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The Journal of Immunology, 2000, 164: 5482-5491.
Copyright © 2000 by The American Association of Immunologists

Impact of HIV-1 Infection on VH3 Gene Repertoire of Naive Human B Cells1

Ronald W. Scamurra*, Darren J. Miller*, Linda Dahl*, Mitchell Abrahamsen{dagger}, Vivek Kapur{dagger}, Sharon M. Wahl{ddagger}, Eric C. B. Milner§ and Edward N. Janoff2,*

* Center for Mucosal and Vaccine Biology, Infectious Disease Section, Veteran Affairs Medical Center, University of Minnesota School of Medicine, Minneapolis, MN 55417; {dagger} Department of Veterinary Pathobiology, University of Minnesota College of Veterinary Medicine, St. Paul, MN 55108; {ddagger} Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892; and § Virginia Mason Research Center, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1–6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean ± SD, 57.1 ± 9.7 vs 61.1 ± 8.7%). Similarly, within VH(1–6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG "memory" B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Humoral immune abnormalities are prominent and have substantial clinical impact during HIV-1 infection. In one context, evidence of overactivity of humoral immunity includes hypergammaglobulinemia (1), lymphadenopathy (2), and the frequent development of B cell lymphomas (3, 4) in HIV-1-infected patients. Alternatively, B cell responses in this population are depressed to T-independent Ags (5), T-dependent recall and neoantigens in vivo (6, 7), and mitogens in vitro (7). These defects precede significant depletion of CD4+ T cells (8) and are not corrected by addition of normal T cells (7, 9), suggesting that intrinsic B cell defects may contribute to impaired humoral responses during HIV-1 infection.

Protective humoral immunity is conferred by Abs whose specificity is dictated by the variable regions of the heavy (VH) and light (VL) chains, which combine to form the Ag binding site. The selection of a specific VH gene, which is subsequently linked to specific diversity (D), and joining (JH) region genes, is a principle determinant of the structure of the Ag binding site (10). The ~50 functional VH genes in humans are divided into 7 families (VH1–7), each of which is distinguished by a >80% nucleotide homology in certain regions (11). The VH3 gene family, with 22 functional genes, is the largest of the 7 families and comprises about half of the expressed VH repertoire in adult peripheral B cells (12, 13, 14, 15, 16). Furthermore, VH3 genes dominate the repertoire of Abs specific for bacterial polysaccharide Ags which are important for the control of, for example, Streptococcus pneumoniae, a common cause of pneumonia and bacteremia in HIV-1-infected patients (17).

Several reports have suggested that VH3-expressing B cells may be depleted during the late stages of HIV-1 disease (18, 19, 20, 21 , and reviewed in Ref. 22). VH3 Abs have been shown to bind to HIV-1 surface envelope glycoprotein gp120 in a nonclassical interaction outside the normal Ag-binding pocket (23, 24). In addition, gp120 stimulates Ig production from VH3 B cells from HIV-1-seronegative donors (25). Based on these findings, HIV-1 gp120 has been proposed to act as a "superantigen," binding to VH3-expressing B cells, inducing their activation and eventual depletion (reviewed in Ref. 26). Since VH3 Abs are important for defense against a variety of bacterial (27, 28) and viral (29, 30) pathogens, many of which commonly affect HIV-1-infected patients, deletion of VH3-expressing B cells may contribute to HIV-1-associated humoral immune dysfunction and to the increased incidence of secondary infections observed in these patients.

To characterize the magnitude and isotype specificity of defects in VH3 Ab expression among HIV-1-infected patients and to delineate whether these changes are present in the naive and/or memory B cell compartment, we examined VH3 gene expression in patients with advanced HIV-1 disease (CD4+ T cells < 200/µl, and high HIV-1 plasma viral loads). We specifically characterized the frequency of expression of individual VH3 genes (V3–23 and V3–30/3–30.5) proposed as candidates to encode HIV-1 gp120 binding sites and for selective deletion (31). We show that VH3 mRNA is normal in resting naive peripheral blood IgM and IgD B cells, both by VH family and gene-specific analyses compared with that in seronegative control subjects. However, the VH3 family is underrepresented in peripheral memory IgG B cells and in IgM Abs in serum. Together, these results suggest that whereas VH3 expression is decreased in differentiated cells (e.g., memory IgG cells and those actively secreting IgM) of HIV-1-infected patients, the naive VH3 IgM and IgD repertoires are relatively intact.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient samples

HIV-1-infected patients (n = 24) and low-risk HIV-1-seronegative control subjects (n = 17) matched for age and race were enrolled following written informed consent in protocols approved at the Minneapolis Veterans Affairs Medical Center and the University of Minnesota. Clinical data from HIV-1-infected patients, including antiretroviral medications, CD4+ T cell count, and plasma viral RNA load as determined by the Amplicor HIV-1 monitor test (Roche Molecular Systems, Pleasanton, CA), are shown in Table IGo. Serum and heparinized blood were collected, and PBMC were separated by density gradient centrifugation. PBMC (107) were dissolved in 1 ml of Trizol (Life Technologies, Rockville, MD) and frozen at -80°C until RNA was extracted.


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  		Table I. Patient clinical data

 
B cell lines

VH1 (21/28) and VH3 (18/2) B cell hybridomas were obtained from Dr. D. Stollar (Tufts University, Boston, MA). VH5 and VH6 EBV-transformed B cell lines were obtained from Dr. R. Insel (University of Rochester, Rochester, NY). The EBV-transformed VH4 cell line was prepared in our laboratory. All cells were grown in RPMI 1640 medium supplemented with antibiotics and 10% FBS and prepared for RNA extraction in Trizol (Life Technologies) as above.

Analysis of serum Abs

Levels of total IgG, IgM, and IgA in sera were determined by nephelometry. The proportion of VH3 family within total serum IgM was determined by its differential binding to staphylococcal protein A (SpA)3 by ELISA (32). With affinity-purified goat anti-human IgM as the capture Ab, total serum IgM was measured as described previously (33) and SpA-reactive IgM was detected with SpA coupled to biotin N-hydroxysuccimide (Sigma, St. Louis, MO) by standard procedures and avidin-horse radish peroxidase (Zymed, San Francisco, CA). Conditioned media from the 18/2 IgM VH3 B cell line was used as a standard. SpA-reactive IgM values were taken as a percentage of total IgM values obtained on the same plate.

Isolation of total cellular RNA

Previously homogenized PBMC and cloned B cell samples were thawed on ice and RNA was extracted by the standard Trizol procedure (Life Technologies). RNA was stored at -80°C until use. Six micrograms of RNA was treated with RNase-free DNase I (Life Technologies) in a final reaction volume of 30 µl that contained 3 U of DNase I. The reaction was stopped by addition of 3 µl of 25 mM EDTA and DNase I was inactivated by heating at 65°C for 10 min. DNase I-treated RNA was used immediately for RT.

RT

First-strand cDNA synthesis was performed according to the manufacturer’s recommendation in a 60-µl reaction containing 21 µl of DNase I-treated RNA, 0.5 mM each dNTP, 7.5 µg/ml random hexamers (Promega, Madison, WI), 8.3 µg/ml oligo(dT) (Life Technologies), and 10 U/µl Moloney murine leukemia virus RT (Life Technologies). The samples were incubated at 37°C for 1 h, heated at 70°C for 15 min, and stored at -20°C.

PCR

Primers required to amplify VH family-specific PCR products for a given Ig isotype (34, 35, 36) (Table IIGo) were commercially synthesized. VH7 was omitted from our analysis because it contains a single member that cross-reacts with VH1 primers and is expressed at very low levels. For each cDNA sample, six 20-µl PCR reactions (one for each VH family) were assembled. To each PCR reaction, the following was added from a Master mix: 0.5 µl of cDNA product, 2.0 mM (IgM) or 2.5 mM (IgG and IgD) MgCl2, 150 mM each dNTP, 0.4 µM 3' constant region isotype-specific primer, 0.02 µCi/µl [{alpha}-32P]dCTP (3000 Ci/mmol), and 0.25 U Amplitaq Gold (Perkin-Elmer, Norwalk, CT). The appropriate 5' VH family-specific primer at 0.4 µM was added last. As negative controls, a Moloney murine leukemia virus-RT-negative cDNA control was assayed for each sample as well as a water blank in reactions with a single 5' VH3 leader primer and an appropriate 3' constant region primer. No signal was ever detected with these controls. Samples were heated to 95°C for 10 min in a Perkin-Elmer 9700 thermocycler, before initiating cycling ("hot start"). Cycling conditions were empirically determined with a standard control PBMC sample for each Ig isotype (see below). Each cycle consisted of a 30-s denaturation at 95°C, a 30-s annealing at variable temperatures per isotype (60°C, IgM and IgD; 62°C, IgG), and a 30-s extension at 72°C. Following amplification, samples were separated on a native 5% polyacrylamide gel, dried on Whatman 3 MM paper (Whatman, Clifton, NJ), and exposed to a phosphor storage screen (Molecular Dynamics, Sunnyvale, CA) for 16–22 h. The relative radioactivity in the VH family-specific PCR bands was measured with a PhosphorImager (Molecular Dynamics). All samples were assayed in duplicate and results were expressed for each VH family as a percentage of the total signal obtained for all six VH families. Data for expression of VH2 and VH6 families, which consistently accounted for <5% of the total signal in all subjects, are not shown.


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  		Table II. PCR primers used for VH family analyses

 
The specificity of the 5' VH family-specific primers was confirmed with a panel of B cell hybridomas and EBV-transformed B cells. For each cell line, a specific PCR product was only amplified with primers specific for the Ig VH family expressed by that cell line with little or no cross-amplification of other VH families (data not shown). No VH2 Ig-expressing cell line was available, but expression of VH2 is also low. In addition, no cross-priming occurred between the 3' Ig isotype-specific primers. When PBMC cDNA was amplified with each of the 3' primers (IgM, IgG, and IgD), each PCR product only bound the probe that matched the isotype of the 3' primer used in the amplification (data not shown). Thus, no cross-amplification was found with either our 3' Ig isotype-specific primers or our 5' VH family-specific primers.

Cycle curve analyses for all three Ig isotypes showed that the relative VH gene expression levels measured reflected the actual proportions of VH mRNA in PBMC samples. As shown for IgM, the linear relationship between signal and cycle number included the 24 cycles used in this assay (Fig. 1GoA); the VH3 curve began to flatten at 27 cycles. Analyzed as a calculated percentage of the total signal for all VH families, 24 cycles also fell in the plateau in which the relative percentages for all VH families remained relatively constant (Fig. 1GoB). Similar data and optimal cycle choices were derived for IgD (28 cycles) and IgG (24 cycles). Finally, we assayed cDNA mixtures from clones that contained constant amounts of VH1 and VH5 with increasing amounts of VH3 (Fig. 2Go). The measured changes in the proportion of VH3 family expression closely approximated the predicted values. Thus, our VH RT-PCR assay reliably detected changes in the proportion of individual VH family expression.



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  		FIGURE 1. Cycle curve analysis of a control PBMC cDNA sample for IgM VH(1–6). An IgM VH RT-PCR reaction was set up with negative control PBMC cDNA sample as described in Materials and Methods except that the volume was increased 4-fold. After dividing the reactions equally into eight tubes, PCR cycling was initiated. A single tube was removed for each VH family at two-cycle intervals between 19 and 33 and analyzed by polyacrylamide gel electrophoresis as described in Materials and Methods. In A, the raw phosphor imager values are plotted on a log scale vs cycle number. In B, the percentage contributed by each VH family member to the total signal was calculated at each cycle. These values were plotted vs cycle number. Dashed vertical line in both panels is the cycle number chosen for IgM VH analysis.

 


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  		FIGURE 2. Assay of mixtures of VH cell lines with comparisons of measured frequencies to predicted values. Increasing concentrations (20% increments) of VH3 cDNA were added to fixed concentrations of VH1 and VH5 cDNA and assayed by the VH RT-PCR protocol. The dashed lines show how the relative percentages of each VH signal are predicted to change as the amount of VH3 cDNA is decreased. Actual measurements were performed in triplicate and are plotted with SE bars.

 
Cloning of VH3 IgD PCR products

VH3 IgD PCR products were generated in a nonradioactive PCR reaction similar to that described above. The reaction was stopped at 24 cycles to maximize the number of independent clones. Pfu polymerase (Stratagene, La Jolla, CA) was used to improve fidelity. Following amplification, PCR products were purified using the Promega PCR wizard kit (Promega). To enable TA cloning, a single dATP was added in a reaction containing native Taq polymerase (Life Technologies) and dATP. The PCR product was again purified with a Promega PCR wizard kit and cloned using the pGEM Easy-T-vector kit (Promega).

Screening of VH3 clones with gene-specific oligonucelotides

Plasmids from individual colonies were purified as per the kit instructions (Qiagen 96-Turbo miniprep kit; Qiagen, Valencia, CA). After alkaline denaturation, plasmids were dot blotted onto replicate Magnacharge nylon membranes (Micron Separations, Westborough, MA) using a 96-well dot blotter (Bio-Rad, Hercules, CA) and cross-linked by UV irradiation. Membranes were hybridized to a set of oligonucleotide probes (M18, M16, M76, M8, E182, M19, and M41) that discriminate between several VH3 genes (V3–23, V3–30/3–30.5, V3–30.3, and V3–33) as described previously (37, 38, 39, 40, 41, 42). Since Abs with HIV-1 gp120-binding activity can harbor gene products derived from either the V3–30 or the V3–30.5 locus (genes at these loci may have the same sequence) (31), we scored clones that bound probes diagnostic of either locus together as V3–30/3–30.5. A full-length cDNA probe (V3–15, obtained from Dr. R. Insel, University of Rochester, Rochester, NY) (43) that is cross-reactive with all VH3 genes was used to identify the total number of VH3+ clones.

Sequencing VH3 clones

Plasmids containingVH3 IgD inserts were sequenced using SequiTherm Excel II kits (Epicentre Technologies, Madison, WI). Both strands were sequenced simultaneously in differentially labeled two-primer reactions (simultaneous bi-directional sequencing, SBS; Li-Cor, Lincoln, NE). IRD-labeled primers were commercially prepared (Li-Cor). Reaction products were separated on a Li-Cor gene reader 4200 series DNA analyzer and the sequence was read using BaseImagIR 4.1 software (Li-Cor). Sequences were compared with VH sequences contained in two different on-line VH databases (V BASE: http://www.mrc-cpe.cam.ac.uk/imt-doc, and IMGT: http://imgt.cnusc.fr:8104/), and alignments were performed using DNAplot software accessed at these sites.

Statistical analyses

Unpaired t tests were used for comparisons between two groups and ANOVA for comparisons of more than two groups. When significant main effects were obtained, means were tested by Fisher’s probable least-square difference test. Statview computer program (SAS Institute, Cary, NC) was used for all analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Patient clinical parameters

Of the 13 HIV-1-infected patients receiving antiretroviral therapy (patients 1–13), all had CD4+ T cell counts <=200/µl, about half had AIDS (7 of 13, Centers for Disease Control stage C3), and 10 had plasma viral loads >10,000 viral particles/ml (Table IGo). Most patients were on combination therapy with protease inhibitors and RT inhibitors (9 of 13), many of whom retained high viral loads. Of the 11 HIV-1-infected untreated patients (patients 14–24), 3 had AIDS, 2 had CD4+ T cell counts <200/µl, and 8 patients were asymptomatic.

Serum Ig levels

Levels of total serum IgG were elevated ~2-fold in HIV-1-infected patients compared with those in control subjects (p < 0.05, Table IIIGo). Although levels of IgM appeared to be similarly elevated, it was not statistically different form controls due to the large coefficient of variation for these data. However, the proportion of total IgM reactive with SpA, an index of VH3 Ig protein expression, showed an inverse correlation with the CD4+ T cell count. Compared with values in control subjects, SpA/VH3 IgM values in sera from HIV-1-infected patients showed a 45% decrease in those with <200 CD4+ T cells/µl (p < 0.001), a 29% decrease in those with 200–500 cells/µl (p = 0.07), but no significant difference in those with counts >500/µl (Table IIIGo). Thus, serum IgM Abs encoded by genes of the VH3 family show a stage-specific decrease among HIV-1-infected patients. Antiretroviral therapy had no significant impact on the proportion of SpA-reactive IgM in HIV-1-infected patients (data not shown). Finally, the 11 patients with the highest viral load values (>100,000 viral particles/ml), but not those with lower HIV-1 levels (data not shown), also had a 45% decrease in SpA-reactive IgM compared with values in control subjects (p < 0.01).


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  		Table III. Levels of serum Igs by isotype and IgM reactive with SpA1

 
VH family expression of HIV-1-infected patients

To determine whether the deficit in serum VH3 IgM Abs of HIV-1-infected patients was accompanied by parallel changes in VH3 IgM mRNA expression in circulating B cells, an RT-PCR-based assay was developed to assess the Ig VH family repertoire of PBMC (see Materials and Methods). In contrast to the decrease in serum SpA-reactive VH3 IgM protein, we identified no significant changes in the relative expression of VH3 IgM mRNA in PBMC of HIV-1-infected patients compared with that of control subjects (mean ± SD, 57.1 ± 9.7 vs 61.1 ± 8.7%, respectively; Fig. 3Go, top panel). To control for the potential inclusion of Ab-producing plasma cells, which may lead to overrepresentation of selected genes by a minority population, we measured the IgD VH expression, which more closely resembles naive resting B cells. Again, relative expression of VH3 IgD mRNA, as well as other VH families, was comparable in peripheral B cells from HIV-1-infected patients and control subjects (53.4 ± 5.8 vs 52.1 ± 4.2%, respectively; Fig. 3Go, middle panel). Moreover, we identified no independent effect of antiretroviral therapy or patient viral load on IgM- or IgD-VH3 mRNA expression between HIV-1-infected patients and control subjects (data not shown). Taken together, the IgM and IgD VH repertoire analyses indicate that HIV-1 infection has little effect on the naive VH family repertoire, even in these patients with advanced HIV-1 disease.



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  		FIGURE 3. Analysis of isotype-specific VH family distribution by RT-PCR in PBMC from control subjects and HIV-1-infected patients by CD4+ T cell count. Top panel, IgM expression; middle panel, IgD expression; and bottom panel, IgG expression. Only VH1, VH3, VH4, and VH5 gene families are shown since VH2 and VH6 routinely comprised <5% of total signal in all isotypes and patients. The n values for the bottom panel reflect that IgG VH family-specific PCR products could not be amplified from three control subjects and one HIV CD4+ T cell <200 patient. The coefficient of variation for a standard PBMC sample repeated 11–13 times was <12% and <11% for VH3 and VH4 families and all Ig isotypes, respectively. **, Significant difference (p < 0.01) compared with control subjects. The single horizontal bar denotes the median of each group.

 
Unlike results with naive IgM and IgD VH3 mRNA expression, the relative expression of VH3 mRNA within the IgG class, which comprises previously activated memory cells, was significantly decreased in HIV-1-infected patients with CD4+ T cell counts <200/µl compared with that in control subjects (48.9 ± 6.3 vs 59.3 ± 8.4%, respectively, p < 0.01; Fig. 3Go, bottom panel). Conversely, the relative expression of VH4 IgG mRNA was increased in this group (35.6 ± 7.5 vs 26.7 ± 7.2%, p < 0.01; Fig. 3Go, bottom panel). No significant changes in the expression of VH3 or VH4 IgG mRNA were found for the HIV-1-infected 200–500 and the >500 CD4+ T cell groups compared with control subjects. HIV-1-infected patients with plasma viral loads >100,000 viral particles/ml had similar changes in the relative expression of VH3 and VH4 IgG mRNA (VH3: 46.9 ± 5.4 vs 59.3 ± 8.4; VH4: 36.0 ± 5.3 vs 26.7 ± 7.2, p < 0.01, HIV-1-infected vs control) as patients with low CD4+ T cell counts. This difference in relative VH3 IgG mRNA expression also persisted when including all HIV-1-infected patients as a group (49.6 ± 6.2% vs 59.3 ± 8.4%, p < 0.01).

To exclude the possibility that our analysis of VH families in HIV-1-infected patient samples may have been out of the linear amplification range, we performed IgM, IgD, and IgG cycle curves on randomly selected samples from three HIV-1-infected patients and three seronegative control subjects. For all six samples, the cycle curves for VH1, 3, 4, and 5 families were linear across the cycle numbers used for quantitation (IgD, Fig. 4Go) (IgM and IgG, data not shown). Thus, the VH RT-PCR assay appears to not overrepresent VH3 in our HIV-1-infected PBMC samples.



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  		FIGURE 4. IgD cycle curves for three HIV-1-infected patients and three seronegative controls. Cycle curves were performed as described in Fig. 1Go. Dashed vertical lines are the cycle number chosen for IgD VH analysis.

 
VH3 gene-specific frequencies in the IgD repertoire of HIV-1-infected patients

The apparently discrepant deficiency noted in serum VH3 IgM Abs but not in IgM or IgD mRNA may have resulted from a selective deficit in binding of certain VH3 Abs by SpA, whereas the mRNA detects all VH3 genes. Not all Abs derived from each of the 22 individual VH3 genes bind to SpA (32). In addition, only a limited number of VH3 genes (V3–23, V3–30/3–30.5 and, infrequently, V3–73, most of which are SpA reactive) code for HIV-1 gp120 binding sites (31). A superantigen-like interaction between gp120 and VH3 family Abs, particularly products of the candidate genes cited above, has been invoked as a potential stimulus for the VH3 deletion mechanism (25, 26, 44). Therefore, we compared the frequency of the two most abundantly expressed genes (V3–23 and V3–30/3–30.5) in the naive VH3 repertoire of nine HIV-1-infected patients with advanced AIDS (Table IGo) and six control subjects.

We constructed VH3 RT-PCR cDNA libraries for each of the selected samples for IgD, again to most closely represent naive unstimulated B cells with relatively few mutations in their VH genes. Based on the binding of diagnostic oligonucleotides either alone or in combination, we could discriminate four individual VH3 genes, V3–23 and V3–30/3–30.5 as described above, as well as two genes which encode non-gp120-binding products, V3–30.3, and V3–33. The differential probe hybridization patterns which characterize these VH3 genes have been described previously (40, 41). Of 1382 clones probed (an average of 92 per individual), 60 clones (4.3%) had ambiguous oligonucleotide hybridization patterns, each of which were sequenced to confirm their identity. The performance characteristics of this approach were evaluated by sequencing all clones from a single control subject that were identified by hybridization to a cDNA probe which recognizes all VH3 genes. Of the 75 VH3+ clones sequenced for this subject, two were not detected by probe because each had mutation(s) in the binding site for the appropriate diagnostic oligonucleotide(s) (data not shown). This result highlights the fact that this procedure detects predominantly unmutated VH3 genes. Indeed, most of the clones were in near germline configuration (99.37 ± 2.12% compared with their closest germline counterpart). Finally, all rearrangements for each VH3 gene, including 23 V3–23 clones, arose from unrelated B cells each with a unique VHDJH combination and CDR3 sequence (data not shown). These results highlight the robust performance characteristics of the method, the naive unmutated germline configuration of the transcripts, as well as the success of the cloning to produce only unique clones (no selective overrepresentation).

Of 22 known VH3 genes, 17 were represented in the 75 sequenced VH3+ clones (Fig. 5Go). Consistent with previous results, ~60% of VH3+ clones were identified by the oligonucleotide hybridization (40). The gp120-associated candidate genes (V3–23 and V3–30/3–30.5) comprised a substantial proportion (37.2 ± 8.5%) of all VH3 genes in all groups (Table IVGo; Fig. 6Go). The frequencies of these individual genes and two control genes (V3–30.3, V3–33) were indistinguishable among HIV-1-infected patients and control subjects, although the range of frequencies for each of the genes was broad (Fig. 6Go). Similarly, the combined frequencies of the gp120-binding candidate genes were not different in HIV-1-infected patients and control subjects (Fig. 6Go, far right). In addition, when we divided the HIV-1-infected group into those with normal and those with low serum levels of SpA+ IgM (an index of VH3 IgM protein expression), no statistically significant differences were found in the frequencies of our candidate genes within mRNA expressed by peripheral B cells (Table IVGo). These detailed gene-specific analyses confirmed the VH family results that VH3 mRNA expression was comparable in control subjects and HIV-1-infected patients, despite a deficiency of VH3 IgM protein in sera from the patients with advanced HIV-1 disease.



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  		FIGURE 5. Distribution of VH3 gene segments for a control subject. All VH3+ clones from the VH3 IgD gene rearrangement library for a control subject were analyzed by dsDNA sequencing. The sequences were then aligned to their closet germline counterpart using two different on-line databases (see Materials and Methods). , Genes which encode putative gp120-reactive Abs (31 ). Based on the correlation between the results of sequencing and probe identification of individual clones, the sensitivity and specificity of the probe system were 95.6 and 100%, respectively. The predictive value of a positive result was 100%, whereas the predictive value of a negative result was 93.5%. The lower sensitivity and predictive value of a negative result were due to two false negative clones (one V3-30.3 and one V3-30/3-30.5 clone) that had mutations in their probe binding sites. Thus, these clones were not detected until they were sequenced.

 

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  		Table IV. Expression of selected VH3 genes in HIV-1-infected patients with low and normal levels of serum SpA+ IgM

 


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  		FIGURE 6. Analysis of VH3 gene segment usage in HIV-1-infected patients. VH3 IgD gene rearrangement libraries were constructed for nine HIV-1-infected patients (see Table IGo) and six seronegative control subjects. The libraries were screened with VH3 gene-specific oligonucleotides. The percentage for each gene is determined by calculating as follows: (number of clones identified for specific gene)/(the total number of VH3+ clones for that library) x 100. Frequency of putative gp120 binders = frequency of (V3–23 + V3–30/3–30.5).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified a striking discrepancy between expression of VH3-family encoded Abs (SpA-binding IgM) in serum and expression of VH3 mRNA in peripheral blood B cells of HIV-1-infected patients. Although levels of serum VH3 IgM protein showed a significant CD4+ T cell-dependent decrease in patients with HIV-1 disease, we found no evidence of corresponding decreases in VH3 mRNA expression for IgM or IgD in peripheral blood B cells from the same patients. The number and surface IgM+ and IgD+ distribution also have been similar for peripheral B cells from HIV-1-infected and control subjects studied to date (A. Lindstrom and E. N. Janoff; manuscript in preparation). We characterized VH3 family mRNA as a proportion of all VH families, as well as by quantitating the frequency of expression of individual gp120-associated candidate genes (V3–23 and V3–30/3–30.5). Ab products of these candidate genes are reported to interact with HIV-1 gp120 in a nonclassical superantigen-like fashion through framework-encoded epitopes (31). Our data with serum IgM are consistent with published results (45) and hypotheses that VH3 B cells are deleted or suppressed during HIV-1 infection (26, 44). Our mRNA data address the question of when such abnormalities are introduced in the course of B cell ontogeny.

Technically, our assays were extensively validated. The RT-PCR method, using Ig isotype- and VH family-specific primers, was performed within the linear range of our assay based on cycle curve analyses of control and patient samples (Fig. 4Go). In addition, the vast majority of clones represented independent rearrangements of VH genes in near germline configuration rather than duplications. Moreover, consistent with results by others (40), the cloning data also confirmed that the putative gp120-associated candidate genes (V3–23 and V3–30/3–30.5) comprised from 40 to 50% of VH3 gene products from HIV-1-seronegative healthy donors. Thus, the probability that VH3 mRNA is overrepresented in our analysis of the naive repertoire of our HIV-1-infected patients is low.

One hypothesis to explain the discrepant results between protein and mRNA is that IgM in serum is synthesized by differentiated B cells in bone marrow and secondary lymphoid organs (e.g., lymph nodes or spleen) and not by the resting circulating B cells examined. In support of this idea, we find relatively few IgM-secreting B cells among freshly isolated PBMC from HIV-1-infected patients or control subjects tested either in bulk culture or by enzyme-linked immunospot assay (E. N. Janoff, unpublished observation). Furthermore, others have reported that VH3 genes are underrepresented in bone marrow (46), lymph node, and spleen (18) of HIV-1 infected patients. Another possibility is that VH3 B cells are not in fact clonally deleted, but are selectively impaired in their ability to synthesize Ab upon stimulation and differentiation. Regardless, our data indicate that patients with advanced HIV-1 disease contain naive IgM/IgD peripheral blood B cells that harbor relatively normal proportions of VH3 genes.

Our results are consistent with those in primates infected with a simian immunodeficiency virus-expressing human gp120 on its surface in which no consistent changes were detected in the VH3 repertoire over several months (47), although the short-term results with this viral construct have been questioned (44). In humans, others have reported a selective VH3 mRNA deficit of about 70% in the naive peripheral VH repertoire of a limited number of HIV-1-infected patients (7), only two of whom had CD4+ T cells < 200/µL) compared with healthy control subjects (21). Similarly, Braun and coworkers (18, 20) also reported a striking deficit in VH3 expression in HIV-1-infected patients, but their methods did not discriminate between the naive and activated or expressed repertoires . The differences between these reports and our findings may be related to the different methodologies used; however, it is difficult to assess the validity of these methods since no assay validation data were given in these reports. For example, David et al. (21) amplified all VH families in a single PCR product using an anchored RT-PCR protocol rather than performing VH family-specific reactions. Berberian et al. (18) amplified DNA using specific 5' VH primers and a conserved 3' J region consensus primer rather than the isotype-specific mRNA analysis we performed. Alternatively, the previous studies were conducted before the introduction of highly active antiretroviral therapy, and, thus, their patients may have had more advanced disease.

Recent reports have indicated that lowering the plasma HIV-1 viral burden to undetectable levels for extended periods can normalize some B cell properties, such as levels of spontaneous Ig production (48) and VH3 IgG expression (49). We addressed this issue by examining the naive VH repertoire of patients on no antiretroviral therapy (patients 14–25, Table IGo), and found no differences in IgM- or IgD-VH3 mRNA expression compared with those in controls. Because our patients did not all have advanced disease, we also selectively examined VH3 mRNA expression in the naive repertoire of our patients with the most advanced disease (Centers for Disease Control stage C3) and found no differences compared with controls. Because of these unresolved discrepancies within the naive VH3 repertoire of HIV-1-infected patients, we chose to carefully examine the contribution of specific VH3 genes to the naive repertoire of our advanced HIV-1-infected patients.

To our knowledge, this is the first report to characterize the relative frequency of specific genes within the VH3 gene family in HIV-1-infected patients. We found no differences in the frequencies of genes which encode putative gp120-reactive Abs within the naive VH3 repertoire of advanced HIV-1-infected patients and control subjects. However, our advanced patients may represent an immunologically diverse group. Indeed, five of nine HIV-1-infected patients for whom VH3 IgD was cloned had frequencies of putative gp120-reactive genes (<33%) that were appreciably lower than those of the remaining four patients (>41%) and six control subjects (Table IVGo). However, neither CD4+ T cell number, HIV-1 viral loads, disease stage, nor antiretroviral therapy differentiated these two groups of HIV-1-infected patients. Furthermore, their levels of SpA+ IgM did not correlate with their frequencies of gp120-reactive candidate genes, suggesting that distinct populations of cells (e.g., activated and differentiated IgM-secreting cells vs resting IgM/IgD-positive cells) contribute to these two different observations. Thus, VH3 genes, including the putative gp120-associated genes V3–23 and V3–30/3–30.5, are present in relatively normal proportions in the naive repertoire of advanced HIV-1-infected patients.

In contrast to VH3 representation in the naive repertoire, we found evidence suggestive of deletion of VH3 B cells in the pool of IgG memory cells. Unfortunately, somatic mutation in memory B cells precluded determination of individual IgG VH3 gene frequency by the oligonucleotide hybridization procedure, which detects primarily unmutated germline configurations in the naive repertoire. The reduction we observed in VH3 IgG mRNA expression (17%), although statistically significant, was not as prominent as in previous reports (50–85%) (18, 21, 50) nor as dramatic as the 40% decrease in VH3 SpA-reactive serum IgM. Moreover, the decrease in VH3 IgG mRNA expression may be offset by the increase in total IgG in these patients (Table IIIGo); a more subtle deficiency in expression of specific VH3 genes cannot be ruled out. In addition, others have reported approximately a 50% decrease in VH3 IgG serum protein levels (49). The reduction we observed in the relative expression of VH3 IgG mRNA was accompanied by an increase in the relative expression of VH4 IgG mRNA. These differences may have arisen due to a decrease in the levels of VH3 IgG mRNA, or an increase in the levels of VH4 IgG mRNA, or a combination of both of these events. We are currently confirming and extending these observations in B cell populations from other sites. Nevertheless, in our study, the VH-associated changes were most apparent in previously activated IgG memory cells and in the actively produced IgM in serum rather than in the resting non-Ig-producing peripheral IgM- and IgD-bearing naive B cells.

The hypothesis that HIV-1 gp120 could bind preferentially to previously activated IgG B cells, rather than to resting naive cells, seems unlikely. Such differentiated cells accumulate somatic mutations, and gp120-binding activity is reported to be more limited with V3–23 and V3–30 mAbs with somatic mutations than with those in germline configuration (31). Thus, if activation and subsequent deletion of naive VH3 B cells by gp120 contributes to the depletion of VH3 IgG memory B cells, then our data suggest that replenishment of naive VH3 B is adequate to maintain the naive pool in HIV-1-infected patients. Whether soluble gp120 alone is sufficient to initiate and sustain the VH3 family B cell deletion process or requires cross-linking, costimulatory activity, or other independent antigenic stimuli (e.g., specific acute or chronic infections) is currently under investigation. Of particular clinical relevance, such preferential deletion of Ag-specific cells following stimulation may underlie the recent observation that significantly increased rates of pneumonia were observed in patients randomized to receive pneumococcal vaccination among HIV-1-infected patients in Africa compared with those in unvaccinated patients (51).

Clearly, given the high levels of HIV-1 detected in the plasma of our patients and others, HIV-1 gp120 has ample opportunity to interact with host B cells in vivo. Moreover, persuasive evidence supports a direct interaction between gp120 and VH3 Ig in vitro (23, 24, 25, 31). However, there is currently no direct evidence that gp120, acting as a superantigen, is responsible for the in vivo deletion of human VH3 B cells. Similarly, experimental evidence delineating the mechanism(s) of such deletion is currently unavailable in humans (26, 44, 52). Nevertheless, in neonatal mice administered the related VH3-specific B cell superantigen SpA, >80% of all SpA-binding splenic B cells (clan III, the murine equivalent of VH3) were deleted (53, 54). However, this effect on conventional (B-2) B cells is transient since SpA-binding B cells return to nearly normal levels in the spleen soon after SpA treatment is terminated (53). In more recent studies, SpA treatment was shown to result in a permanent loss of IgM clan III B cells that derive from the self-renewing B-1 cell pool (55). These changes were proportional to the affinity of the SpA-clan III IgM interaction. Interestingly, these animals also had a concurrent loss of circulating clan III IgM Ab 53, 55 . If these murine models of in vivo B cell superantigen properties are relevant, it would suggest that in vivo B cell activation may be required for gp120-associated B cell dysfunction and VH3 B cell depletion.

In summary, we have shown that defects in VH3 gene expression are more readily apparent in actively produced serum IgM protein and in mRNA from previously activated memory IgG B cells than within the naive IgM and IgD peripheral B cell repertoire of advanced HIV-1-infected patients. These data suggest that activation and differentiation of B cells may be a requisite component of the process leading to HIV-1-related diminution of VH3 Abs. The study of VH genes is important as the sequence of the hypervariable regions provides both diversity and specificity to the humoral response. The recognition of conserved VH3 framework regions by gp120 has been implicated in the pathogenic interaction, resulting in the selective deletion of the VH3 gene family (31), which code for up to 40–50% of all Abs (12, 13, 14, 15, 16). Thus, the genetic substrate for the majority of all Ag-specific Abs produced may be compromised. Of particular relevance, Abs to polysaccharide Ags are encoded predominantly by VH3 family genes (28, 56, 57, 58, 59). These Abs serve as a primary mechanism of defense against Streptococcus pneumoniae, Haemophilus influenzae, Salmonella spp., and Cryptococcus neoformans (17), each of which is a common cause of serious infections during HIV-1 disease. Thus, selective deletion of VH3 pathogen-specific Abs may underlie, in part, the high rates of these often invasive and fatal infections and may contribute to the impaired ability to respond to vaccines available to prevent them (5, 6, 28, 51, 60). Current efforts are directed to determine whether a paucity of VH3 Abs predisposes to these infections and whether such specific infectious stimuli contribute to the proposed activation-associated deletion of VH3 genes in HIV-1-infected patients, further compromising their ability to resist invasive opportunistic infections.


   Acknowledgments
 
We thank Erin Lorenz for excellent clinical support and Dr. Brian Van Ness for thoughtful advice and comments.


   Footnotes
 
1 This work was supported by National Institutes of Health Contracts and Grants DE42600, DE72621, A139445, and HL-96008 and by a grant from the Veteran Affairs Research Service (to E.N.J.). R.W.S. was supported by National Research Service Award Training Grant DE05703 from the National Institute of Dental and Craniofacial Research. Back

2 Address correspondence and reprint requests to Dr. Edward N. Janoff, Infectious Disease Section (111F), Veteran Affairs Medical Center, One Veterans Drive, Minneapolis, MN 55417. Back

3 Abbreviation used in this paper: SpA, staphylococcal protein A. Back

Received for publication October 13, 1999. Accepted for publication March 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Immunol., October 1, 2002; 169(7): 4008 - 4016.
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