JASN Express. Published on January 9, 2008 as doi: 10.1681/ASN.2007030386

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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

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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.

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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). (I through L) ϩ CD11c and PD-L1 staining, with some positive cells before injection (I and J) and an increased proportion of CD11c cells that are also ϩ ϩ PD-L1 after OVA injection (K and L). (M through P) CD11c and PD-L2 staining, with no major changes in the proportion of CD11c ϩ cells that are also PD-L2 between normal kidneys (M and N) and injected kidneys (O and P). Magnification, ϫ400. stitium. For determination of the extent of infiltration, OVA- rescein was observed (Figure 1). Negative controls did not ex- fluorescein (400 ␮g, 8 ␮l of PBS) was injected into two mice, hibit fluorescence (Figure 1, A and B). In OVA-fluorescein– and 18 h after injection, the degree of diffusion of OVA-fluo- injected mice, fluorescence was observed throughout the

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Figure 3. Intrarenal injection of OVA with subcutaneous CFA induces proliferation of naı¨ve OT-II cells. A discrete population of ϩ ϩ ϩ ϩ CD4 V␣2 cells was identifiable in recipients 72 h after OVA injection (A; CD4 V␣2 cells marked by a black box in top right quadrant). This region allows identification of proliferating antigen-specific cells by CFSE fluorescence. The level of CFSE expression of these cells ϩ was assessed (B; illustrative single-parameter histograms). In mice injected with PBS intrarenally, CFSE cells were almost all expressing high levels of CFSE, indicating no proliferation. In OVA-injected mice, a series of peaks were identified indicating the serial halving of Ϫ CFSE. The dotted vertical lines in B represent the degree of CFSE intensity of nonproliferating cells. CFSE recipient cells (recipient ϩ ϩ CD4 V␣2 cells, fluorescence intensity Յ11) were removed for clarity in Figures 3 through 6. After OVA injection, total numbers of OVA cells in the draining LN were increased (C), and the numbers in each division could be determined (D). Ⅺ, Mice injected with OVA intrarenally, CFA subcutaneously; u, mice injected with PBS intrarenally, CFA subcutaneously. tubulointerstitial compartment (Figure 1, C and D) in multiple PD-L2ϩ DC were not confined to any particular interstitial or sections, including cortex and medulla, but no fluorescence perivascular area. was observed in glomeruli (Figure 1D, arrows); therefore, di- rect intrarenal injection delivers antigen to the tubulointersti- Antigen-Specific CD4؉ T Cells Proliferate in the tial compartment. Draining LN In response to Antigen Deposited Intrarenally -(Renal CD11c؉ Cells Express Co-stimulatory Molecules 5,6-Carboxyfluoresceine-diacetate-succinimidyl-ester (CFSE at Rest and after Immune Activation labeled naı¨ve OT-II cells were transferred into C57BL/6 mice. Within the unmanipulated kidney, 10 to 20% of DC constitu- After transfer, naı¨ve OT-II cells comprised an average of 0.4% of tively expressed at least one of the B7 family molecules, CD80, resting LN cells in unmanipulated transfer recipients (consistent CD86, PD-L1, or PD-L2 (Table 1). Only a small proportion of with previous studies11). OVA was injected intrarenally 1 to 3 d CD11cϩ cells were positive for both CD80 and CD86. After after OT-II cells. Studies examining CD4ϩ cell responses after intrarenal injection, the proportion of CD86ϩ cells and cells subcutaneously antigen have used complete Freund’s adjuvant positive for both CD80ϩ and CD86ϩ increased, but intrarenal (CFA). Because CFA is too viscous to inject intrarenally, CFA was DC CD80 expression was not increased. Intrarenal DC expres- injected subcutaneously and OVA intrarenally. A population of sion of PD-L1 but not PD-L2 was increased. After OVA injec- CD4ϩ V␣2ϩ cells was identified in the renal draining LN (Figure tion, a higher proportion of DC from the draining LN ex- 3A). From this population, CD4ϩ cell proliferation was measured pressed CD86 (and possibly PD-L1). Confocal microscopy by serial halving of fluorescence. Administration of subcutaneous (Figure 2) revealed that DC were present predominantly in the CFA with intrarenal PBS did not result in proliferation (Figure 3, interstitium, including within the interstitial space, around B through D). Proliferation 72 h after OVA injection was measur- small and large vessels, and in periglomerular region. DC were able and reproducible with most cells having undergone prolifer- only rarely seen in glomeruli. CD80ϩ, CD86ϩ, PD-L1ϩ,or ation but only a minority to a point at which CFSE was below

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OT-II cells and the degree of antigen-specific CD4ϩ cell pro- liferation after intrarenal antigen injection were profoundly suppressed by neutralization of both CD80 and CD86 (Figure 5, A through C). After intrarenal OVA injection, in control Ab–treated mice, the number of cells that had divided five or six times was substantial, whereas numbers in combined anti- CD80 and anti-CD86 Ab–treated mice were negligible. Of those cells remaining in the absence of functional CD80 and CD86, the majority were in divisions 2 to 4 (Figure 5D), sug- gesting that CD80/CD86-CD28 interactions reduce the time taken for cells to enter division, as well as the numbers of cells entering each division.

CD86 Plays a Dominant Role in Antigen-Specific CD4؉ Cell Activation to Planted Renal Antigens Inhibition of either CD80 or CD86 revealed a dominant role for CD86 in antigen-specific CD4ϩ cell activation and prolif- eration after intrarenal OVA (Figure 6). OT-II cell numbers were not reduced when CD80 signaling was inhibited. OT-II accumulation was reduced when CD86 signaling was inhib- ited, demonstrating that in response to intrarenal OVA, CD86

ϩ is more important than CD80 for OT-II cell activation. Of the Figure 4. Comparison of early antigen-specific CD4 cell prolifer- cells present, similar proportions of cells were in each division Ⅺ f ation when antigen is injected intrarenally ( ) or subcutaneously ( ). in each experimental group. The same preparation of CFSE-labeled OT-II cells was transferred into C57BL/6 mice, which were then injected with the same dosage Interactions between PD-1 and Its Ligands Limit Early of OVA either intrarenally or subcutaneously, both with CFA subcu- ؉ taneously at a distant site. At 40 h, proliferation was less advanced in CD4 Cell Activation mice receiving antigen intrarenally (illustrative single-parameter his- PD-1 signaling was inhibited during OT-II cell activation tograms [A]; proportion of OT-II cells in each division [B]). after intrarenal OVA. Blocking PD-1 was hypothesized to enhance OT-II cell activation; therefore, 48-h responses detection. For determination of whether antigen-specific CD4ϩ (Figure 7A) as well as 72-h responses (Figure 7B) were ex- cell activation was slower when antigen was deposited intrare- amined after intrarenal OVA injection. At 48 h, increased nally, antigen-specific CFSE-labeled OT-II cell proliferation was OT-II cell accumulation in the renal LN was seen, also ob- assessed after either intrarenal or subcutaneous antigen injection served at 72 h. At both time points, the proliferation profile (using cells from the same preparation, with CFA injected subcu- was similar (peak of three divisions at 48 h and five at 72 h); taneously at a distant site). OT-II cell proliferation after intrarenal therefore, PD-1 does not influence the time for antigen- antigen injection was delayed compared with the skin. Forty specific CD4ϩ cells to become activated and undergo first hours after intrarenal injection, draining LN CD4ϩ cell prolifera- division but may limit the survival of antigen-specific CD4ϩ tion was less advanced (Figure 4), indicating that antigens deliv- cells once dividing. The proportion of IFN-␥–producing ered to the kidney induce slower CD4ϩ cell proliferation com- OT-II cells was assessed by intracellular cytokine staining. pared with subcutaneous delivery. In the absence of concurrent At 48 h, in OVA injected, control Ab–treated mice, 0.9% of CFA, OT-II cells proliferated 3 d after intrarenal injection (see OT-II cells from the renal LN produced IFN-␥, but in anti– supplementary information online), but after 5 d, more activated PD-1–treated mice, 4.0% were positive for IFN-␥. Similar OT-II cells were present in the renal draining LN of mice given experiments 72 h after injection showed that a lower pro- subcutaneous CFA and intrarenal OVA; therefore, in most fur- portion of OT-II cells from anti–PD-1–treated mice ther studies, proliferation was assessed in the draining LN 72 h expressed IFN-␥ (control Ab 4.8%, anti–PD-1 Ab 2.8%; a after concurrent subcutaneous CFA and intrarenal OVA. repeat experiment gave similar results).

CD80/CD86-CD28 Interactions Are Required for Antigen-Specific Cells Accumulated in Injected Antigen-Specific CD4؉ Cell Activation to Planted Kidneys 7 D after Antigen Injection ϩ Renal Antigens Naı¨ve OT-II cells (CD45.2 ) were transferred into congenic For determination of the requirement of CD80 and CD86 in CD45.1 mice, OVA was injected intrarenally, and intrarenal antigen-specific CD4ϩ cell proliferation to an intrarenal anti- OT-II cell numbers were assessed after 7 d. OT-II cells returned gen, both CD80 and CD86 were neutralized in vivo with mAb. to kidneys into which OVA had been injected, and CD86 Compared with control Ab–injected mice, total numbers of or PD-1 inhibition for 7 d had some effect on numbers of

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ϩ Figure 5. CD80 and CD86 interactions with CD28 induce antigen-specific CD4 T cell proliferation in the renal draining node. Illustrative single-parameter histograms (A) show that 72 h after OVA, substantial proliferation of OT-II cells is seen in mice given control Ab, but proliferation is almost abrogated after functional inhibition of CD80 and CD86, demonstrated by total numbers of OT-II cells (B) and the numbers of cells in each division (C). In addition, of the few OT-II cells still present in anti-CD80– and anti-CD86–treated mice, the highest proportion were in divisions 3 and 4 (versus division 6 for control Ab–treated mice), suggesting a delay in the onset of rate of proliferation in the absence of CD80 and CD86 (D). The dotted vertical lines in the single-parameter histograms (A) represent the degree of CFSE intensity of nonproliferating cells. (B and C) Ⅺ, Mice injected with control Ab; f, mice injected with anti-CD80 and CD86 Ab.

OT-II cells returning to kidney as the site of antigen injection DISCUSSION (Figure 8). Adaptive immune responses provide specificity, power, and Effect of Co-stimulatory Molecules on Antigen-Specific memory to the immune system and are directed by small num- ϩ Responses to Subcutaneous Antigen Administration bers of antigen-specific CD4 cells that are activated by DC For determination of whether the roles of co-stimulatory expressing co-stimulatory molecules. TcR transgenic mice molecules in early CD4ϩ cell activation are similar when have allowed in vivo studies of antigen presentation, T cell pro- antigen is delivered intrarenally compared with subcutane- liferation, death, migration, tolerance induction, memory cell ously, studies were performed in mice receiving OVA sub- generation, and effector cell movement.1,15,16 These studies es- cutaneously (CFA subcutaneously at a distant site; Figure tablish an in vivo technique for examining antigen-specific 9). After subcutaneous OVA, antigen-specific CD4ϩ cell CD4ϩ cell activation and responses to antigens in the kidney proliferation was attenuated in the absence of CD80 and and allow application of this technique to define the role of the CD86 (Figure 9A), although the attenuation was less pro- key co-stimulatory molecules in CD4ϩ cell activation. They found compared with intrarenal OVA. CD86 was the dom- demonstrate that antigen-specific CD4ϩ cell proliferation inant CD28 ligand in early CD4ϩ cell proliferation after when antigen is in the kidney lags behind that seen when anti- antigen subcutaneously. Inhibition of CD86 resulted in an gen is in the skin. They demonstrate the importance of the approximately 60% reduction in cell numbers, but anti- CD80/CD86-CD28 system in early CD4ϩ cell activation; de- CD80 injection had no effect (Figure 9B). At 72 h, there fine CD86 as a key molecule in CD4ϩ cell proliferation; and were more OT-II cells in the absence of PD-1 (Figure 9C), indicate that even early in the process of activation, interac- and, in contrast to the renal LN, a higher proportion of tions between PD-1 and its ligands are a brake on CD4ϩ cell OT-II cells made IFN-␥ (control Ab 0.8%, anti–PD-1 proliferation, consistent with their limiting excessive, inappro- Ab 5.5%). priate responses. There were several differences between re-

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ϩ Figure 6. CD86 is the dominant CD28 ligand in early antigen-specific CD4 cell proliferation in the renal draining LN. Illustrative single-parameter histograms (A) show that 72 h after intrarenal OVA injection, substantial proliferation of OT-II cells is seen in mice given control Ab. Proliferation is diminished after inhibition of CD86 but not CD80, quantified in the total numbers of OT-II cells (B) and the number of cells in each division (C). Compared with combined CD80 and CD86 neutralization, individual blockade of either CD80 or CD86 did not alter the proportion of OT-II cells in each division, suggesting that time to onset of proliferation is similar in all three groups (D). Experiments were conduced at the same time as simultaneous CD80 and CD86 blockade; the control Ab data are shown again in this figure for clarity. The dotted vertical lines in the single-parameter histograms (A) represent the degree of CFSE intensity of nonproliferating cells. (B and C) Ⅺ, Mice injected with control Ab; f, mice injected with anti-CD80 Ab; o, mice injected with anti-CD86 Ab. sponses when antigen was deposited in the kidney, compared Interactions between PD-1 and its ligands help maintain with responses after subcutaneous injection. Antigen-specific peripheral tolerance and limit tissue injury in inflammation.6 CD4ϩ cell proliferation was slower in the kidney draining LN; PD-1 limits some of CD28’s effects, including those on Bcl-xL the initial proliferation was more profoundly CD80/CD86- expression.22 In vivo studies confirm a negative regulatory role CD28 dependent; and, at least at 72 h, PD-L1/PD-L2/PD-1 for this system in adaptive immunity.2,6 These studies extend interactions were not such a break on the acquisition of effec- PD-1’s functional involvement to primary immune responses tor function, as assessed by single-cell IFN-␥ production. initiated in the renal parenchyma and draining LN. Effects on CD28 ligation by CD86 (or potentially CD80) on DC proliferation were seen at both 48 and 72 h, although the mag- reduces the number of TcR needing to be bound to peptide/ nitude of the effect was greater when T cells had been activated MHC II to trigger activation,17 increases IL-2 gene transcrip- for an additional 24 h, consistent with the increasing expres- tion/mRNA stability, enhances CD4ϩ cell IL-2R expres- sion of PD-1. Increasing degrees of proliferation enable cells to ϩ sion,18,19 and promotes CD4 cell survival via increased Bcl- produce effector cytokines. Endogenous PD-1 limited the abil- xL.20 There were fewer surviving OT-II cells in renal LN of ity of OT-II cells to make IFN-␥ at 48 h but not at 72 h. mice given anti-CD80 and anti-CD86 Ab, compared with sim- Whereas at 72 h the proportion of OT-II cells producing IFN-␥ ilar experiments harvesting cells from LN draining the skin, was lower in renal LN from anti–PD-1 Ab–treated mice, total consistent with cells undergoing increased apoptosis in the re- OT-II cell numbers were increased in LN from anti–PD-1 Ab– nal LN without CD28 engagement. Furthermore, the remain- treated mice, meaning that the total number of IFN-␥–produc- ing cells were delayed in their degree of proliferation, blocking ing cells at 72 h is similar in both groups. Whereas some studies ϩ CD28’s effect on cell-cycle progression.21 showed that the capacity of CD4 cells to secrete IFN-␥ in-

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Figure 7. The effect of PD-1 blockade on antigen-specific OT-II cell proliferation 48 h (A) and 72 h after intrarenal OVA injection (B). At 48 h, proliferation has commenced in mice injected with control Ab, and there is some effect of PD-1 at this early time point. After ϩ 72 h, the effect is more pronounced. Consistent with the lack of expression of PD-1 on naı¨ve CD4 cells, similar proportions of cells were in each division, suggesting that the time to first division is not affected by PD-1. Because these studies were performed by transferring CD45.2ϩ OT-II cells into congenic CD45.1 mice, the single-parameter histograms are from cells that are ϩ ϩ ϩ CD45.2 CD4 Va2 . The dotted vertical lines in the single-parameter histograms (A and B) represent the degree of CFSE intensity of nonproliferating cells. Ⅺ, Mice injected with control Ab; f, mice injected with anti–PD-1 Ab.

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BALB/c T cells),13 and the other transferred DO11.10 cells into BALB/c mice and injected OVA under the kidney capsule.14 In summary, these studies demonstrate that antigen-specific CD4ϩ cell proliferation is slower after antigen is deposited in the kidney compared with the skin as a reference organ. They determine patterns of co-stimulatory molecule expression on renal DC and define the roles of co-stimulatory molecules in early CD4ϩ cell activation after antigen is delivered to the kid- ney. Compared with responses in the skin, qualitatively, the roles of B7 family members are similar in early CD4ϩ cell ac- tivation, but responses via the kidney are more CD80/CD86 dependent, whereas PD-1 plays less of a role in inhibiting early IFN-␥ production in secondary lymphoid organs.

CONCISE METHODS

Intrarenal and Subcutaneous OVA Injection OT-II mice and congenic CD45.1 mice were bred at Monash Medical Centre SPF Facility (Clayton, Victoria, Australia), and SPF C57BL/6 mice were purchased from Monash University Animal Services. Mice Figure 8. OT-II cells return to the kidney 7 d after antigen were anesthetized with 0.2 mg/g ketamine and 10 ␮g/g xylazine intra- injection. OT-II cells (expressing the CD45.2 allele) were trans- peritoneally. The left kidney was exteriorized through a posterior ap- ferred into congenic CD45.1 mice that were injected intrarenally proach, and 8 ␮l of OVA solution or PBS was injected over 4 to 8 min with OVA. Mice were given control Ab, anti-CD86 Ab, or anti– ϩ ϩ using a customized syringe pump–driven system via a 30-G needle PD-1 Ab. CD4 CD45.2 (OT-II) cells were identified in digested kidneys, with more OT-II cells in the injected kidney and changes inserted into the interstitium, allowing diffusion into the tissue with- in intrarenal cell numbers on anti-CD86 and anti–PD-1–treated out leakage. The kidney was replaced, the incision was sutured, and ϩ mice, consistent with their roles in early CD4 cell proliferation. buprenorphine was administered (Reckitt Benchiser, West Ryde, NSW, Australia; 15 ␮g/mouse intraperitoneally). CFA (Sigma, Castle creases with increasing proliferation,23,24 others suggested that Hill, NSW, Australia) was administered subcutaneously. Studies ad- the IFN-␥ secretion is acquired by some cells within 24 h of hered to the National Health and Medical Research Council of Aus- antigen exposure.25 In the kinetics of the immune response tralia guidelines for animal experimentation. Results are expressed as after antigen is deposited in the kidney, blocking PD-1 has an means Ϯ SEM. The unpaired t test was used for statistical analysis. effect on the early burst of IFN-␥ but little net effect on IFN-␥ at a later time point. In experiments using subcutaneous anti- Antibodies for Flow Cytometric Assessment of Cell gen, both the proportion and the numbers of antigen-specific Surface Markers ϩ CD4 cells producing IFN-␥ were increased at 72 h, showing The following Ab were used in flow cytometry (all BD Biosciences that the PD-1 has less effect on suppressing IFN-␥ production unless otherwise specified): Anti–CD4-FITC, -PE, or –APC-Cy7 after intrarenal injection, at least in the draining LN. (GK1.5; ATCC, Manassas, VA; RM4–4), anti–CD11c-PE (HL3), an- ϩ Antigen-specific CD4 cell proliferation in the draining LN ti-CD80 Alexa Fluor-488 (16-A410), anti–CD86–647 (GL-1), anti– is slower after intrarenal antigen injection compared with sub- PD-L1–488 (MIH5, a gift form Prof. M. Azuma, Tokyo Medical and cutaneous antigen injection. The delay in proliferation might Dental University, Tokyo, Japan26), anti–PD-L2–488 (TY2527), anti– reflect differences in numbers of DC trafficking to draining V␣2-647 (B20.1), anti–V␤5-488 (MR9), anti–CD45.2-APC, and an- LN, because the skin is a primary immune barrier. Qualita- ti–CD44-PE. Clone 2.4G2 (anti-CD16/CD32) blocked Fc receptors. tively, members of the B7 family on dermal and renal DC play Ab grown in house were conjugated to Alexa Fluor kits (Molecular similar roles in early proliferation, although renal LN re- Probes, Eugene, OR). Flow cytometric analyses were performed on a sponses were more CD80/CD86-CD28 dependent. These Mo-Flow flow cytometer (Dako, Botany, NSW, Australia). studies demonstrated proof of principle that antigen-activated OT-II cells return to the site of antigen exposure (i.e., the kid- Analysis of Expression of Co-stimulatory Molecules on ney) after 7 d and that neutralizing CD86 or PD-1 can affect DC and Confocal Microscopy this return. Kidneys or the renal draining LN (the murine renal draining LN lie Two studies that examined primary T cell responses trig- posterior to the medial portion of the upper pole) were digested using gered by renal DC suggested that renal DC are not less efficient DNAse I and collagenase D (Roche Diagnostics, Castle Hill, NSW, ϩ than splenic DC in initiating CD4 cell proliferation. One used Australia), enriched for CD11cϩ cells using CD11c microbeads a one-way mixed lymphocyte reaction (C57BL/6 renal DC- (N418; Miltenyi, North Ryde, NSW, Australia), and CD11cϩ cells

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Figure 9. Studies in subcutaneous antigen presentation 72 h after injection of OVA subcutaneously with CFA injected at a distant site, subcutaneously. Studies were performed to neutralize both CD80 and CD86 (A), either CD80 or CD86 (B), and PD-1 (C). Qualitatively, co-stimulation blockade results were similar to studies in intrarenal antigen presentation, although the effects of combined CD80 and CD86 were less profound and the effects of neutralizing PD-1 may be more substantial. Ⅺ, Mice injected with control Ab; f, mice injected with anti-CD80 and CD86 Ab (A), anti-CD80 Ab (B), and anti–PD-1 Ab (C); and o, mice injected with anti-CD86 Ab (B). were phenotyped using anti-CD80, anti-CD86, anti–PD-L1, and an- brachial, pancreatic, mesenteric, inguinal, lumbar, and caudal) were ti–PD-L2 Ab. In analyses, leukocyte populations were gated from collected from naı¨ve OT-II mice into HF2.5 (HBSS, 2.5% FBS) and an other contaminating cell types and cellular debris from forward angle aliquot stained for CD4, V␣2, and V␤5. OT-II cells comprise 91.5% light scatter (cell size) and right angle side scatter (cell granularity). Pro- (n ϭ 3) of CD4ϩ cells in LN. For CFSE labeling,31 cells were centri- pidium iodide–positive cells were excluded from analyses. For confocal fuged and resuspended in PBS 0.1% BSA (107 cells/ml), CFSE (Sigma; microscopy, cryostat cut sections (4 ␮m) of snap-frozen renal tissue were 1 ␮l/ml) was added, the suspension was vortexed and incubated, the blocked (anti-CD16/CD32, 10% rat serum, 10% guinea pig serum in 5% tube was filled with HF2.5 and centrifuged, then cells were resus- BSA/PBS), then incubated with Ab anti–CD11c-FITC (HL3) and anti- pended in PBS for transfer. V␣2ϩCD4ϩ cells (3 to 5 ϫ 106) were CD80 (16-A410), anti-CD86 (GL-1), anti–PD-L1 (MIH5), or anti– injected intravenously (300 ␮l of PBS) into C57BL/6 mice or, in some PD-L2 (TY25), all conjugated to Alexa Fluor-568. Confocal images were experiments, congenic CD45.1 mice. OVA was injected 1 to 3 d after collected using a Leica SP5 confocal microscope with Argon and HeNe transfer. CD4ϩ V␣2ϩCFSEϩ and propidium iodide–negative OT-II lasers (Leica Microsystems, Wetzlar, Germany). cells were identified in recipient LN. The number of OVA-specific CD4ϩ (OT-II) cells was determined by multiplying the percentage ؉ Using OT-II Cells to Determine CD4 Cell Proliferation of CD4ϩV␣2ϩCFSEϩ cells by the total number of LN cells. Recipient An adoptive transfer model was established on the basis of the system CD4ϩV␣2ϩCFSEϪ cells were observed in experiments. At 72 h, these of Jenkins and colleagues,11,28,29 with modifications. OT-II mice30 are not OT-II cells that have undergone more than six divisions and b ϩ Ϫ ϩ have I-A -restricted naı¨ve CD4 T cells recognizing OVA323–339,ex- become CFSE (studies transferring CD45.2 OT-II cells into con- pressing the ␣␤TcR, V␣2, and V␤5. Lymph nodes (cervical, axillary, genic CD45.1ϩ recipients determined that CD4ϩVa2ϩCFSEϪ cells

10 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: –, 2008 www.jasn.org BASIC RESEARCH express CD45.1 (i.e., they were recipient cells [data not shown]). For Rev Immunol 23: 515–548, 2005 clarity, these irrelevant CFSEϪ cells are omitted from single parameter 3. Harris NL, Ronchese F: The role of B7 costimulation in T-cell immunity. histograms in Figures 3 through 6. Experiments used 3 to 5 recipient Immunol Cell Biol 77: 304–311, 1999 4. Salomon B, Bluestone JA: Complexities of CD28/B7: CTLA-4 costimu- mice per group. latory pathways in autoimmunity and transplantation. Annu Rev Im- For determination of the functional role of CD80, CD86, and munol 19: 225–252, 2001 PD-1, neutralizing Ab to each of the molecules were administered. Ab 5. Green JM, Noel PJ, Sperling AI, Walunas TL, Gray GS, Bluestone JA, 16-A410 (CD80) and GL-1 (CD86) were administered (100 ␮gin200 Thompson CB: Absence of B7-dependent responses in CD28-defi- ␮l of PBS intraperitoneally) every second day from the day before cient mice. Immunity 1: 501–508, 1994 6. Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ: The function of pro- 32 OVA injection. For PD-1, Ab clone J43 was given intraperitoneally, grammed cell death 1 and its ligands in regulating autoimmunity and 500 ␮gin200␮l of PBS the day before OVA injection followed by 250 infection. Nat Immunol 8: 239–245, 2007 ␮g 2 d later.33 For 7-d studies, administration of either anti-CD86 or 7. Nishimura H, Honjo T: PD-1: An inhibitory immunoreceptor involved in anti–PD-1 Ab continued (250 ␮g every second day). Nonimmune rat peripheral tolerance. Trends Immunol 22: 265–268, 2001 or guinea pig Ab were administered as control Ab. Parallel studies 8. Nishimura H, Nose M, Hiai H, Minato N, Honjo T: Development of lupus-like autoimmune diseases by disruption of the PD-1 gene en- were conducted with the same dosage of subcutaneous OVA, with coding an ITIM motif-carrying immunoreceptor. Immunity 11: 141– CFA at a distant subcutaneous site and the draining LN studied. 151, 1999 9. Khoury SJ, Sayegh MH: The roles of the new negative T cell costimu- ,Antigen-Specific CD4؉ Cell IFN-␥ Production latory pathways in regulating autoimmunity. Immunity 20: 529–538 2004 OT-II cells were transferred into congenic CD45.1 mice. Single-cell 10. Schoop R, Wahl P, Le Hir M, Heemann U, Wang M, Wuthrich RP: suspensions from renal draining LN were prepared. Because of the Suppressed T-cell activation by IFN-gamma-induced expression of small numbers of cells recovered and the ex vivo stimulation, cells PD-L1 on renal tubular epithelial cells. Nephrol Dial Transplant 19: were pooled from mice in the same experimental group, then resus- 2713–2720, 2004 pended (1 ϫ 106 in 100 ␮l). OVA (1 ␮M; Auspep, Parkville, 11. Kearney ER, Pape KA, Loh DY, Jenkins MK: Visualization of peptide- 323–339 specific T cell immunity and peripheral tolerance induction in vivo. VIC, Australia) was added or omitted (negative control). Cells were Immunity 1: 327–339, 1994 incubated (37°C, 2h); brefeldin A (5 ␮g/ml, Sigma) was added (4 h); 12. Soos TJ, Sims TN, Barisoni L, Lin K, Littman DR, Dustin ML, Nelson PJ: then cells were harvested and incubated with Fc Block (20 min, on CX3CR1ϩ interstitial dendritic cells form a contiguous network ice), washed, and stained for CD4 and CD45.2 (30 min, on ice). After throughout the entire kidney. Kidney Int 70: 591–596, 2006 washing, cells were fixed, permeabilized (BD Cytofix/Cytoperm Kit; 13. Kruger T, Benke D, Eitner F, Lang A, Wirtz M, Hamilton-Williams EE, ␥ Engel D, Giese B, Muller-Newen G, Floege J, Kurts C: Identification BD Biosciences), and stained with anti–IFN- -PE (XMG1.2, BD Bio- and functional characterization of dendritic cells in the healthy murine sciences). kidney and in experimental glomerulonephritis. J Am Soc Nephrol 15: 613–621, 2004 14. Dong X, Swaminathan S, Bachman LA, Croatt AJ, Nath KA, Griffin MD: Antigen presentation by dendritic cells in renal lymph nodes is linked ACKNOWLEDGMENTS to systemic and local injury to the kidney. Kidney Int 68: 1096–1108, 2005 These studies were supported by a Program Grant from the National 15. Reinhardt RL, Bullard DC, Weaver CT, Jenkins MK: Preferential accu- Health and Medical Research Council of Australia. mulation of antigen-specific effector CD4 T cells at an antigen injec- tion site involves CD62E-dependent migration but not local prolifer- Parts of these studies were published in abstract form (JAmSoc ation. J Exp Med 197: 751–762, 2003 Nephrol 17: 798A, 2006; Nephrology 11[Suppl 2]: A5, 2006). 16. Catron DM, Rusch LK, Hataye J, Itano AA, Jenkins MK: CD4ϩ T cells We thank Prof. Bill Heath (Walter and Eliza Hall Institute, Mel- that enter the draining lymph nodes after antigen injection participate bourne, Australia) for the OT-II mice, Prof. Miyuki Azuma (Tokyo in the primary response and become central-memory cells. J Exp Med Medical and Dental University, Japan) for the MIH5 mAb, Alice 203: 1045–1054, 2006 17. Wells AD, Gudmundsdottir H, Turka LA: Following the fate of individ- Wright for technical assistance and Assoc. Prof. Michelle Leech, As- ual T cells throughout activation and clonal expansion. Signals from T soc. Prof. David Finkelstein, and Dr. Brian Howden for advice in the cell receptor and CD28 differentially regulate the induction and du- technique of intrarenal injection. ration of a proliferative response. J Clin Invest 100: 3173–3183, 1997 18. Lindstein T, June CH, Ledbetter JA, Stella G, Thompson CB: Regula- tion of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway. Science 244: 339–343, 1989 DISCLOSURES 19. Fraser JD, Irving BA, Crabtree GR, Weiss A: Regulation of interleukin-2 None. gene enhancer activity by the T cell accessory molecule CD28. Sci- ence 251: 313–316, 1991 20. Boise LH, Minn AJ, Noel PJ, June CH, Accavitti MA, Lindsten T, Thompson CB: CD28 costimulation can promote T cell survival by REFERENCES enhancing the expression of Bcl-XL. Immunity 3: 87–98, 1995 21. Bonnevier JL, Mueller DL: Cutting edge: B7/CD28 interactions regu- 1. Jenkins MK, Khoruts A, Ingulli E, Mueller DL, McSorley SJ, Reinhardt late cell cycle progression independent of the strength of TCR signal- RL, Itano A, Pape KA: In vivo activation of antigen-specific CD4 T cells. ing. J Immunol 169: 6659–6663, 2002 Annu Rev Immunol 19: 23–45, 2001 22. 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