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Keywords:

  • activation-induced cytidine deaminase;
  • apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G;
  • B-cell agonists;
  • HIV infection

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

B cells express two critical deaminases in the development of adaptive and innate immunity. Activation-induced cytidine deaminase (AID) functions in class switch recombination, somatic hypermutation and may result in affinity maturation of antibodies. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G; A3G) is an innate anti-retroviral factor that inhibits HIV replication. We have studied a number of B-cell agonists with the aim of identifying the most effective agents that will up-regulate both deaminases and thereby enhance adaptive and innate immunity. CD40 ligand (CD40L) with interleukin-4 or HLA-class II antibodies significantly up-regulated both AID and A3G in isolated human CD19+ B cells. The functions of these deaminases were demonstrated by enhancement of B-cell surface expression of IgA and IgG and inducing significantly higher IgA and IgG4 antibodies. An enhanced A3G function was then demonstrated by inhibition of HIV-1 replication in co-culture of CD4+ T cells with autologous B cells, treated with CD40L and CD4 or HLA antibodies, compared with unstimulated human B cells. The dual B-cell-induced deaminase functions may be critical in IgA and IgG antibodies inhibiting pre-entry and A3G that of post-entry HIV-1 transmission and suggests a novel strategy of immunization, especially relevant to mucosal infections.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA-enzyme catalytic polypeptide-like 3G (APOBEC3G) are members of the APOBEC cytidine deaminase family of proteins.1,2 AID and APOBEC1 show significant homology and although APOBEC3G (A3G) appears to be a gene-duplication of AID protein3 there is limited homology between the two. AID is expressed in B cells inducing class switch recombination of the μ constant region to γ, α and ε, thereby changing the antibody isotype from IgM to IgG, IgA and IgE. AID is also essential in somatic hypermutation, introducing point mutations at the immunoglobulin gene variable region, which is responsible for affinity maturation and memory.4–6 Deamination is involved not only in antibody gene diversification by AID, but also in protection against retroviral DNA by A3G, mostly studied in CD4+ T cells, dendritic cells and macrophages as a mechanism against retroviral infections.1,7 Although A3G has been reported in B cells and higher levels were found in B cells than in monocytes,8 an anti-HIV-1 function of A3G in B cells, which lack the CD4 receptor for HIV-1, is unlikely.

Although the anti-viral function of secretory IgA at mucosal surfaces is well recognized, the anti-viral function of A3G produced by B cells has not been studied. For A3G produced by B cells to prevent HIV-1 infection it must be transmitted to CD4+ T cells, macrophages or dendritic cells. A potential route is via exosomes, as A3G is a major exosomal component responsible for anti-HIV-1 activity, conferring virus-restricted replication on CD4+ recipient cells.9 Although the A3G-containing exosomes were derived from CD4+ T cells, B cells are a major in vivo source of exosomes, stimulated by CD40 ligand (CD40L) + interleukin-4 (IL-4).10 As most HIV-1 infections are transmitted at mucosal surfaces (cervico-vaginal, rectal and penile foreskin), a dual function of B cells, generating AID, which enhances IgA and IgG antibody development, and A3G, having innate anti-viral activity, may exert pre- and post-entry anti-viral functions, at the most vulnerable mucosal site of infection.

The objectives of this study were (i) to demonstrate in vitro in primary human CD19+ B cells that both AID and A3G mRNA and protein can be up-regulated by stimulating with selected B-cell agonists; (ii) to determine if up-regulation of AID with B-cell agonists will increase IgA and IgG isotype production; and (iii) to establish if the increased A3G will exert anti-HIV-1 function when activated B cells are co-cultured with HIV-1-infected CD4+ T cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Preparation of B cells from human PBMC

Peripheral blood mononuclear cells (PBMC) were isolated either from buffy coats or from apheresis cones (National Blood Service Tooting, London, UK) by centrifugation on Ficoll-Paque PLUS density gradients (GE Healthcare UK Ltd., Little Chalfont, UK). The B cells were prepared from PBMC by magnetic bead separation using positive selection with CD19 MicroBeads (Miltenyi, Bisley, UK). The cells were suspended at 2 × 106 to 5 × 106 per ml in RPMI-1640 with 10% fetal calf serum and stimulated with the following agents for 2–3 days: transforming growth factor-β (TGF-β), B cell activating factor belonging to the TNF family (BAFF), IL-4 and a proliferation inducing ligand (APRIL) (all from R&D Systems, Oxford, UK), anti-HLA Class II DR antibody L234 (BioLegend Ltd, Cambridge, UK), anti-CD45RA and anti-IgM antibodies (from BD Biosciences, Oxford, UK), CD40L trimer (a kind gift from Dr F. Villinger), or lipopolysaccharide from Sigma (Poole, UK).

Selection of the most effective agent that will up-regulate AID and A3G in B cells determined by intracellular immunofluoresence

B cells were stimulated with 100 U/ml IL-4 (R&D Systems) and 100 ng/ml CD40 ligand trimer. After 3 days the cells were washed in PBS with 1% BSA and 0·1% sodium azide and then surface stained with anti-CD19 antibody coupled to allophycocyanin (Serotec, Oxford, UK). After 20 min the cells were washed and fixed lightly by addition of fixation buffer containing formaldehyde for 10 min (eBioscience Ltd, Hatfield, UK). The cells were then washed using permeabilization buffer (eBioscience). Goat antibody to AID (AICDA, Dundee Cell Products, Dundee, UK) or rabbit antibody to A3G (Immunodiagnostics Inc., Woburn, MA) was added at 2 μg/ml in permeabilization buffer. After 20 min cells were washed and FITC-labelled secondary antibody (Sigma-Aldrich, Poole, UK) was added at 1 : 100 dilution, again in permeabilization buffer. After a further 20 min the cells were washed and resuspended in PBS with 0·5% formaldehyde. The cells were analysed by flow cytometry on a FACSCanto II (BD Biosciences) using FACS Diva software. Specificity of signal was determined by inhibition of staining using purified AID (AICDA, Dundee Cell Products) or A3G (Immunodiagnostics Inc.). Where cells were double-labelled for AID and A3G a monoclonal antibody (mAb) to A3G (Immunodiagnostics Inc.) was used and the secondary antibodies were FITC anti-goat immunoglobulin (Sigma-Aldrich) and a phycoerythrin-conjugated anti-mouse immunoglobulin antibody (Southern Biotechnology Associates, Birmingham, AL).

Western blots of A3G protein in B cells

To detect A3G 10 × 106 cells were lysed in 1 ml RIPA buffer for 30 min on ice, cleared by centrifugation and equal volume of 2 × SDS sample buffer was added under reducing conditions before SDS–PAGE. After transfer of proteins to a PVDF membrane, Western blotting was carried out with mouse mAb to A3G (#7105; ImmunoDiagnostics) and β-actin (clone AC-15; Sigma), using horseradish peroxidase-conjugated anti-mouse IgG antibody and Immobilon Western HRP Substrate (Millipore, Watford, UK).

Real-time PCR of AID and A3G in B cells

About 5 × 106 B cells were harvested and washed with PBS, before extraction of RNA with the Promega SV Total RNA Isolation System. RNA was reverse transcribed according to the manufacturer’s instructions with oligo(dT) primers and AMV reverse transcriptase (Promega, Southampton, UK). Real-time PCR with cDNA as template was performed with Sigma’s SYBR Green JumpStart Taq Ready Mix (Sigma-Aldrich) and gene-specific primers for A3G (5′-TTGTTGCCCGCCTCTACTAC-3′, 5′-TTGGCTGTACACGAA CTTGC-3′), AID (5′-AGAGGCGTGACAGTGCTACA-3′, 5′-TGTAGCGGAGGAAG AGC AAT-3′) and glyceraldehyde 3-phosphate dehydrogense (GAPDH: 5′-CTTTTGCGTCGCCAGCCGAG-3′, 5′-ACCAGGCGCCC AATACGACC-3′) as housekeeping control. A Corbett Rotor-Gene 6000 PCR cycler was used to run the reactions and manufacturer’s software to analyse data with the ‘Two Standard Curve’ method. The resulting data were normalized to the housekeeping gene GAPDH and expressed relative to unstimulated cells which were accorded an arbitrary value of 100. To determine the concentrations of CD40L + IL-4 that will induce optimum AID and A3G mRNA relative to unstimulated cells, these were titrated from 50 to 200 ng/ml CD40L and 20–100 units/ml IL-4. The results suggested that 100 U/ml IL-4 with 100 ng/ml CD40L were most consistent in eliciting AID and A3G.

The functional effects of AID on antibody switching and A3G on HIV-1 infectivity

The effect of AID expression was examined in isolated CD19+ B cells by flow cytometry analysis for cell surface IgM, IgG and IgA isotypes using the following fluorochrome-conjugated mAbs: anti-IgG-FITC and anti-IgM-phycoerythrin (BD Pharmingen, Oxford, UK), anti-IgE-FITC and anti-IgA-FITC (Miltenyi Biotec; Bisley, Surrey, UK). The corresponding secreted antibodies with IgG subtypes were then assayed in the culture supernatants after 4 days culture by the Bio-Plex Pro Human 7-Plex Panel (Bio-Rad, Hemel Hempstead, UK), which contains specific antibodies to IgM, IgG1, IgG2, IgG3, IgG4, IgA and IgE.

The effect of B-cell-generated A3G on HIV-1 infection of autologous CD4+ T cells was then studied. CD4-positive cells were activated with 10 μg/ml of phytohaemagglutinin and 20 IU of IL-2 in culture medium (RPMI-1640 medium) with 10% fetal calf serum (Biosera Ltd, Ringmer, UK), penicillin at 100 U/ml, streptomycin at 100 μg/ml and 2 mm l-glutamine (Sigma) for 3 days and then washed with medium. The cells were infected with HIV-1 BaL (R5 strain) or LAI (×4 strain), incubated for 2 hr, washed three times with medium and cultured in triplicate at 0·5 × 105 cells per well in the culture medium containing 100 IU IL-2. CD19+ B cells were activated with CD40L and IL-4 or HLA-class II (DR) antibodies for 3 days. They were then washed thre times, counted and distributed into the wells of 96-well culture plates containing infected cells. Every 3 days, 100 μl of culture supernatant was replaced with 100 μl of supplemented medium. On day 9, the culture supernatants were assayed for p24 using HIV-1 p24 antigen ELISA (ZeptoMetrix Corp., Buffalo, NY) and the results are presented as a mean of two readings.

Statistical analysis

The data are presented as mean (± SEM) and statistical analysis was carried out by the paired or one-sample t-test.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Up-regulation of AID and A3G expression in human B cells by stimulating with B-cell agonists

CD19+ B cells were isolated to > 90% purity from human PBMC and stimulated for 3 days with seven single and 11 combined B-cell agonists (Tables 1 and 2). Comparative immunofluorescence with mAb to AID and A3G showed that stimulation with CD40L failed to induce a significant increase in AID (P = 0·07) compared with A3G (P = 0·014) expression. In contrast IL-4 or HLA-II antibodies induced a significant increase in AID but not A3G (Table 1). Combined CD40L + IL-4, however, stimulated significant up-regulation of both AID (P = 0·004) and A3G (P = 0·048), as did CD40L + HLA-II antibody (AID (P = 0·001) and A3G (P = 0·027) (Table 1). CD40L + IgM antibodies also induced significant increase of both AID (P = 0·002) and A3G (P = 0·001). Lipopolysaccharide with IL-4 or IgM antibodies yielded significant increases in A3G and AID but this was not pursued, because lipopolysaccharide would not be suitable to administer in vivo. CD40L + IgM antibodies were also very effective in stimulating AID and A3G but this was not pursued as we had to focus on a small number of B-cell agonists. Limited or no significant changes were noted with the other B-cell agonists (Table 2). Representative illustration of CD40L + IL-4 stimulation is shown in Fig. 1(a,b). To demonstrate that AID and A3G are expressed in the same cells, purified CD19+ B cells were double-stained with mAb to AID and A3G. Whereas < 5% of the untreated B cells stained with both antibodies to AID and A3G, about 70% reacted with both antibodies after stimulation with CD40L + IL-4 (Fig. 1d,e). These results suggest that the B-cell agonists up-regulated both principal deaminases in the same B cells.

Table 1.   The effect of selected CD19+ B cell agonists on activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (A3G) mRNA and protein (A) and mRNA (B) expression determined by real-time PCR and immunofluorescence with the specific antibodies, respectively
 Mean (± SEM)tPMean (± SEM)tP
(A)AID-MFI proteinA3G-MFI protein
  1. CD40L, CD40 ligand; IL-4, interleukin-4; MFI, mean fluorescence intensity.

  2. AID and A3G mRNA were assayed relative to GAPDH (100). Independent experiments were carried out on B cells with each agonist from four to six subjects. For analysis of AID and A3G protein the paired t-test and for mRNA the one-sample t-test were used; *P < 0.05, **P < 0.005.

Untreated956 (97)  472 (79)  
CD40L1471 (187)4.400.07769 (104)3.670.014*
IL-41660 (271)2.820.037500 (138)0.320.760
CD40L + IL-41820 (246)5.020.004**894 (226)2.600.048*
HLA-II antibodies1249 (139)3.340.021*612 (117)0.990.353
CD40L + HLA-II antibodies1784 (185)6.620.001**1046 (238)3.090.027*
(B)AID mRNAA3G mRNA
CD40L258 (131)1.210.16128 (13)2.210.057
IL-41958 (686)2.710.037*110 (17)0.580.29
CD40L + IL-42093 (771)2.590.041*156 (18)3.120.026*
HLA-II antibodies101 (29)0.030.49138 (22)1.720.092
CD40L + HLA-II antibody295 (128)1.530.11177 (25)3.100.027*
Table 2.   The effect of single or combined B-cell agonists stimulating activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA-enzyme catalytic polypeptide-like 3G (A3G) expression by isolated human B cells was compared with untreated B cells and determined by immunofluorescence with anti-AID or A3G antibodies
B-cell agonist (n)AID-MFIA3G-MFI
Mean (± SEM)tP(n)Mean (± SEM)tP
  1. LPS, lipopolysaccharide; BAFF, B cell activating factor belonging to the TNF family; APRIL, a proliferation inducing ligand; CD40L, CD40 ligand; IL-4, interleukin-4; MFI, mean fluorescence intensity.

  2. The results are presented as mean (± SEM) MFI; the number of independent experiments are shown as (n) and the paired Student’s t-test was used for statistical analysis.

Single B-cell agonists
 Untreated (6)956 (97)  3472 (79)  
 IgM antibody (3)1591 (541)1.160.3675668 (72)1.580.078
 LPS (6)1375 (221)2.460.0575774 (183)1.320.110
 BAFF (6)953 (146)0.030.9755519 (84)0.410.34
 APRIL (6)913 (119)0.730.4985504 (81)0.280.391
Combined B-cell agonists
 CD40L + IgM antibody (4)1905 (136)5.670.00231124 (32)7.710.0001
 CD40L + CD45RA (3)1477 (393)1.290.32251076 (75)5.580.003
 CD40L + LPS (3)1066 (190)1.820.1473350 (92)1.00.375
 IL-4 + IgM antibody (4)2330 (776)1.760.1773691 (67)1.780.059
 IL-4+LPS (4)1681 (206)3.180.03431216 (160)4.180.025
 HLA-II antibody + LPS (6)1710 (295)2.930.0336871 (262)1.970.106
 anti-IgM + LPS (4)2533 (499)3.100.05331092 (115)4.450.021
 anti-IgM + BAFF (3)988 (110)0.190.4243661 (78)1.200.136
 anti-IgM + APRIL (3)1053 (230)0.580.2873638 (75)1.030.170
image

Figure 1.  Representative illustrations of activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G; A3G) expression in isolated CD19+ B cells stimulated with CD40 ligand (CD40L; 100 ng/ml) and interleukin-4 (IL-4; 100 units/ml) bold lines (a, b). Western blot of A3G (c) and expression of both AID and A3G in the same cells (d, e).

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To confirm A3G protein we subjected lysates from unstimulated CD19+ B cells to SDS–PAGE, transferred the proteins to PVDF membranes and blotted with mAb to A3G (Immunodiagnostics Inc.). A band with a molecular weight of about 46 000 with a faint band underneath appeared, which was greatly enhanced when B cells were activated with CD40L + IL-4 (Fig. 1c).

Up-regulation of AID and A3G mRNA expression in human B cells by stimulating with B-cell agonists

AID and A3G mRNA expression were then evaluated by real-time PCR. All results were expressed as mean (± SEM) relative to unstimulated cells, which were accorded an arbitrary value of 100. CD40L induced an increase in AID mRNA from 100 to 258 (± 131) and to a lesser extent in A3G mRNA to 128 (± 13), these failed to reach the 5% level of significance (Table 1). However, IL-4 significantly up-regulated AID (P = 0·037) but not A3G (P = 0·29). The combined CD40L + IL-4 B-cell agonists up-regulated significantly both AID and A3G mRNA (P < 0·05), and this was much greater for AID than A3G mRNA (Table 1). CD40L + HLA class II antibodies (177 ± 25) was more effective for A3G mRNA (P = 0·027) than that for AID mRNA (295 ± 128, P = 0·11). As with immunofluorescence the other B-cell agonists were not pursued further. The results suggest that CD40L + IL-4 yielded the most consistent and significant increases in both mRNA and protein of AID and A3G.

B-cell agonist-mediated up-regulation of AID is translated to switching of immunoglobulin isotypes

We have evaluated a major function of AID in B cells by demonstrating a significant increase in the cell-surface expressions of IgG (P < 0·001) and IgA (P < 0·0001) (Fig. 2b,c) when stimulated with CD40L + HLA-II mAb and to a lesser extent with CD40L + IL-4 (P < 0·03). The IgM also increased but to a greater extent with CD40L + IL-4 than CD40L + HLA-II mAb (Fig. 2a). These studies were then extended to the culture supernatants of the B-cell-agonist-stimulated cells. Using the Luminex bead technology confirmed the increase in IgA antibodies in the 4-day culture supernatants of CD40L + IL-4-stimulated B cells (Fig. 3). The 6·4-fold higher concentration of IgA compared with IgG1 was surprising as the reverse is normally found in serum. This might be related to the shorter half-life of IgA (about 9 days) compared with that of IgG (about 21 days). After 7 days of culture, the supernatants showed a significant increase in IgG4 by stimulation with CD40L + IL-4 (P = 0·01), though the total concentration was moderate (12·6 ± 5·4 ng/ml) (Fig. 3b).

image

Figure 2.  CD19+ B-cell expression of the mean (± SEM) of IgM, IgG and IgA (a, b, c) before and after stimulation for 4 days with CD40 ligand (CD40L; 100ng/ml) + HLA class II antibodies (5 μg/ml) or CD40L + interleukin-4 (IL-4; 100 units/ml), and the corresponding flow cytometry illustrations; *P < 0.05, **P < 0.01, ***P < 0.0001.

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image

Figure 3.  Culture supernatants analysed for IgM, IgG1, IgG2, IgG3, IgG4 and IgA (×10−1) by the Luminex assay; the data are presented as mean (± SEM) ng/ml of four independent experiments of 4 days cultures, and 7 days culture for IgG4; *P < 0.05, **P < 0.01.

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Functional effect of B-cell-generated A3G on HIV infection of autologous CD4+ T cells

A functional effect of up-regulation of A3G by stimulating primary B cells with the selected agonists was studied in HIV-1 (BaL) infectivity of autologous CD4+ T cells. Isolated B cells were stimulated with CD40L + IL-4 or HLA-II mAb for 3 days, followed by co-culturing the B cells with autologous CD4+ T cells (activated with phytohaemagglutinin and IL-2 for 3 days) and infected with serial dilution HIV-1 (BaL) for 9 days. The results showed dose-dependent inhibition of HIV-1 replication with the B cells pre-treated with either of the B-cell agonists, compared with the untreated B cells (Fig. 4a,b). The possibility that the small number of contaminating cells might have inhibited HIV-1 is unlikely, because the evidence of B-cell-dependent inhibition rests on A3G rich exosomes (see Discussion). Hence, B-cell agonists that up-regulate A3G on culture with HIV-1-infected autologous CD4+ T cells significantly inhibit HIV-1 replication and the mechanism involved is suggested in the Discussion.

image

Figure 4.  CD19+ B cells isolated from peripheral blood mononuclear cells (PBMC) were activated with (a) CD40L + IL-4 and (b) CD40L + HLA-class II antibodies for 3 days (open circles) and then co-cultured with autologous CD4+ T cells (phytohaemagglutinin + IL-2 activated) infected with HIV-1 (BaL) for 9 days and compared with co-cultures in which the B cells were not activated (filled circles). Infectivity was assayed by ELISA of HIV p24 and the multiplicity of infection. The data are presented as mean (± SEM) of four independent experiments in PBMC from different subjects in a and b; *P < 0.05.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Investigation of a number of B-cell agonists for their potential ability to up-regulate both AID and A3G deaminases has identified a combination of CD40L with IL-4 or HLA-II antibodies to be effective. However, single B-cell agonists yielded inconsistent results, which was the rationale for using two B-cell agonists. The other B-cell agonists showed variable increases in these deaminases, with the exception of CD40L + IgM antibodies, but this was not studied further.

The two deaminases were demonstrated in the same B cells, by double staining with mAb to AID and A3G. This association has not been studied in the past, though independently AID has been extensively investigated for its essential functions in class switch recombination and somatic hypermutation. These functions are especially significant in mucosal immunity, because of isotype switching from IgM to IgG, IgA and IgE, as well as affinity maturation and memory are essential manifestations of adaptive immunity.4–6 There is clear evidence that B cells residing in human mucosa responding to allergens in vivo undergo direct or sequential class switch recombination from IgM to IgG, IgA and IgE.11 Furthermore, A3G is found in the lungs of mice,12 and lung epithelial cell line,13 suggesting that an adaptive AID-driven antibody mechanism and an innate A3G anti-retroviral factor might be generated at local mucosal sites. Whether IgA and A3G can be similarly induced in vaginal or rectal mucosa remains to be demonstrated. This would be especially important as the innate B-cell-derived A3G is probably produced earlier than IgA antibodies and this may inhibit HIV-1 replication until effective IgG and IgA antibodies develop in the mucosal tissues. Examination of IgG subclass antibodies was surprising, as only IgG4 was significantly up-regulated. The concentration of IgG4 antibodies is the lowest among the IgG subclasses, but of great interest because it is unique in combining two different specificities (H + L chain) in a single antibody molecule, termed Fab-arm exchange.14 This makes IgG4 monovalent and may act as a blocking antibody, engaging two antigens. The Fc portion interacts poorly with complement or Fc receptors on monocytes, thereby being free of these effector activities. It is not clear what role the IgG4 antibodies might play in HIV-1 pathogenesis. However, it was reported recently that in acute HIV infection half of the cohort have gp41 Env-specific and p55 gag-specific IgG4 detectable antibodies, though all subjects showed corresponding IgG1 and IgG3 antibodies.15

It has been well established that HIV-1 transmission through the cervico-vaginal or rectal mucosa leads to infection and destruction of more than 50% of CCR5+ CD4+ T cells within 14 days of infection.16–19 The innate A3G response is surprisingly long-lasting following immunization in macaques20,21 and this has been attributed to A3G being expressed in CD4+ CD95+ CCR7 effector memory T cells.20 Up-regulation of A3G stimulated by CD40L is mediated by ligation of CD40 cell-surface molecules on dendritic cells22 and this is also likely to account for A3G regulation in B cells expressing CD40. However, B-cell-derived A3G in vivo has not been studied previously.

The signalling pathway following engagement of CD40 by CD40L elicits phosphorylation of IκB kinase complex followed by nuclear translocation of nuclear factor-κB (NF-κB), which initiates class switch recombination by binding to the κB site on IH promoters.23,24 CD40L-bound CD40 also activates extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase inducing A3G mRNA and protein expression.22 Interleukin-4 bound to IL-4 receptor induces phosphorylation of Jak1 and Jak3 kinases, followed by phosphorylation and nuclear translocation of the transcription factor signal transducer and activator of transcription (STAT6) leading to class switch recombination.24

Transforming growth factor-β is another B-cell agonist critical in switching IgM to IgA.25 We have pursued a report that appeared after we had completed the project that the AID encoding gene (Aicda) responds to activation with CD40L, IL-4 and TGF-β.26 We confirmed this using human B cells, which showed maximal activation of AID mRNA with the combined three agents (2665 ± 1150), compared with TGF-β alone (80·5 ± 18) and extended it to A3G mRNA from 118 ± 45 to 495 ± 88 (P = 0·030) (data not presented). Flow cytometry studies also demonstrated a significant increase in AID expression by the combined TGF-β + CD40L + IL-4-stimulated B cells. The mechanism advanced26 was that region 4 of the AID encoding gene (Aicda) contains the functional binding sites for NF-κB, STAT6 and Smad 3/4, which are response elements to CD40L, IL-4 and TGF-β, respectively.26 This may lead to de-repression of silencers by B-lineage-specific and stimulation-responsive enhancers. Whether this mechanism might also apply to A3G, another deaminase belonging to the same family produced by B cells, needs to be verified.

We postulate that A3G produced by B cells is transmitted to CD4+ T cells probably via exosomes, in which A3G is a major component.10 B cells are significant producers of exosomes following activation of cell-surface CD40 and IL-4 receptors27 or interaction with T cells via CD40–CD40L molecules.28 Inhibition of HIV replication has been demonstrated between monocyte-derived exosomes and CD4+ T cells.9 Alternatively, B cells might produce intercellular nanotubes which establish contact with CD4+ T cells.29 The adaptive function of AID switching IgM to IgG and IgA and affinity maturation of antibodies and innate A3G antiviral activity in mucosal tissues may play an important role in inhibiting pre-entry and post-entry HIV-1 infection.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

This work was supported by grants from the European Community to TL; Network of Excellence Europrise (LSHP-CT-2006-037611) and MUVAPRED (LSHP-CT-2003-503558).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References