Biology of Blood and Marrow Transplantation
Volume 14, Issue 3 , Pages 290-300, March 2008

Up-regulation of NK Cell Activating Receptors Following Allogeneic Hematopoietic Stem Cell Transplantation under a Lymphodepleting Reduced Intensity Regimen is Associated with Elevated IL-15 Levels

  • Michael Boyiadzis

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • Division of Hematology and Oncology, Department of Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA
    • Corresponding Author InformationCorrespondence and reprint requests: Michael Boyiadzis, University of Pittsburgh School of Medicine, Division of Hematology and Oncology, UPMC Cancer Pavilion, 5150 Centre Ave, Suite 572, Pittsburgh, PA 15232, Phone: 412-648-6589, Fax: 412-648-6579
    • ,
  • Sarfraz Memon

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • ,
  • Jesse Carson

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • J.C.: In Memoriam: 1978-2005
    • ,
  • Kenton Allen

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • ,
  • Miroslaw J. Szczepanski

      Affiliations

    • Division of Hematology and Oncology, Department of Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA
    • ,
  • Barbara A. Vance

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • ,
  • Robert Dean

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • ,
  • Michael R. Bishop

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • ,
  • Ronald E. Gress

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • R.E.G. and F.T.H. both contributed as senior authors to the development and support of this work
    • ,
  • Frances T. Hakim

      Affiliations

    • Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD
    • R.E.G. and F.T.H. both contributed as senior authors to the development and support of this work

Article Outline

Abstract 

Because natural killer (NK) cells can be potent anti-tumor effectors after allogeneic stem cell transplantation, we investigated NK reconstitution and receptor expression in patients undergoing allogeneic hematopoietic stem cell transplantation, focusing on the activating receptors that trigger anti-tumor responses. We determined that NK levels in the peri-transplant period were inversely proportional to the dramatic rise and fall in plasma levels of the NK homeostatic cytokine IL-15, which increased more than 50-fold from pretreatment to the day of transplant during the lymphoreductive preparative regimen. Furthermore, in NK cells cultured with IL-15, we observed an up-regulation of the activating receptors NKG2D, NKp30, and NKp46, associated with an increase in anti-tumor lytic activity. Similarly, the expression of these activating receptors increased significantly during the early post-transplant period, concurrent with a rapid increase in total NK cells and a shift toward increased expression of CD56. These data suggest that the cytokine milieu of transplants, in particular elevated levels of IL-15, may contribute to anti-tumor efficacy post-transplant by enhancing the recovery of NK subsets and modulating expression of activating receptors.

Key Words: Natural Killer Cells, IL-15, Natural Cytotoxicity Receptors, NKG2D, Transplantation

 

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Introduction 

Natural killer (NK) cells play a critical role in the innate immune response through their capacity to lyse virally-infected or malignant cells without prior antigen-specific priming. The triggering of this cytotoxicity, however, is tightly controlled through a balance of signals from the mix of functionally opposing receptors found on each cell [1]. This balance acts as a fail-safe wherein engagement of activating receptors identifies potential targets, but engagement of inhibitory receptors blocks killing of normal autologous cells. Inhibitory receptors, such as the CD94–NKG2A C-type lectin receptor heterodimers, recognize leader peptides derived from HLA–A, –B, or –C presented by the non-classical class I molecule HLA–E. The families of killer immunoglobulin-like receptors (KIR) recognize polymorphic determinants of HLA–A, –B, or –C. Normal autologous cells that express major histocompatibility complex (MHC) class I are protected from NK-mediated attack, but cells that have reduced expression because of malignant transformation or infection with viruses are then subject to attack 2, 3.

Two classes of activating receptors have been linked to NK-mediated killing of tumor and virally infected cells: NKG2D (CD314) and natural cytotoxicity receptors (NCR). NKG2D binds to two distinct families of ligands, the MHC class I chain-related peptides (MICA and MICB) and the UL-16 binding proteins (ULBP). These ligands are up-regulated in cells that have undergone neoplastic transformation; NK cytotoxicity on tumor cells correlates with tumor expression of MICA and ULBP 4, 5, 6. The NCRs, Nkp30 (CD337) and NKp46 (CD335), bind to as yet unidentified tumor antigens; are all activating receptors; and are constitutively expressed on both resting and activated NK cells. These receptors have been demonstrated to be involved in NK cell-mediated killing of multiple cancer cell lines [7]. Blockade of NKG2D, especially in combination with NCR, reduces killing of a wide range of tumor cell lines 4, 5, 7. Down-regulation of NKG2D and NCR receptors by TGFβ, moreover, has been associated with reduced NK cytotoxicity 8, 9, 10, 11. These experiments demonstrate: that NKG2D and NCR are critical to NK anti-tumor cytotoxicity; that the expression of these receptors can be modulated by the cytokine milieu; and that such modulation can regulate the levels of NK cytotoxicity.

Following either myeloablative or reduced intensity (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT), NK cells recover rapidly, returning to normal levels within weeks 12, 13, 14, 15, 16. Functional assessments have shown that the rapid NK cell reconstitution post-transplant is associated with a rapid recovery of normal levels of NK cytotoxic activity 14, 17. Peripheral blood NK cells can be divided into two functional subsets based on surface expression of CD56 [1]. NK cells that express high levels of the CD56 molecule (termed CD56bright) constitute about 10% of the NK cells in the peripheral blood and play an immunoregulatory role through production of cytokines [1]. NK cells expressing lower levels of CD56 (CD56dim) and high levels of CD16 (the Fcγ receptor III) constitute the remainder and are primarily cytotoxic effectors [1]. An early increase in CD56bright NK cells has long been reported [13], but recovery of NK subsets and expression of receptors may reflect multiple factors including: NK differentiation paths; cytokine milieu; and graft-versus-host disease (GVHD) prophylaxis 15, 18, 19, 20. Receptor repertoire is in flux during NK recovery; CD94/NKG2A inhibitory receptors recover early to elevated levels, whereas killer immunoglobulin-like receptors (KIR) may require long periods to return to normal levels 15, 21, 22. Studies following allo-HSCT have focused on the role of inhibitory receptors, particularly KIR receptors capable of distinguishing allogeneic MHC antigens 23, 24, 25, with little information about the role of activating receptors in anti-tumor activity [15]. The balance of signals from the NK cell receptor repertoire determines overall NK cell activity. Therefore, NK activity against tumors, during the critical period of minimal residual disease post-transplant, may be dependent not only upon the rate of reconstitution of NK cell numbers, but also upon the recovery of the repertoire of activating as well as inhibitory receptors.

In this study, we analyzed the reconstitution of NK cells and their receptor repertoire in adult patients undergoing allo-HSCT under an RIC regimen involving doses of cytoxan and fludarabine specifically designed to deplete lymphocytes 26, 27. We determined that the levels of IL-15, a critical cytokine in NK differentiation, activation and homeostasis, underwent dramatic changes in the peri-transplant period, in an inverse relationship to the numbers of NK cells present. Furthermore, we determined that, in normal NK cells, IL-15 rapidly up-regulated the levels of critical activating receptors: NKG2D, NKp30, and NKp46. This IL-15 induced increase in expression was correlated with marked increases in cytotoxic activity, which could be blocked by neutralizing antibodies against these receptors. Finally, we determined that the expression of these key activating receptors (ie NKG2D, NKp30 and NKp46) was increased in the early post-transplant period and that these increases were maintained for at least 3 months post-transplant. Therefore, the IL-15 rich cytokine milieu post-transplant may drive both the recovery of NK populations and the elevated expression of their activating receptors.

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Materials and Methods 

Patients and Donors 

Fourteen patients (11 males, 3 females) underwent reduced intensity allogeneic transplants from HLA-identical related donors for treatment of hematologic malignancies (Table 1). The study was approved by the Institutional Review Board of the National Cancer Institute and informed consent was obtained from all participants.

Table 1. Patient Characteristics
PatientAgeSexDiagnosisStagePrior TherapiesInduction Cycles
161mHDIV23
250mNHLII61
353mNHLIV41
471fCLLIV23
531mHDIII23
639mHDIVB33
739mNHLIV21
869mNHLIV62
932mNHLIII42
1059fNHLIIIB51
1152mCLLII43
1233fHDIV53
1360mMDS 41
1458mMy 13

HD indicates Hodgkin Disease; NHL, Non-Hodgkin Lymphoma; CLL, Chronic lymphocytic leukemia; MDS, Myelodysplastic syndromes; My, Myelofibrosis.

Preparative Regimen 

The goal of the induction regimen was to achieve severe, targeted host immune T cell depletion prior to allogeneic HSCT in order to attain rapid conversion to full donor chimerism and to facilitate the development of a graft versus tumor effect soon after transplantation 26, 27. To reduce CD4 counts to fewer than 100 cells/μl prior to the transplant preparative regimen, all patients received from 1 to a maximum of 3 cycles of an induction regimen of fludarabine (25mg/m2 i.v. daily, days 1-4) in combination with the agents contained in the EPOCH regimen (etoposide 50 mg/m2 i.v. days 1-4, doxorubicin 10 mg/m2 i.v. days 1-4, vincristine 0.4 mg/m2 i.v. days 1-4, cyclophosphamide 750 mg/m2 i.v., day 5 and prednisone 60 mg/m2 PO daily, days 1-5). Patients with CD20+ lymphoid malignancies also received rituximab 375 mg/m2 i.v. on day 1 of each treatment cycle. The transplant preparative chemotherapy regimen consisted of fludarabine (30 mg/m2 per day i.v. infusion daily for 4 days) and cyclophosphamide (1200 mg/m2 per day i.v. infusion daily for 4 days). On transplant day 0, patients received non-manipulated peripheral blood progenitor cells from their HLA-matched sibling donors [27].

Prophylaxis Against GVHD 

Patients received cyclosporine (dose adjusted to 200 ng/ml) beginning at day -1, and gradually tapering from day 100 to day 180 post-transplant. Patients also received intravenous methotrexate 5 mg/m2 on days +1, +3, +6, and +11 post-transplant.

Sample Collection and Evaluation 

Peripheral blood was collected prior to the initiation of the induction regimen, the day of the transplant, and on days 14, 28, 60, 90, 180, 270, and 365 days after transplantation. Flow cytometry was performed using whole blood lysis in a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory (Science Applications International Corporation, Frederick, MD, USA). The absolute number of NK cells (CD3-and CD56+) per liter of blood was calculated based upon the percentages of these cells in the lymphocyte gate, the percentage of lymphocytes (defined as CD45bright, CD14-, low side scatter) and the white cell blood counts.

Assessment of Chimerism 

Chimerism analysis was performed by assessing the variable number of tandem repeats by polymerase chain reaction method in a CLIA-certified laboratory at the Blood Center of Southeastern Wisconsin (Milwaukee, WI, USA).

Interleukin-15 Levels 

The IL-15 QuantiGlo ELISA (R&D Systems) was used to measure the patient's levels of plasma IL-15 prior to therapy with the induction regimen, the day of the transplant, and on days 14, 28, 60, 90, 180, 270, and 365 days after transplantation.

Cell Culture and IL-15 Stimulation 

NK cells were isolated from peripheral blood mononuclear cells of healthy donors using magnetic beads (Miltenyi Biotec). NK cells were cultured in complete medium (CM – RPMI-1640 [InVitrogen-Lifetech] supplemented with 10% FCS, L-glutamine, sodium pyruvate, non-essential amino acids and penicillin/streptomycin) and were stimulated with rhIL-15 (Peprotech) at concentrations from 10-100 ng/ml. Cells were harvested after 1, 3, 7, and 14 days and the NK receptor repertoire were assessed by multi-parameter flow cytometry.

Immunophenotyping Analysis 

The reconstitution of the two NK subsets (CD56dim CD16+ and CD56brightCD16–) and their receptor expression were evaluated at 28, 90, 180, and 365 days post-transplant and was compared to that found in the peripheral blood stem cells (PBSC) apheresis as a standard control. Monoclonal PE conjugated antibodies to NKp30, NKp46, (Beckman-Coulter-Immunotech), and NKG2D (R&D systems) were used in combination with anti-CD3 APC and anti-CD16 FITC and biotinylated anti-human CD56 (BD Biosciences-Pharmingen) followed by streptavidin PE-Cy5 (Invitrogen-Caltag). Flow cytometric analysis was performed on a FACSort flow cytometer (Becton Dickinson) using CellQuest software. Further analysis was performed using Flow-Jo software (Treestar).

NK Cytotoxicity Assay 

NK activity was assessed in 4-h 51Cr release assays using K562 targets as previously described 28, 29. Briefly, target cells were labeled with 100 μCi of 51Cr for 1 h at 37°C at 5% CO2. The labeled cells were washed twice in CM, re-suspended in CM, and the viable cell counts were performed. Cells were co-incubated for 4 h at 37°C at effector to target (E:T) ratios of 6, 3, 1.5, and 0.75 to 1. Co-cultures were set up in triplicate. Controls included: targets incubated in medium alone for spontaneous release; and targets in 5% (v/v) Triton X-100 in PBS for maximum release. Radioactivity was measured by Wallac Wizard 1470 Automatic Gamma Counter. The percentage of cytotoxic activity was calculated using the following formula: % specific lysis = (sample cpm–spontaneous cpm)/(maximal cpm–spontaneous cpm) × 100%. Lytic Units (LU) were calculated as the number of effector cells needed to lyse 20% of 5 X 103 target cells, and expressed as LU/107 cells. For the blocking assays, blocking antibodies to NKG2D, NKp46, and NKp30 were added to the NK cultures 1 hour prior to conducting the NK cytotoxicity assays.

Statistical Analysis 

Because the cell population counts were generally not normally distributed, we report medians and ranges for parameter values. We used non-parametric statistical methods in the analyses. All paired differences between cells measured from the same patients at two different time points were tested using the Wilcoxon signed rank test. For comparison of NK cells between two groups of patients, the exact Wilcoxon rank sum test was used. Correlations were performed using the Spearman rank correlation.

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Results 

NK Cell Reconstitution Following HSCT is Inversely Correlated to Plasma IL-15 Levels 

On the day of the transplant in all patients, the NK levels had been reduced by the lymphodepleting regimen of fludarabine-cytoxan to 0 cells/μl. Subsequent NK cell recovery was rapid. By 4 weeks, the NK cell levels reached a peak level higher than those in the original host (226 cells/μl; range 63-701 cells/μl) and, by the second month post-transplant, the median NK levels had decreased to host pre-transplant levels (77 cells/μl, range 22-374). These levels were maintained through the first post-transplant year (Figure 1A). By 1 month post-transplant, all patients had greater than 90% donor chimerism in the lymphoid compartment.

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  • Figure 1 

    Correlation of NK cell recovery with IL-15 during the post-transplant period. A) Time courses of NK levels (cells/μl) (white squares) and plasma IL-15 levels (pg/ml) (black circles). NK cells recover rapidly post transplant, reaching a peak at 1 month and declining to pretreatment levels for the first year post-transplant. The plasma levels of IL-15 in the peri-transplant period reached a peak at the day of transplant and declined sharply in the first month. B) Inverse relationship overall between NK cell numbers and plasma IL-15 levels at all time points in the first year. C) Strong inverse correlation between NK cell numbers and plasma IL-15 levels at 2 weeks post-transplant, a time when NK populations were rapidly recovering and IL-15 levels varied over a 10-fold range. Spearman non-parametric correlation r values are shown.

Because of the rapid recovery of NK cells following this lympho-depleting transplant regimen, we investigated the levels of IL-15 in the peri-transplant period. On the day of transplant, the IL-15 levels were increased an average of 50-fold from the pre-treatment levels, to a median of 57.2 pg/ml (range 30.3-147.2 pg/ml). Concurrent with the rapid recovery of NK cell levels at 2 weeks, the IL-15 levels in the peripheral blood declined to median level of 17.2 pg/ml (range, 5.5-54.2). By 4 weeks post-transplant, the IL-15 levels had fallen to 2.95 pg/ml (range, 0.2-20.0) and, by 6 months post-transplant, IL-15 levels returned to and maintained pre-transplant levels. (Figure 1A). NK levels assessed during the transplant and year-long recovery process correlated inversely with the levels of IL-15 measured in the plasma (Figure 1B).

All lymphocyte populations are depleted during transplant conditioning, however, so the specific correlation of IL-15 and NK populations was tested during a period of rapid early NK recovery, when other populations remained low. NK numbers and IL-15 levels in individual patients were compared at 2 weeks post-transplant, a time point of rapid change and a broad variation in individual NK recovery (Figure 1C). A strong inverse relationship was found (Spearman correlation r = −0.805, P = .0037). This inverse relationship between circulating cytokine levels and cytokine-consuming NK levels is consistent with a homeostatic relationship, that is, plasma IL-15 levels are defined primarily by consumption, rising during lympho-depletion and falling upon NK recovery.

IL-15 Increases the Expression of Activating NK Cells Receptors 

To assess the effect of IL-15 on the NK cell receptor expression, we cultured NK cells sorted from normal healthy donors with IL-15. We observed an up-regulation of the NK cell activating receptors NKG2D, NKp30, and NKp46, encompassing both increases in the percentages of cells expressing the receptors and the MFI of each receptor. These changes were evident as early as 24 h after the start of culture, reached a maximum within 72 h, and were maintained as long as 14 days in culture (Figure 2A). Expression of CD56 increased on all NK cells in these cultures. Based on expression of CD16 to distinguish NK subsets, comparable levels of activating receptor expression were achieved in both the CD56+CD16+ and the CD56brightCD16– NK subsets (Figure 2B). When sorted CD56bright and CD56dimCD16+ NK were separately cultured with IL-15, the CD56bright NK cells expanded more than CD56dimCD16+ NK cells in culture, but both NK populations up-regulated activating receptor expression.

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  • Figure 2 

    IL-15 up-regulation of expression of activating receptors associated with increased NK activity. A) Contour plots of the expression of NKG2D and NCR receptors in normal donor NK cells cultured with IL-15. Activating receptor expression increased along with a shift toward increased expression of CD56. B) Overlay histograms illustrating increased expression of NKG2D and NCR receptors in both NK subsets when cultured with IL-15. Gray-filled histogram shows day 0 expression; black line indicates expression after 48 h culture; and gray line indicates isotype control. C) NK cytotoxicity on K562 targets after culture with IL-15. Culture of CD56-bead purified NK cells for 72 h with IL-15 (100ng/ml) increased cytotoxicity (lytic units) compared to NK cells prior to culture (∗P < .0001). Addition of neutralizing antibodies to activating receptors NKG2D, NKp30, or NKp46 individually or in combination reduces NK cytotoxicity compared to assays containing an isotype control antibody (∗∗ P < .01; ∗∗∗ P < .001 relative to IL-15).

Concomitant with the up-regulation of the activating receptors following 3 days of IL-15 stimulation, there was a significant increase (P < .0001) in NK cell cytotoxicity on K562 targets (1647 vs 5477 LU) (Figure 2C). Expression of multiple activating and inhibitory receptors is altered in response to culture in IL-15, however, so the contribution of the changes in activating receptors was assessed by the addition of neutralizing antibodies to the cytotoxicity assays. Addition of antibodies to any of the 3 receptors reduced the level of cytotoxicity and addition of all 3 antibodies blocked much of the increase in NK cytotoxicity (5477 to 2434 LU, P < .001) (Figure 2C). Therefore the increase in cytotoxic activity following IL-15 culture was to a large extent dependent upon the increase in expression of NKG2D and NCR activating receptors.

Activating Receptor Expression Post-Transplant 

We then examined the changes in the expression of the 3 activating receptors in 14 donor/recipient pairs, examining the NK cells in donor PBSC aphereses and in patient peripheral blood collected in the year following transplant. We observed the greatest changes in the early post-transplant months, the period in which the highest levels of IL-15 were observed.

Expression of the activating receptors was significantly increased in the early months post-transplant as compared with the expression in the donor PBSC. As in the cultured NK cells, there was a significant increase in the level of expression of CD56. The greatest expression of the activating receptors was observed on those cells with the highest levels mean fluorescence intensity (MFI) of expression of CD56 (Figure 3).

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  • Figure 3 

    Expression of NK receptors during the first year post-transplant. Contour plots of gated NK cells from patient #14 over a time course of the first year post-transplant. At 1 month post-transplant, the proportion of CD56brightCD16– cells within the NK population increased. In addition, an overall increase in the expression of CD56 was observed on both NK subsets. Expression of the activating NKG2D, NKp30, and NKp46 receptors increased on NK cells in the early months post-transplant relative to that of the donor PBSC NK cells. The highest level (MFI) of activating receptor expression was found on cells that had up-regulated CD56 expression.

Expression of the homodimeric activating receptor NKG2D increased significantly from a median of 41% (range 14 – 80%) of NK cells in PBSC to 60% (range 24 – 89) of the NK cells at 1 month. Meanwhile the MFI of NKG2D increased from 12 (range 6-35) in the donor inoculum to 21 (range 10-41) at 1 month (Figure 3, Figure 4) Although NK cells in some individuals continued to express high levels of NKG2D (Figure 3), in general this marker declined by 3 months. Similarly the frequency of NK cells expressing NKp30 (CD337) at 1 month post-transplant was twice that of NK cells in the PBSC, but expression of this marker remained significantly elevated at 3 months. Expression of NKp46 (CD335) showed the greatest change. Expression tripled from 20% (range 8-57) of the total NK cells in the donor apheresis to a median of 63% (range 35-84) at 1 month and remained elevated in both frequency and MFI at 3 months (P = .003) (Figure 3, Figure 4). By 6 months the median expression of these markers had returned to levels in the donor apheresis, but remained elevated as long as 1 year in some individuals.

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  • Figure 4 

    Percentile plots of NK receptor expression in the first year post-transplant. Box plots of percentages of NK cells expressing each receptor, showing the median (central bar), 25th and 75th percentile (box) and 10th and 90th percentile (bar) from the patients. Wilcoxon paired signed rank P values comparing expression in donor PBSCT and at 1 and 3 months post-transplant are indicated.

No significant increases were observed in KIR or NKG2C expression in these patients (data not shown). Expression of NKG2A and CD94 increased (data not shown), consistent with previous reports 21, 30.

Receptor Association with NK Subset Recovery 

We found that the MFI of the 3 activating NK receptors was usually observed on those cells with the highest CD56 expression (Figure 3). As has previously been observed in transplant regimens [13], there was a disproportionate increase of the CD56brightCD16– subset of NK cells at 1 month compared to the increase of the CD56dimCD16+ subset. By 3 months post-transplant, the proportion of NK cells in the CD56brightCD16– subset was declining toward normal ratios, which were then maintained for the first year (Figure 5A). We therefore examined whether or not there was a differential expression of activating NK receptors in the 2 subsets, such that the increase in CD56brightCD16– would be responsible for the increased frequency of activating receptors. We determined that a higher frequency of NK cells in the CD56brightCD16– subset expressed NKG2D at 1 and 3 months, but the increase in expression of the NCR receptors were comparable in both subsets. Furthermore, the MFI of NKp46 was much higher in the CD56brightCD16– subset (Figure 5B, C), but other receptors were expressed at comparable intensities. Overall, therefore, both the CD56brightCD16– subset and the CD56dimCD16+ subset had increased expression of activating receptors in the first months post-transplant.

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  • Figure 5 

    NK subset and receptor recovery. A) Stacked bar graph of the average number of CD56brightCD16– and CD56dim CD16+ NK cells/μl of peripheral blood in donor peripheral blood and in recipients post-transplant. At 1 month post-transplant the CD56brightCD16– subset (black bar) was disproportionately increased compared to the CD56dim CD16+ (white bar). Standard error bars for each subset are shown. B) Overlay histograms illustrating increased expression of activating receptors in both NK subsets in a representative patient (#2) at 1 and 3 months post-transplant. Gray filled histogram shows donor PBSC NK cells; black line shows 1 month post-transplant; thick black line shows 3 months post-transplant. C) Scatterplots of the expression of the activating receptors in NK subsets in donor PBSC and patient peripheral blood following transplant. Horizontal bars represent the median expression of the receptors in the donor group, and at 1 and 3 months post-transplant.

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Discussion 

In this study, we examined the reconstitution of NK cells and their receptor repertoire in patients with hematologic malignancies who had undergone allo-HSCT under a lympho-depleting, non-myeloablative regimen. We demonstrated that the expression of the activating receptors on NK cells increased during the early post-transplant period, at the time when NK cells not only constituted a significant percentage of the lymphocyte population, but also achieved circulating levels not achieved for the remainder of the first post-transplant year. We have documented significantly elevated IL-15 levels in all patients in this study on the day of transplant and a subsequent decline correlated with NK recovery, consistent with homeostatic dynamics. Furthermore, we have demonstrated that the increased expression of activating receptors and the concomitant increase in NK cytotoxicity can be induced in vitro by IL-15. We propose that the cytokine milieu present during the early post-transplant period may underlie the rapid changes in the NK populations and activating receptor expression. Because of the critical role these activating receptors have in anti-tumor reactivity, these changes may underlie NK efficacy on residual disease post-transplant.

Plasma IL-15 levels have been demonstrated to increase significantly following cytoreductive therapy 31, 32. As IL-15 is constitutively produced [33], the elevated plasma IL-15 levels could be related to transplant regimen-induced depletion of lymphoid populations that normally consume circulating IL-15, paralleling the postulated homeostatic relationship of serum IL-7 levels and T cell reconstitution [34]. Miller et al [32] have demonstrated that a high dose fludarabine/cytoxan chemotherapy regimen can result in elevated serum IL-15 levels that were not observed in regimens producing less lympho-depletion.

In the current study, the non-myeloablative regimen specifically focused upon the use of inductive chemotherapy and preparative regimens designed to deplete host lymphocyte populations and produce rapid donor engraftment 26, 27. Depletion of NK cells was particularly effective; no hosts had residual NK cells at day 0. IL-15 could also have been augmented in response to inflammatory stimulation induced by the transplant regimen 35, 36. Both may have contributed to the highly elevated levels of IL-15 were found in all patients on the day of transplant.

Elevated levels of IL-15 at the time of transplant could support both NK differentiation and expansion. The growth, differentiation, and survival of NK cells have been found to be dependent upon IL-15. Mice lacking expression of either IL-15 or the IL-15Rα chain fail to develop NK cells; NK cells transferred into IL-15–/– hosts fail to survive 37, 38, 39. In humans, IL-15 induces the differentiation of NK cells in stroma-free cultures from CD34+ progenitor cells and expands mature NK cells in culture 1, 19, 20. NK clones administered as adoptive therapy persisted and expanded only in patients receiving a lympho-depletive regimen resulting in high IL-15 levels [32]. In our study, the rapid decline in IL-15 levels we observed was strongly correlated with the rapid recovery of NK levels. This was particularly evident at 2 weeks, when the broad range of IL-15 levels present in the patients demonstrated a strong inverse correlation with NK recovery in individual patients. This is the first demonstration that the decline in IL-15 following transplantation is specifically correlated with NK recovery.

Both generation of new NK cells from donor stem cells and expansion of NK cells transferred in the donor inoculum may have contributed to the NK populations present at 1 month. The CD56bright NK subset was disproportionately increased at 1 month relative to the CD56dimCD16– subset, consistent with previous reports 13, 18. In vitro IL-15 has been demonstrated to induce differentiation of hematopoietic progenitor cells into NK cells that primarily express the CD56bright phenotype 1, 19, 40. The early increase in CD56bright NK cells is consistent with evidence that donor stem cells rapidly differentiate into NK cells post-transplant 12, 15. CD56bright cells present in the donor PBSC inoculum may also have expanded disproportionately in the presence of high endogenous levels of IL-15. We and others have observed that CD56bright NK cells proliferate more than the CD56dim subset in IL-15 stimulated cultures [18]. Thus, whether derived from hematopoietic stem cells or from CD56bright NK cells in the donor inoculum, the shift in the NK subset proportions could have been driven by the elevated levels of IL-15 observed during the early post-transplant period.

Culture of progenitor cells in IL-15 has also been demonstrated to promote the step-wise differentiation of inhibitory and activating NK receptors 18, 19, 40, 41. We demonstrated that mature circulating NK cells will also up-regulate the expression of these receptors under the influence of IL-15, with a concomitant increase in receptor-dependent cytotoxic activity on an NK tumor target cell line. The NCRs, NKp30 and NKp46, and the C-type lectin receptor NKG2D were significantly elevated at 1 month post-transplant and elevated levels of expression persisted in some patients up to 12 months post-transplant. Furthermore both the CD56brightCD16– and the CD56dimCD16+ populations demonstrated increases in both the frequency of cells expressing the receptors and the MFI level of expression/cell.

An additional transplant regimen-related factor that might have contributed to the rapid expansion of NK cells and the shift in receptor expression is the use of cyclosporin as a primary prophylactic measure for acute GVHD (aGVHD). Patients were maintained on cyclosporin at a dose adjusted level of 200 ng/ml for the first 100 days, with subsequent tapering till 180 days. Addition of Cyclosporin A to IL-15-stimulated NK cultures has been found to synergize with many of the effects of IL-15 alone, including increased expression of CD56bright NK cells and increased expression of NKp30, although not NKG2D [20]. The contribution of cyclosporin as GVHD prophylaxis to NK efficacy on residual disease remains undetermined. Nevertheless, this work suggests that multiple transplant-regimen related factors–severe host lympho-depletion elevating endogenous IL-15 levels, non-manipulated PBSC infusion containing mature NK cells, and post-transplant prophylaxis supporting IL-15 effects–may work together to establish a rapid engraftment of donor NK cells with elevated expression of tumor-reactive activating receptors.

NK cells have long been found to be effective in killing a broad range of tumor cells. The role of NK cells in enhancing graft-versus-leukemia (GVL) in allogeneic transplants has been demonstrated in both in vivo preclinical and clinical studies 42, 43. Recent studies have shown that transplantation from T cell-depleted donors, whose NK cells were reactive against haploidentical host MHC, protected patients with acute myelogenous leukemia (AML) from relapse 24, 25, 44. The post-transplant period provides an opportunity for enhanced NK-mediated attack on residual tumor cells. NK cell numbers are elevated, particularly the CD56bright populations. Expression of the critical anti-tumor receptors NKp30, NKp46, and NKG2D are elevated. The evidence presented here suggests that these population and receptor shifts are not random, but are driven by the cytokine milieu of transplant, specifically the elevated plasma levels of IL-15. This model linking homeostatic cytokine levels, NK expansion, and expression of activating receptors targeting tumor antigens, therefore supports the use of transplant-based regimens as a platform for NK-based therapies and provides insights into the biology that may direct intervention strategies.

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PII: S1083-8791(07)00653-2

doi:10.1016/j.bbmt.2007.12.490

Biology of Blood and Marrow Transplantation
Volume 14, Issue 3 , Pages 290-300, March 2008

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