International Immunology

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (38) Request Permissions Google Scholar Articles by Obata-Onai, A. Articles by Matsushima, K. Search for Related Content PubMed PubMed Citation Articles by Obata-Onai, A. Articles by Matsushima, K. Social Bookmarking          What's this?

International Immunology, Vol. 14, No. 10, pp. 1085-1098, October 2002
© 2002 Japanese Society for Immunology

Comprehensive gene expression analysis of human NK cells and CD8⁺ T lymphocytes

Aya Obata-Onai¹, Shin-ichi Hashimoto¹, Nobuyuki Onai¹, Makoto Kurachi¹, Shigenori Nagai¹, Ken-ichi Shizuno¹, Tomoyuki Nagahata¹ and Kouji Matsushima¹

¹ Department of Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Correspondence to: K. Mathushima; E-mail: koujim{at}m.u-tokyo.ac.jp
Transmitting editor: M. Miyasaka

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Cytotoxic lymphocytes, NK cells and CD8⁺ T cells play a pivotal role in the host defense. To reveal the biological function of these cells through establishing a comprehensive gene expression profile, serial analysis of gene expression was performed in human peripheral blood NK cells and CD8⁺ T cells. In total, 85,848 tags corresponding to >20,000 different transcripts were sequenced. The genes expressed abundantly in these libraries mostly consisted of genes encoding MHC class I and molecules related to protein synthesis. Among gene transcripts which related to cytotoxicity, granulysin, perforin, granzyme B and -defensin 1 were highly expressed in NK cells. Resting CD8⁺ T cells did not express the genes related to cytotoxicity, but expressed abundantly the genes encoding chemokines, tumor necrosis factor family. When CD8⁺ T cells were sorted into naive, memory and effector subsets based on the expression of CD45RA and CD27, perforin and granzyme B were expressed in the CD45RA⁺CD27^– effector subset. -Defensin 1, one of the selectively expressed genes in NK cells, induced migration of naive CD8⁺CD45RA⁺CD27⁺ T cells, but not memory CD8⁺CD45RA^–CD27⁺ or effector CD8⁺CD45RA⁺CD27^– T cells. Furthermore, treatment with IL-15, a stimulator of NK cell development, differentiation, survival and cytotoxicity, rapidly enhanced the expression of -defensin 1 in NK cells. The identification of the genes preferentially expressed in NK and CD8⁺ T cell subsets may give important insights into the functions of these cells against virus infection and in tumor immunity.

Keywords: cytotoxic T lymphocyte, cytotoxicity, defensin, serial analysis of gene expression, NK

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Lymphocyte-mediated cytotoxicity is the principal mechanism for eradicating tumor cells, virus-infected cells and intracellular pathogens. This process is mainly mediated by NK cells and cytotoxic T lymphocytes, which have cytotoxic granules containing various cytotoxic effecter molecules such as perforin and granzymes (1). These cells also express Fas ligand and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) (2), through which they induce apoptosis in Fas- and TRAIL receptor-expressing target cells (3,4).

NK cells were originally described on a functional basis according to their ability to lyse certain tumors without prior antigenic stimulation (5). It is generally accepted that NK cells provide the first line of defense against certain tumors or viral infections. NK cells do not express conventional receptors for antigens, i.e. surface Ig or TCR. The molecular mechanism allowing NK cells to discriminate between normal and tumor cells has recently been clarified (6). NK cells recognize MHC class I molecules through surface receptors delivering signals that inhibit NK cell function. Triggering of NK cells results not only in cytotoxicity, but also in the production of cytokines and chemokines that exert a regulatory role in the immune response, inflammation and hematopoiesis (7).

CD8⁺ T cells are important mediators for adaptive immunity against certain viral, protozoan and bacterial pathogens. During the initial encounter with a microbe, CD8⁺ T cells bearing TCR specific for pathogen-derived antigens are selected to undergo clonal expansion. As a result, pathogen-specific CD8⁺ T cells rapidly increase from virtually undetectable in the naive host to levels that are easily detectable [1–2% of splenic CD8⁺ T cells in primary responses to certain bacterial infections (8) or even dominate the repertoire, reaching 50% splenic CD8⁺ T cells in the primary response to certain viral infections (9,10)]. These expanded populations of ‘effector’ CD8⁺ T cells contribute to clearance of the pathogen and then decline in numbers to a memory level that may be maintained at 5–10% of the initial clonal burst size (9). Memory CD8⁺ T cells may be present through the life of the host and are able to mount rapid, heightened responses to reinfection with the specific pathogen (11). Thus, NK cells and CD8⁺ T cells belong to different lymphocyte lineages, but share common mechanism to exert their cytotoxic function.

Defensins, comprising a family of small (3.5–4.5 kDa) cationic antimicrobial peptides with three to four intra-molecular cysteine disulfide bonds, are widely distributed in mammals, insects and plants (12,13). Human -defensins 1, 2, 3 and 4 are expressed in neutrophils, and thus are termed human neutrophil peptides (14). Furthermore -defensins 1, 2 and 3 exist in T cells, CD19⁺ B cells, CD56⁺ NK cells and monocytes/macrophages (15). In addition to their antimicrobial effects, it was reported that defensin is chemotactic for human naive CD4⁺ T cells and immature dendritic cells (DC) (16).

Serial analysis of gene expression (SAGE) allows the establishment of both a representative and comprehensive different gene expression profile in various cell types and organs under physiological and pathological conditions (17–21). Since each template contains identifiable tags corresponding to many genes, this method allows global gene expression profiling including unknown genes.

In this study, we have analyzed the expression profiles of genes in freshly isolated, circulating NK cells and CD8⁺ T cells using SAGE, and identified numerous genes of which expression is selective in either population.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Purification of human NK cells and CD8⁺ T lymphocytes
To prepare NK cells, peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Ficoll-Metrizoate density gradient (d = 1.077g/ml, Lymphoprep; Nycomed, Oslo, Norway) from the venous blood drown from three healthy volunteers and were suspended in labeling buffer (0.5% BSA and 2 mM EDTA containing PBS). PBMC were incubated with a cocktail of anti-CD3, anti-CD19 and anti-CD14 mAb-coated microbeads, and NK cells were isolated by passing the PBMC through a magnetic cell separation system (MACS; Miltenyi Biotec, Bergish Gladbach, Germany) with column type VR. The total number of negatively selected NK cells was 2 x 10⁷. More than 95% of the cells were confirmed to be CD56⁺ (PharMingen, San Diego, CA) NK cells by flow cytometry analysis. To prepare human CD8⁺ T cells, PBMC from three healthy volunteers were suspended in labeling buffer and incubated with anti-CD8 mAb-coated microbeads. CD8⁺ T cells were isolated using a magnetic cell separation system with column type VR. The total number of isolated CD8⁺ T cells was 3 x 10⁷. More than 95% of the cells were confirmed to be CD8⁺ (Dako, Kyoto, Japan) T cells by flow cytometry.

SAGE protocol
The total RNA was isolated by direct lyses using RNAzol B (Cinna/Biotex, Tel-Test, Friendswood, TX). Poly(A)⁺ RNA was further isolated using the µMACS mRNA isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. SAGE was performed as described previously (17–21). Poly(A)⁺ RNA (200 ng) was converted to cDNA with a Superscripts Choice System for cDNA synthesis (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol with the inclusion of biotin-conjugated 5'-T18-3' primer.

Double-stranded cDNA was cleaved with NlaIII and the 3'-terminal cDNA fragments were bound to streptavidin-coated magnetic beads (Dynal, Oslo, Norway). After ligation of oligonucleotides containing recognition sites for BsmFI, the linked cDNA were released from the beads by digestion with BsmFI. The released tags were ligated to each other, concatemerized and cloned into the SphI site of pZero 1.0 (Invitrogen). Colonies were screened by PCR using M13 forward and M13 reverse primers. PCR products containing inserts of >600 bp were sequenced with the TaqFS Dye Terminator kit version 2 and analyzed using a 377 ABI automated sequencer (Perkin-Elmer, Branchburg, NJ). All electrograms were reanalyzed by visual inspection to check for ambiguous bases and to correct misreads.

The SAGE was performed on mRNA from human NK cells and CD8⁺ T cells.

Sequence files were analyzed with SAGE software, CGAPSAGE database (http://www.ncbi.nlm.nih.gov/SAGE/) and NCBI’s sequence search tool (Advanced BLAST search, http://www.ncbi.nlm.nih.gov/BLAST/). After elimination of linker sequences and repeated ditags, a total of 85,848 tags representing human NK cells and CD8⁺ T cells were analyzed. To compare these two SAGE libraries, each tag number was normalized to 51,017 using SAGE software.

Statistical analysis
Statistical significance among these samples was calculated as described previously (22). To analyze the correlation coefficients between the different libraries, tags from NK cells, CD8⁺ T cells, T_h1, T_h2, monocytes, GM-CSF-induced macrophages, mature DC and immature DC were normalized to 51,017, and all pairwise Pearson correlation coefficients for each library-to-library comparison were calculated using all normalized gene expression measurements (23).

RT-PCR
The RNA was reverse transcribed using a random hexamer and Moloney murine leukemia virus reverse transcriptase for 1 h at 42°C. cDNA was amplified using AmpliTaq (Perkin-Elmer) and PCR profile consisted of 30–35 cycles (denaturation for 60 s at 94°C, annealing for 60 s at 58°C and extension for 120 s at 72°C). Primers were as follows. Ferritin heavy chain: sense 5'-AGCTGCAGAACCAACGAGG-3', antisense 5'-GGCCAGTTT GTGCAGTTCC-3'; -defensin 1: sense 5'-ATGAGGACCCT CGCCATC-3', antisense 5'-CTCAGCAGCAGAATGCCCA-3'; granulysin: sense5'-AAATCCTGCCCGTGCCT-3', antisense 5'-GGGTCGCAGCATTGGAAA-3'; prostaglandin D₂ synthase: sense 5'-AGAAGAAGGCGGCGTTG-3', antisense 5'-TACAG CAGCGCGTACTGGT-3'; granzyme B: 5'-TCCCCCATCCAG CCTATAA-3', antisense 5'-TGAGACATAACCCCAGCCA-3'; perforin: sense 5'-GCCCAGGTCAACATAGGCA-3', antisense 5'-ATCCCGAACAGCAGGTCGT-3'; CX₃CR1: sense 5'-AGCA TGGCGTCACCATCA-3', antisense 5'-TTCCACATTGCGGA GCAC-3'; LARC: sense 5'-TGCGGCGAATCAGAAGC-3', antisense 5'-TGGATTTGCGCACACAGAC-3'; CCR7: sense 5'-TGGTGATCGGCTTTCTGGT-3', antisense 5'-CCATTGTAGG GCAGCTGGA-3'; hypothetical protein MGC11104: sense 5'-ATGCAGGCGGCCCTAGAG-3', antisense 5'-TCAGGAGGC AGGAAGTGG-3'; MGC13240: sense 5'-ATGACGGAGACC TTT-3', antisense 5'-CTATCTCTTGCTGCTCCT-3'; MGC915: sense 5'-ATGATGGGCGGAGAGTCT-3', antisense 5'-TCAC TGAAACCACCGGAA-3'; FLJ12443: sense 5'-ATGGCCGAG GCCTTGGGT-3', antisense 5'-CTAATCCAGCTTCTTGCG-3'. The relative value of the expression level of each gene was analyzed by Quantity One software (Toyobo, Kyoto, Japan).

Purification of CD45RA⁺CD27⁺ naive, CD45RA^–CD27⁺ memory and CD45RA⁺CD27^– effector CD8⁺ T lymphocytes
For subset purification, CD8⁺ T cells were prepared by positive enrichment using the MACS system. CD8⁺ T lymphocytes were stained with phycoerythrin-conjugated CD45RA (PharMingen) and FITC-conjugated CD27 (PharMingen), and sorted into CD45RA⁺CD27⁺, CD45RA⁺CD27^– and CD45RA^–CD27⁺ populations (purity >98%) on an Epics Elite ESP cell sorter (Beckman Coulter, Fullerton, CA) (24)

Chemotaxis assay
Chemotaxis assays were performed using a 96-well chemotaxis chamber (Neuroprobe, Pleasanton, CA) with a polycarbonate filter (5-µm pore size). Naive (CD45RA⁺CD27⁺), memory (CD45RA^–CD27⁺) and effector (CD45RA⁺CD27^–) subsets of CD8⁺ T cells were highly purified by a cell sorter, and were suspended at a density of 1 x 10⁶/ml in RPMI 1640 medium containing 20 mM HEPES, pH7.2 and 0.5% BSA (Sigma, St Louis, MO). Aliquots of 25 µl of cell suspensions were added into upper chambers and diluted -defensin 1 (PeproTech EC, London, UK; final volume, 29 µl) was added into lower chambers. Chemotaxis chambers were incubated for 3 h at 37°C in 5% CO₂. The number of migrated cells was determined by an Epics Elite ESP cell sorter.

NK cell preparation and culture
NK cells were prepared by negative enrichment using the MACS system as described previously. Cells were cultured in the presence of recombinant human IL-15 (10 ng/ml; R & D systems, Minneapolis, MN) or recombinant human IL-2 (50 ng/ml; R & D systems) in RPMI medium supplemented with 2 mM L-glutamine and 10% FBS (ICN, Aurora, OH). The cells were harvested 1, 2, 3, 6, 12 and 24 h after stimulation, and subjected to RNA preparation and subsequent RT-PCR analysis for the gene expression of ferritin heavy chain and -defensin 1.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Gene expression in NK cells and CD8⁺ T lymphocytes
A total of 85,848 tags, including 34,831 and 51,017 tags from NK cells and CD8⁺ T cells respectively allowed identification of 24,363 different transcripts. The expressed genes were searched through the GenBank database to identify individual genes. Table 1 shows the top 50 transcripts in NK cells. Except for ribosomal proteins, the most expressed genes were identified as ß₂-microgloblin, with an expression frequency of 1.42%, followed by MHC class I, C (0.65%). Other highly expressed genes were RANTES (0.64%), thymosin ß 10 (0.51%), profilin (actin binding protein) (0.34%), NK cell group 7 sequence (0.34%), cofilin 1 (actin-binding protein) (0.33%), cystatin F (hematopoietic cell-specific cysteine proteinase inhibitor) (0.32%), granulysin (antimicrobial peptide) (0.31%) and cytochrome b245 polypeptide (0.27%). Table 2 shows the top 50 transcripts in CD8⁺ T cells. The most expressed transcripts were ß₂-microgroblin (0.63%, except for ribosomal proteins). Other highly expressed genes were MHC class I, C (0.45%) and ferritin heavy polypeptide 1 (0.38%).

View this table: [in this window] [in a new window]   Table 1. Transcriptional profile in human NK cells

 

View this table: [in this window] [in a new window]   Table 2. Transcriptional profile in human CD8+ T lymphocytes

 
Comparison of gene expression profile between NK cells and CD8⁺ T lymphocytes
Figure 1 shows the summary of comparison of gene expression profiles. The expression levels of most transcripts in these cells were similar; however, there were 532 genes with statistically significant differences (P < 0.01) between NK and CD8⁺T cell libraries. Tables 3 and 4 show the genes selectively expressed in NK cells and CD8⁺ T cells respectively .

View larger version (36K): [in this window] [in a new window]   Fig. 1. Distribution of the different tags from NK cells and CD8+ T lymphocytes. The number of times each unique SAGE tag appeared was plotted on a logarithmic scale using normalized tags from CD8+ T lymphocytes (x-axis) versus normalized tags from NK cells (y-axis). The majority of the tags were expressed at similar levels in the two samples (gray diamonds); however, there were 532 tags with statistically significant differences (P < 0.01) between NK and CD8+ T cell libraries (closed triangles).

 

View this table: [in this window] [in a new window]   Table 3. Increased transcripts expressed in human NK cells compared with CD8+ T lymphocytes

 

View this table: [in this window] [in a new window]   Table 4. Increased transcripts expressed in human CD8+ T lymphocytes compaired with NK cells

 
Ksp37 was the most selectively expressed transcript in NK cells (31-fold). Other selectively expressed transcripts were cystatin F (29-fold), actin-related protein 2/3 complex subunit 1A (26-fold), killer cell Ig-like receptor (24-fold), hypothetical protein FLJ10688 (24-fold), protein phosphatase 1 regulatory (inhibitor) subunit 16B (23-fold), likely ortholog of mouse SH3 gene SLY (21-fold), LIM and SH3 protein 1 (20-fold), serine/threonine kinase 10 (20-fold), and -defensin 1 (19-fold: anti microbial peptide). v-jun avian sarcoma virus 17 oncogene homologue (c-jun) was the most selectively expressed gene in CD8⁺ T lymphocytes (31-fold). Other selectively expressed transcripts in CD8⁺ T cells were IL-8 (31-fold), unknown gene (EST, 30 fold), B cell translocation gene 1 anti-proliferative (23.8-fold), hypothetical protein FLJ14058 (22-fold), PHD finger protein (20-fold: contains a zinc finger-like PHD finger), TRAIL (18-fold: TNF family), dual specificity phosphatase 1 (18-fold), human TCR active chain mRNA from JM cell line complete cds (18-fold) and chemokine receptor CCR 7 (18-fold).

Categorized differentially expressed genes
The expressed genes mentioned above could be classified into a number of functional categories. A large number of MHC class I molecules were expressed by either NK cells or CD8⁺ T cells (Table 5). NK cells expressed higher levels of granulysin, granzymeB, -defensin 1, perforin, DNAX-activating protein (DAP) 10 (NKG2D-associated molecules) and NK receptor, which are related to cytotoxicity. With regard to cytokines or cytokine receptors, NK cells expressed high level of RANTES, IL-2 receptor ß, transforming growth factor-ß1 and specifically expressed CX₃C receptor 1, although not at very high levels. CD8⁺ T cells expressed high levels of CC chemokine RANTES, CXC chemokine IL-8, CXCR4, CCR7, and although not at very high levels, specifically expressed IL-4 receptor, IL-7 receptor and CC chemokine LARC. Adhesion molecules galectin 1, integrin ß₇ and integrin ß₂ were selectively expressed in NK cells, but CD8⁺ T cells did not have prominent expression of adhesion molecules. These cells also expressed different kinds of apoptosis-related molecules. Apoptosis-associated speck like proteins containing CARD was selectively expressed in NK cells. TNF ligand superfamily member 12; TRAIL, TNF receptor- associated factor; TRAF 4, apoptosis-related cystain protease; caspase 8 and TRAF 5 were selectively expressed in CD8⁺ T cells. The genes encoding signaling molecules and transcriptional factors are also categorized in Table 5. Although not at very high levels, CD8⁺ T cells selectively expressed STAT6 compared with NK cells. Based on these results, it can be concluded that both NK cells and CD8⁺ T cells play an important role in host defense, but have different gene expression profiles.

View this table: [in this window] [in a new window]   Table 5. Categorized transcripts expressed in human NK cells and CD8+ T lymphocytes

 
Correlation coefficients for all pairwise comparisons of libraries
To estimate the extent of similarity between any two libraries (NK cells, CD8⁺ T cells, T_h1, T_h2, monocytes, macrophages, mature DC and immature DC) (18–21), we calculated each bivariate correlation coefficients. The correlation coefficients for all comparisons are shown in Table 6. Pearson correlation coefficients between NK cell and CD8⁺ T cell libraries showed a high similarity at 0.779. In addition, the CD8⁺ T cell library showed a high similarity to T_h2 cells at 0.806.

View this table: [in this window] [in a new window]   Table 6. Correlation coefficients between the different librariesa

 
RT-PCR of genes represented in the SAGE
Although we pooled peripheral blood from three healthy volunteers to find the average gene expression, there could be differences in gene expression among individual donor-derived cells. To justify the SAGE results, we picked up 13 genes for which expression was distinct between NK cells and CD8⁺ T cells, and analyzed their expression by RT-PCR. Relative intensity of each gene was analyzed and shown under each gel image (Fig. 2). The expression levels of each transcript were compared to SAGE data (Tables 3 and 4). Granulysin [NK cells (NK) 159:CD8⁺ T lymphocytes (CD8⁺) 12], perforin (NK 21:CD8⁺ 4), granzyme B (NK 38:CD8⁺ 2), -defensin 1 (NK 19:CD8⁺ 0), prostaglandin D₂ synthase (NK 11:CD8⁺ 0), CX₃CR1 (NK 5:CD8⁺ 0), hypothetical protein MGC11104 (NK 17:CD8⁺ 0), hypothetical protein MGC13240 (NK 11:CD8⁺ 1), hypothetical protein MGC915 (NK 8:CD8⁺ 0) and hypothetical protein FLJ12443 (NK 51:CD8⁺ 5) were selectively expressed in NK cells; whereas CCR7 (NK 1:CD8⁺ 18) and LARC (NK 0:CD8⁺ 5) were selectively expressed in CD8⁺ T cells. Individual differences in the gene expression of -defensin 1 and granulysin from NK cells or LARC from CD8⁺ T cells (Fig. 2) were observed. A similar expression level of ferritin heavy chain was detected (NK 130:CD8⁺ 193). Each relative intensity was almost consistent with results of SAGE analysis. These results validate our SAGE data for unstimulated NK cells and CD8⁺ T cells, and establish the general expression profiles of the cytotoxic lymphocytes.

View larger version (52K): [in this window] [in a new window]   Fig. 2. RT-PCR analysis of genes expressed differentially in NK cells and CD8+ T lymphocytes. RT-PCR was performed on total RNA isolated from human NK cells and CD8+ T lymphocytes. (A–C) Different donors. ‘SAGE Tag.No’ is the number of the tag which is shown in Tables 3–5. Relative intensity of each band was analyzed and is shown under each gel image.

 
Circulating CD8⁺ T cells can be classified into three major subsets based on the expression of CD45RA and CD27: CD45RA⁺CD27⁺ naive, CD45RA^–CD27⁺ memory and CD45RA⁺CD27^– effector subsets (24). Therefore, we investigated the expression level of the genes in these three subsets, which were identified to be differentially expressed in NK cells and CD8⁺ T cells by RT-PCR, and relative intensity of each gene was analyzed and shown under each gel image (Fig. 3). Perforin was strongly expressed in purified CD45RA⁺CD27^– effector CD8⁺ T cells, but not in CD45RA⁺CD27⁺ naive or CD45RA^–CD27⁺ memory CD8⁺ T cells. CCR7 was selectively expressed in naive and memory subsets, but not in effector CD8⁺ T cells (Fig. 3). Furthermore, according to SAGE data, the genes encoding hypothetical protein FLJ12443 and MGC915, which were selectively expressed in NK cells, were also expressed in memory and effector subsets (Fig. 3).

View larger version (39K): [in this window] [in a new window]   Fig. 3. RT-PCR analysis of the genes expressed differentially in NK cells, naive, memory and effector CD8+ T lymphocytes. RT-PCR was performed on total RNA isolated from (NK) NK cells, (N) naive, (M) memory and (E) effector CD8+ T lymphocytes. ‘SAGE Tag.No’ is the number of the tag which is shown in Tables 3–5. Relative intensity of each band was analyzed and is shown under each gel image. The results from a representative experiment out of three are shown.

 
Chemotactic activity of -defensin 1 against CD8⁺ T lymphocytes and the effect of IL-15 on the enhancement of -defensin 1 gene expression in NK cells
We examined the biological significance of -defensin 1 expression in NK cells, which was one of the most selectively expressed transcripts in NK cells (Tables 3 and 5, and Fig. 2). -Defensin 1 has been known as a chemoattractant for human immature DC and CD4⁺ CD45RA⁺ naive T_h cells (16). To investigate the effect of -defensin 1 on CD8⁺ T cell subsets, chemotactic analysis was performed. -Defensin 1 induced selective migration of naive CD8⁺ T cells, but not the memory or effector subset (Fig. 4). This finding may indicate that there are mechanisms whereby NK cell-produced -defensin 1 regulates the migration of specific CD8⁺ T cell subsets.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Chemotactic responses of three subsets of CD8+ T lymphocytes to -defensin 1. Naive (open columns), memory (closed columns) and effector (hatched columns) CD8+ cells were highly purified by a cell sorter and stimulated with the indicated concentration of -defensin 1 in a 96-well chemotaxis chamber. The assay was performed in duplicates and the number of migrated cells was counted by a flow cytometer. Each point represents mean values ± SE from three independent experiments. Differences were analyzed using Student’s t-test. *P < 0.01.

 
IL-15 is an important factor in the development, differentiation and survival of NK cells (25–28). Indeed, IL-15-treated NK cells show increased killing potentials against a variety of cells, including virus-infected target cells (29). We investigated whether treatment with IL-15 or IL-2 affects the expression of -defensin 1 in NK cells (Fig. 5). Treatment with IL-15 (10 ng/ml) rapidly enhanced expression of -defensin 1 and the expression level was gradually decreased (Fig. 5). In the same experiment, treatment with IL-2 also enhanced the expression of -defensin 1, but the effect was marginal.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Kinetics of mRNA expression of -defensin 1 in IL-15- or IL-2-stimulated NK cells. NK cells were stimulated with IL-15 (10 ng/ml) or IL-2 (50 ng/ml) for 0, 1, 2, 3, 6, 12 and 24 h. RT-PCR was performed on total RNA isolated from IL-15- or IL-2-stimulated NK cells. Relative intensity of each band was analyzed and is shown under each gel image. The results from a representative experiment out of three are shown.

 

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous studies by others on the gene expression of NK and CD8⁺ T cells provided limited information about known genes. In this study, we have applied the SAGE method to perform quantitative analysis of a large number of transcripts in human NK and CD8⁺ T cells. Among differentially expressed genes, transcripts related to cytotoxicity such as granulysin, killer cell Ig-like receptor, -defensin 1, granzyme B, perforin, CD16 and DAP 10 were expressed at higher levels in NK cells than in resting CD8⁺ T cells. These molecules may play a central role in the initiation of cytotoxic activity against viruses or microbes. Without antigen stimulation, CD8⁺ T cells did not express the cytotoxicity-related genes. These results suggest that NK cells can respond more rapidly than resting/circulating CD8⁺ T cells for virus infection. On the other hand, CD8⁺ T cells need priming by antigen-pulsed antigen-presenting cells such as DC and reactivation by the antigen to become mature cytotoxic T lymphocytes.

Fractalkine receptor, CX₃CR1, was selectively expressed in NK cells. A recent study describes fractalkine-mediated endothelial cell (EC) injury by NK cells. Fractalkine is expressed on activated EC, and plays an important role in leukocyte adhesion and migration. Soluble fractalkine or anti-CX₃CR1 antibody markedly suppressed NK cell adhesion to fractalkine-transfected cells (30). Virus, bacteria and their products activate EC, and induce cytokine (31) and fractalkine production as well as ICAM-1 and VCAM-1 on the EC membrane. Although the mechanisms are unclear, NK cells damage the vascular endothelium despite the presence of autologous MHC (30).

CD8⁺ T cells selectively expressed several chemokines and chemokine receptors such as IL-8, LARC and CCR7. CD8⁺ T cells are classified into naive, memory and effector cells on the basis of CD45RA, CD62L and CCR7 expression (32). Sequential engagement of cell-surface CD62L and CCR7 receptor is required for efficient trafficking to lymphoid tissue by means of high endothelial venules. Naive and memory CD8⁺ T cells express CCR7; on the other hand, effector cytotoxic T cells are thought to be CCR7^– (32). In our result, CCR7 was confirmed to be selectively expressed in naive and memory, but not in effector CD8⁺ T cells, consistent with the previous report (32).

In normal mice, LARC mRNA is detected in epithelial cells of intestinal tissues, especially in those lining lymphoid follicles by in situ hybridization (33). LARC efficiently induced migration of T cells, IgM⁺IgD^– naive B cells, CD8⁺ T cells and CD11C⁺ DC derived from Peyer’s patches (33). Our result demonstrated that CD8⁺ T cells selectively expressed LARC mRNA, although the biological role of LARC expression remains to be established.

Defensins were originally described in granulocytes and macrophages, but it has been recently reported that they are also expressed in mucosal epithelia and induced in keratinocytes. They may contribute to host defense by disrupting the cytoplasmic membrane of microbes. Here, we demonstrated the selective expression of -defensin 1 in NK cells compared with CD8⁺ T cells. Furthermore, the expression was enhanced by stimulation with IL-15.

IL-2 and IL-15 belong to four-helix bundle cytokines and share a common receptor, IL-2Rß. These cytokines have also their own receptors, IL-2R and IL-15R respectively. IL-2 and IL2R are mainly expressed by activating T cells, suggesting a role of this cytokine–receptor system in the maintenance of adaptive immunity. In contrast, IL-15 is produced by a variety of cell types, including monocytes/macrophages, bone marrow stromal cells, keratinocytes, DC and synovial-derived cells from patients with rheumatoid arthritis (25,27,34,35). IL-2 and IL-15 stimulate NK cell proliferation and activation. Further more, IL-15 promotes the development and survival of NK cells (28). It is also reported that IL-15 can activate NK cell-mediated antiviral response (29). In this study, we demonstrated that IL-15 enhanced the expression of -defensin 1 in NK cells. Our data suggest an additional role of IL-15 in the function of NK cells. However, IL-2 did not enhance the expression level of -defensin 1.

Defensins are also known to induce the chemotaxis of human CD4⁺CD45RA⁺ naive T cells and immature DC (16). Here, we showed that -defensin 1 induced selective migration of naive CD8⁺CD45RA⁺CD27⁺ T cells, but not of memory or effector subsets. It is still unclear how NK cells are recruited under physiological conditions. NK cells are composed of CD56^bright and CD56^dim subsets. The function of CD56^bright NK cells is different from that of CD56^dim NK cells. CD56^bright NK cells can produce cytokines more abundantly, and express various cytokine receptors and the C-type lectin NK receptor. Furthermore they express CCR7 and L-selectin, which may be important for these cells to migrate into lymph nodes (36). After stimulation, NK cells increased the expression of -defensin 1 which induces migration of naive CD8⁺ T cells. Although the receptor for -defensin 1 is still unclear, naive CD8⁺ T cells but not memory or effector CD8⁺ T cells should express the receptor. A previous report described that preincubation of peripheral blood T cells with pertussis toxin completely blocked defensin-induced migration of peripheral blood T cells, indicating the involvement of G_i protein-coupled receptor(s) in the chemotactic effect (16). We speculate that -defensin 1 from NK cells provides a signal to bridge innate and adaptive immunity by recruiting naive CD8⁺ T cells and DC.

Many unknown genes were detected in our SAGE results. One of them, clone FLJ 12443, shared weak homology with the calcineulin ß subunit based on homology search. Although MGC 915 did not have homology with any known human genes, the gene was highly expressed in effector CD8⁺ T cells and therefore this gene may be important for cytotoxic effects.

In conclusion, identification of the genes selectively expressed in human NK cells and CD8⁺ T cells would provide useful information to clarify the function of these cells. Furthermore, cloning of numerous unknown genes should provide further understanding of molecular mechanisms of cytotoxicity of NK and CD8⁺ T cells.

	Acknowledgements

 
This work was supported by CREST, SORST, and Grant-in-Aid for Scientific Research on Priority Areas (C) ‘Medical Genome Science’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Abbreviations

 
DC—dendritic cell

EC—endothelial cell

PBMC—peripheral blood mononuclear cell

SAGE—serial analysis of gene expression

TNF—tumor necrosis factor

TRAIL—TNF-related apoptosis inducing ligand

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Berke, G. 1994. The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Annu. Rev. Immunol. 12: 735.[Web of Science][Medline]
2. Zamai, L., Ahmad, M., Bennett, I. M., Azzoni, L., Alnemri, E. S. and Perussia, B. 1998. Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. J. Exp. Med. 188: 2375.[Abstract/Free Full Text]
3. Nagata, S. and Golstein, P. 1995. The Fas death factor. Science 267: 1449.[Abstract/Free Full Text]
4. Griffith, T. S. and Lynch, D. H. 1998. TRAIL: a molecule with multiple receptors and control mechanisms. Curr. Opin. Immunol. 10: 559.[Web of Science][Medline]
5. Bancroft, G. J. 1993. The role of natural killer cells in innate resistance to infection. Curr. Opin. Immunol. 5: 503.[Web of Science][Medline]
6. Ljunggren, H. G. and Karre, K. 1990. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today 11: 237.[Web of Science][Medline]
7. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. and Salazar-Mather, T. P. 1999. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17: 189.[Web of Science][Medline]
8. Busch, D. H., Pilip, I. M., Vijh, S. and Pamer, E. G. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8: 353.[Web of Science][Medline]
9. Murali-Krishna, K., Altman, J. D., Suresh, M., Sourdive, D. J., Zajac, A. J., Miller, J. D., Slansky, J. and Ahmed, R. 1998. Counting antigen-specific CD8⁺ T cells: a reevaluation of bystander activation during viral infection. Immunity 8: 177.[Web of Science][Medline]
10. Butz, E. A. and Bevan, M. J. 1998. Massive expansion of antigen-specific CD8⁺ T cells during an acute virus infection. Immunity 8: 167.[Web of Science][Medline]
11. Ahmed, R. and Gray, D. 1996. Immunological memory and protective immunity: understanding their relation. Science 272: 54.[Abstract]
12. Lehrer, R. I., Ganz, T. and Selsted, M. E. 1991. Defensins: endogenous antibiotic peptides of animal cells. Cell 64: 229.[Web of Science][Medline]
13. Broekaert, W. F., Terras, F. R., Cammue, B. P. and Osborn, R. W. 1995. Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol. 108: 1353.[Web of Science][Medline]
14. Ganz, T., Selsted, M. E., Szklarek, D., Harwig, S. S., Daher, K., Bainton, D. F. and Lehrer, R. I. 1985. Defensins. Natural peptide antibiotics of human neutrophils. J. Clin. Invest. 76: 1427.[Web of Science][Medline]
15. Agerberth, B., Charo, J., Werr, J., Olsson, B., Idali, F., Lindbom, L., Kiessling, R., Jornvall, H., Wigzell, H. and Gudmundsson, G. H. 2000. The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood 96: 3086.[Abstract/Free Full Text]
16. Yang, D., Chen, Q., Chertov, O. and Oppenheim, J. J. 2000. Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J. Leukoc. Biol. 68: 9.[Abstract/Free Full Text]
17. Velculescu, V. E., Zhang, L., Vogelstein, B. and Kinzler, K. W. 1995. Serial analysis of gene expression. Science 270: 484.[Abstract/Free Full Text]
18. Hashimoto, S., Suzuki, T., Dong, H. Y., Nagai, S., Yamazaki, N. and Matsushima, K. 1999. Serial analysis of gene expression in human monocyte-derived dendritic cells. Blood 94: 845.[Abstract/Free Full Text]
19. Hashimoto, S. I., Suzuki, T., Nagai, S., Yamashita, T., Toyoda, N. and Matsushima, K. 2000. Identification of genes specifically expressed in human activated and mature dendritic cells through serial analysis of gene expression. Blood 96: 2206.[Abstract/Free Full Text]
20. Hashimoto, S. I., Suzuki, T., Dong, H. Y., Yamazaki, N. and Matsushima, K. 1999. Serial analysis of gene expression in human monocytes and macrophages. Blood 94: 837.[Abstract/Free Full Text]
21. Nagai, S., Hashimoto, S. I., Yamashita, T., Toyoda, N., Satoh, T., Suzuki, T. and Matsushima, K. 2000. Comprehensive gene expression profile of human activated T_h1-and T_h2-polarized cells. Int. Immunol. 13: 367[Abstract/Free Full Text]
22. Madden, S. L., Galella, E. A., Zhu, J., Bertelsen, A. H. and Beaudry, G. A. 1997. SAGE transcript profiles for p53-dependent growth regulation. Oncogene 15: 1079.[Web of Science][Medline]
23. Anbazhagan, R., Tihan, T., Bornman, D. M., Jhonston, J. C., Saltz, J. H., Weigering, A., Piantadosi, S. and Gabrielson, E. 1999. Classification of small cell lung cancer and pulmonary carcinoid by gene expression profiles. Cancer Res. 59: 5119.
24. Hamann, D., Baars, P. A., Rep, M. H., Hooibrink, B., Kerkhof-Garde, S. R., Klein, M. R. and van Lier, R. A. 1997. Phenotypic and functional separation of memory and effector human CD8⁺ T cells. J. Exp. Med. 186: 1407.[Abstract/Free Full Text]
25. Mrozek, E., Anderson, P. and Caligiuri, M. A. 1996. Role of interleukin-15 in the development of human CD56⁺ natural killer cells from CD34⁺ hematopoietic progenitor cells. Blood 87: 2632.[Abstract/Free Full Text]
26. Mingari, M. C., Vitale, C., Cantoni, C., Bellomo, R., Ponte, M., Schiavetti, F., Bertone, S., Moretta, A. and Moretta, L. 1997. Interleukin-15-induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor. Eur. J. Immunol. 27: 1374.[Web of Science][Medline]
27. Carson, W. E., Ross, M. E., Baiocchi, R. A., Marien, M. J., Boiani, N., Grabstein, K. and Caligiuri, M. A. 1995. Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by natural killer cells in vitro. J. Clin. Invest. 96: 2578.[Web of Science][Medline]
28. Carson, W. E., Fehniger, T. A., Haldar, S., Eckhert, K., Lindemann, M. J., Lai, C. F., Croce, C. M., Baumann, H. and Caligiuri, M. A. 1997. A potential role for interleukin-15 in the regulation of human natural killer cell survival. J. Clin. Invest. 99: 937.[Web of Science][Medline]
29. Gosselin, J., TomoIu, A., Gallo, R. C. and Flamand, L. 1999. Interleukin-15 as an activator of natural killer cell-mediated antiviral response. Blood 94: 4210.[Abstract/Free Full Text]
30. Yoneda, O., Imai, T., Goda, S., Inoue, H., Yamauchi, A., Okazaki, T., Imai, H., Yoshie, O., Bloom, E. T., Domae, N. and Umehara, H. 2000. Fractalkine-mediated endothelial cell injury by NK cells. J. Immunol. 164: 4055.[Abstract/Free Full Text]
31. Cines, D. B., Pollak, E. S., Buck, C. A., Loscalzo, J., Zimmerman, G. A., McEver, R. P., Pober, J. S., Wick, T. M., Konkle, B. A., Schwartz, B. S., Barnathan, E. S., McCrae, K. R., Hug, B. A., Schmidt, A. M. and Stern, D. M. 1998. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91: 3527.[Free Full Text]
32. Chen, G., Shankar, P., Lange, C., Valdez, H., Skolnik, P. R., Wu, L., Manjunath, N. and Lieberman, J. 2001. CD8⁺ T cells specific for human immunodeficiency virus, Epstein–Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection. Blood 98: 156.[Abstract/Free Full Text]
33. Tanaka, Y., Imai, T., Baba, M., Ishikawa, I., Uehira, M., Nomiyama, H. and Yoshie, O. 1999. Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans. Eur. J. Immunol. 29: 633.[Web of Science][Medline]
34. Jonuleit, H., Wiedemann, K., Muller, G., Degwert, J., Hoppe, U., Knop, J. and Enk, A. H. 1997. Induction of IL-15 messenger RNA and protein in human blood-derived dendritic cells: a role for IL-15 in attraction of T cells. J. Immunol. 158: 2610.[Abstract]
35. McInnes, I. B., al-Mughales, J., Field, M., Leung, B. P., Huang, F. P., Dixon, R., Sturrock, R. D., Wilkinson, P. C. and Liew, F. Y. 1996. The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis. Nat. Med. 2: 175.[Web of Science][Medline]
36. Cooper M. A., Fehniger T. A. and Caligiuri M. A. 2001. The biology of human natural killer-cell subsets. Trends Immunol. 22: 633[Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				C. L. Wilson, A. P. Schmidt, E. Pirila, E. V. Valore, N. Ferri, T. Sorsa, T. Ganz, and W. C. Parks Differential Processing of {alpha}- and {beta}-Defensin Precursors by Matrix Metalloproteinase-7 (MMP-7) J. Biol. Chem., March 27, 2009; 284(13): 8301 - 8311. [Abstract] [Full Text] [PDF]

				D. Kumar, J. Hosse, C. von Toerne, E. Noessner, and P. J. Nelson JNK MAPK Pathway Regulates Constitutive Transcription of CCL5 by Human NK Cells through SP1 J. Immunol., January 15, 2009; 182(2): 1011 - 1020. [Abstract] [Full Text] [PDF]

				D. Tran, P. Tran, K. Roberts, G. Osapay, J. Schaal, A. Ouellette, and M. E. Selsted Microbicidal Properties and Cytocidal Selectivity of Rhesus Macaque Theta Defensins Antimicrob. Agents Chemother., March 1, 2008; 52(3): 944 - 953. [Abstract] [Full Text] [PDF]

				L. Furci, F. Sironi, M. Tolazzi, L. Vassena, and P. Lusso {alpha}-defensins block the early steps of HIV-1 infection: interference with the binding of gp120 to CD4 Blood, April 1, 2007; 109(7): 2928 - 2936. [Abstract] [Full Text] [PDF]

				A. R. Rodriguez, B. P. Arulanandam, V. L. Hodara, H. M. McClure, E. K. Cobb, M. T. Salas, R. White, and K. K. Murthy Influence of interleukin-15 on CD8+ natural killer cells in human immunodeficiency virus type 1-infected chimpanzees J. Gen. Virol., February 1, 2007; 88(2): 641 - 651. [Abstract] [Full Text] [PDF]

				L. P. Huang, S.-C. Lyu, C. Clayberger, and A. M. Krensky Granulysin-Mediated Tumor Rejection in Transgenic Mice J. Immunol., January 1, 2007; 178(1): 77 - 84. [Abstract] [Full Text] [PDF]

				Y. Meng, H. Harlin, J. P. O'Keefe, and T. F. Gajewski Induction of Cytotoxic Granules in Human Memory CD8+ T Cell Subsets Requires Cell Cycle Progression J. Immunol., August 1, 2006; 177(3): 1981 - 1987. [Abstract] [Full Text] [PDF]

				A. Deng, S. Chen, Q. Li, S.-c. Lyu, C. Clayberger, and A. M. Krensky Granulysin, a Cytolytic Molecule, Is Also a Chemoattractant and Proinflammatory Activator J. Immunol., May 1, 2005; 174(9): 5243 - 5248. [Abstract] [Full Text] [PDF]

				A. Chalifour, P. Jeannin, J.-F. Gauchat, A. Blaecke, M. Malissard, T. N'Guyen, N. Thieblemont, and Y. Delneste Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers {alpha}-defensin production Blood, September 15, 2004; 104(6): 1778 - 1783. [Abstract] [Full Text] [PDF]

				L. L. Ma, C. L. C. Wang, G. G. Neely, S. Epelman, A. M. Krensky, and C. H. Mody NK Cells Use Perforin Rather than Granulysin for Anticryptococcal Activity J. Immunol., September 1, 2004; 173(5): 3357 - 3365. [Abstract] [Full Text] [PDF]

				S.-i. Hashimoto, S. Nagai, J. Sese, T. Suzuki, A. Obata, T. Sato, N. Toyoda, H.-Y. Dong, M. Kurachi, T. Nagahata, et al. Gene expression profile in human leukocytes Blood, May 1, 2003; 101(9): 3509 - 3513. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (38) Request Permissions Google Scholar Articles by Obata-Onai, A. Articles by Matsushima, K. Search for Related Content PubMed PubMed Citation Articles by Obata-Onai, A. Articles by Matsushima, K. Social Bookmarking          What's this?

Online ISSN 1460-2377 - Print ISSN 0953-8178

Copyright © 2009 Japanese Society for Immunology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics