Human NKp44⁺ Group 3 Innate Lymphoid Cells Associate with Tumor-Associated Tertiary Lymphoid Structures in Colorectal Cancer

Innate lymphoid cells (ILC) are tissue-resident lymphocytes and classified as natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer (LTi) cells (1). ILC3s that express the transcription factor RORγt and produce IL17 and IL22, are abundant in the intestine and important for host defenses against invading pathogens (2–5). ILC3s can regulate the responses of IL17- or IFNγ-producing CD4⁺ T cells to commensal bacteria through antigen presentation via MHC class II. These cells are decreased in pediatric patients with Crohn disease (6). ILC3s promote regulatory T-cell–mediated oral tolerance by producing IL2 and this ILC3-mediated regulation is disturbed in the small intestine of patients with Crohn disease (7). Human ILC3s possess LTi cell activity, which can induce ectopic lymphoid aggregates (8). As ILC3s can act as an important regulator of intestinal immune responses, dysregulated ILC3-mediated immune responses may be implicated in the pathogenesis and/or severity of several intestinal diseases.

Studies have identified immune cells, including CD8⁺ T cells, regulatory T cells, and tumor-associated macrophages, infiltrating colorectal tumors and their functions in the suppression or progression of tumor growth (9–11). Accumulating evidence for tumor-infiltrating immune cells has led to immunotherapies for multiple types of solid cancers. However, current immunotherapies for colorectal cancer have not demonstrated excellent clinical efficiency. To identify a novel therapeutic target for colorectal cancer, more extensive characterization of the immune cells infiltrating tumors is required.

Here, we reported that human NKp44⁺ ILC3s, which express genes related to the formation of lymphoid structures, were abundant in normal colon compared with other ILC subsets and infiltrate colorectal cancer tissue. We found that the abundance of NKp44⁺ ILC3s and their expression of tertiary lymphoid structure (TLS) formation–related genes were markedly reduced in T3/T4 tumors. In addition, a decreasing number of NKp44⁺ ILC3s during tumor progression correlated with the TLS density.

Normal colonic mucosa and colorectal tumor samples were obtained from 28 patients with colorectal cancer; these patients received intestinal resections for colorectal cancer at Osaka University (Suita, Osaka, Japan). Patient characteristics are provided in Supplementary Table S1. Patients were excluded when they had a history of inflammatory bowel disease, or autoimmune disease, or when they received a neoadjuvant chemotherapy. Normal colonic mucosa was obtained from a macroscopically intact area ≥5 cm from the tumor. Both normal and tumor tissues were obtained immediately after surgical resection and cell isolations were started subsequently. Blood samples were obtained from healthy adults. This study was approved by the Ethical Committee of Osaka University School of Medicine (Suita, Osaka, Japan). Written informed consent was obtained from all patients for the use of their samples and data.

Human lamina propria cells (LPC) and tumor-infiltrating lymphocytes (TIL) were isolated using a previously described protocol (12) with slight modifications. Briefly, both normal mucosa and tumor tissues were washed in PBS to remove feces and then weighed. The normal mucosa sample was placed in Hank's balanced salt solution containing 5 mmol/L ethylenediaminetetraacetic acid and incubated for 5 minutes with shaking. After washing with PBS, the tissue samples were minced mechanically into small pieces. For both tissues, the small pieces were enzymatically digested with 1 mg/mL collagenase II (Worthington Biochemical Corporation) and 80 U/mL DNase I (Sigma-Aldrich) in RPMI1640 containing 4% FBS for 60 minutes in a 37°C shaking water bath. The digested tissues were filtered through a 40-μm cell strainer. The isolated cells were washed with PBS, resuspended in 7 mL 20% Percoll (GE Healthcare), and overlaid on 2 mL of 40% Percoll in a 15-mL tube. Percoll gradient separation was performed by centrifugation at 500 × g for 30 minutes at 4°C. LPCs and TILs were collected at the Percoll gradient interface and washed with PBS containing 2% FBS.

Isolated cells were stained with surface antibodies for 30 minutes at 4°C followed by 7AAD staining (BD Bioscience) before analysis of flow cytometry. For intranuclear transcription factor staining, cells were fixed, permeabilized, and stained using Foxp3/Transcription Factor Staining Buffer Kit (eBioscience) according to the manufacturer's instructions. Flow cytometric analysis and cell sorting were conducted using a FACSAriaII (BD Bioscience). The data were analyzed using FlowJo Software (Tree Star).

The following antibodies were purchased from BioLegend: anti-human CD3-FITC (HIT3a), CD11c-FITC (Bu15), CD14-FITC (HCD14), CD19-FITC (HIB19), CD20-FITC (2H7), CD45-APC/Cy7 (HI30), CD117-PE (104D2), NKp44-APC (P44-8), GATA3-APC (16E10A23), CD31-Pe/Cy7 (WM59), CD90-BV421 (5E10), ICAM-1-PE (HA58), and VCAM-1-APC (STA). Anti-human CD127-BV421 (HIL-7R-M21) was purchased from BD Biosciences. Anti-human CRTH2-PE (BM16) was purchased from Miltenyi Biotec. Antihuman EpCAM-Alexa Fluor 488 (VU1D9) was purchased from Cell Signaling Technology. Anti-human T-bet-PE (4B19) and ROR gamma t-PE (AFKJS-9) were purchased from eBioscience. Phorbol myristate acetate (PMA) was purchased from Sigma-Aldrich. Ionomycin was purchased from Merck. Recombinant human IL-12p40 was purchased from PeproTech.

Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and cDNAs generated using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). qRT-PCR was performed on a Step One Plus Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems). The amplification conditions were 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. All data were normalized to the expression of GAPDH and expressed as relative expression using the ΔΔC_t method. The following primer sets were used: GAPDH, 5′-GTCGGAGTCAACGGATT-3′ and 5′-AAGCTTCCCGTTCTCAG -3′: CCL19, 5′-CCTGCTGGTTCTCTGGACTT-3′ and 5′-GTACCCAGGGATGGGTTTCT-3′; CCL21, 5′-GTTGCCTCAAGTACAGCCAAA-3′ and 5′-AGAACAGGATAGCTGGGATGG-3′; CSF2, 5′-CACTGCTGCTGAGATGAATGAAA-3′ and 5′-GTCTGTAGGCAGGTCGGCTC-3′; CXCL13, 5′-CAGCCTCTCTCCAGTCCAAG-3′ and 5′-TGAGGGTCCACACACACAAT-3′; ICAM1, 5′-CGATGACCATCTACAGCTTTCCGG-3′ and 5′-GCTGCTACCACAGTGATGATGACAA-3′; IL17A, 5′-GAAGGCAGGAATCACAATC-3′ and 5′-GCCTCCCAGATCACAGA-3′; IL22, 5′-CCCATCAGCTCCCACTGC-3′ and 5′-GGCACCACCTCCTGCATATA-3′; IFNG, 5′-CCAGGACCCATATGTAAAAG-3′ and 5′-TGGCTCTGCATTATTTTTC-3′; LTA, 5′-CCAGCAAGCAGAACTCA-3′ and 5′-ATGGGCCAGGTAGAGTG-3′; LTB, 5′-AGTGCCCCAGGATCAG-3′ and 5′-GCCGACGACACAGTAGAG-3′; RORC, 5′-CCCGTCAGCAGAACTG-3′ and 5′-AGCCCCAAGGTGTAGG-3′; TBX21, 5′-GATGTTTGTGGACGTGGTCTTG-3′ and 5′-CTTTCCACACTGCACCCACTT-3′; and TNF, 5′-CGCTCCCCAAGAAGAC-3′ and 5′-AGGGCTGATTAGAGAGAGGT.

For morphologic analyses, isolated LPCs and TILs were spread on glass slides with cytospin, air-dried, and fixed with methanol for 5 minutes. After air dry, cells were stained with Giemsa (diluted 1:20 with deionized water) for 15 minutes. The stained slides were rinsed in deionized water, air-dried, then evaluated. Images were obtained by the BZ-X710 Microscope System.

We used the miRNeasy Mini Kit (Qiagen) to extract the total RNA from ILC1s (CD117⁻NKp44⁻), NKp44⁻ ILC3s (CD117⁺NKp44⁻), and NKp44⁺ ILC3s (CD117⁺NKp44⁻⁺) in normal colonic mucosa from individual patients with colorectal cancer (n = 10) as directed by the manufacturer. Each cDNA was generated using a SMART-Seq HT Kit (Takara Clontech). Each library was prepared using a Nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer's instructions. Whole-transcriptome sequencing was performed with the RNA samples using the HiSeq 2500 Platform (Illumina) in 75-base single-end mode. Sequenced reads were mapped onto the human reference genome sequences (hg19) using TopHat ver. 2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 1.0. The number of fragments per kilobase of exon per million mapped fragments was calculated by Cufflinks ver. 2.2.1. Extracted transcript reads were normalized to the area and uploaded to ClustVis, a web tool for visualizing the clustering of multivariate data using heatmaps and principal component analysis (Gene Expression Omnibus accession number GSE137564).

Viable EpCAM⁻ CD45⁻ CD31⁻ CD90⁺ stromal cells were isolated from colorectal cancer tissue using a FACSAriaII. These cells were cocultured with NKp44⁺ ILC3s from the lamina propria of normal colon tissue from the same patients at a 1:1 ratio in the presence of IL2 (10 ng/mL; PeproTech) and IL7 (10 ng/mL; PeproTech) for 24 hours and gene expression was analyzed.

Double IHC was performed on formalin-fixed, paraffin-embedded tissue sections (3.5 μm). After deparaffinization, antigen retrieval was performed using 10 mmol/L citrate buffer (pH 6); intrinsic peroxidase activity was blocked with 3% H₂O₂ for 20 minutes, followed by nonspecific interaction blocking with Background Sniper (Biocare Medical) for 10 minutes. The slides were then incubated with primary antibodies for 2 hours at room temperature, including anti-CD3 (SP7, Abcam, rabbit, diluted 1:150) and anti-RORγt (MABF81, Sigma-Aldrich, mouse, diluted 1:50). After staining with primary antibody cocktails, we used MACH2 Double Stain2 (Biocare Medical) as the secondary antibody. Anti-CD3 (F7.2.38, Abcam, diluted 1:250), anti-CD20 (L26, eBioscience, diluted 1:250), and anti-CD21 (2G9, Leica Biosystems, diluted 1:100) were used for IHC staining of TLSs.

For TLS quantification, three hematoxylin and eosin–stained, formalin-fixed, paraffin-embedded tumor tissue sections that contained representative areas of the tumor with adjacent nontumorous tissue were selected. The total area of the tumor (in mm²) within selected sections was measured using the BZ-X710 Microscope System (Keyence). The number of TLSs was counted in the tumor area of all selected sections. The TLS density was then calculated by the ratio of total TLSs to the total tumor area. An expert pathologist observed the whole procedure.

Statistical analyses were performed using GraphPad Prism, version 8.0 (GraphPad). Differences between two groups were calculated by two-tailed Student t test. Differences with P < 0.05 were considered significant.

To characterize ILCs infiltrating colorectal cancer tissue, we first examined ILCs in the normal colons (Supplementary Fig. S1). Among LPCs, CD45⁺ Lin⁻ cells were divided into CD127⁻ cells (subset I) and CD127⁺ ILCs (subset II; Supplementary Fig. S1A and S1B). Subset II comprised CD117⁻ NKp44⁻ ILCs (subset III), CD117⁺ NKp44⁻ ILCs (subset IV), and CD117⁺ NKp44⁺ ILCs (subset V; Supplementary Fig. S1A and S1C). Subset IV and subset V highly expressed RORγt, which is encoded by RORC and responsible for the development of ILC3s, whereas subset III expressed a master regulator of ILC1, T-bet, which is encoded by TBX21 (ref. 1; Supplementary Fig. S1D and S1E). RNA-sequence analysis also demonstrated that higher expression of TBX21 was restricted to subset III (Supplementary Fig. S2A). In addition, subset III profoundly expressed IFNG compared with other subsets, indicating that this subset may be an ILC1 population. Although promoted expression of RORC was common among subsets IV and V, these populations exhibited different patterns of gene expression (Supplementary Fig. S2A and S2B). Regarding the ILC3-related cytokines, IL17A was highly expressed in subset IV, whereas expression of IL22 was higher in subset V (Supplementary Fig. S2A and S2B). Subset V expressed several genes associate with the formation of lymphoid structures in the neonatal or postneonatal period, including LTA, LTB, TNF, CXCR5, ITGB1, ITGAL, and NRP1 (8, 13). These findings suggest that subsets IV and V are distinct subpopulations of ILC3s. Hereafter, we defined subset IV as NKp44⁻ ILC3s and subset V as NKp44⁺ ILC3s.

Next, we investigated the three subsets of ILCs in tumor tissues from patients with T3/T4 stage colorectal cancer. The three ILC subsets were observed at the tumor site, similar to normal mucosa (Fig. 1A). The ILC1 subset expressed higher T-bet than the other two populations, whereas both NKp44⁻ and NKp44⁺ ILC3s highly expressed RORγt (Fig. 1B and C). In addition, the three ILC subsets from tumor tissue exhibited lymphoid morphologies similar to ILCs in the normal mucosa (Fig. 1D). These findings demonstrated that the same three ILC subsets (ILC1, NKp44⁻ ILC3, and NKp44⁺ ILC3) were present in progressed tumor tissues and normal colonic mucosa.

CD127⁺ ILCs contain ILC2s, which express CRTH2 and the transcription factor GATA3 (14, 15). Therefore, we evaluated the presence of ILC2 in the normal colon and tumors from patients with colorectal cancer (Supplementary Fig. S3). The frequency of CD127⁺ CRTH2⁺ ILCs with lymphoid morphology was much lower than the frequency of CD127⁺ CRTH2⁻ ILCs, in both the normal mucosa and T3/T4 tumors, but was abundant among peripheral blood ILCs. In all three tissues, CD127⁺ CRTH2⁺ ILCs expressed higher GATA3 than CD127⁺ CRTH2⁻ ILCs (Supplementary Fig. S3). These findings indicate that fewer CRTH2⁺ GAT3⁺ ILC2s are present among CD127⁺ ILCs in normal colon and progressed tumor tissue.

To investigate the role of ILCs in the tumor immune microenvironment of colorectal cancer, we analyzed the three ILC subsets in normal colon and different stages of colorectal cancer (Fig. 2). The frequencies of ILC1s and NKp44⁻ ILC3s increased gradually, consistent with tumor progression (Fig. 2A, B, and D), whereas the frequency of NKp44⁺ ILC3s decreased as the tumor progressed (Fig. 2C and D). The numbers of all three subsets peaked in the T1/T2 tumors similar to CD45⁺ cells (Supplementary Fig. S4A). In the T3/T4 tumors, NKp44⁺ ILC3s were greatly reduced compared with the normal colon (Fig. 2C), but the other two subsets were sustained at higher numbers than in normal colon (Fig. 2A and B). An analysis of samples from 1 patient who had two synchronous colorectal cancers of T1 and T3 stage revealed a typical alteration of NKp44⁺ ILC3s during tumor progression (Supplementary Fig. S4B). These results indicated that the composition of the three ILC subsets was altered during tumor progression in patients with colorectal cancer, and that NKp44⁺ ILC3s were preferentially reduced in more progressed tumors.

The accumulation of NKp44⁺ ILC3s is necessary for antitumor immunity in human lung cancer by inducing TLSs via the expression of several molecules, including LTα, LTβ, and TNFα (16). NKp44⁺ ILC3s present in normal colon highly expressed LTA, LTB, and TNF (Supplementary Fig. S2). Therefore, we attempted to investigate whether the function of NKp44⁺ ILC3s changed during tumor progression in patients with colorectal cancer. In T3/T4 tumors, the expression of LTA, LTB, and TNF was markedly reduced in NKp44⁺ ILC3s compared with normal colon and T1/T2 tumors (Fig. 3A). However, NKp44⁺ ILC3s in T3/T4 tumors normally expressed RORC, IL22, IL17A, and CSF2 (Supplementary Fig. S5A). A previous murine study showed that proinflammatory cytokine IL-12p40 induces expression of Lt-α in macrophages (17). In T3/T4 tumor NKp44⁺ ILC3s stimulated with IL-12p40 increased the expression of LTA and LTB, whereas TNF expression was upregulated in response to PMA plus ionomycin stimulation (Supplementary Fig. S5B).

Interaction of LTα1β2 with LTβR on stromal cells promotes the expression of CXCL13, CCL19, CCL21, and adhesion molecules, including VCAM-1 and ICAM-1, in stromal cells during the generation of lymphoid structures (13). We isolated stromal cells from T2 and T3/4 colorectal cancer tumors and analyzed the expression patterns of these molecules (Fig. 3B and C; Supplementary Fig. S6A). In T3/T4 tumors, expression of ICAM-1 and VCAM-1 on stromal cells significantly decreased compared with T2 tumors (Fig. 3B). The expression of CXCL13 and CCL21 drastically decreased in stromal cells from T3/T4 tumors relative to T2 tumors, and CCL19 expression was partially reduced in stromal cells from T3/T4 tumors (Fig. 3C). Increased expression of ICAM1, CXCL13, and CCL21 was found in stromal cells from T3/T4 tumors by coculturing with normal colonic NKp44⁺ ILC3s (Supplementary Fig. S6B). These findings suggested that NKp44⁺ ILC3s in T3/T4 colorectal cancer have lower expression of TLS formation–related genes, leading to decreased expression of lymphoid chemokines and adhesion molecules in stromal cells.

As the number of NKp44⁺ ILC3s and expression of lymphoid structure formation–related genes in these cells was diminished in T3/T4 colorectal cancer tissues, we evaluated whether an altered abundance of NKp44⁺ ILC3s correlated with the density of TLSs, which are organized at peritumoral and intratumoral sites in patients with colorectal cancer (18). TLSs comprised of a network of CD20⁺ B cells and CD21⁺ follicular dendritic cells surrounded by CD3⁺ T cells; TLSs were more abundant in the early stages of tumor progression, with numbers gradually reducing as tumor stage progressed (Fig. 4A). CD3⁻ RORγt⁺ cells were distributed at the edge of lymphoid aggregates in peritumoral and intratumoral TLSs (Fig. 4B, i–iii) and around small blood vessels in the intratumoral TLSs (Fig. 4B, iv). The number of CD3⁻ RORγt⁺ cells gradually decreased as tumors progressed (Fig. 4B). In colorectal tumor tissues, the TLS density correlated with the frequency of tumor-infiltrating NKp44⁺ ILC3s among CD127⁺ ILCs (R = 0.62; P < 0.005) and the number of NKp44⁺ ILC3s/mg of tissue (R = 0.64; P < 0.005; Fig. 4C). These findings demonstrated that the presence of NKp44⁺ ILC3s correlated with the density of TLSs during tumor progression in colorectal cancer.

Here, we identified that the same three ILC subsets (ILC1, NKp44⁻ ILC3, and NKp44⁺ ILC3) were present in normal colonic mucosa and colorectal cancer tumor tissue. In normal colon tissue, NKp44⁺ ILC3s were abundant compared with ILC1s, ILC2s, and NKp44⁻ ILC3s and expressed genes related to the formation of lymphoid structures. However, their number and expression of a subset of genes, including LTA, LTB, and TNF, decreased with tumor progression. In addition, a decreased number of NKp44⁺ ILC3s was associated with the density of TLSs in colorectal cancer.

NKp44⁺ ILC3s are the most prevalent ILC subset in the human intestine (14, 19, 20), whereas human ILCs in colorectal cancer remain poorly understood. Here, we found that NKp44⁺ ILC3s were present in colorectal cancer tumors, but decreased as the tumors progressed; conversely ILC1s and NKp44⁻ ILC3s increased during tumor progression. Increasing evidence indicates a plasticity in ILC populations (20, 21). We observed that NKp44⁺ ILC3s in T3/T4 tumors expressed higher ILC1-specific genes, including IFNg, IL10, and CCL20, and NKp44⁻ ILC3-specific genes, including HLA-DRB1 and IL17A, than NKp44⁺ ILC3s in normal tissues (Supplementary Fig. S7). In addition, NKp44⁺ ILC3s from T3/T4 tumors were reactivated, including the upregulation of LTA, LTB, and TNF transcription by exogenous stimuli. These findings suggest that the distinct microenvironment during tumor progression may provide NKp44⁺ ILC3s with ILC1-like and NKp44- with ILC3-like profiles, whereas it leads to a decrease in TLS formation–related genes. Thus, future studies should analyze whether the tumor microenvironment affects the differentiation, plasticity, survival, and activation of ILCs in colorectal cancer.

Effector immune responses are one of the cell types responsible for the elimination of tumor cells in the early phase of the tumor (22). The number of CD45⁺ cells increased in T1/T2 tumors at similar rates to NKp44⁺ ILC3s. Adaptive T lymphocytes, including activated memory CD4⁺ T cells, CD8⁺ T cells, and Foxp3⁺ regulatory T cells, are increased in T1/T2 colorectal cancer compared with the normal colon and T3/T4 colorectal cancer (23, 24). ILCs promote both Th cell and Foxp3⁺ regulatory T-cell responses (7, 25). Thus, coordinated interaction between ILCs and T cells may evoke both innate and adaptive immune responses and correlate to the high density of hematopoietic cells in early stage tumors.

Here, we observed that TLSs were increased in early-stage colorectal cancer tumors with abundant NKp44⁺ ILC3s, whereas the density was reduced in advanced-stage tumors with fewer NKp44⁺ ILC3s. Growing evidence indicates that TLSs play a major role in the inhibition of tumor invasion and metastasis, which results in a favorable prognosis in various types of human cancers, including colorectal cancer (18). ILC3s expressing Neuropilin 1 (NRP1) in human lung mediate the formation of ectopic lymphoid aggregates (8), which is associates with antitumor responses in lung cancer (16). We found a decreased frequency of NKp44⁺ cells among NRP1⁺ ILC3s in T3/T4 colorectal cancer tumors (38.6% ± 4.4%) compared with normal colon (78.0% ± 2.4%, P < 0.0001). Therefore, analysis of whether TLS induction mediated by NKp44⁺ ILC3s contributes to promoting the antitumor immune response in human colorectal cancer is needed in future studies.

Taken together, our results indicated an alteration of ILC subsets during tumor progression in patients with colorectal cancer. In T3/T4 colorectal cancer tumors, the number of NKp44⁺ ILC3s was decreased, with an accompanying decrease in TLS density and expression of lymphoid structure formation–related genes in these cells. These findings shed light on the role of ILCs in the tumor immune microenvironment of colorectal cancer. It will be important to analyze the relevance of less NKp44⁺ ILC3s during tumor progression to antitumor immune responses in the future.

No potential conflicts of interest were disclosed.

Conception and design: A. Ikeda, T. Ogino, H. Kayama, J. Nishimura, C. Matsuda, Y. Doki

Development of methodology: A. Ikeda, T. Ogino, J. Nishimura, H. Takahashi, C. Matsuda, Y. Doki

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Ikeda, T. Ogino, S. Fujino, H. Takahashi, C. Matsuda

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Ikeda, T. Ogino, H. Kayama, D. Okuzaki, N. Miyoshi, H. Yamamoto

Writing, review, and/or revision of the manuscript: A. Ikeda, T. Ogino, H. Kayama, N. Miyoshi, M. Uemura, T. Mizushima

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Ogino, J. Nishimura, K. Takeda, M. Mori

Study supervision: T. Ogino, H. Kayama, J. Nishimura, H. Takahashi, C. Matsuda, H. Yamamoto, K. Takeda, T. Mizushima, M. Mori, Y. Doki

This work was supported by a research grant from the Osaka Medical Research Foundation for Intractable Disease (to T. Ogino), PRIME, Japan Agency for Medical Research and Development (19gm6210016; to H. Kayama), JSPS KAKENHI Grant Number 17K10631 (to T. Mizushima), a research grant from the Princess Takamatsu Cancer Research Fund 18-25031 (to T. Mizushima), and Astellas Research Support (to T. Mizushima).

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