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The Journal of Immunology, 2008, 181, 4507 -4515
Copyright © 2008 by The American Association of Immunologists, Inc.
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Umbilical Cord Blood T Cells Express Multiple Natural Cytotoxicity Receptors after IL-15 Stimulation, but Only NKp30 Is Functional1

Qin Tang*, Bartosz Grzywacz*, Hongbo Wang*, Nandini Kataria, Qing Cao{ddagger}, John E. Wagner*, Bruce R. Blazar*, Jeffrey S. Miller{dagger} and Michael R. Verneris2,*

* Department of Pediatrics and {dagger} Department of Medicine, Division of Blood and Marrow Transplantation, and {ddagger} Biostatistics Shared Resource, Cancer Center, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The natural cytotoxicity receptors (NCRs) NKp30, NKp44, and NKp46 are thought to be NK lineage restricted. Herein we show that IL-15 induces NCR expression on umbilical cord blood (UCB) T cells. NCRs were mainly on CD8+ and CD56+ UCB T cells. Only NKp30 was functional as demonstrated by degranulation, IFN-{gamma} release, redirected killing, and apoptosis. Since NCRs require adaptor proteins for function, the expressions of these adaptors were determined. The adaptors used by NKp30 and NKp46, Fc{epsilon}R1{gamma} and CD3{zeta}, were detected in UCB T cells. There was a near absence of DAP12, the adaptor for NKp44, consistent with a hypofunctional state. NKp46 was on significantly fewer UCB T cells, possibly accounting for its lack of function. Adult peripheral blood (PB) T cells showed minimal NCR acquisition after culture with IL-15. Since UCB contains a high frequency of naive T cells, purified naive T cells from adult PB were tested. Although NKp30 was expressed on a small fraction of naive PB T cells, it was nonfunctional. In contrast to UCB, PB T cells lacked Fc{epsilon}R1{gamma} expression. These results demonstrate differences between UCB and PB T cells regarding NCR expression and function. Such findings challenge the concept that NCRs are NK cell specific.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Umbilical cord blood (UCB)3 is emerging as a preferred stem cell source for allogeneic transplantation because: 1) it is rich in hematopoietic progenitor cells; 2) it is easily collected and cryopreserved at the time of delivery, with no apparent risks to the donor (infant); 3) it undergoes infectious disease screening and HLA typing at the time of collection, allowing for rapid donor identification and transplantation; 4) it is associated with low rates of both acute and chronic graft-vs-host disease, despite HLA mismatch; and 5) it shows similar rates of leukemia relapse relative to other hematopoietic cell sources, such as bone marrow or peripheral blood (PB) (reviewed in Ref. 1). Thus, it is important to understand both the similarities and differences between various effector cell populations in UCB relative to PB.

Although UCB and PB do not differ with regard to the percentages of T cells, T cell subsets (CD4 and CD8), or the proportions of {alpha}β and {gamma}{delta} T cells (2, 3), functional differences are commonly observed. For instance, most UCB T cells are naive, while most adult PB T cells are Ag experienced (4). Compared with PB T cells, UCB T cells express less of the transcription factor NFAT2c, which plays an important role in cytokine gene expression following immune activation (5). Accordingly, CD3-stimulated UCB T cells differ from PB, producing less Th1 (IL-2, TNF-{alpha}, IFN-{gamma}) and Th2 cytokines (IL-4, IL-10, IL-13) (6, 7, 8, 9, 10). Likewise, proteins associated with both activation (CD40L, CD25) and cytotoxicity (perforin and FasL) are reduced in UCB T cells relative to PB T cells (7, 11, 12). Although such differences may suggest immaturity, UCB T cells are capable of functional responses. Ag-specific T cells can be found following in utero infections (13) or in the cord blood of HA-1– infants born to HA-1+ mothers (14).

A subset of PB T cells can express receptors that are mainly found on NK cells. Included are NK cell inhibitory receptors, such as killer Ig-like receptors (KIRs) (15) and CD94/NKG2A (16, 17, 18). In addition to these, PB T cells can also express NK cell activating receptors, such as activating KIR (19, 20), CD94/NKG2C (17, 21, 22), and NKG2D (23). In some studies, engagement of these NK cell-associated receptors can either negatively or positively modulate TCR triggering (cytotoxicity and cytokine secretion) (24, 25, 26). We and others have also shown that triggering of receptors, such as NKG2D, can induce TCR-independent cytotoxicity on IL-2- or IL-15-stimulated T cells (27, 28). As for PB T cells, UCB T cells can also express KIR (CD158a, CD158b, and CD158e1) and NKG2A/CD94, but at significantly lower frequencies (29, 30). These results are in line with the supposition that NK-associated receptors are mainly expressed on effector T cells from adult PB (28, 31, 32, 33). UCB-derived T cells that express NK receptors can be expanded after culture with IL-15 (30). Importantly, the chemotherapy commonly used before allogeneic hematopoietic cell transplantation results in lymphodeletion and an increase system IL-15 levels. This has been linked to the success of clinical trials using adoptively transferred PB T and NK effector cell populations (34, 35).

Recently, three NK activating receptors (NKp30, NKp44, and NKp46) have been identified. Collectively, they have been referred to as the "natural cytotoxicity receptors (NCRs)" because they play a significant role in the killing of malignant targets (reviewed in Ref. 36). The initial reports describing NCRs showed that unlike other NK cell-associated receptors, NCRs were restricted to NK cells and not expressed on PB T cells (37, 38, 39, 40, 41). Herein, we show that a small percentage of freshly isolated UCB T cells coexpress NKp30. We further demonstrate that, unlike PB T cells, UCB T cells can acquire NKp30, NKp44, and NKp46 following culture with IL-2 or IL-15; however, only NKp30 is functional. Lastly, small amounts of naive adult PB T cells can acquire NKp30 following IL-15 stimulation, but this receptor is not functional on these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell isolation and culture

UCB and PB were obtained from healthy donors. Mononuclear cells were prepared by density gradient centrifugation using lymphocyte separation medium (Mediatech). T cells were isolated using CD3 microbeads (Miltenyi Biotec). Purity was assessed by flow cytometry and was >97% (data not shown). Isolated T cells were cultured at 1 x 106 cells/ml in Ham’s F12 plus DMEM (1:2 ratio) with 10% male AB human serum (SeraCare Life Sciences), ethanolamine (50 µmol/L), ascorbic acid (20 mg/L), 5 µg/L sodium selenite (Na2SeO3), 2-ME (24 µmol/L), and penicillin (100 U/ml)–streptomycin (100 U/ml). Recombinant cytokines IL-2 (Chiron), IL-15 (PeproTech), IL-4 (R&D Systems), and IL-7 (gift from National Institutes of Health) were added as indicated.

Flow cytometry analysis

Before staining, cells were washed in PBS with 2% FBS and 0.2% NaN3 and then stained with mAbs for 30 min at 4°C. Samples were analyzed on a FACSCalibur using CellQuest software (BD Biosciences). FlowJo 5.7.0 software was used for data analysis. For intracellular staining, the cells were fixed and permeabilized with BD Cytofix/Cytoperm solution and washed with BD Perm/Wash buffer (BD Biosciences) and stained according to the manufacturer’s specifications. The following mouse anti-human Abs were used: CD3-FITC, CD4-FITC, CD8-FITC, CD62L-PE, CD4-APC, CD8-APC, CD56-APC (BD Biosciences), NKp30-PE, NKp44-PE, NKp46-PE, and TCR{zeta}-PE (Beckman Coulter), CCR7-APC (R&D systems, Minneapolis, MN), and CD45RA-FITC (eBioscience).

Cell proliferation assay

Freshly isolated UCB T cells were washed and resuspended in prewarmed PBS at 5 x 106/ml containing 5 µM CFSE (Molecular Probes) and incubated for 15 min at 37°C. Following this, cells were washed again with prewarmed PBS, then resuspended in prewarmed medium and incubated for another 30 min and again washed before cell culture.

Degranulation assay

Twenty-four-well plates were coated with mAbs against human NKp30 (clone 210847), NKp44 (clone 253415), NKp46 (clone 195314) (R&D Systems), CD3 (OKT3), or isotype control (Sigma-Aldrich), all at 5 µg/ml in PBS for 4 h at 37°C. Plates were washed and UCB or PB T cells at day 14 of culture were added at 0.3 x 106 in 300 µl medium. Anti-CD107a-FITC (BD Biosciences) was added to the culture before incubation. After 8 h of incubation at 37°C, 5% CO2, cells were harvested and analyzed for CD107a expression.

Apoptosis assay

Ninety-six-well plates were coated with Abs against human NKp30, NKp44, NKp46, CD3 (OKT3) mAbs, or isotype control Ab at 5 µg/ml in PBS for 4 h at 37°C. Plates were washed and cells were added (0.2 x 106 cells in 200 µl medium). After 16 h of incubation at 37°C, 5% CO2, cells were harvested and washed with cold PBS, resuspended in 1x binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and stained with annexin V-FITC (BD Biosciences) for 15 min at room temperature and and propidium iodide (Sigma-Aldrich) was added. Cells were analyzed by flow cytometry.

IFN-{gamma} ELISA

Cells (0.2 x 106) in 200 µl medium were stimulated with plate-bound Ab-coated 96-well plates for 24 h. Supernatants were harvested and assayed for IFN-{gamma} production by ELISA (R&D Systems) according to the protocol of the manufacturer.

Redirected killing assay

The Fc{gamma}R-positive cell line P815 (murine mastocytoma) was used as a target for redirected killing assays. P815 cells were labeled with 51Cr (DuPont/NEN) by incubating 1 x 106 cells in 300 µCi (11.1 MBq) 51Cr for 1 h at 37°C, 5% CO2. Cells were then washed three times with PBS, resuspended in culture medium, and added in triplicate to 96-well plates at 104 cells/well. Effector cells were added at the specified ratios and incubated at 37°C, 5% CO2. Effector cells were preincubated in PBS in the presence of soluble Abs (5 µg/1 x 106 cells in 100 µl PBS) for 30 min and washed once with PBS before coincubation with tumor cell targets. After 4 h, 100 µl of supernatant was counted using a gamma counter. The percentage of specific lysis was calculated using the following equation: % specific 51Cr release = 100 x [(test release) – (spontaneous release)]/[(maximal release) – (spontaneous release)].

Polymerase chain reaction

Total RNA was isolated using the RNeasy Mini kit (Qiagen) and reverse transcribed to cDNA (iScript, Bio-Rad). cDNA (2 ng) was amplified using recombinant Taq polymerase (Invitrogen) for 30 cycles. The following primers were used: DAP12 forward CCGCA AAGAC CTGTA CGCCA, reverse TGGAC TTGGG AGAGG ACTGG; Fc{epsilon}RI{gamma} forward ATGAT TCCAG CAGTG GTCTT G, reverse GTGCT CAGGC CCGTG TAAA; CD3{zeta} forward GCACAG TTGCC GATTA CAGA, reverse GGTTC TTCCT TCTCG GCTTT. PCR products of DAP12 (650 bp), CD3{zeta} (273 bp), and Fc{epsilon}RI{gamma} (209 bp) were resolved on 2% agarose gel, and bands were visualized with ethidium bromide.

Western blots

UCB T cells at day 14 of culture were lysed using freshly prepared lysis buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, protease inhibitor cocktail (complete protease inhibitor; Roche), PMSF, and 1 mM Na3VO4. Samples were separated on a 15% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% nonfat dry milk and probed with anti-actin (Santa Cruz Biotechnology), anti-Fc{epsilon}RI{gamma} (Upstate Biotechnology), or anti-DAP12 (generous gift from Dr. Paul Leibson, Rochester, MN) followed by a species-specific secondary HRP-conjugated Ab (Santa Cruz Biotechnology). Blots were developed using chemiluminescence (Pierce).

Statistical analysis

Statistical analyses were performed with Statistical Analysis System statistical software version 9.1 (SAS Institute). For non-normally distributed data, the Mann-Whitney rank sum test was used in the evaluation of the statistical differences between UCB and PB T cells, CD4+ vs CD8+, and CD56– vs CD56+ subpopulations. One-way ANOVA was used when the data were approximately normally distributed with the general linear models procedure (PROC GLM; SAS Institute). Adjustments for multiple comparisons were done with the Tukey’s method. Groups with p values of ≤0.05 were considered to be statistically different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of NCRs on UCB T cells, but not on PB T cells

A small fraction of freshly isolated UCB T cells expressed NKp30 (1.7 ± 0.9%), but not NKp44 (0.4 ± 0.4%) or NKp46 (0.5 ± 0.4%) (Fig. 1, A and C). Such results suggested that UCB T cells may acquire NCRs after activation with cytokines such as IL-15. Purified CD3+ cells form either adult PB or UCB were cultured in IL-15 and analyzed for NCR expression by flow cytometry after 0, 7, 14, or 21 days. As shown in Fig. 1B, NKp30, NKp44, and NKp46 were markedly increased on UCB T cells with only marginal increases noted on PB T cells (Fig. 1B). These results were consistent over a series of donors, resulting in a significant increase in NKp30 (36.8 ± 9.6%, p = 0.01), NKp44+ (40.9 ± 19.5, p = 0.01), and NKp46 (12.9 ± 6.9%, p = 0.01) on UCB T cells compared with PB at day 14 (Fig. 1C). Interestingly, at day 14 the percentage of NKp30- and NKp44-expressing UCB T cells was relatively similar; however, in comparison, the percentage of NKp46-expressing T cells was significantly lower (Fig. 1C). Thus, freshly isolated UCB T cells show rare expression of NKp30, but after culture in IL-15, expression of NKp30, NKp44, and NKp46 is observed. CD3-positive selection may result in signals that induce NCR expression; however, T cells in from unmanipulated UCB lymphocyte cultures (i.e., not positively selected) also showed NCR up-regulation following IL-2 or IL-15 exposure (data not shown). Collectively, such results show significant differences between UCB and PB T cells in NCR acquisition following culture with IL-15.


Figure 1
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  		FIGURE 1. Comparison of NCR acquisition on UCB or PB T cells induced by IL-15. CD3+ T cells from UCB and PB were isolated from healthy donors on day 0 and cultured with IL-15 (50 ng/ml). At days 0, 7, 14, and 21 of culture, NKp30, NKp44, and NKp46 expression were examined with flow cytometry. Representative dot plots of NCR expression on UCB and PB T cells at (A) day 0 and (B) day 14 are shown. Percentages of NCR-expressing cells are indicated. Data are summarized from 10 UCB donors and 5 PB donors (C). p value of <0.05 for the differences between UCB and PB T cells, except at day 0.

 
NCRs are acquired on UCB T cells

As shown above, small numbers of freshly isolated UCB T cells express NKp30, and at day 14 of culture, NKp30 was present on a significant proportion of UCB T cells (Fig. 1, B and C). Such findings suggest NKp30 acquisition after culture, or alternatively the T cells expressing NKp30 could be preferentially expanded in culture. To address this, freshly isolated UCB T cells were stained with CD3 and NKp30 and the CD3+NKp30– and CD3+NKp30+ fractions were purified by FACS sorting (Fig. 2A, left). After 14 days of culture with IL-15, cells from both fractions showed similar expansion and both subsets expressed NKp30, indicating that NKp30 is acquired after culture rather than preferential expansion of NKp30+ cells (Fig. 2A, right). To further investigate whether NCR acquisition was related to cell proliferation, freshly isolated UCB T cells were labeled with the membrane dye CFSE and cultured for 14 days. As shown in Fig. 2B, expression of NCRs is abundant on cells that underwent multiple rounds of divisions, whereas these receptors are absent from nonproliferating cells. Comparing NKp30 with NKp44 and NKp46, there were subtle but reproducible changes in NCR acquisition as it related to CFSE dilution. UCB T cells acquired NKp30 after as few as one cell division. In contrast, NKp44 or NKp46 were only acquired on those cells that showed significant proliferation and loss of CSFE.


Figure 2
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  		FIGURE 2. UCB T cells show de novo NKp30 expression and NCR acquisition are linked with cell division. A, Freshly isolated UCB T cells were sorted into CD3+NKp30– or CD3+NKp30+ cells subsets. NKp30 expression was measured with flow cytometry on cells right after sorting or 14 days of culture with IL-15. Results are representative of two donors. B, Freshly isolated UCB T cells were labeled with CFSE and cultured with media containing IL-15 (50 ng/ml). At day 14 cells were tested for NKp30, NKp44, and NKp46 expression vs CFSE. Results are representative of three independent experiments.

 
Induction of NCRs on UCB T cells by cytokines

IL-2, IL-4, IL-7, and IL-15 all signal through the common {gamma}-chain and act upon T cells. We therefore tested whether these cytokines could induce NKp30, NKp44, and NKp46 expression on UCB T cells. After 14 days of culture with increasing doses of IL-2 and IL-15, there was a dose-dependent induction in the surface expression of all NCRs tested (Fig. 3, A and D). In contrast, no NCR expression was observed when cells were cultured in IL-4, despite robust proliferation (data not shown) (Fig. 3B). Interestingly, IL-7 led to a partial induction of NKp30, but not NKp44 or NKp46, and, unlike IL-2 and IL-15, there was no clear dose-dependent induction at the doses tested (Fig. 3C). Combining IL-4 and IL-15 resulted in a nearly complete inhibition in NCR expression (data not shown). Removing the IL-4 after 7 days of culture and maintaining IL-15 allowed partial acquisition of NCRs (data not shown).


Figure 3
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  		FIGURE 3. Influence of different IL-2 family cytokines on NCR acquisition in UCB T cells. Freshly isolated CD3+ T cells from UCB were cultured in the presence of different concentrations of (A) IL-2, (B) IL-4, (C) IL-7, and (D) IL-15. At day 14, NKp30, NKp44, and NKp46 expression was tested by FACS. Percentages of positive cells are indicated. Results are representative of three independent experiments.

 
Phenotype of NCR-expressing T cells

As shown in Fig. 4A, at day 14 after culture with IL-15, both CD4 and CD8 T cell populations could be observed. A considerable proportion of the CD8+ T cells showed NCR expression. About half of CD8+ T cells expressed NKp30 or NKp44 (Fig. 4, A and B). Similar to our observations in bulk cultures, the percentage of NKp46-expressing CD8+ T cells was considerably lower than that of NKp30 and NKp44. In contrast, a significantly lower fraction of CD4+ T cells showed NKp30 expression (p = 0.01), whereas NKp44 or NKp46 were absent (Fig. 4, A and B).


Figure 4
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  		FIGURE 4. Comparison of NCR expression on UCB T cells subsets. A, Representative dot plot of IL-15 cultured UCB T cells at day 14 of culture stained for CD4+ and CD8+ expression and histograms for NKp30, NKp44, and NKp46 expression on the gated populations. C, Representative dot plot of CD3 vs CD56 of cultured UCB T cells at day 14 of culture and histograms for NKp30, NKp44, and NKp46 expression on the gated populations. E, Representative dot plot of CD3 vs TCR {gamma}{delta} of cultured UCB T cells at day 14 of culture and histograms for NKp30, NKp44, and NKp46 expression on the gated populations. Results are representative of four, four, and three donors, respectively. Shaded histograms represent isotype control and percentages of gated NCR-positive cells are indicated. Summary data from all donors were summarized and Mann-Whitney rank sum test was performed (B, D, and F) with p values indicated.

 
Both freshly isolated and ex vivo-activated T cells can express CD56 (27, 42). The vast majority of freshly isolated UCB T cells were did not express CD56, but after culture in IL-15, such T cells acquired CD56. Gating on the CD3+CD56– and CD3+CD56+ cells after 14 days of culture showed that NCRs were found at higher density on CD56-expressing T cells relative to CD56– T cells (Fig. 4C). Comparing the NCR expression of CD3+CD56+ cells to CD3+CD56– across a series of donors (n = 4), we found a significantly higher frequency of NKp44 and NKp46 (p = 0.05 and 0.02, respectively) on CD3+CD56+ cells, but a nonsignificant trend was observed for NKp30 expression between CD3+CD56– and CD3+CD56+ subpopulations (p = 0.07) (Fig. 4D).

Only a minority of CD3+ T cells in culture expressed TCR {gamma}{delta} at day 14 of culture (average = 8.4% (range = 1.4–22%), n = 6). As shown in Fig. 4E, gating on CD3+{gamma}{delta}+ T cells showed that NKp30, NKp44, and NKp46 were expressed. Comparing the NCR expression on CD3+{gamma}{delta}+ T cells to CD3+{gamma}{delta}– T cells showed no differences between the two cell types for NKp30 (p = 0.42), NKp44 (p = 0.14), or NKp46 (p = 0.25) (Fig. 4F).

Functional evaluation of NCRs on UCB T cells

The ligands recognized by NCRs have not been definitively determined. Thus, to evaluate whether NCRs were functional on UCB T cells, assays were performed using agonist mAb. Controls included OKT3 (positive) and murine IgG (negative). To assay for cytotoxic granule release following receptor engagement, we used a mAb directed against lysosomal-associated membrane protein-1 (LAMP-1, CD107a). CD107a is normally found on the internal membrane of cytotoxic vesicles and is translocated to the cell surface upon degranulation (43). As expected, CD3 ligation (OKT3) resulted in a significant increase in the percentage of cells undergoing degranulation (p = 0.01) (Fig. 5A). Likewise, ligation of NKp30 on UCB T cells resulted in significant degranulation (p = 0.01), while ligation of NKp44 and NKp46 did not (p = 1 for both) (Fig. 5A). In further studies IFN-{gamma} production was investigated following receptor engagement (Fig. 5B). Cells were cultured in the presence of plate-bound Abs for 16 h, and supernatant was collected for ELISA. Similar to degranulation, crosslinking of either NKp30 or OKT3 resulted in IFN-{gamma} production (p = 0.15 and 0.03, respectively). In contrast, little or no IFN-{gamma} was detected in supernatants after stimulation with agonist NKp44 and NKp46 Abs (p = 1 for both, Fig. 5B).


Figure 5
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  		FIGURE 5. Function of NCRs on UCB T cells. IL-15-expanded UCB T cells at day 14 of culture were triggered with different plate-bound mAbs and then assayed for (A) degranulation (summary of three independent experiments), (B) apoptosis (representative of five independent experiments), and (C) IFN-{gamma} release (summary of two independent experiments). Tukey’s method was used to make multiple comparisons with p values indicated. D–G, Redirected killing assay using IL-15-expanded UCB T cells at day 14 of culture and after labeling with agonist Abs as described in Materials and Methods. mAb Ab-coated UCB T cells were combined with 51Cr-labeled P815 cells and 51Cr was measured after 4 h. Each plot is representative of more than three independent experiments with separate donors.

 
Activation-induced cell death (AICD) occurs following prolonged TCR ligation (44), and similar events occur in NK cells after NCR ligation (45). Thus, we tested whether NCR-expressing, activated T cells at day 14 of culture with IL-15 undergo AICD following NCR ligation. As shown in Fig. 5C, the number of apoptotic cells did not change in the presence of agonist NKp44 or NKp46 mAbs. In contrast, prolonged NKp30 crosslinking induced annexin V staining and loss of membrane integrity (propidium iodide+) by a significant proportion of cells, consistent with AICD. Similar results were obtained in control (OKT3)-treated cells.

Next, we investigated whether NCR ligation on UCB T cells could induce cytotoxicity using a reverse Ab-dependent cellular cytotoxicity assay. As shown in Fig. 5D, after 14 days of culture, T cells show potent CD3-redirected lysis (OKT3, positive control). Crosslinking of NKp30 on UCB T cells led to redirected cytolysis. In contrast, a nonspecific IgG Ab or agonist Abs against NKp44 or NKp46 showed no redirected cytolysis. These Abs triggered redirected cytolysis using NK cells, proving that they can induce reverse Ab-dependent cellular cytotoxicity (data not shown). We next investigated whether signaling through NKp44 and NKp46 might provide costimulation and increase cytotoxicity relative to NKp30 alone; however, no enhancement in killing was observed (Fig. 5E). Previous studies have shown that other NK cell-activating receptors, such as NKG2D, can costimulate TCR signaling in CD8+ T cells (24). Thus, we tested whether signaling through both CD3 and NKp30 would augment redirected lysis. Compared with CD3 signaling alone, the addition of NKp30 did not further enhance cytotoxicity (Fig. 5F). We and others have shown that both freshly isolated and cultured T cells express NKG2D and that signaling through this receptor can trigger TCR-independent cytotoxicity (27, 46, 47). We tested whether NKG2D could cooperate with NKp30 in this setting. Signaling through the combination of NKp30 and NKG2D resulted in higher cytotoxicity relative to either NKp30 or NKG2D alone (Fig. 5G). Collectively, these results demonstrate that signals through NKp30, but not NKp44 or NKp46, lead to UCB T cell activation that result in granule exocytosis, cytokine secretion, cytotoxicity, and apoptosis.

Expression of adaptor proteins associated with NCR signaling

NCRs are unable to directly transmit intracellular signals and rely upon adaptor proteins for signal transduction. NKp30 and NKp46 signal through CD3{zeta} and Fc{epsilon}RI{gamma} (48, 49), while NKp44 uses DAP12 (39). To investigate whether these adaptors are expressed by UCB T cells after culture, we performed RT-PCR. Using small quantities of starting mRNA (2 ng), message for all three adaptor proteins were readily amplified (Fig. 6A). To evaluate protein expression, flow cytometry and Western blotting were used. CD3{zeta} was found in the vast majority of T cells, as well as NK cells (Fig. 6B). In contrast, DAP12 was abundantly expressed in NK cells, with a near absence of the protein in UCB T cell lysate (Fig. 6C). However, overexposure of Western blots did demonstrate small quantities of DAP12 in UCB T cells (data not shown). Similarly, Fc{epsilon}RI{gamma} could be detected in UCB T cells, but the quantities were significantly less than in NK cells (Fig. 6D). Collectively, these data show that the adaptor proteins are controlled at the level of translation.


Figure 6
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  		FIGURE 6. Expression of DAP12, CD3{zeta}, and Fc{epsilon}RI{gamma} in IL-15-expanded UCB T cells. A, RT-PCR for DAP12 (650 bp), CD3-{zeta} (273 bp), and Fc{epsilon}RI{gamma} (209 bp) was performed on IL-15-expanded UCB T cells at day 14. Results are representative of three independent experiments. B, CD3{zeta} expression was examined with intracellular staining. The number indicates the percentage of cells in the quadrant gate. Protein extracts of IL-15-expanded UCB T cells at day 14, and NK cells were separated on 15% acrylamide, then transferred to polyvinylidene difluoride membrane and probed with (C) anti-DAP12 and (D) Fc{epsilon}RI{gamma}. Actin was used as a loading control.

 
Expression of NCRs on PB vs UCB T cells subsets

Considering that UCB is composed mainly of naive T cells, while PB contains a mixture of naive and memory cells, we hypothesized that the relative differences in naive T cells may account for the disparity in NCR expression between the two cell sources. T cell subsets from the PB were sorted into naive (CD3+CD62LhighCCR7+CD45RA+), central memory (CM, CD3+CD62LhighCCR7+CD45RA–), effector memory CD45RA+ (EMRA+, CD3+CD62LlowCCR7–CD45RA+), and effector memory CD45RA– (EMRA–, CD3+CD62LlowCCR7–CD45RA+) (reviewed in Ref. 50) and cultured in IL-15 for 14 days. A small fraction of naive, but not memory PB T cells acquired NKp30 after culture (Fig. 7A). Acquisition of NKp30, NKp44, and NKp46 by purified naive and memory T cell subsets after culture with IL-15 is shown in Fig. 7B. Redirected killing assays with cultured naive cells from three donors (with one donor having >30% NKp30+ cells) showed no cytotoxicity after NKp30 engagement. As a positive control, OKT3 engagement induced killing (Fig. 7C). To investigate whether differences in the adaptor proteins used by NKp30 accounted for these differences, we investigated CD3{zeta} and Fc{epsilon}RI{gamma} expression in purified PB fractions after culture. CD3{zeta} was present in all subsets of PB T cells (Fig. 7D). However, Fc{epsilon}RI{gamma} was missing in all subsets of cultured PB T cells, including naive cells, whereas it was abundant in NK cells and detectable in UCB T cells (Fig. 7E).


Figure 7
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  		FIGURE 7. Acquisition of NCRs and function of NKp30 on sorted PB T cell subsets. Freshly isolated PB T cells were sorted into naive, CM, EMRA+, EMRA–, and bulk (unsorted) subsets and cultured in IL-15 for 14 days as described in Materials and Methods. Cells were then assayed for NCR expression with FACS at day 14. A, Representative histograms are shown. Shaded plots represent isotype controls and percentage of positive cells is indicated. B, Summary of the percentage of NCR-positive cells after 14 days of culture with IL-15 are shown from five donors. C, Redirected killing assay (reverse Ab-dependent cellular cytotoxicity) for sorted, naive PB T cells. Results are representative of three donors. D, Expression of CD3{zeta} by FACS in PB populations that were sorted at day 0 and cultured for 14 days with IL-15. E, Expression of Fc{epsilon}RI{gamma} in UCB NK cells, T cells, and PB T cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrate for the first time that a small number of freshly isolated UCB T cells express NKp30. Following culture with either IL-2 or IL-15, UCB T cells acquire NKp30, NKp44, and NKp46. IL-7 induced NKp30, but not the other NCRs. In contrast, the NCRs were not observed when UCB T cells were cultured with IL-4. These cytotoxic triggering receptors were mainly expressed by expanded CD8+ T cells that coexpressed CD56. CD3+CD56+ T cells account for a minor fraction of freshly isolated UCB T cells; however, after culture with IL-2 and/or IL-15, T cells can acquire CD56 after activation and proliferation (30, 51). Similarly, NKp30, NKp44, and NKp46 were expressed mainly on UCB T cells after proliferation. Consistent with the results of others (48, 52, 53), we observed essentially no expression of NKp30, NKp44, or NKp46 on freshly isolated adult PB T cells. In most experiments with adult PB we could, however, detect a small fraction of NCR+ T cells after 14 days of culture with IL-15. The percentage of such cells varied depending upon the individual donor and receptor tested (see Figs. 1B and 7B). Given that UCB is mainly composed of naive T cells (4), we reasoned that naive PB T cells might also have the capacity to acquire NCRs following cytokine stimulation and that these naive PB T cells might be overgrown by the expansion of effector T cells. Indeed, purified naive PB T cells contained a small, but reproducible CD3+NKp30+ fraction after 14 days of culture with IL-15. In some donors we could also detect NKp44-expressing cells in the naive fraction. In contrast to naive cells, purified EMRA+, EMRA–, and CM PB T cell populations showed essentially no NCR acquisition after culture. Such results are remarkable considering that other NK cell-associated receptors (KIR, NKG2A/C, and NKG2D) are preferentially expressed by Ag-experienced T cells and that such cells increase with age (28, 31, 32, 33). In contrast, NCRs appear to be acquired by UCB T cells and naive PB T cells.

Using cytokine secretion, degranulation assays, redirected killing, and AICD, we show that NKp30 on UCB T cells is functional. Interestingly, CD3 triggering induced much higher cytotoxicity in redirected killing compared with NKp30. Such results are not surprising given that all the cells express CD3, while only a fraction (typically 30–50%) expressed NKp30. Interestingly, the difference between these two stimuli (CD3 vs NKp30) was not apparent following plate-bound mAb-induced degranulation. This may be due to the magnitude of response induced by CD3 stimulation in the context of a cellular target (i.e., redirected killing) compared with isolated receptor triggering by plate-bound mAb. In fact, our results with CD107a show that it is a rare subset of T cells (~10%) that degranulate in response to single receptor triggering (either CD3 or NKp30). Upon further investigation we found that most of the cells that degranulate following CD3 ligation are, in fact, NKp30+ (data not shown). Thus, NKp30 may mark activated T cells, poised to degranulate.

In contrast to NKp30, NKp44 and NKp46 were not functional. The proximal adaptor protein for NKp44, DAP12, is only minimally expressed by IL-15-expanded UCB T cells, likely explaining the lack of NKp44 signaling. The lack of NKp46 function is more difficult to understand. The percentage of NKp46 expressing UCB T cells was consistently lower than those expressing NKp30 and NKp44; however, we would have expected to see some evidence of NKp46 function in degranulation, IFN-{gamma}, or redirected killing assays, but this was not observed. The proximal adaptor proteins used by NKp46 (CD3{zeta} and Fc{epsilon}RI{gamma}) are expressed by UCB T cells at levels apparently sufficient to permit signaling via NKp30. Since UCB T cells consistently showed lower levels of Fc{epsilon}RI{gamma} compared with NK cells, it can be hypothesized that Fc{epsilon}RI{gamma} is relatively more important for NKp46 signaling as compared with NKp30. Alternatively, NKp46 could require distinct, and yet undefined, downstream signaling components that are present in NK cells, but not in UCB T cells. Lastly, naive T cells from adult PB can also acquire NCRs, but at a lower proportion than their UCB counterparts. However, these receptors (on naive PB T cells) were not functional, possibly owing to the absence of Fc{epsilon}RI{gamma} in these cells.

At present, we do not understand the mechanism for the observed differences between UCB and PB T cells with respect to NCR acquisition and function. One explanation may be that the hormonal and/or cytokine milieu found in placental tissues favors NCR acquisition. Whether certain placental derived factors "prime" UCB T cells to express NCRs is conceivable. However, we were unable to induce NCR expression following culture of PB T cells in cord blood sera and IL-15 (data not shown). Likewise, it is possible that the hormonal changes that occur at the time of childbirth or the stress associated with delivery may in some way influence the ability of UCB T cells to acquire NCRs. Because UCB units were donated in a de-identified manner, we were not able to determine whether the mode of childbirth (caesarean section vs vaginal delivery) played any role in either de novo NKp30 expression (i.e., day 0) or NCR expression after cytokine stimulation. Since NCRs may be involved in antiviral immunity (54, 55, 56), another possible explanation may be that the ability to acquire NCRs by UCB T cells is a form of evolutionary protection against infectious organisms. Such findings would support the concept that newborns rely more upon their innate immune system until the adaptive immune system fully develops. Whether infants or young children also express NKp30 or acquire NCRs following IL-15 stimulation is not known. Another possible explanation for the differences between NCR expression from PB and UCB is that the cells present in UCB are not the true counterparts of those found in PB. In fact, T cells other than those commonly found in the PB have recently been show to express NCRs. For instance, the Jabri group (57) has demonstrated that intestinal epithelial lymphocytes (T cells) express functional NKp44 and NKp46 in patients suffering from celiac disease. Interestingly, while PB T cells only express CD3{zeta}, thymic-independent T cells isolated from the murine gastrointestinal tract express adaptors both for NKp30 and NKp46 (CD3{zeta} and Fc{epsilon}RI{gamma}) (58), perhaps supporting this view. In summary, the ability to acquire NCRs appears to be dependent upon the maturational status of the T cells. As stated above, such findings are in stark contrast to the expression of other NK cell-associated receptors on T cells. Collectively, these results show that there are fundamental differences between PB and UCB T cells with respect to NCR acquisition and function. Such findings challenge the dogma that NCRs are uniquely expressed by NK cells.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Q.T. designed, performed, and analyzed experiments and wrote the paper. B.G. designed, performed, and analyzed experiments and wrote the paper. H.W. designed, performed, and analyzed experiments. N.K. designed, performed, and analyzed experiments. Q.C. performed statistical analyses. J.E.W. provided essential reagents, analyzed experiments, and contributed to the writing of the paper. B.R.B. designed experiments and contributed to the writing of the paper. J.S.M. designed experiments and contributed to the writing of the paper. M.R.V. designed and analyzed experiments and wrote the paper.

1 This work was supported by Children’s Cancer Research Fund (to M.R.V.) and by National Institutes of Health Grants P01 CA65493 (to J.S.M.), R01 HL55417 (to J.S.M.), R01 AI34495 (to B.R.B.), and R01 CA72669 (to B.R.B.). Back

2 Address correspondence and reprint requests to Dr. Michael R. Verneris, Suite 660, 425 East River Road, Minneapolis, MN 55455. E-mail address: Verneris{at}umn.edu Back

3 Abbreviations used in this paper: UCB, umbilical cord blood; AICD, activation-induced cell death; CM, central memory; EM, effector memory; KIR, killer Ig-like receptors; NCR, natural cytotoxicity receptor; PB, peripheral blood. Back

Received for publication January 22, 2008. Accepted for publication July 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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