Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2013  |  Volume : 9  |  Issue : 7  |  Page : 162--168

Immune responsiveness in a mouse model of combined adoptive immunotherapy with NK and dendritic cells


Feng Cui1, Jingjing Ji2, Huifang Lv1, Di Qu1, Changhua Yu1, Yu Yang1, Yuqing Xu1,  
1 Department of Oncology, The Second Affiliated Hospital of Haerbin Medical University, China
2 Department of Pathology, The Second Affiliated Hospital of Haerbin Medical University, China

Correspondence Address:
Yuqing Xu
Department of Oncology, The Second Affiliated Hospital of Haerbin Medical University, 150086, 246 Xuefu Road, Nangang District, Haerbin, Heilongjiang
China

Abstract

Objective: To determine the tumoricidal ability of combined immunotherapy of natural killer (NK) cells and dendritic cells (DCs) in a melanoma mouse model and the functions of tumor-associated effector cells. Materials and Methods: A C57BL/6 mouse model of subcutaneous melanoma and lung metastasis was established. NK cells and DCs were cultured and labeled in vitro. Varying frequencies of both NK cells and DCs were adoptively transferred into tumor-bearing mice. Tumor, liver, spleen, and lung were studied for the number and distribution of effector cells. Additionally, CD8+T cell numbers in the lung and numbers of metastatic lung nodules were determined. Results: Co-culture of NK cells and DCs might maintain and promote NK cell activity without exogenous cytokines. Both NK cells and DCs were distributed in the tumor microcirculation and parenchyma. We found significant time-dependent differences in the numbers of infiltrating NK cells and DCs (P < 0.01), which stimulated the highest frequencies of effector cells 4 h after transfer and the lowest at 12 h. Low NK cell numbers were found in the spleen, and fewer numbers were found in liver and lung. Infiltration of tumors with effector cells was greatest following mixed cell transfers as compared to single transfers, and markedly increased CD8+T cells were associated with significant decreases in lung metastases. Conclusion: NK cells and DCs adoptive immunotherapy targeted the tumor and exhibited improved therapeutic efficacy as compared to that of the cells given alone. This strategy could induce tumorigenic immunological memory and suggests that mixed NK cells and DCs adoptive immunotherapy offers therapeutic options against cancer.



How to cite this article:
Cui F, Ji J, Lv H, Qu D, Yu C, Yang Y, Xu Y. Immune responsiveness in a mouse model of combined adoptive immunotherapy with NK and dendritic cells.J Can Res Ther 2013;9:162-168


How to cite this URL:
Cui F, Ji J, Lv H, Qu D, Yu C, Yang Y, Xu Y. Immune responsiveness in a mouse model of combined adoptive immunotherapy with NK and dendritic cells. J Can Res Ther [serial online] 2013 [cited 2019 Nov 18 ];9:162-168
Available from: http://www.cancerjournal.net/text.asp?2013/9/7/160/122516


Full Text

 Introduction



Natural killer (NK) cells and dendritic cells (DCs) are two distinct components of the protective immune response and play a vital role during immune regulation and in establishing adaptive immune responses. NK cells are capable of killing tumor cells directly. Some studies have revealed that the interaction between NK cells and DCs plays an important role in antitumor and antiviral responses. [1],[2],[3]

We assessed the pre-clinical efficacy of an adoptive immunotherapy strategy in an attempt to observe the effects of NK cells in combination with DCs in the context of targeting malignant tumors in vivo, and to determine the murine cellular and humoral immune responses of tumor-bearing mice.

In the present study, we have explored the interaction between NK cells and DCs and assessed their potential applicaiton in the immunobiological treatment of malignant tumors.

 Materials and Methods



Ethics statement

The study was conducted in accordance with the guidelines of the Regulations for the Administration of Affairs Concerning Experimental Animals, and was approved by the Animal Ethical Committee of Harbin Medical University.

Animals and tumor cell lines

Healthy male C57BL/6 mice aged 8-12 weeks and obtained from Beijing Weitong Lihua Company were used for this study. B16F10 melanoma cells were bought from Shanghai Wu Li cell bank (Shanghai, China). YAC-1 cells were a gift from Prof. H. W. Xu (Harbin, China). Cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. B16F10 cells were used to induce lung metastasis by tail vein injection of 5 Χ 10 5 cells in 0.3 ml of RPMI 1640 into C57BL/6 mice, and to induce subcutaneous melanoma by subcutaneous injection of the same cells.

In vitro induction, proliferation, and marking of NK cells

NK cells were prepared as previously described. [4] Briefly, spleens were removed aseptically from mice and a single-cell suspension was prepared. Erythrocytes were lysed at room temperature for 8 min and the spleen cells were subsequently washed twice in phosphate-buffered saline (PBS). Cells were transferred to plastic flasks and cultured at 37΀C in an atmosphere of 5% CO 2 in RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum (FCS) and 5% normal human serum. Also, 6000 IU/ml of recombinant human interleukin (IL)-2 (a gift from Prof. Kalinski, Pittsburgh, USA) was added. After 3 days of incubation, CD8-positive cells were magnetically removed with Dynabeads M4502CD8 (Dynal Biotech, Oslo, Norway). The CD8-depleted cells were resuspended in fresh medium containing 6000 IU/ml IL-2 and returned to culture flasks. Fresh medium containing 6000 IU/ml IL-2 was added every 2-3 days. After an additional 3 days of culture, non-adherent cells were decanted and the plastic-adherent cells were harvested. For some experiments, NK cells were labeled with 4,6-united amidine-2-phenylindole DAPI (4',6-diamidino-2-phenylindole) at 50 mg/ml (Sigma, St. Louis, MO, USA) to observe the infiltration in the tumor-bearing mice before injection.

In vitro induction, proliferation, and marking of DCs

DCs were generated from C57BL/6 mice. as previously described. [5] Bone marrow cells obtained from the femurs and tibias of mice were incubated at 5 Χ 10 5 cells per well in 6-well plates at 37΀C in an atmosphere of 5% CO 2 in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 5 10 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, United states), and IL-4 (Peprotech). On day 3, non-adherent cells were discarded and fresh DC culture medium was given. On day 7, non-adherent cells were harvested. For some experiments, DCs were labeled with 5-bromodeoxyuridine (BrdU; Sigma) to observe the infiltration in the tumor-bearing mice before injection.

Different doses of cytokine and DCs administered by tail vein injection to subcutaneous melanoma mice

5 Χ 10 6 NK cells marked by DAPI were administered by tail vein injection to subcutaneous melanoma mice. Then, IL-2 at a dose of 5000 U, 10,000 U, and 50,000 U was administered by intraperitoneal injection, and IL-15 at a dose of 0.2 μg, 1 μg, and 2 μg was administered by tail vein injection. 5 Χ 10 5 , 5 Χ 10 6 , 5 Χ 10 7 DCs, respectively, were administered by tail vein injection. Mice from each group were sacrificed 24 h after injection to obtain the tumor tissue, followed by fixation and freezing the sections. NK cell infiltration was observed by fluorescence microscopy.

Killing activities of NK cells by MTT assay

B16F10 cells were targeted with the following four groups: NK cells alone (control group), NK cells plus IL-2 (1000 IU/ml) group, NK cells plus IL-15 (10 ug/ml) group, and NK cells plus DCs (in the absence of a cytokine biological response modifier) group, wherein the ratio of NK cells to DCs was 10:1, 1:1, and 1:10, respectively. The effector to target cell ratio was 5:1, 10:1, and 20:1, respectively. Effector cell control well(E), the targeting cell control well (T), and the reaction well (E + T) were set with three wells for each group. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)was dissolved in PBS, at a concentration of 5 mg/ml, and a volume of 10 ul MTT was added into each well. After an incubation of 4h, the medium in each well was aspritated as completely as possible without disturbing the cells. A 100 ul volume of dimethyl sulfoxide (DMSO) as a solvent was added to each well. The plates was agitated on a shaker for 10min, and then the optical density (OD) read on an Enzyme-linked Immuno sorbent Assay (ELISA) reader at 570 nm (Model DG-3022, Nanjing Vacuum Tube Manufacturer, China). The mean OD value calculated with three wells was used in the following formula.

killing activity of NK cells (%) = [1 - (OD E + T − OD E )/OD T ]Χ100%

Toxicity determination of NK cells regulated by DCs which are activated by necrotic tumor cells

NK cell cytotoxicity was examined using a CytoTo Χ 96; non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin,USA, BD Pharmingen, San Diego, USA). Briefly, the combined suspension of DCs and NK cells at a ratio of 1:5 or NK cells alone or DCs alone was used as effector cells for cytotoxicity assays. The target cells, namely B16F10 and YAC-1, were suspended in 10 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum and then plated in 96-well plates at 1 Χ 10 5 cells/100 μl per well in triplicate. Then, the effector cells were added to the 96-well plate at different E: T ratios and incubated for 4 h. Fifty microliters of the supernatant was transferred to a new 96-well plate, and then 50 μl of reconstituted Substrate Mix was added to each well and incubated for 30 min. Subsequently, 50 μl of Stop Solution was added, and after another 30 min, the absorbance was measured at wavelength 490 nm. The percentage of specific lysis was calculated according to the following equation: % cytotoxicity = (experimental − effector spontaneous − target spontaneous)/(target maximum − target spontaneous) Χ 100.

NK cells and DCs distribution in vivo following combined infusion

Sixty subcutaneous melanoma established mice were divided into three groups: NK plus DCs group (NK: DCs ratio was 1 Χ 10 7 : 5 Χ 10 7 ), NK cells group (1 Χ 10 7 ), and DCs group (5 Χ 10 7 , plus addition of 50 ng rmGMS-CSF (recombinant mouse granulocyte/macrophage-colony stimulating factor)), and administered by tail vein injection. Groups containing NK cells were subcutaneously injected with rhIL-2 (recombinant human interleukin-2) of 200,000 IU, 6 h before injection and at 0 h, 6 h, and 12 h after injection. Each group of 20 mice was randomly divided into four subgroups; mice from each of the subgroups were sacrificed 1 h, 2 h, 4 h, and 12 h after injection to obtain the tumor tissue, lung, liver, and spleen specimens, followed by fixation and paraffin-embedded sectioning of specimens. Staining was performed by the streptavidin-biotin complex (SABC) immunohistochemical method. Then, the distribution of DCs and NK cells was observed under the fluorescence microscope, and pathological changes were observed under the microscope after hematoxylin-eosin (HE) staining of the specimens.

Influence of a combined transfer of NK cells and DCs on CD8+T cells and lung metastasis nodules

Mice with lung metastases were divided into four groups: NK plus DCs group (NK:DCs ration was 1Χ107 : 5Χ107), NK cell group (1Χ107), DCs group (5Χ107, plus addition of 50 ng rmGM-CSF), and PBS control group. And NK for NK cell group, DC for DC cell group, both cells for NK plus DCs group,and PBS for control group were administered respectively by tail vein injection. Some mice from NK plus DCs group and PBS group were sacrificed on day 5 to obtain the lung tissues from which single-cell suspensions were prepared (five animals per group). Lung tissues were digested three times by shaking for 30 min at 37΀C in RPMI 1640 medium containing 1 mg/ml collagenase VII (Sigma) and 2% FBS. Lung cells were passed through a 70-μm nylon filter, erythrocytes were lysed, and the total number of cells was assessed. The lung cells were incubated with antibodies, CD3-PE (145-2C11, BD Pharmingen, San Diego, USA) and CD8-FITC (53-6.7, BD Pharmingen). One million lung cells were washed once in PBS and resuspended in 100 μl of PBS, and then incubated with various conjugated monoclonal antibodies for 30 min at 4΀C. After washing twice in PBS, the cells were analyzed on FACSCalibur (BD Biosciences, San Jose, CA, USA).

Mice from each of the subgroups (five animals per group) were sacrificed on day 14 to obtain the lung tissues that were then fixed, following which the number of lung metastatic nodules was determined under a surgical dissecting microscope.

Additionally, 14 days after the establishment of lung metastases in C57BL/6 mice, intravenous inoculation of 0.3 ml of DC and NK cell suspensions or PBS (five animals per group) was performed via the lateral tail vein. To test for long-lasting protective immune responses, tumor-bearing mice (1 Χ 10 5 B16F10 cells/mouse) were treated with intravascular injection of DCs and NK cells on days 14 and 21. After 60 days, mice that rejected melanoma cells were further challenged with B16F10 cells via tail vein (5 Χ 10 5 cells/mouse).

Statistics analysis

Data were expressed as Mean ± SD. Statistical analysis was performed using the Student's t-test. For the antitumor activity of NK cells ex vivo, the q-test was used. Results were considered statistically significant at P < 0.05.

 Results



DCs maintain and promote NK cells activity

Infiltration of NK cells in tumor tissue induced by IL-2 might vary according to the dose of IL-2 or IL-15 co-administered. With increasing doses of IL-2 or IL-15 injected, the number of NK cells infiltrating the tumor tissues increased. The number of infiltrating NK cells might also significantly increase by the action of DCs. When the ratio of DCs and NK cells was 1:10, the number of infiltrating NK cells was approximately 75/mm 2 . In addition, when the ratio of DCs and NK cells was 1:1, the number of infiltrating NK cells increased. However, further increasing the number of adoptively transferred DCs did not coordinately increase infiltration of the tumor site by NK cells. When the ratio of DCs and NK cells was 10:1, the number of infiltrating NK cells did not significantly increase [Figure 1]. Our study thus suggested that co-culture of NK cells and DCs might maintain and promote NK cell activity without the requirement for exogenous cytokines.{Figure 1}

Under conditions when the effector to target cell ratio was 20:1, the killing activity of NK cells from the experimental groups was higher than that of the control group. When the ratio of NK cells and DCs was 1:1, the killing activity was significantly increased as compared with a ratio of 10:1 (P < 0.05). However, further increasing the dose of DCs did not significantly enhance the killing activity of NK cells. Also, when the NK cell and DCs ratio was 1:1, the killing activity of NK cells increased significantly as compared with that found for the IL-2 and IL-15 groups. However, we did not confirm any statistical significance (P > 0.05) [Table 1].{Table 1}

DCs activated or not with necrotic tumor cells were incubated with NK cells, and subsequently NK cell activity was tested against target cells. As shown in [Figure 2], DCs activated by necrotic tumor cells induced a higher cytotoxic activity of NK cells against YAC-1 and B16F10 than the DCs that are not activated by necrotic tumor cells. The activity of NK cells against syngeneic splenocytes was not affected by activated or unactivated DCs (data not shown). These data indicate that DCs activated by necrotic tumor cells have an effect on NK cells and promote their cytotoxicity.{Figure 2}

Aggregation of NK cells and DCs about tumor tissues following combined adoptive transfer and their tumoricidal activity against tumor cells

NK cells and DCs were distributed in vascular-rich regions of the tumor tissue and could escape from the microcirculation to infiltrate the tumor parenchyma after infusion to subcutaneous melanoma mice [Figure 3] and [Figure 4]. In addition, there was evidence of NK cell infiltration in the spleen sections of mice from groups 1 and 2. There was also evidence of rare NK cell distribution in lung and liver tissues. These data indicate that both NK cells and DCs were initially distributed in the microcirculation of tumor tissues after intravenous injection, following which some cells escaped from the peripheral blood vessels and infiltrated the tumor parenchyma.{Figure 3}{Figure 4}

Infiltration of effector cells into tumor tissues reached its maximum level 4 h after adoptive transfer and decreased to its lowest level approximately 12 h after injection. There were also significant differences in the infiltration numbers of NK cells and DCs at each time point measured (P < 0.01). There were also significant differences among different NK cells and DCs adoptive treatment groups, which was dependent on the various NK-DC mixing ratios (P < 0.01). Effector cells from the mixed cell adoptive transfer group achieved greater infiltration into tumor tissues than that seen for the single adoptive transfer group [Table 2].{Table 2}

Host immune response after combined adoptive transfer of DCs and NK cells

To analyze the effect of co-injected DCs and NK cells on lung metastasis of B16F10 in C57BL/6 mice, preparations of isolated cells were transferred through tail vein into tumor-bearing mice. After 2 weeks, the mice were sacrificed and the number of metastatic nodules in the lungs was assessed. It was observed that lungs of mice treated with both DCs and NK cells contained fewer metastatic nodules than the control mice [Figure 5].{Figure 5}

To identify the immune cells responsible for the therapeutic effect, we analyzed the size of the CD8+T cell population in the lung. As compared to the control group, there was an increased number of CD8+T cells observed in the lungs of the experimental mice 5 days after intravenous transfer of DCs and NK cells [Figure 6]. The mean number of CD8+T cells present in the lungs of experimental mice increased from 16.13 ± 2.51% to 39.5 ± 1.57%. This result suggests that the reduced metastasis may be associated with the effect of CD8 + T cell population in the lung.{Figure 6}

Adoptively transferred DCs and NK cells offered protection against B16F10 challenge. To prove whether this treatment would induce a long-lasting immunologic memory, mice bearing B16F10 tumors were treated with two simultaneous intravascular injections of DCs and NK cells on days 1 and 8. After 60 days, the surviving mice were challenged with living B16F10 cells. The mice treated with DCs and NK cells had a prolonged survival [Figure 7]. It suggests that the strategy of using DCs and NK cells through an intravascular way may cure mice with lung metastasis.{Figure 7}

 Discussion



Adoptive cellular immunotherapy is a relatively common biological treatment option for malignant tumor, which also displays a wide antitumor spectrum and almost achieves certain therapeutic effects against many different tumors. In addition, cellular immunotherapy alleviates the side effects associated with traditional radiotherapy and chemotherapy.

Currently, adoptive cellular immunotherapy mainly includes cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, DCs and some other approaches. Therapeutic effects and technology of this biological treatment need to be improved. We found that there was a complicated and unique interaction between DCs and NK cells, and this interaction played a vital role in the tumor immune process.

Some studies have revealed that DCs and NK cells might interact to generate a secondary immune response. The contact of NK cells with mature DCs results in activation of these two cells and production of inflammatory cytokines, including NK cell proliferation and DCs maturation. [6],[7],[8] DCs might secrete cytokines such as IL-12, IL-1, IL-15, IL-18, etc., The secretion of IL-12, for example, can increase NK cell-mediated cytotoxicity and interferon (IFN)-g production, whereas IL-15 can improve the survival and differentiation of NK cells. [9]

DCs can also enhance the expression of antigen-specific T cells, and IL-2 secretion by T cells might also activate NK cells. [10] Activated NK cells are capable of driving adaptive immunity by inducing the producing of IFN-g. [11] NK cells can also activate and specify DCs' functional behaviors, which are then able to induce T helper cell type 1 (Th1)- type immunity. Antigen exposure could trigger the memory of Th1, [6],[12],[13] which can provoke the differentiation of DCs and stimulation of CD8+cytotoxic T lymphocytes which are more effective as antitumor effectors. In addition, secretion of GM-CSF by NK cells might enhance the survival of DCs and the differentiation of monocytes into DCs.

Previous studies have suggested that survival and activation of NK cells depend on cytokines. Our study revealed that DCs could promote the killing ability of NK cells without the requirement for exogenous cytokines. Within 24 h, NK cells were transferred into tumor tissues to kill tumor cells, and their effects were similar to that of cytokines. Ferlazzo et al. [12] cultured DCs with NK cells derived from peripheral blood for 7 days and found proliferation of NK cells and secretion of IFN-g. Co-culture of DCs and NK cells activated by IL-2 may achieve similar results. Therefore, co-culture of NK cells and DCs could maintain and promote the activity of NK cells without depending on the addition of exogenous cytokines.

Co-culture of NK cells and DCs in different proportions achieved quite different outcomes. Our study revealed that a low ratio of NK cells and DCs might enhance the functions of DCs, whereas high levels of NK cells may inhibit the effects of their interaction. IFN-g secreted by NK cells might promote the maturation of DCs. By contrast, NK cells might kill immature DC (iDC), [14] a process that might be regulated by the cell surface NKp30 receptor. Therefore, appropriate co-culture frequencies of NK cells and DCs were able to produce the most effective immune effects.

Other studies have found that DCs sensitized by tumor cells and then co-cultured with NK cells could enhance the secretion of IFN-g, as compared with untreated DCs. [15] IFN-g plays a vital role in the antitumor response. Pre-inoculation of DCs sensitized by tumor cells might activate NK cells to trigger CTL responses and inhibit the metastasis of tumor cells. When pre-sensitized DCs were inoculated into tumor-bearing mice whose NK cells had been removed, metastasis of the tumor cells was not inhibited. Therefore, NK cells were shown to be essential for the antitumor immune response caused by DCs. Our study also confirmed that NK cells in combination with DCs that were initially sensitized by necrotic tumor cells might significantly enhance the immunological activity of NK cells and eventually improve their antitumor killing ability.

Many studies have demonstrated that DCs and lymphocytes infiltrate tumor tissues. However, cell number was significantly inadequate, and the number of DCs and lymphocytes infiltrating the tumor mass was associated with the patient's prognosis. Owing to the awareness that NK cells and DCs played a vital role in the antitumor immune responses, our study adopted the optimal proportion of NK cells and DCs (which was found to be 1:5) in accordance with the in vitro results, so that we could complete combined adoptive therapy. [6] Based on the same cell number, infiltration of NK cells and DCs in the tumor tissues was higher in the mixed injection group as compared with of the injection group, which was contributed by the interaction of NK cells and DCs. Our study revealed that interaction of NK cells and DCs might promote the gathering of NK cells around tumor tissue sites to exert their antitumor effects. Moreover, cell debris from tumor cells targeted for destruction by NK cells could be taken up by DCs, where subsequently the antigenic payload and information could be cross-presented to the T cells to mount a secondary immune response against the tumor.

Intravenously injected effector cells were found to be distributed outside the tumor tissues following the circulatory system. However, these cells were still mostly distributed in tumor tissues, which could confirm that NK cells and DCs might gather around the tumor tissue voluntarily to therapeutically target tumor cells.

In addition, Basse et al.[4],[16] performed adoptive immunotherapy by NK cells to treat lung metastases in mice, which confirmed that NK cells might gather and infiltrate metastatic tumors of the lung and liver, and did so even inside the tumor tissue. In our study, we used a subcutaneous tumor mouse model to observe the distribution of NK cells and DCs. One objective of this study was to eliminate cell distribution in certain tissues (such as in the lung and liver) due to the blood circulation and then to histologically characterize and demonstrate that NK cells and DCs had distinctive targeting abilities.

The mechanism by which NK cells and DCs gathered around tumor tissues remains unknown. However, we hypothesized the following possibilities. Firstly, NK cells might exert their effects by the same pathway as a secondary immune cell signaling response. Genomic analysis and immunological detection methods have clearly demonstrated that the receptor Lg-49H was activated by NK cells, and was associated with DAP 12 molecules containing ITAM, an interaction which might mediate transduction of the activation signal. [17] Secondly, inflammatory cells, tumor invasion cells, and stromal cells in the tumor microenvironment could produce various inflammatory cytokines and inflammatory mediators such as heat shock proteins. The presence of heat shock proteins can not only stimulate specific immune responses but also interact with antigen presenting cells to trigger non-specific immune responses. After adoptive immunity, the antitumor killing ability of NK cells, coupled with their interaction with DCs, it is formally possible that NK cells might enhance local inflammatory reactions and thus induce local accumulation and retention of more NK cells and DCs at the local sites of the tumor. Thirdly, many tumor cells can secrete and express chemokines and their receptors. Chemokines, for instance, play decisive roles in the migration of immune cells to the sites of inflammation and indeed tumor metastases.

In the process of the antitumor immune response, tumor cells receive and transmit various extracellular signals from their interaction with NK cells and DCs to elicit a corresponding response. This was in fact one of the most important activities in the antitumor immune response. Based on our study, we propose that it is feasible to combine NK cells with DCs in order to perform a biologically meaningful adoptive transfer strategy in the treatment of malignant tumors. This notion is based on the observations that NK cells interact with DCs, and that the therapeutic effects of this combined therapy might offer greater superiority than either NK cells or DCs alone. Moreover, observations made from the in vivo interaction of NK cells with DCs, coupled with considering whether long-term combined adoptive immunotherapy can offer improved therapeutic gains clearly require further study.

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