Results pVL in Chinese RMs after infection with SIVmac.
Seven SIVinfected LTNPs [21
] and 5 progressors were compared. All animals were inoculated with either SIVmac251 or SIVmac239, and pVLs are shown for individual animals in . Macaque L01 had been depleted of CD8+
cells in an earlier experiment ~60 months after SIV infection [21
], and although this animal displayed transient viremia during the depletion, viremia was undetectable thereafter. Although some LTNPs had pVL “blips,” all maintained the criteria for macaque LTNPs, defined here as persistently low (
copies/mL) to undetectable plasma viremia for months and survival for >36 months after infection. The 5 progressors had pVLs that consistently ranged from 104
copies/mL throughout chronic infection. Animal P03 had a transient period of viral control in the first 5 months of infection, but pVL increased thereafter, and thus was included as a progressor.
Figure 1 Plasma viral loads in simian immunodeficiency virus (SIVmac)-infected rhesus macaques of Chinese origin. Animal numbers of animals with long-tern nonprogressive infection are shown in bold and underlined. Animal L01 had a transient peak of viremia ~60 (more ...) Comparison of cellular viral DNA in different tissues of LTNPs.
To compare levels of cellular SIV viral DNA between different tissue compartments, viral DNA was quantified by real-time PCR ( and ). In the LTNP group, 3 macaques had undetectable viral DNA in
1 compartment (L04, L06, and L07). Data were not available for LNjej for L02, L03, L05, and L07, and data were not available for LNcol for L02 and L07. As a group, mean levels of SIV viral DNA were 103
copies per 106
cells or lower in all compartments, yet with a trend (not statistically significant) for consistently higher levels in jejunum and colon, compared with PBMCs, LNjej, and LNcol (). In the progressor group, all tissues had mean viral DNA >103
copies per 106
cells, with no statistically significant differences detected between compartments among progressors.
Figure 2 Levels of cell-associated simian immunodeficiency virus (SIV) DNA and RNA in periphereal blood mononuclear cells (PBMCs) from peripheral blood, lamina propria lymphocytes from jejunum, colon, jenunal mesenteric lymph node (LNjej), and colonic mesenteric (more ...)
Interestingly, there were no significant differences between the viral DNA levels in the colon and jejunum of progressors and levels in the same compartments in LTNPs. However, progressors had significantly higher viral DNA levels in blood, compared with LTNPs (P=.006), LNjej (P = .029), and LNcol (P = .016) ().
Comparison of viral RNA in different tissues of LTNPs. Cellular viral RNA levels were also compared in the same tissues of progressors and LTNPs ( and ). In the LTNP group, 5 of 7 animals had undetectable cell-associated viral RNA (<10 copies per 106 cells) in PBMCs. However, viral RNA was consistently detected in colon specimens and was found in 5 of 7 jejunum specimens from LTNPs (). Three LTNPs had undetectable viral RNA in LNjej (L04), jejunum (L06), or both jejunum and LNjej (L03) specimens. Data were not available for LNjej for L02, L05, and L07 and were not available for LNcol for L02 and L07. Quantitatively, mean viral RNA levels were also significantly higher in colon specimens (767 copies per 106 cells) and LNcol (486 copies per 106 cells) than PBMCs (38 copies per 106 cells) ().
In contrast, all progressors had detectable viral RNA in all compartments () with levels that were consistently and significantly higher in intestinal tissues than in PBMCs (). As expected, viral RNA in LTNPs was significantly lower in all tissue compartments when compared with corresponding tissues of progressors but was always detected in the colon, regardless of viremia. Combined, these results confirm that the intestine, and particularly the large intestine, is a major reservoir for viral persistence in LTNPs even when viral RNA or DNA is undetectable in peripheral blood ().
Comparison of intestinal SIV-target cells in chronic SIV infection.
Intestinal memory CD4+
T cells are the major early targets for SIV infection. Because the colon and jejunum have different ratios of immune-inductive to immuneeffector sites reflected in different ratios of CD4+
T cell subsets, we hypothesized that there would be differences in levels of viral target cells in the jejunum and colon and that this could be associated with viral persistence. Thus, we compared expression of memory CD4+
T cells in these compartments using slight modifications of a previously described index that accounts for both the total proportion of CD4+
T cells obtained in a sample and the percentage of those that are memory CD4+
T cells [21
]. Basically, this “target cell index” is the percentage of total CD4+
T cells in the sample (gated through CD3) multiplied by the percentage of CD4+
T cells co-expressing CD95 and CCR5 (target cells) ×100. demonstrates the gating strategy used to define memory CD4+
T cells in blood and tissues. Because there were no significant differences in CCR5 expression on blood and LN CD4+
T cells (data not shown), comparisons shown focus on the jejunum and colon. shows the levels of SIV target cells in the jejunum and colon in each group of animals. In the control and LTNP groups, colon specimens had significantly higher levels of target cells than did jejunum specimens (P
< .05). In contrast, in progressors, few target cells remained in jejunum and colon specimens and these levels were significantly lower than levels in matched samples from controls (P
< .05 for both jejunum and colon specimens) and LTNPs (P
< .05 in jejunum specimens and P
< .01 in colon specimens). Moreover, when all samples were analyzed together, there was a strong positive correlation between levels of target cells in the 2 compartments (R
= 0.8286; P
< .001) (). However, when analyzed separately by group, the correlation was not significant in the controls and in progressors, possibly because of the small sample size in the control group and because of severe depletion in both compartments in progressors (data not shown).
Figure 3 Comparison of mucosal memory CD4+CCR5+ T cells and proliferation in jejunum and colon. A, memory CD4+CCR5+ T cell expression in blood, jejunum, and colon in a representative normal Chinese rhesus macaque (RM). Cells were first gated on lymphocytes, CD3 (more ...)
Proliferation of memory CD4+ T cells was also examined by Ki-67 expression on cells in various compartments (). In both groups, the colon had significantly higher levels of Ki-67+ T cells than did peripheral blood (P = .03), and the jejunum in those with progressive disease also had significantly higher rates of proliferation than did peripheral blood (P = .020) (). In LTNPs, the colon had the highest rate of CD4+ T cell proliferation of all tissues examined ( and ).
Figure 4 Frequency of naive (CD95-negative), central memory (TCM), and effector memory CD4+ T cells (TEM) in tissue compartments of normal Chinese rhesus macaques (RMs) (A), animals with long-term nonprogression of disease (LTNPs) (B) and progressors (C). Proliferation (more ...)
Percentages of CD4+ T cell subsets were also compared between the 5 compartments in normal Chinese RM, LTNPs, and progressors. The distribution of naive (CD28+CD95−), central memory (CD28+CD95+) and effector memory (CD28−CD95+) CD4+ T cells in the blood, jejunum, and colon were similar between LTNPs and progressors ( and ). However, LTNPs had higher percentages of naive CD4+ T cells and fewer TCM cells in LNjej and LNcol than did progressors ( and ). In general, naive CD4+ T cells were predominant in lymph nodes and blood, whereas most CD4+ T cells in gut had a central memory (TCM) phenotype (). In the gut, TCM were predominant in both jejunum and colon but were not significantly different between the 2 groups ( and ). However, percentages of proliferating (Ki-67+) TEM were significantly higher in the colon than in any other compartment in LTNPs (). In addition, percentages of Ki-67+ TEM were higher in all gut and LN compartments than in peripheral blood specimens in progressors (). In summary, colon samples from LTNPs had significantly higher rates of CD4+ T cell proliferation and higher percentages of naive CD4+ T cells than did samples from other compartments, both of which suggest that a continual source of resting and proliferating CD4+ cells may contribute to viral persistence and ongoing viral replication in the colon of LTNPs.
Cellular activation in jejunum and colon during SIV infection.
It is generally accepted that viral replication is associated with immune activation [25
], which correlates with disease progression in HIV and SIV infection [25
]. To examine whether there was any difference in immune activation among different compartments, we examined CD69 (an early activation marker) and HLA-DR (late activation) to compare the frequency of activated CD4+
T cells between jejunum and colon. Most CD4+
T cells in the colon and jejunum of both progressors and LTNPs co-expressed CD69 with no significant differences, but both were significantly higher than in uninfected Chinese RM (). However, percentages of CD4+
T cells were low in all compartments of LTNPs and comparable to the control group, whereas progressors had significantly higher levels of HLA-DR+
T cells in both jejunum and colon, particularly in the latter, where >20% of CD4+
cells had an activated (HLA-DR+
) phenotype (). Thus, high levels of CD4+
T cell activation and cellular proliferation, specifically in GALT, may be associated with maintenance of the CD4+
T cells supportive of continual infection and dissemination.
Figure 5 Comparison of T cell activation in different tissue compartments in normal Chinese rhesus macaques, animals with long-term nonprogression of disease (LTNPs), and progressors, as determined by early activation marker CD69+ on CD4+ T cells (A) and HLA-DR (more ...)