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. 1998 Aug;72(8):6315-24.
doi: 10.1128/JVI.72.8.6315-6324.1998.

Inhibition of simian immunodeficiency virus (SIV) replication by CD8(+) T lymphocytes from macaques immunized with live attenuated SIV

M C Gauduin  1 , R L Glickman, R Means, R P Johnson
Affiliations

Inhibition of simian immunodeficiency virus (SIV) replication by CD8(+) T lymphocytes from macaques immunized with live attenuated SIV

M C Gauduin et al. J Virol. 1998 Aug.

Abstract

Characterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8(+) T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Deltanef or SIVmac239Delta3 to inhibit SIV replication. CD8(+) T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4(+) T cells. Suppression of SIV replication by unstimulated CD8(+) T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8(+) T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8(+) T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8(+) cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1alpha (MIP-1alpha), or MIP-1beta from stimulated CD8(+) T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8(+) T cells of control animals. However, addition of antibodies that neutralize these beta-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8(+) T cells. Our results indicate that inhibition of SIV replication by CD8(+) T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1alpha, and MIP-1beta.

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Figures

FIG. 1
FIG. 1
Inhibition of SIV replication by CD8+ T lymphocytes from macaques immunized with SIVmac239Δnef or SIVmac239Δ3. (A) Transmembrane experiments using PBMC from macaques infected with either wild-type SIV or live attenuated SIV and from uninfected controls. PBMC were separated into CD4+ and CD8+ populations by using immunomagnetic beads. CD4+ T cells were ConA stimulated overnight and acutely infected with SIVmac239 at an MOI of 0.01 TCID50/cell. Infected CD4+ T cells were then cultured in the lower well either alone, in direct contact with CD8+ T cells, or with stimulated or unstimulated CD8+ T cells placed in the upper well. Inhibition of SIV replication by CD8+ T cells was assessed by measuring the production of SIV p27 Ag over a period of 10 days. Representative assays are shown for individual animals infected with the indicated SIV strain and for an uninfected control. (B) Quantitative analysis of suppression of SIV replication by CD8+ T lymphocytes from uninfected animals and animals infected with pathogenic or live attenuated strains of SIV. Experiments were carried out as described above, and log reduction of SIV replication (90% inhibition = 1 log, 99% = 2 logs, etc.) was analyzed on days 6 and 10. The data represent the means ± standard deviations for a total of 16 animals: 5 SIVmac239Δnef-, 5 SIVmac239Δ3-, and 2 wild-type (SIVmac239 and SIVmac251)-infected animals and 4 uninfected animals. Suppression of SIV replication by CD8+ T cells from animals infected with either SIVmac239Δnef or SIVmac239Δ3 was statistically significant compared with inhibition by either wild-type SIV-infected animals or normal controls (P < 0.01), for both cells in direct contact and for soluble factors. The difference between wild-type SIV-infected animals and normal controls was not significant.
FIG. 2
FIG. 2
Suppression of SIV replication by unstimulated CD8+ T lymphocytes is MHC restricted. Acutely SIV infected CD4+ T cells were cocultured in direct contact with MHC class I-mismatched allogeneic CD8+ T lymphocytes isolated from uninfected animals or animals infected with pathogenic or live attenuated strains of SIV. Inhibition of SIV replication by allogeneic CD8+ T cells was assessed by measuring the production of SIV p27 Ag for 10 days.
FIG. 3
FIG. 3
Suppression of SIV replication by soluble factors secreted from CD3-activated CD8+ T lymphocytes is not MHC restricted. CD4+ T cells from animals (118.87 and 267.89) immunized with SIVmac239Δnef were infected with SIVmac239 and then cultured (lower well) with autologous (○) and allogeneic (▴) CD8+ T cells (mismatched; 118.87 and 267.89) stimulated with CD3-specific beads. Similar data were obtained in two independent experiments. ▪, no CD8+ cells.
FIG. 4
FIG. 4
Stimulation of CD8+ T cells with cognate CTL epitopes results in production of soluble factors that suppress SIV replication. Autologous CD4+ T cells were acutely infected with SIVmac239 (lower well) and cocultured with stimulated CD8+ T cells from animals (267.89 and 118.87) immunized with SIVmac239Δnef (upper well). Stimulation of CD8+ T cells included either CD3-specific immunomagnetic beads or specific peptides (7G for animal 267.89; 11C for animal 118.87) previously shown to represent CTL epitopes for these animals. A previously reported SIV CTL epitope (12K) not recognized by these animals was used as a negative control. All assays were performed in triplicate.
FIG. 5
FIG. 5
SIV replication is inhibited by soluble factors from activated CD8+ T cells from immunized macaques and by β-chemokines RANTES, MIP-1α, and MIP-1β. (A) Inhibition of SIV replication in SIV-infected C8166-45 cells (SEAP activity) by soluble factors secreted by CD8+ T cells from macaques immunized with either SIVmac239Δnef (118.87) or SIVmac239Δ3 (437.91). C8166-45-SEAP cells were acutely infected with SIVmac251 at decreasing concentrations (from 5.9 to 0.3 ng/ml) and then incubated in the presence of soluble factors (1:2 dilution) secreted by CD3-activated CD8+ T cells from macaques immunized with either SIVmac239Δnef (118.87) or SIVmac239Δ3 (437.91). SEAP activity in cell-free supernatants (Sup.) was measured on day 4 after infection. (B) Inhibition of SIV replication (SEAP activity) in infected C8166-45 and CEMx174 cells by β-chemokines. C8166-45-SEAP and CEMx174-SEAP cell lines were acutely infected with SIVmac251 (0.3 ng/ml) and SIVmac239 (1.47 ng/ml), respectively. Infected cells were then incubated with decreasing concentrations (fivefold dilutions starting from 500 ng/ml) of RANTES, MIP-1α, or MIP-1β. SEAP activity was measured from cell-free medium collected on day 4. All SEAP assays were performed in quadruplicate.
FIG. 6
FIG. 6
Neutralizing antibodies to β-chemokines do not block the ability of soluble factors from CD8+ T cells to suppress SIV replication. C8166-45-SEAP cells were infected with SIVmac251 (0.3 ng/ml) and cultured either with supernatants (1:2 dilution) from CD3-activated CD8+ T cells from a macaque immunized with SIVmac239Δnef or with RANTES, MIP-1α, or MIP-1β (500 ng/ml) added individually or in combination. In addition, in some cases, neutralizing antisera to human RANTES, MIP-1α, and MIP-1β were preincubated at 200, 80, and 100 μg/ml, respectively, with β-chemokines and then were added to the indicated cultures (cross-hatched bars). Similar data were obtained in two independent experiments.
FIG. 7
FIG. 7
Soluble factors from CD3-activated CD8+ T cells from SIVmac239Δnef-infected macaques inhibit HIV-1 replication. (A) Replication of both HIV-1Ba-L and HIV-1JR-CSF in PM1 cells is inhibited by SIVmac239Δnef-infected CD8+ T-cell-mediated soluble factors. PM1 cells were acutely infected with various dilutions of primary isolates HIV-1Ba-L and HIV-1JR-CSF and then incubated in the presence of either soluble factors (1:2 dilution) or RANTES (500 ng/ml). In addition, in some cases, neutralizing antisera to human RANTES, MIP-1α, and MIP-1β were preincubated at 200, 80, and 100 μg/ml, respectively, with soluble factors or RANTES and then added to the indicated cultures. (B) Soluble factors inhibited HIV-1 replication in a dose-dependent manner. In the same conditions, PM1 cells were acutely infected with primary isolates HIV-1Ba-L and HIV-1JR-CSF (dilution of 1/1,000) and then incubated in the presence of soluble factors at various concentrations. RANTES (500 ng/ml) was used as a control. (C) HIV-1NL4-3 replication in CEMx174-SEAP cells is inhibited by SIVmac239Δnef-infected CD8+ T-cell-mediated soluble factors. CEMx-174-SEAP cells were infected with various dilutions of HIV-1NL4-3 and cultured either with supernatants (1:2 dilution) from CD3-activated CD8+ T cells from a macaque immunized with SIVmac239Δnef or with RANTES (500 ng/ml). In addition, in some cases, neutralizing antisera to human RANTES, MIP-1α, and MIP-1β were preincubated at 200, 80, and 100 μg/ml, respectively, either alone or in combination with soluble factors or RANTES, and then were added to the indicated cultures. Similar data were obtained in two independent experiments.
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