With minimal systemic T-cell expansion, CD8+ T Cells mediate protection of rhesus macaques immunized with attenuated simian-human immunodeficiency virus SHIV89.6 from vaginal challenge with simian immunodeficiency virus

Abstract

The presence, at the time of challenge, of antiviral effector T cells in the vaginal mucosa of female rhesus macaques immunized with live-attenuated simian-human immunodeficiency virus 89.6 (SHIV89.6) is associated with consistent and reproducible protection from pathogenic simian immunodeficiency virus (SIV) vaginal challenge (18). Here, we definitively demonstrate the protective role of the SIV-specific CD8(+) T-cell response in SHIV-immunized monkeys by CD8(+) lymphocyte depletion, an intervention that abrogated SHIV-mediated control of challenge virus replication and largely eliminated the SIV-specific T-cell responses in blood, lymph nodes, and genital mucosa. While in the T-cell-intact SHIV-immunized animals, polyfunctional and degranulating SIV-specific CD8(+) T cells were present in the genital tract and lymphoid tissues from the day of challenge until day 14 postchallenge, strikingly, expansion of SIV-specific CD8(+) T cells in the immunized monkeys was minimal and limited to the vagina. Thus, protection from uncontrolled SIV replication in animals immunized with attenuated SHIV89.6 is primarily mediated by CD8(+) T cells that do not undergo dramatic systemic expansion after SIV challenge. These findings demonstrate that despite, and perhaps because of, minimal systemic expansion of T cells at the time of challenge, a stable population of effector-cytotoxic CD8(+) T cells can provide significant protection from vaginal SIV challenge.

FIG. 1.

Experimental design, SIVmac239 plasma vRNA levels and T-cell counts in SHIV-immunized and unimmunized rhesus macaques. (A) Timing of interventions and necropsy for SHIV89.6-immunized and unimmunized macaques. (B) Plasma vRNA levels at 7 and 14 days after vaginal SIVmac239 challenge. (C) T-cell numbers before and after SIVmac239 challenge. The red symbols represent unimmunized monkeys (SIV controls), the blue symbols represent SHIV89.6-immunized monkeys (SHIV plus SIV) and the green symbols represent SHIV89.6-immunized and CD8⁺ T-cell-depleted (SHIV plus SIV αCD8) monkeys. Uninfected monkeys are labeled “SHIV day of SHIV” or “SIV day of SIV.” The P values are based on ANOVA analyses and Tukey's multiple-comparison post hoc test.

FIG. 2.

Increased activation and cell death in peripheral CD8⁺ T cells of unimmunized macaques but not in T cells of SHIV-immunized macaques after SIV challenge. Shown are the frequencies on days 0, 7, and 14 p.c. of CD8⁺ T cells expressing the activation/proliferation marker Ki-67 in PBMC (A) and LN (B) and the frequency of CD8⁺ T cells in LN expressing the proapoptotic marker caspase 3 (C). “Pre-SHIV” is the time point prior to SHIV infection. The P values are based on ANOVA analyses and Tukey's multiple-comparison post hoc test.

FIG. 3.

Polyfunctional SIV-specific CD8⁺ T cells in blood and LN of SHIV-immunized macaques, but not of unimmunized macaques, after vaginal challenge with SIVmac239. The frequency and functional capacity of SIV-specific CD8⁺ T cells after stimulation with a p27-SIV peptide pool as described in Materials and Methods are shown. The samples included PBMC and LN mononuclear cells from unimmunized, SIV control, or SHIV89.6-immunized macaques at days 0, 7, and 14 p.c. Each pie chart represents the average of the positive responses of the animals in a group. The empty circles indicate that there were no positive responses in those samples. Below each pie chart is shown the number of monkeys with a positive response over the number of monkeys tested. For positive responses, the frequency of SIV-specific CD8⁺ T cells was normalized to 10⁵ CD8⁺ T cells, and the mean frequency is shown, along with the standard error, in white at the center of the pie chart. Each colored portion of a pie chart indicates the percentage of SIV-specific CD8⁺ T cells that responded with one, two, or three functions, and the colored arcs around the pie show the function or combination of functions to which the specific response corresponds.

FIG. 4.

SIV-specific CD8⁺ T cells in tissues of SHIV-immunized CD8-depleted animals after challenge. The frequency and functional capacity of SIV-specific CD8⁺ T cells after stimulation with a p27-SIV peptide pool as described in Materials and Methods are shown. The samples included LN mononuclear cells (A) and vagina and cervix (B) from SHIV89.6-immunized macaques depleted of CD8 alpha-chain-positive T cells at 14 days p.c. Each pie chart represents the average of the positive responses of the animals in a group. The empty circles indicate that there were no positive responses in those samples. Below each pie is shown the number of monkeys with a positive response over the number of monkeys tested. For positive responses, the frequency of SIV-specific CD8⁺ T cells was normalized to 10⁵ CD8⁺ T cells, and the mean frequency is shown, along with the standard error, in white at the center of the pie chart. Each colored portion of a pie chart indicates the percentage of SIV-specific CD8⁺ T cells that responded with different numbers of functions (note that the legends are different for LN and genital mucosa), and the colored arcs around the pie show the function or combination of functions to which the specific response corresponds.

FIG. 5.

Polyfunctional and cytotoxic CD8⁺ T cells in the genital tracts of SHIV-immunized macaques after challenge. The frequency and functional capacity of SIV Gag-specific CD8⁺ T cells after stimulation with a p27-SIV peptide pool as described in Materials and Methods are shown. The samples included vagina (A) and cervix (B) of unimmunized SIV control monkeys or of SHIV89.6-immunized macaques at days 0, 7, and 14 p.c. Below each pie chart, the percentage of responders is shown with the fraction of the positive samples in parentheses. The empty circles indicate that there were no positive responses in those samples. See the Fig. 3 legend for an explanation of the pie charts.

FIG. 6.

Highly polyfunctional responses to an immunodominant Gag epitope. The frequency and functional capacity of SIV Gag CM9⁺ CD8⁺ T cells after stimulation with the CM9 SIV Gag peptide as described in Materials and Methods are shown. Samples from Mamu A01⁺ SIV control monkeys and SHIV89.6-immunized monkeys at days 0, 7, and 14 p.c. in PBMC and LN mononuclear cells (A) and vagina and cervix (B) are included. For an explanation of the pie charts, see the Fig. 4 legend.

FIG. 7.

Frequencies of CD8⁺ T cells binding SIV peptide/MHC class I tetramers in blood and LN of the SHIV-immunized and SIV control macaques before and after SIVmac239 vaginal challenge. (A) Percentages of the most frequent tetramer-binding cells at 0, 7, and 14 days p.c. in blood of Mamu A*01⁺ and/or Mamu A*02⁺ SHIV-immunized (n = 6) and unimmunized (n = 2) macaques necropsied at 14 days p.c. The lines connect the data points for the only SHIV-immunized monkey (28850) with an unequivocal expansion of tetramer-binding CD8⁺ T cells in PBMC after SIV challenge. (B and C) The most frequent epitopes binding Mamu A*01 and Mamu A*02 alelles are shown in the vagina and Gen, Ax, and Mes LN at 0, 7, and 14 days p.c. of the immunized macaques (B) and at 7 and 14 days p.c. of the unimmunized macaques (C). The red symbols represent the YY9 and GY9 SIV epitopes binding Mamu A*02, and the blue symbols represent the CM9 and SL8 SIV epitopes binding Mamu A*01. The shaded areas of the graphs denote the frequency range of tetramer-binding CD8⁺ T cells in blood or tissues of a single SHIV-immunized monkey on the day of challenge. In panel A, the red symbols represent unimmunized monkeys (SIV controls) and the blue symbols represent SHIV89.6-immunized monkeys. For the tissues, the number of epitopes for which specific tetramer-binding cells were detected divided by the number of epitopes tested at each time point is shown in parentheses below the x axis.

FIG. 8.

SL8 and CM9 tetramer⁺ CD8⁺ T cells are abundant in the tissues of SHIV-immunized, but not of SIV control, animals. Tetramer⁺ CD8⁺ T cells were detected in situ on vagina and LN tissue samples. The representative images are from animals necropsied at 14 days post-SIVmac239 challenge: unimmunized SIV control monkey 31391 and SHIV-immunized monkey 28850. Shown are SL8⁺ tetramer⁺ CD8⁺ T cells in the vagina (upper panels) and CM9⁺ tetramer⁺ CD8⁺ T cells in Ax LN for 28850 and genital LN for 31391 (lower panels). In each image, anti-CD8 staining is green and Mamu A*01/SL8 (upper panels) and Mamu A*01/CM9 (lower panels) tetramer staining is red, indicated by arrowheads. The images are 400× confocal z series projections spanning 10 to 15 μm into the sections. Scale bar, 50 μm.

FIG. 9.

Increased survival potential of SIV Gag-stimulated CD8⁺ T cells in immunized macaques. The ratios of CD8⁺ T cells expressing only survival signals (Bcl-2⁺ caspase 3⁻) to cells with proapoptotic signals (Bcl-2⁺ caspase 3⁺) are shown in the immunized and unimmunized macaques on the day of challenge and 7 and 14 days post-SIVmac challenge in peripheral blood (A), Gen LN (B), and vagina (C). PBMC or LN mononuclear cells were stimulated for 6 h with a Gag p27 peptide pool. The P values were determined by Kruskal-Wallis and Dunn's multiple-comparison post hoc tests.