Innate control of adaptive immunity via remodeling of lymph node feed arteriole Kelly A. Soderberg*, Geoffrey W. Payne†‡, Ayuko Sato*, Ruslan Medzhitov*§, Steven S. Segal†, and Akiko Iwasaki*¶ *Section of Immunobiology, †John B. Pierce Laboratory and Department of Cellular and Molecular Physiology, and §Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520 Edited by Douglas T. Fearon, University of Cambridge, Cambridge, United Kingdom, and approved September 22, 2005 (received for review July 21, 2005) The adaptive immune system relies on rare cognate lymphocytes to propriate microbial peptides must occur rapidly to provide the host detect pathogen-derived antigens. Naı¨ve lymphocytes recirculate with survival advantage. Thus, we hypothesize that such a process through secondary lymphoid organs in search of cognate antigen. must be inducible upon infection and regulated by the innate Here, we show that the naı¨ve-lymphocyterecirculation pattern is immune system. controlled at the level of innate immune recognition, independent In the present study, we tested these hypotheses using a combi- of antigen-specific stimulation. We demonstrate that inflamma- nation of approaches involving minimal TLR agonists and a tion-induced lymphocyte recruitment to the lymph node is medi- physiologically relevant model of mucosal viral infection with ated by the remodeling of the primary feed arteriole, and that its herpes simplex virus 2 (HSV-2). We report that during the initiation physiological role is to increase the efficiency of screening for rare of an adaptive immune response, a large number of naı¨ve lympho- antigen-specific lymphocytes. Our data reveal a mechanism of cytes are recruited specifically to the LNs draining the site of innate control of adaptive immunity: by increasing the pool of immunization or infection, a process we call ‘‘inflammation- naı¨velymphocytes for detection of foreign antigens via regulation induced recirculation.’’ We demonstrate that inflammation- of vascular input to the local lymph node. induced naı¨ve lymphocyte recruitment to the dLN occurs via remodeling of the primary arteriole feeding the LN to a larger o mount protective immunity, the adaptive immune system diameter, and that its physiological role is to increase the efficiency Trelies on the detection of foreign antigens by rare lymphocytes of screening for rare antigen-specific lymphocytes. possessing appropriate antigenic specificity. After local infection, antigens from the pathogen are taken up by dendritic cells (DCs) Methods (1). Concomitant recognition of pathogen-associated molecular Cells, Viruses, and Reagents. Thymidine kinase mutant HSV-2 patterns (PAMPs) by the Toll-like receptors (TLRs) induces DC strains 186TK⌬Kpn (13) were used to infect mice. Depo-Provera- activation and migration into the draining lymph nodes (dLNs), treated female mice were infected intravaginally (ivag) with 106 where they present antigenic peptides on MHC molecules to naı¨ve PFU of HSV-2 or with uninfected Vero cell lysate (mock infection) T lymphocytes (1–3). TLR-mediated recognition of PAMPs is as described in ref. 14. All antibodies were obtained from eBio- required for the initiation of Th1 immunity (4–7), and incorpora- science. All other reagents were from Sigma, unless otherwise tion of TLR agonists in vaccine adjuvants is often required for the indicated. generation of robust adaptive immunity. TLR signals contribute to the generation of adaptive immunity by several distinct mechanisms Mice. Female C57BL6 ϫ 129 F2, C57BL6, and BALB͞c mice, 6–8 including the activation of antigen presenting cells (APC) (2) and wk old, were purchased from The Jackson Laboratory. TLR9Ϫ/Ϫ stromal cells (7). TLR pathways are also required for the activation (15) or TLR2Ϫ/Ϫ (16) mice were bred and maintained in our of adaptive responses by blocking the suppressive effect of regula- facilities under standard conditions. All procedures used in this tory T cells (8). study complied with federal guidelines and institutional policies set A major unresolved mystery of the adaptive immune system by the Yale animal care and use committee. IMMUNOLOGY relates to how the rare cognate lymphocytes are screened for specificity toward pathogen-derived antigens in a timely manner. Immunization. TLR agonists CpG (50 ␮g) or LPS (5 ␮g) were Although several in vivo studies have elegantly demonstrated the injected into the hind footpad with or without OVA323–339 dynamic interactions of naı¨ve cognate T cells and antigen-loaded peptide in saline (Fig. 1A) or in incomplete Freund’s adjuvant (IFA) DCs in the lymph node (LN) (9–12), to visualize such events, these (Fig. 1B). Four days later, popliteal LNs were collected and experimental approaches often require introduction of a large analyzed. number of lymphocytes bearing a specific T cell receptor. In a naı¨ve mouse, each LN contains Ϸ106 cells, of which only 1 in 106 to 105 Measurement of Cell Proliferation. Mice were continuously fed has the specificity for a given antigen. This screening process BrdUrd at a concentration of 0.8 mg͞ml in their drinking water for represents a daunting task for the antigen-presenting DC to seek the duration of infection. All dLNs (inguinal and iliac) were out the rare cognate lymphocytes within the dLN. We hypothesized harvested, and cell suspensions were stained for BrdUrd and that a mechanism must exist that facilitates the search for cognate lymphocytes within the secondary lymphoid organs. In this regard, the encounter of cognate T cells with the APC can be enhanced in Conflict of interest statement: No conflicts declared. the dLN by several mutually nonexclusive processes. These include This paper was submitted directly (Track II) to the PNAS office. increases in (i) the motility of the APC and͞or T cells within the Abbreviations: ACh, acetylcholine; APC, antigen-presenting cells; CFSE, carboxyfluorescein dLN, (ii) the retention of naı¨ve lymphocytes in the dLN, and (iii) diacetate succinimdyl ester; DC, dendritic cell; dLN, draining lymph node; HEV, high endothelial venule; IFA, incomplete Freund’s adjuvant; ivag, intravaginal(ly); LN, lymph the recruitment of additional naı¨ve lymphocytes to the dLN. The node; ndLN, nondraining LN; NOS, NO synthase; PAMP, pathogen-associated molecular latter two processes would result in cellular accumulation and pattern; p.i., postinfection; SNP, sodium nitroprusside; TLR, Toll-like receptor. enlargement of the dLN. Thus, we propose that an important ‡Present address: University of Northern British Columbia, Northern Medical Program, 3333 immunological purpose of the LN hypertrophy is to facilitate University Way, Prince George, BC, Canada V2N 4Z9. screening for cognate lymphocytes during the initiation of adaptive ¶To whom correspondence should be addressed at: Yale University School of Medicine, immune responses. Moreover, in the face of invasion by a replicat- 300 Cedar Street, TAC S655B, New Haven, CT 06520. E-mail: [email protected]. ing pathogen, the search for lymphocytes that recognize the ap- © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506190102 PNAS ͉ November 8, 2005 ͉ vol. 102 ͉ no. 45 ͉ 16315–16320 Downloaded by guest on September 26, 2021 Fig. 1. TLR agonists alone are suf- ficient to induce LN hypertrophy. (A) The number of cells in popliteal LN was measured in WT or TLR9Ϫ/Ϫ mice (n ϭ 4 per condition) 4 days after footpad injection of TLR li- gands in saline. (B) The number of cells in popliteal LN was measured in WT, TLR9Ϫ/Ϫ, or TLR2Ϫ/Ϫ mice (n ϭ 3 per condition) 4 days after footpad injection of TLR ligands with or without OVA323–339 pep- tide in IFA. (C) The total cell number of dLNs (inguinal and iliac) after ivag HSV-2 (106 PFU) infection was measured at the indicated time points. (D) Photographic depiction of dLNs of HSV-2-infected mice. Similar results were obtained in five separate experiments. *, P Ͻ 0.05, significant difference between the indicated groups. surface lymphocyte markers according to the manufacturer’s in- Results structions (BD Pharmingen). For analysis of DNA content, dLNs Innate Signals Are Sufficient to Induce Lymph Node Hypertrophy. To were harvested at indicated time points after HSV-2 infection, and test our hypothesis that selective accumulation of naı¨ve lympho- single cells were labeled with propidium iodide according to a cytes to the dLN would facilitate screening for cognate lymphocytes standard protocol. The percentage of cells incorporating propidium during the initiation of an immune response, and that such process ͞ iodide at 2N (G1)or4N(G2 M) was determined by flow cytometry must be induced rapidly by the innate immune system, we first according to the manufacturer’s instructions (Becton Dickinson). examined the nature of the stimulus required to promote cellular accumulation in the dLN. Specifically, we asked whether cellular Adoptive Transfer. Single-cell suspensions were prepared from the accumulation depends on antigen, TLR signals, or both. Mice were Ϫ Ϫ LNs of WT or CD62L / mice. The cells were labeled with 0.5 ␮M injected s.c. with LPS or CpG, agonists for TLR4 and TLR9, carboxyfluorescein diacetate succinimdyl ester (CFSE) (Molecular respectively. This procedure resulted in LN hypertrophy and an Probes), and 107 cells were transferred into the lateral tail vein of increase in cellularity (Fig. 1A). Importantly, inoculation of TLR the recipient mice. Mice were infected2hafteradoptive transfer agonist alone induced cellular accumulation