CD58/CD2 Is the Primary Costimulatory Pathway in Human CD28−CD8+ T Cells Judith Leitner, Dietmar Herndler-Brandstetter, Gerhard J. Zlabinger, Beatrix Grubeck-Loebenstein and Peter This information is current as Steinberger of September 29, 2021. J Immunol 2015; 195:477-487; Prepublished online 3 June 2015; doi: 10.4049/jimmunol.1401917

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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 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD58/CD2 Is the Primary Costimulatory Pathway in Human CD282CD8+ T Cells

Judith Leitner,* Dietmar Herndler-Brandstetter,†,1 Gerhard J. Zlabinger,‡ Beatrix Grubeck-Loebenstein,† and Peter Steinberger*

A substantial proportion of CD8+ T cells in adults lack the expression of the CD28 molecule, and the aging of the immune system is associated with a steady expansion of this T cell subset. CD282CD8+ T cells are characterized by potent effector functions but impaired responses to antigenic challenge. CD28 acts as the primary T cell costimulatory receptor, but there are numerous additional receptors that can costimulate the activation of T cells. In this study, we have examined such alternative costimulatory pathways regarding their functional role in CD282CD8+ T cells. Our study showed that most costimulatory molecules have a low capacity to activate CD28-deficient T cells, whereas the engagement of the CD2 molecule by its ligand CD58 clearly costimulated proliferation, cytokine production, and effector function in this T cell subset. CD58 is broadly expressed on APCs including Downloaded from dendritic cells. Blocking CD58 mAb greatly reduced the response of human CD282CD8+ T cells to allogeneic dendritic cells, as well as to viral Ags. Our results clearly identify the CD58/CD2 axis as the primary costimulatory pathway for CD8 T cells that lack CD28. Moreover, we show that engagement of CD2 amplifies TCR signals in CD282CD8+ T cells, demonstrating that the CD2–CD58 interaction has a genuine costimulatory effect on this T cell subset. CD2 signals might promote the control of viral infection by CD282CD8+ T cells, but they might also contribute to the continuous expansion of CD282CD8+ T cells during chronic stimulation by persistent Ag. The Journal of Immunology, 2015, 195: 477–487. http://www.jimmunol.org/

ging and repeated Ag encounter eventually leads to individuals aged 30 y can have 30% or even a higher percentage a state known as replicative senescence or immune se- of CD282CD8+ T cells (9, 10). By contrast, the large majority A nescence. This state is associated with deterioration of human CD4 T cells maintain CD28 expression even in cen- of the effectiveness of immune responses (1–4). In human tenarians (10). CD8 T cells have a central role in antiviral defense T lymphocytes, this state is characterized by the loss of CD28, and chronic infection with herpes viruses; especially CMV leads caused by transcriptional silencing (5–7). Whereas in young to a gradual increase of CD28-deficient CD8 T cells (11–13). Such infants virtually all human T cells express CD28, up to 50% of the an increase is also observed in individuals infected with other by guest on September 29, 2021 CD8 T cell compartment of elderly or chronically infected indi- persistent viruses such as HIV or HCV (14, 15). 2 2 viduals are CD28 (8). Importantly, the appearance of CD28 The CD282 population is characterized by high resistance to T cells in the CD8 compartment is not restricted to elderly as apoptosis (16, 17), enhanced production of proinflammatory cyto- kines, and increased cytotoxicity (12, 18). Moreover, functional im- pairment and a low proliferative capacity have been attributed to *Division of Immune Receptors and T Cell Activation, Institute of Immunology, 2 Center for Pathophysiology, Infectiology and Immunology, Medical University of CD28 T cells (19). Vienna, 1090 Vienna, Austria; †Institute for Biomedical Aging Research, University Importantly, efficient activation of T cells that encounter Ag ‡ of Innsbruck, 6020 Innsbruck, Austria; and Institute of Immunology, Center for critically depends on second signals, generated upon interaction of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria costimulatory receptors with their cognate ligands, in most cases 1Current address: Department of Immunobiology, Yale University School of Medi- provided by the accessory cells that present Ag. Professional APCs cine, New Haven, CT. upregulate B7 molecules upon pathogen uptake and can thus ef- Received for publication July 28, 2014. Accepted for publication May 5, 2015. ficiently induce responses in T cells that harbor the primary co- 2 This work was supported by the Austrian Science Fund (Grant P 21964-B20), the stimulatory receptor CD28 (20). Because CD28 T cells cannot Austrian National Bank (Grant ONB12731 to P.S.), a Theodor Ko¨rner Award (to J.L.), receive signals via this pivotal costimulatory pathway, insufficient and a Future Leaders of Ageing Research in Europe fellowship funded by the Austrian Federal Ministry of Science and Research (to D.H.-B.). activating signals may contribute to functional impairment of this J.L. performed the majority of experiments and analyzed data; J.L. and P.S. designed T cell subset. Importantly, a number of alternative costimulatory experiments and wrote the manuscript; B.G.-L. and D.H.-B. provided T cells and ligands have the property to provide potent activating signals to designed experiments; G.J.Z. provided essential reagents; and all authors critically T cells that recognize Ag. Among these is ICOS-ligand (ICOSL; revised the manuscript and approved the final version. CD275), another member of the B7 family, which ligates the Address correspondence and reprint requests to Judith Leitner and Peter Steinberger, Institute of Immunology, Medical University of Vienna, Lazarettgasse 19, 1090 CD28 superfamily molecule ICOS (21). Several alternative cos- Vienna, Austria. E-mail addresses: [email protected] (J.L.) and timulators such as 41BB-ligand (4-1BBL), OX40L, GITRL, and [email protected] (P.S.) CD70 belong to the TNF-superfamily, which engage the T cell– The online version of this article contains supplemental material. expressed TNFR-superfamily members 4-1BB, OX40, GITR, and Abbreviations used in this article: 4-1BBL, 41BB-ligand; Bw, Bw5417; Bw-CD58, CD27, respectively (22–24). MICA is an MHC class I–like ligand Bw cell expressing only human CD58; DC, dendritic cell; gMFI, geometric mean fluorescence intensity; ICOSL, ICOS-ligand; iDC, immature DC; LPS-DC, LPS- that induces activating signals on various cells expressing its re- activated monocyte-derived DC; mb-aCD3, membrane-bound anti-human CD3 ceptor CD314 (NKG2D) including CD8 T cells (25). Finally, the single-chain Ab fragment; TCS, T cell stimulator cell; TCS-ctrl, control TCS. ligation of several adhesion molecules to their receptors has been Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 shown to generate intracellular signaling events in T cells. Con- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401917 478 CD2 COSTIMULATES CD282CD8+ T CELLS sequently, the CD54/LFA-1, CD58/CD2, and CD166/CD6 inter- TCSs actions can also be regarded as genuine T cell costimulatory TCSs are Bw5417 (Bw) cells (a mouse thymoma cell line) stably expressing pathways (26–28). a membrane-bound anti-human CD3 single-chain Ab fragment (mb-aCD3) Although alternative costimulatory pathways, for example, the (33). Human costimulatory ligands (CD80, CD58, 4-1BBL, MICA, ICOS-L, 4-1BBL/4-1BB interaction (29–31), have been shown to promote CD54, CD166, CD70, OX40L, GITRL) were retrovirally expressed on the the activation of CD282 human T cells, comprehensive informa- TCS. Cells expressing no human costimulatory molecule were used as control TCS (TCS-ctrl). For some experiments, different costimulatory tion on the costimulatory requirements of this T cell subset was ligands were coexpressed on the TCS. Surface expression of mb-aCD3 and lacking. In this study, we have analyzed the expression of co- the expressed molecules was confirmed by FACS analysis. stimulatory receptors on freshly isolated and in vitro–activated CD282 CD8+ T cells. Moreover, we have tested the major alter- Activation of T cells using TCSs native costimulatory ligands regarding their capacity to enhance For T cell proliferation assays, human CD282 or CD28+ CD8+ T cells (1 3 5 6 the activation of this T cell subset. Our results identify the CD58/ 10 /well; 1 3 10 /ml) were cocultured with irradiated (6000 rad) TCSs 3 4 3 5 CD2 axis as the major costimulatory pathway for CD282CD8+ expressing different costimulatory molecules (2 10 /well; 4 10 /ml) in 96-well flat-bottom plates for 72 h, unless indicated otherwise. In some T cells. experiments, the parental Bw cell line without membrane-bound anti-CD3 expressing no human costimulatory molecule (Bw-ctrl) or Bw cells expressing only human CD58 (Bw-CD58) also were included. To assess Materials and Methods T cell proliferation, we added methyl-[3H]thymidine (final concentration: Sample collection 0.025 mCi; Perkin Elmer/New England Nuclear Corporation, Wellesley, MA) for the last 18 h before harvesting of the cells. Methyl-[3H]thymidine Informed, written consent was obtained from the donors, and the ethical uptake was measured as described previously (34). In some experiments, Downloaded from review board of the General Hospital and the Medical University of Vienna IL-2 and IL-15 were added at a final concentration of 10 U/ml (both and the Innsbruck Medical University approved the human studies per- Peprotech, Rocky Hill, NJ). . formed within this work. In indicated experiments, elderly donors (age 65 y) For cytokine measurement, supernatants of T cell proliferation assays were used. were collected after 48 h (unless indicated otherwise) of coculture and 2 pooled from triplicate wells. IFN-g, IL-2, and IL-13 were measured in the Cell purification and isolation of CD28 cells supernatants using the Luminex System 100 (Luminex, TX) as described

PBMCs were isolated from heparinized whole blood of healthy volunteer previously (35). http://www.jimmunol.org/ donors by standard density-gradient centrifugation with Lymphoprep (Axis- Shield PoC AS, Oslo, Norway). Bulk T cells were purified using MACS T cell responses to allogeneic DCs (Miltenyi, Bergisch Gladbach, Germany). For experiments with T cell + 2 + + Generation of immature DCs (iDCs) and LPS-activated monocyte-derived stimulator cells (TCS), CD8 CD28 and CD8 CD28 T cells were puri- DCs (LPS-DC) was done as previously described (36). Differentiation and fied using MACS as described in detail previously (32). Populations maturation of DCs was confirmed by analyzing expression of CD1a and showed .95% purity. CD83. Purified human bulk T cells or purified CD8+ T cells were CFSE For analyses of surface-marker expression, in MLR, in peptide- labeled (1 3 105/well; 1 3 106/ml) and then cocultured with different 2 + stimulation experiments, and in signaling experiments, CD28 or CD28 numbers of allogeneic monocyte-derived DCs in round-bottom 96-well + 2 + + cells or CD8 CD28 and CD8 CD28 , respectively, were purified from plates in the presence or absence of blocking CD58 mAbs. After 6 d of

PBMCs using the appropriate Abs by flow sorting using a FACSAria (BD coculture, cells were harvested, pooled from triplicate wells, and stained by guest on September 29, 2021 Biosciences, Palo Alto, CA). Cells were used immediately after sorting. with mAbs to CD28 and CD8. Proliferative response was analyzed by Populations showed .99% purity. FACS. In cases where bulk T cells were used, CD28+ T cells were ex- cluded by gating. Flow cytometry analysis and Abs Cytotoxicity assay Six-color flow-cytometry analysis was performed to assess surface-marker expression of T cells. For multicolor staining, the following mAbs were To generate effector T cells, we cocultured purified CD282CD8+ T cells used: CD2-PE #TS1/8, CD6-PE #BL-CD6, CD11a-PE #TS2/4, CD27-PE (1 3 105/well; 1 3 106/ml) with irradiated stimulator cells (2 3 104/well; #O323, CD137-PE #4B4-1, CD314-PE #1D11, ICOS-PE #C398.4A, 4 3 105/ml) for 5 d in a 96-well flat-bottom plate with or without IL-2 CD28-PE/Cy7 #CD28.2, CD57-FITC #HCD57, CD69-brilliant violet 510 (final concentration: 10 U/ml). #FN50, and CD8-allophycocyanin #SK1 were all purchased from Bio- Effector T cells were harvested, counted, and incubated with target cells legend. Appropriate isotype control Abs were used: isotype control-PE (ratio 5:1) for 4 h at 37˚C and subsequently analyzed by FACS. GFP+ #MOPC-21 and isotype control-PE #HTK888 (all from Biolegend). stimulator cells (Bw cells expressing membrane-bound-aCD3 but no hu- CD3-brilliant violet 421 #UCHT1 was bought from BD Pharmingen (Palo man costimulatory molecules) were used as model target cells. To deter- Alto, CA). Resting PBMCs or PBMCs stimulated for 24 h with PMA/ mine the specific cell lysis, we also added an equal number of APC+ Bw Ionomycin (final concentration: both 100 nM; both from Sigma-Aldrich, cells. The APC+ Bw cells do not express an anti-CD3 Ab, and thus they are St. Louis, MO) were analyzed with an LSRFortessa flow cytometer (BD not subject to specific lysis by the effector T cells. A total of 5 3 103 APC+ Biosciences). CD3+CD8+ cells (resting cells) or CD69+CD8+ cells (acti- cells was acquired from each sample. vated cells) were separated in respect to their CD28 expression. Data were Percentage of specific lysis was calculated using the following formula: analyzed using the FlowJo software (version 10.0.6; Tree Star, Ashland, þ OR). Fluorescence intensity is shown on a standard logarithmic scale. number of live GFP cells 3 100 100 2 For FACsorting of the indicated populations, CD8-allophycocyanin number of live APCþcells #SK1, CD28-PE, or CD28-PE/Cy7 (both #CD28.2) from Biolegend were used. For peptide-stimulation assays, CD58 mAbs LEAF #TS2/9 and Propidium-iodide was used to identify dead cells. Measurements were done on a FACSCalibur supported by CellQuest Pro Software (BD Biosciences). appropriate isotype control Abs LEAF #MOPC-21 (both Biolegend) were + used at a final concentration of 8 mg/ml. CD86-allophycocyanin #IT2.2 To obtain APC Bw cells, we stained Bw cells expressing human CD86 (Biolegend) was used for standardization in cytotoxicity assay. For den- with an anti-human CD86-allophycocyanin mAb. dritic cell (DC) staining, the following mAbs were used: CD1a #Vit6b, Ag-specific T cell responses CD80 #7-480 were produced in our laboratory; CD86 #IT2.2, CD58 #TS2/ 9, 4-1BBL #5F4 (all from Biolegend); CD83 #HB15e, CD166 #3A6, For peptide stimulation experiments, PBMCs were CFSE (Molecular ICOSL #2D3/B7H2, CD70 #Ki24 (all from BD Pharmingen); and MICA Probes, Eugene, OR) labeled as described previously (37) and cultured in #159207 (R&D, Minneapolis, MN). Bound primary Ab was detected using AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with PE-conjugated goat anti-mouse IgG-Fcg secondary reagent (Jackson 1.5% human serum (Octapharma, Lachen, Switzerland) for 7 d. HLA- Immunoresearch, West Grove, PA). For surface staining in the phosflow B8–restricted immunogenic peptides derived from Influenza (ELRSRY- experiments, the following mAbs were used: CD8-BV421 #RPA-T8, WAI); EBV (RAKFKQLL, FLRGRAYGL, QAKWRLQTL) and CMV CD28-allophycocyanin #28.2, and anti-mouse (m) CD45-FITC #104 (all (ELRRKMMYM, QIKVRVDMV) were obtained from Peptide 2.0 (Chantilly, from Biolegend). VA) at a purity .80% and were used at a final concentration of 2 mg/ml for The Journal of Immunology 479 each peptide as described previously (35). A blocking CD58 mAb was used at a final concentration of 8 mg/ml. Proliferation of the CD8+CD282 cells to antigenic responses was analyzed using a Calibur flow cytometer equipped with CellQuest Pro (version 6.0; BD Biosciences). In additional experiments, CD28+ cells were depleted from PBMCs by flow sorting, and CD282PBMCs were used as described earlier. In another set of experiments, we used the K562 cell line, which was retrovirally transduced to express HLA-B8 molecules. These cells were irradiated (4500 rad) and HLA-B8–restricted antigenic peptides were added (each peptide at a final concentration of 2 mg/ml). CD8+CD282 and CD8+CD28+ T cells were purified from PBMCs of HLA-B8+ donors. For T cell proliferation assays, CFSE-labeled T cells (1 3 105/well; 1 3 106/ml) were cocultured with the K562-HLA-B8 (3 3 104/well; 6 3 105/ml) in 96- well flat-bottom plates for 7 d. CFSE dilution was assessed on a LSRFortessa flow cytometer (BD Biosciences). A total of 50,000 cells was acquired, and data were analyzed with FACSDiva software (v6.2).

Flow-cytometric analyses of intracellular signaling pathways PBMCs or sorted CD8+CD282 T cells (1.5 3 106/well; 5 3 106/ml) were cocultured in the presence of TCS-ctrl, TCS-CD58, Bw-ctrl, and Bw-CD58, respectively (3 3 105/well; 3 3 106/ml), in a 24-well plate (ratio 5:1). After

20 h, cells were harvested and stained for surface expression of CD8, CD28, Downloaded from and mCD45. mCD45 was used to exclude the TCSs from the analyses. Subsequently, cells were fixed in prewarmed Phosflow Fix Buffer I for 10 min at 37˚C. After washing in PBS supplemented with 0.5% BSA, 0.05% NaN3 cells were permeabilized for 30 min on ice using prechilled (220˚C) Phosflow PERM Buffer III (both BD Biosciences). Followed by two washing steps, cells were incubated with one of the following PE-conjugated mAbs for 1 h at room temperature: mouseIgG1 #MOPC21, Zap70 (pY319)/Syk (pY352) #17A/P-Zap70, SLP-76 (pY128) #J141-668.36.58, CD247 (pY142; http://www.jimmunol.org/ CD3z) #K25-407.69, MEK1 #J114-64, S6 (pS240) #N4-41, erk1/2 (pT202/ pY204) #20A (all BD Phosflow; BD Biosciences). Data were analyzed using the FlowJo software. A total of 40,000 cells was acquired. Geometric mean fluorescence intensity (gMFI) values are shown.

Statistics Means and 6 SD are shown. A p value ,0.01 was considered to be sta- tistically significant. Two-tailed Student t test was used to assess signifi- cance in Figs. 3 and 7. SPSS Statistics software version 21 (IBM, Chicago, IL) was used for box plot and ANOVA, followed by Tukey–honestly by guest on September 29, 2021 significant difference correction, in Fig. 2.

Results Expression of alternative costimulatory receptors on CD282CD8+ T cells It is well established that most CD8+ T cells that have lost the CD28 molecule also lack CD27 expression, whereas CD28+ T cells are mostly CD27+. However, it is currently not known whether there are additional differences in the expression and regulation of costimulatory receptors between these two subsets. Therefore, we performed multicolor FACS analysis to compare the expression of costimulatory receptors on resting and in vitro– activated CD28+ and CD282 CD8 T cells. We found that CD2, CD6, CD11a (LFA-1), and ICOS are constitutively expressed on both subsets (Fig. 1). CD27 is expressed by the majority of CD28+ 2 FIGURE 1. Expression and regulation of costimulatory receptors on T cells, whereas most CD28 T cells also lack CD27 expression. CD282 and CD28+CD8+ T cells. (A) Gating strategy used for FACS CD137 (4-1BB) and OX40 were absent in resting T cells and in- analysis. FACS-purified CD282 and CD28+ cells were either used freshly duced in both subsets upon in vitro activation (Fig. 1, Supplemental or activated for 24 h using PMA/Ionomycin. (B) Surface expression Fig. 1). The MICA receptor, CD314, is similarly expressed on both of costimulatory molecules and CD57 on resting and PMA/Ionomycin- subsets in the resting state, but stronger expressed on activated activated CD8+ T cells (left panels,CD8+CD282 T cells; right panels, CD282 cells when compared with CD28+ cells. In line with pre- CD8+CD28+ T cells). Shown are 1 3 105 and 1 3 104 events in case of vious reports and the well-established role of CD57 as marker for resting and activated T cells, respectively. Experiment shown is repre- senescent T cells (38, 39), we found that the majority of CD282 sentative for five independently performed. T cells express CD57, whereas CD28+ T cells were mainly CD572. Moreover, CD27+ cells were overrepresented in the CD572 subset 2 Alternative costimulatory signals in the activation of human of CD28 T cells (Supplemental Fig. 1). Taken together, our data + 2 indicate that, except for the expression of CD27, there are little CD8 CD28 T cells differences regarding the expression and regulation of costimulatory To assess the role of different alternative costimulatory signals in receptors on CD28+ and CD282 CD8 T cells. the activation of CD282 T cells, we used our previously described 480 CD2 COSTIMULATES CD282CD8+ T CELLS system of TCSs (33). In brief, TCS are cells that coexpress expression levels of the costimulatory molecules and homogenous membrane-bound CD3 (mb-aCD3) Abs and human costimulatory expression of mb-aCD3 on the TCS were confirmed by FACS ligands of choice. Upon interaction with human T cells, they analysis (Supplemental Fig. 2). For control purposes, we also trigger their TCR complex and engage distinct costimulatory generated TCSs expressing anti-CD3 Abs, but no human costim- receptors. By using TCS lines expressing different ligands, the ulatory molecule. impact of different costimulatory signals on the activation of CD8+CD282 T cells purified from elderly donors (.65 y) were T cells can be analyzed (Fig. 2A). cocultured with this set of stimulator cells, and proliferation and For this study, we engineered our TCS cell line to express one of cytokine production were assessed (Fig. 2B, 2C). In all experi- the following human costimulatory ligands at high levels: CD80, ments, CD8+CD28+ T cells, purified from the same donor, were CD58, 4-1BBL, MICA, ICOS-L, CD54, CD166, CD70. High stimulated with the same set of stimulator cells in parallel. Iso- Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Alternative costimulatory signals activate CD8+CD282 T cells. (A) Scheme of a TCS–T cell coculture: T cells received Signal 1 and distinct costimulatory signals (Signal 2) upon coculture with TCSs, which coexpress mb-aCD3 and human costimulatory ligands of choice. (B) CD8+CD28+ (left panel) and CD8+CD282 (right panel) T cells were stimulated in the presence of TCSs expressing the indicated molecules or no costimulatory molecule (ctrl) for 72 h. T cell proliferation was assessed by measuring methyl-[3H]thymidine uptake. (C) Cytokine concentrations were measured in the coculture supernatants sampled after 48 h using a Luminex-based system. (B and C) Data show 6SD of triplicates from one experiment representative for nine independently performed. (D) Box plot shows a summary of all experiments performed (n =9;*p , 0.01 compared with response in presence of CD58). Circles indicate outliers. (E) CFSE-labeled mononuclear cells were cocultured with the indicated Bw cells in the presence (white bars) or absence (gray bars) of blocking CD58 mAb. After 5 d of stimulation, proliferative response was assessed by FACS. % CFSElow of CD8+CD282 cells are shown (left panel). In addition, IFN-g content of coculture supernatant was sampled after 5 d using a Luminex-based system (right panel). Data show 6SD of triplicates from one experiment representative for three independently performed. ns, not significant. The Journal of Immunology 481

FIGURE 3. Synergy between costimulatory signals and exogenous cytokines in the activation of CD282 T cells. (A) CD8+CD28+ (left panel) and CD8+CD282 (right panel) T cell subsets were activated with stim- ulator cells expressing CD80, CD58, 4-1BBL, and MICA or combinations thereof. Cytokine content was analyzed after 48 h of coculture using a Luminex-based system (lower panel). (B) CD8+CD28+ (left panel) and CD8+CD282 (right panel) T cell subsets were stimu- lated with stimulator cells expressing CD80, CD58, 4-1BBL, ICOS-L, or no costimulatory molecule (ctrl) in the presence or absence of IL-2 (final concentra- tion: 10 U/ml). (A and B) Proliferative response was determined upon methyl-[3H]thymidine uptake after

72 h of coculture. Data show 6SD of triplicates from Downloaded from one experiment representative for three independently performed. ns, not significant. http://www.jimmunol.org/ lated T cell populations showed .95% purity (Supplemental Fig. much less cytokine production and proliferation than CD2 co- 3). All costimulatory ligands tested clearly enhanced the prolif- stimulation. To exclude that CD2 on its own induces proliferation eration in the CD28+CD8+ subset. CD58 and 4-1BBL had a very and cytokine production in CD282 cells, we cocultured CD282 high capacity to induce proliferation and cytokine production in T cells with the parental Bw cells (which is devoid of membrane- this T cell subset. As expected, the strongest response was ob- bound anti-CD3) and Bw-CD58. CD2 signals alone did not en- served by engagement of the primary T cell costimulatory re- hance T cell proliferation or cytokine production compared with 2 + ceptor CD28 via CD80 (Fig. 2B, 2C). Stimulation of CD28 CD8 stimulation via CD3 alone or CD3/CD2 (Fig. 2E). by guest on September 29, 2021 T cells from the same donors under the same conditions generally Importantly, the potent costimulatory capacity of CD58 is a gen- resulted in much lower proliferation and cytokine production. In eral phenomenon for CD8+CD282 T cells, because it was also ob- this T cell subset, most of the costimulatory ligands had little served when we analyzed such T cells purified from young donors effect on the proliferative response. Interestingly, we observed that (Supplemental Fig. 4). CD58/CD2 costimulation consistently resulted in a very strong 2 Synergy between costimulatory signals and exogenous enhancement of proliferation and cytokine production in CD28 2 T cells (Fig. 2B–D). Whereas in CD28+CD8+ T cells there was cytokines in the activation of CD28 T cells little difference in the costimulatory effects exerted by 4-1BB and In the previous experiments, we found that besides CD58, the CD2 engagement, in CD282 T cells, triggering of 4-1BB induced presence of 4-1BBL and MICA also costimulates CD8+CD282

FIGURE 4. In vitro–activated CD8+CD282 T cells acquire potent cytotoxic capacity. CD8+CD282 T cells were preactivated with stimulator cells expressing CD58 (TCS-CD58), 4-1BBL (TCS-41BBL), or no costimulatory molecule (TCS-ctrl) in absence (upper panel)orpresence(lower panel)ofIL-2. These effector cells were then incubated with GFP+ target cells (stimulator cells expressing mb-aCD3 but no costimulatory molecules). APC+ cells were used to standardize the acquired volume. Percentage of specific lysis is shown. Propidium-iodide+ (FL-3+) cells were excluded. Fluorescence intensity is shown on a logarithmic scale. Experiment shown is representative for three independently performed. 482 CD2 COSTIMULATES CD282CD8+ T CELLS

T cells, albeit to a much lower extent. To assess additive effects of these costimulatory signals, we established TCSs coexpressing combinations of CD58, 4-1BBL, and MICA. High expression levels of these molecules were confirmed by FACS analysis (Supplemental Fig. 2). In CD8+CD28+ T cells, combinations of costimulatory signals clearly acted synergistic. The concomitant triggering of 4-1BB (by 4-1BBL) and CD314 (by MICA) was very effective in inducing proliferation and cytokine production (Fig. 3A). By contrast, we found that in CD8+CD282 T cells the presence of additional costimulators had little capacity to enhance CD58 effects. More- over,comparedwithCD58costimulation, the combination of 4-1BBL and MICA induced a much weaker response, which further underlines that CD8+CD282 T cells strongly depend on costimu- lation via CD58/CD2 (Fig. 3A). In a next set of experiments, we analyzed the effects of exog- enous cytokines on the activation of CD8 T cells that were stimulated in the presence of different costimulatory ligands. We found that exogenous cytokines had a strong effect on proliferation, Downloaded from and costimulatory signals generally had low potential to further enhance proliferation. Interestingly, stimulation via CD58/CD2 pathway and exogenous IL-2 both had a strong impact on the proliferation of CD8+CD282 T cells, but no additive effects were observed (Fig. 3B). When tested in this experimental setting IL-2

and IL-15 had similar effects, and the use of both cytokines did http://www.jimmunol.org/ not further enhance the proliferative response of CD8 T cells (data not shown). In vitro–activated CD8+CD282 T cells acquire potent cytotoxic capacity Cytotoxicity is the major function of CD8 T cells. Consequently, we assessed the impact of CD58 costimulation on the generation of cytotoxic effector T cells from the CD282 subset. For effector cell + 2 differentiation, CD8 CD28 T cells were stimulated with TCSs by guest on September 29, 2021 expressing CD58, 41BB-L, or no costimulatory molecule in the presence or absence of IL-2. After 5 d of stimulation these effector cells were cocultured with GFP+ mb-aCD3–expressing target cells and subsequently analyzed by FACS. We found that during the in vitro activation, all tested CD282CD8+ T cells acquired potent effector function: .70% of the target cells were lysed. Nevertheless, preactivation via CD58 induced the highest specific lysis (91%). Presence of IL-2 during effector cell differentiation strongly enhanced the cytotoxic capacity of CD282CD8+T cells. CD58 costimulation only slightly increased the cytotoxic capacity of effector cells generated in the presence of IL-2 (Fig. 4). These data indicate that in vitro–stimulated CD8+C282 T cells can still acquire and exert potent cytotoxic function. Costimulation via CD58 resulted also in a higher number of effector cells (data not shown), which had a stronger capacity to kill target cells. FIGURE 5. Stimulation of CD282CD8+ T cells by allogeneic DCs. 2 (A) iDCs and LPS-DCs were analyzed for expression of CD1a and CD83 DCs costimulate CD28 T cells via CD58/CD2 and the indicated costimulatory ligands (open histogram: isotype control, DCs are generally regarded as the primary APCs. We assessed the filled histogram: mAb as indicated). (B) CFSE-labeled human bulk expression and regulation of costimulatory ligands on monocyte- T cells or purified CD8+ Tcells(C)(13 105/well) were cocultured with derived DCs. We found that in addition to CD80 and CD86, indicated numbers of iDCs or LPS-DCs in the absence (control, upper these cells also express CD58 and CD166. All these costimu- panel)orpresenceofneutralizingCD58mAbs(lower panel). After 6 d latory ligands are upregulated upon LPS treatment. By contrast, of coculture, T cells were stained for CD8 and CD28 expression. Dot blots depict CD282 T cells. Numbers indicate percentage CFSElow cells. monocyte-derived DCs do not express appreciable amounts of Results are representative for three (B)orfive(C) experiments. (D) ICOSL, MICA, 4-1BBL, CD70, OX40L, or GITRL (Fig. 5A and Summary of all experiments is shown. Bar diagrams depict percentage of data not shown). When we analyzed the allostimulatory capacity CFSElow CD282CD8+T cells in the presence of CD58 Abs (filled bars) + 2 of DCs on CD8 CD28 T cells in the presence of a neutralizing normalized to CFSElow CD282CD8+T cells in control-treated samples CD58 Ab, we found that blockade of CD58 greatly reduced the (open bars). Left panel represents bulk T cells; right panel represents proliferative response in this T cell subset up to 80% (Fig. 5B, CD8+ Tcells. The Journal of Immunology 483

5D). The effect of CD58 blockade is independent of CD4 T cells because it was also observed when purified CD8+ T cells instead of bulk T cells were stimulated with allogeneic DCs (Fig. 5C, 5D). The CD58/CD2 axis in the immune response to pathogen Ags Because signals generated upon CD58/CD2 interaction elicit the strongest proliferative response in the CD282 subset, we analyzed the role of CD58–CD2 signaling in the immune response to prevalent pathogens. CFSE-labeled PBMCs were stimulated with a mix of immunogenic peptides from CMV, EBV, and Influenza. The addition of a blocking CD58 mAb to the stimulation cultures greatly reduced the percentage of CFSElow cells among the CD8+ CD282 subset (Fig. 6A). To rule out that CD58 blockade acts on CD28+ T cells, which might lose CD28 during the course of stimulation, we also used PBMCs depleted of CD28+ cells. These experiments clearly show that response of CD8+CD282 cells to frequent pathogenic Ags strongly depends on CD2 costimulation (Fig. 6B).

In an additional set of experiments, we used K562 cells Downloaded from expressing high levels of human HLA-B8. The cells were loaded with HLA-B8–restricted peptides derived from common path- ogens and used to stimulate CFSE-labeled CD8+CD282 and CD8+CD28+ T cells of an HLA-typed donor. K562 cells are devoid of CD80 and most costimulatory molecules, but they express CD58

(data not shown). These experiments also confirmed the impor- http://www.jimmunol.org/ tance of CD2 costimulation for CD8+CD282 T cells: blocking of CD58 resulted in a strongly reduced number of CFSElow T cells, whereas in CD8+CD28+ T cells this effect was less pronounced (Fig. 6C). Signaling pathways triggered by CD2 costimulation We wanted to determine whether the stimulating effects of CD2– CD58 interaction that were observed in our experiments are

merely owed to enhanced adhesion or if triggering of CD2 via by guest on September 29, 2021 CD58 acts to enhance TCR signals in the CD282CD8+T cells. Based on a previous study from Ska˚nland et al. (40), six different signaling molecules were selected, namely, S6-ribosomal protein, SLP76, CD247 (CD3z), MEK1, erk1/2, and ZAP70, and analyzed regarding their role in the CD2-based activation of CD282 T cells. FIGURE 6. The CD58–CD2 axis in the immune response to common In line with previous studies on CD2 costimulation (40, 41), A 2 + 2 pathogen Ags. Mononuclear cells ( ) and sorted CD28 mononuclear stimulation of gated or FACS-purified CD8 CD28 T cells via cells (B) were left unstimulated or cocultured in the presence of a HLA-B8 CD2 leads to a stronger phosphorylation of S6 ribosomal protein peptide mix with or without blocking CD58 Abs (anti-CD58) for 7 d. compared with stimulation with CD3 alone (Fig. 7A: n = 10, Proliferation of the CD282 cells was assessed by CFSE dilution. Fluo- Fig. 7B: n =5;p , 0.01, ns: not significant). We also observed rescence intensity is shown on a logarithmic scale. Numbers indicate a trend toward enhanced phosphorylation of SLP76, CD247 (CD3z), percentage within quadrants. Experiment shown is representative for 10 + 2 MEK1, erk1/2, and ZAP70 in the presence of CD58, but this did independently performed. (C) FACS-purified CD8 C28 (upper panel) + + not reach statistical difference. Similar results were obtained when and CD8 CD28 (lower panel) T cells were stimulated with K562- primary human T cells were stimulated by cross-linking of CD3/ expressing HLA-B8 in the absence or presence of HLA-B8 peptide mix, in conjunction with control Abs (control) or CD58 blocking Abs. Prolifera- CD2 mAbs (data not shown). tion was assessed by CFSE dilution upon 6 d of coculture. CD8+CFSElow To verify that CD2 engagement enhances TCR signals, coculture events are shown. experiments with CD58-transductants that do not express stimu- lating CD3 Abs were also performed. These experiments indicated pathways in this subset was lacking. To the best of our knowledge, that CD2 engagement costimulates TCR signaling but does not this study is the first to compare the major alternative costimula- generate signals in the absence of TCR engagement (Fig. 7C). tory molecules regarding their capacity to support proliferation and cytokine production on CD282 CD8+ T cells. Our results Discussion clearly identify the CD58–CD2 axis as the primary costimulatory CD282CD8+ T cells are functionally distinct from their CD28- pathway for these cells, whereas all other molecules tested showed sufficient counterparts because they show reduced responses to only modest effects. Azuma et al. (9) were the first to perform CD3/TCR signals (9) and are unable to receive signals via the a phenotypic and functional analysis on CD282CD8+ human primary costimulator CD28. Therefore, we reasoned that the ac- T cells. They state that these cells could not be costimulated by tivation of these cells might critically depend on alternative sig- CD58, but their data indicate that in all three CD282CD8+ T cell nals. Several reports have demonstrated the capacity of 4-1BB clones they have tested, the presence of CD58 clearly enhanced engagement to costimulate human CD282 CD8+ T cells (29, 31, proliferation, although the effect appears to be less pronounced 37), but a comprehensive analysis of alternative costimulatory compared with our study. They have used T cell clones and the 484 CD2 COSTIMULATES CD282CD8+ T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. Signaling events triggered by CD2 costimulation. PBMCs (A) or FACS-purified CD8+CD282 T cells (B) were cocultured in the presence of TCS-control (ctrl) or TCS-CD58. After 20 h of stimulation, six intracellular signaling molecules were analyzed by FACS. (A) Upper panel illustrates the gating strategy of CD8+CD282 T cells. Anti-mouse CD45 Ab (mCD45) was used to exclude TCS. Histograms show the phosphorylation pattern of six key signaling molecules of gated CD8+CD282 T cells stimulated in the presence or absence of CD58. Filled histograms: TCS-control; open histograms: TCS- CD58. gMFI values are shown. Lower panel, Summary of all experiments performed is shown. Each symbol represents one donor (n = 10). (B) Upper panel depicts the sorting strategy and postsort purity of CD8+CD282 T cells. Histograms show the phosphorylation pattern of six key signaling molecules of FACS-purified CD8+CD282 T cells stimulated in the presence or absence of CD58. Filled histograms: TCS-control; open histograms: TCS-CD58. gMFI values are shown. Lower panel, Summary of all experiments performed is shown. Each symbol represents one donor (n = 5). (Figure legend continues) The Journal of Immunology 485 functional changes associated with the cloning procedure might Collectively, our results indicate that CD2 has an essential role in account for the low response to stimulation with CD58 or IL-2 the maintenance and expansion of CD8+ T cells that lack the they have observed in their study. primary costimulatory receptor CD28. Although CD28-deficient Although the CD2–CD58 interaction might promote T cell T cells may contribute to control viral infection, the elevation of activation by enhancing adhesion, it has been clearly demon- CD282CD8+ T cells is generally seen as a significant predictor of strated that CD2 engagement selectively enhances distinct TCR immune senescence (5, 17, 46). Moreover, CD282CD8+ T cells signaling pathways (40–43). To our knowledge, our study is the directly contribute to a defective and aberrant immune function in first to investigate the intracellular signaling events induced in elderly or chronically infected individuals by producing proin- CD282CD8+ T cells upon engagement of CD2 by its natural flammatory cytokines and by exerting immune-suppressive ligand CD58. Our results demonstrate that this engagement effects (12, 18). Most importantly they impair the adaptive im- amplifies TCR signals, thus indicating that intracellular signals mune responses by taking up the available space (“immunological generated by CD2 triggering significantly contributed to the effects niches”), which compromises T cell homeostasis and constrains observed in our study. Signaling in T cells is mostly studied by the expansion of T cells in response to pathogenic challenges or using agonistic Abs or immobilized proteins representing costim- vaccination (18, 45, 47). In light of our data, it can be envisioned ulatory ligands. We applied our cellular system of TCSs, which that CD2 signals might promote immune senescence by providing allows studying the engagement of costimulatory receptors by their essential signals for activation and expansion to CD28-deficient natural cell-expressed ligands and might thus generate intracellular T cells. Thus, blocking CD2 signals might counteract the accu- signals that more closely resemble the effects mediated by co- mulation of CD28 null T cells in elderly individuals, as well as in stimulatory ligands upon APC–T cell interactions. In line with patients suffering from chronic virus infection. However, from our Downloaded from previous results on CD2 costimulation, we have found that also in data it is also evident that CD2 signals are not only important for CD282 T cells CD2 engagement strongly enhanced the phosphor- CD282 T cells but also for CD28-expressing CD4+ and CD8+ ylation of the S6-ribosomal protein (40). In addition, we also ob- T cells. Therefore, it can be expected that strategies to ameliorate served enhanced phosphorylation of other signaling molecules like immune senescence by blocking CD2 signals bear the danger of SLP76, CD247 (CD3z), MEK1, erk1/2, and ZAP70, but the effect compromising protective T cell immunity.

was less pronounced. Finally, we show that CD2–CD58 signals are Given the prominent role of CD2 signals for the activation of http://www.jimmunol.org/ only operative in the presence of TCR signals, which is also con- CD282 T cells, it might be interesting to address whether they can sistent with classical costimulation of CD282CD8+ TcellsviaCD2. overcome additional constraints that contribute to a functional We and others have previously demonstrated that combinations impairment of this subset. Defective Akt (ser473) phosphorylation of different costimulatory ligands act synergistically to promote and lack of telomerase activity have been shown to be overcome potent activation of T cells (37, 44). Unexpectedly, our data do not by blockade of inhibitory costimulatory receptors (48, 49). This support such an effect in CD282 T cells because the presence of indicates that coinhibitory pathways have a prominent role in additional alternative costimulatory ligands like 4-1BBL or MICA the functional impairment of this T cell subset. Many different did not further enhance the response of cells receiving CD2 co- coinhibitory pathways have the potential to reduce T cell re- stimulation. Moreover, in the absence of CD2 signals, the com- sponses (50). In preliminary experiments, we have observed that by guest on September 29, 2021 binations of alternative costimulatory signals yielded responses CD282CD8+ T cells express significant levels of coinhibitory that were considerably lower than those observed in CD282CD8+ receptors on their surface. Compared with their CD28-sufficient T cells activated in the presence of the CD2 ligand CD58, which counterparts, they tend to express more PD-1 and CD160 (data not further underlines the significance of CD2 signals for this T cell shown). Studies on the contribution of distinct pathways to the subset. Cytokines like IL-2 or IL-15 promote survival and induce functional impairment of CD28-deficient T cells are highly war- strong proliferative responses in CD282 T cells (30). We found ranted. In this context it will also be interesting to address whether that IL-2 and CD2 costimulation had similar capacities to induce and to which extent strong CD2 signals can override coinhibition in proliferation in this T cell subset. However, significant synergistic this T cell subset. effects between costimulatory signals and activating cytokines The most widely used model for investigating immune aging is on T cell proliferation were not observed in our experiments. the mouse model, and there are many characteristics of aging of CD282CD8+ T cells express high levels of perforin and gran- human T cells that are well represented by mouse models (51, 52). zymes, and they exert strong cytotoxic effects (45). We found However, there are also profound disparities regarding the immune that preactivation of these cells in the presence of CD58 yielded aging in these species that have to be taken into consideration (52, potent killer cells, indicating that CD2 signals also promote ef- 53). One striking difference between humans and mice is that in fector functions in highly differentiated human CD8+ T cells. mice, CD28-deficient T cells are rare even in old animals (54–57). Given their strong cytotoxic potential, the CD282 T cell subset The fact that mice lack CD58 could be seen as another lead for might have an important role in controlling persistent or recurrent a close functional interrelationship between CD28-deficient T cells viral infections especially in individuals with low numbers of and the CD58–CD2 costimulatory pathway. CD28+CD8+ T cells (18). We demonstrate that blocking CD58 on autologous or engineered APCs led to a profound reduction of 2 Acknowledgments the proliferative response of CD28 T cells to viral Ags. CD58 is We appreciate the excellent technical assistance of Claus Wenhardt, broadly expressed on accessory cells including DCs, which are the Margarethe Merio, and Petra Waidhofer-So¨llner. We thank Dieter Printz primary APC. Our study provides evidence that efficient activation (Children’s Cancer Research Institute, St. Anna, Vienna, Austria) for 2 + of CD28 CD8 T cells by DCs also critically depends on CD2 FACSorting. We thank Dr. Wolfgang Paster for critical comments on the costimulation. manuscript.

(C) PBMCs were cocultured in the presence of TCS-control, Bw-control, and Bw-CD58. After 20 h of stimulation, phosphorylation of S6 ribosomal protein of gated CD8+CD282 T cells was analyzed by FACS. One representative histogram of four independent experiments is shown. ns, not significant. 486 CD2 COSTIMULATES CD282CD8+ T CELLS

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