JASN Express. Published on January 9, 2008 as doi: 10.1681/ASN.2007030386 BASIC RESEARCH www.jasn.org Intrarenal Antigens Activate CD4؉ Cells via Co-stimulatory Signals from Dendritic Cells Kristy L. Edgtton,* Joshua Y. Kausman,* Ming Li,* Kim O’Sullivan,* Cecilia Lo,* Paul Hutchinson,* Hideo Yagita,† Stephen R. Holdsworth,* and A. Richard Kitching* *Centre for Inflammatory Diseases, Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia; and †Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan ABSTRACT Dendritic cells in the kidney take up antigens, but little is known about their role in providing co- ϩ stimulatory signals for the activation of CD4 cells. This study examined the phenotype of dendritic cells in the renal interstitium and in the lymph node draining the kidney before and after intrarenal ovalbumin injection. After intrarenal injection of the antigen, expression of the co-stimulatory molecules CD86 and programmed cell death ligand 1 (PD-L1) increased on renal dendritic cells, whereas expression of only CD86 increased on dendritic cells of the draining lymph node. The activation and proliferation of ϩ antigen-specific CD4 cells in the lymph node were assessed by transfer of naı¨ve,fluorescently labeled ovalbumin-specific T cell receptor transgenic cells to mice before antigen administration. Blocking both ϩ CD86 and CD80 profoundly inhibited CD4 cell proliferation, but CD86 was the dominant CD28 ligand ϩ in the early proliferative response of CD4 cells. Conversely, activation of PD-1, the receptor expressed ϩ ϩ on CD4 cells that binds PD-L1 and PD-L2, reduced the proliferation of CD4 cells in the draining lymph ϩ node. Comparing subcutaneous and intrarenal administration of antigen, it was found that CD4 cell activation was slower and the effects of combined CD80 and CD86 blockade were more profound when antigen was presented via the kidney compared with the skin. In summary, renal dendritic cells take up ϩ antigen and participate in the control of antigen-specific CD4 cell proliferation by upregulating ϩ co-stimulatory molecules such as CD86 that stimulate CD4 cell proliferation and by signaling through PD-1, which prevents an inappropriately exuberant immune response. J Am Soc Nephrol ●●: –, 2008. doi: 10.1681/ASN.2007030386 Because CD4ϩ cells direct adaptive immune re- response. Antigen presentation in the absence of sponses in infection, autoimmunity, and allogeneic co-stimulatory signals results in anergy and is responses they are relevant to renal infection, important in peripheral tolerance. DC co-stimu- pathologic renal inflammation, and renal trans- latory molecule expression is an integral part of plantation. CD4ϩ cells are activated in local drain- DC maturation and largely defines their capacity ing lymph nodes (LN) after encounters with acti- to activate naı¨ve T cells. The B7 family of co- vated and licensed dendritic cells (DC) presenting relevant antigenic peptides. Licensed DC that have altered their phenotype in inflammation migrate Received March 30, 2007. Accepted September 21, 2007. from tissues to present antigen. T cells proliferate, Published online ahead of print. Publication date available at acquire effector functions, and leave the LN several www.jasn.org. days after antigen is presented.1 Co-stimulatory sig- Correspondence: Dr. Richard Kitching, Centre for Inflammatory nals are important for effective immune responses, Diseases, Monash University Department of Medicine, Monash ϩ Medical Centre, 246 Clayton Road, Clayton, VIC 3168, Australia providing the “second signal” for CD4 cell activa- Phone: 61-3-9594-5520; Fax: 61-3-9594-6495; E-mail: richard. tion. Co-stimulatory molecules provide positive or [email protected] negative signals to enhance or inhibit the immune Copyright © 2008 by the American Society of Nephrology J Am Soc Nephrol ●●: –, 2008 ISSN : 1046-6673/●●00- 1 BASIC RESEARCH www.jasn.org lation of activated T cells. Generally speaking, PD-L2 is ex- pressed largely by DC, whereas PD-L1 may also be expressed by tissue cells.2,6,10 Despite their central role in the immune response, CD4ϩ cells specific for an individual antigen exist at low frequency, even in active immune responses. This low frequency of anti- gen-specific CD4ϩ cells makes the proliferation and behavior of antigen-specific CD4ϩ cells difficult to study in vivo. The development of T cell receptor (TcR) transgenic mice in which the majority of T cells express a single TcR allows study of how immune signals induce antigen-specific responses by in vivo assessment of relatively large numbers of antigen-specific cells.1,11 Because the skin is a primary site of entry of infectious threats and subcutaneous injection is simple, studies are per- formed by injecting antigen subcutaneously. The kidney, a solid internal organ, performs different functions compared with the skin. Whereas the tubulointerstitium may be exposed Figure 1. Intrarenal antigen injection results in diffusion of anti- to foreign antigens via ascending infection, proteins processed gen throughout the interstitium. Whereas fluorescence was not present in control mice (A and B), 18 h after direct injection of by renal DC are more likely to be self-antigens or innocuous OVA-fluorescein into the kidney, fluorescence was observed foreign antigens. Renal DC form a relatively extensive network throughout the tubulointerstitium (C and D), excluding large ves- in the tubulointerstitium12 but may not be as potent at induc- sels and glomeruli (arrows, D). Magnifications: ϫ100 in A and C; ing T cell proliferation as splenic DC.13,14 ϫ200 in B and D. These studies sought to define the response of renal DC exposed to antigen in the kidney parenchyma. The timing of stimulatory molecules includes the prototypic positive sig- proliferation was compared with subcutaneous antigen injec- nals B7-1 (CD80) and B7-2 (CD86).2 Their ligands are tion, the expression of co-stimulatory molecules and capacity ϩ CD28, which is constitutively expressed on CD4ϩ T cells to present antigen and activate antigen-specific CD4 T cells in and induces activation,3 and CTLA4 (CD152), which is ex- the renal draining LN was assessed, and DC co-stimulatory pressed after T cell activation and limits T cell activation.4 molecules were inhibited for better definition of the mecha- The absence of signaling through CD28 impairs naı¨ve CD4ϩ nisms of immune recognition and antigen presentation in the T cell activation.5 kidney. A new model of antigen presentation in the renal node The immune receptor programmed cell death 1 (PD-1) and was established using direct intrarenal injection and in vivo ϩ its ligands, PD-L1 and PD-L2, B7 family members, provide assessment of transgenic ovalbumin (OVA)-specific CD4 negative co-stimulatory signals. They limit immune responses cells. Experiments on dermal presentation of the same antigen to avoid an overexuberant response and may prevent the de- allowed comparison of antigen presentation in the kidney with velopment of pathogenetic self-reactivity.2,6 PD-1–deficient skin DC. mice have enhanced immune responses with autoimmunity,7,8 and evidence is accumulating that PD-1 and its ligands regulate immune responses in peripheral tissues.6 Information on the RESULTS role of PD-1 in the early activation of CD4ϩ T cells is limited ϩ because PD-1 expression is delayed after CD4 cell activation.9 Direct Intrarenal Antigen Injection Expression persists on the cell surface, suggesting involvement A method of injecting antigen into the kidney was established in a later phase of CD4ϩ T cell activation, including downregu- by injecting OVA (400 ␮g, 8 ␮l of PBS) into the cortical inter- ϩ Table 1. Changes in co-stimulatory molecule expression on CD11c DC within the kidney and within the renal draining ϩ LN before and after intrarenal antigen injection, expressed as a percentage of CD11c cells Kidney Renal LN Parameter 0 h 24 h 0 h Renal LN 24 h CD80 19.0 Ϯ 2.0 28.0 Ϯ 7.0 17.0 Ϯ 2.0 22.0 Ϯ 1.0 CD86 10.0 Ϯ 0.2 29.0 Ϯ 5.0a 31.0 Ϯ 4.0 65.0 Ϯ 4.0a Both CD80 and CD86 3.0 Ϯ 0.3 16.0 Ϯ 5.0a 13.0 Ϯ 2.0 16.0 Ϯ 1.0 PD-L1 10.0 Ϯ 1.0 29.0 Ϯ 2.0a 35.0b 74.0 Ϯ 3.0 PD-L2 20.0 Ϯ 1.0 28.0 Ϯ 3.0 39.0b 52.0 Ϯ 3.0 aP Ͻ 0.05. bPooled renal LN samples. 2 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: –, 2008 www.jasn.org BASIC RESEARCH ϩ Figure 2. Confocal microscopic images of CD11c DC in the kidney (green fluorescence) and expression of co-stimulatory molecules (CD80, CD86, PD-L1, or PD-L2; red fluorescence) in normal mouse kidneys (A, B, E, F, I, J, M, and N) and in mice 24 h after intrarenal OVA injection with subcutaneous FCA (C, D, G, H, K, L, O, and P). Each pair of images represents a single section stained with both ϩ anti-CD11c (green) and an Ab against the relevant co-stimulatory molecule (red). Arrows represent CD11c DC that are not positive for the co-stimulatory molecule of interest; arrowheads denote DC that are positive for both CD11c and the relevant co-stimulatory molecule. In general, DC were found most commonly in the interstitium, at times perivascular or periglomerular, and only uncommonly in glomeruli. There was no distinct anatomic distribution for DC positive for any particular co-stimulatory molecule. (A through D) ϩ ϩ Ϫ CD80 DC, with no particular change between normal (A and B) and injected (C and D) kidneys. A CD11c CD80 cell is visible in C, ϩ centrally within a glomerulus. (E through H) CD86 DC, with some positive cells before injection (E and F) and an increased proportion ϩ ϩ of CD11c cells that are also CD86 after OVA injection (G and H, including a double-positive cell within a glomerulus).