Representative medullary thymic sections of (C) and (D) infected thymi stained for iNOS (brown). previously showed that experimental contamination with and prospects to the establishment of thymic contamination in chronically infected mice (18, 19). Now we statement that contamination of the thymus is usually followed by the establishment of protective immunity within the thymus, characterized by the appearance of both CD4+ and CD8+ T cells specific for mycobacterial antigens. These antigen-specific T cells do not originate from the pool of differentiating T cells in the thymus, but are instead T cells that recirculate from peripheral organs back to the infected thymus to control contamination. Their recruitment to the thymus correlates with the expression of the chemokine receptor CXCR3 and the production of CXCL9 and CXCL10 by the infected thymus. These data are the first to show that T cell recirculation to the thymus is usually a mechanism used by the immune system to survey and safeguard the thymus from contamination and maintain thymic integrity. Materials and Methods Mice and contamination C57BL/6 (WT) mice were purchased from Charles River Laboratories (Barcelona, Spain) or from Jackson Laboratories (Bar Harbor, ME) and CD45.1 mice (B6.SJL-Ptprca Pepcb/BoyJ)(32) and TCR KO (B6.129S2-Tcratm1Mom/J) from your Jackson Laboratory (Bar Harbor, ME)(33). RAG-GFP mice (34) were kindly provided by Dr. Rabbit Polyclonal to PIK3C2G Antnio Bandeira (Pasteur Institute, Paris, France). Both TCR KO and RAG-GFP mice were bred in our facilities. Mice were 7 to 10 weeks aged at the start of the experiments. All animal experiments were performed in accordance with National and European Commission guidelines for the care and handling of laboratory animals and were approved by the National Veterinary Directorate and by the local Animal Ethical Committee or by the Dana Farber Malignancy Institute Animal Care and Use Committee (Animal Welfare Assurance no. A3023-01), under Public Health Service assurance of Office of Laboratory Animal Welfare guidelines. Mice infected with were housed in a biosafety level 3 facility under specific pathogen-free conditions at the STA-21 Animal Biohazard Containment Suite (Dana Farber Malignancy Institute, Boston, MA). Experimental contamination (strain 2447, provided by Dr. F. STA-21 Portaels, Institute of Tropical Medicine, Antwerp, Belgium) contamination was performed intravenously through the lateral tail vein delivering 106 CFU per mouse. For each (Erdman strain) contamination, a bacterial aliquot was thawed, sonicated twice for 10 s in a cup horn sonicator, and then diluted in 0.9% NaClC0.02% Tween 80. A 15 ml suspension of was loaded into a nebulizer (MiniHEART nebulizer; Vortran Medical Technologies) and mice were infected via the aerosol route using a nose-only exposure unit (Intox Products) and received 100C200 CFU/mouse. At different times post-infection, mice were euthanized by carbon dioxide inhalation or by decapitation and organs were aseptically removed, individually homogenized and viable bacteria were enumerated by plating 10-fold serial dilutions of organ homogenates onto 7H10 or 7H11 agar plates for and and colonies were counted after 7 and 21 d, respectively. Gene expression analysis Total RNA was isolated from thymi, spleens and lungs using TRIZOL reagent or TRIZOL Plus RNA purification system (Invitrogen, CA, USA). Five hundred nanograms of total RNA were amplified using the Superscript RNA amplification system (Invitrogen CA, USA) according to the manufacturer’s instructions. mRNA transcripts were assessed by quantitative real-time PCR (qPCR) using SsoFast? EvaGreen Supermix? (BioRad, CA, USA) in a BioRad CFX96? Real-Time System with a C1000? Thermal Cycler or a Stratagene Mx3005P Thermal Cycler. The hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as reference gene. Specific oligonucleotides were utilized for (sense: 5′-GCT GGT GAA AAG GAC CTC T-3′; antisense: 5′-CAC AGG Take action AGA ACA CCT GC-3′), (sense: 5′-TGC CTA TGT CTC AGC CTC TTC-3′; antisense: 5′-GAG GCC ATT TGG GAA CTT CT-3′), (sense: 5′-CTT TTC CTC TTG GGC ATC AT-3′; antisense: 5′-GCA TCG TGC ATT CCT TAT CA-3′), (sense: 5′-GCT GCC GTC ATT TTC TGC-3′; antisense: 5′-TCT CAC TGG CCC GTC ATC-3′), (sense: 5′-AGC ACC AAT GGG CTC TGA-3′; antisense: 5′-TTT GGT CAG GAA TAC CAC AGC -3′) and inducible nitric oxide synthase (sense: 5′-CTC GGA GGT TCA CCT CAC TGT-3′; antisense: 5′-GCT GGA AGC CAC TGA CAC TT-3′). The cDNA was denatured for 1 min at 95 C, followed by 40 cycles of 95 C for 15 s, incubation at the optimized melting heat for 20 s, and 72 C for 20 s. Optimized melting temperatures were 57 C for and 58 C for and 59 C for activation and IFN measurement by ELISA Cell suspensions from thymus and spleen were prepared STA-21 by gentle STA-21 disruption of the organs between two notched slide glasses or by forcing organs through a 70 m nylon strainer (Fisher). For lung preparations, tissue was digested for 1 h at 37 C in 1 mg/mL collagenase (Sigma) prior to straining. Erythrocytes were lysed using a hemolytic answer (155 mM.