This is confirmed by the experimental evidence showing that mature NK cells are nearly absent in mice lacking any of the 3 different subunits that compose the IL-15 heterotrimeric receptor (IL-15R) complex: CD215, CD122, and CD132 (γc chain) (97– 101). Accordingly, patients showing an X-linked gene mutation in the γc gene (il2rg) are affected by a severe combined immunedeficiency characterized by a high susceptibility to infections due to developmental defects of lymphocytes (including NK cells) (100). Despite the γc chain of IL-15R is shared by other relevant cytokines (i.e., IL-2, IL-4, IL-7, IL-9, and IL-21) for their downstream signaling (102), dysfunctions of this subunit affects only the IL-15 pathway. Indeed, knockout mice lacking IL-2, IL-2Rα, IL-7, IL-7Rα, and IL-21R have normal frequencies of mature circulating NK cells (103–106).

The production of IL-15 at NK cell developmental site is mainly exerted by BM stromal and myeloid cells (107). The binding of soluble IL-15 to CD215 on the surface of surrounding cells mediates the trans-presentation of this complex to NK cells expressing CD122 and CD132 heterodimer (108–110). This engagement of IL-15R on NK cells induces the activation of JAK1/3 downstream cascade that, in turn, activates STAT3/5 and the mitogen-activated protein kinase (MAPK). These signaling pathways mediate NK cell survival via both the up-regulation of anti-apoptotic B cell lymphoma 2 (BCL-2) family members and the down-regulation of pro-apoptotic proteins (111–114). Indeed, both Stat5-deficient and NK cell-specific Stat5-deficient mice show a marked reduction of circulating mature NK cells (115, 116). In humans, a similar severe reduction in NK cells is observed in patients with a mutation of STAT5b, one of the two highly conserved Stat5 human genes (117, 118).

NK cellresponsiveness to IL-15 during NK cell development is also influenced by the expression of phosphoinositide-dependent kinase-1 (PDK1) that connects IL-15 signaling to the activation of both E4BP4 (also known as Nfil3) and Inhibitor of DNA-binding 2 (ID2) transcription factors (119–121).

An additional cytokine playing a critical role in the development of NK cells is IL-21. Indeed, IL-21 stimulation in vitro, together with FLT3 and IL-15, promotes the differentiation and the expansion of cytotoxic CD56dim NK cells from BM progenitors (104, 122). In addition, IL-21 induces rapid maturation of human NK cells and the acquisition of a KIRpos mature phenotype from CD34pos cell precursors (123). On the other side, IL-7 is key in promoting the survival and early differentiation of NKPs (106). As a matter of fact, although mice lacking IL-7 or its receptor (CD127) keep a relatively normal NK cell development (105), the correct engagement of CD127 expression is key in the early stages of NK cell differentiation and in the retention of NKPs in SLTs (52). Moreover, those NK cells enriched in thymus are characterized by high constitutive expression of CD127 and require IL-7 for their homeostasis (100).

IL-4 has been recently described of being able to induce the development of tissue-resident NK lymphocytes in mice by converting CD49apos/Eomesneg NK cells into their functional CD49apos/Eomespos counterparts (124). Since CD49apos/Eomes neg NK cell subset is considered a liver-resident NK cell subset in mice, these findings could be relevant for a better understanding of the specific tissue-resident generation of NK cells (125). However, other than expressing high levels of Eomesodermin (Eomes) transcripts, human liver-resident NK cells appear to be much more phenotypically heterogeneous compared to their murine counterparts (126).

IL-12 can also promote differentiation of NK cells and can enhance their cytotoxicity in vitro (127). Interestingly, an alternative pathway of NK cell development that bypasses the above-mentioned γc-signaling relies on the engagement of IL-12 in response to viral infections. Indeed, the stimulation of NKPs with this pro-inflammatory cytokine in the BM generates an unconventional but yet functional NK cell subset. However, this pathway is still not exploited in humans and might be highly relevant in patients with SCID (128). IL-12, together with IL-18, has been also described for its ability to induce the differentiation of ml-NK cells. In this regard, IL-12 and IL-18 have been studied as co-stimulatory factors for the generation of CMV-specific murine Ly49Hpos ml-NK cells (129). In particular, the IL-12/STAT4 signaling pathway is required for the formation and the expansion of these NK cells with adaptive traits (130). Similarly, the expansion of NKG2Cpos ml-NK cells in humans upon HCMV infection has been shown to be IL-12- plus IL-18-dependent (131, 132). In this regard, in vitro activation of both murine and human NK cells with IL-12, IL-18, and IL-15 supports the generation of cytokine-induced ml-NK cells (133, 134). This mechanism has been recently employed in a clinical trial to boost the expansion of adaptive NK cells showing enhanced anti-tumor responses against myeloid leukemia (135).

### Transcription Factors

The commitment and differentiation of hematopoietic stem cells/precursors toward NK cell lineage require the expression of specific transcription factors (TFs). In this context, the current knowledge on NK cell development derives from experimental findings mainly generated either in vitro or in animal models and very little is known in human setting in vivo. However, it is widely accepted that several TFs are required by CLPs for their transition to both NKPs and iNK cells. These mechanisms are not specific for NK cell development as the same TFs are used to commit CLPs toward different cell lineages (136, 137). Ets-1 and PU.1, members of the Ets TF family, are involved in the transition of CLPs to NKPs and they are broadly expressed in multiple hematopoietic-derived lineages (138–141). Interestingly, Ets-1 deficient mice have a severe decrease of circulating NK cells, while knocking out PU.1 in murine models does not affect the frequency of NK cells in PB. These latter animals are also characterized by an up-regulation of Ets-1, thus suggesting the existence of compensatory mechanisms to ensure a correct ontogenesis and maturation of NK cells (142, 143).

As previously mentioned, the transition to NKPs also requires the expression of CD122 that induces STAT5 phosphorylation, dimerization and nuclear translocation (115–117). Although the specific gene targets of STAT5 in NK cells have not yet been clarified, more than 15,000 STAT5 DNA binding sites have been identified in T cells, including genes required for lymphocyte proliferation and survival (144). In addition, the expression of CD122 in NK cells is regulated by the Runx family of TFs that represent key regulators of lymphocyte lineagespecific gene expression (145, 146). In particular, Runx3 has been reported to play an important role both in NK and CD8pos T cell development, thus indicating its specific involvement in transcriptional programs of cytolytic lymphocytes (146). Similarly, Thymocyte selection-associated high mobility group box protein (Tox) and the interferon-regulatory factor (IRF) families regulate the transition toward NKPs as well as toward B and T cells, ILCs and myeloid lineages (147–149). On the contrary, several other TFs regulating the early steps of NK cell differentiation are much more restricted to the development of innate lymphocytes. Indeed, E4BP4-deficient mice lack only NK cells and ILCs, as the expression of this TF is required to tune the expression of Eomes and ID2 in early progenitor cells (150– 154). Other reports also claimed the existence of alternative and E4BP4-independent development pathways for immature and tissue-resident NK cells (125, 155, 156).

Another TF required for the differentiation of ILCs is ID2, whose expression is controlled by Ets-1 (138). ID2-deficient mice show a block of NK cell development between NKPs and mature NK cells with the subsequent lacking of circulating NK cells (120, 121). Recently, it has been also reported that ID2 regulates NK cell responsiveness to IL-15 through the modulation of DNA-binding helix-loop-helix E proteins (E2A) (157).

In later stages of NK cell maturation, T-box protein 21 (Tbet) and Eomes play a major role in promoting the expression of cytolytic and IFN-γ production machineries (158, 159). Indeed, mice deficient for both these latter TFs have a systemic lack of circulating mature NK cells (160, 161). However, whether or not Eomes and T-bet act in the same pathway is not yet clear as these two members of the T-box family are believed to function in a sequential manner during NK cell maturation. T-bet is required for the production of iNK and it is detectable just prior this development stage (154). Indeed, T-bet deficiency results in an accumulation of iNK cells in BM (161, 162). On the contrary, Eomes tunes the differentiation of mature NK cells from iNK cells and it is also critical to discriminate between NK and ILC1 subsets (158, 159). In addition, T-bet and Eomes have been reported to regulate NK cell development at different anatomical site as T-bet is primarily required for the production of NK cell at extramedullary sites (125, 163, 164).

### SURFACE MOLECULES REGULATING NK CELL TRAFFICKING AND MATURATION

Very little is known about the mechanisms orchestrating the trafficking of human NKPs and mature NK cells from PB to tissues/organs and vice versa (165). Several lines of evidence indicate that this trafficking is governed by several adhesion molecules, such as integrins, selectins, and chemokine/cytokine receptors. Among them, CXCR4, the alpha-chemokine receptor specific for the stromal derived factor-1, has a role in maintaining HSCs in the BM niche (166–168). Indeed, it has been shown that the treatment with a CXCR4 antagonist promotes the progenitor mobilization from the BM (169, 170). CXCR4 appears to also play a key role in the first steps of NK cell ontogenesis since it is highly expressed by NKPs and iNK cells, while its surface levels gradually decrease during NK cell maturation (171).

Differently from CXCR4, the down-modulation of CX3C chemokine receptor 1 (CX3CR1) in response to stimulation with transforming growth factor (TGF)-β prevents the NK cell egress from the BM (172, 173). Furthermore, CX3CR1, together with CC chemokine receptors (CCRs)-7 and−5, tunes NK cell maturation as the acquisition of a CD56dim phenotype is associated to its induced expression (174, 175). Similar to CX3CR1, the sphingosine-1-phosphate receptor 5 (S1P5) is involved in the NK cell release in the bloodstream and in NK cell differentiation. Indeed, terminally differentiated stage 6 NK cells up-regulate S1P5 and migrate in response to sphingosine-1-phosphate (S1P) (176). The active role of this bioactive sphingolipid in determining the NK cell trafficking is also suggested by the observation that S1P creates a gradient with highest concentrations in the blood and lymph, while its levels are maintained low in tissue parenchyma (177).

Several other adhesion molecules and chemokine receptors regulate the preferential localization of CD56br and CD56dim NK cells in SLTs, PB and inflamed tissues (178). Indeed, while S1P5 seems to be involved in retaining CD56dim NK cells in the bloodstream, CD62L, CCR7, and CXCR3 are involved in the selective homing of CD56br NK cell to LNs. Indeed, these latter surface molecules are either absent or expressed at very low levels on CD56dim NK cells (30, 62, 179, 180). Furthermore, CD69 is now considered not only as a marker of cell activation, but also as a tissue-residency one. Indeed, CD56br NK cells in tissues (i.e., liver, uterus, LNs) express high levels of CD69, while their counterparts in PB are CD69neg (38, 181). Moreover, highly cytotoxic CD56dim cells infiltrating metastatic LNs express CD69 and CCR7 and can upregulate CXCR1 (182–184). CD103 and CD49a are other tissue-residency markers that are upregulated by NK cells in response to TGF-β (185, 186). CD103 heterodimerized with β7 and binds to E-cadherin on epithelial cells, thus retaining NK cells in tissues (187). Moreover, the heterodimer β1-CD49a is involved in the tissue retention of NK cells via the binding to collagen (188). Those CD56dim NK cells preferentially migrating toward inflamed tissues, express a different patterns of cytokine/chemokine receptors that include CXCR1, CXCR2, and ChemR23 (59, 175, 189).

### NK CELL EDUCATION AND ACQUISITION OF TOLERANCE TO SELF

Although NK cell ontogenesis and education are two separated processes, there are quite a few interconnections between these two key mechanisms of NK cell homeostasis. The acquired expression of iNKRs together with their binding to self-HLA molecules in BM during NK cell development represent the mechanism generating functional NK cells that are also tolerant against autologous targets (190). Indeed, the direct cell-to-cell interactions with "self "-MHC-I educate NK cells to sense the down-regulation or lack of matched HLA alleles on target cells in order to mount an efficient effector-responses only against threatening viral-infected or tumor-transformed or allogeneic targets (190–192). Hence, the so-called process of "NK cell education" relies on the avidity of binding between NKRs and self-HLA molecules and on the level of response of an NK cell to activating signals (i.e., stress ligands, inflammatory cytokines, and Fc receptor engagement) (193, 194).

In addition to the recognition of self-HLA/MHC antigens in trans on neighboring cells, the expression of MHC-I molecules on the NK cell itself has been shown to play an important role in regulating NK cell activity and licensing in mice, by Ly49 interaction in cis (195, 196). In agreement, evidences in literature indicate that KIR:HLA interactions could occur both in trans and in cis in humans too. While the HLA-I trans-presentation seems to be mainly involved in NK cell education, the cis interaction in humans could play a major role in the maintenance of NK cell effector potential (190). However, since, unlike Ly49, KIRs do not have a flexible stalk, it has been proposed that this cis interaction between HLA-C and KIR2DL could occur in endosomes rather than on the cell surface (197).

Each individual shows a highly stochastic but tolerant HLA-I specific repertoire of iNKRs, which can be shaped by the subjectspecific immunological history. These phenomena are regulated by several "licensing" iKIRs, that recognize HLA-A/B/C, and by CD94/NKG2A, that binds HLA-E (9, 198, 199). During NK cell maturation, the NKR repertoire is selected to the expression of at Di Vito et al. NK Cell Ontogenesis

least one iNKRs specific for self-HLA-I haplotype on each mature NK cell. This makes NK cells able to recognize target cells, thus avoiding autoreactivity (200). CD94/NKG2A is the first HLA-Ispecific iNKR expressed on differentiating NK cells prior to the appearance of KIRs. Indeed, it is present on CD56br NK cells and to a less extent on CD56dim NK cells (201). Moreover, in vitro differentiating NK cells from immature post-natal thymocytes express high levels of CD94/NKG2A that prevents the lysis of autologous cells expressing self-HLA-I alleles (202). However, it is widely accepted that iKIRpos cells represent the main subset of "educated" NK lymphocytes. Indeed, during NK cell differentiation, the surface levels of CD94/NKG2A decrease while the expression of KIRs increases only on terminally differentiated CD56dim NK cells (201). In this context, the human KIR gene family shows a certain degree of diversity due to both the high variability of KIR gene contents and allelic polymorphisms (203, 204). KIR and HLA genes are located on different chromosomes, and are inherited independently. This phenomenon might affect the selective evolutionary pressure as well as the NK cellmediated susceptibility toward infections and diseases, as it is possible that KIR genes can be inherited in the absence of the cognate HLA ligand. Moreover, as previously mentioned, only NK cells expressing at least one KIR can be considered fully "licensed" (200, 205).

Although the mechanism tuning the process of NK cell education has not yet been fully disclosed, NK cell responsiveness is acquired in a finely regulated manner through KIR–KIR ligand interactions during development and several working hypotheses are currently being discussed on this matter. The first one relies on the concept of "arming" in which a given iKIR recognizes its cognate self-HLA-I allele, thus allowing the fully maturation of NK cells. In this regard, an NK cell expressing more than one iKIR should receive a stronger inhibitory signal, but this cell should also mediate a more potent alloreactivity when encountering a non-self-target (206). An opposite theory is instead based on the idea that NK cells expressing iKIRs mismatched with self HLA-I alleles are not clonally deleted, but are rather kept "unlicensed" both in PB and tissues in a state of hypo-responsiveness to ensure self-tolerance (194, 207, 208). This so called "disarming" working hypothesis states that, in the absence of self-iKIRs, the chronic stimulation of a still undetermined aNKR is associated with NK cell anergy (207).

An additional iNKR involved in NK cell education is ILT2/LIR1, which recognizes HLA-G and other shared epitopes present in all human HLA-I molecules. It has been shown that the expression of LIR1 by NK cells is able to prevent the secretion of IFN-γ (199). This iNKR appears to be expressed by mature NK cells and its surface level increases upon cytokine stimulation or HCMV infection, thus representing a possible escape mechanism from NK cell immune-surveillance (209–211). Moreover, the LIR1-mediated inhibition of NK cell effector-functions has been proposed to be also important in regulating the maternal-fetal immune tolerance during pregnancy (212).

Besides HLA-I specific iNKRs, several additional surface molecules have been reported to be involved in NK cell licensing to prevent their cell activation against self-cells. These additional mechanisms likely ensure a multi-layered and complementary system of immune tolerance and education of NK cells. In this context, the appearance of NKp46 and NKp30 before HLA-I specific iNKRs during development could ensure an HLA-I independent self-tolerance at early stages of NK cell differentiation (198, 213, 214). This hypothesis is also supported by clinical evidence in human HLA-I-deficient individuals, in which NK cells do not kill autologous cells although the engagement of HLA-specific iNKRs is either impaired or lacking (215, 216). In line with this last theory, it has been demonstrated that 2B4 could be involved in NK cell education by being expressed early on the CD34pos NKPs. As a matter of fact, although 2B4 is an aNKR in mature NK cells, it can exert an inhibitory function when expressed on immature NK cells (198, 213).

Finally, another mechanism possibly preventing NK cell autoreactivity relies on the differential/asynchronous expression of NK cell receptors and ligands. A classic example is the expression of NKG2D, an aNKR able of modulating NK cell receptor activation with different thresholds (217). In adults, it has an important role in eliminate potentially dangerous cells expressing NKG2D ligands including tumor-transformed and viral-infected target cells. On the contrary, NKG2D is not expressed in embryonic life, when the soluble and exosomebound ligands MIC-A and MIC-B are produced by human placenta. This mechanism thus contributes to prevent the activation of mother NK cells against the fetus (218, 219).

### CONCLUDING REMARKS

Although our current knowledge on the mechanisms tuning human NK cell ontogenesis greatly advanced over the past 2 decades, several questions still remain unanswered. In particular, the signals and pathways involved in NK cell development in SLTs and in other anatomic compartments remain to be clarified. Furthermore, the intracellular processes by which an NK cell is able to discern between self and non-self are still elusive. Emerging evidence from high-throughput technologies highlighted that NK cell diversity is more complex than expected and it is determined by genetic and environmental determinants. Thus, it is possible that this phenotypic NK cell diversity and apparent redundancy, within the same tissue and between the different tissues, could be the result of NK cell plasticity and could mirror the different NK cell functional properties rather than mere developmental intermediates.

Future efforts in understanding NK cell differentiation, effector-functions and heterogeneity in both physiological and pathological conditions will provide insights for the prevention and the treatment of human diseases. In particular, a better understanding of NK cell development in malignancies and other diseases will facilitate the design and implementation of NK cell-mediated immunotherapies.

## AUTHOR CONTRIBUTIONS

CD, JM, and DM wrote the manuscript and approved the final version.

### FUNDING

This work was supported by Fondazione Cariplo (2015/0603 to DM), Associazione Italiana per la Ricerca sul Cancro (IG-14687 and IG 21567 to DM), Italian Ministry of Health (Bando Ricerca Finalizzata PE-2016- 02363915), and Intramural Research Funding of Istituto Clinico Humanitas (5 × 1000 project to DM). CD was recipient of the post-doctoral fellowships from the

### REFERENCES

Fondazione Umberto Veronesi (2017-1464, 2018-1974, and 2019-2563).

### ACKNOWLEDGMENTS

The present review paper is dedicated to the memory of Alessandro Moretta, a great mentor and a pillar on the field of NK cell biology. We will always miss his friendship and enthusiasm for our work.

generates all human innate lymphoid cell subsets. Immunity. (2016) 44:1140– 50. doi: 10.1016/j.immuni.2016.04.007

in the human KIR repertoire and involve activating KIRs. Blood. (2013) 121:2678–88. doi: 10.1182/blood-2012-10-459545

cytokine responses and normal immune function. Immunity. (2012) 36:586–99. doi: 10.1016/j.immuni.2012.02.017

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Di Vito, Mikulak and Mavilio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Alessandro Moretta and Transporter Associated With Antigen Processing (TAP) Deficiency: On Giant's Shoulders

#### Jacques Zimmer\*

Laboratory of Innate Cellular Immunity and Chronic Inflammation, Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg

The laboratory hosting me for my Ph.D. described in 1994 the first human cases of

#### Edited by:

Eric Vivier, INSERM U1104 Centre d'immunologie de Marseille-Luminy, France

#### Reviewed by:

Teresa Bellon, Hospital La Paz Institute for Health Research, Spain Christelle Retière, Établissement Français du Sang (EFS), France

> \*Correspondence: Jacques Zimmer<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to NK and Innate Lymphoid Cell Biology, a section of the journal Frontiers in Immunology

> Received: 03 July 2019 Accepted: 25 September 2019 Published: 15 October 2019

#### Citation:

Zimmer J (2019) Alessandro Moretta and Transporter Associated With Antigen Processing (TAP) Deficiency: On Giant's Shoulders. Front. Immunol. 10:2404. doi: 10.3389/fimmu.2019.02404 TAP deficiency in two siblings with recurrent bacterial airway infections and a negative Human Leukocyte Antigen class I (HLA) serotyping. At this time, it became clear that natural killer (NK) cells interact with HLA class I molecules which inhibit them. Inhibitory receptors were postulated, and Alessandro Moretta was the first to generate monoclonal anti-human NK cell antibodies that bound to such molecules, which he characterized in detail (Killer Immunoglobulin-like receptors—KIR). Natural killer cells from healthy donors preferentially kill targets with absent HLA class I molecules ("missing self" concept), whereas we observed that the NK cells from the TAP-deficient patients were hypo-responsive and did not lyse the HLA class I-negative leukemia cell line K562. Moreover, they were not very active in antibody-dependent cellular cytotoxicity assays. To address the question if such NK cells would express KIR or not, my thesis supervisor requested the anti-KIR antibodies from Alessandro Moretta, who was kind enough to provide us generously with aliquots. It turned out that the NK cells from the TAP-deficient individuals expressed most of these inhibitory receptors normally. We then had the privilege to receive almost every new antibody generated in the Moretta lab and to complete the phenotypic studies of the NK cells from our patients. I had the great chance to meet Alessandro Moretta at several occasions. He deeply impressed me each time and strongly influenced my way of thinking.

Keywords: TAP deficiency, natural killer cells, HLA class I, cytotoxicity, antibodies

## INTRODUCTION

It is likely that most scientists meet, especially during the early phase of their career, more advanced colleagues that guide them and leave a deep impression on them for the rest of their life. My case was no exception, as I was lucky enough to find two of such mentors, namely my Ph.D. thesis supervisor, Dr. Henri de la Salle, and Professor Alessandro Moretta.

In 1995, after my medical studies in Strasbourg, France, and 2 years working at an Emergency Department, I realized that I would not want to continue spending my lifetime under such conditions, and decided to give a new orientation to my career by acquiring a Master Degree in Immunology, a field in which I always had been interested. I found an

internship position at the Blood Transfusion Center in Strasbourg, in the lab of Daniel Hanau and Henri de la Salle. They had described in 1994 two siblings with a very low cell surface expression of HLA class I molecules (negative serotyping), chronic bacterial infections of the respiratory tract and bronchiectasis, found to have a homozygous mutation in the TAP2 gene (1, 2). The transporter associated with antigen processing (TAP) is a heterodimer (TAP1 and TAP2 subunits) inserted into the membrane of the endoplasmic reticulum (ER) that transports endogenous peptides from the cytosol into the ER lumen, where they are loaded onto newly synthesized HLA class I molecules. Peptide acquisition is necessary for their stabilization, export from the ER and migration to the cell surface (3). Consequently, in the absence of a functional TAP, these processes do not efficiently occur.

In 1994, it was already established, in the context of the "missing self-hypothesis" formulated by Ljunggren and Kärre, that natural killer (NK) cells, the third type of lymphocytes besides B and T cells, preferentially killed targets not expressing HLA class I molecules (4). However, peripheral blood mononuclear cells from the TAP-deficient patients (containing the NK cell fraction), which contained a normal percentage of NK cells compared to healthy donors, were not cytotoxic to HLA class I negative K562 leukemia cells (1). Mouse models of TAP1- and β2-microglobulin (β2m) deficiency confirmed the observation, as NK cells from these animals were likewise hyporesponsive (5, 6).

### NK CELLS FROM TAP-DEFICIENT PATIENTS EXPRESS INHIBITORY NK CELL RECEPTORS

The project for my master thesis aimed at studying NK cells from the two TAP-deficient patients in more detail, as Alessandro Moretta previously had described several monoclonal anti-human antibodies of murine origin that identified NK cell subpopulations and whose target molecules were clonally distributed (indeed, the Moretta lab was successful in cloning NK cells) (7–9). This work later revealed that the structures recognized by the antibodies were inhibitory NK cell receptors (IR), able to negatively regulate NK cell functions.

Henri de la Salle asked Alessandro Moretta for these antibodies (GL183, EB6, Z27, and XA185), which were not yet commercially available, and received generous amounts of hybridoma cell culture supernatants in the context of a scientific collaboration. My role was first to check if the patient's NK cells, drawn from peripheral blood, did express the IR recognized by the antibodies. We did not purify the NK cells but identified them by three-color flow cytometry after staining with anti-CD3 (specific T cell marker), anti-CD19 (specific B cell marker), and anti-IR antibodies. Natural killer cells were considered as the CD3-CD19- events in the lymphocyte gate which allowed to evaluate the percentages of cells positive for the different IR. As controls, we used PBMC from the unaffected father of the patients (heterozygous for the TAP mutation) in addition to those from 10 unrelated healthy donors. The results clearly showed that the percentages of IR+ NK cells were in the same range for the patients, their father and the healthy donors (10), of course by taking into consideration the substantial interindividual variability in IR expression. Patient's NK cells were no outliers, although the percentages of cells positive for the antibody XA185 were rather high (10).

A second readout for receptor expression in flow cytometry is the mean fluorescence intensity (MFI), which reflects the density of expression of a given molecule. Regarding this parameter, it turned out that the MFI for the Killer Immunoglobulin-like Receptors (KIR) recognized by the antibodies EB6 and Z27 was again in the normal range, whereas for GL183, the MFI was slightly higher for the NK cells from the patients and their father, suggesting rather a genetic cause than a relationship to the TAP deficiency. However, and interestingly, the MFI of XA185 was much more pronounced in the case of the patients' NK cells than in their father and the ten normal donors (10).

In the meantime, Prof. Moretta had developed additional anti-NK cell antibodies called Q66, FES172, and Z270. Whereas, the former two were directed against a KIR and an activating isoform, respectively, the latter was specific for CD94/NKG2A only and not for all CD94-bound receptors like XA185. CD94 is a chaperone protein necessary for the surface expression of different NKG2 molecules, and among them, the inhibitory receptor NKG2A (8). Again, we received aliquots of these antibodies, and they confirmed prior results in that the anti-KIR Q66 antibody and the anti-NKG2A Z270 antibody stained percentages of NK cells in the patients comparable to those of the father and the healthy volunteers, whereas the frequency of FES172+ cells was extremely low except in three healthy donors (10). Such a pattern was compatible with the previously observed fact that only one third of the analyzed donors had a subset of FES172+ NK cells. It is now clear that this receptor is an activating KIR isoform which binds to a bacteria-derived epitope on HLA-C (11). Furthermore, even if the MFI of Q66 was in the lower normal range, the one of Z270 was strongly increased on patients' NK cells (even slightly more than for XA185) (10).

**Table 1** summarizes the different IR and, if applicable, their activating counterparts. The presence or absence of activating KIR isoforms depend on the KIR haplotype that an individual has inherited (12).

### FIRST MEETING WITH ALESSANDRO MORETTA

The first time I had the honor and pleasure to meet Alessandro Moretta was in November 1996, at the Annual Congress of the French Society for Immunology held at the Pasteur Institute in Paris, France. Henri de la Salle was present too, and we listened to the brilliant presentation of Alessandro Moretta about the antibodies allowing to distinguish different NK cell subpopulations and clones. During the coffee break, he was literally assaulted by colleagues who wanted his antibodies. Fortunately, Henri de la Salle had fixed an appointment during the lunch break, and there I was introduced to Alessandro. I had read almost all his papers and, as lots of young researchers,

List of monoclonal antibodies generated in the Moretta lab and used for the study of NK cells in TAP-deficient patients. The molecules recognized by these antibodies are members of the killer immunoglobulin-like receptor (or KIR) family (first five cells in column 2) or of the C-type lectin superfamily (last two cells in column 2). All the receptors mentioned are IR, except p50.1, p50.2, and p50.3, which are AR.

thought that I was already very good in my field. Nevertheless, I was nervous when I was introduced to him, but his kindness and nonchalance together with an impressive scientific rigor, had an anxiolytic effect on me. He gave us interesting ideas to try out. Then, I had to present my poster describing my data. Alessandro followed with interest and asked a lot of challenging questions.

### INVESTIGATION OF THE CYTOTOXICITY OF PATIENTS' NK CELLS

Back in Strasbourg, we continued the investigation of the cytotoxic properties of patients' non-activated NK cells by using 51Chromium release assays. Natural killer cells were purified after depletion of monocytes and T cells with anti-CD14 and anti-CD3 magnetic beads, respectively. They confirmed the absence of K562 killing, but in addition, antibody-dependent cellular cytotoxicity (ADCC) against rabbit antiserum-coated Raji Burkitt's lymphoma cells was also quite low compared to the NK cells from a healthy donor. Raji without antiserum was used as a negative control due to its resistance to unstimulated NK cells, and indeed neither the NK cells from the healthy donors nor those from the patients were cytolytic to this target. The EBV-transformed B lymphoblastoid cell lines (B-EBV) from the patients were killed to a substantial amount by healthy donor NK cells, fully in accordance with the "missing self " concept [(10) and unpublished data].

It had been described that the stimulation of NK cells with the cytokine interleukin (IL)-2 leads not only to their proliferation, but also to a strong increase in their cytotoxic properties toward targets already killed by their non-activated counterparts, and in addition to an extension of the spectrum of susceptible cell lines. Thus, Raji and Daudi, which both resist to ex vivo NK cells, are killed by activated ones (10).

The most efficient method for NK cell expansion and activation at that time was the one described by Perussia et al.: total PBMC are co-cultured with irradiated Burkitt's lymphoma Daudi feeder cells (at a 5:1 ratio) and IL-2 for 6 days, identically re-stimulated and then grown for a few additional days (13). We usually harvested the cells at day 12, purified the NK cells with anti-T cell immunomagnetic beads (as some T cells likewise expand under these conditions) and then used them in cytotoxicity assays. In preliminary experiments, we noticed that the method worked even better with B lymphoblastoid cell lines like ST-EMO derived from one of the patients. Later, we found that the best stimulator cell line was TND-3 (unpublished data), derived from a Japanese patient with a TAP1 deficiency (14). We have to date no clear explanation for the phenomenon that TAPdeficient B-EBV cell lines induce higher expansion rates than cell lines derived from healthy volunteers.

When we cultured normal and TAP-deficient PBMC under these conditions, we observed that the latter expanded much less well (10). Normal activated NK cells very significantly killed K562, Daudi and Raji and performed a strong ADCC against Raji. Interestingly, the TAP-deficient activated NK cells were likewise efficient in terms of natural cytotoxicity and ADCC against the same target cell lines, which was a bit surprising for us. We then tested the killing of autologous B-EBV cell lines from five healthy donors and from the patients. These experiments revealed that the percentage of lysis of the normal B-EBV cells never exceeded 15 %, whereas it reached 35% in the case of patients' cell lines (10). This observation suggests some degree of auto-aggressivity exerted by the activated NK cells of these individuals.

### NK CELL-MEDIATED KILLING OF TAP-DEFICIENT FIBROBLASTS

Having demonstrated this autologous killing performed by TAPdeficient NK cells (in the meantime I had become a PhD student), we went on to check if it was also the case against primary cells, namely skin fibroblasts. We cultured them with HLA class Iinducing cytokines (IFN-α, IFN-γ, IFN-γ + TNF-α) and used them as targets for purified activated autologous NK cells from three healthy donors and one of the patients (15). As expected, the patient's fibroblasts only marginally increased their surface HLA class I expression (W6/32 antibody), in contrast to the normal fibroblasts. As a consequence, the former were strongly lysed by their autologous NK cells, whereas healthy fibroblasts were protected either completely or at least to a significant extent. We then thought to demonstrate that the masking of HLA class I by the A6-136 antibody (IgM isotype) from the Moretta lab restored (normal cells) or increased (patient's cells) the killing of the fibroblasts This was clearly the case for the healthy fibroblasts but not for the TAP-deficient ones, for whom no effect could be observed (15).

In the next step, T lymphoblasts from phytohemagglutinin (PHA)-stimulated cultures were used as targets. When originating from healthy donors exposed to autologous activated NK cells, almost no lysis was observed, whereas such NK cells efficiently killed TAP-deficient PHA-blasts. The latter were surprisingly only killed by healthy volunteers' NK cells but not by autologous patients' NK cells, in contrast to our previous observations with B-EBV lymphoblastoid cell lines and skin fibroblasts. When the A6-136 antibody was added to the cytotoxicity assays, autologous T-PHA blasts from the healthy donors were significantly lysed by their autologous activated NK cells. Inversely, the patients' activated NK cells did not attack autologous T-PHA blasts.

During all this work, Henri and me regularly discussed the data by phone (Skype didn't yet exist) with Alessandro who was very interested in the topic and gave useful advice. To facilitate the exchanges, I traveled twice to his lab where I received a very warm welcome from him and his collaborators. We shipped PBMC and fibroblasts to Genova to be able to do further experiments there. I still precisely remember the scientific discussions we had at these occasions, because they represented high quality lessons in immunology during which I learned a lot. On the last evening of my stays, Alessandro took us to nice restaurants where I became aware even more what a fascinating personality Alessandro had and where I was delighted to enjoy local pasta specialties in a lot of different variations.

### EXPRESSION OF ACTIVATING RECEPTORS

It had become clear that NK cells need not only IR, but also activating receptors (AR) to efficiently kill targets. Here again, Alessandro was the first to identify a family of them, namely the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46 (16). We received the corresponding antibodies before their appearance on the market and could show that these molecules were expressed totally normally by the NK cells of the patients. Regarding NKG2D, Alessandro was scooped by another group (from whom we could not obtain an antibody aliquot), although his lab later developed antibodies against this AR, too. It is likewise expressed by TAP-deficient NK cells.

The work in Alessandro's lab further allowed to demonstrate that TAP-deficient activated NK cells are able to produce IFNγ but difficult to clone (17), and we completed together the phenotypic studies of our patients in 2007, focusing this time on the expanded CD56bright population and the overexpression of the broad-spectrum IR, NKG2A, and ILT2 (18).

### FURTHER CASES OF TAP DEFICIENCY

Other groups identified TAP-deficient patients as well and overall confirmed our data (19, 20). We then described a patient with cytotoxic NK cells at the baseline (21). This individual was heavily infected in the airways at the time of blood drawing, and thus her NK cells might have been activated in vivo. Over the years, several additional patient observations appeared in the literature, focusing more on the clinical aspects (22–29). Based on the number of published cases, TAP deficiency is extremely rare, but there are probably much more patients, as the frequent diagnosis of idiopathic bronchiectasis only seldomly leads to a check for normal HLA class I expression (30), and as the diagnosis is probably often not made in regions with a difficult access to medical structures and a high consanguinity rate.

The last time I met Alessandro was in 2012, when I organized a scientific meeting in Luxembourg, entitled "HLA

class I molecules in Health and Disease," bringing together an impressive international speaker panel composed of first-class immunologists. There was a session exclusively dedicated to TAP deficiency, whereas Alessandro gave a talk about his current work. For the gala dinner, I arranged to sit at his table, and once more, we had very interesting and stimulating discussions.

### SUMMARY AND OUTLOOK

In the light of the vertiginous evolution Immunology has taken in recent years, can we give a new interpretation to our findings about TAP deficiency? It is now obvious that NK cells from TAPdeficient patients are not educated (not licensed), but Henri de la Salle and myself were not creative enough to go deeper into the hyporesponsiveness mechanisms of these cells, and we also did not resolve the molecular basis of the cytotoxic activity after cytokine stimulation.

Most of the patients described to date have serious and chronic bacterial infections of the respiratory tract (leading to bronchiectasis), whereas viral infections of course occur but are not exceptionally severe (3). In addition, most of them also suffer from deep skin ulcers on the legs that are healing very slowly and are difficult to treat (3, 27, 29). They might even develop granulomatous lesions of the nose, leading to the complete destruction of the nasal cartilage. It is known that NK cells are involved in antibacterial defense but that their effects can be beneficial as well as deleterious, depending on the pathogen and the precise context (31–33). Natural killer cells in tissues are different, at least in part, from those circulating in peripheral blood (34). Therefore, we cannot say for the moment if lung NK cells from the patients are dysfunctional in terms of antibacterial defense and/or if they destroy bronchial tissues due to their hypothetical (over-)activation in the infectious situation. Likewise, skin NK cells might be at the origin of the skin ulcers, where they have indeed been found (19). The nasal lesions resemble lethal midline granuloma which is now called NK/T cell lymphoma, nasal type, and this suggests that, once activated, NK cells destroy autologous tissues which in the case of TAP deficiency do not express sufficient amounts of HLA class I molecules to inhibit these cytolytic effectors. Recently, we found that TAP-deficient patients have a higher percentage of the newly described CD56dimCD16dim NK cell subset than healthy controls (35), and this might be the result of a slightly delayed maturation of patients' NK cells. The Malmberg group, in collaboration with our lab, also showed that a polyclonal expansion of adaptive NKG2C+ NK cells occurs in the patients, which might partially explain their resistance to severe viral infections (36). Furthermore, we analyzed in detail the repertoire of the HLA class I-binding receptors KIR, NKG2A and CD8 and found a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors (37).

In summary, Alessandro Moretta has made substantial contributions to the in-depth description of TAP-deficient NK cells. Without his antibodies, I might not have been able to publish in high quality journals during my PhD thesis and to find a postdoc position in the lab of Prof. Werner Held at the Ludwig Institute in Lausanne, Switzerland. Interestingly, Alessandro had started his career at this same Institute, in the lab of his brother Lorenzo.

His work has not only theoretical but also practical clinical implications, as illustrated by the well-known phenomenon of KIR-ligand mismatch in semi-allogeneic bone marrow grafts (38). Very recently, a NK cell engager targeting NKp46, CD16 and a tumor antigen was described by the Vivier group (39), and this tri-specific engager holds quite some promise for cancer immunotherapy.

 I am sad that we lost him, but I am proud and honored to have known him.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the manuscript/supplementary files.

### REFERENCES

### AUTHOR CONTRIBUTIONS

JZ conceived and wrote the article.

### FUNDING

This article was a personal initiative of JZ and received no dedicated funding.

### ACKNOWLEDGMENTS

JZ acknowledges the Department of Infection and Immunity of the Luxembourg Institute of Health, and particularly its Director, Prof. Markus Ollert, for ongoing support. He is also grateful to the patients who generously agreed to provide blood for our studies, and to Dr. Henri de la Salle, Etablissement Français du Sang, Strasbourg, France, for his kind but rigorous and stimulating supervision during the Ph.D. thesis.

**Conflict of Interest:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zimmer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


*2.2. Experimental Equipment*

The following devices were employed for the adsorption and desorption kinetic aspects of Pb2+: a digital water bath oscillator, high-speed desktop centrifuge, and microwave.

The experimental devices for water table fluctuations include a 90 cm long column, water tank with 25 L volume, peristaltic pump with 100 rpm speed, piezometer tube, and soil solution sampler. We set eight openings for sampling in every column at heights of 20, 30, 35, 40, 45, 50, 55, and 60 cm. The experimental devices are shown in Figure 1.

**Figure 1.** Experimental devices for the water table fluctuations. (**a**) The schematic diagram. (**b**) The field experiment diagram.


**Author details**

Bentahar Attaouia1 \*, Kandouci Malika2 , Ghouali Samir3 and Dinar Amina Elbatoul3

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Waveguide Amplifier for Extended Reach of WDM/FSO DOI: http://dx.doi.org/10.5772/intechopen.104790*


*Article* **Estimation of Daily Potential Evapotranspiration in Real-Time from GK2A/AMI Data Using Artificial Neural Network for the Korean Peninsula**

**Jae-Cheol Jang \* , Eun-Ha Sohn, Ki-Hong Park and Soobong Lee**

National Meteorological Satellite Center, Korea Meteorological Administration, Jincheon 27803, Korea<EMAIL_ADDRESS>(E.-H.S<EMAIL_ADDRESS>(K.-H.P<EMAIL_ADDRESS>(S.L.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +82-07-7850-5915

**Abstract:** Evapotranspiration (ET) is a fundamental factor in energy and hydrologic cycles. Although highly precise in-situ ET monitoring is possible, such data are not always available due to the high spatiotemporal variability in ET. This study estimates daily potential ET (PET) in real-time for the Korean Peninsula, via an artificial neural network (ANN), using data from the GEO-KOMPSAT 2A satellite, which is equipped with an Advanced Meteorological Imager (GK2A/AMI). We also used passive microwave data, numerical weather prediction (NWP) model data, and static data. The ANN-based PET model was trained using data for the period 25 July 2019 to 24 July 2020, and was tested by comparing with in-situ PET for the period 25 July 2020 to 31 July 2021. In terms of accuracy, the PET model performed well, with root-mean-square error (RMSE), bias, and Pearson's correlation coefficient (*R*) of 0.649 mm day−1 , −0.134 mm day−1 , and 0.954, respectively. To examine the efficiency of the GK2A/AMI-derived PET data, we compared it with in-situ ET measured at flux towers and with MODIS PET data. The accuracy of the GK2A/AMI-derived PET, in comparison with the flux tower-measured ET, showed RMSE, bias, and Pearson's *R* of 1.730 mm day−1 , 1.212 mm day−1 , and 0.809, respectively. In comparison with the in-situ PET, the ANN model produced more accurate estimates than the MODIS data, indicating that it is more locally optimized for the Korean Peninsula than MODIS. This study advances the field by applying an ANN approach using GK2A/AMI data and could play an important role in examining hydrologic energy for air-land interactions.

**Keywords:** evapotranspiration; GK2A/AMI; artificial neural network; Korean Peninsula

## **1. Introduction**

Evapotranspiration (ET) reflects fundamental components of hydrologic and energy cycles of the Earth and is a key element in hydrological resource management [1]. As climate change has progressed, trends in drought and flood have shown different spatial variability, and the importance of hydrological system monitoring has been emphasized [2]. Accordingly, it is fundamental to quantify and monitor ET. However, since water resources are directly affected by regional hydrologic systems and meteorology, ET shows high spatial and temporal variability [3].

A major application of ET is drought monitoring. Climate change has altered drought trends, increasing the intensity, frequency, and extent of droughts [4]. Thereafter, numerous indices for drought monitoring have been developed, with several, such as the standardized precipitation evapotranspiration index [5], precipitation evapotranspiration difference condition index [6], reconnaissance drought index [7], and combined terrestrial evapotranspiration index [8], directly associated with ET. Based on these drought indices, many studies were conducted to investigate the long-term variability of water budget under specific climate change conditions [9], effects of climate elasticity of ET on water balance [10], spatiotemporal variability of drought characteristics [11], and impacts of

**Citation:** Jang, J.-C.; Sohn, E.-H.; Park, K.-H.; Lee, S. Estimation of Daily Potential Evapotranspiration in Real-Time from GK2A/AMI Data Using Artificial Neural Network for the Korean Peninsula. *Hydrology* **2021**, *8*, 129. https://doi.org/10.3390/ hydrology8030129

Academic Editors: Aristoteles Tegos and Nikolaos Malamos

Received: 14 July 2021 Accepted: 23 August 2021 Published: 27 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

drought events on agricultural production [12]. In addition to various applications of ET, the methods to estimate ET with higher accuracy and spatiotemporal resolution have also been studied.

ET can be classified depending on the soil moisture condition. Potential ET (PET) is defined as the water vapor transpired and evaporated from vegetation and soil in unlimited soil moisture conditions [13]. Actual ET (AET) represents the water vapor transferred from a surface under limited soil moisture conditions. Weighing lysimeters are the most accurate AET measuring instruments [14]. Although they measure AET directly, the available AET data are substantially limited for end-users [15]. To ensure the versatility of ET data, various ET estimating models have been developed that can be broadly classified into three types [16]: (1) fully physical combination models that deal with mass and energy transfer principles [17,18]; (2) semi-physical models that account for mass or energy transfer principles and are based on temperature and radiation [19,20]; and (3) black-box models that are based on empirical relationship, artificial neural networks (ANN), fuzzy, and genetic algorithm. Although there are various ET estimating models, the most widely used method is the Penman–Monteith (PM) method [21,22]. The PM method is a fully physical model developed by Penman [17] and later modified by Monteith [18]. This model is recommended as the global reference model for ET monitoring by the Food and Agriculture Organization of the United Nations (FAO).

Although in-situ ET measurements are highly precise, the spatial variability of ET is high, and the availability of in-situ ET measurements is limited [23]. Remotely sensed data have been used to address this problem. Satellite data have broad spatial coverage with high temporal resolution and produce reliable products [24]. MODerate resolution Imaging Spectroradiometer (MODIS) derives the operative ET products with 500 m spatial and 8 days temporal resolution [25]. Several studies have estimated the spatial distribution of ET using low Earth orbit (LEO) satellites with optical-infrared and microwave sensors [26,27]. When calculating ET using LEO satellites, external input data, such as meteorological data, are generally necessary [28]. In particular, because LEO satellites observe the Earth's surface at specific local times, it is difficult for the instantaneous observation to monitor the environmental conditions all day and all weather [29]. Therefore, due to the high temporal variability of ET, LEO satellite-derived ET has inevitable limitations for routine monitoring of daily ET and surface energy fluxes [11,30]. In addition, since LEO satellites apply the physical-based model or energy conservation-based model for estimating ET, there exist uncertainties of external input data for applying the model [25–27]. Using geostationary orbit (GEO) satellites data can compensate for the limitations associated with the temporal resolution of LEO satellite data. However, it is difficult to resolve the uncertainties of external input data and the data contaminated by weather conditions, including clouds and aerosols [29].

The Korean Peninsula is located on the margin of Northeast Asia, bordering the northwest Pacific Ocean (Figure 1a). Since it is located in a monsoon region, where meteorological droughts occur during the summer monsoon, the droughts tend to propagate into agricultural or hydrological droughts [31]. In particular, the Korean Peninsula land cover type showed complex spatial distribution comprising of diverse vegetation cover types (Figure 1b). Furthermore, in the Korean Peninsula, the drought frequency has increased, and drought trends and characteristics vary regionally [32]. The Korean Peninsula has various land cover types and specific terrain properties; these factors make it particularly difficult to monitor daily ET even employing both in-situ measurement and remotely sensed data. Due to frequent cloud cover and rainfall, it is challenging to observe the land surface using optical-infrared satellites in the summer monsoon season [33]. Therefore, to overcome this limitation, numerical model data and ancillary data have been used to retrieve ET [34,35].

**Figure 1.** MODIS land cover from the Annual International Geosphere-Biosphere Programme over (**a**) Northeast Asia and (**b**) the Korean Peninsula in 2019. **Figure 1.** MODIS land cover from the Annual International Geosphere-Biosphere Programme over (**a**) Northeast Asia and (**b**) the Korean Peninsula in 2019.

In order to manage hydrological resources over the Korean Peninsula, Korea Meteorological Administration (KMA) monitors the ET in real-time using in-situ measurements and numerical model data. In-situ measurements exhibit good performance with high temporal resolution every hour; however, its availability is limited due to the point observation. For complementing the limitation of in-situ measurements, KMA calculates the spatial distribution of ET using numerical model data based on geophysical models. Numerical model data-derived ET is suitable for analyzing droughts with a large time scale. In contrast, the accuracy of the ET changes depending on the numerical model data, In order to manage hydrological resources over the Korean Peninsula, Korea Meteorological Administration (KMA) monitors the ET in real-time using in-situ measurements and numerical model data. In-situ measurements exhibit good performance with high temporal resolution every hour; however, its availability is limited due to the point observation. For complementing the limitation of in-situ measurements, KMA calculates the spatial distribution of ET using numerical model data based on geophysical models. Numerical model data-derived ET is suitable for analyzing droughts with a large time scale. In contrast, the accuracy of the ET changes depending on the numerical model data, and it is difficult to calculate the ET that reflects various topographical characteristics of the Korean Peninsula due to the sparse spatial resolution of the numerical model data.

and it is difficult to calculate the ET that reflects various topographical characteristics of the Korean Peninsula due to the sparse spatial resolution of the numerical model data. Although physical-based models show good performance, due to numerous associated meteorological parameters, it is difficult to estimate accurate ET, especially in remote sensing applications. Then, over the last few decades, many researchers have identified that machine learning (ML) approaches were an effective method to overcome the complexity of ET estimation [29]. Because ML techniques solve the non-linear relationship between input and output variables, a lot of ML techniques have been proposed to estimate Although physical-based models show good performance, due to numerous associated meteorological parameters, it is difficult to estimate accurate ET, especially in remote sensing applications. Then, over the last few decades, many researchers have identified that machine learning (ML) approaches were an effective method to overcome the complexity of ET estimation [29]. Because ML techniques solve the non-linear relationship between input and output variables, a lot of ML techniques have been proposed to estimate ET for hydrological applications [36], such as k-nearest neighbors [37], support vector machine [38], random forest [37], and artificial neural network (ANN) [39]. Previously, most studies applied ML approaches to in-situ measurements; however, many recent studies have also applied ML approaches to remote sensing data [40–42].

ET for hydrological applications [36], such as k-nearest neighbors [37], support vector machine [38], random forest [37], and artificial neural network (ANN) [39]. Previously, most studies applied ML approaches to in-situ measurements; however, many recent studies have also applied ML approaches to remote sensing data [40–42]. In this study, considering the spatiotemporal variability in ET, we developed a model that estimates daily PET based on ANN using the GEOstationary Korea Multi-Purpose In this study, considering the spatiotemporal variability in ET, we developed a model that estimates daily PET based on ANN using the GEOstationary Korea Multi-Purpose SATellite 2A (GEO-KOMPSAT 2A, GK2A). The objective was to retrieve real-time daily ET with a spatial resolution of 1 km for hydrological resource monitoring on the Korean Peninsula. To reflect the complex relationships and nonlinearity between the GK2A-derived data and ET, we used precipitation data and the digital elevation data as input data for the ANN. Daily PET from KMA was used as reference data for ANN model training.

SATellite 2A (GEO-KOMPSAT 2A, GK2A). The objective was to retrieve real-time daily

rived data and ET, we used precipitation data and the digital elevation data as input data for the ANN. Daily PET from KMA was used as reference data for ANN model training. The accuracy of the model was verified by comparing modeled data with ET from in-situ measurements of the KMA and National Institute of Forest Science (NIFoS) for the period

excluding the period of training data.

The accuracy of the model was verified by comparing modeled data with ET from in-situ measurements of the KMA and National Institute of Forest Science (NIFoS) for the period excluding the period of training data.

#### **2. Data and Methods**


Different Aspects of Behçet's Disease

*Edited by Müzeyyen Gönül and Arzu Kılıç*

Published in London, United Kingdom

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**Chapter 6**

*Aortic Aneurysm and Aortic Dissection*

stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways. Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;**28**(8):1511-1518

[51] Curci JA. Digging in the "soil" of the aorta to understand the growth of abdominal aortic aneurysms. Vascular.

[52] Powles J et al. Global, regional and national sodium intakes in 1990 and 2010: A systematic analysis of 24 h urinary sodium excretion and dietary surveys worldwide. BMJ Open.

[53] Whelton PK et al. Sodium, blood pressure, and cardiovascular disease: Further evidence supporting the American Heart Association sodium reduction recommendations. Circulation. 2012;**126**(24):2880-2889

[54] Appel LJ et al. The importance of population-wide sodium reduction as a means to prevent cardiovascular disease and stroke: A call to action from the American Heart Association. Circulation. 2011;**123**(10):1138-1143

[55] Golledge J et al. Reported amount of salt added to food is associated with increased all-cause and cancer-related mortality in older men in a prospective Cohort study. The Journal of Nutrition, Health & Aging. 2015;**19**(8):805-811

[56] Maron BA, Leopold JA. Aldosterone

receptor antagonists: Effective but often forgotten. Circulation.

2010;**121**(7):934-939

2009;**17**(Suppl 1):S21-S29

2013;**3**(12):e003733

**88**


*3.6. XRD Analysis*

Figure 8 presents XRD patterns of non-cement blended samples prepared using various S/FA ratios at 28 days. Hydration products appeared in various phases, including Ca(OH)2, Ca3SiO5, Ca6Al2(OH)12(SO4)3-26H2O and CaFe2O5. The non-cement blended specimens displayed roughly the same CaSO4-2H2O and Ca6Al2(OH)12(SO4)3-26H2O (ettringite) [31] peaks compared to the control groups. Note that the non-cement blended specimens also presented relatively small Ca(OH)2 peaks and slightly high Ca3SiO5 peaks compared to the P specimens. It also indicated that the hydration products of non-cement blended specimens were similar to the P specimens and almost less calcium hydroxide (Ca(OH)2) was found in non-cement blended specimens. It indicated that the quartz (SiO2) in the FA reacted with lime (CaO) and Ca(OH)2, resulting in the formation of C-S-H and C-A-S-H gel, which are the key factors in strength development in cementitious materials [32,33]. The FA also had higher proportions of f-CaO and CaSO4, which has been shown to activate cementing processes and hydration reactions [34]. It also confirmed that the strength of non-cement blended composites can be achieved to conventional cement-based materials at the age of 56 days.

**Figure 8.** XRD patterns of various samples at 28 days.