To determine whether pathogenic SIV infection affects Treg
proportions in lymphoid tissues, we used a cynomolgus macaque (Macaca fascicularis
) model and examined tissues at different stages after intrarectal infection with the pathogenic SIV/DeltaB670 isolate (18
). Axillary LNs were examined from acutely infected (2wk post-infection [PI]) and AIDS-developing (defined by decreasing CD4 counts, opportunistic infections, and wasting) animals, as well as uninfected controls (). As FOXP3 is considered to be a highly specific marker for Treg
), changes in its expression were measured at the RNA and protein levels. FOXP3 mRNA levels in LNs were decreased both early and late after SIV infection (). Immunofluorescence staining for FOXP3+
cells in LN tissue sections also revealed a 60-70% decrease in the proportion of cells that were FOXP3+
after SIV infection ( and ),which was highly correlated with the mRNA measurements (r=0.721, P=0.008). Additionally, flow cytometric analyses of LN single cell suspensions confirmed an approximately 50% decrease in the FOXP3+
proportion of total live cells following SIV infection (). These findings indicate there was a loss of FOXP3+
in LNs after pathogenic SIV infection of cynomolgus macaques.
Study animals and clinicovirologic states
FIGURE 1 Changes in FOXP3 and cytokine levels in LN tissues during SIV infection of cynomolgus macaques. A, Real-time RT-PCR was used for measurement of FOXP3 mRNA in LN tissues from uninfected macaques or macaques in the early (Acute) or late (AIDS) stages of (more ...)
The cytokine TGF- β1 is expressed by Treg
, acts in concert with IL-2 in the differentiation and survival of inducible Treg
), and suppresses IFN-γ expression (22
). Measurement of TGF-β1 and IL-2 mRNA levels in macaque LNs indicated that they also were decreased following SIV infection ( and ), and were highly correlated with FOXP3 levels (, r=0.91). Decreased expression of TGF-β1 and IL-2 could contribute to the reduction in FOXP3+
levels in LNs during SIV infection by reducing differentiation of naïve T cells into inducible Treg
. Although there were decreases in FOXP3, TGF-β1, and IL-10 (data not shown) expression, another immunosuppressive element, indoleamine 2,3-dioxygenase (IDO) was significantly increased after SIV infection, as was IFN-γ, an upstream inducer of IDO (23
)( and ). This is consistent with reports that IDO is increased in tonsils of HIV-1-infected patients (24
) and LNs of SIV-infected rhesus macaques (16
Loss of Treg would be expected to lead to increased immune activation and this was observed. Immunofluorescence detection and enumeration of CD3+ cells also positive for the proliferation marker Ki67 in macaque LNs revealed that the percentage of CD3+ cells that were also Ki67+ increased 3-5 fold after SIV infection (). Overall proportions of Ki67+ cells were also significantly increased after SIV infection (data not shown). These data indicated that T cell activation levels increased concordantly with loss of Treg.
highly express CCR4 (7
) and CCR7 (5
), and thus their ligands, CCL17 and CCL21, could modulate homing of Treg
to lymphoid tissues. Measurement of mRNA levels of these and other chemokines in macaque LNs revealed that mRNAs encoding ligands for CCR4 (CCL17 and CCL22) and CCR7 (CCL21) decreased (-) following SIV infection, whereas mRNAs encoding IFN-γ-inducible CXCR3 ligands (CXCL9-11) increased (-). The expression levels of the cognate chemokine receptors changed in parallel with their respective ligands (-). The changes in CCR4 and CXCR3 ligand expression were likely driven by the increased IFN-γ levels (), because ex vivo
treatment of macaque LN cells with IFN-γ led to simultaneous induction of CXCR3 ligands and decrease of CCR4 ligands ( and ). Correlation analyses revealed that in LNs, levels of CCL17 and CCL22 were positively correlated with those of their receptor, CCR4 (r=0.761 and r=0.736, respectively), and levels of CCL21 were positively correlated with CCR7 (r=0.646). In situ
hybridization and immunostaining of tissue sections confirmed these changes in chemokine and chemokine receptor expression primarily in paracortical regions ().
FIGURE 2 Changes in the relative expression levels of chemokine and chemokine receptor mRNAs during pathogenic SIV infection. A-I, Real-time RT-PCR was used to measure the levels of expression of the indicated mRNA in LNs from animals in the same disease states (more ...)
FIGURE 3 In situ detection of chemokine and chemokine receptor levels in axillary LN tissue sections from SIV-infected and uninfected cynomolgus macaques. A-F, In situ hybridization was performed for the indicated chemokine mRNA in axillary LN tissue sections (more ...)
More extensive correlation analyses revealed that FOXP3 mRNA levels were positively correlated with CCL22 (r=0.627) and CCL21 (r=0.682) levels ( and ), and their cognate receptors CCR4 (r=0.493) and CCR7 (r=0.745), respectively, but negatively correlated with CXCL9 (r=-0.615), CXCL10 (r=-0.552), CXCL11 (r=-0.584) levels (-) and local SIV viral RNA loads (r=-0.735). These findings indicate that loss of Treg in macaque LNs during SIV infection is associated with multiple changes in chemokine, cytokine and SIV levels.
FIGURE 4 Association between CXCR3 ligand and CCR4 ligand expression and function, and FOXP3 levels in macaque LNs. Linear (A and B) and nonlinear (C-E) regression analysis plots of FOXP3 mRNA versus the indicated chemokine mRNA levels are shown. F, Flow cytometry (more ...)
Given that CCR4 and CCR7 are expressed by a large proportion of Treg
) and have been shown to be important in Treg
homing to LN and other tissues (5
), we used flow cytometry to examine their expression on FOXP3+
cells in uninfected macaque LNs, the strategy of which is outlined in . Approximately 25% of CD3+
cells expressed CCR4 or CCR7(), whereas 35% expressed CXCR3 and 10% expressed CCR6. To determine whether CCL17, CCL21, and CXCL11 recruit FOXP3+
cells via CCR4, CCR7, and CXCR3, respectively, we performed chemotaxis with uninfected macaque LN cells. Immunostaining of FOXP3 in the input and migrated cells revealed that CCL17 and CCL21 recruited a population of cells that had a greater proportion of FOXP3+
cells than that recruited by CXCL11 (), despite clear expression of CXCR3 (). The lack of recruitment by CXCL11 could represent the net effect of positive and negative signaling through different receptors or possibly uncoupling of CXCR3 at the intracellular interface (25
). These chemotactic data support the interpretation that CCL17 and CCL21 contribute to homing of FOXP3 +
into LNs and the reduced expression of these chemokines () could contribute to loss or redistribution of FOXP3+
cells after SIV infection.
FIGURE 5 Flow cytometric gating strategy used to measure chemokine receptor expression on macaque FOXP3+CD4+ T-lymphocytes. Shown is the successive gating strategy used to ultimately visualize chemokine receptor levels on cynomolgus macaque LN Treg. Details are (more ...)
Given that ligands for CXCR3 antagonize the type 2 chemokine receptor CCR3 (26
) and are upregulated during SIV infection (), we examined whether a CXCR3 ligand would antagonize the type 2 and Treg
chemokine receptor, CCR4. The migration of cells stably expressing CCR4 in response to CCL17 or CCL22 was inhibited by a 100-fold excess of CXCL11 (), whereas 100-fold excess of the CXCR2 ligand, CXCL8, did not antagonize CCR4. CXCL11 alone did not induce chemotaxis of the CCR4+
cells (data not shown). Therefore, simultaneous antagonism of CCR4 by increased CXCR3 ligand expression and loss of CCR4 ligand expression could contribute to reduced homing of FOXP3+
cells to LNs during SIV infection. The nonlinear, negative correlations between CXCR3 ligand and FOXP3 levels (-) indicate that even moderate changes in CXCR3 ligand expression likely have potent inhibitory effects on homing of FOXP3+
to LNs. Finally, consistent with the antagonism of CCR4 by CXCR3 ligands, the proportion of Treg
expressing CCR4 in macaque LNs decreased during the course of SIV infection ().