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Development of Peptide-Selected CD8 T Cells in Fetal Thymic Organ Culture Occurs via the Conventional Pathway¹

Department of Laboratory Medicine and Pathology and the Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455

	Abstract

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Fetal thymic organ culture of TCR transgenic (Tg) tissue has been used to study issues of timing and specificity in T cell development. Because most TCR Tgs express a rearranged ß TCR on the cell surface at an earlier stage in development than normal mice, there is a possibility that the conclusions of studies using TCR Tg cultures may not apply to normal development. In particular, in our studies of peptide-induced development of CD8 T cells, it is possible that the peptide acts on the immature double-negative cell, driving development of CD8 T cells without passing through a double-positive stage. This issue was examined by asking whether MHC class I restriction was required and by analyzing CD8ß levels and endogenous TCR chain rearrangements. We found that if nonstimulatory peptides were used in fetal thymic organ culture, CD8 T cells developed via the conventional pathway, transiting through a double-positive stage. However, we could not rule out that cells selected in the presence of stimulatory peptides (agonists) did not develop directly from double-negative precursors.

	Introduction

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The development of ß T cells in the thymus proceeds from CD4, CD8 double-negative (DN)³ precursors to double-positive (DP) intermediates. At the DP stage, the cells are subject to positive and negative selection, which are events dictated by the specificity of the ß TCR (1). Further development and commitment to the CD4 or CD8 lineage occurs as a consequence of positive selection. Since positive selection has not been recapitulated with fidelity by in vitro assays, fetal thymic organ culture (FTOC) has been used to study the timing and specificity of positive selection (2). In particular, several groups have used FTOCs of animals expressing a single TCR to study the peptide specificity of positive selection of MHC class I-restricted CD8⁺ T cells. It was shown that both stimulatory (3, 4, 5) and nonstimulatory (6, 7) peptide ligands for the TCR are capable of inducing the development of CD8 single positive (SP) T cells in organ culture. However, most TCR transgenics (Tgs) express the rearranged ß receptor earlier in development than normal mice (8, 9). In normal mice, DN precursors express a pre-TCR comprised of a rearranged ß-chain paired with an invariant pre-T -chain at the cell surface (10). Because rearrangement of the TCR locus is initiated by signals through the pre-TCR, rearranged TCR chains do not appear on the cell surface until later in development, when the cell is doubly positive for CD4 and CD8. Therefore, in TCR Tg thymi in which peptide ligands are introduced during development, it is possible that the peptide is ligating the Tg TCR on the DN cell and directly causing differentiation to CD8 SP.

In normal mice, the lineage commitment to ß vs occurs at the DN stage (11). In TCR Tg mice, it has been theorized that the presence of an ß TCR at this stage influences the cell to differentiate down the pathway (12). Development of such cells occurs in the absence of a positive-selecting MHC (13) and presumably does not involve a DP intermediate. This development of cells with ß receptors in a lineage is clearly an artifact of TCR Tg animals and does not occur in normal mice. The fact that some T cells express CD8 (14) further supports the possibility that CD8 T cells which are induced by peptide in organ cultures may be developing via an nonconventional pathway and not via a DP intermediate like normal thymocytes.

To address this possibility, we identified several features of CD8 SP T cells that would be predicted to differ if the cells had differentiated via the conventional pathway (involving a DP intermediate) or via a nonconventional pathway (direct from DN cells). First, one might predict that the direct pathway would occur with both class I- or class II-restricted TCRs, while the conventional pathway would occur only with class I-restricted TCRs. This prediction is based on the assumption that class I- and class II-restricted receptors do not transduce qualitatively different signals in the absence of coreceptors. Second, CD8 SP cells that matured via a nonconventional pathway might have decreased biologic responses to Ag. This possibility is based on data from HY TCR Tg mice, where a population of CD8⁺ cells that developed directly from DN cells had an altered functional capacity (12, 13). Third, the isoform of CD8 expressed might be useful in discriminating the two pathways. T cells that express CD8 express exclusively CD8 homodimers, while conventional ß T cells express CD8ß heterodimers (14). Indeed, it was shown in one case that TCR activation caused DN T cells to express CD8 homodimers (15). If a nonconventional development of CD8 SP in TCR Tg mice involved commitment to the lineage by Ag exposure at the DN stage, then it seems possible that the CD8 cells would express CD8 homodimers. Finally, the status of rearrangement at the TCR locus is expected to differ between cells that have arisen by a conventional vs direct pathway. The TCR gene undergoes rearrangement predominantly at the DP stage (16). This is true for the endogenous TCR genes, even in TCR Tg mice, despite the fact that positive and negative selection trigger the down-regulation of the recombinase-activating genes (RAGs) (17). Cells that did not transit through a DP intermediate would not have rearrangements at the TCR locus. Thus, the presence or absence of endogenous -chain rearrangements is the most definitive way to discriminate between conventional and direct differentiation of CD8 T cells from DN precursors. In this paper, we tested whether CD8 T cells selected with different peptide ligands in FTOCs exhibited these four characteristics.

	Materials and Methods

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Mice and peptides

OT-I (6), TAP° (a gift of Anton Berns, Netherlands Cancer Institute), ß₂-microglobulin° (ß₂m°) (18), and DO11.10 (19) mice and their various crosses were maintained in our colony at the University of Minnesota. Peptides were synthesized by standard fluorenylmethoxycarbonyl chemistry at the University of Minnesota Biomedical Engineering Division or by Research Genetics (Huntsville, AL). Typically, peptides were purified by reverse phase HPLC and analyzed by time-of-flight mass spectrometry. The sequences of the K^b binding peptides used in this work are as follows: OVA_257–264 or OVAp, SIINFEKL; A2, SAINFEKL; E1, EIINFEKL; R4, SIIRFEKL; V-OVA, RGYNYEKL; and CP1, ISFKFDHL. All peptides used were determined to bind H-2K^b similarly using the RMA-S stabilization assay (20). OVA_323–339 was a gift of Marc Jenkins (University of Minnesota).

FTOCs

Organ cultures were performed essentially as described previously (5, 6), except that both OT-I ß₂M° and OT-I TAP° animals were used. Briefly, the thymic lobes were removed from animals at gestational day (gd) 16 and placed on cellulose ester filters resting on top of Gelfoam sponges (Upjohn, Kalamazoo, MI). A total of 2 ml of media containing various concentrations of peptide was added and was replenished daily. When OT-I ß₂m° animals were used, 5 µM human ß₂m was included. After 7 days, thymocytes were harvested and analyzed by flow cytometry or tested for their capacity to respond to Ag in a 2-day proliferation assay determined by [³H]thymidine incorporation as described previously (5).

Flow cytometry and Abs

Thymocytes were harvested and stained with various combinations of biotin-B20.1 (V2: the OT-I Tg -chain), biotin-KJ126 (the DO11.10 clonotype), biotin-H57-597 (pan TCRß), phycoerythrin-RM4-5 (CD4), FITC-53.6 (CD8), and biotin-53-5.8 (CD8ß). All Abs were purchased from PharMingen (San Diego, CA) except KJ126 (a gift of Marc Jenkins). Tricolor-streptavidin (Caltag, San Francisco, CA) was used as a secondary reagent. Cells were analyzed on a FACScan using CellQuest analysis software and sorted using a FACSCalibur on the "exclusion" mode (Becton Dickinson, Mountain View, CA). A live gate was established using forward and side scatter properties. The purity of the sorted populations was 92 to 97%.

Assay for endogenous TCR chain rearrangements

Endogenous TCR chain rearrangements were detected by PCR and hybridization essentially as described previously (21). Briefly, 0.5 µg of DNA from sorted or control cells was subjected to PCR using a mixture of three V primers and one J primer (listed 5' to 3': V8, ACCCAgACAgAAggCCTggTCACT; V1, CAgAAggTgCAgCAgAgCCCAgAA; V3, ACTgTCTCTgAAggAgCCTCTCTg; and J28, gAAgACACACTTACATggTAgACATgg) After 28 cycles of amplification, the DNA was electrophoresed on a 1.8% agarose gel, and transferred to a nitrocellulose filter. The filter was hybridized with a ³²P-labeled oligonucleotide probe internal to the J28 primer (gTgCCAgATCCAAATgTCAgCgCA). Primers specific to elongation factor-1 (EF-1) were used as a control (CTgCTgAgATgggAAAgggCT and TTCAggATAATCACCTgAgCA).

	Results

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Effect of different TCR ligands on CD8 development in OT-I FTOCs

The TCR Tg strain OT-I bears an ß receptor specific for K^b plus OVA_257–264 (6). It requires K^b plus self peptide(s) for positive selection. We have used this strain in combination with ß₂m° or TAP° mice to define synthetic or naturally occurring peptides that can act to drive development of CD8 T cells in FTOCs. Table I shows a summary of the effect of several peptides in this system, some of which are data from previous publications. The antigenic peptide itself (Table I, OVAp) induces a profound negative selection even at low peptide concentrations and did not induce a net increase in the absolute number of CD8 SP at any concentration (5). A2, a variant of OVAp with strong agonist activity, induced positive selection at low doses but negative selection at high doses. However, the cells selected in the presence of A2 were not responsive to Ag stimulation through the TCR (5). This finding is similar to data reported using P14 TCR Tgs (3, 4), where a lack of function was also seen with agonist-selected cells (22). The most efficient peptides for inducing CD8 SP cells in the OT-I system are nonstimulatory variant peptides such as V-OVA (6) or the self peptide CP1 (23). The CD8 SP cells that develop in these cultures make proliferative responses comparable with that seen with OT-I wild-type (wt) FTOC cells (Table I).

Some Tg strains express an ß TCR at the cell surface at the DN stage

On day 0 of the cultures, which was gd 16 of the mouse, the OT-I fetal thymus contained predominantly DN cells (66%) (see Fig. 1). In this Tg, 36% of the cells expressed the Tg TCR on the cell surface. These values are similar to a class II-restricted TCR Tg DO11.10 (Fig. 1) and to several other TCR Tgs (Ref. 8 and B.J. Fowlkes, personal communication). In fact, both TCR Tgs express significant levels of the Tg receptor at an age (gd 15) when only DN thymocytes are present in the lobes (Fig. 1).

View larger version (43K): [in this window] [in a new window]   FIGURE 1. TCR Tg animals express the TCRß on the surface of thymocytes early in gestation. Thymic lobes from mice at gd 15 or 16 were analyzed for expression of CD4 and CD8 (dot plots). The percentage of cells in each quadrant is indicated. Data are from either a class I- (OT-I) or a class II- (DO11.10) restricted TCR Tg. The level of Tg receptor after gating on the DN cells is shown in the histograms.

Expression of CD8ß in CD8⁺CD4^- cells

If a signal through the Tg receptor on the DN cell leads to differentiation of CD8 SP in the lineage, then it would be predicted that the CD8 SP cells would express CD8 heterodimers, as do some cells. On the other hand, the majority of conventional TCRß⁺ CD8 T cells express CD8ß heterodimers. Thus, we asked whether peptide-selected CD8 SP cells in FTOCs bear CD8 or CD8ß dimers. We performed OT-I FTOCs with animals on either a B6 background (TAP^wt; Fig. 2) or a TAP° background, supplemented with K^b binding peptides daily for 7 days. At the end of the culture period, the thymocytes were harvested, counted, and stained for CD4, CD8, and CD8ß. Figure 2 shows the level of CD8ß on CD8⁺CD4^- (SP) cells. The level of CD8ß on DP cells is also shown as a control for the gating, since the plots are from different experiments. Indeed, most (84%) of the CD8 SP cells that developed in OT-I wt FTOCs (where the animal is K^b+ and no peptide is added) expressed CD8ß heterodimers (Fig. 2). On the other hand, CD8 T cells that developed in the presence of a strong agonist ligand, A2, bore mostly (70%) CD8 homodimers. The peptide E1 is a variant that acts as an antagonist at low concentrations but is stimulatory at high concentrations (agonist/antagonist). This peptide induces positive selection of functional cells as seen here (Table I) but can induce deletion and/or CD8 down-regulation at high density (6). When used at a dose at which it induces positive selection, E1 yielded CD8 SP cells with an intermediate level of CD8ß. Therefore, the agonist-selected cells induced development of a CD8 SP population that was phenotypically distinct from cells developing under normal selection conditions in FTOCs. These same cells were also functionally compromised as judged by their proliferative capacity (Table I), again suggesting that their development may be unconventional in some manner.

View larger version (36K): [in this window] [in a new window]   FIGURE 2. The expression of CD8 and CD8ß on peptide-selected FTOC cells. OT-I FTOC was performed in the presence or absence of various peptides. After 7 days, the cells were stained for CD4, CD8, and CD8ß. The histograms show the level of CD8ß on cells after gating on either CD8+CD4- cells (CD8 SP cells) or CD4+CD8+ (DP cells). The percentage of CD8 SP cells expressing CD8 but not CD8ß is indicated.

Effect of agonist peptide in class II-restricted TCR Tg FTOCs

Conventional positive selection of CD8 SP cells in vivo and in FTOCs requires a class I-restricted receptor. If an agonist peptide ligates the TCR on DN cells in TCR Tg FTOCs and results in a direct differentiation to CD8 SP, it might be predicted that this event, occurring in the absence of either coreceptor, would happen regardless of whether the TCR was class I- or class II-restricted. To test this possibility, we performed FTOCs with a class II-restricted TCR Tg DO11.10. This receptor recognizes OVA_323–339 in the context of I-A^d. Fetal thymic lobes from gd 16 DO11.10 mice on a BALB/c background were cultured in the presence or absence of varying concentrations of the antigenic peptide OVA_323–339. Figure 3A shows the CD4/CD8 phenotype of thymocytes at the end of a 7-day culture period. Untreated cultures have a high percentage of TCR Tg^high CD4 SP cells, indicating the efficient positive selection of thymocytes with this receptor in FTOC. As expected, significant deletion of the CD4 SP subset is seen at increasing concentrations of the peptide. This negative selection is also reflected in the total cell yield from the cultures (Fig. 3B). Of interest to us is whether a population of CD8 SP cells appears as a consequence of culture with the agonist peptide. Figure 3A shows that, indeed, the percentage of CD8 SP increases 3-fold at 50 to 500 nM peptide. Because the overall cell yields decrease with increasing peptide concentration, it is critical to evaluate the absolute numbers of cells in this population as opposed to the percentage. Figure 3B shows that a slight increase in the absolute numbers of CD8 SP cells is seen at 50 nM but not at 500 nM OVA_323–339. While not striking, the data suggest that agonist peptides might drive differentiation of CD8 SP in a nonconventional manner. As seen with the agonist-selected cells in OT-I cultures (see Fig. 2, A2), the agonist-selected CD8 SP cells in DO11.10 cultures are predominantly expressing CD8 homodimers (Fig. 3C, OVA_323–339 panel). This observation is in contrast to the small number of CD8 SP that develop normally in DO11.10 cultures (Fig. 3C, no peptide panel), presumably as a result of endogenous rearrangement at the -chain locus.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. The effect of agonist peptides in class II-restricted organ cultures. FTOC was performed with DO11.10 TCR Tg thymic lobes in the absence or presence of various concentrations of the antigenic peptide (OVA323–339). A, Thymocytes were stained for CD4, CD8, and the Tg TCR after 7 days of organ culture. The dot plots on the left display all live cells. The dot plots on the right show the CD4 and CD8 expression after gating on TCRhigh cells. The numbers indicate the percent of TCRhigh CD8 SP cells of total live cells. B, The numbers of cells in each subset was determined by multiplying the total number of thymocytes recovered per lobe by the percentage of cells in the indicated gate. The numbers are an average of at least four lobes per group and were combined from four separate experiments. C, The level of CD8ß (in the histograms) on cells in the CD8+CD4- gate (gate shown in the dot plots) for cultures with no peptide or 500 nM peptide. Data from representative lobes are shown.

Endogenous -chain rearrangements in CD8 SP from FTOCs

To confirm that the cells selected in FTOCs by nonstimulatory ligands were differentiating via a conventional pathway, we wished to find a way of "marking" cells in the DP stage and determining whether CD8 SP cells from FTOCs retained such markers. Nature has provided such a means, in that TCR chain rearrangement is up-regulated after a cell has differentiated to the DP stage (26). This is particularly true for the fetal thymus, where rearrangement is not detectable until DP cells appear (reviewed in 16 . Thus, CD8 SP cells that have endogenous TCR chain rearrangements have derived from DP precursors.

Rearrangement at the TCR locus is initiated as the cell expresses CD4 and CD8 and is terminated by successful positive selection; thus, TCR Tg animals on a selecting background have fewer endogenous -chain rearrangements compared with cells from a nonselecting background or with normal animals. However, we found that we could easily detect endogenous TCR chain rearrangements from adult OT-I-selecting mice using PCR primers from three different Vs (data not shown). To determine whether CD8 SP cells from FTOCs were derived from DP precursors, we sorted the CD8 SP cells from OT-I wt or peptide-selected cultures, purified DNA, and performed PCR for endogenous -chain rearrangements. The amplification was terminated at 28 cycles, which was on the linear part of the amplification curve, in an attempt to be semiquantitative. The PCR products were resolved by gel electrophoresis, transferred to nitrocellulose filters, and hybridized with a probe internal to the J PCR primer used. PCR of a control gene, EF-1, was also performed for each sample.

Figure 4 shows the detection of endogenous -chain rearrangements in CD8 SP sorted from OT-I wt (K^b+) FTOCs. OT-I RAG° thymocytes do not have such rearrangements, indicating that the primers do not detect the Tg receptor. Importantly, the CD8 SP sorted from cultures in which the nonstimulatory peptide V-OVA induced differentiation also have endogenous -chain rearrangements. In fact, the signal was stronger than that of the wt control. That DN cells do not have such rearrangements is shown in Figure 4, lane 4, which is DNA purified from OT-I FTOC cells cultured in the presence of OVAp. Because such cultures have only DN cells, no sorting was performed. We were unable to sort sufficient numbers of CD8 SP from A2-selected cultures to be included in this type of experiment. In part, the technical difficulty was a result of the very high level of dead cells and debris in A2-selected cultures. Nonetheless, it appears unequivocal from these data that CD8 SP cells selected by nonstimulatory peptide ligands in FTOCs arise from DP precursors.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Endogenous TCR rearrangements were detected in both normal and peptide-selected CD8 SP cells from FTOCs. The top panel shows blot hybridization analysis of PCR products generated using PCR primers specific for three V gene families and one J. The bottom panel is a control showing equivalent levels of PCR amplification of the EF-1 gene. The four DNA samples are from 1) adult OT-I RAG° thymi, 2) sorted CD8 SP cells from OT-I B6 FTOCs, 3) sorted CD8 SP cells from OT-I TAP° FTOCs supplemented with 5 µM V-OVA peptide, and 4) total cells from OT-I TAP° FTOCs supplemented with 10 nM OVAp.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the case in which nonstimulatory peptide ligands are used to drive development of CD8 SP cells in FTOC, we obtained strong evidence that the cells differentiate in a conventional manner (i.e., from a DP precursor). First, such cells exhibit a proliferative response that is comparable with wt cells (selected under normal conditions). Second, these cells express CD8ß heterodimers on their cell surface. These attributes, especially the latter, would not be expected if the cells had selected the lineage. Finally, we tested whether or not these cells differentiated directly from DP precursors. Previously, we had chosen to perform thymic reaggregate assays (27). Here, OT-I DP precursors were purified, mixed with TAP° thymic stromal cells, and allowed to develop for 4 days in special culture conditions. When the nonstimulatory peptide R4 was present, CD8 SP developed; in the absence of this peptide, none accumulated (data not shown). While this finding argues that peptide can induce progression of DP to CD8 SP, it does not prove that this pathway is used in organ cultures. Therefore, we tested whether cells selected by nonstimulatory peptides in FTOCs had endogenous TCR chain rearrangements. The TCR chain locus undergoes rearrangement at the DP stage, which serves to brand cells that have differentiated from such an intermediate. CD8 SP cells were sorted from FTOCs in which development was driven by the nonstimulatory peptide V-OVA. In such cells, endogenous TCR rearrangements were easily detected. In fact, we found a greater level of rearrangements in the peptide-selected cells compared with wt OT-I CD8 SP cells. Because positive selection terminates rearrangement, this result may reflect less efficient selection in response to peptide compared with the natural ligand. This is consistent with our measurement of the numbers of CD8 T cells developing under the two conditions (Table I). Taken together, these results strongly support the conclusion that nonstimulatory peptide ligands promote development of class I-restricted cells via the conventional pathway in organ cultures.

Our analysis of cells selected in the presence of stimulatory or agonist peptide ligands was more ambiguous. Agonist-selected cells have a compromised proliferative response (Table I) and have a greater percentage of CD8 homodimers on their surface (Fig. 2). That agonist treatment in FTOCs results in nonresponsiveness has been reported previously (5, 22, 28). Indeed, the CD8ß^low phenotype was noted when cells developed in the presence of agonist ligands (29). Additionally, the appearance of CD8ß^low cells was reported for thymocytes cultured with agonists in vitro (30). The lower level of CD8ß is a likely explanation for the decreased proliferative response, since CD8ß has been shown to be critical for both TCR signaling in CD8⁺ cells and for thymic development (31). Since altered function and expression of CD8 homodimers are features that might be predicted of TCRß⁺ cells developing in a lineage, the data do not rule out the possibility that such an abnormal pathway of development operates in response to agonist ligands. Further evidence in support of an abnormal pathway was the observation (Fig. 3) that class II-restricted FTOCs also generated CD8 SP cells when treated with agonist peptide, although this occurred only to a very minor extent.

An alternative hypothesis is that agonist treatment promotes positive selection of CD8 SP from DP but also results in the down-regulation of CD8ß as a mechanism of tolerance. We favor this hypothesis because of the result seen with E1 (Fig. 2). Treatment with this peptide generated CD8 SP cells that had an intermediate level of CD8ß. If cells were developing in the lineage, they would be expected to express only CD8 homodimers. Thus, the population seen in FTOCs might be a mix of cells that are positive or negative for CD8ß. That the cells had an intermediate level instead of being a mix is more suggestive of a down-regulatory mechanism than of two developmental lineages. In fact, in the in vitro experiments in which purified DP thymocytes were cultured with stromal cells and antigenic peptide, CD8⁺ cells appear that have down-regulated CD8ß (30). This observation indicates that CD8ß can be down-regulated independently of CD8 on DP cells. Alternatively, as a DN cell acquires expression of CD4 and CD8, those with a high level of CD8ß might be deleted in the presence of agonist ligands, while those with a lower level would be allowed to differentiate. This partial deletion would be consistent with the lower numbers of CD8 SP cells found in agonist-selected FTOCs (Table I). Nonetheless, since we did not obtain TCR rearrangement data on agonist-treated cells, we cannot make a strong statement about their developmental lineage.

To date, there is little evidence that CD8ß down-regulation is a mechanism of thymic tolerance in normal animals. Greater than 99% of lymph node CD8 T cells express CD8ß heterodimers where the level of CD8ß is high. It is interesting to note here that in patients with Wiskott-Aldrich syndrome, an X-linked immunodeficiency, the CD8 molecules on peripheral blood T cells are composed mostly of homodimers (32). Without a greater understanding of the immunologic nature of this disease, however, we cannot know whether the CD8ß^- cells have anything to do with a tolerance mechanism. It is possible that in normal animals CD8ß^low cells could be generated in the thymus but not survive in the periphery. Ultimately, the accumulation of nonfunctional CD8 SP thymocytes in TCR Tg mice as described here and elsewhere (28) may be unique to TCR Tgs and may not occur in animals with a normal repertoire, where the competition by other developing thymocytes is great.

	Acknowledgments

 
We thank Sonya Schober and Scott Pearcy for technical assistance, Steve Jameson and our laboratory colleagues for critical reading of the manuscript, and Marc Jenkins for providing DO11.10 breeders and OVA_323–339 peptide.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI39560 and by an Arthritis Foundation Investigator Award.

² Address correspondence and reprint requests to Dr. Kristin A. Hogquist, Department of Laboratory Medicine and Pathology, University of Minnesota, Box 334 FUMC, 420 Delaware St., SE, Minneapolis, MN 55455. E-mail address:

³ Abbreviations used in this paper: DN, double-negative; DP, double-positive; FTOC, fetal thymic organ culture; SP, single-positive; Tg, transgenic; RAG, recombinase-activating gene; ß₂m, ß₂-microglobulin; gd, gestational day; EF-1, elongation factor-1; wt, wild-type.

Received for publication March 18, 1998. Accepted for publication June 9, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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