CD70 Is Downregulated by Interaction with CD27 Mirela Kuka, Ivana Munitic, Maria Letizia Giardino Torchia and Jonathan D. Ashwell This information is current as of September 25, 2021. J Immunol 2013; 191:2282-2289; Prepublished online 2 August 2013; doi: 10.4049/jimmunol.1300868 http://www.jimmunol.org/content/191/5/2282 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD70 Is Downregulated by Interaction with CD27

Mirela Kuka,1,2 Ivana Munitic,1,3 Maria Letizia Giardino Torchia, and Jonathan D. Ashwell

Engagement of the receptor CD27 by CD70 affects the magnitude and quality of responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic cells (DCs), T cells, and B cells only upon activation. In this article, we show that a second level of regulation also is present. First, although undetectable on the cell surface by flow cytom- etry, immature DCs have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DCs and T cells were higher in mice deficient in CD27, or on DCs for which the interaction between CD70 and CD27 was precluded by blocking Abs. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor. The Journal of Immunology, 2013, 191: 2282–2289. Downloaded from nteraction between the costimulatory receptor CD27 and its restricted, and is barely detectable on the cell surface at steady state, ligand CD70 is required for optimal T cell activation (1–3). and even then only rare cells in the thymic medulla and the lamina I Studies using CD27- and CD70-deficient mice or anti-CD70 propria are CD70+ (17–20). Transient transcriptional upregulation of blocking Abs have found defects in primary and/or secondary T cell CD70 occurs in dendritic cells (DCs) activated via TLR- or CD40- responses in a variety of infectious models (4–8). Furthermore, mediated stimulation and in Ag-activated T and B cells (6, 20). In

manipulations that increase CD27–CD70 interactions have been DCs, where its expression seems to be most relevant, CD70 is http://www.jimmunol.org/ successfully used in experimental vaccination protocols (9, 10). It is transported by the invariant chain to late endocytic structures, where notable that a fine line exists between beneficial and deleterious it colocalizes with MHC-II molecules (21, 22). Upon interaction of CD70-mediated effects. For example, whereas efficient clearance activated DCs with cognate CD4 T cells, CD70 is codelivered to the of acute lymphocytic choriomeningitis virus (LCMV) strains requires immune synapse with MHC-II, ensuring optimal T cell stimulation. CD27 occupancy by CD70, this interaction precludes clearance of Uncontrolled CD27–CD70 interactions have detrimental effects. the chronic LCMV strain (7, 8, 11). Therefore, the existence of In mouse models where CD70 was constitutively expressed on regulatory mechanisms for the CD70–CD27 pathway ensures ef- B cells, DCs, or T cells, a continuous generation of effector T cells fective and prevents deleterious immune responses. was observed, which in B cell and DC CD70 transgenics resulted Normally, tight control of CD27 and CD70 expression avoids in an autoimmune disease and death (23–25). In contrast, consti- by guest on September 25, 2021 excessive T cell activation. CD27, a member of the TNFR family, is tutive CD70 expression on DCs was sufficient to break peripheral constitutively expressed by T cells as a membrane-bound homodimer, tolerance and, among other things, generate tumor-specific respon- and its surface levels change during T cell activation (3). The baseline ses to peptide immunization without the need for adjuvants (24). In level in resting naive and memory T cells is upregulated during the addition to these observations made in transgenic mice, the im- first days after TCR engagement because of increased transcription portance of excessive CD27–CD70 interactions has been demon- (12–14). Notably, surface levels of CD27 are downregulated during strated in a chronic LCMV infection model (11). Continuous CD27 T cell effector differentiation by shedding and/or decreased tran- engagement, likely mediated by a subset of CD70-expressing scription, and some terminally differentiated effector memory T cells B cells, led to T cell cytokine-mediated splenic germinal center and (TEM) retain a CD27-negative phenotype (13–15). CD27 can also be marginal zone destruction, thus precluding the generation of a neu- reversibly downregulated on memory CD8 T cells that enter non- tralizing Ab response. lymphoid organs (16). In contrast, expression of CD70, a homotri- It is generally believed that the downregulation of T cell CD27 meric transmembrane member of the TNF family, is much more levels during persistent stimulation is an activation-intrinsic event. However, some evidence suggests that it is also the interaction with Laboratory of Immune Cell , National Cancer Institute, National Institutes of CD70 that results in decreased CD27levelsintheabsenceofacti- Health, Bethesda, MD 20892 vation. For example, T cell coculture with B cell lines expressing CD70 1M.K. and I.M. contributed equally to this work. triggered CD27 downregulation, and even naive T cells in CD70 Tg 2Current address: Division of Immunology, Transplantation and Infectious Diseases, mice had substantially lower CD27 levels (26, 27). In the course of San Raffaele Scientific Institute, Milan, Italy. studying mice deficient in either CD27 or CD70, we made the un- 3Current address: Department of Biotechnology, University of Rijeka, Rijeka, Croatia. expected observation that in the absence of one the other was up- Received for publication March 29, 2013. Accepted for publication June 26, 2013. regulated. In this article, we show by Ab blocking and genetic This work was supported by the Intramural Research Program of the Center for manipulation that the relationship between CD27 and CD70 expres- Cancer Research, National Cancer Institute, National Institutes of Health. sion is reciprocal and mediated by direct protein–protein interactions. Address correspondence and reprint requests to Dr. Jonathan D. Ashwell, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Building 37, Room 3002C, Bethesda, MD 20892. E-mail address: [email protected] Materials and Methods Mice The online version of this article contains supplemental material. Abbreviations used in this article: CHX, cycloheximide; LCMV, lymphocytic cho- C57BL/6 (B6) mice were obtained from Frederick Cancer Research Facility 2/2 riomeningitis virus; DMFI, change in mean fluorescence intensity; poly(I:C), poly- (Frederick, MD). CD70 mice backcrossed to B6 for 13 generations 2 2 inosinic-polycytidylic acid; WT, wild-type. were described (8). CD27 / mice (4) were a gift from Jannie Borst www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300868 The Journal of Immunology 2283

(The Cancer Institute, Amsterdam, The Netherlands) and were CD272/2, and CD702/2 mice were infected i.p. with 2 3 105 PFU maintained in our facility. The National Cancer institute Animal Care and LCMV. Use Committee approved all animal studies. Cell stimulation and flow cytometry Reagents Splenocytes were cultured at a concentration of 5 3 106 cells per milliliter Abs to CD27, TCR-b, B220, CD16/CD32 clone 2.4G2 (Fc block), iso- and stimulated with poly(I:C) (30 mg/ml) for 22 h or primed with IFN-g type controls, and fixation and permeabilization buffers were obtained (50 ng/ml) for 2 h and then stimulated with LPS (200 ng/ml) for an ad- from BD Biosciences. The unconjugated Ab to CD70 (clone FR70), the ditional 20 h. In some experiments, cells were treated for 15 min with fluorochrome-conjugated Abs to CD70 (clone FR70), CD11c and MHC anti-CD27 blocking Ab (5 mg/ml) or with isotype control prior to stim- class II, the blocking Ab to CD27 (clone LG.3A10), the isotype control, ulation. In internalization experiments, anti-CD70 PE-conjugated Ab was and recombinant IFN-g were purchased from eBioscience. LCMVArmstrong added to the cultures after stimulation at the indicated time points, at a 53b (LCMV) was a gift from Rafi Ahmed (Emory University School of concentration of 2 mg/ml. Cells were treated or not with CHX (20 mg/ml) b Medicine, Atlanta, GA). GP33-41 H-2D (GP33) tetramers were obtained 1 h before adding anti-CD70 PE-conjugated Ab. For staining of surface from the National Institutes of Health Tetramer Core Facility at Emory molecules, CD16/CD32 clone 2.4G2 was added to block Fc-receptor University. Power SYBR Green was obtained from Applied Biosystems. binding prior to the addition of fluorochrome-conjugated Abs. In some Cycloheximide (CHX), LPS, and polyinosinic-polycytidylic acid [poly(I:C)] experiments, cells were incubated with unconjugated anti-CD70 Ab prior were purchased from Sigma-Aldrich. LIVE/DEAD Fixable Dead Cell to surface staining. Detection of intracellular molecules was performed Stain was obtained from Life Technologies. B16-FLT3L murine melanoma after fixation and nuclear permeabilization. Dead cells were excluded cells were a gift from Ulrich H. von Andrian, Harvard Medical School using the LIVE/DEAD Fixable Dead Cell Stain, doublet exclusion gating (Boston, MA). was performed, and splenic DCs were gated as CD11c+MHC-II+ cells. Viral growth and LCMV infection Flow cytometry was done with a BD LSRFortessa cytometer, using BD FACSDiva software (BD Biosciences). The change in mean fluorescence LCMV was grown in our laboratory in baby hamster kidney cells (BHK- intensity (DMFI) was calculated by subtracting the nonspecific back- Downloaded from 21), and viral titers were determined as described (28). Wild-type (WT), ground on cells from CD702/2 mice (for CD70) or CD272/2 mice (for http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. CD70 and CD27 levels inversely correlate during LCMV infection. (A–E) WT, CD702/2, and CD272/2 mice were infected i.p. with 2 3 105 PFU of LCMV Armstrong, and analyzed at the indicated days. CD70 levels detected ex vivo by surface staining on splenic DCs (CD11c+MHC-II+) are shown as one representative experiment (A) or as mean 6 SEM of three to seven mice (B). (C) CD70 levels on splenic T naive CD8+T cells (top)orin GP33-specific T cells (bottom) are shown from one representative experiment. (D and E) CD27 levels detected ex vivo on splenic T cells are shown as mean 6 SEM of three to nine mice (D) or as one representative staining (E). DMFI of CD70 and of CD27 was calculated by subtraction of the background signal in CD702/2 and CD272/2 cells, respectively. *p , 0.05. 2284 ENGAGEMENT BY CD27 DOWNREGULATES EXPRESSION OF CD70

CD27). All flow cytometry data analysis was performed with FlowJo were then normalized to CD70-deficient cells, which were assigned an software (TreeStar). arbitrary unit of 1. Expansion of DCs in vivo Statistical analysis B16-FLT3L cells were cultured in DMEM containing 10% FCS and Statistical analysis was done with GraphPad Prism software, using a Student antibiotics. Mice were injected s.c. with 106 B16-FLT3L cells, and spleens two-tailed unpaired t test. were analyzed 10–13 d later. Cell sorting and purification Results Cell surface levels of CD70 and CD27 are inversely correlated Transcriptional analysis of CD70 was performed either on 107 total splenocytes from mice injected with B16-FLT3L cells or on DC:T (ratio Infection with a variety of pathogens induces CD70 expression 1:5) cocultures. DCs and T cells were isolated by flow cytometry–based within a few days, but the levels detected by flow cytometry are sorting (FACSAria II; BD Biosciences) with a purity .95%. A total of 6 3 relatively low (6). The recent availability of CD70-deficient mice 104 DCs were cultured with 3 3 105 T cells in the absence or presence of stimulus. In some experiments, DCs were isolated with CD11c provided an ideal negative staining control, and revealed that MicroBeads (Miltenyi Biotec) and T cells were purified with the EasySep CD70 expression during acute LCMV infection is barely detect- kit (STEMCELL Technologies) following the manufacturer’s protocol. In able (M. Kuka, I. Munitic, and J.D. Ashwell, unpublished obser- this case, 3.5 3 105 DCs were cultured with 1.75 3 106 T cells in the vations), despite the fact that CD70-deficient mice exhibited a absence or presence of stimulus. defective CD8 T cell response and delayed viral clearance (8). RNA isolation and quantitative RT-PCR Because the relevance of the CD70–CD27 pathway seems to be

highly dependent on the timing and levels of expression of these Downloaded from RNA isolation from total splenocytes or from sorted cells was performed with the RNeasy Mini Kit or RNeasy Micro Kit (QIAGEN), respectively. molecules, we sought to determine the kinetics of expression of The cDNA was prepared with the SuperScript II Reverse Transcriptase Kit CD70 and CD27 on immune cells during acute LCMV infection. (Life Technologies). Real-time PCR for CD70 was performed with SYBR CD70 was undetectable on resting splenic DCs, and only very low Green, using the 7500 Real-Time PCR System by Applied Biosystems levels were detected on day 2 of infection (Fig. 1A, 1B). Of note, (Carlsbad, CA), with the following primers: CD70-Forward, 59-TGCTG- TTGGTTTCATTGTAGCG-39; CD70-Reverse, 59-ATCCTGGAGTTGT- DCs of mice lacking CD27 had higher levels of CD70 on their surface (Fig. 1A, 1B). At day 6, levels of CD70 were downreg- GGTCAAGGG-39. Housekeeping ribosomal 18S RNA was amplified to http://www.jimmunol.org/ normalize RNA content of the lysate and obtain the dCT value. Values ulated in both WT and CD27-deficient mice. Because activated by guest on September 25, 2021

FIGURE 2. CD70 levels are downregulated upon interaction with CD27. (A and B) WT, CD702/2, and CD272/2 splenocytes were stimulated for 20–22 h with poly(I:C). Surface CD70 levels on DCs are shown in one representative experiment (A) or as mean 6 SEM of 15 mice (B). (C) CD70 levels on splenic DCs pretreated with an anti-CD27 blocking Ab (thick solid line) or with an isotype control (dashed line) and stimulated with TLR agonists for 22 h are depicted from one representative experiment. **p , 0.01. The Journal of Immunology 2285

T cells express CD70, we evaluated its levels on Ag-specific Taken together, these data suggest that CD70 and CD27 cell sur- T cells at the peak of the immune response and found that face expression is reciprocally regulated, and raised the possibility CD70 was detectable only in CD27-deficient mice (Fig. 1C). To that they in fact regulated each other. correlate CD27 levels with those of CD70, the kinetics of CD27 expression on T cells during LCMV infection was assessed. As Downregulation of CD70 is mediated by a direct interaction previously described (13), CD27 levels rose slightly in the first with CD27 few days and fell to less than resting levels during the effector To assess whether the CD70 upregulation during LCMV infection differentiation phase (Fig. 1C). Notably, the levels of CD27 on was diminished by direct interaction with CD27, we studied CD70 days 1 and 2 were clearly higher on T cells from CD70-deficient levels on DCs from splenocyte cultures stimulated with TLR mice (Fig. 1C, 1D), whereas during the downregulation phase the agonists. In this system, most of the CD27 is provided by T cells, difference between the two mice strains progressively narrowed. although NK and memory B cells could also participate. In line Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. CD70 is internalized and recycles in DC. (A) Surface and intracellular CD70 levels on untreated or poly(I:C)-stimulated WT or CD702/2 splenocytes are shown in one representative experiment (left) and as the mean 6 SD of two independent experiments (right). Staining was performed with (dashed line) or without (thick solid line) blocking of surface CD70 with an unconjugated anti-CD70 Ab. (B) WT or CD702/2 splenocytes were stimulated and cultured overnight in the presence of anti-CD70 PE-conjugated Ab (dashed line) or of the isotype control (thin solid line). Levels of the accumulated anti-CD70 Ab are shown in DCs, T cells, or B cells in one representative experiment. (C) WT or CD702/2 splenocytes were stimulated for 22 h and cultured in the absence (top row) or presence (bottom row) of anti-CD70 PE-conjugated Ab during the last 8 h. Cells were incubated on ice (dashed line) or at 37˚C (thick solid line). (D) CD70 levels on DC surface or after 4 h of culture with anti-CD70–PE are depicted as mean 6 SEM of two to four independent experiments. (E) WT or CD702/2 splenocytes were stimulated for 22 h (bottom graph) or left untreated (top graph) and cultured for the last 2, 4, or 8 h in the presence of anti-CD70 PE-conjugated Ab (s). At 1 h before adding the Ab, some cells were pretreated with CHX (d). As a control for CHX treatment, splenocytes were pretreated with CHX prior to stimulation. Upregulation of CD80 and CD70 was completely abrogated following such pretreatment, demonstrating that CHX worked (data not shown). DMFI of CD70 was calculated by subtracting the background signal on CD702/2 DCs, as well as the levels of CD70 on parallel samples stained only for surface expression at each time point. Mean 6 SD of two independent experiments is shown. *p , 0.05. 2286 ENGAGEMENT BY CD27 DOWNREGULATES EXPRESSION OF CD70 with the in vivo observations, upon stimulation with poly(I:C) DCs cell culture. Thus, CD70 continuously recycles from the cell from CD27-deficient mice consistently displayed higher levels membrane in both unstimulated and stimulated DCs. of CD70 than those of control mice (Fig. 2A, 2B). An important CD70 transcription, but not internalization, is affected by question was whether this was due to lack of the CD70–CD27 CD27 binding interaction itself or was secondary to some other consequence of CD27 loss. To address this, WT splenocytes were treated with Given that CD70 is constantly recycling from the membrane, we isotype control or blocking anti-CD27 Ab and stimulated with considered the possibility that the rate or extent of internalization either poly(I:C) or IFN-g and LPS. Blockade of the CD27–CD70 was increased by its interaction with CD27. In such a case, pre- interaction resulted in a substantially larger increase in CD70 venting the interaction with CD27 would result in accumulation on expression than that observed in isotype-treated cultures (Fig. 2C, the cell surface. To test this, CD70 internalization was measured in middle row). Notably, this was equal to the CD70 increase found splenocytes cultured in the absence or presence of blocking anti- in DCs from CD27-deficient mice (Fig. 2C, top row). Moreover, CD27 Abs (Fig. 4A, 4B). No difference in anti-CD70 internali- blocking Abs to CD27 in CD27-deficient mice did not result in zation was detected in resting DCs, and the small and not statis- a further increase of CD70, ruling out nonspecific Ab-mediated tically significant increase found in stimulated DCs could be effects (Fig. 2C, bottom row). These results indicated that the accounted for by the increase seen on the cell surface (Fig. 2C). elevated levels of CD70 in CD27-deficient mice were directly These results suggest that CD70 downregulation by CD27 is not caused by enhanced internalization. Another possibility is that the caused by a lack of interaction with its receptor, and that en- decrease in cell surface CD70 could be due to redistribution be- gagement of CD70 by CD27 downregulates CD70 levels on DCs.

tween plasma membrane and cytosolic pools. However, combined Downloaded from CD70 constitutively recycles from the cell surface intracellular and surface staining for CD70 indicated that the total amount of CD70 in DCs, not just the amount on the cell sur- We considered the possibility that CD70 might be undetectable on face, increased after blockade of CD27 (Fig. 5A). The possibility the surface of resting cells because of continuous internalization. that CD27 engagement regulates CD70 levels by suppressing its Notably, most of the CD70 expressed by both resting and stimu- transcription was evaluated. Although several reports have shown lated DCs was intracellular, as detected by specific intracellular

that blocking Abs to CD70 have effects on the proliferation and http://www.jimmunol.org/ staining after blocking surface CD70 with unlabeled anti-CD70 Ab survival of T cells, the ability of CD70 to perform “reverse sig- (Fig. 3A). To determine whether CD70 is turning over on the cell naling” has not been directly demonstrated. To determine if this surface of resting cells, splenocytes were cultured overnight with occurs, splenocyte CD70 mRNA levels were measured in vitro. fluorescently labeled Ab to CD70 in the presence or absence of Because the percentage of DCs in total splenocytes is very low stimulation, and Ab accumulation was measured. The final signal and CD70 mRNA undetectable, DCs were expanded in vivo by detected accounted for both the accumulation of anti-CD70 and injection of B16 melanoma cells engineered to express FLT3 li- the surface CD70 expression. Whereas labeled isotype control was gand (29). WT mRNA in unmanipulated splenocytes was unde- not detectably internalized, the level of anti-CD70 fluorescence in

DCs after overnight culture was easily detectable (Fig. 3B). When by guest on September 25, 2021 internalization from the cell surface was prevented by incubation on ice, the total cellular level of fluorescent signal was similar to that found with surface staining alone (Fig. 3C). Importantly, unlike in DCs, internalization of CD70 was not detected in B or T cells, establishing that CD70 is not expressed by resting lym- phocytes. Therefore, although undetectable by conventional cell surface staining, constitutive expression of CD70 is, in fact, present on resting DCs (Fig. 3B, 3D). Accumulation of Ab was even more prominent in stimulated DCs (Fig. 3B, 3D) and increased over time (Fig. 3E). To determine if CD70 recycles to the plasma membrane after internalization, we assessed the accumulation of fluorescently labeled anti-CD70 Ab under conditions in which new CD70 molecules could not be synthesized. Cells pretreated with the protein synthesis inhibitor CHX prior to stimulation failed to upregulate CD70 and CD80, as expected, demonstrating the efficiency of blockade (Supplemental Fig. 1, top panel). CD80 levels on CHX-treated stimulated cells were substantially below the levels on resting cells, suggesting that newly synthesized protein is the major contributor to surface upregulation. This was not the case for CD70, consistent with a slower turnover. Impor- tantly, inhibition of protein synthesis had little effect on surface CD70 levels when added during the last 9 h of stimulation, indi- cating that after the initial upregulation, CD70 levels are largely maintained in the absence of new protein (Supplemental Fig. 1, bottom panel). Notably, inhibition of de novo protein synthesis FIGURE 4. The higher levels of CD70 upon blocking of CD27 cannot be explained by different rates of internalization. (A and B) WT or CD702/2 did not reduce fluorescent Ab accumulation, indicating that cell splenocytes were stimulated and cultured overnight in the presence of anti- surface CD70 cycles from and to the plasma membrane (Fig. 3E). CD70 PE-conjugated Ab. Cells were pretreated with anti-CD27 blocking This finding was true also for the unstimulated splenocytes, pro- Ab (thick solid line) or with the isotype control (dashed line). Levels of the viding direct evidence that the small steady-state CD70 pool accumulated anti-CD70 Ab are shown as one representative experiment (A) found in untreated DCs was not a result of their maturation during or as mean 6 SEM of two to five independent experiments (B). The Journal of Immunology 2287 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. CD70 is downregulated at the transcriptional level by interaction with CD27. (A) WT or CD702/2 splenocytes were pretreated with anti- CD27 blocking Ab (thick solid line) or with the isotype control (dashed line) and stimulated overnight. Intracellular CD70 levels in DCs are shown in one representative experiment. (B) Splenocytes of WT or CD702/2 mice injected 10–13 d before with B16-FLT3L tumors were pretreated with anti-CD27 blocking Ab (gray bars) or with the isotype control (white bars) and stimulated for 6 or 24 h. CD70 mRNA was quantified by quantitative RT-PCR, normalizing the Ct values to the 18S housekeeping gene and to the values of CD702/2 cells (arbitrarily set to 1). Mean 6 SEM of three independent experiments is shown. *p , 0.05, **p , 0.01. (C) DCs sorted by flow cytometry from WT or CD702/2 mice were cultured with sorted T cells from WT or CD272/2 mice and stimulated for 6 h, as indicated. CD70 mRNA was quantified as in (B). Mean 6 SD of two independent experiments is shown. (D) DCs isolated with magnetic beads from six pooled WT or CD702/2 spleens were cultured with purified T cells from WT or CD272/2 mice and stimulated for 6 h, as indicated. CD70 mRNA was quantified as in (B). tectable (that is, the signal was detected at the same level as in scripts were detected only in cocultures with CD27-deficient T CD70 knockout splenocytes) after 6 h of culture, and was detected cells, confirming that CD70 transcription is enhanced when in- at only very low levels after 24 h (Fig. 5B). In contrast, when teractions with CD27 are removed. CD70 mRNA could not be CD27 was blocked CD70 transcripts increased almost 300- to detected in WT DCs stimulated in the presence of WT T cells, 1000-fold, even in the absence of activation. Stimulation with likely because the low amount of material derived from sorted poly(I:C) or IFN-g and LPS caused large increases in CD70 cells was below the limit of detection (Fig. 5C). Indeed, when mRNA that was further increased by blockade of CD27. The higher numbers of DCs were purified using magnetic beads, we differences between blocked and unblocked splenocytes were were able to detect CD70 mRNA also in WT DCs stimulated in greater at 6 h than at 24 h of stimulation, suggesting that the signal the presence of WT T cells. Substantially higher CD70 levels were triggered by CD27 engagement plateaus. To exclude the possible observed in DC:CD27-deficient T cell cocultures (Fig. 5D), con- contribution of CD70 mRNA from other cell types, DCs were firming the results obtained with sorted cells. Taken together, purified by flow cytometric sorting or isolated by magnetic beads these findings indicate that the interaction of CD27 with CD70 from naive mice and cocultured with WT or CD27-deficient suppresses CD70 transcription, leading to a tight control of CD70 T cells (Fig. 5C, 5D). In the case of sorted DCs, CD70 tran- levels. 2288 ENGAGEMENT BY CD27 DOWNREGULATES EXPRESSION OF CD70

Discussion cell surface levels. This finding implies that CD70 itself has the Costimulation is a key requirement for efficient activation of T capability to transmit a signal (reverse signaling), as has been cells, but it needs to be regulated to prevent an overly aggres- previously suggested for CD70 and other TNF family members (23, sive immune response. Controlled and tissue-specific expression of 32–34). In one case (23), activation of PI3K and MAPKs has been costimulatory molecules is often not sufficient to mediate this implicated in CD70 reverse signaling. An important question is regulation, and other mechanisms are required. For example, ac- whether such reverse signaling has functional repercussions other tivated T cells costimulated through the B7-CD28 ligand/receptor than regulation of the CD70–CD27 pathway. A model in which pair express the coinhibitory receptor CTLA-4, which competes the intracellular tail of CD70 is mutated or deleted would perhaps with CD28 for ligand and suppresses T cell responses (30). In the give some insights on roles of CD70 other than that of a costim- case of the CD70–CD27 pathway, other competing ligands or ulatory ligand for CD27. In any case, the findings in this study receptors have not been described, and CD27 is the only known identify another level of regulation of the CD70–CD27 pathway in receptor for CD70. Therefore, other levels of regulation must which CD70 is actively regulated itself. Given the known func- exist. One mechanism is controlled expression of both the ligand tional consequences of fixed CD70 expression (24, 25, 35), it is and the receptor. For example, CD27 mRNA and protein are dy- likely that reciprocal regulation of CD70 and CD27 expression namically regulated upon T cell activation, being constitutively plays an important role in the optimal function of this costimu- expressed on naive T cells, upregulated in the first few days of latory couple. activation, and transiently downregulated during the effector phase (2). Whereas CD27 downregulation is thought to be a direct result

Acknowledgments Downloaded from of activation-induced signaling, it is also possible that it is a con- We thank Ehydel Castro for assistance with animal experiments; Atef Allam sequence of interactions between CD70 and CD27. Indeed, in this for LCMV preparation; Rafi Ahmed for providing LCMV Armstrong; the report we found that CD70-deficient mice have slightly higher National Institutes of Health Tetramer Core Facility at Emory University, CD27 levels than do WT mice at steady state, and this difference Atlanta, GA, for the generous supply of tetramers; and Ulrich von Andrian increases during the first few days of LCMV infection, when for the B16-FLT3L cell line. CD70 levels on DCs rise. Direct CD70-mediated downregulation of CD27 could provide real-time fine-tuning of ongoing T cell Disclosures http://www.jimmunol.org/ activation processes necessary for controlling immune responses. The authors have no financial conflicts of interest. Formal proof of this hypothesis could be provided by studies investigating the consequences of expressing permanently high References levels of CD27 on T cells, but at this time such transgenic mice are 1. Denoeud, J., and M. Moser. 2011. Role of CD27/CD70 pathway of activation in not available. immunity and tolerance. J. Leukoc. Biol. 89: 195–203. Although CD70 is thought to be constitutively expressed in small 2. Nolte, M. A., R. W. van Olffen, K. P. van Gisbergen, and R. A. van Lier. 2009. Timing and tuning of CD27-CD70 interactions: the impact of signal strength in subsets of nonhematopoietic cells in the thymic medulla and lamina setting the balance between adaptive responses and immunopathology. Immunol. Rev. 229: 216–231. propria, it has not been found on DCs of unmanipulated mice (19, by guest on September 25, 2021 3. Watts, T. H. 2005. TNF/TNFR family members in costimulation of T cell 20). Our data indicate that resting DCs in fact do express low levels responses. Annu. Rev. 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