Lack of clinical AIDS in SIV-infected sooty mangabeys with significant CD4+ T cell loss is associated with double-negative T cells

Abstract

SIV infection of natural host species such as sooty mangabeys results in high viral replication without clinical signs of simian AIDS. Studying such infections is useful for identifying immunologic parameters that lead to AIDS in HIV-infected patients. Here we have demonstrated that acute, SIV-induced CD4(+) T cell depletion in sooty mangabeys does not result in immune dysfunction and progression to simian AIDS and that a population of CD3(+)CD4(-)CD8(-) T cells (double-negative T cells) partially compensates for CD4(+) T cell function in these animals. Passaging plasma from an SIV-infected sooty mangabey with very few CD4(+) T cells to SIV-negative animals resulted in rapid loss of CD4(+) T cells. Nonetheless, all sooty mangabeys generated SIV-specific antibody and T cell responses and maintained normal levels of plasma lipopolysaccharide. Moreover, all CD4-low sooty mangabeys elicited a de novo immune response following influenza vaccination. Such preserved immune responses as well as the low levels of immune activation observed in these animals were associated with the presence of double-negative T cells capable of producing Th1, Th2, and Th17 cytokines. These studies indicate that SIV-infected sooty mangabeys do not appear to rely entirely on CD4(+) T cells to maintain immunity and identify double-negative T cells as a potential subset of cells capable of performing CD4(+) T cell-like helper functions upon SIV-induced CD4(+) T cell depletion in this species.

Figure 1. Passage of multitropic SIV results in dramatic CD4⁺ T cell decline in multiple tissue compartments.

(A) Absolute numbers of peripheral blood CD4⁺ T cells prior to and following passage of multitropic SIV from 1 CD4-low SIV⁺ mangabey to SM7, SM8, and SM9. (B) Plasma viral loads following passage as measured by the number of copies of gag RNA per ml of plasma. (C) Absolute numbers of peripheral blood CD8⁺ T cells following SIV infection. (D) Representative flow data showing the percentage of CD4⁺, CD8⁺, and DN T cells (indicated by boxes in lower right, upper left, and lower left sections of flow plots, respectively) in BAL, rectal biopsy (RB), and LN samples at –60, 14, and 180 dpi in SM7. Events shown were gated through live/dead and CD3⁺ gates.

Figure 2. Rapid decline of peripheral blood CD4⁺ T cells has an impact on the phenotypic distribution of CD4⁺ T cells.

The percentage of peripheral blood CD4⁺ T cells (A–C) and CD8⁺ T cells (D–F) expressing naive, central memory, and effector memory phenotypic markers following passage of multitropic SIV to SM7, SM8, and SM9. Naive cells were identified as CD28⁺CD95^–; memory cells were gated as CD28^–CD95⁺CCR7^– (effector memory) or CD28⁺CD95⁺CCR7⁺ (central memory). Dotted lines represent the average of all animals at baseline.

Figure 3. Levels of immune activation remain low during chronic SIV infection of CD4-low sooty mangabeys.

(A and B) Percentage of proliferating (Ki-67⁺) CD4⁺ T cells and CD8⁺ T cells following passage of multitropic SIV. (C) Comparison of plasma LPS levels in uninfected and chronically SIV-infected sooty mangabeys with absolute CD4⁺ T cell levels greater than 200 cells/mm³ blood (CD4-healthy) or less than 200 cells/mm³ blood (CD4-low; SM1, SM2, SM7, SM8, SM9 shown). Dotted line represents the average of 10 chronically SIV-infected rhesus macaques (20); Uninfected rhesus macaques have an average of 15 pg/ml LPS (20) (not shown). (D) Concentration of plasma LPS during SIV infection of SM7, SM8, and SM9. (E and F) Fold change of occludin mRNA expression in SM7, SM8, and SM9 or RMs 1–6 during acute (2–15 dpi) and chronic (184 dpi) infection or at time of necropsy. Fold change was determined relative to uninfected control animals of the same species; the fold change is considered increased or decreased when it is greater than 2 SD from the mean expression in uninfected animals (represented by the area between the dotted lines).

Figure 4. SIV-specific adaptive immune responses are present in SIV⁺ mangabeys with dramatic CD4⁺ T cell depletion.

(A) Reciprocal endpoint titers of SIV-specific antibodies following passage of multitropic SIV to SM7, SM8, and SM9. (A and B) Percentages of CD8⁺ T cells producing TNF-α (B), or IFN-γ (C) in response to the SIV peptides Gag, Env, Pol, Nef, and Gag consensus peptides (Gag Con) during chronic infection (279 wpi for SM1 and SM2; 52 wpi for SM7, SM8, and SM9).

Figure 5. DN T cells are present at high levels and are capable of producing multiple cytokines throughout SIV infection of sooty mangabeys.

(A) Absolute numbers of DN T cells in uninfected and SIV⁺ sooty mangabeys. (B) Absolute numbers of DN T cells in SM7, SM8, and SM9 are similar prior to and following passage of a multitropic SIV. (C) Percentage of DN T cells expressing a central memory (CM), effector memory (EM), or naive phenotype in SM7, SM8, and SM9 following infection (memory phenotypes determined as described in Figure 2 legend). Data points represent the mean of all 3 animals ± SEM for a particular phenotype. (D) Gene expression of IFN-γ, IL-4, and IL-17 by SIV-infected CD4-low sooty mangabey DN T cells (n = 5). DN T cells were isolated (purity > 97%) from SIV-infected CD4-low mangabeys and stimulated by anti-CD3 and anti-CD28. Fold change of cytokine mRNA expression as measured by real-time PCR is defined as the change in expression of stimulated cells over unstimulated cells from the same animal, with a fold change of 1 indicating no change. Experiments were performed in duplicate. (E) SIV-specific TNF-α responses are detectable in DN T cells from SIV-infected CD4-low sooty mangabeys. PBMCs were stimulated with the SIV peptides Gag, Env, Pol, Nef, and Gag consensus (Gag Con) and the percentage of DN T cells producing TNF-α was measured by intracellular cytokine staining.