All study protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Wuhan University School of Medicine (Wuhan, China) in accordance with the regulations of the National Institute of Health “Guide for the Care and Use of Laboratory Animals” and all details of animal welfare and steps taken to ameliorate suffering were in accordance with the recommendations of the Weatherall report, “The use of non-human primates in research.” The animals were housed in an air-conditioned room with an ambient temperature of 16–26°C, a relative humidity of 40–70%, and a 12-h light–dark cycle at the Animal Bio-Safety Level-III (ABSL-III) laboratory of the Wuhan University School of Medicine, which were monitored in real time by a computer-based recording system. The ABSL-III Laboratory is certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were individually housed in stainless steel wire-bottomed cages with sufficient space (800 mm wide, 800 mm depth, and 1600 mm height) and provided with a commercial monkey diet. In addition to normal pellet food, fresh fruit was provided twice daily, and water was freely available at all times. The study animals were provided with an intellectually and physically enriched environment including rings, perches, forage boxes, puzzle feeders, music, and video in the room. Animal health was monitored daily by the animal care staff and veterinary personnel. Physiological parameters of the animal, such as heart rate, body temperature, and blood pressure, were monitored at constant intervals, and pain was evaluated by veterinarian. All experimental procedures were performed under anesthesia with intramuscular injection of ketamine hydrochloride (10 mg/kg) plus intramuscular injection of atropine (0.04 mg/kg), and all efforts were made to minimize suffering.

Twelve Chinese rhesus macaques (RM; Macaca mulatta) were purchased from Sichuan Ping’an Non-Human Primates Breeding and Research Center (Sichuan Province, China). These animals (females, 5–6 years of age, 4–6 kg of weight) were individually housed and handled at the ABSL-III laboratory of the Wuhan University (Wuhan, China). The study animals were provided with an intellectually and physically enriched environment including rings, perches, forage boxes, and puzzle feeders. The animals were inoculated intravenously (i.v.) or intravaginally (i.vag.) with pathogenic R5 SIV strains (mac239, mac251, or macR71/17E). SIV-uninfected animals in the control group (n = 11) were from parallel studies. Collections and processing of blood were performed, as described previously (3, 17–19). Blood samples were used for routine flow cytometry analysis, and plasma was separated by centrifugation within 1 h of phlebotomy. All experimental procedures were performed under anesthesia with intramuscular injection of ketamine hydrochloride (10 mg/kg) and atropine (0.04 mg/kg), and all efforts were made to minimize suffering of the study animals. The humane endpoint criteria included the presentation of any of the following: (1) loss of 25% body weight from baseline weight when assigned to the protocol, (2) major organ failure or medical conditions unresponsive to treatment, and (3) tumors. If any one of these conditions was present, a humane endpoint was called and the animal was euthanized within 24 h. At necropsy, LNs from different tissues and the intestine tissues (Table 1) were collected from the animals after spontaneous death or euthanasia and subjected to lymphocyte isolation (20, 21). All the specimens were also cryopreserved or fixed in 10% buffered formalin.

Lymph node-derived lymphocytes were mechanically homogenized and passed through a 70-μm cell strainer to remove residual tissue fragments. These LNs were classified into pLNs or GI LNs based on their locations. The pLNs include LNs from the fossa axillaris and inguen where there were available in the SIV-infected animals. The GI LNs include those LNs from the colon, mesentery, and ileum that were closer to the GI tract. Some animals might have several LNs from the same tissue, while some might have no particular LNs (Table 1). To isolate lymphocytes from colon and jejunum intestines, these tissues were cut into small pieces and digested with 1 mg/ml collagenase for 1 h at 37°C with constant shaking, and then passed through a 70-μm cell strainer. Intestinal intraepithelial lymphocytes (IELs) were separated by 30–60% Percoll-plaque density gradient centrifugation, as described previously (22, 23). All samples were processed, cryopreserved, fixed (1% paraformaldehyde), and analyzed within 24 h of collection.

Six-parameter flow cytometric analysis was performed on whole blood, IELs, and T cells from LNs according to standard procedures (24) using a panel of monoclonal antibodies (mAbs) that were originally designed to detect human molecules but that we and others have shown to be cross-reactive with rhesus monkey (3, 19, 25, 26). The antibodies used were as follows: anti-CD3-FITC (clone SP34), anti-CD4-APC-H7 (clone L200), anti-CD8-PE-Cy7 (clone RPA-T8), anti-CD69-PE (clone FN50), and anti-HLA-DR-PerCP-Cy5.5 (clone G46-6) (all from BD Pharmingen). Isotype antibody was used for negative control of CD69 and HLA-DR expression. Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on a BD Verse cytometer driven by the FACS Verse software (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (TreeStar, Ashland, OR, USA). The CD4⁺ and CD8⁺ T cell percentages were based on the CD3⁺ T cells, and the activation markers on CD4⁺ and CD8⁺ T cells were based on CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells, respectively.

Plasma RNA were extracted and reverse transcribed into cDNA, and real-time RT-PCR for SIV gag were performed, as described previously (3). Briefly, SIV RNA copies per milliliter of plasma were determined by comparing sample cycle threshold (Ct) to that of a known standard of SIV gag RNA. Duplicate samples were analyzed, and the limit of detection was 200 SIV RNA copies per milliliter plasma. Real-time PCR to quantify tissue-associated viral loads was also performed on LNs or intestine tissues from animals as shown in Table 1. RNA was extracted from cryopreserved tissues, and 2 μg of RNA was reverse transcribed into cDNA for real-time RT-PCR measurement of SIV gag and host cytokines/chemokines [interleukin (IL)-10, C–C motif ligand 2 (CCL2), interferon (IFN)-γ, and tumor necrosis factor alpha (TNF-α)] using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were used as an endogenous reference to normalize the quantities of mRNAs.

Data are presented as mean ± SD per group as uninfected or SIV-infected monkeys or as pLNs or GI LNs. These data were statistically analyzed using GraphPad Prism software (La Jolla, CA, USA). Data between two groups were compared by a non-parametric Mann–Whitney T test, and p < 0.05 was considered as significant.

We initially infected eight animals with SIV_mac239 and collected LNs from different lymphoid tissues at necropsy with six out of eight animals associated with diarrhea AIDS complication at the end-stage of infection. However, due to the limited numbers of LNs available from these animals, we included the specimens from one animal infected with SIV_mac251 and three animal infected with SIV_macR71/17E. The viral loads in the plasma were monitored periodically. All the animals became infected as evidenced by increased levels of plasma SIV gag RNA (Figure 1A). The CD4/CD8 ratio decreased significantly in these animals 2 weeks after SIV infection (Figure 1C). The viral loads and CD4/CD8 ratios tend to change in opposite directions during the course of infection (Figures 1B,D).

We next examined the extent of CD4⁺ T cell decline in lymphoid tissues of SIV-infected macaques. Figure 2A shows the CD4⁺ T cell percentages in the peripheral blood, inguinal LNs, and colon from one uninfected animal (T096057) and four SIV-infected animals (WSP09, WSN01, WSP11, and WSL03). CD4⁺ T cells were dominant (54–66%) in all these lymphoid tissues from the uninfected macaque. In contrast, SIV-infected animals had much lower percentages of CD4⁺ T cells in these compartments. We next analyzed whether there was significant difference between the two groups in the CD4⁺ T cell abundance in these tissues. As shown in Figure 2B, the CD4⁺ T cell percentages differed all significantly in these compartments between the SIV-infected and -uninfected animals (Figure 2B; p < 0.05). Furthermore, in SIV-infected animals, the depletion of CD4⁺ T cells was more profound in the GI LNs as compared with those in blood (p = 0.047) and pLNs (p = 0.0029). In contrast, in the uninfected animals, the CD4⁺ T cell percentages in GI LNs were comparable to those in pLNs. Consistent with the difference in CD4⁺ T cell percentages between the two groups, SIV-infected animals had significant lower CD4/CD8 ratios than uninfected animals in all the corresponding compartments (p < 0.05; Figure 2C). In addition, in uninfected animals, the mean CD4/CD8 ratio in GI LNs was 1.82 (1.34–2.49), which was comparable to that in the pLNs (mean 1.76; 1.54–1.94; p = 0.60) but was significantly higher than that in the blood (mean 1.24; 0.81–2.03; p = 0.0003). On the contrary, in the SIV-infected monkeys, the mean CD4/CD8 ratio in GI LNs was 0.36 (0.02–1.03), which was significantly lower than those in both blood (mean 0.62, 0.18–1.61, p = 0.0479) and pLNs (mean 0.64, 0.014–1.70, p = 0.0144).

Intraepithelial lymphocytes are lymphocytes in the epithelial layer of mammalian mucosal linings, such as the GI tract. IELs have been reported to be primary targets for HIV acquisition (27). We thus isolated the IELs from different parts of the intestines from SIV-infected and -uninfected macaques. Figure 3A shows the CD4⁺ T cell percentages in IELs from 1 uninfected macaque (T096057; 1 out of 10 animals from which IELs were obtained) and four SIV-infected macaques (WSP09, WSN01, WSP11, and WSL03; 4 out of 6 animals from which IELs were obtained). It was found that in the uninfected macaque (T096057), the jejunum and colon IELs contained 57.1 and 60.7% of CD4⁺ T cells, respectively. In contrast, the CD4⁺ T cells in the IELs from the intestinal tissues of the four SIV-infected macaques were almost depleted (<1% in the jejunum IELs and <5% in the colon IELs). The percentages of CD4⁺ T cells (Figure 3B) and CD4/CD8 ratio (Figure 3C) in IELs from SIV-infected animals were significantly lower than those in IELs from uninfected animals.

Hyperactivation of T cells, particularly CD8⁺ T cells, in the peripheral blood is a hallmark of chronic HIV/SIV infection. However, the activation status of T cells in the LNs of different origins remains unclear. We thus investigated the T cell activation in the different tissues in both SIV-infected and -uninfected animals. As shown in Figures 4A,B, there was no significant difference in the percentages CD4⁺CD69⁺ and CD4⁺HLA-DR⁺ in the three types of lymphoid tissues of the uninfected animals as compared with SIV-infected animals, except for a higher percentage of CD4⁺HLA-DR⁺ in the pLNs of SIV-infected animals than that of uninfected animals (p < 0.0001). On the contrary, the percentages CD8⁺CD69⁺ and CD8⁺HLA-DR⁺ in these tissues of the infected animals were all significantly higher as compared with those of uninfected animals, except for no difference in the peripheral blood CD8⁺HLA-DR⁺ proportions between the infected and uninfected animals (Figures 4C,D).

To determine the contribution of T cells activation on CD4 depletion, we examined whether there is an association between the levels of T cell activation and CD4/CD8 ratios in the peripheral and GI LNs from SIV-infected monkeys. As shown in Figures 5B,D, the CD4/CD8 ratio of these LNs was negatively correlated with CD8⁺ T cell activation in terms of the percentages of CD8⁺CD69⁺ T cells (p = 0.0103) and CD8⁺HLA-DR⁺ T cells (p = 0.0415) in these tissues. In contrast, CD4⁺ T cell activation in terms of the percentage of CD4⁺HLA-DR⁺ T cells was positively correlated with the tissue CD4/CD8 ratio (Figure 5C, p = 0.0042). There was no correlation between CD4/CD8 ratio and the level of CD4⁺CD69⁺ (Figure 5A). Further analysis shows that in pLNs, CD4/CD8 ratio was inversely correlated with CD8⁺CD69⁺ T cell percentage (p = 0.0033), whereas in GI LNs, it was inversely correlated with CD8⁺HLA-DR⁺ T cell percentage (p = 0.0482) (Figure S1 in Supplementary Material).

In HIV infection, LNs are important sites of viral replication, in which the high local density of CD4⁺ T cells and other target cells may favor cell contact-mediated viral spread (28). We next examined the SIV loads and the cytokine expression in the different LNs of SIV-infected monkeys. As shown in Figure 6A, the defined GI LNs had significantly higher viral loads (p = 0.0181) than LNs from peripheral tissues (mainly inguinal and axillary LNs). The expression of TNF-α was also significantly higher in GI LNs than that in pLNs (p = 0.0078; Figure 6B). However, there was no significant difference in the expression levels of CCL2, IL-1β, and IL-8 between the two types of LNs (data not shown).