The HIV-1 envelope protein gp120 impairs B cell proliferation by inducing TGF-β1 production and FcRL4 expression

Katija Jelicic, Raffaello Cimbro, Fatima Nawaz, Da Wei Huang, Xin Zheng, Jun Yang, Richard A Lempicki, Massimiliano Pascuccio, Donald Van Ryk, Catherine Schwing, Joseph Hiatt, Noreen Okwara, Danlan Wei, Gregg Roby, Antonio David, Ii Young Hwang, John H Kehrl, James Arthos, Claudia Cicala, Anthony S Fauci, Katija Jelicic, Raffaello Cimbro, Fatima Nawaz, Da Wei Huang, Xin Zheng, Jun Yang, Richard A Lempicki, Massimiliano Pascuccio, Donald Van Ryk, Catherine Schwing, Joseph Hiatt, Noreen Okwara, Danlan Wei, Gregg Roby, Antonio David, Ii Young Hwang, John H Kehrl, James Arthos, Claudia Cicala, Anthony S Fauci

Abstract

The humoral immune response after acute infection with HIV-1 is delayed and ineffective. The HIV-1 envelope protein gp120 binds to and signals through integrin α4β7 on T cells. We found that gp120 also bound to and signaled through α4β7 on naive B cells, which resulted in an abortive proliferative response. In primary B cells, signaling by gp120 through α4β7 resulted in increased expression of the immunosuppressive cytokine TGF-β1 and FcRL4, an inhibitory receptor expressed on B cells. Coculture of B cells with HIV-1-infected autologous CD4(+) T cells also increased the expression of FcRL4 by B cells. Our findings indicated that in addition to mediating chronic activation of the immune system, viral proteins contributed directly to HIV-1-associated B cell dysfunction. Our studies identify a mechanism whereby the virus may subvert the early HIV-1-specific humoral immune response.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
MAdCAM-1 and HIV-1 gp120 bind to α4β7 in a cation dependent manner. (a) α4β7 expression detected by flow cytometry on freshly isolated primary B cells by an anti-β7 and anti-α4β7 (Act-1). (b) Variability of α4β7 expression on B cells from two separate donors. Flow cytometry shows that the binding of R66M gp120 (derived from a patient within the first month of infection) is proportional to β7 expression. β7 gating is based on an isotype control for each donor. (c) Binding of R66M gp120 to α4β7 on B cells. R66M gp120 binding to B cells in the presence of Ca2+/Mn2+ +/− anti-α4 (2B4), or in the absence of Ca2+/Mn2+ (EDTA buffer). MAdCAM-1 binding to B cells in the presence of Ca2+/Mn2+ +/− anti-α4 (2B4). Values reported indicate % of binding normalized to R66M (or MAdCAM-1) binding (100%), and are derived from 3 independent donors, p<0.0001 (one way ANOVA) (R66M n=3) (MAdCAM-1 n=2). (d) Comparison of the α4β7-reactivity of a panel of five recombinant gp120 preparations. α4β7-reactivity is reported as mean fluorescence intensity (MFI) of gp120s. An unlabeled anti-α4 (2B4) was included as a specificity control. Results are representative of at least three independent experiments using B cells from different donors.
Figure 2
Figure 2
An α4β7-reactive gp120 inhibits α-IgM + CpG induced B cell proliferation. (a) CFSE proliferation assay of α-IgM + CpG induced proliferation of B cells in the presence of gp120s. An α4β7-reactive gp120 (R66M) blocks proliferation, while a gp120 with minimal affinity for α4β7 (92Th14.12) fails to block proliferation. B cells were cultured with B cell stimuli (α-IgM + CpG) with or without gp120s for 96 h. (b) Division Index (FlowJo) indicating the average number of cell divisions in cells from the original population from 5 independent donors. Treatment stimuli denoted below the x-axis, p<0.001 (two-way ANOVA) (n=5). (c) A dose response utilizing three increasing concentrations of a gp120 with a high affinity for α4β7 (R880F) were employed. (d) Light scatter overlay of freshly isolated (red), α-IgM + CpG stimulated (blue), and α-IgM + CpG + gp120 stimulated (green) B cells. (e) Flow-cytometry based cell cycle analysis of α-IgM + CpG stimulated (blue), and α-IgM + CpG + gp120-stimulated (green) B cells stained with PI (x-axis). Data reported are representative of three independent experiments.
Figure 3
Figure 3
HIV-1 gp120s with different affinity for α4β7 affect gene expression of freshly isolated human B cells. (a) Flow cytometry shows the binding to human primary B cells of two gp120s employed for microarray analysis: Z205F 0M with a high affinity for α4β7 and Z205F 38M with a low affinity for α4β7.(b) Heat map visualization by Partek of gene expression modulation in response to treatment with month-0 gp120 (Z205F 0M with a high (H) affinity for α4β7) and month-38 gp120 (Z205F 38M with a low (L) affinity for α4β7) isolated from a single patient. B cells from three normal donors were treated with the envelopes for 0.5, 3 and 6 h. Statistical significance is reported relative to mock-treated B cells. Categories of the genes modulated by gp120-α4β7 interaction are specified (DAVID Bioinformatics Tool). (c) PCR verification of gp120-induced genes in B cells. NT: mock, Low: gp120 with low α4β7 affinity, High: gp120 with high α4β7 affinity. Values are shown in fold increase over mock treated samples. Data reported are representative of three independent experiments.
Figure 4
Figure 4
HIV-1 gp120s with different affinity for α4β7 affect gene expression of α-IgM + CpG stimulated B cells. (a) Flow cytometry shows the binding to human primary B cells of the two gp120s employed for microarray analysis: R880F 0M with a high affinity for α4β7 and 92Th14.12 with a low affinity for α4β7. (b) Heat map visualization by Partek of gene expression modulation in response to treatment with month-0 gp120 (H) with a high affinity for α4β7 (R66M) and a gp120 with a low (L) affinity for α4β7 (92Th14.12). B cells were treated with the envelopes for 6h. Statistical significance is reported relative to mock-treated B cells. Categories of the genes modulated by gp120-α4β7 interaction are specified (DAVID Bioinformatics Tool). (c) FcRL4 mRNA induction at 6h in the first set and second set of microarray analysis shown in fold change (log2). Data reported are representative of three independent experiments.
Figure 5
Figure 5
CD80 induction by α-IgM + CpG stimulation is reduced in presence of an α4β7-reactive gp120. (a) FACS analysis of CD80 surface expression induced by α-IgM + CpG stimulation for 72h, in the presence or absence of gp120s with high (R880F) versus low (92Th14.12) affinity for α4β7. Values reported are average % reactivity normalized to CD80 expression at 72h upon B cell stimulation with α-IgM + CpG alone (100%), p<0.0001 (two way ANOVA) (s.e.m bars) (n=3). (b) FACS analysis of CD80 surface expression over time induced by stimulation with α-IgM + CpG (24, 48, 72 h) in the presence or absence of gp120 with high affinity for α4β7 (R880F) or intermediate affinity for α4β7 (AN1). (c) FACS analysis of CD86 surface expression on B cells activated by α-IgM + CpG for 24, 48, 72 h, in presence of gp120s (R880F, AN1, Z185F) with different affinities for α4β7 (72h). Data reported indicate MFI, and are representative of three independent experiments using different donor B cells.
Figure 6
Figure 6
FcRL4 expression induced by α-IgM + CpG stimulation is increased and prolonged in presence of an α4β7-reactive gp120. (a) FACS analysis of FcRL4 expression over a time course of 0–72h on freshly isolated B cells stimulated with α-IgM + CpG. Isotype control in shown as a grey shade. (b) FACS analysis of FcRL4 expression over time (0–72h) on freshly isolated B cells stimulated with α-IgM + CpG +/− an α4β7-reactive gp120. (c) FACS analysis of FcRL4 expression at 72h on freshly isolated B cells stimulated with α-IgM + CpG in presence or absence of gp120 with a high (R880F) and an intermediate (AN1) affinity for α4β7.(d)In vitro proliferation assays (CFSE) were performed with B cells that were stimulated with α-IgM + CpG for 96h and analyzed by FACS after staining for FcRL4 surface expression. CFSE staining by FcRL4 is presented and is representative of five independent experiments. (e) % FcRL4 expression on B cells stimulated with α-IgM + CpG +/− α4β7-reactive gp120 from 6 independent donors p=0.03 (Wilcoxon matched-pairs signed rank test) (n=6). (f) Histogram of a FACS analysis of an A32 (anti-gp120) staining of HIV-1 infected or uninfected CD4+ T cells that were employed in the co-culture experiment. (g) FACS analysis of FcRL4 expression on B cells co-cultured with infected CD4+ T cells isolated from the same donor. Shown is a representative result from one of ten independent donors of the surface expression of FcRL4 by CD19 detected by FACS after 48h of co-culture. (h) % FcRL4 expression on B cells from 10 donors stimulated with α-IgM + CpG and co-cultured with autologous uninfected or HIV-1 infected donor CD4+ T cells, p=0.002 (Wilcoxon matched-pairs signed rank test) (n=10).
Figure 7
Figure 7
gp120 induces FcRL4 via the induction of TGF-β1. (a) Average TGF-β1 ELISA of supernatants from cultured primary B cells from three separate healthy donors stimulated with α-IgM + CpG in presence of an α4β7-reactive gp120 (R66M), p<0.0001 (unpaired t-test) (s.e.d. bars) (n=3). (b) FACS analysis of % FcRL4 and % CD80 expression on α-IgM + CpG stimulated B cells in presence of gp120 or TGF-β1. Anti-TGF-β1 was employed to block both gp120-mediated and TGF-β1-mediated effects. The average % of cells expressing FcRL4 and CD80, normalized to FcRL4 and CD80 expression at 96h upon B cell stimulation with α-IgM + CpG alone (100%), p<0.0001(one way ANOVA, Bonferroni Multiple Comparison Test) (s.e.m bars) (FcRL4 n=8) (CD80 n=5). (c) CFSE assay of α-IgM + CpG induced B cell proliferation (1st panel), in the presence of: an α4β7-reactive gp120 (2nd panel), an α4β7-reactive gp120 and an anti-TGF-β1 (3rd panel), soluble TGF-β1 (4th panel), and soluble TGF-β1 and an anti-TGF-β1 (5th panel) of a representative donor. Cells were cultured for 96h. (d) Division Index (FlowJo) indicating the average number of cell divisions in three independent donors, p<0.001 (two-way ANOVA) (n=3). Treatments are listed below the x-axis.
Figure 8
Figure 8
A T-dependent stimulation of B cells is suppressed by an α4β7-reactive gp120. (a) FACS analysis over time of the average % CD80 surface expression in three donors, induced by α-IgM + CpG + α-CD40 stimulation (24, 48, 72 h) in presence or absence of an α4β7-reactive gp120. Values reported are average % reactivity normalized to CD80 expression at 72h upon B cell stimulation with α-IgM + CpG + α-CD40 alone (100%), p<0.001 at 72h (two way ANOVA) (s.e.m bars) (n=4). (b) FACS analysis over time of the average % CD86 surface expression in three donors, induced by α-IgM + CpG + αCD40 stimulation (24, 48, 72 h) in presence or absence of an α4β7-reactive gp120. Values reported are average % reactivity normalized to CD86 expression at 72h upon B cell stimulation with α-IgM + CpG + α-CD40 alone (100%) (two way ANOVA) (s.e.m bars) (n=4). (c) FACS analysis for FcRL4 expression over a time course of 0–72h on freshly isolated B cells stimulated with α-IgM + CpG + α-CD40. Isotype control in shown as a grey shade. (d) FACS analysis of the average % FcRL4 and % CD80 expression induced on α-IgM + CpG + α-CD40 stimulated B cells in presence of R66M or TGF-β1. Anti-TGF-β1 was employed to block both gp120-mediated and TGF-β1-mediated effects. The average % of cells expressing FcRL4 and CD80 is reported normalized to FcRL4 and CD80 expression at 96h upon B cell stimulation with α-IgM + CpG + α-CD40 (100%), p<0.001 and p<0.0001(one way ANOVA, Bonferroni Multiple Comparison Test) (s.e.m bars) (FcRL4 n=4) (CD80 n=5). Values are normalized to % FcRL4 expression on α-IgM + CpG + α-CD40 stimulated B cells (100%). (e) CFSE assay of α-IgM + CpG + αCD40 induced B cell proliferation (1st panel), in the presence of: an α4β7-reactive gp120 (2nd panel), an α4β7-reactive gp120 and an anti-TGF-β1 (3rd panel), soluble TGF-β1 (4th panel), and soluble TGF-β1 and an anti-TGF-β1 (5th panel) of a representative donor. Cells were cultured for 96h. (f) Division Index (FlowJo) indicating the average number of cell divisions in three independent donors, p<0.001 (two-way ANOVA) (n=3). Treatments are listed below the x-axis.

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