Regulatory T-cell markers, indoleamine 2,3-dioxygenase, and virus levels in spleen and gut during progressive simian immunodeficiency virus infection

Abstract

High levels of viral replication occur in gut-associated lymphoid tissue (GALT) and other lymphoid tissues (LT) since the early phase of human/simian immunodeficiency virus (HIV/SIV) infection. Regulatory T cells (T(reg)), a subset of immunosuppressive T cells expressing CTLA-4 and the FoxP3 transcription factor, accumulate in LT during HIV/SIV infection. Here we show that FoxP3 and CTLA-4 mRNA are increased in leukocytes from the spleens, lymph nodes (LN), and mucosal sites of chronically SIV-infected macaques with high viremia (SIV(HI)) compared to animals with low viremia (SIV(LO)). FoxP3 and CTLA-4 correlated with SIV RNA levels in tissues; SIV virus levels in the spleen, inguinal LN, mesenteric LN, colon, and jejunum directly correlated with the plasma virus level. Importantly, CTLA-4 and FoxP3 mRNA were predominantly increased in the CD25(-) subpopulation of leukocytes from SIV(HI), further challenging the classical definition of T(reg) as CD4(+) CD25(+) T cells. Similar to CTLA-4 and FoxP3, expression of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme induced by T(reg) in antigen-presenting cells, was increased in the spleens, mesenteric LN, colons, and jejuna from SIV(HI) compared to SIV(LO) and directly correlated to SIV RNA in the same tissues. Accordingly, plasma kynurenine/tryptophan, a marker for IDO enzymatic activity, was significantly higher in SIV(HI) compared to SIV(LO) and correlated with plasma viral levels. Increased T(reg) and IDO in LT of SIV-infected macaques may be the consequence of increased tissue inflammation and/or may favor virus replication during the chronic phase of SIV infection.

FIG. 1.

Longitudinal plasma viral level measurements for the SIV-infected macaques. Plasma viral RNA was measured by NASBA, as described in Materials and Methods, in SIV_LO (A) and SIV_HI (B) animals. Plasma viral levels for the last 26 weeks before euthanasia are shown. For all animals, antiretroviral therapy was terminated at least 52 weeks before euthanasia. Dotted lines represent the 10⁴ copies/ml cutoff value between SIV_LO and SIV_HI. ND*, under detection level.

FIG. 2.

Correlation between plasma viral level and viral RNA expression in lymphoid tissues. The plasma viral RNA was measured by NASBA in all SIV-infected animals. SIVgag RNA was measured in biopsy samples from the spleen (A), inguinal LN (B), mesenteric LN (C), colon (D), and jejunum (E) by real-time PCR as described in Materials and Methods. Diagonal lines represent the best-fit regression line, to which the slope values refer. Dotted lines represent the 10⁴ copies/ml cutoff value between SIV_LO and SIV_HI. ND*, under detection level.

FIG. 3.

CTLA-4 and FoxP3 mRNA expression in blood and tissues from SIV-infected macaques. CTLA-4 (A) and FoxP3 (B) mRNA in leukocytes isolated from peripheral blood (PBMC), spleens, and LN (Mes., mesenteric; Ing., inguinal) of SIV_NEG (U), SIV_HI (H), and SIV_LO (L). In panels A and B horizontal bars within boxes correspond to the median, the box limits correspond to the 25th and 75th percentiles, and the vertical lines extend to the 10th and 90th percentiles. CTLA-4 (C) and FoxP3 (D) mRNA correlated with SIVgag RNA in the spleens and LN from SIV-infected animals (spleens, mesenteric LN, and inguinal LN were grouped together for statistical purposes).

FIG. 4.

CTLA-4 and FoxP3 mRNA expression in CD25⁺ and CD25⁻ cells from blood and tissues of SIV-infected macaques. CD25⁺ and CD25⁻ cells were isolated from peripheral blood (PBMC), spleens, and LN (Mes., mesenteric; Ing., inguinal) of SIV_HI (H) and SIV_LO (L) animals. CTLA-4 (A and C) and FoxP3 (B and D) mRNA in CD25⁺ (A and B) and CD25⁻ (C and D) cells are shown. Horizontal bars within boxes correspond to the median, box limits correspond to the 25th and 75th percentiles, and vertical lines extend to the 10th and 90th percentiles.

FIG. 5.

Flow cytometry analysis of FoxP3 expression in CD25⁺ and CD25⁻ CD4⁺ T cells. (A) Flow cytometry plot of one example inguinal LN showing the gates for CD25⁺ and CD25⁻ CD4 T cells. (B and C) Flow cytometry histograms showing intracellular staining for FoxP3 in CD25⁺ (solid histograms) and CD25⁻ (open histograms) CD4 T cells from the inguinal LN of one SIV_NEG, one SIV_LO, and three SIV_HI (B) and from the spleen of one SIV_NEG and one SIV_HI (C). Numbers beside each histogram indicate the MFI of FoxP3 staining in CD25⁺ and CD25⁻ CD4 T cells and the plasma virus levels of each animal at the time of tissue collection.

FIG. 6.

CTLA-4 and FoxP3 mRNA expression in gut tissues from SIV-infected macaques. CTLA-4 (A and C) and FoxP3 (B and D) mRNA were determined in snap-frozen gut tissues of SIV_NEG (U), SIV_HI (H), and SIV_LO (L) macaques. One sample from colon (A and B) and one sample from jejunum (C and D) were analyzed for each animal. Horizontal bars within boxes correspond to the median, box limits correspond to the 25th and 75th percentiles, and vertical lines extend to the 10th and 90th percentiles. (E) Flow cytometry analysis of CD4 and CD8 in T lymphocytes (left panel) and of CD25 and FoxP3 in the gated CD4⁺ T cells (right panel) in leukocytes from the jejunum of one SIV_HI macaque. Numbers within plots represent the frequencies of the subpopulations identified by the gates.

FIG. 7.

IL-2 mRNA expression in lymphoid tissues from SIV-infected and uninfected animals. IL-2 mRNA was measured in leukocytes from spleens (A) and mesenteric LN (B), as well as in biopsy samples from the colons (C) and jejuna (D) of SIV_NEG (U), SIV_HI (H), and SIV_LO (L) animals. Horizontal bars within boxes correspond to the median, the box limits correspond to the 25th and 75th percentiles, and the vertical lines extend to the 10th and 90th percentiles.

FIG. 8.

IDO mRNA expression in spleen and gut tissues and IDO enzymatic activity in SIV-infected macaques. IDO mRNA was measured in lymphoid tissues (A, B, C, and D), and the Kyn/Trp ratios were determined in the plasma of SIV _NEG (U), SIV_HI (H) and SIV_LO (L) animals (F). One sample from the spleen (A), one from the mesenteric LN (B), one from the jejunum (C), and one from the colon (D) were analyzed for each animal. (E) IDO mRNA correlated with the SIVgag RNA measured in the corresponding tissues. (G) Plasma Kyn/Trp ratio correlated with plasma viral level. Horizontal bars within boxes correspond to the median, box limits correspond to the 25th and 75th percentiles, and vertical lines extend to the 10th and 90th percentiles.