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Keywords:

  • Cytokines;
  • Dendritic cells;
  • Human;
  • Natural killer cells;
  • Th1/Th2 cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Human secondary lymphoid tissues harbor NK cells that predominantly secrete cytokines in response to activation. Here, we demonstrate that these immunoregulatory NK cells assist in the Th1 polarization of primary immune responses, induced by dendritic cells. Tonsilar, but not peripheral blood NK cells enhanced the expansion of IFN-γ-producing CD4+ T cells via their superior ability to produce IFN-γ. Addition of IFN-γ increased Th1 polarization while antibody blocking of this cytokine abolished NK cell-dependent Th1 polarization. Our data suggest that NK cells in secondary lymphoid organs assist priming of Th1 cells via cytokine secretion and this effect should be harnessed during vaccination against viruses and tumors.

Abbreviations:
poly I:C:

polyinosine-polycytidylic acid

SEB:

staphylococcal enterotoxin B

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Natural killer (NK) cells are innate lymphocytes that rapidly respond to pathogenic challenge. They were originally described by their killing of infected and transformed cells 1, but recently it was appreciated that humans harbor in addition a subpopulation of these cells that primarily secretes cytokines upon activation 2. Both functionally distinct human NK cell subsets recognize their target cells with an array of inhibitory and activating receptors. The main activating NK cell receptors 3 are engaged by ligands, which are up-regulated upon virus infection and/or cellular transformation, for example in connection with DNA damage 4. Most of the inhibitory NK cell receptors recognize MHC class I molecules and thereby detect down-regulation of these restriction elements for CD8+ T cells during immune evasion by viruses and tumors 5. However, these elaborate target cell recognition mechanisms only develop their full potential after NK cell activation. IL-2 was initially used to activate human peripheral blood NK cells to investigate their effector functions. However, in humans this cytokine is probably mainly secreted by T cells, and, therefore, restricted to adaptive and not innate immunity.

Instead of IL-2, DC are now thought to primarily activate NK cells during innate immune responses 6, 7. DC were found to induce proliferation, cytokine secretion and increase cytolytic capacity of both mouse and human NK cells 811. Instead of the above listed inhibitory and activating NK cell receptors, this interaction seems to be mediated in part by DC-derived cytokines. IL-12, the signature cytokine of myeloid DC, was described to mediate IFN-γ secretion by NK cells upon DC encounter 12, 13. Type I IFN, the signature cytokines of plasmacytoid DC, increase cytolytic NK cell function during DC stimulation 13. Finally, IL-15, presented on myeloid DC surfaces, was implicated in DC-induced NK cell proliferation and survival 12. These molecular mechanisms of human NK/DC interactions have all been characterized in vitro. However, in order to interpret their role in immune responses the localization of these encounters should be considered.

DC are sentinels of the immune system that patrol peripheral sites and migrate at increased frequency to secondary lymphoid tissue upon pathogen-induced maturation 14. Accordingly, mature DC, which are the superior activators of NK cells, can primarily be found in secondary lymphoid tissues, which drain sites of inflammation. Interestingly, the primarily cytokine secreting CD56brightCD16 NK cell subset, which is preferentially activated by DC 12, 15, is enriched in lymph nodes, spleens and tonsils 16, 17. Therefore, NK cells could be activated by DC at these sites and restrict infection as well as assist in the shaping of the adaptive immune response.

Here, we report experiments addressing the later hypothesis. We show that alloreactive IFN-γ-secreting CD4+ T cells can be more readily expanded by polyinosine-polycytidylic acid (poly I:C)-matured DC in the presence of tonsilar NK cells. This Th1 polarizing effect is dependent on NK cell derived IFN-γ, which is secreted to 50-fold higher levels by tonsilar than peripheral blood NK cells. These data suggest that human NK cells in secondary lymphoid organs are well equipped and positioned to assist Th1 polarization of DC-primed immune responses.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

DC maturation influences Th1 polarization by tonsilar NK cells

CD56brightCD16NK cells, which are enriched in human secondary lymphoid tissues 16, respond to activation mainly with cytokine secretion. Moreover, it has been demonstrated that mature DC selectively stimulate the CD56brightCD16 NK cell subset 12, 15. In order to evaluate if DC-mediated activation of NK cells from secondary lymphoid organs might play an immunoregulatory role in T cell polarization, tonsilar mononuclear cells were stimulated for 1 week with allogeneic peripheral blood monocyte-derived DC. DC were matured using the TLR3 ligand poly I:C (poly I:C DC) or proinflammatory cytokines (cytokine DC) 18. After 1 week of coculture, tonsilar mononuclear cells were restimulated with the allogeneic DC, used for the primary stimulation, and medium alone or staphylococcal enterotoxin B (SEB) as controls. IFN-γ and IL-10 production was assessed by intracellular staining in CD3+CD4+ T cells. A significant percentage of CD4+ T cells producing IFN-γ was only detectable when poly I:C-matured DC were used as stimulus, while cytokine-matured DC expanded only few IFN-γ-producing alloreactive T cells (Fig. 1). Depletion of tonsilar NK cells caused a significant 20% decrease in IFN-γ-producing CD4+ T cell after priming with allogeneic poly I:C-, but not cytokine-matured DC (p = 0.00006) (Fig. 1). The frequencies of IL-10-producing CD4+ T cells were too low to allow interpretation. These results reveal that tonsilar NK cells, upon appropriate activation with DC, polarize alloreactive CD4+ T cell responses towards increased IFN-γ secretion.

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Figure 1. Tonsilar NK cells promote Th1 polarization only when activated by poly I:C-matured DC. (A) Mononuclear cells obtained from tonsils were stimulated with allogeneic peripheral blood monocyte-derived DC at the ratio 1:10 (DC:tonsilar mononuclear cells). DC were matured using poly I:C (poly I:C DC) or a standard mixture of proinflammatory cytokines consisting of IL-1β, IL-6, TNF-α, and PGE2 (cytokine DC). After 1 week, cells were restimulated and IFN-γ and IL-10 production was assessed by intracellular cytokine staining in CD3+CD4+ T cells. Cells stimulated with medium or SEB were used as controls. The percent of CD4+ T cells producing IFN-γ is indicated. The data shown are representative of results obtained in four independent experiments. (B) Tonsilar mononuclear cells were stimulated with cytokine DC or poly I:C DC and differences in percentage of IFN-γ positive CD4+ T cells with (w NK) and without (w/o NK) NK were plotted. Data represent results from four independent experiments, mean ± SEM is shown.

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Peripheral blood NK cells are not able to promote Th1 differentiation

Human PBMC contain around 10% of NK cells and among these 5% belong to the CD56brightCD16 immunoregulatory subset 19. This amounts to a frequency of 0.5% of CD56brightCD16 NK cells in total peripheral blood lymphocytes. Similarly, 0.4% of tonsilar mononuclear cells are NK cells and the majority of these are CD56brightCD1616. As blood and tonsils harbor comparable amounts of CD56brightCD16 NK cells, we investigated whether also peripheral blood NK cells are able, upon activation by DC, to promote Th1 differentiation. Therefore, mononuclear cells from blood and tonsils were stimulated for 1 week with allogeneic poly I:C-matured DC and after restimulation the proportion of blood and tonsilar CD4+ T cells producing IFN-γ and IL-10 was evaluated by intracellular cytokine staining. In contrast to tonsilar mononuclear cell preparations, depletion of NK cells in PBMC did not decrease the expansion of alloreactive IFN-γ-producing CD4+ T cells (Fig. 2). The data presented above show that, despite the presence of similar CD56brightCD16 NK cell frequencies in human peripheral blood and tonsil, only tonsilar NK cells assist in CD4+ T cell polarization. This suggests that blood and tonsilar CD56brightCD16neg NK subset differ functionally.

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Figure 2. Peripheral blood NK cells do not promote CD4+ Th1 polarization. (A) Tonsilar and peripheral blood mononuclear cells, with and without NK, were stimulated for 1 week with allogeneic peripheral blood monocyte-derived DC at the ratio 1:10 (DC:mononuclear cells). DC were matured with poly I:C. After restimulation, IFN-γ and IL-10 production was assessed by intracellular cytokine staining after gating on CD3+CD4+ T cells. The percent of CD4+ T cells producing IFN-γ is indicated. One of four similar experiments is shown. (B) Tonsilar and peripheral blood mononuclear cells were stimulated with poly I:C-matured DC and percentages of IFN-γ+ CD4+ T cells with (w NK) and without (w/o NK) NK cells were plotted. Data represent results from four independent experiments; mean ± SEM is shown.

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Tonsilar NK cells secrete more IFN-γ than peripheral blood NK cells

Peripheral blood CD56brightCD16 NK cells secrete more cytokines than their CD56dimCD16 counterparts 19, but functional differences between CD56brightCD16 NK cells from blood and secondary lymphoid organs have not been described so far. Therefore, we compared the amount of IFN-γ secreted by tonsilar and blood CD56brightCD16 NK cells upon stimulation with allogeneic poly I:C-matured DC by ELISA assays. We adjusted NK cell numbers for the different percentages of CD56brightCD16NK cells present in blood and tonsils. We found that 104 tonsilar NK cells secreted 5-fold more IFN-γ than 105 blood NK cells. When we normalized the detected IFN-γ concentration to 104 CD56brightCD16 NK cells according to the NK cell subset content as determined by flow cytometry, tonsilar CD56brightCD16 NK cells produced up to 5000 pg/mL and blood CD56brightCD16 NK cells up to 1000 pg/mL IFN-γ (Fig. 3A). Stimulation of blood or tonsilar NK cells with cytokine-matured DC yielded only small amounts of NK-secreted IFN-γ (Fig. 3B) and no IFN-γ secretion by blood or tonsilar NK cells was detected without DC addition. Therefore, total tonsilar NK cells produce 50-fold more IFN-γ than total blood NK cells and even the CD56brightCD16 NK cell subset in tonsils secretes 5-fold more IFN-γ than its peripheral blood counterpart. Similarly, we had previously noticed that lymph node NK cells display higher mean fluorescence intensities in intracellular IFN-γ staining after DC activation than peripheral blood NK cells 12. These data indicate that NK cells in secondary lymphoid organs are ideally suited for cytokine mediated immunoregulation.

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Figure 3. Tonsilar NK cells produce more IFN-γ than peripheral blood NK cells after stimulation with poly I:C-matured DC. (A) Tonsilar and blood NK cells were isolated by flow cytometric sorting and stimulated with allogeneic poly I:C-matured DC. According to the different percentages of CD56brightCD16 NK cells in tonsils and peripheral blood, 104 tonsilar NK cells (tonsil) and 105 blood NK cells (blood) were cultured with indicated number of allogeneic poly I:C DC. IFN-γ concentrations were normalized to 104 CD56brightCD16 NK cells according to the NK cell subset content as determined by flow cytometry. After 24 h, supernatants of triplicate cultures were collected and IFN-γ secretion was quantified by ELISA. (B) Poly I:C matured (poly I:C) and cytokine matured (cytokines) DC were compared for stimulation of 105 blood NK cells (blood) and 104 tonsilar NK cells (tonsil). After 24 h, supernatants of triplicate cultures were collected and IFN-γ secretion was quantified by ELISA. IFN-γ concentrations were normalized to 104 CD56brightCD16 NK cells according to the NK cell subset content as determined by flow cytometry. Data in (A) and (B) are representative of two independent experiments.

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NK cell derived IFN-γ assists in Th1 polarization

The above-presented data suggested that Th1 polarization of alloreactive CD4+ T cells might be supported by IFN-γ secretion of tonsilar NK cells. Hence, the addition of recombinant human IFN-γ (rhIFN-γ) to peripheral blood lymphocytes might enhance expansion of IFN-γ-secreting CD4+ T cells by DC. In addition, it had been suggested that DC maturation in the presence of IFN-γ might augment the Th1-polarizing capacity of DC 20. Therefore, we added 4000 pg/mL rhIFN-γ during PBMC stimulation with allogeneic poly I:C-matured DC (poly I:C DC + IFN-γ) and/or together with poly I:C for DC maturation prior to coculture (poly I:C + IFN-γ DC + IFN-γ or poly I:C + IFN-γ DC, respectively). This IFN-γ concentration was chosen because tonsilar NK cells were able to secrete similar amounts after overnight culture with poly I:C-matured DC (Fig. 3). Furthermore, 4000 pg/mL was also detected in the supernatants of tonsilar lymphocyte cultures after 1 week of stimulation with allogeneic DC (data not shown).

When we used allogeneic poly I:C + IFN-γ-matured DC for stimulation of PBMC, we found a slight but not significant (p = 0.2171) increase of IFN-γ+ CD4+ T cells in comparison with PBMC cultures stimulated with poly I:C-matured DC (Fig. 4A). However, we found a significant increase of Th1-committed CD4+ T cells when IFN-γ was added during the coculture of PBMC, both with poly I:C-matured DC (poly I:C DC + IFN-γ) (p <0.03) and poly I:C + IFN-γ-matured DC (poly I:C + IFN-γ DC + IFN-γ) (p = 0.03) (Fig. 4). Stimulation with poly I:C- + IFN-γ-matured DC plus IFN-γ induced the highest increase in the percentage of IFN-γ-producing cells, suggesting that activation of DC with IFN-γ and poly I:C at the same time can boost Th1 polarization, but the presence of IFN-γ during the coculture was essential to reach a significantly increased expansion of IFN-γ+ CD4+ T cells.

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Figure 4. Addition of recombinant human IFN-γ to PBMC enhanced expansion of CD4+ Th1 cell after DC stimulation. (A) PBMC were stimulated with allogeneic poly I:C-matured DC (poly I:C DC) or with DC, which had been matured with both poly I:C and recombinant human IFN-γ (poly I:C + IFN-γ DC). The DC mononuclear cell ratio was 1:10. Where indicated, rhIFN-γ was also added during coculture of PBMC with DC (poly I:C DC + IFN-γ and poly I:C + IFN-γ DC + IFN-γ). After 1 week, cells were restimulated with poly I:C-matured DC, and IFN-γ and IL-10 production was assessed by intracellular cytokine staining gating on CD3+CD4+ cells. Cells stimulated with medium or SEB were used as controls. The percent of CD4+ T cells producing IFN-γ is indicated. The data shown are representative of results obtained in four independent experiments. (B) PBMC were stimulated with poly I:C-matured DC (poly I:C DC) and poly I:C plus IFN-γ-matured DC with additional IFN-γ in the coculture (poly I:C + IFN-γ DC + IFN-γ), and differences in percentage of expanded IFN-γ+ CD4+ T cells were plotted. Data summarize four independent experiments; mean ± SD is shown.

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As direct evidence for the role of IFN-γ secretion by tonsilar NK cells in CD4+ T cells polarization, we blocked IFN-γ during coculture of poly I:C-matured DC with tonsilar mononuclear cells. When an antibody against IFN-γ was used, we found a significant reduction of CD4+ T cells secreting IFN-γ (p <0.01) (Fig. 5), compared to the control cells cultured with an isotype-matched irrelevant antibody. Expansion of alloreactive IFN-γ-producing CD4+ T cells was decreased by antibody blocking of IFN-γ to the level observed after NK cell depletion (Fig. 5). Blocking IFN-γ in NK depleted tonsilar mononuclear cells had no effect. In good agreement with these findings, IFN-γ levels were significantly decreased in supernatants of NK depleted cultures with tonsilar mononuclear cells as measured by ELISA (data not shown). These data suggest that NK cells assist Th1 polarization via IFN-γ secretion in secondary lymphoid organs.

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Figure 5. Blocking IFN-γ caused a significant decrease in proportion of IFN-γ-producing CD4+ T cells. Tonsilar mononuclear cells, with (w NK) or without (w/o NK) NK cells, were stimulated with allogeneic poly I:C-matured DC. Anti-IFN-γ or isotype control (IgG1) antibodies were added on days 0, 2 and 4 at 10 μg/mL. After 1 week, cells were restimulated and IFN−γ and IL-10 production was assessed by intracellular cytokine staining in CD3+CD4+ T cells. Differences in percentage of IFN-γ positive CD4+ T cells were plotted. Data represent results from four independent experiments, mean ± SD is shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Here, we describe that tonsilar NK cells assist in the Th1 polarization of T cell priming by DC and identify NK cell-secreted IFN-γ as the main mediator of this NK cell help. Our data suggest that NK cells in secondary lymphoid organs, when appropriately triggered by DC, shape adaptive immunity and that DC-based immunotherapy should consider this for efficient T cell stimulation. Recent studies suggested that mouse NK cells could mediate similar effects after DC or Leishmania major induced recruitment to lymph nodes 21, 22, or NK cell expansion in secondary lymphoid organs in mice with genetically impaired TGF-β signaling 23. In contrast to Th1 cell polarization after NK cell recruitment in mice, humans have a dedicated NK cell subpopulation that primarily secretes cytokines upon activation and is enriched in secondary lymphoid organs to fulfill this task. While 5% of mononuclear cells in human lymph nodes are predominantly of the immunoregulatory CD56brightCD16 NK cell subset 16, mice have overall only 0.3% NK cells in this tissue 21. In good agreement with cytokine dependence of NK cell help in T cell priming as reported in our study, mouse NK cells recruited by mature DC to lymph nodes facilitated Th1 polarization of ovalbumin-specific T cell responses also primarily via secretion of IFN-γ 21. Therefore, mouse NK cells seem to assist Th1 polarization of DC mediated T cell priming after induced recruitment of blood NK cells to lymph nodes, while specialized NK cells reside at these sites in humans and perform the same task by their superior ability to secrete IFN-γ.

These human CD56brightCD16 NK cells are also optimally located in the parafollicular T cell zone of normal human lymph nodes, where myeloid DC were also found 12, 17. Matured DC, CD56brightCD16 NK cells, naive and central memory T cells seem all to utilize the chemokine receptor CCR7 to home to these sites 24, 25 and this colocalization might accelerate NK cell assisted Th1 cell priming by DC. In humans, a large proportion of NK cells is dedicated to this task, as around 40% of total lymphocytes are thought to be contained in lymph nodes, while only 2% are circulating in peripheral blood 26, 27. With NK cell frequencies of 5% in mononuclear cells of lymph nodes and 10% in peripheral blood 16, ten times more NK cells in the human body are immunoregulatory and assist in Th1 polarization in lymph nodes than circulate and mediate rapid cytolysis, the characteristic that gave these cells originally their name.

This Th1 polarization, assisted by secondary lymphoid organ NK cells, is crucial in anti-viral and anti-tumor immune responses. Th1 polarized T cell responses have been found to be more effective in the immune control of tumors 28 and viral infections 29 in mice. Furthermore, Th1-based immune control has been correlated with protection from the human cytomegalovirus (HCMV) after primary infection in kidney transplant patients 30. Part of the superior capacity of Th1 cells to control infections and tumor growth can be attributed to their ability to home efficiently to sites of inflammation due to CXCR3 expression 29, 31. Moreover, Th1 polarized CD4+ T cells control infections via both IFN-γ secretion 32 and direct cytolysis of infected cells 3335. Therefore, DC-based immunotherapies should harness the Th1 polarizing effect of NK cells by choosing vaccine adjuvants that mature DC optimally for NK cell activation. Our results suggest that the TLR3 agonist poly I:C should be considered in this respect.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Tonsils

All tonsils were obtained as part of Institutional Review Board-approved protocols. They were collected immediately after surgery from patients undergoing tonsillectomy for chronic inflammation. Soon after their removal, tissues were mechanically dissociated to obtain single-cell suspensions and were then filtered through a 75-µm nylon cell strainer to exclude undissociated fragments. Debris and dead cells were eliminated using Ficoll/Hypaque (Amersham Pharmacia) discontinuous gradient centrifugation. Single-cell suspensions were then extensively washed and cryopreserved.

Generation of DC

PBMC were isolated from leukocyte concentrates, provided by the New York Blood Center, by density gradient centrifugation on Ficoll-Paque (Amersham Pharmacia Biotech). Positive selection for CD14+ PBMC was performed using anti-CD14-MicroBeads, LS columns, and MidiMACS separators (Miltenyi Biotec). DC were generated from CD14+ PBMC. CD14+ PBMC (2 × 106/mL) were plated in 6-well plates with RPMI-1640, plus 1% single donor plasma, glutamine, and gentamicin. rhIL-4 (Peprotech) and rhGM-CSF (Immunex) were added to a final concentration of 500 and 1000 U/mL, respectively, at days 0, 2, and 4 in 500µL of fresh medium/well. On day 5, the floating immature DC were transferred to new plates at 1 × 106 cells/well and half of the medium was replaced with fresh medium containing IL-1β (10 ng/mL), IL-6 (1000 U/mL), TNF-α (10 ng/mL) and PGE2 (1 μg/mL) (all from R&D Systems except PGE2 from Sigma-Aldrich) (cytokine DC) or 25 μg/mL poly I:C (Invivogen) (poly I:C DC), to mature the DC for 2 days. In some experiments, 4 ng/mL of rhIFN-γ (Sigma) were added on day 5.

Polarization and IFN-γ production of tonsilar and blood CD4+ T cells

Tonsilar or PBMC were plated at 2 × 106/well in 24-well plates with RPMI-1640, plus 5% pooled human serum, glutamine, and gentamicin. Where indicated, tonsilar or PBMC were depleted of NK cells using anti CD56-MicroBeads, LS columns, and MidiMACS separators (Miltenyi Biotec). Allogeneic poly I:C- or cytokine-matured DC were added to the cultures at 2 × 105/well; in some experiments anti-IFN-γ mAb (IgG1, BioLegend), or isotype-matched irrelevant mAb as control, were added at 10 μg/mL and replaced on days 2, 4 and 6. After 1 week, the cells were transferred to 48-well plates and further restimulated with mature DC derived from the same donor, with 1.5  μg/mL of SEB, or with medium alone. Monensin was added for a final concentration of 2 μM at the same time. After 9–12 h, cells were stained for CD3 and CD4 (PerCP–conjugated anti-CD3 and FITC-conjugated anti-CD4, BD PharMingen) and then fixed using paraformaldehyde (2% in PBS). Cells were permeabilized using saponin (0.1% saponin, 1% BSA in PBS) and stained for intracellular IFN-γ (APC-conjugated anti-IFN-γ, BD PharMingen) and intracellular IL-10 (PE-conjugated anti-IL-10, BD PharMingen), according to the manufacturer's instructions. Negative controls included directly labeled isotype-matched irrelevant mAb. In some experiments, supernatants were collected after 1 week and the secreted IFN-γ was detected by IFN-γ ELISA (Mabtech). Where indicated, 4 ng/mL recombinant human IFN-γ was added at day 0 to the DC/PBMC cell coculture.

IFN-γ secretion by tonsilar and peripheral blood NK cells

NK cells were isolated by flow cytometric sorting of CD3CD56+ cells using a BD FACSVantage SE cell sorter. According to different percentages of CD56brightCD16 NK cell subset present in tonsils and blood, 104 tonsilar NK cells and 105 blood NK cells were cultured in 96-well plates with allogeneic DC at the indicated ratios and in triplicate cultures. After 24 h secreted IFN-γ was detected by IFN-γ ELISA (Mabtech) and values were adjusted to IFN-γ/104 CD56brightCD16 NK cells according to the NK cell subset content as determined by flow cytometry.

Statistical analysis

Statistical analyses were performed using the GraphPad Prism program. Statistical significance was evaluated by paired Student's t-test. The p values are reported.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We thank Till Strowig and Fabienne Brilot for critically reading the manuscript, and Klara Velinzon for expert technical assistance with flow cytometric cell sorting. These studies were in part supported the Arnold and Mabel Beckman Foundation, the Alexandrine and Alexander Sinsheimer Foundation, the National Cancer Institute (R01CA108609) (to C.M.) and in part by a General Clinical Research Center grant (M01-RR00102) from the National Center for Research Resources at the National Institutes of Health (to the Rockefeller University Hospital). B.M. was supported by a fellowship from Fondazione Italiana per la Ricerca sul Cancro. GF is supported by Associazione Italiana Ricerca sul Cancro (AIRC).

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