Polarity of CD4+ T cells towards the antigen presenting cell is regulated by the Lck adapter TSAd

Polarization of T cells towards the antigen presenting cell (APC) is critically important for appropriate activation and differentiation of the naïve T cell. Here we used imaging flow cytometry (IFC) and show that the activation induced Lck and Itk adapter T cell specific adapter protein (TSAd), encoded by SH2D2A, modulates polarization of T cells towards the APC. Upon exposure to APC presenting the cognate antigen Id, Sh2d2a−/− CD4+ T cells expressing Id-specific transgenic T cell receptor (TCR), displayed impaired polarization of F-actin and TCR to the immunological synapse (IS). Sh2d2a−/− T-cells that did polarize F-actin and TCR still displayed impaired polarization of PKCξ, PAR3 and the microtubule-organizing center (MTOC). In vitro differentiation of activated Sh2d2a−/− T cells was skewed towards an effector memory (Tem) rather than a central memory (Tcm) phenotype. A similar trend was observed for Id-specific TCR Sh2d2a−/− T cells stimulated with APC and cognate antigen. Taken together our data suggest that TSAd modulates differentiation of experienced T cells possibly through polarization of CD4+ T cells towards the APC.

Sh2d2a−/− mice displayed reduced clearance of a mutant murine cytomegalovirus which does not activate the NK cell receptor Ly49H 21 . Recently, we found that TSAd affects CD4+ T cell-mediated rejection of experimental multiple myeloma 19 . Wild type mice are highly susceptible to MOPC315 myeloma, while mice carrying transgenic TCR, recognizing a peptide (Id) derived from the variable region of the L chain of the MOPC315 IgA (Id-specific TCR), are partially protected 22 . The Id-peptide is presented on I-E d by tumor-infiltrating macrophages. Rejection is caused by interplay of Th1 T cells and M1-activated macrophages 23 . The Sh2d2a−/− Id-specific TCR mice displayed increased resistance towards myeloma 19 , however the molecular mechanism for the increased resistance remains to be determined.
We here examined the hypothesis that TSAd regulates T cell differentiation through altered polarization of T cells towards APC. Using imaging flow cytometry (IFC), the distribution of signalling molecules towards the IS in T-cell/APC conjugates was assessed 19 . We found that in Id-specific TCR T cells, TSAd affected actin polymerization as well as polarization of TCR, PKCξ and MTOC at the IS. Our results suggest that altered polarization of T cells may influence differentiation of effector and memory T cells.

Results
TSAd is continuously expressed in activated T cells. We previously demonstrated increased resistance towards experimental myeloma in the absence of TSAd 19 . Resistance towards myeloma in this model is driven by tumor-specific Th1 cells which activate tumor-infiltrating antigen-presenting macrophages 24,25 . TSAd expression is induced in T cells upon activation through the TCR/CD3 complex 15,16 . To begin to explore the molecular mechanism for increased resistance to myeloma in the absence of TSAd, we thus first asked whether TSAd continues to be expressed in proliferating T cells. The amount of TSAd in CD3/CD28 activated human CD4+ T cells was measured in dividing cells using CTV dilution combined with intracellular staining for TSAd. TSAd was found to be expressed at the same level over several generations of proliferating human CD4+ cells (Fig. 1A) indicating that this intracellular Lck-adapter potentially could affect T cell proliferation, differentiation or cell survival.

TSAd promotes differentiation of Th1 cells with a central memory phenotype. Having found
that TSAd is continuously expressed in proliferating T cells, we next asked whether TSAd affects the proliferation and phenotype of activated CD4+ T cells. Previous studies of short term stimulated TSAd deficient T cells have revealed conflicting results 15,18,20 . Here we stimulated murine Sh2d2a+/+ or Sh2d2a−/− CD4+ T cells from C57/BL6 mice for three days using anti-CD3/anti-CD28 activator beads as a surrogate for APC, followed by incubation with IL-2 for six days to promote cytokine mediated proliferation 26 (Fig. 1B and C). Live cells were counted at the indicated times, and the frequency of live cells expressing CD44 and CD62L was analysed by flow cytometry in order to assess the size of the T cell populations with a central memory phenotype (i.e. Tcm or CD44high/CD62Lhigh) and an effector memory phenotype (i.e. Tem or CD44high/CD62Low) ( Fig. 1B and C). Over the nine days of IL-2 stimulation, the accumulated proliferation of Sh2d2A−/−CD4+ T cells was significantly lower than that of Sh2d2a+/+ CD4+ T cells (Fig. 1C). Moreover, the cultured Sh2d2a−/− CD4+ T cells were significant skewed towards a Tem phenotype as compared to Sh2d2a+/+ CD4+ T cells (Fig. 1D). Similarly, Sh2d2a−/− Id-specific TCR transgenic CD4+ T cells 19,27 cultured under Th1 stimulating conditions in the presence of Id-peptide loaded APCs from Sh2d2a+/+ spleens 22 , displayed a significantly reduced accumulated proliferative response compared to the corresponding Sh2d2a+/+ Id-specific TCR CD4+ T cells (Fig. 1E). These same cells showed a trend towards skewed Tem differentiation (Fig. 1F). Taken together, these data suggests that TSAd influences cytokine mediated proliferation and differentiation of T cells.

TSAd regulates polarization of F-actin in antigen stimulated CD4+ T cells.
Asymmetric distribution of signalling molecules in APC-conjugated T cells have previously been associated with subsequent differentiation of T cells into Tcm and Tem cells 8 . TSAd regulates actin polymerization at the interphase between T cell and APC 14 . As TSAd is continuously expressed in activated T cells over multiple cell divisions, and since cytokine mediated proliferation of Sh2d2a−/− T cells is diminished compared to Sh2d2a+/+ T cells, we thus asked whether TSAd affects the polarization of critical molecules towards the IS in experienced T cells. Briefly, to generate IS-positive Id-specific T:APC-conjugates in vitro Id-specific TCR CD4+ T cell blasts were used to ensure adequate expression of TSAd (Fig. 1A) and its interaction partner Itk 14 . SNARF-labeled T cells were co-incubated with A20 lymphoma cells or F9 lymphoma cells expressing Id-peptides bound to I-E d (F9 is a derivative of A20) as APC for 30 minutes (for details see Materials and Methods). After staining with Id-specific TCR specific antibody and phalloidin labelling F-actin, cells were analysed by IFC. T-cells conjugated to APC were examined for polarisation of F-actin to the IS ( Fig. 2A and B). Polarisation of F-actin was determined by comparing the signal in the synapse mask to the signal in the whole cell (see Materials and methods for details). In line with our previous finding 14 , significantly fewer conjugated Sh2d2a−/− T cells polarised F-actin to the IS compared to Sh2d2a+/+ T cells ( Fig. 2C and D). However, among conjugated T cells displaying polarized F-actin, the degree of polarized F-actin, as assessed by the median polarisation ratio, was no significant difference in Sh2d2a−/− compared to Sh2d2a+/+ T cells (Fig. 2E).
TSAd regulates polarization of TCR to the IS. Presence of polarized F-actin on the T cell side of the Id-specific T:APC contact area was defined as a marker for IS-positive T cells. TCR is known to accumulate at the centre of the mature IS 13 . We thus considered that accumulation of the Id-specific TCR at the IS may be a marker for a maturing synapse. When analysing IS-positive Id-specific T:APC conjugates, formed after 30 minutes of co-incubation of T cells with APC, for accumulation of TCR at the IS (Fig. 2F), we found that IS-positive Sh2d2a−/− T cells displayed significantly fewer Id-specific T:APC conjugates with polarized TCR than their Sh2d2a+/+ counterpart ( Fig. 2G and H). However, although the median polarisation ratio of TCR also was lower in Sh2d2a−/− T cells compared to Sh2d2a+/+ T cells displaying a maturing IS, this difference did not reach  statistical significance (Fig. 2I). No significant differences in the frequency of T-APC conjugates with polarized F-actin or TCR was observed between Sh2d2a+/+ and Sh2d2a−/− T cells when A20 cells lacking Id was used as APC ( Fig. 2C and G and data not shown). Taken together, these results indicate a deficiency of IS maturation in the absence of TSAd. [28][29][30][31][32] is controlled by highly conserved intracellular polarity proteins known as the Scribble (including Scrib and SAP97) and PAR3 (including PAR3, PKCξ and PKCθ) protein complexes respectively. These polarity complexes define distinct spatial regions of the cell through differential localisation of macromolecules. There is accumulating evidence that the PAR3 complex modulates T cell polarisation, migration and IS formation [29][30][31]33,34 . In order to examine whether polarisation complexes were affected by the absence of TSAd, we analysed Id-specific T:APC conjugates with a maturing IS (displaying accumulation of F-actin and TCR) for distribution of PAR3, PKCξ, PKCθ, Scrib or SAP97 (Fig. 2J). In Id-specific T:APC conjugates having a maturing IS after 30 minutes of co-incubation of T cells with APC, there was no significant difference in the frequency of conjugates displaying asymmetric distribution of polarity complexes within the T cell (Fig. 2K). However, the proportions of PKCξ and SCRIB polarised towards the synapse in the conjugates, as measured by their median polarisation ratios, were significantly lower in Sh2d2a−/− than in Sh2d2a+/+ T cells (Fig. 2L). In Id-specific T:APC conjugates involving A20 cells, there were no significant differences (data not shown).

TSAd promotes re-localization of MTOC to the maturing IS of CD4+ T cells. The relocation of
MTOC to beneath the interface between the T cell and the APC is a hallmark of a mature IS 13 . Having found that Sh2d2a−/− T cells displayed reduced frequency of conjugates with polarised F-actin, TCR and reduced polarisation of the PAR3 complex member PKCξ to the IS, we went on to assess the relocation of the MTOC in Id-specific T:APC conjugates. T cells and APC's were co-incubated for 30 minutes, followed by visualization of MTOC by intracellular staining for γ-tubulin. The maturing IS was defined as previously described, using phalloidin and anti-TCR staining of the conjugates (Figs 2A and 3A). The intensity ratio of γ-tubulin at the synapse was assessed as previously described (Figs 2A, 3B and Materials and methods).The frequency of conjugates displaying MTOC re-localisation beneath the IS in T cells was significantly lower in Sh2d2a−/− compared to Sh2d2a+/+ T cells (Fig. 3C). However, once the MTOC was polarised, the proportion of γ-tubulin signal relocating to the IS was not significantly different in Sh2d2a−/− T cells compared to that observed in Sh2d2a+/+ (Fig. 3D).

TSAd influences the kinetics of F-actin, TCR, PKCξ and MTOC polarisation in CD4+ T cells.
Having found that maturation of the IS was affected in Sh2d2a−/− T cells early after conjugation with APC, we proceeded to analyse the kinetics of IS maturation in Sh2d2a+/+ and Sh2d2a−/− T cells over a 12 hour time course, including also the polarisation of the polarity proteins PAR3 and PKCξ (Fig. 4). As previously, T cells and Id-positive APC (F9 cells) were co-incubated for the indicated time points, followed by staining with the indicated antibodies. The initial reduced frequency of Sh2d2a−/− T cells displaying polarized F-actin as well as the relative amount of F-actin polarized to the IS in conjugated Sh2d2a−/− T cells was not observed at later time points (Fig. 4A and B). Similarly, the difference in frequency of TCR polarised conjugates towards IS was not observed at later time points (Fig. 4C). As already noted, the relative amount of TCR accumulated towards the synapse was not significantly different at early time points and remained so throughout the entire time course (Fig. 4D). The frequency of conjugates with PKCξ polarised towards the mature IS was similar in Sh2d2a−/− and Sh2d2a+/+ T cells over the course of the experiment (Fig. 4E), while the reduced amount of polarized PKCξ in Sh2d2a−/− was only observed for up to 60 minutes of Id-specific T:APC co-culture (Fig. 4F). Both the frequency of conjugates with PAR3 at the IS, as well as the amount of PAR3 polarised towards the mature IS, was significantly lower in Sh2d2a−/− only at the latest time point tested (720 minutes) (Fig. 5G and H). The frequency of conjugates with polarized γ-tubulin was significantly lower in Sh2d2a−/− over the course of the experiment (Fig. 4I). However once polarisation occurred, γ-tubulin was similarly polarised as assessed by polarisation ratio, irrespective of TSAd (Fig. 4J). There were no significant differences in polarisation in the absence of Id presentation (data not shown). Taken together, these results support a role for TSAd in regulating maturation and organisation of the IS at early time points during initial T cell:APC interaction.

IFNγ is reduced in antigen stimulated Sh2d2a−/−Id-specific TCR T cells.
Cytokine secretion is a crucial part of T cell effector function. Several cytokines are polarised and secreted through the IS, including IFNγ 35 . Polarisation of IFNγ to the IS is dependent on polarisation of F-actin, but not MTOC reorientation 35,36 . Having found that TSAd influences accumulation of polarized F-actin as well as members of the PAR3 polarity complex to the IS, we examined whether Sh2d2a−/− also affected IFNγ during T cell:APC interaction.
As above Id-specific TCR T blasts and APC were co-incubated for the indicated time points and subsequently stained intracellularly for IFNγ-F-actin and TCR. IFNγ expression in Id-specific TCR T cells with maturing IS was assessed (Fig. 5A). After 30 minutes incubation, both Sh2d2a+/+ and Sh2d2a−/− Id-specific T:APC (K) Graph shows relative frequencies of Id-specific T: -APC conjugates with polarization of indicated polarity complex molecules towards IS relative to Sh2d2a+/+. (L) Graph show relative values of the intensity ratios in Id-specific T:-APC conjugates of the indicated polarity complex molecules. (j) and (K) Data represents the median values (+95% CI) normalized to those observed in Sh2d2a+/+ Id-specific T: APC conjugates of three independent experiments, n >50 gated events in each experiment. Statistical significance was assessed using paired student's t-test. conjugates displayed intracellular IFNγ (Fig. 5B,C). The number of IFNγ expressing Sh2d2a+/+ T cells varied throughout the experiment, while after 720 minutes of incubation significantly fewer Sh2d2a−/− T cells were expressing IFNγ (Fig. 5C). Taken together, the effect of TSAd on IS-formation and T cell differentiation includes a positive effect on amount of IFNγ-in antigen stimulated CD4+ T cells.

Polarisation of signalling molecules in the triggered T cell towards the APC is crucial for normal activation and function of T cells. Here we have shown that the Lck-adapter TSAd is required in the early phases in the T cell's interaction with APC. Absence of TSAd results in altered differentiation and proliferation of activated T cells in vitro.
In this study, we mainly used imaging flow cytometry, which allows for the rapid and unbiased analysis of large numbers of Id-specific T:APC conjugates. Though the resolution of IFC is inferior to confocal microscopy, the method permits quantitative high throughput analysis of Id-specific T:APC conjugates, without encountering issues of photo-bleaching and observer bias in identifying Id-specific T:APC conjugates associated with confocal microscopy.
A key feature of TSAd is that it is rapidly induced upon activation of the T cells. Resting T cells display low levels, while recently activated T cells express high levels of TSAd. In T cells primed via inflammatory cAMP inducing signals, TSAd protein expression may be induced already 15 minutes after initial TCR triggering of primary resting T cells 37 . Here we show that expression of TSAd is stably maintained over several cell divisions, and that absence of TSAd results in reduced proliferation and a significant skewing towards effector T cell phenotype. This support the notion that TSAd also regulates T cells after the initiation of TCR stimulation. Although reduced proliferation of Sh2d2a−/− T cells in response to TCR-triggered activation was initially reported 15 , a significant difference in short term anti-CD3/anti-CD28 stimulated T cell proliferation has not consistently been observed 19 . Our present data strongly indicates that TSAd may impact the T cell phenotype upon long term or chronic immune challenges.
The main finding of this study is that TSAd regulates polarization of key molecules, including actin and members of the PAR3 complex, to the IS in T cells during early APC interactions. This results in altered MTOC polarisation, as well as IFNγ expression. Secretion of IFNγ at the IS is dependent on actin remodelling 36 . Intracellular vesicles associated to the GTPases Rab3d and Rab19 traffic IFNγ for secretion at the IS 35 . Whether these GTPases are also affected by the presence of TSAd remains to be studied. Altered polarization of T cells towards the APC may represent a possible molecular mechanism for the improved resistance of Sh2d2a−/− Id-specific TCR transgenic mice towards experimental myeloma that we previously observed 19 . IFNγ is a major effector cytokine produced by CD4+ T cells, which can induce tumor-killing macrophages 24,25 . Elimination of the MHC negative plasmacytoma MOPC315 involves indirect recognition by CD4+ T cells of tumor antigen presented by infiltrating macrophages 25,38,39 , followed by differentiation of cytotoxic macrophages through direct contact with the T cells 39,40 . Our data suggest that altered cytoskeletal and polarisation dynamics in TSAd deficient CD4+ T cells during APC interactions could provide reduced secretion of IFNγ at the IS. It is thus possible that the improved tumor rejection in TSAd deficient Id-specific TCR transgenic T cells 19 is caused by diffuse instead of polarized secretion of IFNγ during CD4+ T cell interactions with APC. In conjunction with a more slowly dividing effector T cells population, this may lead to improved and persistent recruitment of cytotoxic macrophages and increased tumor killing 24,38,40,41 . Whether recruitment of cytotoxic macrophages and bystander killing of the plasmacytoma cells is affected by lack of TSAd need to be explored 39,40 .
Our present data show that TSAd modulates early polarisation of the cytoskeleton as well as MTOC re-arrangement in T cells, however the molecular mechanism for how TSAd affects polarity of T cells remains to be determined. TSAd is an adapter for both Lck and Itk and promotes phosphorylation of Itk by Lck 18 and Cdc42 43,44 is a key GTPase for establishing cell polarity 45,46 . Lck and Itk mediated signalling upon TCR stimulation is required for Cdc42-WASP controlled actin polarisation towards the IS 44 . TSAd also promotes recruitment of Lck and SLP-76 to Nck 47 . SLP-76 and Nck coordinates WASP recruitment to Cdc42 43 . Cdc42 controls polarity of cells through activation of multiple polarity pathways, including the PAR3 polarity complex 48 .
It is likely that the alterations in actin and polarity complex polarization in the Sh2d2a−/− T cells are mechanistically linked to the overall reduced MTOC polarisation observed in the same cells. Though the mechanism for MTOC reorientation in T cells remains unclear, it appears to involve motor protein activity and polarity complex interactions 49,50 . It was recently proposed that Arp2/3 mediated actin nucleation tethers MTOC to the nucleus, through the linker of nucleoskeleton and cytoskeleton (LINC) complex 51 . During TCR stimulation Arp2/3 is recruited to the plasma membrane, resulting in local depletion of Arp2/3 and release of MTOC from the nucleus, allowing for its reorientation towards the IS 51 .
Taken together, it is conceivable that the change in TCR induced polarisation dynamics observed in Sh2d2a−/− CD4+ T cells is ultimately linked to altered Lck signalling, and downstream Itk and Nck mediated cytoskeletal polarisation 14,47 (depicted in Fig. 6 52,53 ). However, whether TSAd modulates downstream polarization events through a Cdc42-dependent mechanism remains to be determined.

Conclusion
Polarisation of T cells towards the APC and altered expression on IFNγ in conjugated T cells stimulated with cognate antigen is regulated by the Lck adapter TSAd. This may explain why TSAd deficiency is associated with increased resistance towards tumors where IFNγ dependent macrophage mediated cytotoxicity is crucial.

Materials and Methods
Ethical Statements. All experiments were performed in accordance with relevant guidelines and regulations. The animals were bred under conventional conditions, regularly screened for common pathogens and housed in compliance with guidelines set by the Experimental Animal Board under the Ministry of Agriculture of Norway. All experimental protocols involving transgenic and wild type animals were approved by the National Committee for Animal Experiments (Oslo, Norway). Peripheral blood mononuclear cells were isolated from healthy, adult, anonymous blood donors. Informed consent was obtained from all blood donors. The study was approved by the regional ethics committee (REK no S-09325b). Mice and Cell Lines. The Sh2d2a−/− C57BL/6 and Id-specific TCR transgenic BALB/c mice were previously described 19 . Briefly, Sh2d2a−/− mice on a mixed C57BL/6-129 background were backcrossed to C57BL/6 or BALB/c mice. Sh2d2−/+ BALB/c mice heterozygous for Id-specific TCR were crossed with Sh2d2a−/+ BALB/c mice to generate littermates both for studies of Id-specific TCR transgenic mice and normal BALB/c mice with or without Sh2d2a. The BALB/c MHC class II positive B cell lymphoma cell line A20 and F9 (A20 cells stably expressing Id + λ2 315 L chain 55 ) were cultured in complete medium (RPMI 1640 medium supplemented with 10% fatal calf serum (FCS), 1 mM non-essential amino acids, 1 mM sodium pyruvate, 1 mM L-glutamine, 100 units/ ml penicillin, 100 µg/ml streptomycin (all from GIBCOBRL ® , Life Technologies ™ ) and 50 µM β-mercaptoethanol (Sigma)).

Purification of CD4+ T Cells. Human CD4+ T cells were isolated from PBMC using Dynabeads ® CD4
Positive Isolation Kit, (ThermoFisher). Murine CD4+ T cells were isolated from single cell suspensions of spleens using Dynabeads ® Untouched ™ Mouse CD4 Cells Kit (ThermoFisher). The composition of the recovered population was more than 90% CD4+ T cells as analysed by flow cytometry.
In vitro CD4+ T Cell Stimulation. Human CD4+ T cells were loaded with CTV before being stimulated with plate bound anti-CD3 (OKT3, 5 µg/ml) and soluble anti-CD28 (CD28.2, 1 µg/ml) in complete medium containing 30 U/ml IL-2 for 4 days. Cells were then stained with anti-TSAd-DyLight 488 and analysed by flow cytometry. Dividing cells were identified by CTV dilution. Murine CD4+ T cells were stimulated with Dynabeads ® Mouse T-Activator CD3/CD28 beads (ThermoFisher), bead: cell ratio = 1:1 in complete medium containing 30 U/ml IL-2. CD3/CD28 beads were removed after 3 days in vitro and cultured in the presence of IL2 (30 U/ml) for another 7 days. Live cells were counted by trypan blue dye exclusion using a TC20 automated cell counter (Bio-Rad), and phenotyped by flow cytometry at 0, 3, 7 and 9 days in vitro. Surface staining was performed using CD4-PerCP-Cy5.5, CD44-FITC and CD62L-PE in FACS buffer (2% FCS, 0,1% sodium azide in PBS) at 4 °C. Cells were stained subsequently with LIVE/DEAD Fixable Near-IR to exclude dead cell, fixed for 10 minutes with 2% PFA in FACS buffer, and acquired on a FACSCantoII. Data was analysed using FlowJo version 7.6 (Tree Star Inc.), where live cells were gated on CD4-PerCP-Cy5.5 followed by analysis of CD44-FITC and CD62L-PE. Th1 differentiation assay. Id-specific TCR transgenic Th1 cells were obtained as previously described 27 .
Briefly, splenic CD4+ T cells were cultured with 10 μg/mL anti-IL-4, 20 ng/mL IL-12 together with irradiated wild type splenocytes loaded with Id peptide as APC (1:3 T cell to APC ratio). The Id-peptide comprises residues 91-101 of the λ2 315 L chain corresponding to CDR3. Mutated residues 94, 95, 96 are crucial for I-E d -restricted stimulation of Id-specific TCR transgenic CD4+ T cells 56,57 . Live cells were counted using a TC20 automated cell counter as above, at 0, 2, 5, 7 and 10 days in vitro before being phenotyped as described above on day 10.
Conjugation assay. CD4+ T cells from Id-specific TCR transgenic BALB/c mice expanded for 5 days using CD3/CD28 beads, were rested for 48 hours in the absence of beads before being stimulated with irradiated (2500 rad) F9 or A20 cells. F9 cells presenting Id-peptide on MHC II strongly activates Id-specific TCR transgenic CD4+ T cells 22 . CD4+ T cells were labelled with 0,1 µM SNARF as per manufacturers instructions. The parental A20 cell line was used as a negative control. 1 × 10 6 A20 or F9 target cells were co-cultured with 0,6 × 10 6 Id-specific T cells in complete medium in 96 well U-bottom plates. Cells were centrifuged at 70 × g for 1 minute and incubated for indicated time points at 37 °C before stimulation. All subsequent pipetting was done gently with wide bore 200 µl pipette tips (VWR). Cells were stained with LIVE/DEAD Fixable Near-IR before being fixed with 2% PFA for 10 minutes, or fixed and permeabilised for 5 minutes with Acetone at −20 °C in case of γ-tubulin staining, followed by GB113-PE staining which binds Id-specific TCR (mAb; GB113 54 ), at 10 µg/ml in FACS buffer for 30 minutes. Cells were then permeabilised and stained with FACS buffer containing 0,1% Saponin, 6,25U/ml Phalloidin Alexa Fluor 647 in combination with 1 µg/ml of one of the following antibodies: PAR3, PKCξ, PKCθ, Scrib, SAP97 (Santa Cruz), anti-γ-tubulin (Sigma) or IFNγ-FITC (BD). Cells were then washed and stained when necessary with secondary antibody goat anti-donkey, goat anti-rabbit or isotype specific anti-mouse conjugated to Alexa Fluor 488 (Thermo Fisher Scientific) together with DAPI. Cells were washed and stored in PBS, 0,1% NaAzide at 4 C until run on ImageStream X. Imagestream acquisition and analysis. Samples were acquired at 40x magnification on a four-laser, twelve channel, ASSIST calibrated ImageStream X (Amnis, Seattle, WA) imaging flow cytometer. 405 nm, 488 nm, 561 nm and 658 nm laser excitations were set to avoid pixel saturation. Single stained controls were collected and used to generate a compensation matrix to correct for spectral crosstalk. Data was analysed using IDEAS v6.1, where the T cell mask was defined based on the signal of the cell tracker SNARF. Live cells were identified by viability dye exclusion, and GB113 positive cells identified Id-specific TCR transgenic T cells. The aspect ratio and area of the DAPI signal was used to identify images containing two cells 58 . Selecting images with a high SNARF (T cell) intensity and aspect ratio was then done to identify images containing 1 T cell 58 . Using the "Interface" masking function in IDEAS 58 the T cell synapse of the T cell was defined in all the images ( Fig. 2A). Marker polarisation towards the synapse was quantified by calculating the average fluorescent signal in the synapse masks as a ratio to the average fluorescent signal of the whole T cell. To adjust for cell-to-cell variation in the size of the synapse mask, the signals were normalised against the area of the respective masks. A threshold ratio above 1,5 was defined as the fluorescent marker being polarised to the IS, based on the polarisation ratio of the uniformly distributed cell tracker SNARF (not shown).
Statistical analysis. The data was exported and graphed using GraphPad Prism 6 (GraphPad Software, Inc.). To assess statistical significance, a two-tailed Student's t-test was applied to compare two normally distributed datasets while two-way analysis of variance was applied to datasets with multiple time points. A significance level of 0,05 was used.