Thirty-one adult rhesus macaques (Macaca mulatta) of Chinese origin were included in this study.

Four healthy macaques received a single subcutaneous injection of recombinant glycosylated simian IL-7 (R-sIL-7gly; 80 µg/kg body weight); the IL-7 was provided by Cytheris SA; Issy les Moulineaux; France. The macaques were euthanized at 12 h (n = 1), 1 day (n = 2), or 7 (n = 1) days after IL-7-treatment.

Five uninfected healthy macaques (#1–#5) were euthanized and served as controls.

Twenty-two macaques were infected by intravenous injection of 50 AID₅₀ of the pathogenic SIVmac₂₅₁ isolate (kindly provided by Dr. Anne-Marie Aubertin; INSERM U544, Strasbourg, France). All macaques were productively infected, as demonstrated by positive plasma SIV viral load by days 3–7.

Nine SIV-infected macaques were euthanized at day 3 (n = 3; #6, #7, and #8), 7 (n = 2; #9 and #10), 10 (n = 2; #11 and #12), or 14 (n = 2; #13 and #14). One SIV-infected macaque was euthanized 1-year post-infection because of AIDS-related symptoms (weight loss, cachexia, and prostration). Samples from two other animals euthanized at day 65 were also included in some experiments (samples generously provided by Dr Michaela Müller-Trutwin—Institut Pasteur, Paris).

Organs were collected at necropsy and treated immediately for future analyses. Samples were either frozen at −80°C for future DNA and RNA extraction or snap frozen in liquid nitrogen in optimum cutting temperature (O.C.T.) compound (Labonord, Templemars, France) and preserved at −80°C.

The 10 remaining SIV-infected macaques were longitudinally followed during the acute phase of SIV infection. Blood was sampled every 3–4 days for 3 weeks (10 mL was collected on EDTA).

Frozen intestinal tissue samples (20–30 mg) were directly lysed in 500 µL of ice cold Tris Lysis Buffer (Meso Scale Discovery, Rockville, MD, USA) supplemented with protease inhibitors (4× of complete EDTA-free protease inhibitor cocktail, Roche Diagnostics, Meylan, France). Sample grinding consisted of two rounds of 2 min in the TissueLyser II system at 30 Hz with a 10-min incubation on ice in between the cycles. After 30 min of incubation on ice, lysed samples were centrifuged at high speed for 10 min at 4°C to remove cell debris and aliquots were frozen at −80°C. Total protein concentration was quantified using the Pierce Bicinchoninic Acid Assay (BCA Protein Assay Kit, Thermo Fisher Scientific, Villebon sur Yvette, France). IL-7 quantification was performed using the Quantikine Immunoassay IL-7 HS (R&D Systems, Lille, France), according to the manufacturer’s instructions.

Total mRNA was extracted from homogenized tissue (20–30 mg) using the RNeasy kit (Qiagen, Les Ulis, France), including 1 step of DNase treatment (RNase-Free DNase Set, Qiagen). RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). Specific cDNAs were PCR-amplified using “outer” 3′/5′ primer pairs by 15 min of denaturation at 95°C, followed by 22 cycles of 30 s at 95°C, 30 s at 60°C, and 3 min at 72°C. Each target was coamplified together with HPRT sequences; HPRT was used as a housekeeping gene. Target mRNA and HPRT were quantified within each of the PCR products in LightCycler^® experiments performed on 1/280th of the PCR products; “inner” 3′/5′ primer pairs and the LightCycler^®480 SYBR Green I Master Mix (Roche Diagnostics, Meylan, France) were used. The PCR cycling program consisted of 10 min of initial denaturation at 95°C, 40 cycles of 10 s at 95°C, 6 s at 64°C, and 15 s at 72°C. Fluorescence measurements were performed at the end of the elongation steps. Plasmids containing one copy of both the HPRT gene and of the target amplicons were used to generate standard curves. IL-7, chemokines, and HPRT mRNA quantifications were performed in independent experiments using the same first-round serial dilution standard curve. Quantifications were made in triplicate for all samples studied; three to five samples were used for each tissue from each macaque. The target mRNA concentration was normalized to HPRT mRNA in each sample. The results were expressed as the absolute number of target mRNA copies per HPRT mRNA copy. HPRT-, IL-7-, and chemokine-specific primers are described in Table S1 in Supplementary Material.

A quantification method similar to the one described above for IL-7 and chemokine quantification was used to quantify total SIV RNA; Gag-specific primers were used (Table S2 in Supplementary Material).

For SIV DNA quantifications, tissues were lysed in Tween-20 (0.05%), Nonidet P-40 (0.05%), and proteinase K (100 µg/mL) for 30 min at 56°C, followed by 15 min at 98°C. SIV DNA was directly quantified from the lysate using Gag-specific primers (Table S2 in Supplementary Material). Gag sequences were amplified together with the rhesus macaque CD3γ chain in triplicate using the “outer” 3′/5′ primer pairs and the same conditions as described earlier. SIV Gag and CD3γ were then quantified using the “inner” 3′/5′ primer pairs (Table S2 in Supplementary Material) and a plasmid containing one copy of both the CD3γ and Gag amplicons. The results were expressed as the absolute number of SIV copies per 10⁶ cells.

Peripheral blood cell phenotype was analyzed from frozen whole blood by flow cytometry using a BD FACS CANTO II (BD Biosciences, Le Pont de Claix, France) as described (41). The fluorochrome-conjugated antibodies used for polycromatic flow cytometric analysis are listed in Table S3 in Supplementary Material.

Ileum cryosections 7 μm in thickness were placed on SuperFrost^® Plus slides (Thermo Scientific Menzel, Braunschweig, Germany), fixed for 10 min at 4°C in acetone/methanol (50/50), rinsed in PBS, and blocked with 5% BSA/2% normal goat serum in PBS at room temperature (RT) for 30 min. Sections were incubated overnight with primary antibodies in a 4°C humid chamber. Sections were rinsed in 0.5% Tween20/PBS, incubated at RT in the dark for 30 min with secondary antibodies, and rinsed in 0.5% Tween20/PBS. Finally, sections were washed in PBS alone, counterstained with 4,6-diamidino-2-phenylindol (DAPI; Molecular Probes, Cergy Pontoise, France), and mounted in Fluoromount-G medium (Southern Biotechnology, Birmingham, AL, USA). Cell quantifications in tissue were performed on large (25 mm²) images reconstructions of at least two samples per macaque acquired with 20× oil objective (Leica Microsystems Gmbh, Wetzlar, Germany) using the Yokogawa CSU X1 Spinning Disk (Yokogawa, Tokyo, Japan) coupled with a DMI6000B Leica microscope with MetaMorph 7.7 software (Molecular Devices, Sunnyvale, CA, USA), using the Scan-Slide option (10% overlap). To identify IL-7 in the infected mucosa, immunostaining experiments were performed using polyclonal antibodies directed against IL-7, cytokeratin, and ezrin (polyclonal antiserum obtained from P. Mangeat; CNRS UMR 5539, Montpellier, France). Acquisitions were performed with 20× objective on a DMI6000B Leica microscope with MetaMorph 7.7 software. The antibodies used for immunohistofluorescence labeling are listed in Table S4 in Supplementary Material.

Analyses were performed using the ImageJ software.¹ In order to accurately quantify nuclei (DAPI), CD3⁺ cells (Alexa Fluor^® 546), or CD4⁺, or CD8⁺ cells (Alexa Fluor^® 488), images were acquired over the entire thickness of the sample with 13–20 z-stack images and the best focuses were chosen. In order to count immune cells in large areas, the LP, submucosa (SM), and regions containing epithelial cells (E) or lymphoid follicles (LF) were manually defined. Pixels corresponding to E + LF zones were excluded from the process. These excluded zones were replicated on all treated images. After a denoising process, a manual threshold was applied on CD3⁺, CD4⁺, and CD8⁺ stains to identify immune cell surfaces. CD3⁻CD4⁺ and CD3⁻CD8⁺ stained surfaces, as well as CD3⁺CD4⁺ and CD3⁺CD8⁺ costained surfaces, were quantified. Results were presented as total single-stained or costained surfaces/total surface of LP or SM. At least 1.5 mm² of LP and 1.4 mm² of SM surfaces were analyzed per macaque.

The non-parametric Spearman rank correlation test was used to investigate the relationship between parameters. The non-parametric Mann–Whitney test was used to compare different groups of macaques, and the non-parametric Wilcoxon rank sum test was used to compare data from the same macaques at different time points before and after SIV infection. All statistical analyses were performed with Real Statistics² add-in to Microsoft Excel software. In two-tailed tests, p values of 0.05 or lower were considered significant.

All the animals included in this study were housed, cared for, and handled in BSL2 (uninfected macaques) or BSL3 (SIV-infected macaques) facilities at the Institut Pasteur (Paris, France; accreditation no. A 78-100-3) and IDMIT (“Infectious Disease Models and Innovative Therapies” at the CEA “Commissariat à l’Energie Atomique,” Fontenay-aux-Roses, France; accreditation no. C 92-032-02) NHP facilities, licensed by the French Ministry of Agriculture, in accordance with European guidelines for animal care (European directive 86/609, “Journal Officiel des Communautés Européennes,” L358, December 18, 1986). The CEA is in compliance with Standards for Human Care and Use of Laboratory of the Office for Laboratory Animal Welfare (OLAW, USA) under OLAW Assurance number #A5826-01. This study and the procedures used were approved by the “Comité Régional d’Ethique en Expérimentation Animale Ile-de-France—Paris—Comité 1,” which follows the governance of the “Charte nationale portant sur l’expérimentation animale” according to the EU Directive 2010/63 on the protection of animals used for scientific purposes (notifications no. 2011-0001 and 2012-0003). All experimental procedures were carried out in strict compliance with European guidelines for NHP care (European Directive 86/609 then; as of January 2013, EU Directive N 2010/63/EU) for protection of animals used for experimentation and other scientific purposes and the Weatherall report. Food and water were provided ad libitum. Temperature was maintained constant (22 ± 2°C). Air was renewed at least 20 times per hour. Fluorescent light was provided with a 12:12 h light–dark cycle. All efforts were made to minimize suffering, including improving housing conditions and providing enrichment opportunities (e.g., provision of monkey biscuits supplemented with fresh fruit and constant water access, objects to manipulate, interaction with caregivers and research staff). Animals were kept in cages in compliance with European regulations in terms of floor surface per animal. All manipulations (injections, blood draws, and euthanasia) were performed under anesthesia (tiletamine/zolasepam; 4.6 mg/kg). Euthanasia was performed by intravenous injection of pentobarbital (10 mL). Permanent veterinarians managed the animal facilities and were responsible for the well-being of the experimental animals. The animal facilities were run by teams of animal caretakers under the supervision of animal technicians. According to European and French regulations, animal caretakers have received a specific training in laboratory animal science. Technicians who were doing experiments on live animals had received the appropriate training. Biohazards were handled in BSL2 and BSL3 facilities according to French legislation.

Intestinal tissue and LNs were sampled from five healthy and nine SIV-infected Chinese rhesus macaques sacrificed at days 3 (n = 3), 7 (n = 2), 10 (n = 2), or 14 (n = 2) following intravenous infection with 50 AID₅₀ of SIVmac₂₅₁. In these tissues, we first evaluated IL-7 expression by measuring mRNA levels using a highly sensitive qRT-PCR assay. Interestingly, IL-7 transcription significantly rose by day 3 in the ileum. Maximal IL-7 transcription was observed at day 7–10 post-infection (pi) (p = 0.009; Figure 1A). IL-7 expression returned close to baseline values at day 14. IL-7 mRNA levels were also increased by day 3 in the duodenum and the jejunum, slightly increased at day 10 in the large bowel and remained stable in the LNs, suggesting that the increase in IL-7 transcription was mostly confined to the small bowel in acutely SIV-infected macaques (data not shown). Quantification of IL-7 protein in the ileal tissue also demonstrated that IL-7 expression was higher in macaques sampled throughout acute infection than in uninfected monkeys. Mean local IL-7 concentration was 5.8 pg/mg of total protein from days 3 to 10, as compared to 3.7 pg/mg in uninfected controls (p = 0.014; Figure 1B). Moreover, in the ileum, IL-7 mRNA levels directly correlated with IL-7 protein concentration (r = 0.81; p = 0.002; Figure 1C), suggesting that increased transcription translated in increased protein production. Immunohistochemical staining confirmed epithelial cells as a possible source of intestinal IL-7. Indeed, similar to ezrin staining, IL-7 staining mostly localized at the apical pole of the epithelial cells as demonstrated by cytokeratin staining (Figure 1D).

In order to investigate whether intestinal IL-7 production could be initiated by local infection, we quantified SIV DNA and RNA in tissues from SIV-infected monkeys. At early time points, higher levels of SIV replication were observed in the ileum compared to any other organ, including LNs, with viral DNA being detectable by day 3 in all tested ileal samples (Table 1). Large amounts of SIV DNA were observed in most organs sampled at day 10 or 14 post-infection (pi), reaching 10³ genomes per million cells in mesenteric LNs, in the ileum, and in the large intestine. In the ileum, SIV DNA load correlates with IL-7 expression (r = 0.54, p = 0.047; Spearman’s rank correlation).

Simian immunodeficiency virus replication, defined by the presence of unspliced viral mRNA, was detectable in most organs sampled at days 10 or 14 (Table S5 in Supplementary Material). Sporadic SIV RNA positivity was observed before day 10.

To further characterize the impact of early infection on the mucosal cytokine/chemokine network, we investigated the expression of 13 chemokines (CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL20, CCL21, CCL25, CCL28, CXCL8, CXCL10, and CXCL12) using real-time quantitative RT-PCR. Tissues were sampled from the gut of macaques sacrificed at days 3, 7, 10, or 14 pi and chemokine levels were compared to values from uninfected controls; the values obtained from these controls served as baseline, i.e., pre-infection values. At baseline, a few chemokines were constitutively expressed in the ileal mucosa (CCL5, CCL19, CCL21, CCL25, CCL28, and CXCL12; Figure 2). Higher chemokine transcription was observed in one infected macaque sacrificed at day 7 (CCL3, CCL4, CCL5, CCL20, CCL25, CCL28, and CXCL10; Macaque #9; Figure 2). At day 10, transcription of CCL3, CCL4, CCL5, CCL19, CCL25, CCL28, and CXCL10 was increased in both animals while CCL20 and CXCL8 were overexpressed in macaque #12 (Figure 2). At day 14, most chemokine levels had returned to close to baseline levels, except for CCL3, CCL4, CCL19, and CCL25 which remained high in one of the two macaque (Macaque #14) and CXCL10 levels which remained elevated in both macaques (Figure 2).

Similarly, several chemokines were overexpressed in the other parts of the small intestine of the macaques sacrificed at day 10 or 14 (CXCL10 in both tissues from macaques #11, #12, #13, and #14; CCL11 and CCL20 in the duodenum of macaques #11 and #12; CCL2, CCL3, CCL5, CCL11, and CXCL8 in the duodenum of macaque #14; CCL5, CCL11, and CCL19 in the jejunum of macaque #14; data not shown).

We had previously demonstrated that IL-7 injection triggers chemokine expression in tissues (37). We therefore examined whether, in infected tissues, endogenous IL-7 could be responsible for the observed local increase of chemokine transcription. Accordingly, classifying the infected monkeys based on ileal IL-7 transcription levels, we established that CCL4, CCL5, CCL25, CCL28, and CXCL8 expressions were significantly increased in the ileum of monkeys demonstrating high IL-7 expression (#7, #9, #11, and #12) as compared to macaques with low IL-7 transcription/expression (#1, #2, #3, #4, #5, #6, #8, #10, #13, and #14; Figure 3A). Moreover, at both tissue sample level (data not shown) and animal level (Figure 3B), IL-7 mRNA levels directly correlated with local mRNA expression of CCL4, CCL5, CCL25, and CCL28 in the ileum (r = 0.69, 0.78, 0.82, and 0.55; p = 0.006, 0.001, <0.001, and 0.039, respectively); multivariate analysis showed that viral load had no impact on these correlations. Similar correlations were observed in the duodenum (CCL5; r = 0.60; p = 0.01; Figure S1A in Supplementary Material) and in the jejunum (CCL5, CCL20, and CCL25; r = 0.65, 0.70, and 0.78; p = 0.01, 0.005, and 0.001, respectively; Figure S1B in Supplementary Material). These data suggest that IL-7 is involved in upregulating the transcription of these chemokine in the infected intestine. By contrast, ileal transcription of CCL2, CCL3, CCL19, and CXCL10 correlated with local SIV DNA (r = 0.69, 0.55, 0.76, and 0.61; p = 0.01, 0.04, 0.001, and 0.02, respectively; Figure S2 in Supplementary Material) and SIV RNA loads in SIV-infected monkeys (r = 0.57, 0.64, 0.66, and 0.65; p = 0.03, 0.02, 0.009, and 0.01, respectively; Figure S2 in Supplementary Material), suggesting a direct role for viral infection in increasing the transcription of these chemokines.

To confirm the role of IL-7 in regulating chemokine expression, four uninfected macaques were subcutaneously injected with pharmacological doses of IL-7 (80 μg/kg of body weight) and sacrificed either 12 h, or 1 or 7 days after treatment. Of note, the local IL-7 concentrations measured at day 1 were of the same order of magnitude as those observed in SIV-infected macaques (15–30 pg/mg of total proteins; data not shown). Ileal transcription of CCL3, CCL4, CCL25, CCL28, and CXCL8 was increased in IL-7-treated macaques (p < 0.02 as compared to untreated monkeys; Figure 4), a trend being observed for CCL2 (p = 0.07; Figure 4). Similarly, increased transcription of CCL4, CCL5, and CCL28 in the duodenum, and of CCL4 and CXCL8 in the jejunum, were observed following systemic IL-7 administration (data not shown).

Collectively, these data suggest that locally produced IL-7 acts as a danger signal that triggers chemokine overexpression in the ileum of acutely SIV-infected Chinese macaques. We hypothesize, therefore, that SIV-induced production of IL-7 expression in the gut may participate in the recruitment of immune cells into the infected mucosa, as observed in IL-7-treated humans and macaques (37, 38).

In order to evaluate the possible consequences of local chemokine network changes on immune cell localization in the infected ileum, we quantified immune cell subsets in the ileal mucosa of uninfected macaques or SIV-infected monkeys. Infected macaques were sampled throughout the acute infection, at day 65 pi or at the AIDS disease stage. We observed that despite the relatively homogeneous cellular contents of the LP, as demonstrated by the constant proportion of the mucosal surface that was occupied by nuclei, CD3 surface staining varied considerably in the various LP zones analyzed (Figure S3 in Supplementary Material). In order to minimize the impact of the uneven T-cell distribution observed in both uninfected and infected macaques, immune cell distribution was quantified on entire tissue sections (25 mm² per slide). In SIV-infected macaques, CD3 surface staining increased by day 7 in the ileal LP (9.7, 12.1, 9.8, and 12% of the analyzed surfaces in macaques #9, #11, #12, and #13, respectively, as compared to 4.8 and 7.1% in macaques #1 and #2 sampled at baseline; Figure 5A; left panel). At day 65 pi, CD3⁺ T-cell counts remained high in the ileal LP of one macaque, while it returned to baseline levels in the second macaque. In the monkey with AIDS-related symptoms, 7.2% of the analyzed surface stained for CD3, a value similar to that observed at baseline. The increase in the density of CD3⁺ T-cells observed during the course of the primary infection was almost exclusively due to CD8⁺ T-cells. Indeed, the proportion of the surface area that was stained CD3⁺CD4⁻ increased during acute infection and remained high throughout the study (Figure 5A; central panel), except for the second macaque sampled at day 65 pi. Of note, the values obtained for CD3⁺CD8⁺ cell quantifications of tissues sampled at baseline, days 10 and 14 were very similar to the values obtained for CD3⁺CD4⁻ staining, suggesting that the CD3⁺CD4⁻ cells are in fact CD8⁺ T-cells (hatched bars on Figure 5A; central panel). By contrast, the density of CD4⁺ T-cells (CD3⁺CD4⁺) remained constant until day 65 pi, representing 3–4.2% of the analyzed surfaces at any time point (Figure 5A; right panel). Depletion of ileal LP CD4⁺ T-cells was only observed in both the second macaque sacrificed at D65 pi and the macaque with AIDS-related symptoms (82 and 76% decrease compared to mean values in macaques sampled at baseline, Figure 5A; right panel), consistent with the depletion of gut CD4⁺ T-cells that has been largely described in chronically infected macaques and humans.

Using these same tissue sections, we also analyzed the different cell populations in the submucosa (Figure 5B). Despite low baseline cell concentrations [the median percentage of the tissue surface occupied by nuclei was 31.12% (26.8–63.6)], both CD4⁺ and CD8⁺ T-cell numbers fluctuated over the course of acute infection, with transient declines at days 3 and 10 pi (Figure 5B). At AIDS stage, as well as in the second macaque sampled at D65, CD8⁺ T-cells were almost completely depleted from the submucosa, while CD4⁺ T-cells increased at AIDS stage.

In the ileal mucosa, T-cells represent only a fraction of the total lymphoid cells. In healthy rhesus macaques, all LP CD8⁺ cells were round and more than 70% coexpressed CD3, marking them as T-cells (Figure S4 in Supplementary Material). By contrast, more than half of mucosal CD4⁺ cells did not coexpress CD3; these cells had the typical shape of antigen-presenting cells (APCs) (Figure S4 in Supplementary Material).

Lamina propria tissues were thus also examined for changes in the proportion of CD4-expressing non-T-cells (CD4⁺CD3⁻) during the course of SIV infection. These CD4⁺CD3⁻ cells rapidly expanded/infiltrated the ileal LP during the first 3 days pi (3.7 and 6.5% of tissue surface at day 0 and day 3, respectively; Figure 6A; top panel) and then gradually declined. At day 14 pi, these cells were less abundant in the LP than before infection (2.5% of tissue surface). This effect was only transient in acute infection, as CD4⁺CD3⁻ stained surfaces returned to baseline levels at day 65 pi. Finally, these cells were barely detectable in late stage disease. In the submucosa, CD4⁺ non-T-cells represented more than 90% of the CD4⁺ surface in healthy macaques (data not shown). This population was almost entirely composed of cells with dendrites that expressed PM-2K, CD68, CD169 (Sialoadhesin/Siglec-1), and DC-SIGN, but not CD11c, marking them as macrophages (Figure 6B). Furthermore, these cells expressed low levels of MamuLa-DR, CD83, and CD86 (B7-2) (Figure 6B; data not shown) and did not proliferate as they were Ki67⁻ (data not shown). In the submucosa, the CD4⁺ macrophage subset rapidly declined during the first days of acute infection; thus, the changes in the numbers of this macrophage population mirrored the changes observed in the LP (Figure 6A; bottom panel).

Altogether, our data suggest that cell migration occurred rapidly into and/or within the ileal mucosa during the first days of SIV infection.

Given the presence of important T-cell attracting chemokines in the small intestine of acutely SIV-infected macaques, we investigated whether cell migrations could be observed from the blood. Blood samples were collected over the first 21 days of SIV infection (n = 10). Severe depletion of both CD4⁺ and CD8⁺ T-cells characterized the first 10 days of infection (Figure 7A). Naïve T-cells were the most affected subsets during the early phase of SIV infection, with decreases of 5.8- and 4.8-fold at day 10 in CD4⁺ and CD8⁺ T-cell subsets, respectively. By contrast, effector memory T-cell counts barely changed, while a twofold to threefold reduction was observed for central memory T-cells. Following this initial contraction phase, peripheral T-cell counts rebounded by day 14 pi. At this time point, most of the increases in the CD4⁺ T-cell compartment occurred within the naïve subset, while all CD8⁺ T-cell subset numbers increased (Figure 7A). When we examined Ki-67 proliferation marker within circulating T-cells, we identified a significant transient increase in the frequency of Ki-67⁺ cells at day 7 in all T-cell compartments (Figure 7B). However, given the observed T-cell count decreases, these increased frequencies did not translate into any significant change in the numbers of circulating Ki-67⁺ cells. By day 14 pi, a large proportion of the cells that returned to the peripheral blood expressed Ki-67 antigen. This second wave of Ki-67 expression was observed at day 14 pi in naïve CD4⁺ and CD8⁺ T-cells, as well as in memory CD8⁺ T-cells (Figure 7B). Finally, memory CD4⁺ T-cell cycling was detectable at day 17 pi (Figure 7B).

In the same samples, we evaluated cell surface expression of CCR6 (CCL20 receptor) and CCR9 (CCL25 receptor), both of which are involved in intestinal T-cell homing. We also assessed the cell surface expression of β7 integrin, which, in association with α4 integrin, acts as a specific docking molecule to gut endothelial cells. While the frequency of CCR6⁺β7⁺ cells remained constant in all T-cell subsets, we observed a gradual decrease in the frequency of CCR9⁺β7⁺ cells throughout the follow-up period, leading to a twofold to fourfold reduction at day 14 (Figure 7C). Similarly, a twofold reduction in the frequency of CCR9⁺ cells within β7⁺ CD4⁺ and CD8⁺ T-cells was observed at day 14 (data not shown). Together with CCL25 overexpression in the small bowel, these data suggest that circulating immune cells presenting ileal imprinting preferentially migrated into the small intestine during the first 2 weeks of acute SIV infection.

Altogether, these data suggest that chemokine expression in organs, and in particular in the small intestine, triggers massive recruitment of circulating T-cells from peripheral blood. By day 14, cells migrating back into the circulation express the Ki-67 proliferation marker, suggesting that these cells have undergone either antigen priming or cytokine (IL-7) stimulation, especially within the small intestine.