Animals and ethics statement
Mauritian adult male Cynomolgus macaques (Macaca fascicularis), weighing 4 to 6 kg, were housed each in single cages within level 3 biosafety facilities. Animals were housed and cared for in accordance with the European Guidelines for Animal Care (“Journal officiel des Communautés Européennes” L358, 18 décembre 1986). A regional Animal Care and Use Committee: “Comité Régional d’Ethique sur l’expérimentation animale Ile-de-France Sud”, reviewed and approved all protocols, with the goal of improving animal welfare and limiting unnecessary suffering. The animals were sedated with Ketamine chlorydrate (Rhone-Merieux, Lyon, France) before virus injection, blood sample collection or euthanasia. The animals were inoculated intravenously with 50 AID50 SIVmac251 and were euthanized at 14 (five animals), 21 (five animals) and 28 (three animals) days post-infection (dpi). Blood samples were collected before infection and every 3 dpi thereafter until euthanasia. Plasma samples were kept at −80°C until use. Changes in plasma viral load, CD4+ T-cell counts, circulating and tissue-specific B-cell subsets of these SIV-infected macaques have been previously reported . After euthanasia, duodenal and terminal ileum tissue samples (corresponding to a five cm section before the caecum) were formalin-fixed and paraffin-embedded. Control tissues were collected from three non-infected animals.
Immunohistochemistry (IHC) and image analysis
All paraffin-embedded tissues were cut into (3 μm) sections, perpendicularly to the intestinal wall so that each intestinal compartment could be examined. After antigen retrieval in sodium citrate (pH6) (CD20, Ki67, Buffy2), EDTA (pH 9) (CD3) or EDTA (pH 8) (other Ab), sections were then labeled with optimized concentrations of monoclonal or polyclonal antibodies against: CD20 (L26), IRF4 (MUM-1P), IgA (rabbit F(ab’)2, IgG (rabbit F(ab’)2, IgM (rabbit F(ab’)2, CD45R0 (OPD4), and CD68 (clone KP1) (all from Dako, Glosturp, Danemark), CD4 (1 F6) and CD23 (1B12) (from Novocastra, Newcastle, UK), Ki67 (MIB5, Beckman Coulter, Fullerton), CD3 (SP34-2, Becton Dickinson, Franklin Lakes, NJ) and cleaved caspase-3 (rabbit serum, Cell Signaling Technology Inc., Danvers, MA). Polyclonal Ig and mouse isotype controls were from Dako or R&D systems. Antibody binding was visualized with the Novolink anti-rabbit/mouse secondary Ab Polymer kit (Novocastra Laboratories, Newcastle upon Tyne, UK) according to the manufacturer’s instructions. Binding of Buffy2 mAb (Enzo life Sciences, Villeurbanne, France) was visualized by the EnVision™ + Dual Link Kit from Dako. Nuclei were counter-stained with Hematoxylin QS (Vector Laboratories, Burlingame, CA). Digital images of tissue sections were captured without manipulation using a Zeiss Microscope (Axiophot 2) coupled to a Microfire camera (Optronics, CA) and using the MorphoLite software (Explora Nova, La Rochelle, France).
Image analysis was performed with the Mercator 4.42 software (Explora Nova) on tissue sections from three non-infected animals as controls and from SIV-infected animals euthanized at either 14 or 28 dpi. Epithelium was outlined by hand and excluded in each field. Area from muscularis mucosae to tips of villi was referred to as “total mucosa” whereas CD3+ T-cell areas surrounding B-cell areas, including isolated lymphoid follicles and B-cell follicles in PP, were referred to as “follicular T-cell zones” throughout the manuscript. The LP was defined as total mucosa minus follicular T-cell zones and B-cell areas. The number of B-cell areas was determined by counting CD20+ areas in total mucosa at 100X magnification, corresponding to surface areas of 9,467 to 308,392 (duodenum) and 8,000 to 12,409 (ileum) μm2. Data are expressed as the average number of B-cell areas per μm2 of total mucosa. Immunoreactive cells were counted in all GC present in every section at 100X magnification. By using the Novolink anti-rabbit/mouse secondary Ab Polymer kit, we experienced limited difficulties in discriminating Ig-producing cells from the reticular background possibly observed in GC or sub-epithelial areas. Accordingly, only cells with a clear cytoplasmic staining were taken into account for quantification. Data are expressed as mean (±SD) number of positive cells per GC. Immunoreactive cells were counted in follicular T-cell zones, muscularis mucosae (MM) or LP by analyzing as many fields as possible at 100X magnification. Surface areas of 7,900 to 112,172 (duodenum) and 6,167 to 161,140 (ileum) μm2 for follicular T-cell zones; 1,182 to 71,742 (duodenum) and 29,000 to 51,500 (ileum) μm2 for muscularis mucosae; 147,000 to 900,000 (duodenum) and 235,000 to 750,000 (Ileum) μm2 for LP were analyzed. Data are expressed as the mean density of immunoreactive cells (number of positive cells (±SD)/μm2).