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[Cancer Research 61, 3240-3244, April 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Tumor Cells Regulate the Lytic Activity of Tumor-specific Cytotoxic T Lymphocytes by Modulating the Inhibitory Natural Killer Receptor Function1

Asma Gati, Nadia Guerra, Julien Giron-Michel, Bruno Azzarone, Eric Angevin, Alessandro Moretta, Salem Chouaib and Anne Caignard2

INSERM U487, Institut Fédératif de Recherche (IFR 54) [A. G., N. G., S. C., A. C.], Unité d’Immunologie Clinique [E. A.], Institut Gustave Roussy, 94805 Villejuif, France; INSERM U508, Hôpital Paul Brousse, 94805 Villejuif, France [J. G-M., B. A.]; and Dipartimento di Medicina Sperimentale, Universita di Genova, 16132 Genova, Italy [A. M.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Tumor-infiltrating p58+ T cells from a renal tumor were specifically expanded in response to tumor cell stimulation and cloned. These p58+ T cells were found to express a memory phenotype and corresponded to clonal TCRBV3 T-cell expansion. Functionally, p58+ CTLs displayed a low lytic activity for HLA-A2 tumor and normal cells. However, this lytic activity was significantly increased after blockade of p58 with specific monoclonal antibodies. Interestingly, we demonstrated that stimulation by tumor cells was required to trigger the inhibitory effect of p58 on the lytic activity of antigen-specific CTLs and that stimulation of the inhibitory function of p58 by tumor cells correlated with an inhibition of nuclear factor-{kappa}B activation in p58+ tumor-specific CTLs.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
RCC,3 like melanoma, is considered to be a potentially immunogeneic tumor. In this context, durable tumoral responses were obtained after IL-2 treatment in 15–20% of metastatic patients, some of them resulting from elicitation of T-cell responses (1) . However, despite the presence of a large CD3+ infiltrate, it is difficult to generate stable tumor-specific cytotoxic T cells from RCCs, and the rare specific CTLs described in this model displayed functional alterations that resulted in marginal lytic potential as well as low proliferation efficiency (2) . In an attempt to understand the alteration of in situ amplified T-cell proliferative and cytotoxic responses in vitro, we showed previously that expression of inhibitory NK-Rs may contribute to the local immune suppression detected in RCC (3) . The identification of the NK-R family specific for HLA-I molecules and the characterization of these related inhibitory (KIR) and activatory (KAR) receptors allowed a better understanding of the mechanisms regulating target cell recognition as well as NK and T-cell activation (4) . KIRs have also been detected on peripheral T lymphocytes from healthy donors with a selective expression on minor T-cell subsets, mostly of the CD8+, CD28-, CD45RO+ memory phenotype (5) . It is established that KIR engagement also leads to inhibition of T-cell functions including TCR-mediated cytolytic activity and lymphokine production (6 , 7) . We previously saw evidence for the expression of particular KIRs (p58 receptors specific for HLA-C molecules) by significant subsets of TILs in most RCCs. Among the tumors studied, we have characterized these particular KIR+ T cells as tumor-specific T cells, the activity of which is inhibited by the NK-R (3) . In the present study, we identified a subpopulation of p58+ T cells detectable at the tumor site and that corresponded to potentially autoreactive T cells. Interestingly, we further show here that stimulation by tumor cells is a prerequisite for the functional inhibitory effect of the KIR. Tumor cells presumably induce the recruitment of KIR+ T cells reactive to self antigens at the tumor site and control their lytic activity by regulating the KIR inhibitory function as well. The present studies point to a particular regulation of the KIR functionality expressed on antigen-specific p58+ T cells by autologous renal carcinoma tumor cells and reveal a new mechanism of tumor escape to the host T cell-specific immune response.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Establishment and Culture of Tumor Cell Lines.
The autologous tumor cell lines were derived from the primary tumor as described previously (8) . Briefly, total cell suspensions were centrifuged onto Ficoll Hypaque to eliminate dead cells, and viable cells were cultured in specific culture medium to amplify tumor cells and TILs. For tumor cells, DMEM/HAM F-12 supplemented with 1% glutamine, 10% FCS, and 2% Ultroser G (Life Technologies, Inc., Paisley, Scotland) was used as complete medium. A permanent tumor cell line was derived from patient VM that was genotyped as HLA-A2, A26, B04, B44, Cw05, Cw-. Confluent culture dishes were treated by EDTA/trypsin, and cells were subcultured.

Immunofluorescence Analysis.
The phenotypes of TILs and PBLs were analyzed by indirect two-color fluorescence. Cells (2 x 105) were first incubated for 30 min at 4°C with conjugated mAbs: EB6-PE (anti-p58.1, IgG1), GL183-PE (anti-p58.2, IgG1), CH92-PE (anti-BV3TCR, IgM), CD3-FITC, CD56-PECy5, and CD8-FITC purchased from Immunotech (Marseille, France). Analysis was performed on 5000 gated TILs (appearing in forward scatter/side scatter as small lymphocytes comparable with PBLs) after exclusion of dead cells and debris. Background levels were measured using isotypic controls. Analysis was done on a FACS-Sort (Becton Dickinson, Pont de Claix, France) using Cell Quest software (Becton Dickinson). Compensation was set up with single stained samples.

Derivation of p58+, p58- T-Cell Lines and CTLs.
TILs were stimulated for 2 weeks in MLTC in the presence of 30 units/ml IL-2 before positive selection of p58+ T cells using anti-p58.2 mAb (GL183) and immunomagnetic beads (Dynal, Campiégne, France). After immunoselection, p58+ and p58- T-cell subsets were expanded (3 x 103 cells/well) on irradiated allogeneic feeder cells (7 x 104 peripheral blood mononuclear cells + 3 x 104 LAZ 509 cells/well) and of irradiated autologous tumor cells (2 x 103 cells/well). The p58+ T-cell line was cloned by limiting dilution 0.3–1 cell/well using the above feeder cell conditions. For regulation of KIR function, CTLs were cultured on irradiated LAZ509 cells (HLA-A2, Cw6, Cw12) alone (3 x 104 cells/well) or in presence of 3 x 103 tumor cells/well for 6–8 days.

Assay for Cytolytic Activity.
The cytolytic activity of T-cell lines and clones was measured in a 4-h 51Cr release assay, against autologous PHA blasts, autologous tumor cells (VM), allogeneic tumor cell lines (LR, DM, GE, Caje, and CS), and EBV-transformed B cells (LAZ509 and LCL GE) as described previously (8) . In some experiments, mAbs 11PB6 and EB6 (anti-p58.1), GL183 (anti-p58.2), B1.23.2 (anti-HLA-B/C, IgG2a), Z27 (anti-p70, IgG1), and UCHT1 (anti-CD3, IgG1) were added at predefined saturating concentrations at the onset of the cytolytic assay. Data are expressed as the percentage of specific lysis at the indicated E:T cell ratio.

The cytolytic activity of T-cell lines was also assessed in a CD3-redirected lysis assay using P815 mastocytoma mouse cells. Briefly, 51Cr-labeled P815 (3 x 103) cells coated with anti-CD3{epsilon} (1–100 ng/ml) were incubated with serial dilutions of T cells (E:T ratio ranging from 10:1 to 2:1). CD3-redirected lysis of labeled P815 cells was modulated by the presence of the anti-KIR mAbs (4 µg/well). Results obtained in independent experiments were statistically analyzed using the Fisher test.

TCRBV Gene Segment Usage and Sequencing of TCR Expressed by CTLs.
TCRBV gene segment usage was determined using a semiquantitative PCR analysis as described previously (9) . Briefly, total RNA was extracted from T-cell lines (5 x 106 cells), using a modified guanidinium thiocyanate phenol/chloroform method (Trizol; Eurobio, Les Ulis, France). Amplification of cDNA was performed over 30 cycles with the 5' sense primers specific for the 24 TCRBV subfamilies and one 3' antisense TCRBC-specific primer, and amplimers were detected after electrophoresis on agarose gel. TCRBV3-BC PCR products derived from p58+ CTLs were purified using the Qiagene columns (Qiaquick PCR purification kit; Hilden, Germany) and sequenced using ABI Prism Big Dye sequencing Ready Reactions kits before electrophoresis on an ABI sequencer (Applied Biosystem, Foster City, CA).

Confocal Microscopy Analysis.
Transcription factor NF-{kappa}B was detected using 1 µg/ml of the rabbit polyclonal purified IgG anti-NF-{kappa}B p65 for 30 min at 4°C (Santa Cruz Biotechnology), followed by incubation with Alexa Fluor 488 GAR (Molecular Probes). Nuclei were stained using propidium iodide (red staining). Stained cells were washed with PBS, cytocentrifuged in a cytospin 3 (Shandon, Pittsburg, PA), and analyzed by laser scanning confocal microscopy using a Leica TCS Confocal System (Wetzler, Germany).


   Results
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Clonal p58+ T Cells Expressing a Unique TCR Are Detected at the Tumor Site and Successfully Expanded in Vitro in MLTC.
Analysis of TILs and PBLs from RCC patients was performed by double color immunofluorescence using conjugated anti-p58 and CD3 mAbs to measure expression of p58 on NK and T cells. From dissociated tumor, p58+ T cells represented 9% of the CD3+ T cells, whereas CD56+/CD3- NK cells constituted 9% of the TIL population, and 2.5% were p58+ (Fig. 1A)Citation . NK receptor p58 was expressed by 5% of the CD3+ T cells derived from PBLs (Fig. 1A)Citation . Among the 7% of blood CD56+/CD3- NK cells, 2% coexpressed a p58 receptor.



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  		Fig. 1. A, detection of p58+ NK and T cells in TILs and PBLs in vivo (T0) and after in vitro stimulation of TILs and PBLs by autologous tumor cells (MLTC). B, phenotype of p58+ T-cell line and CTLs. C, detection of p58+ TCRBV3 T cells in situ.

 
After stimulation by tumor cells in MLTC in the presence of low doses of IL-2, TILs proliferated (two to four times) mainly from the CD8 subset, and the percentage of p58+ cells was maintained reaching 10% of the CD3+ T cells (Fig. 1A)Citation . In contrast, PBLs stimulated by autologous tumor cells did not proliferate and remained in a non-activated state with a significant loss of CD56 expression and a subsequent decrease in p58+/CD3+ T-cell subset (Fig. 1A)Citation . To further characterize p58+/CD3+ T cells amplified during MLTC from TILs, p58+ cells were immunoselected and expanded in vitro. As described previously, these cells express CD56 and CD45RO but not CD28. In addition, p58+ T cells displayed a very restricted TCR repertoire expressing only TCRBV3 (Fig. 1B)Citation . Using specific mAbs, double color immunofluorescence analysis was performed to visualize antigen-specific p58+ T cells in situ. In tumor, TCRBV3+ p58+ T cells corresponded to 1% of the mononuclear gated cells but were not detected in PBLs (Fig. 1C)Citation . Analysis by reverse transcription-PCR of TCR repertoire using a panel of 24 BV-specific primers confirmed the presence of unique BV3 transcripts in p58+ T cells, whereas the p58- T-cell line displayed a polyclonal TCR repertoire (data not shown).

p58 Receptor Inhibits the CD3-mediated Lysis of CTL Clones.
To investigate whether the p58 receptor interferes with the lytic activity of CTL clones, blocking experiments with specific mAbs were performed. As depicted in Fig. 2ACitation , after immunoselection, both p58+ and p58- T-cell subsets were found to display low levels of cytotoxic activity against autologous tumor cells. However, when anti-HLA-BC (not recognizing HLA-A molecules) or anti-p58 mAbs were added during the effector phase, a dramatic increase in the lysis of tumor cells by p58+ T cells but not by p58- T cells was observed. Using limiting dilution, from the selected p58+ T-cell line, we derived 12 proliferative T-cell clones displaying a similar lytic potential. They all expressed TCRBV3 chain, p58.1+ and p58.2+ receptors. Sequencing of TCRBV3 chain in three TCRBV3 CTL clones revealed an identical TCRBV3/BJ 1.2 rearrangement, indicating that they derived from the same clone, and CTL 2H7 was further used as a representative clone.



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  		Fig. 2. A, functional analysis of p58+ and p58- T-cell lines. Cytotoxic activity of T-cell lines against autologous tumor cells was tested in a standard chromium release assay at the indicated ratio of E:T cells. Inhibition of the lytic activity was tested after incubation of the cells for 30 min with the indicated mAbs before the test. B, lytic activity of p58+ CTL 2H7 against tumor cells (E:T ratio, 10:1 and 5:1) in the absence and in the presence of anti-p58.1 and anti HLA-BC mAbs. C, CD3-redirected lysis of P815 cells induced by CTL 2H7 was generated by anti-CD3{epsilon} (10 ng/ml) in the presence of anti-KIR mAbs.

 
Although both p58.1 and p58.2 receptors were expressed by p58+ CTLs, tumor cells (HLA-Cw05, Cw-) were found to only express the HLA-Cw4 supertype, the ligand for p58.1 receptor. Accordingly, the lytic activity of CTL 2H7 against autologous tumor cells was inhibited by anti-p58.1 or anti HLA-B/C mAbs added during the effector phase (Fig. 2B)Citation .

The functionality of p58 receptors was also assessed by CD3-redirected lysis of P815 murine cells. These assays revealed that p58 CTLs displayed a high lytic potential because limited quantities of anti-CD3 immunoglobulin (1–10 ng/ml) were able to activate the lytic machinery. Engagement of p58.1 or p58.2 receptor by specific mAbs inhibited the CD3-redirected lysis of mouse FcR+ P815 cells by CTL 2H7, indicating that both p58.1 and p58.2 receptors were functional and inhibitory (Fig. 2C)Citation . It is noteworthy that these tests were performed using serial concentrations of anti-CD3 mAb to obtain optimal triggering of CD3, which could be modulated by the KIR receptor. This also indicates that the outcome of the lysis results most likely from the existence of a balance between positive (CD3) and negative (KIR) signals.

p58+ T Cells Kill Both Normal Autologous Tumor Cells and HLA-A2+ Allogeneic Tumor Targets.
The lytic activity of CTL 2H7 was assessed against different normal and tumor cell lines by cytotoxicity assays performed in presence of mAbs to block p58 receptor. Data shown in Table 1Citation indicate that CTL 2H7 efficiently killed autologous normal PHA blasts and three of four HLA-A2 renal tumor cell lines, whereas one HLA-A2- was not recognized. It also induced the lysis of HLA-A2+ B-cell lines and one T lymphoma with high efficiency. Differences in lysis of HLA-A2+ tumor cells may reflect their different expression levels of HLA-A2 and HLA-C alleles that control, respectively, the positive and negative signals. These results suggest that CTL 2H7 recognizes a ubiquitous antigen, probably presented by the HLA-A2 molecule, and most likely corresponds to an autoreactive T-cell clone functionally regulated by a powerful p58 inhibitory receptor.


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  		Table 1 Cytotoxic activity of p58+ CTL 2H7 against autologous targets and allogeneic normal and tumor cell lines

 
Tumor Cells Trigger p58 Inhibitory Function and Inhibit NF-{kappa}B Activation in Tumor-specific CTLs.
To better understand the local in vivo expansion of these particular CD3+/p58+ T cells, we analyzed the effect of tumor cells on the activation of p58+ antigen-specific CTLs. We showed that stimulation of CTL 2H7 with irradiated stimulating LAZ509 cells (efficiently recognized by CTL 2H7) alone does not interfere with the lytic activity of this clone in the presence or in the absence of anti-p58.1 or anti-BC mAbs. Interestingly, the addition of tumor cells (1 of 10) to LAZ509 turned on the inhibitory function of KIR and resulted in an abrogation of the basal lytic activity of CTL 2H7 (Fig. 3A)Citation . In five independent CD3-redirected lysis experiments, anti-p58.1 mAb did not significantly affect the lysis of CD3-coated P815 cells by LAZ509-stimulated CTLs (P > 0.05), whereas it significantly decreased their lysis by CTLs stimulated in the presence of tumor cells (P = 0.0042) as assessed by Fisher test (Fig. 3B)Citation . It should also be noted that phenotypic analysis of CTLs stimulated under these distinct conditions was similar with respect to the expression of p58 receptors (similar mean fluorescence intensity), suggesting that the effect of the tumor cells does not involve p58 receptor expression but rather its function.



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  		Fig. 3. Modulation of the KIR functionality expressed by CTL 2H7 upon stimulation by tumor cells. CTL 2H7 was stimulated for 6 days in the presence of LAZ 509 (LAZ) or LAZ509 and autologous tumor cells (10:1; LAZ+TUM) in the presence of 100 units/ml IL-2 (one representative experiment of five). A, lytic activity against autologous tumor cells. Bars, SE. B, CD3-redirected lysis of P815 mouse cells in presence of anti-CD3. Bars, SE. C, activation pattern of NF-{kappa}B in CTL 2H7 stimulated by LAZ509 (LAZ) or LAZ509 + VM tumor cells (LAZ+TUM).

 
Because it has been suggested that the alteration of TIL function in RCC correlated with the inhibition of NF-{kappa}B activation (10) , we therefore asked whether under our experimental condition, a correlation exists between KIR inhibitory function and NF-{kappa}B activation. Interestingly, we observed a dramatic decrease of NF-{kappa}B in CTLs after stimulation in the presence of tumor cells that paralleled the inhibitory function of the p58 receptor. Using confocal microscopy, we show the nuclear localization of NF-{kappa}B p65, detected by a yellow nuclear staining in most CTLs stimulated by LAZ509 only, whereas CTLs stimulated by tumor cells exhibited a green cytoplasmic and a red nuclear staining, indicating the absence of nuclear translocation (Fig. 3C)Citation . In addition, cytotoxicity assays indicate that CTLs cultured in both conditions display similar levels of CD3-redirected lysis of P815 in the absence of p58 triggering (Fig. 3B)Citation . Thus, tumor cell stimulation of p58+ CTLs induced a sustained p58-mediated inhibition of the lytic potential, and inactivation of NF-{kappa}B may constitute one possible mechanism to explain this observation.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Inhibitory NK receptors, first described on NK cells, have also been demonstrated on T cells. In normal individuals, small subsets of CD3+ KIR+ cells expressing a memory phenotype and an oligoclonal TCR repertoire are detected in the peripheral blood. These cells may be activated in response to chronically expressed antigens (11) , possibly involved in the maintenance of tolerance or prevention of autoimmunity. In the case of an immune response against tumor, overexpression of self antigens may induce the activation and expansion of these particular T cells at the tumor site (12) . A p58+ CTL that recognized a normal tissue-specific antigen was derived from the blood of a metastatic melanoma patient in response to tumor cells that had lost expression of a complete HLA haplotype (13) . In RCC, we previously saw evidence for the presence of CD3+ p58+ T cells infiltrating most renal tumors and demonstrated that CTL activity was inhibited by p58 receptors, although there was no major alteration of HLA-I molecule expression by the tumor cells (3) .

The present study suggests the presence of autoreactive T cells expressing an inhibitory p58 receptor infiltrating a renal tumor. These T cells display a memory phenotype CD45RO+, CD28-, CD56+, and correspond to clonal T cells. These cells were visualized by double color immunolabeling using anti-BV3 and anti-p58 mAbs in the uncultured TILs, where they represented 1% of CD3+ T cells but were not detected in peripheral T cells. This particular p58+ T-cell subset, specifically expanded from TILs in vitro, corresponds to the main tumor-specific cytotoxic T-cell population because p58- T cells mainly mediated HLA-unrestricted lysis. Thus, it may be hypothesized that the antigen presentation by tumor cells and tumor environment may play a role in the local in vivo expansion of the p58+ T cells, whereas in peripheral blood, these tumor-specific p58+ CTLs are efficiently regulated and not committed to proliferate.

Few, disparate data are yet available concerning the regulation of KIR expression and function on T cells, mainly because of the heterogeneity of the models studied, although recent studies demonstrated that some T cells acquire NK marker expression after activation (14 , 15) . Furthermore, it has been shown recently that TCR triggering restored the expression and function of KIR by human T cells (16) . In our model, antigen presentation by renal tumor cells does influence the fate of the KIR function but not its membrane expression on the antigen-specific T cells. Our data point out that p58 receptor expression does not necessarily correlate with anergic T cells but rather modulate T-cell signaling. In this regard, recently it has been reported that Ly49A receptors, the main inhibitory receptors on murine cells, enhance the amounts of activating signals required for effector cell responses and that inhibition of Ly49A may be overcome by increasing peptide concentration (17) .

Although much data have focused on the ability of NK cell inhibitory receptors to alter T-cell activation and cytotoxicity, recent studies using KIR transgenic mouse models proposed that NK-R expression by CTLs may also represent a checkpoint in T-cell activation after antigen stimulation (18 , 19) . These cells expressing KIR may present a selective advantage to avoid complete exhaustion by overstimulation and thus may correspond to the memory cell pool. Our data are consistent with both of these non-mutually exclusive hypotheses because although we demonstrated a significant inhibition of the lytic potential of CTLs by KIR in vitro, this receptor may play a role in the control of the proliferation or the survival of the corresponding T cells in vivo.

Interestingly, the sustained inhibitory function (KIR on) of p58+ T cells in the presence of tumor cells was correlated with an inhibition of NF-{kappa}B activation. An altered activation of the transcription factor NF-{kappa}B in TILs and PBLs from RCC patients, responsible for the functional alterations of these effectors (20, 21, 22) , was proposed as a general mechanism of immune suppression in these tumors. In addition, shedding of gangliosides from RCC cell lines and extracts able to block NF-{kappa}B nuclear translocation was described recently (23) . In the present study, NF-{kappa}B inactivation by renal tumor cells, probably independent of TCR stimulation, is induced in antigen-specific CTLs. Confocal analysis revealed a similar expression of granzyme B and perforin in the two KIR on/KIR off CTL subsets (data not shown), in accordance with the results of CD3-redirected lysis. This suggests that NF-{kappa}B may be involved in the control of the lytic potential of CTLs by a mechanism independent of these two cytotoxic gene transcriptions. A growing number of genes have been demonstrated to be induced by NF-{kappa}B, including cell adhesion molecules, cytokines, and growth factors (10) . Whether inhibition of NF-{kappa}B activation interferes with some structures involved with the cytotoxic effector phase in our model has to be investigated. Alternatively, it is known that KIR triggering induces the recruitment of protein tyrosine phosphatases, such as SHP-1 (24) , that may interfere with several kinases required for the phosphorylation of I{kappa}B{alpha} and thus blocks NF-{kappa}B activation. It would be of interest to explore the possible antagonism between SHP-1 and kinases involved in the control of I{kappa}B{alpha} phosphorylation. It is also essential to elucidate the relationship between inhibition of NF-{kappa}B and impairment of T-cell responses in KIR+ T cells to get more insights in the understanding of the function of NK-R in vivo.


   ACKNOWLEDGMENTS
 
We thank Yann Lécluse for immunofluorescence analyses, Dr. Ryad Tamouza for HLA genotyping, and Dr. Vladimir Lazar for help in sequencing.


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

1 This work was supported by grants awarded by INSERM and Grant ARC 9702 (to A. C.) from the Association for Cancer Research. Back

2 To whom requests for reprints should be addressed, at Unité INSERM U487 Cytokines et Immunologie des Tumeurs humaines, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, F-94805 Villejuif, France. Phone: 33/1 42-11-50-36; Fax: 33/1 42-11-52-88; E-mail: caignard{at}igr.fr Back

3 The abbreviations used are: RCC, renal cell carcinoma; IL, interleukin; NK, natural killer; NK-R, NK receptor; TIL, tumor-infiltrating lymphocyte; KIR, killer inhibitory receptor; PBL, peripheral blood lymphocyte; mAb, monoclonal antibody; MLTC, mixed lymphocyte tumor cell culture; TCR, T-cell receptor; NF-{kappa}B, nuclear factor-{kappa}B; PHA, phytohemagglutinin. Back

Received 1/21/01. Accepted 2/21/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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