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Bone Marrow Transplantation (2001) 28, 673–680  2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt

Immune modulation Immunomodulation of early engrafted natural killer cells with interleukin-2 and interferon-␣ in autologous stem cell transplantation LF Porrata, DJ Inwards, MQ Lacy and SN Markovic Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN, USA

Summary: High relapse rates during the first year after autologous stem cell transplantation (ASCT) for multiple myeloma or non-Hodgkin lymphoma are due to the failure of high-dose chemotherapy to eradicate minimal residual disease. Post-ASCT immunorecovery studies have shown that quantities of natural killer (NK) cells return to normal within 1 month post-ASCT in contrast to the recovery of T and B cell populations (up to 1 year). Preclinical studies have demonstrated that NK cells have potent antitumor activity. IL-2 and IFN-␣ enhance NKcell activity. We investigated the efficacy of IL-2 and IFN-␣ to up-regulate NK-cell cytotoxicity at 14 days post ASCT. Twenty patients undergoing ASCT had PBMCs collected pretransplantation and at 14 days post transplantation. PBMCs (effector cells) from each blood sample were incubated in vitro with IFN-␣ and IL-2 at 10 000 IU/ml. NK cell activity was determined by sodium chromate 51Cr release assay for lysis of K562 target cells. IL-2 and IFN-␣ each increased lysis of K562 cells compared with placebo (effector-to-target ratio, 50:1, P ⬍ 0.001). Increased NK cell activity occurred in samples from all patients. IL-2 and IFN-␣ up-regulated NK cell activity at 14 days post ASCT. They may be useful as immunomodulators as early as 14 days post ASCT to eradicate or control minimal residual disease. Bone Marrow Transplantation (2001) 28, 673–680. Keywords: interferon-alpha; interleukin-2; natural killer cells; stem cell transplantation

The high relapse rates that occur after autologous stem cell transplantation (ASCT) have been attributed to the failure of high-dose chemotherapy with or without radiotherapy to eliminate minimal residual disease. Recurrences often occur during the first year1,2 and may occur within 100 days post ASCT.3 Results of post-ASCT immunorecovery studies have demonstrated that humoral (B cells)4,5 and cellmediated (T cells)6,7 components of the immune system are not fully recovered at the time of most tumor relapses. This Correspondence: Dr SN Markovic, Division of Hematology and Internal Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA Received 27 April 2001; accepted 18 July 2001

suggests that lack of post-ASCT immunosurveillance may contribute to the risk of tumor relapse. Overwhelming evidence from several post-ASCT immunorecovery studies demonstrated normal quantitative reconstitution of natural killer (NK) cells within 1 month after ASCT.8–13 NK cells comprise 5% to 8% of human peripheral blood lymphocytes and morphologically resemble large granular lymphocytes.14 NK cells are capable of dual cytolytic activity. First, they are capable of spontaneous, antibody-independent, non-major histocompatibility complexrestricted cytotoxicity mediated by the binding of NK cell surface leukocyte function-associated antigen 1 (clusters of differentiation [CD]11aCD18) and CD2 to their target cell ligands, CD54 and CD58, respectively.15 Second, they can mediate antibody-dependent cellular cytotoxicity by the binding of NK cell surface CD16 to their target cell ligands.15 Several studies have shown the presence of infiltrating NK cells in solid tumors and lymphomas.16–20 Preclinical studies have shown that NK cells eradicate and control tumor cell growth of leukemias and lymphoma.21–23 Because NK cells recover early during the period when most tumor relapses occur post ASCT, our interest focused on regulation of NK cells as a method of immunotherapy to improve remission and cure rates by eradicating minimal residual disease by means of stimulated NK cells. IL-2 is a 15-kDa glycoprotein that promotes proliferation and differentiation of helper T cells, cytotoxic T cells, and B cells and is necessary for acute and anamnestic adaptive immune responses.24 IL-2 is an excellent candidate to be an immunomodulator for post-ASCT up-regulation of NK cells, because NK cells are unique in that they constitutively express IL-2 receptors; thus, they always react to IL2.25 NK cells have also interferon receptors in their cell membrane, and interferons have been shown to be effective up-regulators of NK-cell activity.26–29 Results of previous studies have demonstrated that IL-2 and IFN-␣ augment NK cell cytotoxicity.30–34 The aim of our study was to determine whether IL-2 or IFN-␣ can up-regulate NK cell activity in vitro as early as 14 days after ASCT.

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Patients and methods Patient samples This study was approved by the Institutional Review Board of Mayo Foundation. Twenty patients who were candidates for ASCT at Mayo Clinic were entered in the study from October 1999 until April 2000. Of these 20, 10 had multiple myeloma and 10 had non-Hodgkin lymphoma. Preparation of mononuclear cells Venous blood (30 ml) was drawn from every patient just before stem cell mobilization and collection and at 14 days post ASCT. Mononuclear cells were separated by gradient centrifugation on Ficol and washed and resuspended on RPMI 1640. Immunophenotyping Immunophenotyping of PBMCs was performed using a two-color direct staining of cells with mouse monoclonal antibodies. Fluorescein isothiocyanate- or phycoerythrincoupled antibodies (Becton Dickinson PharMingen, San Diego, CA, USA) were directed at CD2, CD3, CD4, CD8, CD16, CD19, CD25, CD56, and human leukocyte antigen D-related. Phenotypic analysis was performed with a FACStar PLUS flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) equipped with CONSORT 40 software (Becton Dickinson Biosciences, San Jose, CA, USA). Fluorescein isothiocyanate- and phycoerythrin-coupled isotype-matched immunoglobulins were used as controls.

NK-92 (American Type Culture Collection, Manassas, VA, USA). Samples in different dose ranges for IL-2 (66 IU/ml– 18 million IU/ml) and IFN-␣ (25 IU/ml–3 million IU/ml) were incubated for 24 h in RPMI 1640, and the NK cell cytotoxicity assay was performed with a 50:1 effector-totarget ratio. NK cell activity NK cells were activated for cytotoxicity assays. For this purpose, PBMCs were cultured in RPMI 1640 for 24 h in the presence of 10 000 U of recombinant IL-2 and IFN-␣. Normal volunteers’ PBMCs and human cell line NK-92 were used as controls with and without cytokines. Maintenance of cell lines Cell line K562 (NK-sensitive target, American Type Culture Collection) was maintained in continuous culture in RPMI 1640 medium supplemented with 1% glutamine and 5% heat-inactivated fetal calf serum. Human NK cell line NK-92 was maintained in continuous culture in RPMI 1640 supplemented with 100 U/ml of IL-2, 1% glutamine, 5% heat-inactivated fetal calf serum, and 1% sodium pyruvate. Statistics Significance levels were determined by the two-tailed Student’s t-test. Results

NK cell cytotoxicity assay

Patient characteristics

The cytotoxicity of the cell preparations was tested against the NK-sensitive myeloid leukemia cell line K-562 in a 4-h sodium chromate 51Cr release assay. Target cells were labeled with 100 ␮Ci of 51Cr for 4 h at 37°C. Washed target cells (1 million per well) were added to the wells of roundbottom 96-well plates (Falcon; Becton Dickinson) and incubated at effector-to-target cell ratios ranging from 50:1 to 6.25:1. Maximum release and spontaneous release were determined by incubating the target cells with 1% Triton X-100 (Sigma-Aldrich, Milwaukee, WI, USA) and medium alone, respectively. All determinations were made in triplicate. Radioactivity was counted with a COBRA gamma counter (Packard Instrument Company, Meriden, CT, USA) after using a Skatron supernatant harvester (Molecular Devices, Sunnyvale, CA, USA). The percentage of specific lysis was determined with this formula: % specific lysis = [mean c.p.m. (experimental release) − mean c.p.m. (spontaneous release)]/[mean c.p.m. (total release) − mean c.p.m. (spontaneous release)] × 100.

Our study included 16 males and four females (Table 1). Median age was 58 years (range, 23–71 years). Diseases for which ASCT was performed included multiple myeloma (10 patients) and non-Hodgkin’s lymphoma (large cell lymphoma, eight patients; T cell-rich B cell lymphoma, one patient; and mantle zone lymphoma, one patient). All patients who had multiple myeloma were conditioned with melphalan, 200 mg/m2, 1 day before transplantation. One patient also received samarium, 12 mCi/kg, on the 10th day before his ASCT. The patients who had non-Hodgkin’s lymphoma were conditioned on the 6th day before ASCT with carmustine, 300 mg/m2; on the fifth to the second day before ASCT with cytarabine, 100 mg/m2 every 12 h, and with etoposide, 100 mg/m2 every 12 h; and on the day before ASCT with melphalan, 140 mg/m2 (BEAM). All patients achieved neutrophil engraftment and had absolute neutrophil counts of at least 0.5 × 109/l day 14 post ASCT. The mean absolute lymphocyte count at day 14 post ASCT was 0.48 × 109/l (range, 0.17–0.85 × 109/l; Mayo Clinic normal value, 0.9–2.9 × 109/l).

IL-2 and IFN-␣ dose–response curves To identify the best in vitro cytokine dose to achieve maximum NK cell cytotoxicity, dose–response curves were determined for IL-2 and IFN-␣ for normal volunteers, for patients post engraftment, and for the human NK cell line Bone Marrow Transplantation

Immunophenotyping Table 2 shows the phenotypic composition of PBMCs in normal controls and patients before transplantation and at day 14 post ASCT. Before transplantation and at day 14,

Immunomodulation of engrafted natural killer cells LF Porrata et al

Table 1

Patient characteristics

Patient

Age, years

Sex

demonstrate that the maximum up-regulation of NK cell cytotoxicity occurred at 10 000 IU/ml for each cytokine. At higher concentrations, each cytokine produced marked down-regulation of NK cell cytotoxicity.

675

Patients who had multiple myeloma (melphalan-conditioning regimen)a 1 2 3 4 5 6 7 8 9 10b

59 71 55 53 63 49 61 60 63 56

M M F M M M M M M M

Patients who had non-Hodgkin’s lymphoma (BEAM-conditioning regimen)c 11 12 13 14 15 16 17 18 19 20

50 46 45 52 23 69 62 63 43 63

M M F M M M M M F F

a

Melphalan, 200 mg/m2. b Conditioning regimen included samarium, 12 mCi/kg, in addition to melphalan. c BEAM, carmustine (BCNU), 300 mg/m2; etoposide, 100 mg/m2; cytarabine (Ara-C), 100 mg/m2; and melphalan, 140 mg/m2.

the percentages of CD3+, CD4+, CD8+, and CD19+ cells were low compared with those of normal controls. In contrast, the percentages of CD16+CD56+CD3− cells (NK cell phenotypic markers) before transplantation were equal to or greater than those of the normal controls. At day 14, the percentages of CD16+CD56+CD3− cells had recovered to levels that were equal to or greater than those of the normal controls. IL-2 and IFN-␣ dose–response curves Figure 1 shows the dose–response curves for IL-2 and IFN-␣ for normal volunteers, for the human NK-cell line NK-92, and for patients 14 days post ASCT. All curves Table 2

NK cell activity The incubation of PBMCs with IL-2 and IFN-␣ markedly increased NK cell activity (Figure 2). When cytokine- and placebo (media)-treated effector PBMCs were compared, the differences in NK cytotoxicity were significant (effector-to-target ratio, 50:1, P ⬍ 0.0001). This effect by each cytokine was observed before transplantation and at 14 days post ASCT for all patients. The controls (normal volunteers and human NK cell line NK 92) demonstrated similar NK cell up-regulation by each cytokine. No statistical difference existed in up-regulation of NK cell activity by either cytokine between patients and controls (effectorto-target ratio, 50:1, P ⬍ 0.66). Discussion The delay in immunorecovery during the first year after transplantation of autologous stem cells supports the hypothesis that the high number of relapses during this period may be due in part to lack of effective immunosurveillance capable of containing or eradicating minimal residual disease during this period. For eight patients with B cell non-Hodgkin lymphoma who had autotransplantations with T cell-depleted CD34+ stem cells, Divine et al35 showed a sustained quantitative deficiency for CD3+, CD4+, CD8+, and CD19+ cells up to 18 months post transplantation. Talmadge et al36 showed a faster recovery for CD4+, CD8+, and CD19+ cells for autologous peripheral stem cell transplantation than for ABMT in the first 100 days post transplantation, although levels were lower than those for normal controls. The T and B cell functional recovery remains deficient for years post transplantation.37–39 The delayed quantitative and functional recovery of humoral (B cells) and cellmediated (T cells) components of the immune system is evidence against their utility in immunotherapeutic strategies early post transplantation.

Phenotypic composition of peripheral blood mononuclear cells

Clusters of differentiation

Positive cells at pretransplantation, %, mean (range)

CD3 CD4 CD8 CD19 CD16+CD56+CD3−

30 22 11 10 14

(10–61)a (8–50)a (1–38)a (0–36)a (9–23)b

Positive cells at 14 days post ASCT, %, mean (range) 28 (10–40)c 21 (5––48)c 13 (2–40)c 13 (0–22)d 18 (10–48)e

Positive cells for normal controls, %, mean (range) 68 46 27 25 8

(52–84) (50–65) (22–42) (20–35) (5–15)

ASCT = autologous stem cell transplantation. a P ⬍ 0.0001 when compared with normals. b P ⬍ 0.1 when compared with normals. c P ⬍ 0.0001 when compared with normals. d P ⬍ 0.02 when compared with normals. e P ⬍ 0.15 when compared with normals. Bone Marrow Transplantation

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NK cell cytotoxicity, %

100

Normals

a

Normals

80

60

40

20

0

18 ´ 106

2.8 ´ 105

1 ´ 104

1.1 ´ 103

Placebo

2 ´ 105 3 ´ 106

1 ´ 104 700 4.7 ´ 104 3 ´ 103

IL-2, IU/ml

NK cell cytotoxicity, %

100

IFN-a, IU/ml

NK-92

b

Placebo 183

NK-92

80

60

40

20

0

1 ´ 106 18 ´ 106

NK cell cytotoxicity, %

100

c

1 ´ 104 7 ´ 105

1000 4 ´ 103

Placebo

3.8 ´ 105 3 ´ 106

68

1 ´ 104 9.4 ´ 104

187 1.5 ´ 103

IL-2, IU/ml

IFN-a, IU/ml

Day 14 post ASCT

Day 14 post ASCT

Placebo

80

60

40

20

0

1.1 ´ 106 18 ´ 106

1 ´ 104 7 ´ 104

IL-2, IU/ml

235 4.4 ´ 103

Placebo

7.5 ´ 105 4.7 ´ 104 1.3 ´ 103 3 ´ 106 2 ´ 105 1 ´ 104 366

92 Placebo

IFN-a, IU/ml

Figure 1 Dose–response graphs of IL-2 and IFN-␣ show an optimal dose of 10 000 IU/ml. (a) Normal volunteers; (b) human natural killer (NK) cell line NK-92; (c) patients at day 14 post ASCT. Maximum NK cell activity occurred after in vitro exposure of peripheral blood mononuclear cells to doses of IL-2 and IFN-␣ of 10 000 IU/ml. Higher or lower doses of IL-2 or IFN-␣ resulted in less efficient NK cell stimulation than did the optimal dose of 10 000 IU/ml. These graphs suggest the existence of an optimal immunostimulatory dose range for NK cell activation induced by IL-2 and IFN-␣. Higher doses of IL-2 or IFN-␣ seem to exert a negative effect on NK cell function. Similar results were observed in all patient samples. ASCT = autologous stem cell transplantation.

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Immunomodulation of engrafted natural killer cells LF Porrata et al

NK cell cytotoxicity, %

a

100 80 60 40 20 0

NK cell cytotoxicity, %

b

NK cell cytotoxicity, %

50:1

25:1

12.5:1

6.25:1

Effector-to-target ratio 100 80

Placebo Pretransplantation 14 days post ASCT

IFN-a

60 40 20 0

c

Placebo Pretransplantation 14 days post ASCT

IL-2

50:1

25:1

12.5:1

6.25:1

Effector-to-target ratio 100 80

Controls

Placebo Normals NK-92

60 40 20 0

50:1

25:1

12.5:1

6.25:1

Effector-to-target ratio Figure 2 Incubation of PBMCs with IL-2 and IFN-␣ markedly increased NK cell activity. (a and b) PBMCs were incubated with plain media (placebo), IL-2, or IFN-␣ for 24 h before the NK assay. PBMCs were reacted against natural killer (NK)-sensitive K562 target cells at effectorto-target ratios ranging from 50:l to 6.25:l. Up-regulation of NK cell activity by IL-2 (10 000 IU/ml) and IFN-␣ (10 000 IU/ml), respectively, pretransplantation and at 14 days post ASCT (P ⬍ 0.001). Similar results have been observed in all 20 patients studied thus far. (c) Results from control samples of above experiments (normal volunteers and human NK cell line NK-92) demonstrated a greater baseline NK cell cytotoxicity compared with that of patients who had undergone transplantation. Upregulation of NK cells with IL-2 and IFN-␣ was similar for controls and for patients who had undergone transplantation (P ⬍ 0.001). ASCT = autologous stem cell transplantation.

In contrast, NK cell quantity and function return to normal within 30 days post transplantation.40–42 The development of NK cells before T cell subsets from stem cells has been demonstrated in normal volunteers. Lotzova et al43

cultured CD34+ stem cells in long-term bone marrow culture from normal donor bone marrow. At 2 weeks, 80% of the lymphocyte subsets were CD56+CD3− (NK cells) compared with 10% of the T cell phenotype. Long-term bone marrow culture without IL-2 did not support the genesis of NK cells. These NK cells showed high cytotoxic efficacy against the K562 cell line. In a stroma-dependent long-term bone marrow culture system, Miller et al44 showed that NK cells developed earlier than T cell subsets from CD34+DR− normal donor stem cells. At 5 weeks, 84.8% of the cells expressed the NK cell phenotype (CD56+/CD3−) compared with 3.1% of T cells expressing CD8. No cultured cells expressed CD3, CD5, T cell receptor ␣/␤, or T cell receptor ␥/␦ T cell markers. The NK cells demonstrated high cytotoxic activity, not only to the K562 cell line, but also to the lymphoma cell line RAJI. The early development of NK cells is attributed to the fact that NK cells do not require thymic priming before their final maturation. The results of our study confirm the early recovery of NK cells post ASCT compared with that of humoral and cell-mediated immunity. However, compared with the results of other studies in which phenotypic NK cell analysis was performed at multiple intervals post transplantation, the results of our study show that quantities and function of NK cells return to normal as early as 14 days post ASCT. Multiple preclinical studies have demonstrated dose– response curves for up-regulation of NK cell activity with either IL-2 or IFN-␣. Talmadge et al45 reported that lowdose IL-2 (100 U) achieved a greater cure rate than did a dose of 10 000 U for mice inoculated with mannan-binding lectin-2 lymphoma. Using murine splenic NK cells against the yeast artificial chromosome 1 lymphoma cell line, Kuribayashi et al46 showed maximum NK cell cytotoxicity (40%) at concentrations of IL-2 and IFN-␣ of 10 000 U/ml. At greater concentrations, NK cell cytotoxicity decreased to 15%. Shaw et al47 reported maximum NK cell activity (100 lytic units) by incubating normal volunteers’ PBMCs with IL-2 at 3000 U/ml against a colorectal carcinoma cell line, LoVo. At higher concentrations, down-regulation of NK cell activity (60 lytic units) occurred. Results of these studies corroborate our dose–response curves for normal volunteers, human NK cell line NK-92, and patients at day 14 post ASCT. Our in vitro data strongly support the concept that low-dose IL-2 and IFN-␣ result in maximum NK cell activity, and higher doses of IL-2 (18 million U) and IFN-␣ (3 million U) produce down-regulation of NK cell activity. Studies with humans have shown that high-dose IL-2 given either intravenously or subcutaneously produces severe lymphopenia. Thompson et al48 reported that nonselective, dose-dependent lymphopenia occurred in 22 patients treated with 2-h or 24-h infusions of IL-2 in doses ranging from 3 million U to 30 million U. The lymphopenia lasted 7 days after the last day of IL-2 therapy. NK cell activity was markedly decreased 24 h after the last dose of IL-2 compared with NK-cell activity before IL-2 therapy. Higuchi et al49 treated 16 patients post ABMT with intravenous induction IL-2 therapy for 5 days, with doses ranging from 3 to 4.5 million U/m2. Marked lymphopenia during the induction therapy was followed by a rebound lymphocytosis 24 h after termination of the induction treat-

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Table 3

Clinical studies of post transplantation administration of low-dose IL-2

Patients, no.

7 26 6 11 29

Type of malignancy

Type of BMT

ALL, HD, NHL ABPSCT MM, ALL, NHL ABMT, ABPSCT NHL, HD ABMT NHL ABMT AML, MDS, NHL, CML Allo-BMT

IL-2 dose, MIU

Duration of therapy, months

Hematologic toxicity (grade 3/4), %a

Nonhematologic toxicity (grade 3/4), %a

Ref.

4.5 0.4–1 0.25–0.5 2–3 0.2–0.4

4 3 3 12 3

6 0 0 0 0

26 0 33 0 0

50 51 52 53 54

ABMT = autologous bone marrow transplant; ABPSCT = autologous blood peripheral stem cell transplant; ALL = acute lymphoblastic leukemia; AlloBMT = allogeneic bone marrow transplant; AML = acute myelogenous leukemia; BMT = bone marrow transplant; CML = chronic myelogenous leukemia; HD = Hodgkin’s lymphoma; IL-2 = interleukin-2; MDS = myelodysplastic syndrome; MIU = million international units; MM = multiple myeloma; NHL = non-Hodgkin’s lymphoma. a Percentage of total number of patients in each study.

ment. The reason why lymphopenia occurs in vivo after high-dose IL-2 is unknown. Lymphopenia may result from margination and extravasation of lymphocytes. However, the dose–response curves of IL-2 and IFN-␣ reported in preclinical studies, including our data, strongly suggest the possibility that the down-regulation of NK cell activity observed at higher doses of these two cytokines could be due to the activation of a down-regulation pathway or to a cytotoxic effect on NK cells. Using IL-2 or IFN-␣, we up-regulated NK cell activity as early as 14 days post ASCT. Up-regulation of NK cell activity by each cytokine was observed in all engrafted samples. This finding is important because up-regulation of NK cell activity can be achieved as early as 14 days post ASCT to target minimal residual disease and possibly to prevent tumor relapses. In addition, the finding that upregulation of NK cell activity occurred with each cytokine pretransplantation supports the argument that NK cells retain their capability to respond to immunomodulators despite being treated with multiple chemotherapy drugs. IL-2 has been used more often than IFN-␣ as a posttransplantation immunomodulator. Unfortunately, higher doses of IL-2 have been associated with serious complications, including death from capillary leak syndrome. Post-transplantation administration of low-dose IL-2 has been considered a means to achieve maximum immunomodulatory effect with minimal side-effects. Table 3 shows results of clinical studies in which low-dose IL-2 was given post transplantation. Results of these studies showed no grade 4 toxicity and minimal grade 3 toxicity. Lauria and colleagues53 reported no relapses after 12 months of IL-2 therapy (median follow-up, 22 months; range, 10–42 months). Two patients who had residual disease after ABMT (one patient had disease in the liver and the second, in the lymph nodes) obtained a complete response after IL2 therapy of 10 and 7 months, respectively. These data suggest that low-dose IL-2 has a low toxicity profile and some efficacy in delaying relapses post transplantation. In summary, our study demonstrates up-regulation of NK cells with low-dose IL-2 or IFN-␣ as early as 14 days post ASCT. The available low toxicity profile data and therapeutic efficacy of low-dose IL-2 post ASCT and the results from our study suggest the rationale to develop studies to assess the efficacy of low-dose cytokines post ASCT to Bone Marrow Transplantation

enhance immunologic recovery and activity (ie NK cells) to target minimal residual disease. Acknowledgements Portions of this study were published as an abstract in Proceedings of ASCO 2000; 19: 61a.

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