Alcohol-sourced acetate impairs T cell function by promoting cortactin acetylation

Summary Alcohol is among the most widely consumed dietary substances. Excessive alcohol consumption damages the liver, heart, and brain. Alcohol also has strong immunoregulatory properties. Here, we report how alcohol impairs T cell function via acetylation of cortactin, a protein that binds filamentous actin and facilitates branching. Upon alcohol consumption, acetate, the metabolite of alcohol, accumulates in lymphoid organs. T cells exposed to acetate, exhibit increased acetylation of cortactin. Acetylation of cortactin inhibits filamentous actin binding and hence reduces T cell migration, immune synapse formation and activation. While mutated, acetylation-resistant cortactin rescues the acetate-induced inhibition of T cell migration, primary mouse cortactin knockout T cells exhibited impaired migration. Acetate-induced cytoskeletal changes effectively inhibited activation, proliferation, and immune synapse formation in T cells in vitro and in vivo in an influenza infection model in mice. Together these findings reveal cortactin as a possible target for mitigation of T cell driven autoimmune diseases.


INTRODUCTION
For appropriate immune response to occur, immune cells, such as T cells, must migrate within and outside of lymphoid organs. [1][2][3][4] The cytoskeleton plays a vital role during T cell migration, scanning for antigens, and cell activation. [5][6][7] To migrate, the cytoskeletal structures must remain highly dynamic. 3 Failure in cytoskeletal machinery leads to immunodeficiencies due to defects in immune synapse formation, chemotaxis, and migration. 8 Modulation of the components of cytoskeleton via post-translational modifications such as acetylation provides further fine-tuning to fit cell's demands. 9 Histone deacetylases (HDAC) reverse acetylation of variety of proteins and have been shown to impact immunomodulatory effects of T cells. 10 HDAC6 is one of the deacetylases acting upon cytoskeletal proteins that are required for proper immune synapse formation and migration. [11][12][13][14][15][16] For example, pharmacological inhibition of HDAC6 in a preclinical model of systemic lupus erythematosus (SLE) reduced infiltrating T follicular helper (T FH ) cells into germinal centers (GCs) with significant consequences on autoantibody titers. 13,14 Similarly, we have previously shown that increased exposure to alcohol and alcohol's main metabolite, acetate, affect T FH cell responses in the collagen induced arthritis (CIA)-a mouse model of inflammatory arthritis. 17 Mice exposed to either alcohol-sourced or directly supplemented acetate exhibited reduced T FH cell infiltration of B cell follicles and destabilized T FH -B cell conjugate formation both in vivo and in vitro. 17 Consequently, we observed acetate's suppression to be specific to T cell dependent humoral responses both in autoimmune and vaccination mouse models. 17 Inhibition of HDAC6 in the context of inflammatory arthritis has been shown to reduce disease severity to levels comparable to dexamethasone treatment. 12 Here, we hypothesized that acetate's inhibitory effect could be due to increased acetylation of the cytoskeletal proteins ultimately affecting T cell migration and function. Our hypothesis could explain alcohol's double-edged sword effect in regard to benefits in autoimmunity and damages to health in general. 18,19 Alcohol consumption has correlated with decreased severity in rheumatoid arthritis (RA), type 1 diabetes, SLE, and multiple sclerosis in humans as well as in disease mouse models. 19 Upon consumption, alcohol is first rapidly metabolized to toxic intermediary metabolite acetaldehyde, and then to acetate, contributing to increased serum concentrations. 20 It is important to note the detrimental health effects and induced molecular changes by acetaldehyde. 19,21 In our previous study where we made use of mouse model of alcohol consumption with RA, we reported that mice consuming 10% alcohol (v/v) have average blood alcohol concentration of about 0.03 g per milliliter (0.65 mM). 17,22,23 We also demonstrated an approximate 2-fold increase in blood acetate concentration from 0.04 mM to 0.08 mM upon chronic alcohol consumption. 17 In contrast to mice, humans demonstrated much higher blood alcohol and consequent blood acetate concentrations reaching approximately 2 mM concentrations upon alcohol consumption. 24,25 Our previous measurements in steady state mice, showed 0.04 mM acetate concentration in serum, while literature reported data in humans, shows about 0.5 mM. 17,24 While there is no data on acetate accumulation in human tissues upon alcohol consumption, akin to our mouse data, there is about 2x fold increase in serum acetate levels of alcoholics. 17,24 Once in the blood, acetate permeates cells where it is converted to acetyl-Co-enzyme A (acetyl-CoA) and used as a donor for protein acetylation. 26,27 Indeed, increased levels of intracellular acetyl-CoA were linked with significantly higher protein acetylation. 28 In 2019, Mews and colleagues showed that alcohol consumption can quickly lead to histone acetylation in the brain. 29 Considering worldwide use of alcohol, its contribution to rise in blood acetate concentrations, and acetate's potential to increase acetylation of intracellular proteins, we set out to investigate whether there is a link between acetate exposure and mitigation of cytoskeletal dynamics in T cells.
Our findings indicate that cortactin, previously not known to play a key role in T cell function, is expressed in T cells. We show that upon alcohol consumption, alcohol metabolism leads to increased acetate levels and accumulation in lymphoid organs. Consequently, T cells that are exposed to increased acetate concentrations, demonstrate increased acetylation of cortactin, leading to decreased F-actin binding. In turn we observed deficiencies in lamellipodia formation, in vitro and in vivo migration, and T cell activation. We also demonstrate reduced T cell function in alcohol consuming influenza infected mice.

Upon alcohol consumption acetate accumulates in lymphoid organs
Building upon our published results, we ventured to study whether the decreased infiltration of GCs by T and B cells along with reduced T FH -B cell contacts was due to cytoskeletal deficiencies following alcohol-sourced acetate exposure. 17 First, we set to quantify the exposure of T cells to acetate upon alcohol consumption by measuring acetate levels in alcohol-impacted and immune relevant lymphoid tissues such as the liver, inguinal lymph node (iLN), and spleen of alcohol-fed mice. We found that acetate indeed accumulates in these organs, especially in lymphoid organs reaching concentrations surpassing 5 mM ( Figures 1A-1C). Next, we quantified whether exposure to 5 mM acetate concentration results in incorporation to cellular metabolism. Previously it was shown that increased in vivo ethanol concentration can lead to an increase in intracellular acetate and citrate. 29 In an in vitro treatment of mouse naive CD4 + CD25 À CD44 low CD62L high T cells, hereafter naive CD4 + T cells, with 13 C labeled acetate, we were able to confirm an increase in 13 C containing citrate starting at exposure to 2 mM acetate concentrations ( Figure 1D). In a subsequent quantification of histone 3 (H3) acetylation levels in the same CD4 + T cells, we observed increased H3 acetylated at lysine 27 ( Figures 1E and S1A). Together, these findings confirm increased exposure of T cells to acetate, leading to increased protein acetylation within cells.

Exposure to acetate reduces T cell migration capacity
We next performed a trans-well migration assay of either mouse CD4 + or human Jurkat T cells in the presence and absence of acetate. Here, we found a 20%-30% decrease in trans-well migration of mouse and human derived CD4 + T cells ( Figures 1F and 1G). To study the potential impact of decreased T cell migration in vivo, primary mouse CD4 + T cells were pre-treated for 4 h with 5 mM acetate in vitro, loaded with CellTrace dye and combined at 1:1 ratio of control to acetate treated CD4 + T cells, to a total of 63 10 7 cells. We, then, adoptively transferred 6310 7 cells to recipient C57BL/6 wild type naive mice not supplemented with alcohol. Two hours post adoptive transfer, we observed about 50% decrease in migration of acetate treated CD4 + T cells to spleens of recipient mice compared to control CD4 + T cells (Figures 1H and S1B). The same observation was true at 48 h post adoptive transfer (Figures 1I and S1B). Next, we asked whether acetate exerts its effects on T cells via G-protein coupled receptor 43 (GPR43), a membrane receptor bound and activated by short-chain fatty acids (SCFA). 30 We found GPR43 knockout (GPR43KO) CD4 + T cells also migrate less in an in vitro trans-well assay in the presence of acetate ( Figure 1J). These data indicate that the exposure to acetate reduces T cell migration capacity in vitro and in vivo. iScience Article T cells exposed to acetate exhibit reduced total filamentous actin T cells require a dynamic cytoskeleton: rapid modifications of filamentous actin (F-actin), for migration, immune synapse formation and activation. 3 Upon quantification of total F-actin levels by Alexa Fluor 488-conjugated phalloidin staining and flow cytometry analysis in mouse naive CD4 + T cells exposed to 5 mM acetate, we found a dose dependent decrease of F-actin median fluorescence intensity (MFI) (Figures 2A and S2). Most of the F-actin is concentrated to the edges of the cell, also referred to as cortical actin or cortical F-actin, where it serves to help cells maintain and modify shape. 31 Hence, F-actin deficiency also manifests itself by increased cell deformability measured by real-time deformability cytometry (RT-DC). 32 We performed RT-DC and found an increase in T cell deformability upon acetate exposure ( Figure 2B). Plotting T cell deformability against cell size sustained the decrease in deformability upon acetate exposure ( Figures 2C and 2D). Together these data reveal a reduction of total F-actin in acetate-exposed T cells.

Exposure to acetate increases acetylation of cortactin in T cells
A recent review article by Mu et al. summarized the acetylation of various components of the cytoskeleton. 9 We identified cortactin, a facilitator and stabilizer of F-actin branching, as a likely candidate for further research as it has acetylation sites in each of its 6.5 repeat regions within the actin-filament binding domain iScience Article (ABD). 9 Initially, it was believed that T cells express hematopoietic homolog of cortactin, hematopoietic lineage cell-specific protein 1 (HS1). 33 But later, cortactin was discovered in dendritic cells (DCs), macrophages, and lymphocytes. 34 HS1, in comparison to cortactin, has 3.5 cortactin repeat regions and acetylation sites on only 2 of those repeats. 35 In addition, cortactin is a direct target of HDAC6. 9,36 As such, treatment of C57BL/6 mice with HDAC6 inhibitor reduced disease severity in an SLE preclinical model and akin to our findings with acetate-exposed CIA and T cell dependent vaccination mouse models. 13,14,17 Cortactin acetylation leads to decreased binding to F-actin and subsequent F-actin branching. 36 We first confirmed cortactin expression in mouse primary CD4 + T cells by RNA sequencing ( Figure S3A). Acetate treatment of Jurkat T cells increased acetylation levels of cortactin as shown by western blotting (Figures 3A and S3B). We then identified cortactin protein with high confidence (protein score À10lgPvalue 189.32) in Jurkat T cells by Mass Spectrometry (ESI MS/MS) and confirmed its acetylation, e.g., at the c-terminal lysine 181 of peptide VDKSAVGFDYQGK (score À10logP 28.34). Evidence of cortactin acetylation by proteomics, compelled us to mutate lysine residues relevant for cortactin-F-actin binding. As such, we mutated 6 lysine residues, acetylation of which previously have been reported to inhibit binding of cortactin to F-actin, to arginine residues at the acetylation sites (CTTN_KR) within cortactin ABD (see Figure S4C for overexpression western blot analysis). 36 Zhang et al., have demonstrated that the mutation of lysine residues to arginine blocks acetylation but still allows binding of cortactin to F-actin. 36 Microscopy analysis of F-actin MFI in Jurkat T cells overexpressing CTTN_KR exhibited increased F-actin amounts . Statistical analyses of panel C were carried out using a one-dimensional linear mixed model that incorporates fixed effect parameters and random effects to analyze differences between cell subsets and replicate variances, respectively. p-values were determined by a likelihood ratio test, comparing the full model with a model lacking the fixed effect term. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. iScience Article even in the presence of acetate ( Figure 3B). Further analysis of F-actin-bound cortactin by Fö rster resonance energy transfer (FRET) revealed reduced F-actin-bound cortactin levels upon acetate treatment, and that Jurkat T cells expressing mutated cortactin (CTTN_KR) were resistant to such reduction ( Figures 3C and 3F). Further, we also quantified acetate's effect on cortactin-F-actin binding in HEK293T cells and found similar results ( Figure 3D). Next, in trans-well migration assays we found that the expression of acetylation-resistant cortactin (CTTN_KR) rescued acetate-induced migration deficiency ( Figure 3E). To rule out the effect of CTTN_KR overexpression, we generated Jurkat T cells overexpressing wild type cortactin ( Figures S4A and S4B). Simultaneous assessment of acetate's effect on F-actin levels in Jurkat T cells overexpressing wild-type or CTTN_KR mutant cortactin revealed decreased F-actin in case of wild type cortactin expressing cells but not the mutant ( Figures S4C-S4E). In addition, acetate exposure of Jurkat T cells overexpressing wild type cortactin led to decreased migratory capacity ( Figure S4F). To recreate the 3-dimensional (3D) environment in which T cells migrate and function, we incorporated Jurkat T cells into Matrigel domes and imaged using Leica Thunder 3D Imaging microscopy (Videos S1 and S2, control and acetate treated, respectively). We observed decreased total distance migrated of acetate treated Jurkat T cells ( Figure 3G). To better understand deficiencies in migration, we performed 2D Jurkat T cell tracking in cell culture treated wells by live-cell microscopy ( Figure 3H). We found that acetate treated Jurkat T cells had a reduced linearity of forward progression and directional change rate, and that cells expressing acetylationresistant cortactin (CTTN_KR) were resistant to the effects of acetate ( Figures 3I and  3J). Modifications to F-actin are required for lamellipodia formation and proper T cell migration. 3 Cortactin has been shown to be required for lamellipodial persistence and cell migration. 37 To study the highly dynamic lamellipodia formation and persistence, we generated Jurkat T cells expressing LifeAct-mScarlet-i_N1, a short peptide that binds F-actin and allows for visualization by confocal microscopy. Analysis of lamellipodia numbers, per cell, for the period of 60 s, revealed reduced lamellipodia formation upon acetate treatment of Jurkat T cells ( Figures 3K, 3L, and Video S3). Finally, western blot analysis of the splenocytes isolated from alcohol-fed mice exhibited increased levels of acetylated cortactin in comparison to water-fed control mice ( Figures 3M and S3C). In a proof-of-concept experiment, primary CD4 + T cells isolated from cortactin knockout mice exhibited severe impairment of migration in an in vitro trans-well assay, exhibiting little known importance of cortactin for proper T cell migration (Figure 3N). These findings indicate that alcohol consumption can directly increase acetylation of cortactin in secondary lymphoid organs (SLOs) such as spleen, and such an increase in T cells impairs T cell migration capacity. iScience Article Acetate-induced F-actin deficiency impairs T cell activation and proliferation F-actin cytoskeleton is required for T cell-antigen presenting cell (APC) interaction and immune synapse formation. [38][39][40] We performed confocal fluorescence microscopy to visualize F-actin at the immune synapse by phalloidin staining and quantified MFI values. For this, we isolated DCs and CD4 + T cells from previously NP-CGG immunized mice and incubated both cell types at 1:1 ratio, at 10 7 cells per mL concentration (total volume 50 mL) following NP-CGG pulse of DCs and treatment of T cells ( Figure 4A). We found a decrease in F-actin accumulation at the immune synapse within T cells between T cells and DCs ( Figure 4B). To rule out the possible effect of acetate on DC immune synapse engagement, we repeated this experiment by using T cell activation beads. Activation of naive CD4 + T cells with anti-CD3 and anti-CD28 antibody coated beads in the presence of acetate also resulted in reduced F-actin amounts, as quantified by Alexa Fluor 488-phalloidin MFI (Figures 4C and S5A). Next, we performed co-culturing experiment of NP-CGG antigen-pulsed DCs and CD4 + T cells to better interrogate if acetate-directed F-actin deficiency affects T cell activation. T cells were either pre-treated with 5 mM acetate or vehicle, while antigen-pulse of DCs with NP-CGG was performed in an absence of acetate. Later upon combining DCs and T cells, acetate treatment was continued in an effort to replicate in vivo acetate exposure within lymphoid organs. Here, flow cytometry analysis revealed reduced surface CD69 levels on acetate treated CD4 + CD25 + T cells indicating reduced activation ( Figures 4D and S5B). In the same experiment, flow cytometry analysis of acetate treated CD4 + T cells revealed that Ki67 levels, a proliferation marker, were reduced ( Figures 4B and S5B). These experiments document the sensitivity of T cells to cytoskeletal deficiencies for migration, immune synapse formation, and activation, and they shed light on our previously published finding of reduced T FH -B cell conjugate formation upon acetate exposure in vivo and in vitro. 17

Acetate-exposed mice exhibit inhibited T cell responses in the lungs upon influenza infection
Since we have witnessed acetate's effect on T cell cytoskeleton, we wondered whether alcohol-sourced acetate will impair T cell migration and activation during viral infection. For this, we utilized mouse influenza infection model. Here, alcohol consuming mice were infected with influenza H1N1 and two weeks later lungs isolated and analyzed. Flow cytometry analysis revealed reduced numbers of CD8 + CD69 + CD103 + resident T cells in the lungs of alcohol consuming influenza infected mice in comparison to water-fed control mice ( Figures 4F and S6). Interestingly, we also found decreased percentage of CD69 + Tet + influenza specific CD8 + T cells and CD69 + CD8 + T cells in alcohol consuming influenza infected mice in contrast to water-fed control mice ( Figures 4G and 4H). In addition, we observed reduced percentage of CXCR3 + CD8 + T cells infiltrating the lungs of alcohol consuming mice in comparison with water-fed control mice ( Figure 4I). Flow cytometry analysis of the CD4 + T cells in the lungs, revealed a reduction in percentage of CD69 + and also CXCR3 + CD4 + T cells in alcohol consuming mice in contrast to water-fed control mice ( Figures 4J and 4K). Together these data reflect deficiencies in T cell migration, activation, and function in vivo and support our earlier adoptive transfer experiments of acetate-treated T cells.

DISCUSSION
Our findings indicate that alcohol-sourced acetate accumulates in lymphoid organs, increases cellular acetylated cortactin levels preventing F-actin branching and impeding T cell migration (overview Figure 5). The Arp2/3 complex is a part of F-actin branching nucleator complex. 40 But it does require cortactin to facilitate its binding to F-actin. Whereas cortactin was shown to increase the affinity of Arp2/3 complex for F-actin by almost 20-fold. 41 In addition, cortactin plays a key role in linking cortical F-actin to the cell membrane. 42 Inhibition of Arp2/3 complex formation reduced T cell deceleration upon encountering high affinity antigens to a level observed for low affinity antigens. 43 This finding potentially explains the weak immune synapse formation in our current study and also reduced T FH -B cell conjugate formation upon acetate exposure in our previously published findings. 17 Currently, there are over dozen nucleating factors known to facilitate Arp2/3 complex binding to F-actin. 44 Albeit, how each and every one of such nucleating factors, including cortactin, play a role in T cells is poorly understood. F-actin rearrangement enables cells to migrate, direct adhesion molecules to cell-cell contact zones, divide, and regulate various other cellular processes. 45,46 Cytoskeleton is one of the central elements for optimum T cell function. 47 For example, functional actin cytoskeleton has been shown to be important for T cell sampling of antigen-MHCII complexes on APCs with subsequent role in immune synapse formation. 38 CD4 + and CD8 + T cell migration patterns through SLOs differ, CD4 + T cells spend more time probing MHCII molecules presented on DCs and are significantly faster in entering and exiting SLOs than CD8 + T cells. [48][49][50] It is possible that diminished F-actin branching can therefore mitigate TCR coupling with high-affinity antigens and reduce T cell arrest and subsequent activation. Interestingly, in a mouse model of type iScience Article with acetate-yielding chow exhibited a reduction in autoreactive T cells (both CD4 + and CD8 + ) and ultimately reduced the incidence of the disease. 51 In another study of experimental autoimmune encephalomyelitis (EAE), cortactin knockout mice exhibited decreased incidence, disease severity, and infiltration of the central nervous system by CD4 + T cells. 52 Interestingly, we observed the same effect in alcohol-fed EAE mice. 17 Similarly,  Figure S5A). (C-G) Flow cytometry analysis of percentage of CD69 + CD25 + CD4 + and (D) Ki67 MFI of CD4 + T cells in vitro co-culture with NP-CGG pulsed DCs in the presence or absence of 5 mM acetate (for gating see Figure S5B). Flow cytometry analysis of CD69 + CD103 + (F) CD8 + T cells and (G) influenza specific tetramer (Tet + ) CD8 + T cells in the lungs of alcohol-fed mice in comparison to water-fed control mice.
(H and I) Flow cytometry analysis of percentage of CD69 + and (I) percentage of CXCR3 + of CD8 + T cells in the lungs of alcohol-fed mice in comparison to water-fed control mice. (J and K) Flow cytometry analysis of percentage of CD69 + and (K) percentage of CXCR3 + of CD4 + T cells in the lungs of alcohol-fed mice in comparison to water-fed control mice (each point represents one mouse. n = 6 control, n = 5 alcohol-fed mice) (for gating strategies of panels F-K see Figure S6). Representative data shown either from one of three independent experiments (D, E), or two independent experiments (A, B, C, F-K) and expressed as mean G SD. Statistical difference was determined by Student's two-tailed t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.  54,55 Identification of an Achille's heal of T cell migration, motility, and immune synapse formation can set the stage for pharmacological and/or natural targeting in cases of autoimmunity. Further, such interventions can also help improve immune responses to vaccinations. In an evolutionary perspective, hominids adapted to alcohol metabolism by feeding on fermenting fruits from the forest floor. 56 It is possible that indirect alcohol consumption mitigated adverse immune responses in early humans under high microbial load. Today, we see the same effects reflected in mitigation of autoimmunity and susceptibility to infections among alcohol consumers. 57

Limitations of the study
Although data shown herein directly link alcohol consumption to increased acetylation of cortactin, it is important to note that alcohol-sourced acetate can have other effects on T cell biology. While our study provides a robust result on the new role of cortactin in T cells, there are currently no mouse strains where cortactin is knocked out specifically in T cells, hence our limited use of in vivo models. Additionally, cortactin is highly conserved among different immune cells and tissues. Therefore, warranting future studies to measure the effect of cortactin acetylation on antigen presenting cells, B cells, macrophages, and other types of cells.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.
Data and code availability d Proteomics data are available via ProteomeXchange with identifier PXD037723. Gene expression data is available on Gene Expression Omnibus with dataset identifier GSE216415. Original Western Blot images are included in Supplemental Information. Microscopy data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code.
d The data that support the findings of this study are available from the corresponding author upon reasonable request by email to mario.zaiss@uk-erlangen.de.

Mice
Female, 6 weeks old C57BL/6NCrl mice were purchased from Charles River (Germany). Mice were cohoused for 3 weeks prior to start of experiments. Cortactin knock out mice were kindly provided by Prof. Dr. Klemens Rottner from Helmholtz Centre for Infection Research (Braunschweig, Germany). All mice were housed, and experiments were conducted under specific pathogen-free conditions. All protocols for animal experiments were approved by the local ethics authorities of the Regierung von Unterfranken

METHOD DETAILS
Adoptive transfer experiments CD4 + T cells (naïve or total) were enriched with naïve or total CD4 + T cell negative enrichment kits (Stemcell, Biolegend) from the spleens of female, 8 weeks old C57BL/6NCrl mice. T cells were then either treated with 5 mM sodium acetate or sodium matched R10 medium (RPMI 1640, 10% v/v Fetal Bovine Serum, supplemented with 50 mM b-mercaptoethanol, non-essential amino acids, 2 mM L-Glutamine, sodium pyruvate, 10 mM HEPES, and 1% penicillin and streptomycin) for 4 hours. Cells were spun down (300xg) and washed two times with PBS at room temperature (RT). Cells were then loaded with CellTraceâ cell dye (CFSE and Violet, for control and acetate treatment) according to manufacturer's instructions (Thermo Fisher) and incubated further 30 minutes with corresponding treatment (control or acetate containing R10 medium). Cells were then counted and adjusted to 3x10 7 cells for each treatment and combined to make 6x10 7 cells in PBS prior to transfer to recipient mice (C57BL/6NCrl, female, matching age) intravenously in the tail vain. Recipient mice were sacrificed 2 hours and 48 hours post adoptive transfer, and spleens collected for flow cytometry.

In vitro migration
Jurkat T cells or naïve CD4 + T cells were treated with 5 mM sodium acetate containing R10 medium for 4 hours prior to Transwell migration assay. T cells were then seeded at 10 5 cells in 100 mL total volume above 3 mm and 8 mm pore size transwell inserts (GreinerBio), mouse CD4 + T cells and Jurkat T cells, respectively. Transwell inserts were placed in 24-well plate containing R10 medium matching the volume height within the insert (600 mL), 20 ng/mL of CCL19 (mouse CD4 + T cells) or 5 ng/mL CCL2 (Jurkat T) added to the bottom well and incubated for 4 hours. Migrated cells were collected from the bottom wells and centrifuged, resuspended in 100 ml FACS buffer, quantified by flow cytometry. In case of primary CD4 + T cells, we have also performed trans-well migration assay through b.End3 endothelial cell barrier.
In vitro co-culture of antigen-presenting cells (APCs) and T cells

RNA sequencing
Naïve CD4 + T cells were treated with 2.0 mM sodium acetate or vehicle (R10 medium) containing R10 medium for 4 hours. RNA was isolated using the Rneasy micro kit (Qiagen). Sequencing was performed on the Illumina platform (Novogene, Europe). Raw reads were processed through fastp (Galaxy). Mapping the processed data to the reference genome (GRCm39) was performed using Star software, and alignment visualization was done with the integrative genomics viewer (IGV). Raw data is available under GEO accession GSE216415.

Immune synapse formation
Dendritic cells (DCs) and CD4 + T cells were enriched from C57BL/6CNrl mice immunized two times, 14 days apart, with 100 mg NP-CGG in 200 mL of Imjectä Alum and sacrificed on day 21 (Stemcell, Thermo Fisher, LGC). CD4 + T cells were pre-treated with 0.5, 2.0, 5.0 mM acetate or vehicle (R10 medium) for 6 hours in R10 medium. Simultaneously, DCs were pulsed with 20 mg/mL NP-CGG in R10 medium for 6 hours. Then, 10 6 pre-treated naïve CD4 + T cells and 10 6 NP-CGG pulsed DCs were combined in 100 mL of R10 medium for 15 minutes at 37 C. Then 1.5 mL of 1.5% paraformaldehyde (PFA) was added to arrest T-DC conjugates. Cells were then washed with PBS+2% FCS (v/v) three times. Cells were then stained for flow cytometry with anti-mouse CD4 BV421ä and anti-mouse CD11c BV510ä, or for microscopy anti-CD4 Alexa Fluor 647ä only in PBS+2% FCS (v/v) for 30 minutes at 4 C. Cells were washed, and fixed with eBiosciencesä IC fixation buffer for 15 minutes at 4 C. Cells were washed with PBS three times and permeabilized with 1x eBiosciencesä permeabilization buffer for 1 hour at room temperature. Phalloidin-iFluor 488 stock solution was diluted in PBS containing 1% w/v BSA 1000x. After permeabilization, cells were washed three times and Phalloidin-iFluor 488 solution added for 2 hours at RT. Then, cells were washed with PBS three times with 5-minute incubations in PBS to remove all unbound Phalloidin-iFluor 488. For microscopy samples, nuclei were staining with DAPI in PBS for 5 minutes and then washed and resuspended in 60 mL of PBS. Drops of 30 mL were placed on microscope slides and allowed to dry in the dark. After, ProLongä Glass Anti-fade mounting medium was used to mount slides and let cure for 24 hours. Samples for flow cytometry were resuspended after washing Phalloidin-iFluor 488 out and resuspending in PBS.

T cell migration within 3D Matrigelä
Jurkat T expressing mScarlet-H cells were maintained in R10 medium at a denstity of 1x10 5 -1x10 6 cells/ml. Jurkat T cells were then split into two groups of untreated and treated cells. Treatment of Jurkat T cells was performed with 5 mM sodium acetate containing R10 medium for a total of 48 hours. Afterwards, approximately 1.5x10 5 cells were spun down, supernatant was aspirated, and cells were resuspended in 25 mL ice cold R10 medium. Then, cells were mixed with 25 mL Matrigel (cold tips) and a dome was pipetted in the middle of a pre-warmed 8-well slide (Ibidi). The slide was then placed in an incubator to let the Matrigel polymerize and after 15 minutes the slide was taken out, and the chambers were filled with 280 ml of warm R10 medium. Additionally, 5 mM acetate was added to the chambers containing acetate-treated Jurkat T cells. Excitation was carried out at 555 nm and emission detected with 572 nm filter, timelapses with approximately 100 mm Z-Stack were acquired for 3-4 hours with Leica Thunder Imager with 20x dry objective with NA 0.8 and instant computational clearing. Timelapses were analyzed with TrackMate plugin from ImageJ software and visualized using VisualizeTracks plugin. 58

Live cell tracking
Jurkat T cells and Jurkat T cells overexpressing acetylation resistant cortactin (CTTN_KR) were cultured in R10 medium supplemented with 5 mM sodium acetate for 4 hours. For acetate treatment control, Jurkat T cells were cultured in R10 medium. As a control for plasmid electroporation, Jurkat T cells electroporated with mScarlet_H plasmid were used. The cells were then transferred to incubated and CO2 gassed Zeiss Cell Discoverer (Carl Zeiss) live imaging microscope. Then images were taken by brightfield illumination with Apochromat 10x objective in 2-minute intervals for a total of 180 minutes. Cells were then tracked using TrackMate plugin for Fiji. 58,59 To visualize individual tracks, tracks were obtained from TrackMate and re-centered at the origin of the plot.

Live cell imaging of lamellipodia
Jurkat T cells overexpressing pLifeAct_mScarlet-i_N1 (see Electroporation methods), were treated with 5 mM sodium acetate or vehicle (R10 medium) containing R10 medium for 4 hours. Later, cells were seeded into m-Slide 8 well microscope slides (ibidi) and placed into incubated microscopy chamber where 5 ng/mL concentration of CCL2 was added to induce polarization. Zeiss Spinning Disc Axio observer Z1 microscope with oil immersed 63x 1.2 NA objective and EVOLVE 512 EMCCD with Yokogawa CSU-X1M 5000 was used to locate cells. Cells were illuminated with a laser at 561 nm wavelength, and emissions captured through band-pass filter 629/62 every 4 seconds for 60 seconds. Images were later processed and analyzed for numbers of lamellipodia at each time point and counted. Statistical analysis was done by area under the curve analysis.

Electroporation of Jurkat T cells
Jurkat T cells were maintained in R10 medium. 10 7 Jurkat T cells were washed two times with RPMI1640 medium only, centrifugation steps at 200xg for 10 minutes, at RT. Jurkat T cells were then washed one more time with 5 mL of OptiMEMâ medium and resuspended in 100 mL of OptiMEMâ medium. 25 mg (1 mg/mL concentration) of plasmid DNA was added to electroporation 4 mm cuvettes (Bio-Rad) first and then cells added to the cuvettes. Electroporation was carried out with Bio-Rad Gene Pulser Xcell Electroporation Systems at Square wave, 500 Volts, 3 ms, Pulse 1, Interval 0). After the electroporation, cells were transferred to T25 cell culture flasks with 5 mL of prewarmed (37 C) R10 medium and incubated overnight. For control purposes, no plasmid DNA containing cell mixture was used for electroporation.