Effect of Anti–IL-15 Administration on T Cell and NK Cell Homeostasis in Rhesus Macaques

The Journal of Immunology

Maren Q. DeGottardi,*,†,1 Afam A. Okoye,*,†,1 Mukta Vaidya,*,† Aarthi Talla,‡ Audrie L. Konfe,*,† Matthew D. Reyes,*,† Joseph A. Clock,*,† Derick M. Duell,*,† Alfred W. Legasse,*,† Amit Sabnis,‡ Byung S. Park,x Michael K. Axthelm,*,† Jacob D. Estes,{ Keith A. Reiman,‖ Raﬁck-Pierre Sekaly,‡ and Louis J. Picker*,†

IL-15 has been implicated as a key regulator of Tand NK cell homeostasis in multiple systems; however, its speciﬁc role in maintaining peripheral T and NK cell populations relative to other g-chain (gc) cytokines has not been fully deﬁned in primates. In this article, we address this question by determining the effect of IL-15 inhibition with a rhesusized anti–IL-15 mAb on T and NK cell dynamics in rhesus macaques. Strikingly, anti–IL-15 treatment resulted in rapid depletion of NK cells and both CD4+ and CD8+ effector memory

T cells (TEM) in blood and tissues, with little to no effect on naive or central memory T cells. Importantly, whereas depletion of NK cells was nearly complete and maintained as long as anti–IL-15 treatment was given, TEM depletion was countered by the onset of massive TEM proliferation, which almost completely restored circulating TEM numbers. Tissue TEM, however, remained signiﬁcantly reduced, and most TEM maintained very high turnover throughout anti–IL-15 treatment. In the presence of IL-15 inhibition, TEM became increasingly more sensitive to IL-7 stimulation in vivo, and transcriptional analysis of TEM in IL-15–inhibited monkeys revealed engagement of the JAK/STAT signaling pathway, suggesting alternative gc cytokine signaling may support TEM homeostasis in the absence of IL-15. Thus, IL-15 plays a major role in peripheral maintenance of NK cells and TEM. However, whereas most NK cell populations collapse in the absence of IL-15, TEM can be maintained in the face of IL-15 inhibition by the activity of other homeostatic regulators, most likely IL-7. The Journal of Immunology, 2016, 197: 1183–1198.

ymphocyte homeostasis and function are tightly regulated cytokines share the gc receptor (CD132), their activity on by the activities of the common g-chain (gc) cytokines, in various lymphocyte populations are often different, differences L particular, IL-2, IL-7, and IL-15. Although these three that are, in part, mediated by differential expression of the a-chain component of the receptor (1–7). For instance, IL-2 is mainly + + *Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, produced by CD4 T cells, and to a lesser degree CD8 T cells, † OR 97006; Oregon National Primate Research Center, Oregon Health & Science NK cells, and NKT cells, and it acts to generally potentiate the University, Beaverton, OR 97006; ‡Department of Pathology, Case Western Re- by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. serve University, Cleveland, OH 44106; xDivision of Biostatistics, Department of expansion of activated T and NK cells (8, 9). However, IL-2 has a Public Health and Preventative Medicine, Oregon Health & Science University, { unique primary role in regulating immune tolerance by promoting Portland, OR 97239; AIDS and Cancer Virus Program, Leidos Biomedical Re- + + + search, Inc., Frederick National Laboratory, Frederick, MD 21702; and ‖MassBiologics, the production and maintenance of CD4 CD25 Foxp3 T reg- University of Massachusetts Medical School, Boston, MA 02126 ulatory cells, which constitutively express IL-2Ra (CD25). In- 2/2 1M.Q.D. and A.A.O. contributed equally to this work. deed, this was demonstrated in early models of IL-2 and 2/2 2/2 ORCIDs: 0000-0002-7994-5458 (A.L.K.); 0000-0002-6868-888X (A.S.); 0000- IL-2Ra /IL-2Rb knockout mice, which manifested rapid 0003-3516-7516 (B.S.P.); 0000-0002-1984-5906 (M.K.A.). lethal autoimmune diseases likely resulting from the failure of T Received for publication January 12, 2016. Accepted for publication June 15, 2016. regulatory cell development and homeostasis (10). IL-7 is pro- This work was supported by the National Institutes of Health (Grants 5R37AI054292, duced by nonhematopoietic cells such as stromal and epithelial https://www.jimmunol.org 5R01A1082529, 5U19AI067854, U42OD010426, and 8P51OD01109255) and the Na- cells, and it is important for thymocyte development and periph- tional Cancer Institute, National Institutes of Health (Contract HHSN261200800001E). Reagents were provided by the Nonhuman Primate Reagent Resource supported by the eral T cell homeostasis. IL-7 signals via the IL-7R, which is a National Institute of Allergy and Infectious Diseases (Contract HHSN2722000130031C) heterodimer consisting of the IL-7Ra (CD127) and CD132. IL-7 and the National Institutes of Health (Grant OD010976). is particularly important for promoting the proliferative expansion The content of this publication does not necessarily reﬂect the views or policies of the and survival of naive T cells (TN) and central memory T cells U.S. Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. (TCM), which maintain high levels of CD127 expression (11). Downloaded from IL-15, in contrast, has been shown to regulate the homeostasis and The gene array data presented in this article have been submitted to the Gene Expres- sion Omnibus microarray repository (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? activation of many cell types throughout the body, including token=mbqtaouqztynzsz&acc=GSE76797) under accession number GSE76797. memory T cells (TM), NK cells, invariant NKT cells, gd T cells, Address correspondence and reprint requests to Dr. Louis J. Picker, Vaccine and Gene and intestinal intraepithelial lymphocytes (12–22). As such, Therapy Institute, Oregon Health & Science University, West Campus, 505 NW IL-15Ra2/2 and IL-152/2 knockout mice typically manifest se- 185th Avenue, Beaverton, OR 97006. E-mail address: [email protected] vere deﬁciencies in these cell subsets (23). IL-15 signals via in- The online version of this article contains supplemental material. teraction with a heterotrimeric receptor complex composed of Abbreviations used in this article: gc, g-chain; GSEA, Gene Set Enrichment Anal- ysis; LN, lymph node; MRM, mitochondrial respiration machinery; NHP, nonhuman IL-15Ra (CD215), IL-2/15Rb (CD122), and CD132 (16, 24–27). primate; RM, rhesus macaque; rsIL-7, recombinant simian IL-7; RT, room temper- IL-15Ra is expressed on APCs (macrophages, monocytes, and ature; TCM, central memory T cell; TEM, effector memory T cell; TM, memory T cell; dendritic cells) and binding of IL-15 to IL-15Ra enables trans T , naive T cell; T , transitional T . N TrM M presentation to a responding cell expressing CD122 and CD132 Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 (28). Because biologically active IL-15 has been shown to exist in

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600065 1184 IMPACT OF ANTI–IL-15 ON LYMPHOCYTE HOMEOSTASIS IN MACAQUES

a soluble form in complex with IL-15Ra (29), it has been ques- have taken the alternative approach of determining the impact of tioned whether IL-15Ra is part of the receptor for IL-15 or part of inhibiting IL-15 activity in vivo with a newly developed rhesu- a heterodimeric cytokine that interacts with the CD122/CD132 sized anti–IL-15 mAb on T cell and NK cell homeostasis in rhesus receptor. In either scenario, the IL-15Ra protein is thought to be macaques (RM). We demonstrate that this rhesusized anti–IL-15 a critical determinant of IL-15 specificity and function. can be repeatedly administered to RM and is highly effective at Although each of these gc cytokines has unique characteristics, long-term inhibition of IL-15 activity in vivo. We further demon- their in vivo activity often manifests considerable overlap. For in- strate that in vivo inhibition of IL-15 activity resulted in a near- stance, IL-2 and IL-15 share the same b receptor (CD122) and are complete depletion of NK cells and a significant decrease in the + + both involved in the initial amplification of Ag-specific T cell re- numbers of circulating CD4 and CD8 TEM with negligible effects sponses, and the regulation of TM development, differentiation, and on the TCM or TN subsets. Strikingly, however, TEM, but not NK cell maintenance (30–32). In addition, both IL-2 and IL-15 induce the numbers, rebounded by proliferative expansion, and in the absence activation and proliferation of NK cells and enhance NK cell cyto- of IL-15 signaling, TEM became increasingly more sensitive to IL-7 lytic activity by inducing the upregulation of effector molecules such signaling. These data suggest that whereas IL-15 signaling is re- as perforin and granzyme B (33–35). Similarly, IL-7 and IL-15 both quired for NK cell homeostasis, TEM can be maintained by other seem to play major, albeit nonexclusive, roles in maintaining pe- cytokines, most likely IL-7, when IL-15 signaling is not available. ripheral TM homeostasis, supporting both TM proliferation and survival (31). Thus, the specific nonredundant roles these gc cytokines Materials and Methods play in controlling various lymphocyte population dynamics in vivo Animals are not completely characterized, a lack of understanding that com- plicates efforts to rationally develop therapeutic strategies based A total of 41 purpose-bred RM (Macaca mulatta) of Indian genetic on their specific biologic activities to enhance immune responses background and free of Macacine herpesvirus 1, D type simian retrovirus, simian T lymphotrophic virus type 1, and SIV infection were used in this to cancer or microbial agents, to promote immune reconstitution study. A group of 30 RM was administered the rhesus recombinant anti– after conditions of lymphopenia (HIV infection, chemotherapy, IL-15 mAb, clone M111 (n = 17) or rhesus recombinant IgG control mAb aging), or to counter pathologic immune responses in the various (n = 13), i.v. once every 2 wk at 20 mg/kg on day 0 and 10 mg/kg on days autoimmune/inflammatory disorders (rheumatoid arthritis, celiac 14 and 21. The neutralizing anti-IL-15 Ab was constructed by grafting the disease, inflammatory bowel disease, multiple sclerosis, and type complementarity-determining regions of mouse anti-human IL-15 mAb, M111 (American Type Culture Collection) into rhesus V region frame- 1 diabetes) linked to dysregulation of these cytokines (36–40). works. mAb was expressed in Chinese hamster ovary cells as full-length Ig Because of its activity on NK cells and Ag-specific cytotoxic with rhesus IgG1 and k constant regions. Two RM were dosed with an T cells, IL-15 is in clinical trials for the treatment of metastatic immunologically silenced form of the same Ab (anti–IL-15 LALA) that malignancies (41). Previous studies have shown that IL-15 can contained the L235A, L236A mutation in the H chain constant region. RM dosed with the anti–IL-15 LALA mAb displayed similar phenotypes to the increase the production of long-lived Ag-specific TM (32, 42, 43) monkeys treated with the nonmutated anti–IL-15 mAb for all parameters and can also induce the migration and redistribution of TM from assayed, and were therefore grouped as anti–IL-15 mAb–treated RM. circulation into tissues (44, 45). In nonhuman primates (NHP), BrdU (Sigma-Aldrich) was prepared as previously described (49) and provision of exogenous IL-15 typically induces an initial brief administered i.v. in three separate doses of 30 mg/kg body weight over a period of lymphopenia followed by lymphocytosis (45–47). Lym- 24-h period before terminal necropsy at $35 d after first anti–IL-15

by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. treatment. A separate group of 11 RM received 6 biweekly doses of rhe- phocytosis is associated with the expansion of NK cells and sus recombinant anti–IL-15 mAb (n = 6) or rhesus recombinant IgG TM (41, 44). However, the TM compartment is quite heterogeneous control (n = 5) at 20 mg/kg on day 0, followed by 10 mg/kg on days 14, 28, and comprises the TCM subset, which is responsible for anam- 42, 56, and 77 concurrently with s.c. administration of rhesus recombinant nestic T cell responses and primarily recirculates between sec- IL-7 at 30 mg/kg on days 35 and 42. All RM were housed at the Oregon National Primate Research Center in accordance with standards of the ondary lymphoid tissues, and the effector-differentiated memory Center’s Institutional Animal Care and Use Committee and the National subsets, transitional TM (TTrM) and effector memory T cells Institutes of Health Guide for the Care and Use of Laboratory Animals. (TEM), which can also migrate to extralymphoid effector sites Measurement of plasma IL-7 concentration (48). In NHP, TEM and TTrM are very responsive to IL-15 in vivo, and studies from our laboratory and others have shown that ex- The concentration of IL-7 in the plasma was measured by ELISA using https://www.jimmunol.org ogenous IL-15 administration can dramatically increase the pro- the IL-7 Quantikine HS kit (R&D Systems) according to manufacturer’s instructions. liferative fraction and absolute numbers of TEM and TTrM in peripheral blood (44, 46, 47). In contrast, TN and TCM are less Flow cytometric analysis and cell sorting responsive to IL-15 administration, and it is still unclear whether Whole blood and mononuclear cells isolated from lymph nodes (LNs), IL-15 plays a direct role in vivo in regulating their homeostasis. bronchoalveolar lavage, bone marrow, spleen, kidney, lung, liver, tonsil, and + Most of these studies have focused on CD8 T cells, and in vaginal and intestinal mucosa were obtained and stained for flow cytometric Downloaded from general, IL-15 has been more closely associated with regulation of analysis as described previously (49, 50). Polychromatic (8- to 12-parameter) CD8+ T than with CD4+ T . However, CD4+ T and T are flow cytometric analysis was performed on an LSR II instrument using M M EM TrM Pacific Blue, AmCyan, FITC, PE, PE-Texas Red, PE-Cy7, PerCP-Cy5.5, also highly responsive to IL-15 in vivo, suggesting that this dis- allophycocyanin, allophycocyanin-Cy7, and Alexa Fluor 700 as the crepancy is largely attributable to the fact that the highly IL-15– available fluorescent parameters. Instrument setup and data acquisition responsive TEM subset comprises a much larger fraction of total procedures were performed as previously described (51). List mode multi- + + circulating T for CD8 than for CD4 lineage cells (44). parameter data files were analyzed using FlowJo software. Delineation of TN M 2 In this study, we sought to determine the specific, nonredundant and TM subsets and criteria for setting + versus markers for CCR5 and Ki- + + 67 expression have been previously described (49, 50, 52–54). In brief, TN role(s) of IL-15 in the regulation of CD4 and CD8 T and NK cell constitute a uniform cluster of cells with a CD28moderate, CCR7+, CCR52, population dynamics in NHP. As indicated earlier, most previous CD95low phenotype, which is clearly distinguishable from the phenotypi- studies addressing IL-15 activity in NHP have assessed the effect cally diverse memory population that is CD95high and may display one or more of the following nonnaive phenotypic features: CD282, CCR72,or of therapeutic administration of IL-15, which results in unnatu- + CCR5 . The TCM,TTrM, and TEM components of the memory subset in the rally high levels of cytokine immediately after administration. blood were further delineated based on the following phenotypic criteria: + + 2 + +/2 + Because such pharmacologic (supraphysiologic) IL-15 levels may TCM (CD28 , CCR7 , and CCR5 ), TTrM (CD28 , CCR7 , and CCR5 ), 2 2 dim not accurately reflect physiologic cytokine function in vivo, we and TEM (CD28 , CCR7 , and CCR5 ). For delineating NK cells in The Journal of Immunology 1185

blood, small lymphocytes were gated to obtain CD32, CD8a+, NKG2a+ extracted from these whole-tissue scans. The percent area of the LN T cell cells that were CD202 and CD142. NK cell subsets were further delin- zone and lamina propria (colon) that stained for each protein/cell type of eated based on CD16 and CD56 expression as previously described (55). interest was quantified using Photoshop CS5 using Fovea tools. For each subset to be quantified, the percentages of the subset within the overall small lymphocyte populations were determined. For quantifica- Microarray analysis tion of peripheral blood subsets, absolute small lymphocyte counts + + For transcriptional analysis, sort-purified CD8 TEM and CD8 TCM from were obtained using an AcT5diff cell counter (Beckman Coulter) and PBMCs, obtained 28–49 d after the first anti–IL-15 or control IgG mAb from these values, absolute counts for the relevant subset were calcu- administration, were resuspended in RLT lysis buffer (Qiagen) and stored lated based on the subset percentages within the light scatter–defined at 280˚C until use. RNA was isolated using RNeasy Micro Kits (Qiagen), small lymphocyte population on the flow cytometer. Baseline values were and the quantity and quality of the RNA was confirmed using a NanoDrop determined as the average of values at days 214, 27, and 0. Results are 2000c (Thermo Fisher Scientific) and an Experion Electrophoresis System. presented as percentage of baseline, with baseline shown as 100%, or Samples (50 ng) were amplified using Illumina TotalPrep RNA amplifi- changes in proliferative fraction, indicated as the difference in the %Ki- cation kits (Ambion). The microarray analysis was conducted using 750 ng + D + 67 ( %Ki-67 ) measured at the designated time points from baseline of biotinylated cRNA hybridized to HumanHT-12_V4 BeadChips (Illu- (0% = no change). mina) at 58˚C for 20 h. The arrays were scanned using Illumina’s iSCAN m For analysis of pSTAT5 expression, whole blood (100 l) was added to and quantified using Genome Studio (Illumina). The analysis of the Ge- polystyrene flow tubes and stained with fluorochrome-conjugated mAbs nome Studio output data were conducted using the R and Bioconductor against CD3, CD4, CD8, CD28, CCR5, CCR7, and CD95, at room tem- software packages. Quantile normalization was applied, followed by a log2 perature (RT) for 30 min. Next, tubes were either left unstimulated or transformation performed using the Bioconductor LIMMA package (57). stimulated with increasing concentrations of IL-7 or IL-15 (ranging from Outlier samples with abnormalities in gene expression based on hierar- 0.5 to 32 ng/ml) for 15 min at 37˚C/5% CO2. Detection of pSTAT5 was chical clustering methods and multidimensional scaling analysis as a assessed with the BD Phosflow staining protocol according to the manu- dimensionality reduction method for the evaluation of similarities or dis- facturer’s instructions. In brief, cells were fixed with the BD Phosflow similarities between samples were identified and removed. The LIMMA Lyse/Fix buffer for 5 min at RT and permeabilized in ice-cold BD Phos- package was used to fit a linear model to each probe and perform (mod- flow perm buffer IV for 5 min at RT. After washing, cells were stained for erated) t tests or F tests on the groups being compared. A Gene Set En- intracellular markers with fluorochrome-conjugated anti-pSTAT5 and anti– richment Analysis (GSEA) (58) using 1000 permutations was performed Ki-67 for 45 min at RT. Cells were washed and flow cytometric analysis on the genes differentially expressed between the groups compared in the was performed on the LSR II flow cytometer. subsets preranked by the decreasing order of the absolute T-statistic. The In vitro cytokine-induced expansion assay canonical pathways of Ingenuity Pathway Analysis software (Ingenuity Systems) were used as the database to perform GSEA. This was followed PBMCs were sort purified using a FACSAria II (BD Biosciences) based on by building modules of related pathways based on at least 25% gene defined phenotypic markers as described earlier and plated in 48-well overlap (Jaccard index [59] .25%) between pathways using the enrich- plates in 1 ml of R10 media (RPMI 1640 [HyClone], 10% FBS, 100 U/ml ment map (60) strategy and representing the genes present in at least 25% penicillin, 10 mg/ml streptomycin [Sigma-Aldrich], 200 mMofL-glutamine of the pathways in the module. To control the expected proportions of false [Sigma-Aldrich]) at a density of 150,000–300,000 cells/ml. IL-7 or IL-15 positives, the FDR for p values was calculated using the Benjamini and was added at a concentration of 50 ng/ml to the cultures and incubated at Hochberg method implemented in LIMMA. The complete data set is + 37˚C/5% CO2 for 14 d alone or in the presence of 10% sort-purified CD14 available at the Gene Expression Omnibus microarray repository (http:// monocytes. After 7 d, the culture was resuspended and 0.5 ml was removed www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=mbqtaouqztynzsz&acc= for phenotypic analysis by flow cytometry. An equal amount of fresh R10 GSE76797), accession number GSE76797. was added back to the remaining culture and incubated at 37˚C/5% CO2 for a further 7 d. On day 14, the entire culture was harvested for phenotypic Statistics analysis by flow cytometry. To investigate whether the administration of anti–IL-15 mAb blocks sig- by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. Abs and cytokines naling in circulating lymphocytes, we estimated the EC50 (the dose where the response is the midpoint between the maximum and minimum) for The following Abs were used for flow cytometry: CD3 Alexa 700 (SP34-2; each RM at given time points (days after mAb) using the four-parameter BD Biosciences), CD4 AmCyan (L200; BD Biosciences), CD8 PerCP- logistic model often referred to as an Emax model, followed by repeated- Cy5.5 (SKI; eBioscience), CD8 AmCyan (SKI; BD Biosciences), CD28 measures ANOVA to evaluate the effect of anti–IL-15 mAb with EC50 as a PE-Texas Red (CD28.2; Beckman Coulter, BD Biosciences), CD95 PE response variable and anti–IL-15 mAb–treated status as between-group (DX2; BD Biosciences, eBioscience), CCR5 allophycocyanin (3A9; BD factor, and days after Ab as within-group factors. Longitudinal analysis Biosciences), Ki-67 FITC (B56; BD Biosciences), CD56 PerCP-Cy5.5 of STAT5 phosphorylation, peripheral blood TN and TM (including TCM, (MEM-188; Invitrogen), CD16 Pacific Blue (3G8; BD Biosciences, Bio- TTrM, and TEM subsets) counts and proliferation, peripheral blood NK cell legend), CD20 allophycocyanin-Cy7 (L27; BD Biosciences), HLA-DR counts, and plasma IL-7 levels were evaluated using repeated-measures PE-Texas Red (TU36 [Invitrogen], Immu357 [Beckman Coulter]), ANOVA with anti–IL-15 mAb and IgG control mAb–treated groups as https://www.jimmunol.org NKG2A PE (Z199; Beckman Coulter), CD14 FITC (M5E2; BD Biosci- between-group factors and time points as within-group factors, because in a ences, R&D Systems), STAT5 PE (47/Stat5 [pY6]; BD Biosciences), BrdU typical experiment using repeated measures two measurements taken at ad- FITC (B44; BD Biosciences), and BrdU allophycocyanin (B44; BD Bio- jacent times are more highly correlated than two measurements taken several sciences). Anti-CCR7 (150503) was purchased as purified Ig from R&D time points apart. Due to the limited sample size, a simpler covariance Systems, conjugated to biotin using a Pierce Chemical biotinylation kit, structure, first order autoregressive, was used as correlation within each an- and visualized with streptavidin–Pacific Blue (Invitrogen). Rhesus recom- imal. Tukey–Kramer adjustment was used to control for multiple compari- binant anti–IL-15 and rhesus recombinant control IgG1 mAb were pro- sons. Differences in the number of pSTAT5+ cells/mm2 in peripheral LNs

Downloaded from vided through the National Institutes of Health’s Nonhuman Primate between pretreatment and posttreatment were evaluated using Wilcoxon Reagent Resource Program. Recombinant simian IL-7 (rsIL-7) was pro- signed rank tests. The difference in CD3+ caspase-3+ in colon and peripheral vided by Cytheris SA (Issy-Les-Moulineaux, France). Rhesus rIL-15 was LN via immunostaining and BrdU incorporation in CD4+ and CD8+ Tcell provided by Francois Villinger (Emory University) through the Resource subsets between anti–IL-15 and IgG control mAb–treated groups was com- for Nonhuman Primate Immune Reagents. pared using Mann–Whitney U test. Data were analyzed using SAS 9.4. The , Immunohistochemistry p values 0.05 were considered to be statistically significant. Immunohistochemistry was performed as previously described (56). Abs Results used in this study were mouse monoclonal anti-human IL-15 (clone Differential signaling of IL-15 and IL-7 on NK and T cell BDI150; Antibodies Online), rabbit monoclonal anti-active caspase-3 subsets in vitro (clone 5A1E; Cell Signaling Technologies), rabbit monoclonal anti- pSTAT5 (1:100; clone C11C5; Cell Signaling Technologies), and rabbit As a first step in characterizing the specific, nonredundant in vivo monoclonal anti-human CD3 (clone SP7; Labvision/Thermo Fisher Sci- role(s) of IL-15 in controlling NHP T cell and NK cell population entific). All stained slides were scanned at high magnification (3200) + + using the ScanScope CS System (Aperio Technologies) yielding high- dynamics, we determined the extent to which RM CD4 and CD8 resolution data from the entire tissue section. Representative regions of T cell and NK cell subsets, studied directly ex vivo in peripheral interest (250–500 mm2) were identified and high-resolution images blood, could respond to IL-15 and IL-7. Because common gc 1186 IMPACT OF ANTI–IL-15 ON LYMPHOCYTE HOMEOSTASIS IN MACAQUES

cytokines mediate signal transduction via the JAK/STAT signaling These results demonstrate that lymphocyte populations differ in pathway leading to the phosphorylation of the transcription factor their response to early signal transduction by IL-7 versus IL-15, and STAT5 (61), we quantified the fraction of cells within each phe- suggest that IL-7 and IL-15 preferentially signal pre-effector and notypically defined subset that phosphorylated STAT5 (e.g., be- effector populations, respectively. We next sought to determine the came pSTAT5+) within 15 min after exposure to increasing degree to which this differential signaling translates to downstream concentrations of these two gc cytokines. As shown in Fig. 1A and gene expression and homeostatic regulation by examining the 1B, both IL-15 and IL-7 induced pSTAT5 in TN and all TM sub- ability of these cytokines to support survival and proliferative sets; however, there were clear differences among these subsets expansion of sort-purified TN,TCM, and TEM during 7 and 14 d of with respect to their dose response to these cytokines, and the in vitro culture. Because the pattern of STAT5 phosphorylation to fraction of responding cells at the optimal dose. TN and TCM (both IL-7 and IL-15 among these differentiation-defined subsets was CD4+ and CD8+) were more responsive to IL-7 than to IL-15, similar between the CD4+ and CD8+ lineages (Fig. 1A, 1B), we with .60% of cells in these subsets expressing pSTAT5 at 4 ng primarily focused on CD4+ T cells for these studies. In this in vitro + of IL-7 versus ,12% of TN and TCM at the same dose of IL-15. In CD4 T cell model system, responses to IL-15 were generally + + contrast, both CD4 and CD8 TEM showed increased respon- more robust than to IL-7, but responsiveness to both cytokines was siveness to IL-15 compared with IL-7, with pSTAT5 expression differentiation dependent (Fig. 2). TEM were again found to be between 60 and 80% at 8 ng, in contrast with ,40% with the same highly responsive to IL-15 compared with IL-7, with .60% of dose of IL-7. This reduced response to IL-7 is likely due to the low IL-15–treated cells expressing the proliferation Ag Ki-67 by day 7 + + expression of CD127 on TEM (62). Interestingly, CD4 and CD8 of culture, compared with background Ki-67 expression after IL-7 TTrM responded to IL-7 and IL-15 at similar levels, reflecting their treatment (Fig. 2A). IL-7 and IL-15 induced similar, modest in- + intermediate phenotype between TCM precursors and effector- creases in Ki67 expression by CD4 TCM, whereas IL-7 was more differentiated TEM (Supplemental Fig. 1) (54). NK cells showed effective than IL-15 in inducing TN proliferation. Next, based on an even larger difference in responsiveness to IL-7 versus IL-15. the concept that IL-15 might be more efficiently presented in trans, Although all circulating NK cell subsets (defined by CD16 and we cultured the same cells with IL-15 or IL-7 in the presence of sort- CD56 [55]) responded robustly to IL-15 with induction of pSTAT5 purified CD14+ monocytes (63, 64). In the presence of monocytes, + expression in .80% of cells at 16 ng (Fig. 1C), only the CD56 we saw modestly enhanced responsiveness of TN,TCM,andTEM to 2 CD16 “regulatory” NK cell subset showed any response to IL-7 IL-7 and profoundly increased responsiveness of TN and TCM to IL-15 + stimulation at the tested doses, and this response required a 4-fold (Fig. 2B). Interestingly, the CD4 TEM proliferative response to IL-15 higher dose of IL-7 than the response of the same cells to IL-15. was similar with or without monocytes, suggesting that trans by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. https://www.jimmunol.org Downloaded from

FIGURE 1. Comparative analysis of IL-7 and IL-15 signaling in T cell and NK cell populations. (A) Representative histograms showing the induction of pSTAT5 in the indicated peripheral blood T cell subsets after ex vivo stimulation with 8 ng/ml IL-7 (blue), 8 ng/ml IL-15 (red), or no stimulation (gray). (B)Dose– response curves showing the induction of pSTAT5 in the indicated peripheral blood T cell subsets after ex vivo stimulation with 0, 0.5, 1, 2, 4, 8, 16, or 32 ng/ml of IL-7 (n = 14) or IL-15 (n = 14). Results (mean + SEM) are shown as change from baseline. (C) Induction of pSTAT5 in the indicated peripheral blood NK cell subsets after ex vivo stimulation with 0, 0.5, 1, 2, 4, 8, 16, or 32 ng/ml of IL-7 (n =8)orIL-15(n = 8). Results (mean + SEM) are shown as change from baseline. The Journal of Immunology 1187

FIGURE 2. Representative dot plots demonstra- tive of eight separate experiments, showing induction + of Ki-67 expression on CD4 TN,TCM, and TEM. CD4+ T cell subsets were puriﬁed from PBMCs by multiparameter cell sorting and cultured in media containing (A) rIL-15 or rIL-7 at 50 ng/ml, alone or (B) in the presence of 10% highly puriﬁed CD14+ monocytes. After 7 and 14 d, cultures were analyzed for Ki-67 expression.

presentation or other monocyte-derived signals are not required on whole blood at various time points posttreatment. From days 1 for maximal TEM responsiveness. Similar IL-15 responsiveness was through 35 posttreatment, the ability of IL-15 to induce pSTAT5 + + + demonstrated for CD8 TEM (Supplemental Fig. 2A). expression in CD4 and CD8 T cells ex vivo in anti–IL-15–treated by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. Collectively, these in vitro studies demonstrate that TN and TCM RM was signiﬁcantly reduced (essentially abrogated) compared are more responsive to IL-7 than TEM, whereas TEM are more with baseline and control IgG-treated RM (p , 0.0001; Fig. 3B, responsive to IL-15. However, IL-15 can induce robust prolifer- Supplemental Fig. 3A, 3B). To further assess the in vivo activity of ation in TN and TCM in the presence of monocytes, suggesting a our anti–IL-15 mAb treatment, we performed immunohistochem- more pleotropic role for this cytokine. Interestingly, culture of ical analysis of LN sections from both control IgG mAb–treated TN and TCM with IL-15 in the presence of monocytes induces and anti–IL-15 mAb–treated RM using the original mouse anti– upregulation of CCR5 and downregulation of CCR7 (Supplemental IL-15 mAb M111. As expected, there was a substantial reduction in Fig. 2B), consistent with induction of effector memory differenti- IL-15 immunoreactivity in LNs of anti–IL-15–treated RM com-

https://www.jimmunol.org ation (50, 65). Thus, IL-15 may both maintain the homeostasis of pared with control RM (Fig. 3C), demonstrating either absence of pre-existing TEM and, under certain conditions, drive TEM differ- IL-15 posttreatment or blocking of immunostaining by the ad- entiation from noneffector-differentiated precursors. ministered function-abrogating mAb (consistent with in situ neu- Anti–IL-15 specifically blocks IL-15 signal transduction in vivo tralization). As a final confirmation of the in vivo effectiveness of our anti–IL-15 mAb, we determined whether anti–IL-15 treatment To investigate the role of IL-15 in T cell and NK cell homeostasis reduced JAK/STAT signaling in situ by measuring pSTAT5 ex- in vivo, we developed a “rhesusized” anti–IL-15 mAb that is pression in LN sections. As shown in Fig. 3D and Supplemental Downloaded from suitable for repeated administration to RM. This mAb was based Fig. 3C, we observed that RM treated with the anti–IL-15 mAb, but on the mouse, anti-human IL-15 mAb clone, M111, which is not the IgG control mAb, had a significant reduction in pSTAT5 cross-reactive with RM IL-15 (66). Rhesusization was accom- detection posttreatment (untreated animals: p = 0.38, anti–IL-15– plished by exchanging the mouse amino acid sequences in the treated RM: p , 0.0001). This decline in pSTAT5 expression in- constant regions and variable binding surfaces for rhesus amino dicates a reduction in JAK/STAT signal transduction specific to acid sequences, leaving V region sequences responsible for IL-15 IL-15. A complete inhibition of pSTAT5 in tissues was not ex- recognition unchanged (67, 68). We hypothesized that repeated pected because other gc cytokines such as IL-2 and IL-7, which are administrations of this rhesusized M111 mAb would block IL-15 not inhibited by M111, also signal via the JAK/STAT pathway. activity in vivo and result in changes to T and/or NK cell pop- Taken together, these results clearly demonstrate that the rhesusized ulation dynamics that would reflect physiologic IL-15 function. To anti–IL-15 mAb can effectively inhibit IL-15 signaling in vivo. initially test this approach, we administered rhesus anti–IL-15 or a rhesus IgG control mAb to RM every other week for 6 wk before IL-15 blockade results in near-complete NK cell depletion necropsy, as shown in Fig. 3A. We assessed IL-15 signaling inhi- Having demonstrated the in vivo activity of the rhesusized anti– bition by performing pSTAT induction analysis with rhesus rIL-15 IL-15 mAb, we next examined the effect of this IL-15 blockade on 1188 IMPACT OF ANTI–IL-15 ON LYMPHOCYTE HOMEOSTASIS IN MACAQUES by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. https://www.jimmunol.org

Downloaded from FIGURE 3. Anti–IL-15 administration speciﬁcally blocks IL-15 signaling in RM. (A) Schematic representation of the anti–IL-15 treatment schedule used in this study. Healthy RM received 20 mg/kg of anti–IL-15 or IgG control Ab on day 0 and 10 mg/kg on days 14 and 28. Twenty-four hours before

necropsy, all RM received three doses of BrdU at 30 mg/kg. (B) Showing the change in pSTAT5 in peripheral blood TN,TCM,TTrM, and TEM, after ex vivo stimulation with 8 ng/ml IL-15 in anti–IL-15 mAb–treated (n = 9) or IgG control mAb–treated (n = 5) RM. Results (mean + SEM) are shown as percentage of baseline, and significance was assessed as described in Materials and Methods.(C) Representative images from immunohistochemical analysis performed on LN sections obtained from IgG control mAb–treated (top panel) and anti–IL-15 mAb–treated (bottom panel) RM using a mouse mAb that is cross-reactive with rhesus IL-15 (original magnification 3200). (D) Quantification of the number of pSTAT5+ cells/mm2 in peripheral LNs of RM treated with anti–IL-15 mAb (n = 15) or IgG control mAb (n = 8). Significance was evaluated using Wilcoxon signed rank tests.

peripheral lymphocyte population dynamics. As shown in Fig. 4A, of baseline through day 35. Because the cytotoxic CD16+ CD562 compared with control mAb treatment, anti–IL-15 mAb admin- NK cell subset constitutes the most abundant population in the istration resulted in a near-complete loss of NK cells in the blood peripheral blood of RM (55), it is not surprising that the post- in the ﬁrst 2 wk of treatment. Importantly, the absolute numbers of treatment dynamics of this subset closely followed the overall total NK cells in anti–IL-15–treated RM were maintained at ,5% NK cell population with absolute counts declining to ,14% of The Journal of Immunology 1189

FIGURE 4. IL-15 blockade induces profound depletion of NK cells in blood and tissues. (A) Quantification of absolute NK cell counts, including CD16+ CD562,CD162 CD56+, and CD162 CD562 subsets in blood after anti–IL-15 mAb (n = 17) or IgG control mAb (n = 13) treatment. Significance was assessed as described in Materials and Methods. Results (mean + SEM) are shown as percentage of baseline. (B) Dot plots showing CD32 NKG2A+ NK cells in peripheral blood, spleen, liver, lung, kidney, and tracheobronchial LN (LN TB) of two representative RM that received either anti–IL-15 mAb or IgG control mAb before necropsy. (C) Lymphocytes isolated from the indicated tissues were analyzed to determine frequencies of total NK cells (CD32, CD8a+, NKG2A+) within the overall small lymphocyte population in anti–IL-15 mAb– treated (n = 9) or IgG control mAb–treated (n =5) RM. Histograms show mean frequencies of the NK cells as a fraction of total lymphocytes in each tissue; error bars show SEM. by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. baselinebyday7and,3% of baseline at day 35. The depletion impact of IL-15 blockade on T cell population dynamics varied by of the other NK subsets was substantial but less complete. The differentiation-defined subset. Anti–IL-15 mAb treatment had a 2 2 + + CD16 CD56 NK subset declined to ∼10% of baseline by day 14, negligible effect on circulating CD4 and CD8 TN, because but recovered to ,30% of baseline by day 35, despite ongoing absolute counts and proliferative responses in anti–IL-15 mAb– therapy. Interestingly, the largely regulatory CD162 CD56+ NK treated RM were not significantly different from what was ob- subset was least affected by IL-15 blockade, showing a maximal served in control RM (Fig. 5A). A similar pattern was observed depletion to ∼25% of baseline by day 28. We would note that with the TCM subsets, because IL-15 blockade had no significant 2 + + + + CD16 CD56 NK cells were the only NK subset to respond to effect on CD4 or CD8 TCM absolute counts and CD8 TCM https://www.jimmunol.org IL-7 signaling in our in vitro assay (Fig. 1C), suggesting the pos- proliferation, and resulted in only marginal enhancement of CD4+ sibility that IL-7 may compensate for IL-15 signaling inhibition to TCM proliferation. However, IL-15 blockade had a profound effect 2 + partially maintain CD16 CD56 NK cell homeostasis (see later). on TEM dynamics, with anti–IL-15 administration resulting in a + + To more comprehensively assess the extent of NK cell deple- rapid reduction in CD4 and CD8 TEM absolute counts to ∼30% tion after anti–IL-15 treatment, we isolated cells from peripheral of baseline by day 14 posttreatment (CD4+: p = 0.0006, CD8+: lymphoid and extralymphoid tissues at necropsy of the anti– p , 0.0001). Strikingly, starting about day 14, this depletion was Downloaded from IL-15–treated versus control-treated RM, and determined the countered by a dramatic increase in the fraction of proliferating 2 + + + + fraction of NK cells (CD3 CD8a NKG2A lymphocytes) among TTrM and TEM, such that absolute CD4 and CD8 TEM counts in total lymphocytes in each tissue. As shown in Fig. 4B and 4C, blood were restored to pretreatment (CD4+) or near-pretreatment NK cells were dramatically reduced in anti–IL-15 mAb–treated levels (CD8+) by day 35. Whereas anti–IL-15 treatment signifi- + + RM compared with controls. Taken together, these data demon- cantly increased the proliferative fraction of CD4 and CD8 TTrM + + strate that anti–IL-15 mAb administration can induce systemic (CD4 : p , 0.0001, CD8 : p , 0.0001), absolute TTrM counts depletion of NK cells in RM, with the implication that both cir- remained unchanged, possibly reflecting a proportion of TTrM culating and tissue-resident NK cells in RM are highly dependent differentiating to TEM. Indeed, we note that the relative propor- on IL-15 signaling for their homeostatic stability. tions of all TM subsets in the blood before anti–IL-15 administration returned to baseline by day 35 posttreatment (Fig. 5B). IL-15 blockade results in selective perturbation of TEM Taken together, these data demonstrate a key, specific role for IL-15 homeostasis signaling for TEM homeostasis, but also indicate that a significant + + As expected based on the earlier-described differential sensitivity proportion of TEM,bothCD4 and CD8 , can persist in the pres- of T cells to IL-15 signaling according to differentiation status, the ence of IL-15 signaling inhibition (similar to the CD162 CD56+ 1190 IMPACT OF ANTI–IL-15 ON LYMPHOCYTE HOMEOSTASIS IN MACAQUES

FIGURE 5. Comparison of T cell dynamics in blood of anti–IL-15– treated RM. (A) Absolute counts and proliferative fraction of CD4+ and + CD8 TM, including the TCM,TTrM, and TEM subsets in blood of RM treated with anti–IL-15 mAb (n =17) or IgG control mAb (n = 13). Results (mean + SEM) are shown as percentage of baseline, or for percentage of Ki-67+, change (D) from baseline with signiﬁcant p values shown as: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. Signiﬁcance of difference between treatment groups was assessed as described in Materials and Methods. (B) Pie charts showing the relative + + proportions of CD4 and CD8 TCM, TTrM, and TEM subsets as fraction of total TM in the blood of anti–IL-15– treated RM (n = 16) before treatment and days 14 and 35 after anti–IL-15 mAb treatment. by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. https://www.jimmunol.org

NK cell subset), suggesting other regulatory molecules could play treatment has negligible effects on steady-state TCM homeostasis a role in modulating peripheral TEM regeneration and stability in (Fig. 5A). There was also no difference between control mAb– and the absence of IL-15. anti–IL-15–treated RM in the frequencies of BrdU-labeled CD8+ + We next determined whether the robust increases in CD4 and TTrM, although we did observe significant differences in the + + + CD8 TEM and TTrM proliferation observed in response to anti– %BrdU CD4 TTrM in the blood (p = 0.018), spleen (p = 0.044), Downloaded from IL-15 mAb treatment were confined to cells in the blood or were a lung (p = 0.024), and liver (p = 0.018) with anti–IL-15 mAb reflection of cells proliferating in tissues before entering into the treatment. Strikingly, however, the TEM compartment saw the most peripheral circulation. To address this question, we used in vivo dramatic increases in cell turnover in the presence of IL-15 signaling BrdU labeling to label all cells in S phase around the time of inhibition, which was apparent for both the CD4+ and CD8+ Tcell peak proliferation (see Fig. 3A). BrdU incorporation offers a more lineages. Anti–IL-15–treated RM showed increased frequencies of + + accurate indication of the location and timing of cell proliferation BrdU CD8 TEM within most peripheral LN (iliosacral: p = 0.024 (specifically delineating S phase of the mitotic cycle) than Ki-67 and tracheobronchial: p = 0.018), as well as within the lung + + expression, which is maintained for several days after cell division (p = 0.045), whereas frequencies of BrdU CD4 TEM were sig- during which time cell migration might occur (50). We adminis- nificantly higher in blood (p = 0.044), lung (p = 0.024), kidney tered three doses of BrdU within 24 h before necropsy to label cells (p = 0.045), and liver (p = 0.018) of anti–IL-15–treated animals. + + synthesizing DNA in this 24-h period. We observed that the fre- Interestingly, the frequencies of BrdU-labeled CD4 and CD8 TEM + + quencies of BrdU-labeled CD4 and CD8 TCM in all tissues were in the intestinal lamina propria (including the colon, ileum, and je- unchanged in anti–IL-15 mAb–treated RM compared with controls junum) and vaginal mucosa were not significantly different between (Fig. 6A), substantiating our previous conclusion that anti–IL-15 anti–IL-15 mAb– and IgG control mAb–treated RM. The Journal of Immunology 1191 by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. https://www.jimmunol.org Downloaded from

FIGURE 6. IL-15 blockade increases TEM turnover in vivo. (A) RM treated with anti–IL-15 mAb (n = 7) or IgG1 control mAb (n = 5) were i.v. administered three doses (30 mg/kg) of BrdU 24 h before necropsy. Lymphocytes were isolated from the indicated tissues and further analyzed for T cell + + + markers and BrdU positivity. Results (mean + SEM) are shown as the percentage of BrdU cells (CD4 or CD8 TCM,TTrM, and TEM) determined for each tissue. Significance was evaluated using Mann–Whitney U test (*p , 0.05). (B and C) Quantification of the T cell population within the colon lamina propria (LP) as determined by (B) the percent area of the LP that stained CD3+ and (C) extent of apoptosis measured by caspase-3+ within the colon and (D) LNs via immunostaining in RM after anti–IL-15 mAb or IgG control mAb treatment. The significance in these parameters between groups was evaluated using Mann–Whitney U test. (E and F) Images show active caspase-3 expression (red, original magnification 3200) in the colon (E) and LNs (F) of three representative RM treated with either the IgG control mAb (top images) or anti–IL-15 mAb (bottom images).

Collectively, these data suggest that, in the presence of IL-15 subsequently are replaced by TEM cells generated by a homeo- blockade, a signiﬁcant proportion of TEM initially undergo apo- static proliferative response that is IL-15 independent (e.g., occurs ptosis because of the abrupt withdrawal of IL-15 signaling, but despite ongoing IL-15 blockade). Although this homeostatic 1192 IMPACT OF ANTI–IL-15 ON LYMPHOCYTE HOMEOSTASIS IN MACAQUES

+ response returns the numbers of circulating TEM back to baseline greater than those differentially expressed in the CD8 TCM (2225 genes) 216 2 levels, it does not appear to restore the TEM compartment to (p , 2.2 3 10 , using a Pearson x test for equality of pro- normal because we observed a significant reduction in CD3+ portions). Strikingly, IL-15 blockade did not induce changes in the T cell density in the colonic lamina propria (p , 0.0001) in anti– expression of genes associated with the MRM pathway in TCM, IL-15 mAb–treated RM compared with control RM (Fig. 6B). suggesting no disruption to their steady-state mitochondrial ac- Notably, the blood TEM compartment is very small compared with tivity. However, as shown by the module enrichment in TCM,we the tissue effector site TEM compartment represented by the co- did observe an upregulation of proapoptotic genes (BID, APAF1, lonic lamina propria, and thus the latter is a much more accurate CASP8, CASP3) in this cell type in anti–IL-15 mAb–treated RM, indicator of the status of the overall TEM population than the former possibly reflecting an increase in cell death in a fraction of TCM (which can be “reconstituted” by relatively few cells). In addition, induced to differentiate in the absence of IL-15 signaling. Interest- we observed a significant increase in caspase-3 expression within ingly, we also observed the enrichment of the S1P1 pathway in TCM lymphocytes of both colonic lamina propria (p = 0.0087) and LN that regulates lymphocyte egress from the LN (72) (Supplemental Tcellzones(p = 0.02) (Fig. 6C–F), suggesting increased and Table I). However, these differences did not translate to signifi- sustained levels of T cell apoptosis. Thus, the compensatory mecha- cant changes in TCM population dynamics in vivo, because absolute + nism that restores circulating TEM numbers in the absence of IL-15 counts and proliferative fractions of CD8 TCM were statistically results in a high turnover state that is unable to completely reestablish indistinguishable between anti–IL-15 mAb–treated RMs and controls normal TEM homeostasis. (Fig. 5A).

IL-15 blockade disrupts pathways that regulate T cell TEM become increasingly sensitive to IL-7 in response to IL-15 activation, cell cycle entry, and survival blockade To further characterize the impact of IL-15 signaling inhibition on Collectively, gene expression profiling demonstrates the dramatic TEM homeostasis, we performed transcriptomic profiling on sort- effect IL-15 blockade has on peripheral TEM,incomparisonwiththe + purified CD8 TEM obtained at the approximate peak of TEM TCM. Somewhat paradoxically, in the absence of IL-15 signaling, + + proliferation in the blood. These studies demonstrated that CD8 circulating CD8 TEM predominantly display the induction of genes TEM from anti–IL-15 mAb–treated RM showed significant dif- involved with T cell activation, cell cycle progression, and mito- ferences with control IgG mAb–treated RM with respect to gene chondrial activity, raising the possibility that in the periphery, TEM expression in several signal transduction pathways and transcrip- are responding to another proproliferative cytokine. In this regard, tional nodes that regulate proximal T cell activation, cell cycle we asked whether IL-7 might be supporting TEM homeostasis in the entry, and cell survival/cell death (Fig. 7A, 7B). With regard to absence of IL-15. IL-7 is the most likely candidate to act as a T cell activation, components of TCR signaling (CD3D, CD3E), compensatory cytokine for IL-15 because of its bioavailability in activation markers such as MHC molecules, and transcription the blood and LN (73–75). Notably, IL-15 blockade did not change factors downstream of TCR signaling (REL, CIITA) (69) were all plasma levels of IL-7 (Fig. 8A), suggesting that anti–IL-15 mAb + upregulated in CD8 TEM from anti–IL-15 mAb–treated RM treatment did not increase overall production of IL-7. We then (Fig. 7A). Genes associated with cell cycle progression (ATM, determined whether TEM proliferating in responsetoanti–IL-15mAb E2F2, PIK3CB, PIK3CG, PIK3R4, PIK3C2A) were also upreg- treatment manifest increased sensitivity to IL-7. To address this by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. ulated in these cells, whereas the cyclin-dependent kinase inhib- question, we performed ex vivo pSTAT induction analysis on whole itor, CDKN2B/p15, which inhibits cell cycle G1 progression, was blood at various time points after anti–IL-15 mAb treatment with 4 downregulated (Fig. 7B). In addition, genes associated with the ng of IL-7 (this dose was selected because it reflected the minimum mitochondrial respiration machinery (MRM) pathway, including dose that elicited a maximal response in most T cell subsets in our NDUFV1, NDUFA2, UQCR1, UQCRB, and ATP5C1, were sig- in vitro assay; Fig. 1B). Interestingly, we observed a significant in- + nificantly upregulated in CD8 TEM from the anti–IL-15 mAb– creaseinpSTAT5expressioninresponsetothisdoseofIL-7inboth + + treated RM (Fig. 7C). This was further supported by the upregulation CD4 and CD8 TEM from anti–IL-15 mAb–treated RM compared of genes for enzymes with antioxidant activity, such as PRDX3 with controls (CD4+: p = 0.0073, CD8+: p = 0.0016) (Fig. 8B). and GPX4, which protect cells from death by their effect on lipid Importantly, this increase in IL-7 responsiveness was observed only https://www.jimmunol.org peroxidation (70). All of these changes in TEM from anti–IL-15 in the TEM subset, because the D pSTAT5 expression in TN,TCM,or mAb–treated RM are consistent with their increased proliferation TTrM after IL-7 stimulation was statistically indistinguishable be- observed in vivo (Fig. 5A). However, we also noted concomitant tween both groups. changes in gene pathways associated with T cell survival, in To determine whether anti–IL-15 treatment increases IL-7 re- particular the upregulation of cells that promote cell death, in- sponsiveness in TEM in vivo, we designed an in vivo experiment cluding Bax, Fas, and Casp6 (p53 pathway) and Cabin1 and Mef2 in which anti–IL-15 mAb–treated or IgG control mAb–treated Downloaded from (Nur77 signaling pathway) (71) (Supplemental Table I). In addi- RM were treated with rsIL-7 (11, 62). RM received three doses of tion, disruption of cell survival mechanisms was further shown by anti–IL-15 or IgG control mAb biweekly followed by two doses the downregulation of BCL-2 (Fig. 7B). Collectively, these ob- of rsIL-7 (30 mg/kg) 1 wk apart and then a further three doses of servations suggest that IL-15 signaling inhibition results in in- anti–IL-15. To ensure a steady-state of IL-15 neutralization at the duction of TEM activation and proliferation, but at the same time time of IL-7 dosing, rsIL-7 was administered between the third and results in transcriptional changes that may limit the long-term TEM fourth doses of anti–IL-15. As shown in Fig. 9A, rsIL-7 induced a + + survival. 2-fold increase in the absolute numbers of CD4 and CD8 TCM in + Transcriptional profiling of CD8 TCM also revealed differences the blood, which was similar between anti–IL-15 mAb–treated RM in gene expression pathways between anti–IL-15 mAb–treated and controls, further supporting our previous observations that and IgG control mAb–treated RM (Fig. 7D); however, these dif- IL-15 blockade has negligible effects on steady-state TCM ho- ferences involved pathways and genes distinct from those ob- meostasis (Figs. 5A, 6A). In contrast, there was a greater increase + + served in TEM. In addition, the number of genes differentially in both CD4 and CD8 TEM absolute counts in the blood of anti– expressed between anti–IL-15 mAb–treated RM versus IgG con- IL-15 mAb–treated RM in response to IL-7, which was significant + trol mAb–treated RM in CD8 TEM (2808 genes) was significantly on days 10 (p = 0.016), 12 (p = 0.003), and 14 (p , 0.001) for The Journal of Immunology 1193 by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. https://www.jimmunol.org

+ + FIGURE 7. Transcriptomic proﬁling on sort-puriﬁed CD8 TEM and CD8 TCM obtained from anti–IL-15 mAb–treated RM and compared with samples taken from RM treated with the IgG control mAb. GSEA was performed to identify pathways that are enriched at p , 0.05 and for grouping pathways into functional modules. The genes represented in the heatmaps are enriched in at least 50% of the gene sets of the module (if the number of gene sets in the module is #4) and genes enriched in at least 25% of the gene sets of the module (if the number of gene sets in the module is .4). The color scale of the + pathways represent the –log10(p value) of the pathway (A, B, and D). (A) Module 1 enriched in CD8 TEM is representative of T cell activation. (B)

Downloaded from + + Module 2 enriched in CD8 TEM is representative of cell cycle progression. (C) Representation of the MRM pathway in CD8 TEM upregulated in the anti– + IL-15 mAb–treated RM compared with IgG control mAb–treated RM. (D) Module enriched in CD8 TCM is representative of upregulation of proapoptotic signaling and downregulation of type I IFN activity with blockade of IL-15.

CD4+ andondays10(p = 0.002) and 12 (p = 0.0069) for CD8+.This of STAT1 and SOCS3 (known to be downstream of IL-7 and other increase in absolute TEM counts peaked by day 14, before declining cytokines), suggesting engagement of this pathway (Fig. 9B). Alto- to levels similar to controls by day 28. These data strongly support gether these data support the involvement of secondary gc receptor the concept that TEM become more responsive to IL-7 in vivo in the cytokine signaling in supporting TEM homeostasis in the presence of absence of IL-15 signaling. This increased sensitivity to IL-7 could IL-15 signaling inhibition. help support the rapid TEM proliferative expansion observed after anti–IL-15 treatment. Consistent with this interpretation, the tran- Discussion + scriptional analysis of peripheral blood CD8 TEM described earlier IL-2, IL-7, and IL-15 share a common gc receptor and mediate also showed an upregulation of genes associated with the JAK/STAT most of their immunoregulatory function through activation of the pathway in anti–IL-15–treated RM, highlighted by the upregulation JAK/STAT signaling pathway. Because of these common features, 1194 IMPACT OF ANTI–IL-15 ON LYMPHOCYTE HOMEOSTASIS IN MACAQUES

FIGURE 8. TEM are more responsive to IL-7 in the presence of IL-15 signaling inhibition. (A) Plasma collected at indicated time points after anti–IL-15 mAb (n = 6) or IgG control mAb (n = 4) treatment was analyzed for IL-7 levels by ELISA. Results (mean + SEM) are shown as percent change from baseline in IL-7 concentration. (B) Change in pSTAT5 expression in

peripheral blood TN,TCM,TTrM, and TEM after ex vivo stimulation with 4 ng of IL-7 in RM treated with anti–IL-15 mAb (n = 9) or IgG control mAb (n = 5). Signiﬁcance of difference in all parameters was assessed as described in Materials and Methods (*p , 0.05).

the downstream activities of these cytokines often overlap, but compensate for IL-15 signaling inhibition, partially supporting nevertheless, each has its own biologic role. In this study, we sought their homeostatic maintenance. + + to characterize the distinct role IL-15 plays in maintaining The impact of IL-15 blockade on both CD4 and CD8 TEM was by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. lymphocyte population dynamics in NHP. To achieve this, we also both dramatic and specific, revealing a unique aspect of TEM developed an RM model of in vivo IL-15 blockade that involved homeostasis. In the absence of IL-15 signaling, absolute CD4+ and + repeated administration of a rhesusized anti–IL-15 mAb. We hy- CD8 TEM (but not TN,TCM,orTTrM) counts in blood rapidly pothesized that blocking IL-15 signaling would result in substantial declined to ,70% of baseline levels. However, starting between changes in lymphocyte population dynamics specific to IL-15 7 and 14 d after treatment initiation, this initial TEM decline was function. Similar to previous reports in humans and cynomolgus countered by a burst of sustained proliferation in both TEM and macaques, anti–IL-15 was well tolerated, with no adverse side ef- TTrM that corresponded with an increase in TEM counts in blood fects observed with repeated dosing. We directly demonstrated that back to baseline or near-baseline levels. Although some anti-gc anti–IL-15 administration was effective at inhibiting IL-15 signal- cytokine Abs that display blocking effects in vitro sometimes https://www.jimmunol.org ing in vivo. become activating (proproliferative) when administered in vivo A major consequence of IL-15 blockade was the systemic, nearly (80–85), we believe that this mechanism is unlikely in our study complete depletion of NK cells, demonstrating the essential role of because the effects of anti–IL-15 included: 1) the ex vivo inhi- IL-15 in the development and survival of most NK cells in RM. A bition of IL-15 signaling as measured by the induction of pSTAT5 recent report by Lebrec et al. (66) suggests that IL-15 may not be expression; 2) the systemic depletion of NK cells; and 3) the se- required for NK cell homeostasis in humans, because a blocking lective effects of anti–IL-15 mAb treatment on TTrM and TEM,but Downloaded from mAb directed against human IL-15 did not decrease circulating not TN or TCM populations. However, we did experimentally ad- NK cell counts. Although it is unclear why anti–IL-15 adminis- dress the issue whether TEM were indirectly induced to proliferate tration did not affect NK cell homeostasis in that study, our data in response to IL-15/anti-immune complexes via an Fc-dependent confirm previous observations in mice and NHP that IL-15 mechanism. To test this, we treated two RM with a variant of the blockade significantly diminishes NK cell populations in vivo rhesus anti–IL-15 mAb that had an inactivating LALA mutation in (23, 34, 76–79). In our study, the CD16+ CD562 NK cell sub- the Fc receptor binding site (86). We found that this Fc inactiva- set, which constitutes the major NK population in the blood of tion had no effect on the in vivo activity of the anti–IL-15 mAb on RM, was severely depleted by IL-15 blockade. In contrast, the T cell and NK cell dynamics (data not shown), and we thus 2 + minor NK cell subset (CD16 CD56 ) was less sensitive to IL-15 concluded that the TEM proliferative response after anti–IL-15 signaling inhibition. Considering CD162 CD56+ NK cells can mAb treatment was most likely not an immune complex–mediated induce pSTAT5 expression in response to both IL-7 and IL-15 artifact, but rather a compensatory, IL-15–independent homeostatic in vitro, whereas CD16+ CD562 NK cells respond only to IL-15 process. (Fig. 1C), it is possible that this less efficient depletion of the This conclusion is also supported by the observations that T cell CD162 CD56+ NK cell subset is due to the ability of IL-7 to densities in colonic lamina propria (an extralymphoid effector site The Journal of Immunology 1195

FIGURE 9. The TEM response to IL-7 in vivo is increased with anti– IL-15 treatment. (A) RM were administered anti–IL-15 mAb (n =6) or IgG control mAb (n = 5) biweekly over 8 wk for a total of five doses. On days 35 and 42 posttreatment, both groups received two s.c. injec- tions of IL-7 at 30 mg/kg. Results (mean + SEM) are shown as percentage of baseline absolute counts of the indicated T cell subsets. The significance in parameters between RM groups was evaluated using rANOVA with Tukey–Kramer adjustment (significant p values shown as: **p , 0.01, ***p , 0.001. (B) Transcriptomic profiling of sort- + purified CD8 TEM showing genes associated with the JAK/STAT pathway (source gene set from MSigDB) at p , 0.05 in anti–IL-15 mAb– treated RM compared with IgG control mAb–treated RM. by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd.

with predominant populations of resident TM with TEM or TTrM IL-15 mAb–treated RM derives from the IL-15–independent phenotypes [48, 87]) remained significantly depleted well after subsets. In this scenario, the incomplete reconstitution observed in normalization of absolute TEM counts in blood, and that high TEM the IL-15–deficient RM might be because of an inability of these proliferation was maintained for the duration of treatment (7 wk), IL-15–independent subsets to effectively fill TEM niches usually associated with increased levels of active caspase-3, consistent occupied by IL-15–dependent cells. Finally, it is possible that the with apoptosis and a high turnover state (88), which was widely TEM regeneration that occurs in the absence of IL-15 derives from https://www.jimmunol.org (although not homogeneously) distributed in peripheral lymphoid TTrM or even TCM precursors that are induced by non–IL-15 sig- + and extralymphoid tissues. Transcriptional profiling of CD8 TEM nals to both proliferate and differentiate toward TEM. As with the in the blood confirmed a predominant upregulation of genes as- second possibility, the observed incomplete TEM reconstitution in sociated with T cell activation and cell cycle progression in anti– this situation might be because of the inability of these non–IL-15 IL-15 mAb–treated RM. In particular, genes involved with mito- regulators to induce full differentiation into the panoply of TEM + chondrial respiration were upregulated in CD8 TEM, indicating needed to fill all TEM niches. Downloaded from that in the periphery, TEM can receive secondary signals that The next question to be addressed concerns the nature of ho- support proliferative expansion and an active metabolic state in meostatic factor(s) responsible for stimulating the TEM prolifera- the absence of IL-15. The cell substrate upon which this homeo- tive expansion in the presence of IL-15 signaling inhibition. In this static mechanism depends remains an open question with three regard, it is interesting that treatment of monkeys with the JAK3 nonmutually exclusive possibilities. First, the homeostatic mech- inhibitor CP-690550, which would inhibit all gc cytokines (90), + anism may simply operate on TEM that are normally maintained results in depletion of NK cells and CD8 TM (particular TEM) by IL-15 but can respond to other cytokines in the absence of similar to what we report in this article for anti–IL-15 mAb + IL-15. Although, in this scenario, we would posit the availability treatment; however, with JAK3 inhibition, CD8 TM rebound or activity of these putative non–IL-15 cytokines is suboptimal, occurs only after drug cessation and there is no evidence of the accounting for the incomplete TEM reconstitution and failure to TEM regenerative process described in this report (43). These achieve (or approach) quiescence during the 7 wk of observation. differences suggest that the TEM regenerative process observed Second, because in mice there is clear heterogeneity in the IL-15 with anti–IL-15 depends upon the JAK3 signaling, and therefore dependence of effector memory CD8+ T cell populations in var- might involve a non–IL-15 member or members of the gc cyto- ious sites (89), it is possible the rebounding population in anti– kine family. 1196 IMPACT OF ANTI–IL-15 ON LYMPHOCYTE HOMEOSTASIS IN MACAQUES

Although expression of the IL-7R CD127 is maintained at low intraepithelial lymphocytes, CD56+ NK cells) for IL-7 or other levels on TEM, the relative abundance of IL-7 in peripheral cir- compensatory gc cytokines when IL-15 is not available. Such culation as well as constitutive expression within lymphoid mi- competition would decrease TEM population stability in tissues croenvironments suggests IL-7 as a primary candidate for this where IL-7 and/or other compensatory gc cytokines are limiting. role. Initial analysis of plasma cytokine levels showed no changes Indeed, the precise role that IL-15 plays in regulating the migration in IL-7 or other immune-modulating cytokines such as TNF-a and subsequent maintenance of TEM in such tissues still needs to be and IL-12 (data not shown). Although this indicated that anti– more clearly defined in primates. IL-15 mAb treatment did not increase IL-7 production, it did lead In summary, this study unequivocally establishes a major in us to ask whether TEM might acquire increased sensitivity to en- vivo role for IL-15 in the development and survival of NK cells dogenous IL-7 in the absence of IL-15 signaling. In line with this, in an NHP model. In addition, this study demonstrates the impor- + + + we observed a significant increase in the fraction of both CD4 tant role IL-15 plays in maintaining peripheral CD4 and CD8 TEM + and CD8 TEM with pSTAT5 expression in response to IL-7 homeostasis, and the complex interaction of IL-15 with other stimulation. Interestingly, this increase in IL-7 responsiveness gc cytokines in the regulation of the TEM compartment. IL-15 is was not observed in other T cell subsets (TN,TCM,orTTrM). This overexpressed in several autoimmune disorders, where it is thought effect was further demonstrated in vivo by administering IL-7 to to drive the activation and proliferation of self-reactive TM (94). monkeys that were being treated with anti–IL-15 versus control Based on this, blockade of IL-15 activity is being evaluated as a + + mAbs. Notably, absolute numbers of CD4 and CD8 TEM, but not therapeutic strategy for these diseases (95, 96). We would propose TCM, were significantly increased in response to IL-7 in anti– that in vivo IL-15 blockade of IL-15 in RM provides a useful model IL-15 mAb–treated RM compared with controls. Although it is for exploring the implications of this therapeutic strategy. As shown important to note that we did not examine whether TEM were more in this study, anti–IL-15 therapy will likely not uniformly deplete responsive to other gc cytokines (i.e., IL-2), our data strongly T and NK cell effectors, but rather will change their balance, po- support the concept that TEM generated in the absence of IL-15 tentially resulting in unexpected, off-target immune reactions. signaling become increasingly more responsive to JAK/STAT These results suggest that further study will be necessary to fully signaling via at least one other gc cytokine (IL-7). Transcrip- characterize the therapeutic potential of anti–IL-15 mAbs in the tional profiling also supported this conclusion by revealing an treatment of autoimmune disorders. increased expression of the transcription factor, STAT1 in TEM. STAT1 is upregulated during conditions of lymphopenia and plays Acknowledgments + a role in regulating the expansion and survival of CD8 T cell We thank Cytheris S.A. for providing rsIL-7, as well as Francois Villinger responses, particularly after viral infection (91, 92). Because IL-7 (Emory University) and the Resource for Nonhuman Primate Immune Re- can activate STAT1 and JAK1/STAT1 signaling has been linked agents for providing rhesus IL-15. We acknowledge the NIH Nonhuman with supporting T cell survival (93), it is possible that increases in Primate Reagent Resource for the rhesus recombinant anti–IL-15 (M111R1) and rhesus IgG control mAbs. We thank S. Planer, J. Turner, P. Jewett, STAT1 expression contributed to TEM becoming more responsive to IL-7 signaling in animals treated with anti–IL-15. Other non- T. Swanson, M. Fischer, and J. Dewane for expert animal husbandry. We mutually exclusive possibilities are that in the absence of IL-15, also thank S. Shiigi, E. McDonald, A. Kiddle, N. Hamilton, C. Pexton, I. Axthelm, S. Hagen, Y. Fukazawa, R. Lum, C. Abana, H. Park, A. Townsend, expanding and differentiating T retain IL-7 sensitivity as they TrM and L. Boshears for technical or administrative assistance. by guest on September 30, 2021. Copyright 2016 Pageant Media Ltd. differentiate into TEM and/or that IL-15–independent TEM subsets have the ability to more efficiently respond to IL-7. Although elucidation of these mechanisms will require further study, the Disclosures data presented in this study strongly support the concept that even The authors have no financial conflicts of interest. when gc cytokines have primary roles in the homeostatic regu- + lation of specific subsets, there is often (TEM; CD56 NK cells), References + but not always (CD16 NK cells), plasticity in this regulation such 1. Kondo, M., T. Takeshita, N. Ishii, M. Nakamura, S. Watanabe, K. Arai, and that other members of this cytokine family can provide “backup” K. Sugamura. 1993. 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