n-3 Polyunsaturated Fatty Acids Impede the TCR Mobility and the TCR–pMHC Interaction of Anti-Viral CD8+ T Cells

The immune-suppressive effects of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) on T cells have been observed via multiple in vitro and in vivo models. However, the precise mechanism that causes these effects is still undefined. In this study, we investigated whether n-3 PUFAs regulated T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) interactions. The expansion of anti-viral CD8+ T cells that endogenously synthesize n-3 PUFAs (FAT-1) dramatically decreased upon lymphocytic choriomeningitis virus (LCMV) infection in vivo. This decrease was not caused by the considerable reduction of TCR expression or the impaired chemotactic activity of T cells. Interestingly, a highly inclined and laminated optical sheet (HILO) microscopic analysis revealed that the TCR motility was notably reduced on the surface of the FAT-1 CD8+ T cells compared to the wild type (WT) CD8+ T cells. Importantly, the adhesion strength of the FAT-1 CD8+ T cells to the peptide-MHC was significantly lower than that of the WT CD8+T cells. Consistent with this result, treatment with docosahexaenoic acid (DHA), one type of n-3 PUFA, significantly decreased CD8+ T cell adhesion to the pMHC. Collectively, our results reveal a novel mechanism through which n-3 PUFAs decrease TCR-pMHC interactions by modulating TCR mobility on CD8+ T cell surfaces.

CD8 + T cells recognize antigens through physical contact between T cell receptors (TCRs) and the peptide-major histocompatibility complex (pMHC) on antigen-presenting cells. This triggers T cell activation and leads to the eradication of the damaged target cells (e.g., tumor and virus-infected cells). Therefore, investigating the mechanism that regulates the physical interaction between the TCR and the pMHC is important in understanding the overall T cell-mediated immune responses. Although TCR affinity cannot be altered by somatic hypermutation, membrane characteristics such as fluidity and lipid rafts can influence the degree of bond formation between the TCR and the pMHC [23][24][25]. Since n-3 PUFAs are known to affect membrane characteristics [21,22], the TCR-pMHC interaction may be regulated by n-3 PUFAs, which may subsequently influence CD8 + T cell functionality.
The lymphocytic choriomeningitis virus (LCMV) is a member of the Arenaviridae family of viruses and has a negative-strand RNA genome [26]. An acute LCMV (strain Armstrong) infection in mice strongly triggers the activation of anti-viral CD8 + T cells, leading to a rapid viral clearance within seven to eight days post-infection [27,28]. Therefore, the infection of laboratory mice with the LCMV is a useful animal model to investigate the underlying mechanisms of the anti-viral CD8 + T cell response.
In this study, we investigated the potential role of n-3 PUFAs in regulating the TCR-pMHC interactions. Remarkably, we found that n-3 PUFAs reduced TCR mobility on the surface of CD8 + T cells, which potentially causes decreased TCR-pMHC adhesion.

Isolation of CD8 + Cells
CD8 + cells were purified using a MojoSort mouse CD8 + T cell isolation kit (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, the splenocytes were incubated with a CD8 + negative selection antibody cocktail and incubated with streptavidin-coated metal beads. The desired cells were purified with a magnet, and the unwanted cells were washed away. CD8 + T cell purity (>95%) was confirmed via flow cytometry.

In Vitro Activation of CD8 + Cells
The splenocytes were incubated in the presence of GP 33-41 peptide (1 µg/mL) and 6 µg/mL of LPS (Sigma-Aldrich, Saint Louis, MO, USA) for six days. Two days after the initial stimulation, 12.5 U/mL of murine IL-2 (Peprotech, Rocky Hill, NJ, USA) was added to the media. The CD8 + cells were isolated with a MojoSort mouse CD8 + T cell isolation kit (Biolegend, San Diego, CA, USA) before use.

Generation of Bone Marrow-Derived Dendritic Cells
The bone marrow cells obtained from the femur of naïve C57BL/6 mice were transferred to a 100 mm petri dish and cultured in an RPMI medium supplemented with 200 U/mL of mGM-CSF (Peprotech, Rocky Hill, NJ, USA). Six days later, the cells were analyzed for the expression of CD11b, CD11c, and MHC II by flow cytometry before further experiments.

Trans-Well Chemotaxis Assay
Purified CD8 + T cells were resuspended in RPMI media (2.0 × 10 6 cells/mL), and 100 µL was added into a SPL Insert™ Hanging well (pore size: 3 um) (SPL, Pocheon, Korea). 300 µL of RPMI media with or without CCL19 (Peprotech, Rocky Hill, NJ, USA) was placed in the bottom chamber. Transferred cell numbers were normalized to the relative cell numbers.

Highly Inclined and Laminated Optical Sheet (HILO) Microscopic Analysis
We diluted the CD8 + T cells that were stained with a PE-conjugated anti-TCR-β antibody in the imaging buffer (4 mM Trolox, 0.8% (w/v) glucose, 50 mM NaCl, 165 U/mL glucose Oxidase, 2170 U/mL catalase) for enhancing the stability of the PE during the imaging acquisition process and added the cells in the imaging chamber for monitoring TCRs of the cells using a highly inclined and laminated optical sheet (HILO) microscope [31]. The homebuilt objective total internal reflection microscope was modified to excite the cells at a highly inclined angle. The 532 nm laser (Cobolt, Sweden) excited PE-conjugated TCRs in the cells through a 60× water immersion objective (Olympus, Japan) that gathered the fluorescence emission of PE to the EMCCD camera (Andor iXon897, Andor Technology, Belfast, UK). Then, the recorded fluorescence movies that were obtained with the EMCCD camera were analyzed using the ImageJ (https://imagej.nih.gov/ij/) software (NIH, Bethesda, MD, USA).

Statistical Analyses
All statistical significances were calculated using Student's t-test. The error bars indicate the SEM (standard error of the mean). The calculated mean values were compared and defined as statically significant or not. All the experiments were repeated independently at least three times.

n-3 PUFAs Reduce in Vivo Expansion of Anti-Viral CD8 + T Cells
In agreement with a previous report [13], the P14 T cells endogenously synthesizing the n-3 PUFAs (FAT-1 P14) that were adoptively transferred into the naïve wild type (WT) mice displayed dramatically reduced expansion capability upon LCMV infection in vivo when compared to WT P14 cells (Figure 1a). Since the n-3 PUFAs are involved in the survival of diverse cell types including tumor cells [32][33][34], we compared the survival rates between the WT P14 and the FAT-1 P14 cells in vivo. The naïve WT P14 and FAT-1 P14 cells were stimulated with the LCMV glycoprotein 33-41 (gp33) peptide in vitro to generate effector cells that were transferred into the naïve WT mice respectively. Seven days later, the numbers of adoptively transferred WT P14 and FAT-1 P14 cells were measured in spleens. However, there was no significant reduction in the number of adoptively transferred FAT-1 P14 cells when compared to WT P14 cells. This indicates that the endogenous n-3 PUFAs did not reduce the survival of the effector CD8 + T cells (Figure 1b). Next, we investigated whether the n-3 PUFAs affected the memory CD8 + T cell response against the LCMV infection. The in vitro-generated effector WT P14 or FAT-1 P14 cells were transferred into the naïve WT mice respectively, and 20 days later, the mice were challenged with the LCMV to measure the expansion of the WT P14 and FAT-1 P14 cells, respectively. The expansion of the FAT-1 P14 cells was greatly reduced compared to WT P14 cells in both the spleen and liver, indicating that the memory anti-viral CD8 + T cell response was reduced by the endogenous expression of n-3 PUFAs (Figure 1c). Collectively, these results suggest that endogenous n-3 PUFAs down-regulate the expansion of CD8 + T cells during an LCMV infection in vivo. n-3 PUFAs may influence the intrinsic activation potential of CD8 + T cells, resulting in a differential expansion pattern between WT P14 and FAT-1 P14 CD8 + T cells. To test this hypothesis, the WT P14 and FAT-1 P14 CD8 + T cells were treated with PMA/ionomycin that diffuses directly into the cytoplasm to activate the Protein Kinase C and NFAT signaling pathways [35]. However, no significant differences between the WT P14 and FAT-1 P14 CD8 + T cell proliferation (CFSE+ cells, WT: 71% ± 4%; FAT-1: 74% ± 1%) were observed (Figure 2c). The chemotactic migration of naïve CD8 + T cells into secondary lymphoid organs is a critical step in initiating an anti-viral CD8 + T cell response. Therefore, we tested whether n-3 PUFAs influence the chemotactic migration ability of CD8 + T cells. The expression level of a major lymph node trafficking receptor, CCR7, was not significantly different between the WT and FAT-1 CD8 + T cells (Figure 2d). Furthermore, when the T cell migration ability was measured with the trans-well assay, the chemotactic migration in response to CCL19 (a CCR7 ligand) was comparable between the WT and FAT-1 CD8 + T cells (Figure 2e). These results indicate that endogenous n-3 PUFAs do not affect the intrinsic activation and migration potential of anti-viral CD8 + T cells.

n-3 PUFAs Decrease TCR Mobility on CD8 + T Cell Membrane
n-3 PUFAs are known to regulate cell membrane fluidity [36], which could affect receptor-ligand interaction [37,38]. Since TCR-pMHC interaction is prerequisite for the activation of T cells, we investigated whether endogenous n-3 PUFAs affect the TCR mobility of CD8 + T cells. To this end, the WT or FAT-1 CD8 + T cells that were stained with the PE-conjugated anti-TCR-β antibody were analyzed by a highly inclined and laminated optical sheet (HILO) microscope (Figure 3a). To visualize TCRs on cells, we used a home-built fluorescence microscope with HILO illumination because it provided an improved signal-to-noise ratio compared to a conventional wide-field microscope. The fluorescent movies of the WT and FAT-1 CD8 + T cells displayed the mobile or static fluorescent spots of the TCRs. Then, we analyzed the fluorescent movies by using a temporal color coding for the first 10 frames of each movie, where each frame has its own unique color. With the temporal color coding, the static spots tended to exhibit white colors in the merged image for the first 10 frames because the fixed spots that located at the same position over multiple time frames displayed the overlapped white colors. However, the mobile spots exhibited the unique color of each frame because it changed its position over time (Figure 3a). The whiter spots from the FAT-1 CD8 + T cells compared with WT cells demonstrated that the TCRs of WT cells possessed a significantly higher fluidity than the ones of the FAT-1 cells (Figure 3b,c). Thus, these results show that n-3 PUFAs reduced TCR mobility on the CD8 + T cell surface.

Discussion
Omega-3 has therapeutic potential against several immune disorders. Although its anti-inflammatory activity on CD4 + and CD8 + T cells is known to play a central role, the detailed underlying mechanism of its therapeutic potential is still unknown. The aim of this study was to identify how n-3 PUFAs reduced the activation of T cell responses. The physical interaction between a TCR and the cognate antigen, in the context of the MHC molecule on the target cell, leads to the activation of the T cell. Surprisingly, the TCR mobility of n-3 PUFA-sufficient FAT-1 CD8 + T cells was significantly lower than that of the WT cells.
Additionally, the n-3 PUFAs reduced the TCR-pMHC bond formation. These results provide novel insight into how n-3 PUFAs regulate T cell activation by interfering with TCR-pMHC interactions.
Incorporation of n-3 PUFAs into the membrane alters the phospholipid composition, lipid raft formation, and cholesterol deposition in the membrane, which can change certain membrane characteristics such as fluidity [41,42]. Indeed, our results indicate that endogenous n-3 PUFAs reduce CD8 + T cell TCR mobility. Since TCR mobility is important for supplying TCRs to the immunological synapse during T cell and target cell interactions [43], TCR-pMHC interactions could be affected by the degree of TCR mobility. Our results demonstrated that TCR-pMHC interactions were significantly reduced in n-3 PUFA-sufficient CD8 + T cells. Therefore, these results support that an n-3 PUFA-mediated reduction in TCR mobility possibly interferes with CD8 + T cell TCR and antigen interactions in the context of MHC molecules on target cells. Although further investigations are required, n-3 PUFAs might also induce other modifications such as changes in cytoskeleton alignment, the motion of other large membrane proteins, and signaling/clustering associated with T cell adhesion, which could influence TCR-pMHC interaction.
Since n-3 PUFAs can affect membrane fluidity [44,45], the mobility of other proteins on the cell surface may also be changed. This might influence the interactions between other surface proteins. For example, the activation of CD4 + T cells also requires physical interaction between TCRs and MHC class II molecules. Therefore, the CD4 + T cell response might be influenced by membrane-incorporated n-3 PUFAs. Indeed, n-3 PUFAs are known to down-regulate the antigen-dependent activation of CD4 + T cells [46,47]. Therefore, n-3 PUFAs could be used to suppress the unwanted hyperactivation of both CD8 + and CD4 + T cells during infections and autoimmune diseases.
In this study, we developed a novel plate-based TCR-pMHC binding assay to confirm whether n-3 PUFAs regulated TCR-pMHC interactions. While the gp 33-41 tetramer staining did not discriminate the TCR-pMHC interactions between the WT P14 and FAT-1 P14 cells, this assay result indicated that the TCR-pMHC interactions of the FAT-1 P14 cells were significantly lower than that of the WT P14 cells. Therefore, this simple assay system is a useful tool for studying the regulation of physical interactions between TCR-pMHC. Furthermore, this assay system could be used to study the interaction between other diverse surface proteins.
Collectively, our results provide novel insight into how antigen recognition by anti-viral CD8 + T cells is regulated along with its clinical application.