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J Immunol. Author manuscript; available in PMC 2017 August 15. Published in final edited form as: J Immunol. 2016 August 15; 197(4): 1183–1198. doi:10.4049/jimmunol.1600065.
Effect of anti-IL-15 administration on T cell and NK cell homeostasis in rhesus macaques 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§, Michael K. Axthelm*,†, Jacob D. Estes¶, Keith A. Reinmann^, Rafick-Pierre Sekaly‡, and Louis J. Picker*,†
Author Manuscript
*Vaccine
and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR 97006
†Oregon
National Primate Research Center, Oregon Health & Science University, Beaverton, OR
97006 ‡Department
of Pathology, Case Western Reserve University, Cleveland, OH 44106
§Division
of Biostatistics, Department of Public Health and Preventative Medicine, Oregon Health & Science University, Portland, OR 97239
¶AIDS
and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD 21702 ^MassBiologics,
University of Massachusetts Medical School, Boston, MA 02126
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Abstract
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IL-15 has been implicated as a key regulator of T and NK cell homeostasis in multiple systems; however, its specific role in maintaining peripheral T and NK cell populations relative to other gamma-chain (γc) cytokines has not been fully defined in primates. Here, 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 naïve 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 γc 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,
Correspondence to: Louis J. Picker, M.D., Vaccine and Gene Therapy Institute, Oregon Health & Science University – West Campus, 505 NW 185th Ave., Beaverton, OR 97006, PHN: (503) 418-2720; FAX: (503) 418-2719, [email protected]. 1M.Q.D. and A.A.O. contributed equally to this work. Disclosures: All authors have no financial conflicts of interest.
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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.
INTRODUCTION
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Lymphocyte homeostasis and function are tightly regulated by the activities of the common gamma chain (γc) cytokines, in particular IL-2, IL-7 and IL-15. While these 3 cytokines share the γc receptor (CD132) their activity on various lymphocyte populations are often different, differences that are, in part, mediated by differential expression of the α-chain component of the receptor (1–7). For instance, IL-2 is mainly produced by CD4+ T cells, and to a lesser degree CD8+ T cells, NK cells and NKT cells, and it acts to generally potentiate the expansion of activated T and NK cells (8, 9). However, IL-2 has a unique primary role in regulating immune tolerance by promoting the production and maintenance of CD4+ CD25+ Foxp3+ T regulatory (Treg) cells, which constitutively express IL-2Rα (CD25). Indeed, this was demonstrated in early models of IL-2−/− and IL-2Rα−/−/IL-2Rβ−/− knockout (KO) mice, which manifested rapid lethal autoimmune diseases likely resulting from the failure of Treg development and homeostasis (10). IL-7 is produced by nonhematopoietic cells such as stromal and epithelial cells and is important for thymocyte development and peripheral T cell homeostasis. IL-7 signals via the receptor IL-7R, which is a heterodimer consisting of the IL-7Rα (CD127) and CD132. IL-7 is particularly important for promoting the proliferative expansion and survival of naïve T cells (TN) and central memory T cells (TCM), which maintain high levels of CD127 expression (11). IL-15, on the other hand, has been shown to regulate the homeostasis and activation of many cell types throughout the body, including memory T cells, NK cells, invariant NKT cells, γδ T cells and intestinal intraepithelial lymphocytes (12–22). As such, IL-15Rα−/− and IL-15−/− KO mice typically manifest severe deficiencies in these cell subsets (23). IL-15 signals via interaction with heterotrimeric receptor complex composed of IL-15Rα (CD215), IL-2/15Rβ (CD122) and CD132 (16, 24–27). IL-15Rα is expressed on antigen-presenting cells (macrophages, monocytes and dendritic cells) and binding of IL-15 to IL-15Rα enables trans-presentation to a responding cell expressing CD122 and CD132 (28). Because biologically active IL-15 has been shown to exist in a soluble form in complex with IL-15Rα (29), it has been questioned whether IL-15Rα is part of the receptor for IL-15 or part of a heterodimeric cytokine that interacts with the CD122/CD132 receptor. In either scenario, the IL-15Rα protein is thought to be a critical determinant of IL-15 specificity and function.
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Although each of these γc cytokines has unique characteristics, their in vivo activity often manifests considerable overlap. For instance, IL-2 and IL-15 share the same β receptor (CD122) and are both involved in the initial amplification of antigen-specific T cell responses, and the regulation of memory T cell development, differentiation, and maintenance (30–32). In addition, both IL-2 and IL-15 induce the activation and proliferation of NK cells and enhance NK cell cytolytic activity by inducing the upregulation of effector molecules such as perforin and granzyme B (33–35). Similarly, IL-7 and IL-15 both seem to play major, albeit non-exclusive, roles in maintaining peripheral TM homeostasis, supporting both TM proliferation and survival (31). Thus, the specific nonredundant roles these γc cytokines play in controlling various lymphocyte population
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dynamics in vivo are not completely characterized, a lack of understanding that complicates efforts to rationally develop therapeutic strategies based on their specific biologic activities to enhance immune responses to cancer or microbial agents, to promote immune reconstitution after conditions of lymphopenia (HIV infection, chemotherapy, aging), or to counter pathologic immune responses in the various autoimmune/inflammatory disorders (rheumatoid arthritis, celiac disease, inflammatory bowel disease, multiple sclerosis and type 1 diabetes) linked to dysregulation of these cytokines (36–40).
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Due to its activity on NK cells and antigen-specific cytotoxic T cells, IL-15 is in clinical trials for the treatment of metastatic malignancies (41). Previous studies have shown that IL-15 can increase the production of long-lived antigen-specific TM (32, 42, 43), and can also induce the migration and redistribution of TM from circulation into tissues (44, 45). In nonhuman primates (NHP), provision of exogenous IL-15 typically induces an initial brief period of lymphopenia followed by lymphocytosis (45–47). Lymphocytosis is associated with the expansion of NK cells and TM (41, 44). However, the TM compartment is quite heterogeneous and comprises the TCM subset, which is responsible for anamnestic T cell responses and primarily recirculates between secondary lymphoid tissues, and the effectordifferentiated memory subsets – transitional memory (TTrM) and TEM - which can also migrate to extra-lymphoid effector sites (48). In NHP, TEM and TTrM are very responsive to IL-15 in vivo, and studies from our lab and others have shown that exogenous IL-15 administration can dramatically increase the proliferative fraction and absolute numbers of TEM and TTrM in peripheral blood (44, 46, 47). In contrast, TN and TCM are less responsive to IL-15 administration, and it is still unclear whether IL-15 plays a direct role in vivo in regulating their homeostasis. Most of these studies have focused on CD8+ T cells, and in general, IL-15 has been more closely associated with regulation of CD8+ TM than with CD4+ TM. However, CD4+ TEM and TTrM are also highly responsive to IL-15 in vivo, suggesting that this discrepancy is largely attributable to the fact that the highly IL-15responsive TEM subset comprises a much larger fraction of total circulating TM for CD8+ than for CD4+ lineage cells (44).
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In this study, we sought to determine the specific, non-redundant role(s) of IL-15 in the regulation of CD4+ and CD8+ T and NK cell population dynamics in NHP. As indicated above, most previous studies addressing IL-15 activity in NHP have assessed the effect of therapeutic administration of IL-15, which results in unnaturally high levels of cytokine immediately after administration. Since such pharmacologic (supra-physiologic) IL-15 levels may not accurately reflect physiologic cytokine function in vivo, we have taken the alternative approach of determining the impact of inhibiting IL-15 activity in vivo with a newly developed rhesusized anti-IL-15 monoclonal antibody (mAb) on T cell and NK cell homeostasis in rhesus macaques (RM). We demonstrate that this rhesusized anti-IL-15 can be repeatedly administered to RM and is highly effective at long-term inhibition of IL-15 activity in vivo. We further demonstrate that in vivo inhibition of IL-15 activity resulted in a near complete depletion of NK cells and a significant decrease in the numbers of circulating CD4+ and CD8+ TEM with negligible effects on the TCM or TN subsets. Strikingly, however, TEM, but not NK cell numbers, rebounded by proliferative expansion, and in the absence of IL-15 signaling, TEM became increasingly more sensitive to IL-7 signaling. These data
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suggest that whereas IL-15 signaling is required for NK cell homeostasis, TEM can be maintained by other cytokines, most likely IL-7, when IL-15 signaling is not available.
MATERIALS AND METHODS Animals
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A total of 41 purpose-bred RM (Macaca mulatta) of Indian genetic background and free of Macacine herpesvirus 1, D type simian retrovirus, simian T-lymphotrophic virus type 1, and SIV infection were used in this study. A group of 30 RM were administered the rhesus recombinant anti-IL-15 mAb, clone M111 (n=17) or rhesus recombinant IgG control antibody (n=13), i.v. once every two weeks at 20mg/Kg on day 0 and 10mg/Kg on days 14 and 21. The neutralizing anti-IL-15 antibody was constructed by grafting the complementarity determining regions of mouse anti-human IL-15 mAb, M111 (American Type Culture Collection) into rhesus variable region frameworks. MAb was expressed in Chinese hamster ovary cells as full-length Ig with rhesus IgG1 and kappa constant regions. Two RM were dosed with an immunologically silenced form of the same antibody (anti– IL-15 LALA) which contained the L235A, L236A mutation in the heavy chain constant region. RM dosed with the anti-IL-15 LALA mAb displayed similar phenotypes to the monkeys treated with the non-mutated anti-IL-15 mAb for all parameters assayed, and were therefore grouped as anti-IL-15 mAb-treated RM. BrdU (Sigma-Aldrich) was prepared as previously described (49), and administered i.v. in three separate doses of 30mg/Kg body weight over a 24-hour period prior to terminal necropsy at ≥35 days post-first anti-IL-15 treatment. A separate group of 11 RM received 6 biweekly doses of rhesus recombinant anti-IL-15 mAb (n=6) or rhesus recombinant IgG control (n=5) at 20mg/Kg on day 0, followed by 10mg/Kg on days 14, 28, 42, 56 and 77, concurrently, with subcutaneous administration of rhesus recombinant IL-7 at 30μg/Kg on days 35 and 42. All RM were housed at the Oregon National Primate Research Center in accordance with standards of the Center’s Institutional Animal Care and Use Committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Measurement of plasma IL-7 concentration The concentration of IL-7 in the plasma was measured by ELISA using the IL-7 Quantikine HS kit (R & D Systems) according to manufacturer’s instructions. Flow cytometric analysis and cell sorting
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Whole blood and mononuclear cells isolated from lymph nodes (LN), bronchoalveolar lavage (BAL), bone marrow, spleen, kidney, lung, liver, tonsil, and vaginal and intestinal mucosa were obtained and stained for flow cytometric analysis as described previously (49, 50). Polychromatic (8–12 parameter) flow cytometric analysis was performed on an LSR II instrument using Pacific blue, AmCyan, FITC, PE, PE–Texas red, PE-Cy7, PerCP-Cy5.5, Allophycocyanin, Allophycocyanin-Cy7, and Alexa Fluor 700 as the available fluorescent parameters. Instrument set-up and data acquisition procedures were performed as previously described (51). List mode multiparameter data files were analyzed using FlowJo software. Delineation of naive and memory T cell subsets and criteria for setting + versus − markers for CCR5 and Ki-67 expression have been previously described (49, 50, 52–54). In brief, TN J Immunol. Author manuscript; available in PMC 2017 August 15.
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constitute a uniform cluster of cells with a CD28moderate, CCR7+, CCR5−, CD95low phenotype, which is clearly distinguishable from the phenotypically diverse memory population that is CD95high and may display one or more of the following non-naive phenotypic features: CD28−, CCR7−, or CCR5+. The TCM, TTrM, and TEM components of the memory subset in the blood were further delineated based on the following phenotypic criteria: TCM (CD28+, CCR7+ and CCR5−), TTrM (CD28+, CCR7+/− and CCR5+), and TEM (CD28−, CCR7− and CCR5dim). For delineating NK cells in blood, small lymphocytes were gated to obtain CD3−, CD8α+, NKG2a+ cells that were CD20− and CD14−. NK cell subsets were further delineated based on CD16 and CD56 expression as previously described (55). For each subset to be quantified, the percentages of the subset within the overall small lymphocyte populations were determined. For quantification of peripheral blood subsets, absolute small lymphocyte counts were obtained using an AcT5diff cell counter (Beckman Coulter) and from these values, absolute counts for the relevant subset were calculated based on the subset percentages within the light scatter–defined small lymphocyte population on the flow cytometer. Baseline values were determined as the average of values at days −14, −7 and 0. Results are presented as percentage of baseline, with baseline shown as 100%, or changes in proliferative fraction, indicated as the difference in the %Ki-67+ (Δ%Ki-67+) measured at the designated time points from baseline (0% = no change).
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For analysis of pSTAT5 expression, whole blood (100μL) was added to polystyrene flow tubes and stained with fluorochrome-conjugated mAbs against CD3, CD4, CD8, CD28, CCR5, CCR7 and CD95, at room temperature (RT) for 30 minutes. Next, tubes were either left unstimulated or stimulated with increasing concentrations of IL-7 or IL-15 (ranging from 0.5 – 32ng/mL) for 15 min at 37°C/5% CO2. Detection of pSTAT5 was assessed with the BD Phosflow staining protocol according to the manufacturer’s instructions. Briefly, cells were fixed with the BD Phosflow Lyse/Fix buffer for 5 min at RT and permeabilized in ice-cold BD Phosflow perm buffer IV for 5 min at RT. After washing, cells were stained for intracellular markers with fluorochrome-conjugated anti-pSTAT5 and anti-Ki-67 for 45 min at RT. Cells were washed and flow cytometric analysis was performed on the LSR II flow cytometer. In vitro cytokine-induced expansion assay
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PBMCs were sort purified using a FACS Aria II (BD Biosciences) based on defined phenotypic markers as described above and plated in 48-well plates in 1mL of R10 media [RPMI (HyClone), 10% Fetal Bovine Serum (FBS), 100units/mL Penicillin, 10mg/mL Streptomycin (Sigma-Aldrich), 200μM L-glutamine (Sigma-Aldrich)] at a density of 150,000 to 300,000 cells/mL. IL-7 or IL-15 were added at a concentration of 50ng/mL to the cultures and incubated at 37°C/5% CO2 for 14 days alone or in the presence of 10% sort purified CD14+ monocytes. After 7 days, the culture was resuspended and 0.5mL was removed for phenotypic analysis by flow cytometry. An equal amount of fresh R10 was added back to the remaining culture and incubated at 37°C/5% CO2 for a further 7 days. On day 14, the entire culture was harvested for phenotypic analysis by flow cytometry.
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Antibodies and cytokines
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The following antibodies were used for flow cytometry: CD3 Alexa 700 (SP34-2 BD Biosciences), CD4 AmCyan (L200 BD Biosciences), CD8 PerCP-Cy5.5 (SKI eBiosciences), CD8 AmCyan (SKI BD Biosciences), CD28 PE-Texas Red (CD28.2 Beckman Coulter, BD Biosciences), CD95 PE (DX2 BD Biosciences, eBiosciences), CCR5 Allophycocyanin (3A9 BD Biosciences), Ki-67 FITC (B56 BD Biosciences), CD56 PerCPCy5.5 (MEM-188 Invitrogen), CD16 Pacific Blue (3G8 BD Biosciences, Biolegend), CD20 Allophycocyanin-Cy7 (L27 BD Biosciences), HLA-DR PE-Texas Red (TU36 Invitrogen, Immu357 Beckman Coulter), NKG2A PE (Z199 Beckman Coulter), CD14 FITC (M5E2 BD Biosciences, R&D Systems), STAT5 PE (47/Stat5 (pY6) BD Biosciences), BrdU FITC (B44 BD Biosciences), BrdU Allophycocyanin (B44 BD Biosciences). Anti-CCR7 (150503) was purchased as purified immunoglobulin from R&D Systems, conjugated to biotin using a Pierce Chemical Co. biotinylation kit, and visualized with streptavidin–Pacific Blue (Invitrogen). Rhesus recombinant anti-IL-15 and rhesus recombinant control IgG1 mAb were provided through the National Institutes of Health’s Nonhuman Primate Reagent Resource Program. Simian recombinant IL-7 was provided by Cytheris SA (Issy-LesMoulineaux, France). Rhesus recombinant IL-15 was provided by Francois Villinger (Emory University) through the Resource for Nonhuman Primate Immune Reagents. Immunohistochemistry
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Immunohistochemistry was performed as previously described (56). Antibodies used in this study were mouse monoclonal anti-human IL-15 (Antibodies Online; clone BDI150), rabbit monoclonal anti-active caspase-3 (Cell Signaling Technologies; clone 5A1E), rabbit monoclonal anti-phosphorylated STAT5 (1:100; clone C11C5; Cell Signaling Technologies, Inc.), and rabbit monoclonal anti-human CD3 (clone SP7; Labvision/Thermo Fisher Scientific). All stained slides were scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies) yielding high-resolution data from the entire tissue section. Representative regions of interest (ROIs; 250 to 500 mm2) were identified and high-resolution images extracted from these whole-tissue scans. The percent area of the lymph node T cell zone and lamina propria (colon) that stained for each protein/cell type of interest were quantified using Photoshop CS5 using Fovea tools. Microarray analysis
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For transcriptional analysis, sort-purified CD8+ TEM and CD8+ TCM from PBMC, obtained 28–49 days post-first anti-IL-15 or control IgG mAb administration, were resuspended in RLT lysis buffer (Qiagen) and stored at −80°C until use. RNA was isolated using RNeasy Micro Kits (Qiagen), and the quantity and quality of the RNA was confirmed using a NanoDrop 2000c (Thermo Fisher Scientific) and an Experion Electrophoresis System. Samples (50ng) were amplified using Illumina TotalPrep RNA amplification kits (Ambion). The microarray analysis was conducted using 750ng of biotinylated complementary RNA hybridized to HumanHT-12_V4 BeadChips (Illumina) at 58°C for 20h. The arrays were scanned using Illumina’s iSCAN and quantified using Genome Studio (Illumina). The analysis of the Genome Studio output data was conducted using the R and Bioconductor software packages. Quantile normalization was applied, followed by a log2 transformation
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performed using the Bioconductor LIMMA package (57). Outlier samples with abnormalities in gene expression based on hierarchical clustering methods and multidimensional scaling analysis as a dimensionality reduction method for the evaluation of similarities or dissimilarities between samples were identified and removed. The LIMMA package was used to fit a linear model to each probe and perform (moderated) t tests or F tests on the groups being compared. A Gene Set Enrichment Analysis (GSEA) (58) using 1000 permutations was performed on the genes differentially expressed between the groups compared in the subsets pre-ranked by the decreasing order of the absolute T-statistic. The canonical pathways of Ingenuity Pathway Analysis software (IPA, Ingenuity Systems) were used as the database to perform GSEA. This was followed by building modules of related pathways based on at least 25% gene overlap [Jaccard index (59) > 25%] between pathways using the enrichment map (60) strategy and representing the genes present in at least 25% of the pathways in the module. To control the expected proportions of false positives, the FDR for p-values was calculated using the Benjamini and Hochberg method implemented in LIMMA. The complete dataset is available at the Gene Expression Omnibus microarray repository (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? token=mbqtaouqztynzsz&acc=GSE76797), accession number GSE76797.
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Statistics
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To investigate if the administration of anti-IL-15 mAb blocks signaling in circulating lymphocytes, we estimated the EC50 (the dose where the response is the midpoint between the maximum and minimum) for each RM at given time points (days post-mAb) using the four-parameter logistic model often referred to as an Emax model, followed by repeated measures ANOVA to evaluate the effect of anti-IL-15 mAb with EC50 as a response variable and anti-IL-15 mAb-treated status as between group factor, and days post-antibody as within group factors. Longitudinal analysis of STAT5 phosphorylation, peripheral blood TN and TM (including TCM, TTrM and TEM subsets) counts and proliferation, peripheral blood NK cell counts and plasma IL-7 levels were evaluated using repeated-measures ANOVA with antiIL-15 mAb and IgG control mAb-treated groups as between group factors and time points as within group factors; since in a typical experiment using repeated measures, two measurements taken at adjacent times are more highly correlated than two measurements taken several time points apart. Due to the limited sample size, a simpler covariance structure, first order auto-regressive (AR1), was used as correlation within each animal. Tukey-Kramer adjustment was used to control for multiple comparisons. Differences in the number of pSTAT5+ cells/mm2 in peripheral lymph nodes between pre-treatment and posttreatment were evaluated using Wilcoxon Signed rank tests. The difference in CD3+ caspase-3+ in colon and peripheral LN via immunostaining and BrdU incorporation in CD4+ and CD8+ T cell subsets between anti-IL-15 and IgG control mAb-treated groups was compared using Mann-Whitney U test. Data was analyzed using SAS 9.4. The p-values 80% of cells at 16ng (Fig. 1C), only the CD56+ CD16− “regulatory” NK cell subset showed any response to IL-7 stimulation at the tested doses, and this response required a 4-fold higher dose of IL-7 than the response of the same cells to IL-15.
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These results demonstrate that lymphocyte populations differ in their response to early signal transduction by IL-7 vs. IL-15, and suggest that IL-7 and IL-15 preferentially signal pre-effector and effector populations, respectively. We next sought to determine the degree to which this differential signaling translates to downstream gene expression and homeostatic regulation by examining the ability of these cytokines to support survival and proliferative expansion of sort-purified TN, TCM and TEM during 7 and 14 days of in vitro culture. Since the pattern of STAT5 phosphorylation to IL-7 and IL-15 among these differentiation-defined subsets was similar between the CD4+ and CD8+ lineages (Fig. 1A– B), we primarily focused on CD4+ T cells for these studies. In this in vitro CD4+ T cell model system, responses to IL-15 were generally more robust than to IL-7, but responsiveness to both cytokines was differentiation dependent. TEM were again found to be highly responsive to IL-15 compared to IL-7, with over 60% of IL-15-treated cells expressing the proliferation antigen Ki-67 by day 7 of culture, compared to background Ki-67 expression after IL-7 treatment (Fig. 2A). IL-7 and IL-15 induced similar, modest increases in Ki67 expression by CD4+ TCM, whereas IL-7 was more effective than IL-15 in inducing TN proliferation. Next, based on the concept that IL-15 might be more efficiently presented in trans, we cultured the same cells with IL-15 or IL-7 in the presence of sort-
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purified CD14+ monocytes (63, 64). In the presence of monocytes, we saw modestly enhanced responsiveness of TN, TCM, and TEM to IL-7, and profoundly increased responsiveness of TN and TCM to IL-15 (Fig. 2B). Interestingly the CD4+ TEM proliferative response to IL-15 was similar with or without monocytes, suggesting that trans-presentation or other monocyte-derived signals are not required for maximal TEM responsiveness. Similar IL-15 responsiveness was demonstrated for CD8+ TEM (Supplementary Figure 2A).
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Collectively, these in vitro studies demonstrate that TN and TCM are more responsive to IL-7 than TEM, whereas TEM are more responsive to IL-15. However, IL-15 can induce robust proliferation in TN and TCM in the presence of monocytes, suggesting a more pleotropic role for this cytokine. Interestingly, culture of TN and TCM with IL-15 in the presence of monocytes induces up-regulation of CCR5 and down-regulation of CCR7 (Supplementary Figure 2B), consistent with induction of effector memory differentiation (50, 65). Thus, IL-15 may both maintain the homeostasis of pre-existing TEM and, under certain conditions, drive TEM differentiation from noneffector-differentiated precursors. Anti-IL-15 specifically blocks IL-15 signal transduction in vivo
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To investigate the role of IL-15 in T cell and NK cell homeostasis in vivo, we developed a “rhesusized” anti-IL-15 mAb that is suitable for repeated administration to RM. This mAb was based on the mouse, anti-human IL-15 mAb clone, M111, which is cross-reactive with RM IL-15 (66). Rhesusization was accomplished by exchanging the mouse amino acid sequences in the constant regions and variable binding surfaces for rhesus amino acid sequences, leaving variable region sequences responsible for IL-15 recognition unchanged (67, 68). We hypothesized that repeated administrations of this rhesusized M111 mAb would block IL-15 activity in vivo and result in changes to T and/or NK cell population dynamics that would reflect physiologic IL-15 function. To initially test this approach, we administered rhesus anti-IL-15 or a rhesus IgG control mAb to RM every other week for 6 weeks prior to necropsy, as shown in Fig. 3A. We assessed IL-15 signaling inhibition by performing pSTAT induction analysis with rhesus recombinant IL-15 on whole blood at various time points post-treatment. From day 1 through day 35 post-treatment, the ability of IL-15 to induce pSTAT5 expression in CD4+ and CD8+ T cells ex vivo in anti-IL-15-treated RM was significantly reduced (essentially abrogated) compared to baseline and to control IgG-treated RM (p
J Immunol. Author manuscript; available in PMC 2017 August 15. Published in final edited form as: J Immunol. 2016 August 15; 197(4): 1183–1198. doi:10.4049/jimmunol.1600065.
Effect of anti-IL-15 administration on T cell and NK cell homeostasis in rhesus macaques 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§, Michael K. Axthelm*,†, Jacob D. Estes¶, Keith A. Reinmann^, Rafick-Pierre Sekaly‡, and Louis J. Picker*,†
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*Vaccine
and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR 97006
†Oregon
National Primate Research Center, Oregon Health & Science University, Beaverton, OR
97006 ‡Department
of Pathology, Case Western Reserve University, Cleveland, OH 44106
§Division
of Biostatistics, Department of Public Health and Preventative Medicine, Oregon Health & Science University, Portland, OR 97239
¶AIDS
and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD 21702 ^MassBiologics,
University of Massachusetts Medical School, Boston, MA 02126
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Abstract
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IL-15 has been implicated as a key regulator of T and NK cell homeostasis in multiple systems; however, its specific role in maintaining peripheral T and NK cell populations relative to other gamma-chain (γc) cytokines has not been fully defined in primates. Here, 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 naïve 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 γc 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,
Correspondence to: Louis J. Picker, M.D., Vaccine and Gene Therapy Institute, Oregon Health & Science University – West Campus, 505 NW 185th Ave., Beaverton, OR 97006, PHN: (503) 418-2720; FAX: (503) 418-2719, [email protected]. 1M.Q.D. and A.A.O. contributed equally to this work. Disclosures: All authors have no financial conflicts of interest.
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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.
INTRODUCTION
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Lymphocyte homeostasis and function are tightly regulated by the activities of the common gamma chain (γc) cytokines, in particular IL-2, IL-7 and IL-15. While these 3 cytokines share the γc receptor (CD132) their activity on various lymphocyte populations are often different, differences that are, in part, mediated by differential expression of the α-chain component of the receptor (1–7). For instance, IL-2 is mainly produced by CD4+ T cells, and to a lesser degree CD8+ T cells, NK cells and NKT cells, and it acts to generally potentiate the expansion of activated T and NK cells (8, 9). However, IL-2 has a unique primary role in regulating immune tolerance by promoting the production and maintenance of CD4+ CD25+ Foxp3+ T regulatory (Treg) cells, which constitutively express IL-2Rα (CD25). Indeed, this was demonstrated in early models of IL-2−/− and IL-2Rα−/−/IL-2Rβ−/− knockout (KO) mice, which manifested rapid lethal autoimmune diseases likely resulting from the failure of Treg development and homeostasis (10). IL-7 is produced by nonhematopoietic cells such as stromal and epithelial cells and is important for thymocyte development and peripheral T cell homeostasis. IL-7 signals via the receptor IL-7R, which is a heterodimer consisting of the IL-7Rα (CD127) and CD132. IL-7 is particularly important for promoting the proliferative expansion and survival of naïve T cells (TN) and central memory T cells (TCM), which maintain high levels of CD127 expression (11). IL-15, on the other hand, has been shown to regulate the homeostasis and activation of many cell types throughout the body, including memory T cells, NK cells, invariant NKT cells, γδ T cells and intestinal intraepithelial lymphocytes (12–22). As such, IL-15Rα−/− and IL-15−/− KO mice typically manifest severe deficiencies in these cell subsets (23). IL-15 signals via interaction with heterotrimeric receptor complex composed of IL-15Rα (CD215), IL-2/15Rβ (CD122) and CD132 (16, 24–27). IL-15Rα is expressed on antigen-presenting cells (macrophages, monocytes and dendritic cells) and binding of IL-15 to IL-15Rα enables trans-presentation to a responding cell expressing CD122 and CD132 (28). Because biologically active IL-15 has been shown to exist in a soluble form in complex with IL-15Rα (29), it has been questioned whether IL-15Rα is part of the receptor for IL-15 or part of a heterodimeric cytokine that interacts with the CD122/CD132 receptor. In either scenario, the IL-15Rα protein is thought to be a critical determinant of IL-15 specificity and function.
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Although each of these γc cytokines has unique characteristics, their in vivo activity often manifests considerable overlap. For instance, IL-2 and IL-15 share the same β receptor (CD122) and are both involved in the initial amplification of antigen-specific T cell responses, and the regulation of memory T cell development, differentiation, and maintenance (30–32). In addition, both IL-2 and IL-15 induce the activation and proliferation of NK cells and enhance NK cell cytolytic activity by inducing the upregulation of effector molecules such as perforin and granzyme B (33–35). Similarly, IL-7 and IL-15 both seem to play major, albeit non-exclusive, roles in maintaining peripheral TM homeostasis, supporting both TM proliferation and survival (31). Thus, the specific nonredundant roles these γc cytokines play in controlling various lymphocyte population
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dynamics in vivo are not completely characterized, a lack of understanding that complicates efforts to rationally develop therapeutic strategies based on their specific biologic activities to enhance immune responses to cancer or microbial agents, to promote immune reconstitution after conditions of lymphopenia (HIV infection, chemotherapy, aging), or to counter pathologic immune responses in the various autoimmune/inflammatory disorders (rheumatoid arthritis, celiac disease, inflammatory bowel disease, multiple sclerosis and type 1 diabetes) linked to dysregulation of these cytokines (36–40).
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Due to its activity on NK cells and antigen-specific cytotoxic T cells, IL-15 is in clinical trials for the treatment of metastatic malignancies (41). Previous studies have shown that IL-15 can increase the production of long-lived antigen-specific TM (32, 42, 43), and can also induce the migration and redistribution of TM from circulation into tissues (44, 45). In nonhuman primates (NHP), provision of exogenous IL-15 typically induces an initial brief period of lymphopenia followed by lymphocytosis (45–47). Lymphocytosis is associated with the expansion of NK cells and TM (41, 44). However, the TM compartment is quite heterogeneous and comprises the TCM subset, which is responsible for anamnestic T cell responses and primarily recirculates between secondary lymphoid tissues, and the effectordifferentiated memory subsets – transitional memory (TTrM) and TEM - which can also migrate to extra-lymphoid effector sites (48). In NHP, TEM and TTrM are very responsive to IL-15 in vivo, and studies from our lab and others have shown that exogenous IL-15 administration can dramatically increase the proliferative fraction and absolute numbers of TEM and TTrM in peripheral blood (44, 46, 47). In contrast, TN and TCM are less responsive to IL-15 administration, and it is still unclear whether IL-15 plays a direct role in vivo in regulating their homeostasis. Most of these studies have focused on CD8+ T cells, and in general, IL-15 has been more closely associated with regulation of CD8+ TM than with CD4+ TM. However, CD4+ TEM and TTrM are also highly responsive to IL-15 in vivo, suggesting that this discrepancy is largely attributable to the fact that the highly IL-15responsive TEM subset comprises a much larger fraction of total circulating TM for CD8+ than for CD4+ lineage cells (44).
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In this study, we sought to determine the specific, non-redundant role(s) of IL-15 in the regulation of CD4+ and CD8+ T and NK cell population dynamics in NHP. As indicated above, most previous studies addressing IL-15 activity in NHP have assessed the effect of therapeutic administration of IL-15, which results in unnaturally high levels of cytokine immediately after administration. Since such pharmacologic (supra-physiologic) IL-15 levels may not accurately reflect physiologic cytokine function in vivo, we have taken the alternative approach of determining the impact of inhibiting IL-15 activity in vivo with a newly developed rhesusized anti-IL-15 monoclonal antibody (mAb) on T cell and NK cell homeostasis in rhesus macaques (RM). We demonstrate that this rhesusized anti-IL-15 can be repeatedly administered to RM and is highly effective at long-term inhibition of IL-15 activity in vivo. We further demonstrate that in vivo inhibition of IL-15 activity resulted in a near complete depletion of NK cells and a significant decrease in the numbers of circulating CD4+ and CD8+ TEM with negligible effects on the TCM or TN subsets. Strikingly, however, TEM, but not NK cell numbers, rebounded by proliferative expansion, and in the absence of IL-15 signaling, TEM became increasingly more sensitive to IL-7 signaling. These data
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suggest that whereas IL-15 signaling is required for NK cell homeostasis, TEM can be maintained by other cytokines, most likely IL-7, when IL-15 signaling is not available.
MATERIALS AND METHODS Animals
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A total of 41 purpose-bred RM (Macaca mulatta) of Indian genetic background and free of Macacine herpesvirus 1, D type simian retrovirus, simian T-lymphotrophic virus type 1, and SIV infection were used in this study. A group of 30 RM were administered the rhesus recombinant anti-IL-15 mAb, clone M111 (n=17) or rhesus recombinant IgG control antibody (n=13), i.v. once every two weeks at 20mg/Kg on day 0 and 10mg/Kg on days 14 and 21. The neutralizing anti-IL-15 antibody was constructed by grafting the complementarity determining regions of mouse anti-human IL-15 mAb, M111 (American Type Culture Collection) into rhesus variable region frameworks. MAb was expressed in Chinese hamster ovary cells as full-length Ig with rhesus IgG1 and kappa constant regions. Two RM were dosed with an immunologically silenced form of the same antibody (anti– IL-15 LALA) which contained the L235A, L236A mutation in the heavy chain constant region. RM dosed with the anti-IL-15 LALA mAb displayed similar phenotypes to the monkeys treated with the non-mutated anti-IL-15 mAb for all parameters assayed, and were therefore grouped as anti-IL-15 mAb-treated RM. BrdU (Sigma-Aldrich) was prepared as previously described (49), and administered i.v. in three separate doses of 30mg/Kg body weight over a 24-hour period prior to terminal necropsy at ≥35 days post-first anti-IL-15 treatment. A separate group of 11 RM received 6 biweekly doses of rhesus recombinant anti-IL-15 mAb (n=6) or rhesus recombinant IgG control (n=5) at 20mg/Kg on day 0, followed by 10mg/Kg on days 14, 28, 42, 56 and 77, concurrently, with subcutaneous administration of rhesus recombinant IL-7 at 30μg/Kg on days 35 and 42. All RM were housed at the Oregon National Primate Research Center in accordance with standards of the Center’s Institutional Animal Care and Use Committee and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Measurement of plasma IL-7 concentration The concentration of IL-7 in the plasma was measured by ELISA using the IL-7 Quantikine HS kit (R & D Systems) according to manufacturer’s instructions. Flow cytometric analysis and cell sorting
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Whole blood and mononuclear cells isolated from lymph nodes (LN), bronchoalveolar lavage (BAL), bone marrow, spleen, kidney, lung, liver, tonsil, and vaginal and intestinal mucosa were obtained and stained for flow cytometric analysis as described previously (49, 50). Polychromatic (8–12 parameter) flow cytometric analysis was performed on an LSR II instrument using Pacific blue, AmCyan, FITC, PE, PE–Texas red, PE-Cy7, PerCP-Cy5.5, Allophycocyanin, Allophycocyanin-Cy7, and Alexa Fluor 700 as the available fluorescent parameters. Instrument set-up and data acquisition procedures were performed as previously described (51). List mode multiparameter data files were analyzed using FlowJo software. Delineation of naive and memory T cell subsets and criteria for setting + versus − markers for CCR5 and Ki-67 expression have been previously described (49, 50, 52–54). In brief, TN J Immunol. Author manuscript; available in PMC 2017 August 15.
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constitute a uniform cluster of cells with a CD28moderate, CCR7+, CCR5−, CD95low phenotype, which is clearly distinguishable from the phenotypically diverse memory population that is CD95high and may display one or more of the following non-naive phenotypic features: CD28−, CCR7−, or CCR5+. The TCM, TTrM, and TEM components of the memory subset in the blood were further delineated based on the following phenotypic criteria: TCM (CD28+, CCR7+ and CCR5−), TTrM (CD28+, CCR7+/− and CCR5+), and TEM (CD28−, CCR7− and CCR5dim). For delineating NK cells in blood, small lymphocytes were gated to obtain CD3−, CD8α+, NKG2a+ cells that were CD20− and CD14−. NK cell subsets were further delineated based on CD16 and CD56 expression as previously described (55). For each subset to be quantified, the percentages of the subset within the overall small lymphocyte populations were determined. For quantification of peripheral blood subsets, absolute small lymphocyte counts were obtained using an AcT5diff cell counter (Beckman Coulter) and from these values, absolute counts for the relevant subset were calculated based on the subset percentages within the light scatter–defined small lymphocyte population on the flow cytometer. Baseline values were determined as the average of values at days −14, −7 and 0. Results are presented as percentage of baseline, with baseline shown as 100%, or changes in proliferative fraction, indicated as the difference in the %Ki-67+ (Δ%Ki-67+) measured at the designated time points from baseline (0% = no change).
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For analysis of pSTAT5 expression, whole blood (100μL) was added to polystyrene flow tubes and stained with fluorochrome-conjugated mAbs against CD3, CD4, CD8, CD28, CCR5, CCR7 and CD95, at room temperature (RT) for 30 minutes. Next, tubes were either left unstimulated or stimulated with increasing concentrations of IL-7 or IL-15 (ranging from 0.5 – 32ng/mL) for 15 min at 37°C/5% CO2. Detection of pSTAT5 was assessed with the BD Phosflow staining protocol according to the manufacturer’s instructions. Briefly, cells were fixed with the BD Phosflow Lyse/Fix buffer for 5 min at RT and permeabilized in ice-cold BD Phosflow perm buffer IV for 5 min at RT. After washing, cells were stained for intracellular markers with fluorochrome-conjugated anti-pSTAT5 and anti-Ki-67 for 45 min at RT. Cells were washed and flow cytometric analysis was performed on the LSR II flow cytometer. In vitro cytokine-induced expansion assay
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PBMCs were sort purified using a FACS Aria II (BD Biosciences) based on defined phenotypic markers as described above and plated in 48-well plates in 1mL of R10 media [RPMI (HyClone), 10% Fetal Bovine Serum (FBS), 100units/mL Penicillin, 10mg/mL Streptomycin (Sigma-Aldrich), 200μM L-glutamine (Sigma-Aldrich)] at a density of 150,000 to 300,000 cells/mL. IL-7 or IL-15 were added at a concentration of 50ng/mL to the cultures and incubated at 37°C/5% CO2 for 14 days alone or in the presence of 10% sort purified CD14+ monocytes. After 7 days, the culture was resuspended and 0.5mL was removed for phenotypic analysis by flow cytometry. An equal amount of fresh R10 was added back to the remaining culture and incubated at 37°C/5% CO2 for a further 7 days. On day 14, the entire culture was harvested for phenotypic analysis by flow cytometry.
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Antibodies and cytokines
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The following antibodies were used for flow cytometry: CD3 Alexa 700 (SP34-2 BD Biosciences), CD4 AmCyan (L200 BD Biosciences), CD8 PerCP-Cy5.5 (SKI eBiosciences), CD8 AmCyan (SKI BD Biosciences), CD28 PE-Texas Red (CD28.2 Beckman Coulter, BD Biosciences), CD95 PE (DX2 BD Biosciences, eBiosciences), CCR5 Allophycocyanin (3A9 BD Biosciences), Ki-67 FITC (B56 BD Biosciences), CD56 PerCPCy5.5 (MEM-188 Invitrogen), CD16 Pacific Blue (3G8 BD Biosciences, Biolegend), CD20 Allophycocyanin-Cy7 (L27 BD Biosciences), HLA-DR PE-Texas Red (TU36 Invitrogen, Immu357 Beckman Coulter), NKG2A PE (Z199 Beckman Coulter), CD14 FITC (M5E2 BD Biosciences, R&D Systems), STAT5 PE (47/Stat5 (pY6) BD Biosciences), BrdU FITC (B44 BD Biosciences), BrdU Allophycocyanin (B44 BD Biosciences). Anti-CCR7 (150503) was purchased as purified immunoglobulin from R&D Systems, conjugated to biotin using a Pierce Chemical Co. biotinylation kit, and visualized with streptavidin–Pacific Blue (Invitrogen). Rhesus recombinant anti-IL-15 and rhesus recombinant control IgG1 mAb were provided through the National Institutes of Health’s Nonhuman Primate Reagent Resource Program. Simian recombinant IL-7 was provided by Cytheris SA (Issy-LesMoulineaux, France). Rhesus recombinant IL-15 was provided by Francois Villinger (Emory University) through the Resource for Nonhuman Primate Immune Reagents. Immunohistochemistry
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Immunohistochemistry was performed as previously described (56). Antibodies used in this study were mouse monoclonal anti-human IL-15 (Antibodies Online; clone BDI150), rabbit monoclonal anti-active caspase-3 (Cell Signaling Technologies; clone 5A1E), rabbit monoclonal anti-phosphorylated STAT5 (1:100; clone C11C5; Cell Signaling Technologies, Inc.), and rabbit monoclonal anti-human CD3 (clone SP7; Labvision/Thermo Fisher Scientific). All stained slides were scanned at high magnification (x200) using the ScanScope CS System (Aperio Technologies) yielding high-resolution data from the entire tissue section. Representative regions of interest (ROIs; 250 to 500 mm2) were identified and high-resolution images extracted from these whole-tissue scans. The percent area of the lymph node T cell zone and lamina propria (colon) that stained for each protein/cell type of interest were quantified using Photoshop CS5 using Fovea tools. Microarray analysis
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For transcriptional analysis, sort-purified CD8+ TEM and CD8+ TCM from PBMC, obtained 28–49 days post-first anti-IL-15 or control IgG mAb administration, were resuspended in RLT lysis buffer (Qiagen) and stored at −80°C until use. RNA was isolated using RNeasy Micro Kits (Qiagen), and the quantity and quality of the RNA was confirmed using a NanoDrop 2000c (Thermo Fisher Scientific) and an Experion Electrophoresis System. Samples (50ng) were amplified using Illumina TotalPrep RNA amplification kits (Ambion). The microarray analysis was conducted using 750ng of biotinylated complementary RNA hybridized to HumanHT-12_V4 BeadChips (Illumina) at 58°C for 20h. The arrays were scanned using Illumina’s iSCAN and quantified using Genome Studio (Illumina). The analysis of the Genome Studio output data was conducted using the R and Bioconductor software packages. Quantile normalization was applied, followed by a log2 transformation
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performed using the Bioconductor LIMMA package (57). Outlier samples with abnormalities in gene expression based on hierarchical clustering methods and multidimensional scaling analysis as a dimensionality reduction method for the evaluation of similarities or dissimilarities between samples were identified and removed. The LIMMA package was used to fit a linear model to each probe and perform (moderated) t tests or F tests on the groups being compared. A Gene Set Enrichment Analysis (GSEA) (58) using 1000 permutations was performed on the genes differentially expressed between the groups compared in the subsets pre-ranked by the decreasing order of the absolute T-statistic. The canonical pathways of Ingenuity Pathway Analysis software (IPA, Ingenuity Systems) were used as the database to perform GSEA. This was followed by building modules of related pathways based on at least 25% gene overlap [Jaccard index (59) > 25%] between pathways using the enrichment map (60) strategy and representing the genes present in at least 25% of the pathways in the module. To control the expected proportions of false positives, the FDR for p-values was calculated using the Benjamini and Hochberg method implemented in LIMMA. The complete dataset is available at the Gene Expression Omnibus microarray repository (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? token=mbqtaouqztynzsz&acc=GSE76797), accession number GSE76797.
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Statistics
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To investigate if the administration of anti-IL-15 mAb blocks signaling in circulating lymphocytes, we estimated the EC50 (the dose where the response is the midpoint between the maximum and minimum) for each RM at given time points (days post-mAb) using the four-parameter logistic model often referred to as an Emax model, followed by repeated measures ANOVA to evaluate the effect of anti-IL-15 mAb with EC50 as a response variable and anti-IL-15 mAb-treated status as between group factor, and days post-antibody as within group factors. Longitudinal analysis of STAT5 phosphorylation, peripheral blood TN and TM (including TCM, TTrM and TEM subsets) counts and proliferation, peripheral blood NK cell counts and plasma IL-7 levels were evaluated using repeated-measures ANOVA with antiIL-15 mAb and IgG control mAb-treated groups as between group factors and time points as within group factors; since in a typical experiment using repeated measures, two measurements taken at adjacent times are more highly correlated than two measurements taken several time points apart. Due to the limited sample size, a simpler covariance structure, first order auto-regressive (AR1), was used as correlation within each animal. Tukey-Kramer adjustment was used to control for multiple comparisons. Differences in the number of pSTAT5+ cells/mm2 in peripheral lymph nodes between pre-treatment and posttreatment were evaluated using Wilcoxon Signed rank tests. The difference in CD3+ caspase-3+ in colon and peripheral LN via immunostaining and BrdU incorporation in CD4+ and CD8+ T cell subsets between anti-IL-15 and IgG control mAb-treated groups was compared using Mann-Whitney U test. Data was analyzed using SAS 9.4. The p-values 80% of cells at 16ng (Fig. 1C), only the CD56+ CD16− “regulatory” NK cell subset showed any response to IL-7 stimulation at the tested doses, and this response required a 4-fold higher dose of IL-7 than the response of the same cells to IL-15.
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These results demonstrate that lymphocyte populations differ in their response to early signal transduction by IL-7 vs. IL-15, and suggest that IL-7 and IL-15 preferentially signal pre-effector and effector populations, respectively. We next sought to determine the degree to which this differential signaling translates to downstream gene expression and homeostatic regulation by examining the ability of these cytokines to support survival and proliferative expansion of sort-purified TN, TCM and TEM during 7 and 14 days of in vitro culture. Since the pattern of STAT5 phosphorylation to IL-7 and IL-15 among these differentiation-defined subsets was similar between the CD4+ and CD8+ lineages (Fig. 1A– B), we primarily focused on CD4+ T cells for these studies. In this in vitro CD4+ T cell model system, responses to IL-15 were generally more robust than to IL-7, but responsiveness to both cytokines was differentiation dependent. TEM were again found to be highly responsive to IL-15 compared to IL-7, with over 60% of IL-15-treated cells expressing the proliferation antigen Ki-67 by day 7 of culture, compared to background Ki-67 expression after IL-7 treatment (Fig. 2A). IL-7 and IL-15 induced similar, modest increases in Ki67 expression by CD4+ TCM, whereas IL-7 was more effective than IL-15 in inducing TN proliferation. Next, based on the concept that IL-15 might be more efficiently presented in trans, we cultured the same cells with IL-15 or IL-7 in the presence of sort-
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purified CD14+ monocytes (63, 64). In the presence of monocytes, we saw modestly enhanced responsiveness of TN, TCM, and TEM to IL-7, and profoundly increased responsiveness of TN and TCM to IL-15 (Fig. 2B). Interestingly the CD4+ TEM proliferative response to IL-15 was similar with or without monocytes, suggesting that trans-presentation or other monocyte-derived signals are not required for maximal TEM responsiveness. Similar IL-15 responsiveness was demonstrated for CD8+ TEM (Supplementary Figure 2A).
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Collectively, these in vitro studies demonstrate that TN and TCM are more responsive to IL-7 than TEM, whereas TEM are more responsive to IL-15. However, IL-15 can induce robust proliferation in TN and TCM in the presence of monocytes, suggesting a more pleotropic role for this cytokine. Interestingly, culture of TN and TCM with IL-15 in the presence of monocytes induces up-regulation of CCR5 and down-regulation of CCR7 (Supplementary Figure 2B), consistent with induction of effector memory differentiation (50, 65). Thus, IL-15 may both maintain the homeostasis of pre-existing TEM and, under certain conditions, drive TEM differentiation from noneffector-differentiated precursors. Anti-IL-15 specifically blocks IL-15 signal transduction in vivo
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To investigate the role of IL-15 in T cell and NK cell homeostasis in vivo, we developed a “rhesusized” anti-IL-15 mAb that is suitable for repeated administration to RM. This mAb was based on the mouse, anti-human IL-15 mAb clone, M111, which is cross-reactive with RM IL-15 (66). Rhesusization was accomplished by exchanging the mouse amino acid sequences in the constant regions and variable binding surfaces for rhesus amino acid sequences, leaving variable region sequences responsible for IL-15 recognition unchanged (67, 68). We hypothesized that repeated administrations of this rhesusized M111 mAb would block IL-15 activity in vivo and result in changes to T and/or NK cell population dynamics that would reflect physiologic IL-15 function. To initially test this approach, we administered rhesus anti-IL-15 or a rhesus IgG control mAb to RM every other week for 6 weeks prior to necropsy, as shown in Fig. 3A. We assessed IL-15 signaling inhibition by performing pSTAT induction analysis with rhesus recombinant IL-15 on whole blood at various time points post-treatment. From day 1 through day 35 post-treatment, the ability of IL-15 to induce pSTAT5 expression in CD4+ and CD8+ T cells ex vivo in anti-IL-15-treated RM was significantly reduced (essentially abrogated) compared to baseline and to control IgG-treated RM (p
