The Chemokine Receptor CXCR6 Is Required for the Maintenance of Liver Memory CD8+ T Cells Specific for Infectious Pathogens

MAJOR ARTICLE The Chemokine Receptor CXCR6 Is Required for the Maintenance of Liver Memory CD8+ T Cells Specific for Infectious Pathogens Sze-Wah Tse,1,a Andrea J. Radtke,1 Diego A. Espinosa,1 Ian A. Cockburn,1,2 and Fidel Zavala1 1 Department of Molecular Microbiology and Immunology and Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland; and 2Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Canberra Keywords. malaria; memory CD8+ T cells; Plasmodium; chemokine receptor; liver. CD8+ T-cell responses represent a major mechanism of immune protection against intracellular infections caused by viruses, bacteria and parasites. These T-cell responses are induced by natural exposure to infectious pathogens or vaccination, and while their protective efficacy is well-recognized, the development of robust CD8+ T-cell responses is still not an integral part of most vaccine development programs. In part, this is due to significant knowledge gaps regarding the biology of these T-cell responses and the mechanisms involved in the maintenance of long-term protective memory responses. Received 11 March 2014; accepted 29 April 2014; electronically published 13 May 2014. Presented in part: Malaria Keystone Symposia, New Orleans, Louisiana, January 2013. a Present affiliation: Program in Cellular and Molecular Medicine of Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts. Correspondence: Fidel Zavala, MD, Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N Wolfe St, Baltimore, MD 21205 (). The Journal of Infectious Diseases® 2014;210:1508–16 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: . DOI: 10.1093/infdis/jiu281 1508 • JID 2014:210 (1 November) • Tse et al CD8+ T cells play a significant protective role in a number of infections affecting the liver, such as viral hepatitis, listeriosis, and malaria. Numerous studies in experimental models and humans have clearly shown that CD8+ T cells specific for antigens expressed by Plasmodium parasites during the liver stages are an important component of the antiparasite immune response induced after immunization with radiationattenuated malaria sporozoites (γ-spz) [1, 2]. Immunization with sporozoites through mosquito bites or intradermal injection induces CD8+ T-cell responses in the skin-draining lymph nodes [3]. After priming, activated CD8+ T cells migrate to the liver, where they develop a memory transcriptional signature that is unique, compared with that of splenic memory cells; in particular, these cells have increased expression of the chemokine receptors CXCR3 and CXCR6, as well as differences in expression of the transcription factor Eomes and cell cycle genes, such as Ki67 [4]. These liver-homing antigen-specific memory CD8+ T cells are capable of recognizing parasite antigen presented by hepatocytes [5] and eliminating Plasmodium-infected cells [6]. It is well established that immunization with attenuated malaria sporozoites induces CD8+ T cells that eliminate parasite-infected hepatocytes. Liver memory CD8+ T cells induced by immunization with parasites undergo a unique differentiation program and have enhanced expression of CXCR6. Following immunization with malaria parasites, CXCR6-deficient memory CD8+ T cells recovered from the liver display altered cell-surface expression markers as compared to their wild-type counterparts, but they exhibit normal cytokine secretion and expression of cytotoxic mediators on a per-cell basis. Most importantly, CXCR6-deficient CD8+ T cells migrate to the liver normally after immunization with Plasmodium sporozoites or vaccinia virus, but a few weeks later their numbers severely decrease in this organ, losing their capacity to inhibit malaria parasite development in the liver. These studies are the first to show that CXCR6 is critical for the development and maintenance of protective memory CD8+ T cells in the liver. MATERIALS AND METHODS the Institutional Animal Care and Use Committee of Johns Hopkins University. Parasites, Immunizations, Quantification of Parasite Burden, and Adoptive Transfer The generation of Plasmodium berghei CS5M parasites and irradiation and immunization of mice was performed as described elsewhere [5]. Quantification of liver-stage parasites was performed as previously described [24]. For immunization with vaccinia-OVA, mice were given 2 × 106 plaque-forming units (PFU) of virus intravenously following adoptive transfer of OT-I, as described elsewhere [5]. Adoptive transfer of T cells was performed by intravenous injection of 5000 TCR-transgenic Cxcr6+/− (CXCR6+) or Cxcr6−/− (CXCR6−) OT-I cells T cells (CD45.1+) from spleen into naive CD45.2+ Cxcr6+/− heterozygous C57Bl6 recipients. For the in vivo homing assay, mice that received naive CXCR6+ or CXCR6− OT-I were subsequently immunized with 1 × 105 γ-spz or 2 × 106 PFU rVV-OVA. Between 6 and 8 days after immunization, spleens were harvested, and numbers of CD45.1+ OT-I were estimated by flow cytometry with anti-CD45.1 and anti-CD8 antibodies. Equal numbers of OT-I CD8+ T cells (approximately 0.5 × 106) were then adoptively transferred to naive CD45.2+ recipient mice. Twenty hours after transfer, spleen and liver were excised, and the numbers of donor CD8+CD45.1+ cells in these organs were determined by flow cytometry. Lymphocyte Isolation and Ex Vivo Stimulation Tissues were harvested on day 3–90 after immunization as indicated. Lymphocytes were isolated as described previously [25]. In brief, liver was perfused with Hank’s balanced salt solution (HBSS), homogenized, and resuspended in 35% Percoll gradient (GE Healthcare). Lungs were treated with collagenase II (1 mg/mL) at 37°C for 30 minutes, followed by Percoll gradient as described above. Lymphocytes were cocultured with El4 cells pulsed with or without SIINFEKL peptide (10 µg/mL) for 4 hours at 37°C in the presence of brefeldin A and monensin. Intracellular staining was performed using Cytofix/perm kit (BD Biosciences). Mice C57Bl/6 mice aged 5–8 weeks were purchased from the National Cancer Institute (Frederick, MD). Ccr5−/− (N10) and Cxcr6−/− (N10) [21, 22] were purchased from Jackson Laboratory (Bar Harbor, ME). Cxcr3−/− mice were obtained from Dr Craig Morrell [23]. OT-1 (SIINFEKL-specific TCR) transgenic mice were obtained from Dr David Sacks (National Institute of Allergy and Infectious Diseases, Bethesda, MD). Cxcr6−/− mice were bred with OT-1 (CD45.1+) mice to generate Cxcr6−/− OT-1 transgenic mice. Age-and sex-matched Cxcr6+/− mice were used as recipient mice in adoptive transfer experiments. All mice were housed and bred in the animal facility at Johns Hopkins University. Experiments involving mice were approved by Antibodies (...truncated)