Tumors develop numerous mechanisms through which they evade NK cell attack. Several molecules secreted by YAC-1 cells and P815 cells were evaluated for their interaction with NK cell receptors. The affinity characterization of NK cell receptor with tumor expressed ligands will help in understanding the binding patterns required for the activation of NK cell activity. A detailed understanding of the mechanisms of tumor evasion and tumor susceptibility in the tumor microenvironment is essential for developing effective cancer therapies. The multi-faceted recognition pattern of tumor derived factors by NK activating receptors and every accessible surface involved in this binding event has been explored. Interaction between the known factors (galactin-3, CD63 (AD1 antigen), gp70 and p30) present on the surface membrane of mastocytoma P815 cell with NK inhibitory receptor Ly49A on NK cell was explored for evaluating the basis for tumor resistance to NK mediated lysis and interaction between T-cell lymphoma YAC-1 cell derived ligands(p30CA, p12, p15MA and p15E) with NK activating receptor Ly49H was evaluated for potential basis for NK susceptibility by YAC cells addressed by computational approaches which further help to develop NK based cancer therapeutic strategies (Graphic 1).

•    P815 cell
•    Natural killer cells receptors
•    YAC-1 cells
•    Ligand interactions

Mishra R, Verma R, Shreni S, Kaur J, Joshi D, et al. (2016) Evaluating the Basis for NK Sensitivity and NK Resistance in Prototypic NK Sensitive and Resistant Cell Line. JSM Chem 4(4): 1035

NK cells are large, granular, cytotoxic lymphocytes of innate immune system. NK cells have natural capacity to kill tumor cell. They do not require any prior antigenic sensitization and is capable of dealing with a broad range of virus infected cells and tumor cells. NK cell activation and function are strongly influenced by the interplay of inhibitory and activating signals [1].

Tumor cells have evolved various mechanisms by which they can evade NK cell attack. These mechanisms include interference of NK cell activation, inducing inhibition as well as modulation of NK receptor expression pattern and thus regulating NK cell function. Tumor derived factors may cause modulation of costimulation, adhesion or susceptibility to apoptosis [2].

NK cells recognize target cells in two different ways i.e. lack of MHC-class I expression and absence of expression of appropriate ligands for NK cell receptor which renders target cells susceptible to NK mediated lysis. NK cell receptors utilize several parallel recognition patterns that enable them to distinguish between abnormal cell and healthy cell.

YAC-1 is a murine T-cell lymphoma cell line which is susceptible to NK-mediated killing. MHC-I molecule is a ligand for inhibitory receptors on NK cells [3], thus causing susceptibility of YAC-1 cells to NK cell cytotoxicity, while P815, which is a mastocytoma cell line expresses reduced activating ligands. Inhibitory receptor present on NK cells MHC molecule and upon engagement of these receptors, block the ability of NK cells to attack target cells, hence if inhibitory ligand is not present on the target cell, NK cell will cause lysis of the target cell.

However in the present study we tried to explore if in the addition to modulation of MHC I (ligand for inhibitory receptor) direct binding to tumor expressed ligand with other NK inhibitory or activating receptors do indeed play a role in NK resistance and susceptibility.

Structure of various ligands expressed on YAC-1 and P815 cell were modeled using Phyre 2 and molecular docking was performed using PATCHDOCK between YAC-1 ligands (namely, p30CA,p12,p15MA and p15E) and NK activating receptor, Ly49H,similarly P815 ligands (namely, galectin-3, CD63, gp70 and p30) were studied for molecular docking with NK inhibitory receptor Ly49A.

In the present study we tried to elucidate the molecular basis of susceptibility of YAC tumor cells and resistance of P815 cells to NK mediated cytotoxicity. YAC 1 cells express low level of MHC I (ligand for inhibitory receptors on NK) and P815 cells on the other hand express high level of MHC I molecules thus causing susceptibility of YAC and resistance of P815 to NK mediated cytolysis. However, in the present study in addition to above expression pattern of MHC I, we show direct binding efficiency of tumor expressed ligands with activating and inhibitory NK receptors. Furthermore, D-complex energy was calculated and some significant interaction between ligand and receptor, which highlighted the binding pattern required for the activation of NK cell effecter function has been reported in present study.

Receptor Modeling

Ly49H: A 265 amino acid sequence of Ly49H of mouse origin was retrieved from NCBI (accession no. = AAR03586.1) and Ly49A: A 262 amino acid sequence of Ly49A of mouse origin was retrieved from NCBI (accession no = AAF99547.1). Since the crystal structure of Ly49H and Ly49A was not available, the protein was modeled using a hybrid modeling server named Phyre2. The platform incorporates ab initio folding simulation called Poing 2to model regions of proteins with no detectable homology to the known structure [4-6].

Ligand preparation

Ligands expressed on NK sensitive YAC-1 cell line, viz., p30CA, p12, p15MA, p15E and NK resistant p815 cell line namely, Galectin-3, CD63 (AD1 Antigen), gp70 and p30 were studied.

The following information about p30CA (Accession no.= NP_955585.1, 263 amino acids, of Moloneymurine leukemia virus origin), p12 (Accession no.= 0804277A, 84 amino acids, of Moloneymurine leukemia virus origin), p15MA (Accession no.= NP_955583.1, 130 amino acids, of Moloneymurine leukemia Virus origin), p15E (Accession no.= NP_955589.1, 196 amino acids, of Moloneymurine leukemia virus origin) and Galectin-3 (Accession no. NP_034835.1), 264 amino acids of MusMusculus origin), CD63 (Accession no. NP_001036045.1), 238 amino acids of MusMusculus origin), gp70 (Accession no. CAA41992.1), 644 amino acids of Murine Leukemia Virus, p30 (Accession no. AAA46522.1), 160 amino acids of Murine Leukemia Virus were retrieved from NCBI. The crystallized structure of above mentioned ligands were not available in RCSB Protein Data Bank , so the structure of these ligands were modeled using a hybrid modeling server named Phyre2. The platform incorporates into folding simulation called Poing2to model regions of proteins with no detectable homology to the known structure [7-10].

Molecular docking using patchdock (an automatic server for molecular docking)

Patch Dock is an algorithm used for molecular docking. Here, the input is two molecules of any type e.g., proteins, DNA, drugs. The output obtained is a list of potential complexes that are sorted by the shape complementarity criteria. The Patch Dock algorithm has been inspired by image segmentation and object recognition techniques that are used in Computer Vision. If two molecules are given, then their surfaces are divided into patches depending upon the surface shape. These patches correspond to the patterns that distinguish between the puzzle pieces. Once these patches are identified and then they can be superimposed using the shape matching algorithms (Figure 1). The algorithm has three main stages:

• Molecular Shape Representation 
• Surface Patch Matching 
• Filtering and Scoring

The receptor molecule along with its ligand molecule was uploaded in the PATCHDOCK server, the respective e-mail id was entered and then the results were noted (Figure 2,3).

Refining models by FIREDOCK

After running PATCHDOCK, the top 10 results were refined by using FIREDOCK. The FireDock server then addresses refinement problem of the protein-protein docking solutions. This method simultaneously targets problem of flexibility along with the scoring of solutions that are produced by fast rigid-body docking algorithms. Given up to a set of 1000 potential docking candidates, it can refine and score them based on energy function. This is the first web server that allows scoring of docking solutions and performing large-scale flexible refinement online (Figure 4,5). The results with hydrogen bonds, the D COMPLEX predict the binding affinity of the protein complex, determining the energy in kcal/mol.

3D structure of NK activating receptor Ly49H

The 3D structure of NK activating receptor Ly49H is not available in PDB, so it was predicted using hybrid modeling server named Phyre2 (Figure 6) [11,12].

Structures of NK inhibitory receptor Ly49A

The crystal structure of Ly49A was not available on PDB, so the protein was modeled using a hybrid modeling server named Phyre2 (Figure 7).

Predicted structure of YAC-1 surface ligands

For all the YAC-1 surface ligands including, p30CA, p12, p15MA, p15E, the structures were not present in PDB. So the structures were also predicted with Phyre 2 (Figure 8: A-D).

Predicted structure of P815 surface ligands

The structures of CD63, galectin-3, p30, gp70, these are the p815 surface expressed molecules were not available in PDB so, the protein structures were modeled using a hybrid modeling server named Phyre 2 (Figure 9: A-D) (Table 1).

Predicted structure of P815 surface ligands

The structures of CD63, galectin-3, p30, gp70, these are the p815 surface expressed molecules were not available in PDB so, the protein structures were modeled using a hybrid modeling server named Phyre 2 (Figure 9: A-D) (Table 1).

Ly49H-p15E

The results obtained after docking of receptor Ly49H with p15E were observed. For each complex obtained, following data was analyzed. Here, the complex with minimum energy is the second one with D-complex energy -12.518432Kcal/mol and the complex representing minimum energy is shown below (Figure 10) (Table 2).

Ly49H-p30CA

The complex with minimum energy is the first one as depicted in Table (3) with D-complex energy - 11.061380 Kcal/ mol and the complex representing minimum energy is shown in (Table 3) (Figure 11).

Ly49H-p15MA

The complex with minimum energy is the first one as depicted in Table (4) with D-complex energy - 12.205013 Kcal/mol and the complex representing minimum energy is shown in Table (4) (Figure 12).

Ly49H-p12

The complex with minimum energy is the tenth one as depicted in Table (5) with D-complex energy - 8.455273 Kcal/ mol and the complex representing minimum energy is shown in Table (5) (Figure 13).

To understand the interaction between Ly49H, activating receptor (chain-X) and the ligands (chain-Y) present (p15E, p30CA, p15MA, p12) on the surface of YAC-1 cell line, docking was performed and the D-complex energies for each receptor– ligand pair was studied. The result shows significant interaction between each of the YAC ligands with Ly49H.

PATCHDOCK was used to perform molecular docking of murine inhibitory surface receptor Ly49A with the surface

PATCHDOCK was used to perform molecular docking Ly49H with the YAC-1 ligands; p15E, p30CA, p15MA, p12. PATCHDOCK determines top 10 complexes and for each of the complexes, the D-complex energy was calculated.

Ly49H-p15E

The results obtained after docking of receptor Ly49H with p15E were observed. For each complex obtained, following data was analyzed. Here, the complex with minimum energy is the second one with D-complex energy -12.518432Kcal/mol and the complex representing minimum energy is shown below (Figure 10) (Table 2).

Ly49H-p30CA

The complex with minimum energy is the first one as depicted in Table (3) with D-complex energy - 11.061380 Kcal/ mol and the complex representing minimum energy is shown in (Table 3) (Figure 11).

Ly49H-p15MA

The complex with minimum energy is the first one as depicted in Table (4) with D-complex energy - 12.205013 Kcal/mol and the complex representing minimum energy is shown in Table (4) (Figure 12).

Ly49H-p12

The complex with minimum energy is the tenth one as depicted in Table (5) with D-complex energy - 8.455273 Kcal/ mol and the complex representing minimum energy is shown in Table (5) (Figure 13).

To understand the interaction between Ly49H, activating receptor (chain-X) and the ligands (chain-Y) present (p15E, p30CA, p15MA, p12) on the surface of YAC-1 cell line, docking was performed and the D-complex energies for each receptor– ligand pair was studied. The result shows significant interaction between each of the YAC ligands with Ly49H.

Ly49A-gp70 The results obtained after docking of receptor Ly49A with gp70 were observed. For each complex obtained, following data was analyzed (Table 9) (Figure 17). Here, the complex with minimum energy is the first one with D-complex energy -11.879547 Kcal/mol and the complex representing minimum energy is shown above.

Table 1: p815 surface expressed ligands.

Table 2: Representing minimum energy for Ly49H-p15E.

Table 3: Representing minimum energy Ly49H-p30CA.

Table 4: Representing minimum energy Ly49H-p30CA.

Table 5: Representing minimum energy Ly49H-p12.

Table 6: D-complex energies for Ly49A forLy49A-CD63.

Table 7: D-complex energies for Ly49A-galectin-3.

The evasion of tumors in spite of host immune defenses is by the interaction of various tumor ligands with their receptors on immune cells that affects the function of host cells involved in immune responses. So, different tumor-derived factors or ligands may affect the function of natural killer (NK) cells by upregulatory and down-regulatory modulation of receptors [4].

The present study aimed at identifying the multiple factors derived from NK sensitive cell line (YAC-1) and NK resistant cell line (P815) that might be involved in bringing about changes in the NK cytotoxicity outcome. Ly49H is a prototypic activating receptor while Ly49A has been taken as a prototypic inhibitory receptors found in mouse NK cells. Sequences of respective ligands from YAC-1 and P815 cells were obtained from NCBI. Further, PATCHDOCK was used to perform molecular docking of Ly49H with the YAC-1 ligands and Ly49A with P815 ligands.

PATCHDOCK determines top 10 complexes for each of the ligandreceptor complexes from which the D-complex energy was calculated.

The ligands expressed by YAC tumor cells p30CA,p12,p15MA and p15E show significant binding efficiency with Ly49H and the ligands expressed by P815 cells line galactin-3,CD63 (AD1 antigen),gp70 and p30show significant binding efficiency with Ly49A.

Our study provides direct evidence that in case of YAC1 cell line which was known to show susceptibility to NK cytotoxicity owing to its low level of expression of MHC I thus preventing engagement of inhibitory receptors on NK cells is not the only reason for its susceptibility. Yac1 cell expressed ligands, p30CA, p12, p15MA and p15E all show significant binding to activating NK cell receptor thus leading to activation signal transduction for cytotoxicity.

P815 cell line known to be resistant to NK cell mediated cytotoxicity has been known to express high levels of MHCI and hence engage inhibitory receptors on NK cells resulting in escape from NK mediated killing. However, in the present study, we have also shown that P815 expressed ligands galactin-3,CD63 (AD1 antigen),gp70 and p30, all show direct significant binding to inhibitory Ly 49 receptor thus providing direct evidence that engagement of tumor expressed ligands by inhibitory receptors are significant in providing resistance of P815 from NK mediated cell lysis.

Table 8: D-complex energies for Ly49A-p30.

Table 9: D-complex energies for Ly49A-gp7.

Killing of tumor cells by NK cells is a dynamic interaction and signaling process in which binding of activating ligands to NK activating receptors induce target cell mediated killing by initiating cascade of signaling reactions resulting in cytolysis by NK cells. Susceptibility of tumor cells to NK may often be due to reduced engagement of inhibitory receptors by down regulation of MHC I ligand for NK inhibitory receptors. However in case of YAC-1 cells, we have shown conclusively that it is not only down regulation of inhibitory signal that results in activation but direct engagement of activating receptors by tumor expressed ligands on YAC-1 cells results in upregulation of activation signals in NK resulting in susceptibility of YAC cells to NK mediated cytotoxicity. In case of NK resistant cell line, P815 engagement of specific tumor expressed ligands by inhibitory receptors in NK cells results in interference of the activating signaling cascade resulting in target cell being spared by NK cells. Each NK cell expresses a multitude of activating and inhibitory receptors. The sum total of these opposing signals results in the outcome of the NK target interaction.

In the present study, surface expressed molecules present on p815 and YAC-1 cells were studied for binding with inhibitory receptors and activating receptors respectively. The results obtained show that the binding affinities obtained with docking of murine NK inhibitory receptor Ly49A with the surface ligands of mastocytoma p815 were quite significant (CD63 with -6.579559, galectin-3 having -10.283447, p30 with -11.074407 and gp70 having -11.879547 binding affinities) this tumor is itself known to modulate NK by changing the receptor profile of it and hence, further increasing the inhibition. Multiple inhibitory ligands were engaged by p815 cells and further studies need to be carried out whether these ligands of NK resistant cell line cause modulations in inhibitory receptor profiling, while docking results of NK activating receptor Ly49H with the surface ligands of T-lymphoma YAC-1 were also significantly marked (p15E with -12.518432, p30CA with -11.061380, p15MA with -12.205013 and p12 with -8.455272), which activate the NK effector function and tumor cells could not resist themselves anymore and thus are killed.

In the same way, other important surface proteins can be taken in future studies like Moloney leukemia virus-determined cell surface antigen (MCSA) [5], NKTS [6], p71 [5,6,13-15], H-2a , etc., once their sequences are deduced.

In the present studies we tried to elucidate the molecular basis of susceptibility of YAC tumor cells and resistance of P815 cells to NK mediated cytotoxicity.

We are grateful to Department of Science and Technology, SERB Young Scientist Award which supported the present work. All the present work was done in Delhi Technological University, Department of Biotechnology.

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