T cell immunity which microbial DNA normally supplies. Previous work demonstrated that CD86 was also not increased on the surface of CVID B cells cultured with Staphylococcus aureus + IL-2, or after ligation of the BCR, even though other activation markers were demonstrated (70, 71). The studies here show that this defect in CVID B cells cannot be overcome by TLR9 triggering. On exposure to CpG-DNA, IL-6 and IL-10–cytokines important in the growth and maturation of B cells–are not produced, and CpG ligands could not provide the survival advantage afforded to normal B cells (72). Defects of TLR9 would also lead to reduced B cell proliferation, abrogated IgG class switch, and loss of memory B cells. If TLR9 plays a role in long-term retention of serologic responses, patients with CVID would also be deprived of this biologic benefit. Although the result of TLR9 dysfunction on PDC-driven immune responses is less clear, PDCs contribute most of the type I IFN produced in viral infections (73). As a result, one would hypothesize that TLR9 defects might lead to viral infections; however, these are not especially characteristic of CVID (1, 5, 6). Recent studies show that mice lacking the TLR adaptor My88 have only a partial reduction in IFN responses and normal viral resistance, indicating that there are TLR-independent pathways to IFN production which may compensate for TLR defects and permit effective viral immunity (73, 74).

Although deficiencies of adaptive immune responses have been investigated in many studies in CVID, our data demonstrate that an integral component of the innate immune system–TLR9 protein-, mRNA-, and CpG-DNA-induced TLR9 activation of B cells and PDCs–is significantly impaired in this immune deficiency disorder. Whether primary or secondary, TLR9 defects could lead to a loss of normal B cell and PDC function.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported National Institutes of Health Grants AI 101093, AI-467320, AI-48693, and National Institute of Allergy and Infectious Diseases Contract 03-22. Back

2 Address correspondence and reprint requests to Dr. Charlotte Cunningham-Rundles, Department of Medicine, Mount Sinai Medical Center, 1425 Madison Avenue, New York, NY 10029. E-mail address: Charlotte.Cunningham-Rundles{at}MSSM.edu Back

3 Abbreviations used in this paper: CVID, common variable immune deficiency; ODN, oligodeoxynucleotide; PDC, plasmacytoid dendritic cell; MFI, mean fluorescence intensity; XLA, X-linked agammaglobulinemia. Back

Received for publication July 29, 2005. Accepted for publication November 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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