Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo

Abstract

Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.

FIG. 1.

Infectious or AT-2-inactivated X4 HIV-1 variants inhibit B-cell responses in human lymphoid tissue ex vivo in a dose-dependent manner. Tissue blocks challenged with TT and PWM were inoculated with AT-2-inactivated or infectious virus X4_LAV.04. Inactivated virus was applied either for the entire culture period of 15 to 18 days (continuous) or for 3 h (pulse). The antibody response was evaluated as anti-TT or total IgG (mean ± standard error) released by 27 to 36 identically treated tissue blocks from each donor and expressed as percent relative to similarly challenged matched sham-treated tissue. Concentrations of inactivated virus are indicated: 1× = 15-ng/ml p24^gag, the peak concentration in the medium of productively infected tissue cultures. (a) Production of anti-TT IgG in tissues challenged with TT and inoculated with infectious X4_LAV.04 or AT-2-inactivated X4_LAV.04, either continuously or as a pulse (n = 10, 5, and 9 donor tissues, respectively). (b) Production of IgG in tissues challenged with PWM and inoculated with infectious X4_LAV.04 or AT-2-inactivated X4 _LAV.04, either continuously or as a pulse (n = 10, 10, and 5 donor tissues, respectively). (c) Production of anti-TT IgG in TT-challenged tissues inoculated with one dose (1×) of AT-2-inactivated X4_LAV.04 or dilutions thereof (n = 3). (d) Production of IgG in PWM-challenged tissues inoculated with one dose (1×) of AT-2-inactivated X4_LAV.04 or dilutions thereof (n = 3).

FIG. 2.

AT-2-inactivated or infectious R5 HIV-1 does not inhibit B-cell responses in human lymphoid tissue ex vivo. Tissue blocks challenged with TT and PWM were inoculated with AT-2-inactivated or infectious R5_SF162 virus. The antibody response was evaluated as anti-TT or total IgG (mean ± standard error) released by 27 to 36 identically treated tissue blocks from each donor and expressed as percent relative to similarly challenged matched sham-treated tissue. Concentrations of inactivated virus are indicated: 1× = 15-ng/ml p24^gag, the peak concentration in the medium of productively infected tissue cultures. (a) Production of anti-TT IgG in tissues challenged with TT and inoculated with infectious R5_SF162 or AT-2-inactivated R5_SF162 at 1× or 10× (n = 6, 6, and 7 donor tissues, respectively). (b) Production of IgG in tissues challenged with PWM and inoculated with infectious R5_SF162 or AT-2-inactivated R5_SF162 at 1× or 10× (n = 9, 9, and 7 donor tissues, respectively).

FIG. 3.

AT-2-inactivated X4_LAV.04 does not inhibit mitogenic responses of tissue lymphocytes. Cells were mechanically isolated from control or AT-2-inactivated X4_LAV.04-exposed tissue blocks after 3 days and incubated for another 3 days with PWM or PHA. Cell proliferation was measured at 72 h in cultures pulsed with [³H]thymidine for the last 12 h. The results are expressed as SI (mean ± standard error of 16 replicates). Results are representative of three experiments.

FIG. 4.

Production of ISF by tissue exposed to AT-2-inactivated X4_LAV.04. Medium was conditioned by tissue blocks exposed to AT-2-inactivated X4_LAV.04 for the indicated periods (days 1 to 3, 4 to 6, 7 to 9, and 10 to 12 postexposure). The conditioned media were tested for immunosuppressive activity on fresh tonsil cultures challenged with TT and PWM. Anti-TT IgG (a) and total IgG (b) are expressed as percent relative to similarly challenged matched tissue treated with conditioned medium samples (mean ± standard error, n = 3).

FIG. 5.

Immunosuppressive activity of size-fractionated medium conditioned by AT-2-inactivated X4_LAV.04-exposed tissue. Medium conditioned by tissue blocks exposed to AT-2-inactivated X4_LAV.04 was collected between days 6 and 9 postexposure and size fractionated with centrifugal concentrators. Immunosuppressive activity of each fraction was evaluated on fresh tonsil cultures challenged with TT and PWM. Anti-TT IgG(a) and total IgG (b) are expressed as percents relative to those of similarly challenged matched tissue treated with correspondent fractions of control conditioned medium (mean ± standard error of the mean; n = 4).