Viral interactions with B-cells contribute to increased regulatory T-cells during chronic HCV infection

Abstract

Hepatitis C virus (HCV) has a propensity to establish chronic infection that is characterized by attenuated virus-specific T-cell responses. Mechanisms leading to T-cell attenuation are poorly understood and likely involve dysfunctional interactions between antigen-presenting cells (APC) and effector/regulatory T-cells. Reports on dendritic cells (DC) have described only minor dysfunction during HCV infection. However, there is a paucity of reports regarding B-cell function, despite clear associations with B-cell-related secondary sequelae. In this study we evaluated the state of B-cells during chronic HCV infection, and observed a diminished ability to respond to mitogenic stimuli, correlating with increased apoptosis. This was in contrast to their ex vivo phenotype, which indicated ongoing chronic activation in vivo. There was a high association of HCV-positive strand RNA with B-cells in a subset of HCV patients. Interestingly, ex-vivo-derived HCV RNA-positive B-cells induced significantly greater proliferation in allogeneic T-cells than in HCV-negative B-cells, correlating with an increased generation of CD4(+)CD25(+)FOXP3(+) regulatory T-cells (Tregs). In-vitro exposure of healthy peripheral blood mononuclear cells (PBMC) to HCV resulted in robust activation of resting B-cells. These HCV-exposed B-cells also showed an enhanced ability to generate Tregs. Our results provide strong evidence for a novel and paradoxical link between HCV-induced enhanced APC function and the generation of Tregs.

FIG. 1.

B-cells from HCV patients have an attenuated activation potential. (A) B-cells from chronic HCV patients or healthy donors were isolated using CD19 magnetic microbeads. First, 2 × 10⁵ B-cells (triplicate cultures in 96-well plates) were stimulated for 4 d with a 1:1000 dilution of pansorbin (protein A). ³H-thymidine was added in the last 16 h of culture and the scintillation counts were measured. The data are representative of replicate experiments from three separate donor HCV pairs. (B) CD19⁺ B-cells from chronic HCV patients or healthy donors were incubated for 2 d with a 1:5000 dilution of pansorbin (protein A). The cells were then stained with annexin V-FITC, propidium iodide (PI), and CD19-APC. Flow cytometry dotplots indicate the percent expression of annexin V versus PI, and are representative of five separate experiments. (C) Cumulative data of pansorbin-stimulated CD19⁺ B-cells, depicting percentages of annexin V⁺/PI⁻ B-cells, and indicating cells undergoing early apoptosis, showing significant differences in healthy versus HCV B-cells (p < 0.01). (D) Bulk PBMC from chronic HCV patients or healthy donors were incubated for 24 h with 5 μg/mL of pokeweed mitogen. The cells were subsequently stained with the following three panels of activation markers: (1) CD19-PECy7, CD25-APC, and CD69-PE; (2) CD19-PECy7, CD27-FITC, and CD23-PE; and (3) CD19-PECy7, CD71-FITC, CD86-PE, and CD38-APC. Representative dotplots from a single healthy donor and an HCV patient are shown. Numbers indicate percentages of expression of each marker on gated B-cells. (E) Cumulative data from nine HCV patients and four healthy control subjects, depicting percentage expression of the indicated markers. There was an overall trend toward lower induced activation on HCV B-cells. Statistical significance was observed for CD23 differences between healthy subjects and HCV patients (p < 0.05).

FIG. 2.

HCV RNA⁺ B-cells induce significantly greater MLR responses and exhibit an activated phenotype. (A) Microbead-purified B-cells (top panels), and monocytes (bottom panels), from up to 13 healthy donors and 13 HCV-infected patients were used to stimulate purified allogeneic CD3⁺ T-cells from one of two healthy third-party subjects. Every experimental set-up consisted of at least one healthy-HCV pair against the same third-party donor. Allogeneic cultures were pulsed with ³H-thymidine on day 5 and harvested on day 6. Responses of T-cells at a stimulator:responder ratio of 1:10 are represented as CPM on the y-axis. Data are separated based on HCV RNA status of the patients' B-cells: patients with HCV RNA⁺ B-cells are represented in the left two panels, whereas those with HCV RNA⁻ B-cells are represented in the right two panels. (B) B-cell data from panel A were normalized for responses generated from healthy control subjects (assigned as 100). Responses induced by HCV RNA⁺ B-cells were significantly greater than those from HCV RNA⁻ B-cells (p < 0.001). (C) PBMC from 14 HCV patients (6 B-cell HCV RNA⁺ and 8 HCV RNA⁻), and 8 healthy donors were stained ex-vivo for HLA-DR, CD23, CD25, CD27, CD38, CD71, and CD86. The expression level of each marker was evaluated on a gated CD19⁺ B-cell population. Isotypic controls were used to determine cutoffs. Mean fluorescence intensity (MFI) levels were used for HLA-DR and CD86, whereas percentage expression was determined for the remaining markers.

FIG. 3.

Exposure of healthy PBMC to HCV results in robust B-cell activation. (A) PBMC from healthy donors were exposed to JFH-1 virus (or mock media) for 3 h, washed, and cultured for 7 d. B-cell activation was monitored at various time points (days 1 and 7 are shown). CD19 is shown on the x-axis, and staining for the indicated activation markers is represented on the corresponding y-axes. The percentages of B-cells that were positive for each marker (i.e., within gated CD19⁺ cells) is indicated. The data are representative of replicate experiments from five separate donors. (B) PBMC were incubated with viable virus, UV-inactivated virus (UV virus), or were pre-incubated with anti-CD81 (αCD81), or anti-E2 antibody (αE2⁺), for 1 h prior to the addition of viable virus for 3 h at 37°C, followed by washing and culture for 7 d. PBMC were then harvested and analyzed for B-cell activation (CD71 is shown as a representative activation marker). Data are representative of three replicate experiments, using PBMC from different donors. (C) Bulk PBMC, monocyte-depleted PBMC (PBMC-M), T-cell-depleted PBMC (PBMC-T), purified B-cells + monocytes (B + M), B-cells + T-cells (B + T), and purified B-cells + monocytes + T-cells (B + M + T), were incubated with viable JFH-1 virus at a 0.01 virus:cell ratio. All cell combinations were incubated for 7 d, and gated B-cells were subsequently analyzed for activation (represented by CD71-FITC and CD86-PE expression). Data are representative of three replicate experiments, using PBMC from different donors.

FIG. 4.

HCV-modified B-cells are potent inducers of regulatory T-cells. (A) Bead-sorted B-cells from healthy subjects and treatment-naïve HCV patients were used as APC in CFSE-based MLR assays, using sorted third-party CD4⁺CD25⁻ T-cells as responders. The top panels show representative CFSE histograms obtained from assays with healthy B-cells, HCV RNA⁺ B-cells, and HCV RNA⁻ B-cells as APC. The percentages of proliferation are indicated, demonstrating a significantly greater response from HCV RNA⁺ B-cells. The bottom panels demonstrate CD25 versus FOXP3 staining on gated CD4⁺ T-cells from the same cultures. The percentages of CD25⁺ and FOXP3⁺ are indicated, showing higher Treg generation in HCV RNA⁺ MLR. Data are representative of 11 replicate experiments, using different healthy and HCV donors [a total of 11 healthy and 11 HCV (6 HCV⁺ and 5 HCV⁻) subjects]. (B) Cumulative graph of the CFSE-based MLR assays described in panel A. Results are represented as percentages of CFSE^low(proliferating)/CD25⁺/FOXP3⁺ cells induced by HCV B-cells relative to corresponding healthy donor B-cells (normalized to 100%). These data confirm a trend toward higher proliferation and increased CD25/FOXP3 induction by HCV⁺ B-cells (*p = 0.06). (C) B-cell-induced MLR were set up as described for panel A. Activated CD25⁺ T-cells from these MLR were then tested for suppressor activity, using flow cytometry–based, anti-CD3-stimulated suppression assays. Autologous T-cells were incubated with increasing numbers of putative suppressor T-cells, derived from MLR. The percentage of proliferation was normalized to the proliferation seen in the absence of any suppressors (designated as 100). Both healthy (black bars) and HCV (gray bars) B-cell-induced CD4⁺CD25⁺FOXP3⁺ T-cells demonstrated comparable Treg activity on a per-cell basis. These data are representative of five separate MLR-induced suppressor pair replicate experiments. (D) JFH- or mock-exposed B-cells were used as APC in allogeneic MLR with CD4⁺25⁻ T-cells. The percentages of CD4⁺CD25⁺FOXP3⁺ Tregs from the indicated cultures are demonstrated. Data are representative of three replicate experiments.