B Lymphocytes, but Not Dendritic Cells, Efficiently HIV-1 Trans Infect Naive CD4+ T Cells: Implications for the Viral Reservoir

Abstract

Insight into the establishment and maintenance of HIV-1 infection in resting CD4⁺ T cell subsets is critical for the development of therapeutics targeting the HIV-1 reservoir. Although the frequency of HIV-1 infection, as quantified by the frequency of HIV-1 DNA, is lower in CD4⁺ naive T cells (T_N) than in the memory T cell subsets, recent studies have shown that T_N harbor a large pool of replication-competent virus. Interestingly, however, T_N are highly resistant to direct (cis) HIV-1 infection in vitro, in particular to R5-tropic HIV-1, as T_N do not express CCR5. In this study, we investigated whether T_N could be efficiently HIV-1 trans infected by professional antigen-presenting B lymphocytes and myeloid dendritic cells (DC) in the absence of global T cell activation. We found that B cells, but not DC, have a unique ability to efficiently trans infect T_Nin vitro In contrast, both B cells and DC mediated HIV-1 trans infection of memory and activated CD4⁺ T cells. Moreover, we found that T_N isolated from HIV-1-infected nonprogressors (NP) harbor significantly disproportionately lower levels of HIV-1 DNA than T_N isolated from progressors. This is consistent with our previous finding that antigen-presenting cells (APC) derived from NP do not efficiently trans infect CD4⁺ T cells due to alterations in APC cholesterol metabolism and cell membrane lipid raft organization. These findings support that B cell-mediated trans infection of T_N with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression.IMPORTANCE The latent human immunodeficiency virus type 1 (HIV-1) reservoir in persons on antiretroviral therapy (ART) represents a major barrier to a cure. Although most studies have focused on the HIV-1 reservoir in the memory T cell subset, replication-competent HIV-1 has been isolated from T_N, and CCR5-tropic HIV-1 has been recovered from CCR5^neg T_N from ART-suppressed HIV-1-infected individuals. In this study, we showed that CCR5^neg T_N are efficiently trans infected with R5-tropic HIV-1 by B lymphocytes, but not by myeloid dendritic cells. Furthermore, we found that T_N isolated from NP harbor no or significantly fewer copies of HIV-1 DNA than those from ART-suppressed progressors. These findings support that B cell-mediated trans infection of T_N with HIV-1 has a more profound role than previously considered in establishing the viral reservoir and control of HIV-1 disease progression. Understanding the establishment and maintenance of the HIV-1 latent reservoir is fundamental for the design of effective treatments for viral eradication.

Keywords: B lymphocytes; HIV-1; dendritic cells; naive CD4+ T cells; trans infection.

FIG 1

B cells trans infect CD4⁺ T cells with higher efficiency than DC. (A) B cells or immature DC (iDC) were pulsed with HIV-1^BaL (10⁻³ MOI) and cocultured with PHA/IL-2-activated autologous CD4⁺ T cells at a 1:10 ratio for up to 12 days as described in Materials and Methods (trans infection). PHA/IL-2-activated CD4⁺ T cells were also pulsed with HIV-1^BaL (10⁻³ MOI) and cultured alone (cis infection). Coculture supernatants were tested at the indicated time points for HIV-1 Gag p24 levels by ELISA. (B) PHA/IL-2-activated CD4⁺ T cells were pulsed with HIV-1^BaL (10⁻¹ MOI) and cultured up to 12 days. Culture supernatants were tested at the indicated time points for HIV-1 Gag p24 levels by ELISA. Data are mean ± SE; n = 8; *, P < 0.05; **, P < 0.005.

FIG 2

Only B cells trans infect T_N. (A) B cells or iDC were pulsed with HIV-1^BaL (10⁻³ MOI) and cocultured with PHA/IL-2-activated purified naive (T_N; n = 5) or central memory (T_CM; n = 4) CD4⁺ T cells. Coculture supernatants were tested at the indicated time points for HIV-1 Gag p24 levels by ELISA. Data are mean ± SE. (B) T_N were treated with CCL-19, washed, and cocultured with HIV-1^BaL (10⁻³ MOI)-pulsed B cells, iDC, or mature DC (mDC). Coculture supernatants were tested at the indicated time points for HIV-1 Gag p24 by ELISA. Data are mean ± SE, n = 6. (C) Total CD4⁺ T cells or T_N were treated with PHA/IL-2 or CCL-19, pulsed with HIV-1^BaL (10⁻¹ MOI), and cultured alone. Culture supernatants were tested at the indicated time points for HIV-1 Gag p24 by ELISA. Data are mean ± SE, n = 4. (D) B cells pulsed with HIV-1 92FR_BX08 (10⁻³ MOI) were cocultured with CCL-19-treated total CD4⁺ T cells or T_N for up to 12 days. Coculture supernatants were tested at the indicated time points for HIV-1 Gag p24 by ELISA. Data are mean ± SE, n = 3 replicates from 2 donors. (E) iDC pulsed with HIV-1 92FR_BX08 (10⁻³ MOI) were cocultured with CCL-19-treated total CD4⁺ T cells or T_N for up to 12 days. Coculture supernatants were tested at the indicated time points for HIV-1 Gag p24 by ELISA. Data are mean ± SE, n = 3 replicates from 2 donors. Data were analyzed by one-way ANOVA followed by ad hoc Student’s t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.0001.

FIG 3

Coculture with B cells or DC does not affect T_N phenotype. (A) CCL-19-treated T_N were cultured alone (top row) or cocultured with HIV-1^BaL (10⁻³ MOI)-pulsed B cells or iDC (middle and bottom rows, respectively), sampled at the indicated time points, stained with anti-CCR5 and CD27 MAb, and analyzed by fluorescence-activated cell sorting (FACS) as described in Materials and Methods. Representative data from 3 independent experiments. (B) CCR5 and CD27 percent positive cells in control T_N cultures or cocultures. Data are mean± SE, n = 3.

FIG 4

Detection of HIV-1 p24 antigen in trans infection coculture with B cells or DC. (Left) Trans infection. B cells or iDC were pulsed with HIV-1^BaL (10⁻³ MOI) and cocultured with CCL-19-treated total CD4⁺ T cells or T_N. Cocultures were sampled after 8 days, stained with anti-Kc57, -CD4, -CD3, or -CD19, and analyzed by FACS as described in Materials and Methods. (Right) Cis infection. CCL-19-treated total CD4⁺ T cells or T_N were pulsed with HIV-1^BaL (10⁻¹ MOI) and cultured alone. Cultures were sampled after 8 days, stained, and analyzed by flow cytometry in parallel to the trans infection cocultures. Controls represent uninfected cultures. Representative data from 2 independent experiments.

FIG 5

B cell-mediated trans infection of T_N is inhibited by anti-DC-SIGN. (A) B cells were incubated with 20 μg/ml anti-DC-SIGN MAb for 30 min at 4°C prior to pulsing with HIV-1^BaL (10⁻³ MOI) and then cocultured with CCL-19-treated T_N for trans infection as described in Materials and Methods. Supernatants were collected after 12 days and tested for HIV-1 Gag p24 by ELISA. B cells treated with mouse IgG (20 μg/ml) were used as the control. Data are mean ± SE, n = 3 replicates from 2 donors. (B) In parallel cultures, CCL-19-treated T_N were subsequently treated with maraviroc (1 μM) as described in Materials and Methods and cocultured with HIV-1^BaL (10⁻³ MOI)-pulsed B cells for trans infection. Supernatants were collected after 12 days and tested for HIV-1 Gag p24 by ELISA. Untreated T_N were used in the control cocultures. Data are mean ± SE, n = 3 replicates from 2 donors. Data were analyzed by one-way ANOVA followed by ad hoc Student’s t test. **, P < 0.005; ns, not significant. (C) Anti-DC-SIGN- or maraviroc-treated cells and the corresponding controls from the cocultures in panels A and B, as well as uninfected cocultures, were sampled after 12 days, stained with anti-Kc57, -CD4, -CD3, or -CD19, and analyzed by FACS as described in Materials and Methods.

FIG 6

Detection of virus after LRA reactivation. (A) Schematic representation of the experimental approach to measure reversal of HIV-1 latency in T_N trans infected by B cells or iDC. (B) Cocultures were treated with LRAs at day 8 and then sampled at the indicated time points after reactivation. Supernatants were tested for HIV-1 Gag p24 by ELISA.

FIG 7

Quantification of total HIV-1 DNA in CD4⁺ total T cells and T_N. DNA was quantified by qPCR as described in Materials and Methods. Each dot represents a unique donor. Statistical comparison was analyzed using a Wilcoxon matched-pairs signed-rank test. P < 0.05 was considered significant.