Nuclear Envelope Lamin-A Couples Actin Dynamics with Immunological Synapse Architecture and T Cell Activation

Lamin-A and lamin-C are transiently increased in abundance upon T cell activation

To investigate the expression of the gene encoding A-type lamins before and after T cell activation, we analyzed human peripheral blood lymphocytes (PBLs) and T lymphoblasts as well as mouse splenocytes and T lymphoblasts (fig. S1). We first analyzed primary immune cells from mice or human donors by confocal microscopy to detect A-type lamins. We found that lamin-A and lamin-C (collectively referred to hereafter as lamin-A/C) were detectable in only a small fraction of CD4⁺ T cells in preparations of human PBLs and mouse splenocytes (Fig. 1A), whereas we detected B-type lamins in all CD4⁺ T cells (fig. S2A). Flow cytometric studies demonstrated that lamin-A/C proteins were present in less than 10% of CD4⁺ T cells from human peripheral blood (Fig. 1B).

To assess whether T cell activation resulted in increased lamin-A/C abundance, we analyzed the amounts of these proteins before and after TCR activation in PBLs and T lymphoblasts from humans and mice. Flow cytometry, confocal microscopy, and Western blotting analyses showed a transient and marked increase in lamin-A/C abundance upon TCR activation with Staphylococcus enterotoxin E (SEE) (Fig. 1C), SEE-loaded Raji cells (a human B lymphoblastoid cell line), SEE-loaded dendritic cells (DCs) (fig. S2B), or phytohemagglutinin (PHA) (Fig.1D). Consistent with these findings, CD4⁺ T lymphoblasts from OTII transgenic mice bearing OTII TCRs specific for the ovalbumin (OVA) antigen displayed enhanced lamin-A/C abundance upon activation with OVA-loaded DCs (Fig. 1E).

To further investigate the time course of the increase in lamin-A/C abundance upon T cell activation, we performed quantitative, reverse transcriptase quantitative polymerase chain reaction (qRT-PCR) and Western blotting analyses of human T lymphoblasts at different times after stimulation with Raji cells alone (which served as a negative control) or SEE-loaded Raji cells. Previous studies showed that APC–T cell interactions predominantly occur between 5 and 24 hours after exposure of T cells to antigen-presenting DCs (23-26). Compared with that in T cells incubated with Raji cells alone, LMNA mRNA abundance in T cells activated with SEE-loaded Raji cells was increased, with a peak at 4 hours after stimulation (fig. S2C), which was followed by the accumulation of lamin-A/C protein (fig. S2D). Appropriate T cell activation after their stimulation with SEE-loaded Raji cells was confirmed by detection of a marked increase in the amounts of mRNAs for the T cell activation markers interleukin-2 (IL-2) and CD25, which was not observed in cells stimulated with Raji cells alone (fig. S2E). Together, these data suggest that A-type lamins are rapidly and transiently increased in abundance upon activation of T cells through antigen presentation.

Lamin-A and lamin-C are required for optimal activation of T cells in vitro and in vivo

We next investigated whether A-type lamins regulated T cell function. Compared with wild-type splenocytes, those from Lmna^−/− mice, which lack lamin-A/C proteins, had markedly reduced abundance of CD25 mRNA upon stimulation in vitro with anti-CD3 and anti-CD28 antibodies, phorbol myristate acetate (PMA) and ionomycin (a calcium ionophore), or concanavalin A (Fig. 2A). Similarly, CD4⁺ T cells isolated from the spleens of Lmna^−/− mice and stimulated with anti-CD3 and anti-CD28 antibodies displayed a substantial reduction in the cell-surface abundances of the T cell activation markers CD69 and CD25, as observed by flow cytometric analysis (Fig. 2B). Moreover, compared with wild-type controls, CD4⁺ T cells from OTII transgenic mice lacking lamin-A/C (Lmna^−/− OTII mice) displayed reduced cell-surface CD25 abundance upon stimulation with OVA-loaded DCs (Fig 2C).

To assess the role of lamin-A/C proteins as regulators of the immune response in vivo, we used a hapten-induced contact hypersensitivity (CHS) model (27). The immune response in this model starts with the application of the hapten oxalazone to the ear skin of mice, which triggers a sensitization phase during which specific T cells proliferate and migrate out of the lymph node upon recognition of hapten-carrier complexes presented by APCs. After re-exposure of the mice to oxalazone, an elicitation phase is initiated during which specific CD4⁺ and CD8⁺ T cells are activated in the dermis and trigger the inflammatory process (27, 28). As expected, we found that the elicitation phase after application of oxalazone to the ears of wild-type mice induced the progressive recruitment of CD4⁺ T cells to the draining cervical lymph nodes over a period of 3 days (fig. S3, top). Furthermore, CD4⁺ T cells recruited to lymph nodes in wild-type mice after challenge with oxalazone showed increased lamin-A/C abundance, with a peak at 2 days after challenge (fig. S3, bottom).

To investigate whether this increase in lamin-A/C abundance played any role in this model, we performed studies with lethally-irradiated wild-type mice transplanted with bone marrow cells from either wild-type mice or Lmna^−/− mice. Irradiation and reconstitution was used to avoid any potential interference from non-immune cells and from the defects in T cell and B cell development that have been reported to occur in the thymus and spleen of Lmna^−/− mice (15). Ear inflammation increased in both groups of mice during the first 2 days after challenge with oxalazone and then was progressively reduced (Fig. 2D). However, ear swelling was substantially reduced at all of the time points analyzed in mice reconstituted with immune cells lacking lamin-A/C (Fig. 2D). To assess whether the reduced response observed in mice reconstituted with bone marrow cells from Lmna^−/− mice was a result of decreased numbers of CD4⁺ T cells in the ears, we performed adoptive transfer of naïve CD4⁺ T cells from CD45.2⁺ wild-type or CD45.2⁺ Lmna^−/− mice into recipient CD45.1⁺ wild-type mice. Oxazolone was then applied to the abdomens of the recipient mice, and we quantified the percentages of CD4⁺ T cells in the ears three days after the second application of oxalazone to the ears. We found that the percentages of transferred Lmna^−/− CD45.2⁺ CD4⁺ T cells in the ears of recipient mice were reduced compared to those of transferred wild-type CD45.2⁺ CD4⁺ T cells (Fig. 2E).

We next performed competition experiments by adoptively transferring a 1:1 mixture of naïve CD4⁺ T cells from CD45.1⁺CD45.2⁺ wild-type and CD45.2⁺ Lmna^−/− mice into CD45.1⁺ wild-type recipient mice (Fig. 2F). Under these conditions, the numbers of transferred CD45.2⁺ Lmna^−/− T cells that accumulated in the ears, lymph nodes, and spleens of recipient CD45.1⁺ wild-type mice were reduced compared to those of the transferred CD45.1⁺ CD45.2⁺ wild-type T cells (Fig. 2F). The reduction in the percentage of transferred Lmna^−/− CD4⁺ T cells in the spleen (Fig. 2F) suggests that the absence of lamin-A/C might affect T cell proliferation upon recognition of the hapten. Together, these results demonstrate that loss of lamin-A/C in the mouse impairs T cell responses in vitro and in vivo.

To assess whether lamin-A enhanced the activation of human T cells, we performed gain-of-function studies with J77 cells, a human Jurkat T cell line derivative that does not have detectable endogenous LMNA mRNA or protein under basal conditions and which failed to exhibit increases in LMNAmRNA abundance upon activation with SEE-pulsed Raji cells (fig. S4, A and B). As expected, J77 cells stably expressing GFP (J77-GFP cells) (fig. S4C) were activated upon incubation with SEE-loaded Raji cells compared with control J77-GFP cells incubated with non-loaded Raji cells, as determined by detection of increased amounts of CD69 and CD25 mRNAs (Fig. 3A) and the increased cell-surface abundance of CD69 (Fig. 3B). Moreover, J77 cells stably expressing GFP–Lamin-A (J77-GFP-Lamin-A; fig. S4, C and D) that were incubated with SEE-loaded Raji cells showed enhanced activation when compared to conjugated J77-GFP cells, as revealed by the presence of increased amounts of CD69 and CD25 mRNAs (Fig. 3A) and increased cell-surface abundance of CD69 (Fig. 3B). Treatment with anti-CD3 and anti-CD28 antibodies also enhanced the cell-surface abundance of CD69 on J77-GFP-Lamin-A cells compared to that on J77-GFP cells (Fig. 3C). Moreover, transient expression of dsRED-Lamin-A increased the cell-surface abundance of CD69 in J77-GFP cells incubated with SEE-loaded Raji cells compared to that on J77-GFP cells transfected with plasmid expressing dsRED alone (fig. S4E).

A-type lamins accelerate T cell–APC interaction dynamics and TCR-CD3 mobility in the plasma membrane and modulate TCR signaling

We next sought to elucidate the mechanisms through which A-type lamins enhanced T cell activation. Immunological synapse formation and T cell activation are finely regulated processes that entail multiple changes in the plasma membrane, cytoplasm, and nucleus (20). We hypothesized that lamin-A/C could regulate immunological synapse formation, orchestrating different processes required for T cell activation. To assess this possibility,we studied the effect of lamin-A on T cell-APC interactions and on the movement of molecules involved in immunological synapse formation. J77-GFP or J77-GFP-Llamin-A cells were incubated with non-loaded or SEE-loaded Raji cells for different times and then were plated onto poly-L-lysine (PLL)-coated coverlips for immunofluorescence analysis. These studies revealed that J77-GFP-Lamin-A cells formed a greater percentage of conjugates than did J77-GFP cells at all of the time points analyzed (Fig. 4A), suggesting that lamin-A facilitates and stabilizes the formation of T cell–APC interactions.

To study this in more detail, we examined immunological synapse formation by time-lapse confocal microscopy (Fig. 4B and movie 1). Stably-transfected J77 cell lines were stained with different fluorescently labeled dyes and then were added on top of SEE-loaded Raji cells that had been stained with the cell tracker CMAC (7-amino-4-chloromethylcoumarin) and then allowed to attach to PLL–coated coverslips. We did not observe any differences in the times it took the J77-GFP and J77-GFP-Lamin-A cells to reach the focal plane (Fig. 4C). However, compared with J77-GFP controls, J77-GFP-Lamin-A cells formed more conjugates with SEE-loaded Raji cells at all of the time points tested (Fig. 4D), and these interactions were protracted (Fig. 4E).

After TCR engagement, TCR-CD3 complexes and co-stimulatory receptors accumulate at the cSMAC before being internalized (20). To study the effect of A-type lamins on the dynamic redistribution of receptors and co-receptors at the plasma membrane, we analyzed J77 cells transiently co-transfected with plasmids encoding CD3ζ-EGFP and either dsRED or dsRED-Lamin-A. GFP-rich populations were obtained by cell-sorting (fig. S5A) and then were activated by being plated onto coverslips coated with anti-CD3 antibody. Although the stimulating ligand was immobile on the glass and the TCR microclusters remained stationary, under these conditions, there is protein flux into and out of the assembled clusters (29, 30). We then analyzed activated cells by total internal reflection fluorescence (TIRF) microscopy at a penetration depth of ~90 nm, which enabled the observation of CD3 microclusters at the plasma membrane (Fig. 5A). At the initial time points, J77-dsRED-Lamin-A cells exhibited an increased number of CD3-containing microclusters and a larger cSMAC area than did J77-dsRED cells, and these parameters were progressively reduced over time in both cell types (Fig. 5, B and C; movie 2).

We noted that the disappearance of CD3-containing microclusters from the TIRF detection depth range was faster in J77-dsRED-Lamin-A cells than in J77-dsRED cells (Fig. 5D), which suggested increased internalization. Accordingly, confocal microscopic analysis of J77-GFP-Lamin-A cells 30 min after conjugation with SEE-loaded Raji cells revealed that they had smaller cSMAC diameters and reduced numbers of CD3-containing microclusters at the membrane contact area compared to those of J77-GFP control cells (fig. S5, B and C). These results, which suggest that the presence of lamin-A accelerates both the dynamic redistribution of CD3 at the contact area during immunological synapse maturation as well as concomitant CD3 internalization, prompted us to investigate the effects of the presence of lamin-A on different signaling pathways downstream of TCR-CD3 activation. Western blotting analysis of J77-GFP-lamin-A cells conjugated for different times with SEE-loaded Raji cells revealed increased amounts of phosphorylated (activated) Vav1, myosin IIA, and extracellular signal–regulated kinase 1 (ERK1) and ERK2 (ERK1/2) compared to those of similarly treated J77-GFP cells (Fig. 5E). Moreover, treatment with the ERK1/2 inhibitor U0126 substantially reduced the cell-surface amount of CD69 in J77-GFP cells and J77-GFP-lamin-A cells conjugated with SEE-loaded Raji cells, and almost blunted the increase in CD69 abundance elicited by lamin-A (Fig. 5F).

A-type lamins promote MTOC translocation and F-actin polymerization in activated T cells

We hypothesized that differences in lamin-A/C–dependent signaling in activated T cells might be related to changes in the tubulin and actin cytoskeleton. To address this possibility, we first examined the translocation of the MTOC toward the immunological synapse, an important step in lymphocyte activation initiated by TCR signaling (31). Immunofluorescence and confocal microscopy revealed that the MTOC translocated faster to the immunological synapse in J77-GFP-Lamin-A cells than in control J77-GFP cells (Fig. 6A). Moreover, J77-GFP-Lamin-A cells exhibited increased F-actin polymerization induced by anti-CD3 and anti-CD28 antibodies, as measured by flow cytometric analysis of phalloidin-stained cells (Fig. 6B), as well as increased F-actin relocalization to the immunological synapse with respect to the rest of the membrane in cells conjugated with SEE-loaded Raji cells, as detected by confocal microscopy (Fig. 6C). Accordingly, human primary lamin-A/C–expressing T lymphoblasts subjected to knockdown of lamin-A/C by small inhibitory RNA (siRNA) (Fig. 6D) exhibited reduced F-actin polymerization upon stimulation with SEE-loaded Raji cells (Fig. 6E) or anti-CD3 and anti-CD28 antibodies (Fig. 6F). Together, these results indicate that A-type lamins promote F-actin polymerization and MTOC translocation during immunological synapse formation.

The lamin-A–mediated increase in T cell activation requires a physical connection between the nucleus and the cytoskeleton through the LINC complex

Together with nesprins and SUN proteins, lamin-A/C proteins mediate the connection between the nuclear lamina and the cytoskeleton (3-5). SUN proteins interact with lamin-A, and their SUN domain extends into the perinuclear space to form contacts with the KASH (for Klarsicht/Anc-1/SYNE homology) domains of nesprins, which are located at the outer nuclear membrane and connect with cytoplasmic microtubules, actin, and intermediate filaments (4, 5). To assess whether these connections were required for optimal T cell activation and F-actin polymerization, we transfected J77-GFP-Lamin-A cells with plasmids encoding either a dominant-negative nesprin construct containing the C-terminal KASH domain fused to an N-terminal mCherry (DN KASH) (32), or a dominant negative SUN1 luminal domain construct (DN SUN) (33, 34). Overexpression of these proteins disrupts the LINC complex and the connections between A-type lamins and the cytoskeleton (32). Consistent with our earlier results, we found that J77-GFP-Lamin-A cells conjugated with SEE-loaded Raji cells showed increased cell-surface expression of CD69 compared with J77-GFP cells (Fig. 7A), a response that was blunted upon overexpression of DN KASH (Fig. 7A). Furthermore, the presence of DN KASH did not affect CD69 cell-surface abundance in J77-GFP cells lacking lamin-A/C (Fig. 7A). Moreover, compared with cells transfected with control plasmids, J77-GFP-Lamin-A cells transfected with plasmids encoding DN KASH (Fig. 7B) or DN SUN (Fig. 7C) displayed reduced F-actin content upon stimulation with SEE-loaded Raji cells. Together, these results suggest that lamin-A–mediated increases in F-actin polymerization and T cell activation depend, at least in part, on the physical connection between the nucleus and the cytoskeleton mediated through physical interactions between lamin-A, SUN1, Nesprin, and the cytoskeleton.

Plasmids

The plasmids coding for mCherry and EGFP were purchased from Clontech. The plasmids coding for CD3ζ-EGFP (66), DN KASH (32), DN SUN (33, 34) dsRED and dsRED-Lamin-A (67) and EGFP-lamin-A (68) were previously described.

Cells and mice

Vβ8 Jurkat J77cl20 (J77) T cells, Vβ3 Jurkat (CH7C17) T cells, and lymphoblastoid Raji B cells were cultured in complete medium [RPMI 1640, 10% fetal bovine serum (FBS, Sigma)]. Human PBLs were isolated from freshly prepared buffy coats obtained from healthy donors. PHA-activated and SEE-specific human T lymphoblasts were obtained as described previously (69). When indicated, human T lymphoblasts were co-cultured with SEE-loaded monocyte-derived DCs, which were obtained as described previously (70). Stable J77 cell populations overexpressing GFP or GFP-Lamin-A were generated by transfection of plasmids encoding EGFP or EGFP-lamin-A, respectively, followed by selection with G418 (1 mg/ml, Invitrogen). Lmna^−/− mice were previously described (71). C57BL/6-Tg (TcraTcrb)425Cbn/J mice (OTII mice) expressing a TCR specific for the OVA peptide (amino acid residues 323 to 339) in the context of I-Ab were purchased from the Jackson Laboratory (stock number 004194). Lmna^−/− OTII mice were generated by crossing OTII mice with Lmna^−/− mice. CD45.1⁺ B6.SJL mice were used as the recipients for adoptive transfers. CD45.1⁺CD45.2⁺ wild-type mice were generated by crossing C57BL/6 CD45.2⁺ mice with C57BL/6 CD45.1⁺ mice. To obtain T lymphoblasts from wild-type and OTII mice, naïve CD4⁺ T cells (10⁶ cells/ml) were cultured in the presence of irradiated APCs (T cell-depleted splenocytes) and OVA peptide (10 μg/ml). After 1 day, cells were washed, and IL-2 (10 ng/ml) was added to the medium. Seven days later, irradiated APCs and OVA peptide (10 μg/ml) were added to stimulate the CD4⁺ T lymphoblasts.

Antibodies and reagents

Staphylococcus enterotoxin E (SEE) was purchased from Toxin Technology. The CellTracker Blue CMAC, Alexa Fluor 647-conjugated anti-α-tubulin antibody, Alexa Fluor 488-conjugated phalloidin, and Alexa Fluor 647-conjugated phalloidin were obtained from Invitrogen. Human fibronectin, PLL, anti-α-tubulin antibody, and U0126 were obtained from Sigma-Aldrich. Primary antibodies include SPV-T3b (anti-human-CD3) (72), TP1/55 (anti-human CD69) (73), and HP2/6 (anti-human CD4) (74). The monoclonal antibody specific for Vav1 phosphorylated at Tyr¹⁷⁴ (pVav1 Y174) was a kind gift from X. Bustelo (Centro de Investigación del Cáncer, Salamanca). Anti-Vav and anti-ERK1/2 antibodies were from Millipore. Anti-phosphorylated MLC, anti-MLC, anti-lamin-A/C, anti-lamin-A, and anti-ERK2 antibodies were obtained from Santa Cruz Biotechnology. Anti–PLC-γ1 (Y783), anti–PLC-γ1, Alexa Fluor 488-conjugated anti-lamin-A/C, and Alexa Fluor 488-conjugated mouse immunoglobulin G1 (IgG1) isotype control were obtained from Cell Signaling. Allophycocyanin (APC)-conjugated anti-human CD69, fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD69, anti-mouse CD3, anti-CD28, PerCPCy5.5-conjugated anti-CD45.1, Alexa Fluor 647-conjugated anti-CD45.1, anti-CD45.2-V450, and biotinylated antibodies against B220, CD19, MHCII, CD11c, CD11b, CD44, and CD8α were from BD Biosciences.

Cell transfection and gene silencing

J77 cells or primary human T lymphoblasts (2 × 10⁷ cells) were washed twice with Hanks’ balanced salt solution (HBSS, Cambrex) and transiently transfected by electroporation with plasmids (20 μg) or 1 μM siRNA in OPTIMEM medium (Gibco, Invitrogen) at 240 V and 32 ms (Gene Pulser II, Bio-Rad). The plasmids coding for EGFP-lamin-A, dsRED and dsRED-Lamin-A were used . Specific double-stranded siRNAs against human lamin-A/C or negative control siRNAs were purchased from Eurogentec. The efficiency of gene silencing was assessed by Western blotting, flow cytometric, and confocal microscopic analysis 24 hours after transfection.

Cell-cell conjugate formation

Raji cells were incubated with CMAC in HBSS for 30 min and then with or without SEE (0.5 μg/ml), centrifuged at low speed, washed, and then allowed to form conjugates with J77 cells or CD4⁺ human T lymphoblasts obtained from SEE-treated PBLs at 37°C for the times indicated in the figure legends.

Flow cytometry

Cells were cocultured in 96-well plates at 37°C for the times indicated in the legends. After cells were incubated and stained with specific antibodies, data were acquired on FACSCantoII or LSRFortessa flow cytometers (BD Biosciences) and analyzed with BD FACSDIVA (BD Biosciences) or FlowJo (Treestar Inc) software. Raji cells were distinguished from other cells by CMAC staining. To measure F-actin content, cells were stimulated for different times with purified anti-CD3 antibody (10 μg/ml, T3b) or SEE-loaded, CMAC-stained Raji cells, fixed in 2% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100, and labeled with phalloidin. For experiments in which ERK1/2 signaling was blocked, J77 cells were preincubated for 1 hour with the MEK1/2 inhibitor U0126 (which prevents the activation of ERK1/2), and then were allowed to form conjugates with Raji cells in the presence of the inhibitor for the times indicated in the figure legends.

Western blotting analysis

J77 cells were allowed to form conjugates with SEE-loaded Raji cells (at a ratio of 10:1) for the times indicated in the figure legends, after which they were lysed at 4°C for 40 min with 50 mM Tris-HCl (pH 7.5), 1% NP-40, 0.2% Triton X-100, 150 mM NaCl in phosphate-buffered saline (PBS) containing phosphatase and protease inhibitors (Roche). Cell lysates were centrifuged at 2500g for 10 min to remove cellular debris and nuclei. Whole-cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and incubated with the appropriate primary antibodies in Tris-buffered saline, 1% Tween 20. Bound antibodies were detected with horseradish peroxidase -conjugated secondary antibodies, and membranes were developed by enhanced chemiluminescence with Super-Signal West Pico or Femto chemiluminescent substrate (Pierce Chemical). Band intensities were quantified with Metamorph software, and the intensities of bands of interest were normalized to those of the appropriate controls, for example, total proteins in the case of analysis of phosphorylated proteins.

Fluorescence confocal microscopy

Confocal images were obtained with a Leica TCS-SP5 confocal scanning laser unit attached to an inverted epifluorescence microscope (DMI6000B) and fitted with an HCX PL APO lambda blue 63X/1.4 oil immersion objective, or with a Zeiss LSM700 confocal scanning laser unit attached to an inverted epifluorescence microscope (Observer.Z1) fitted with a Pan APO Chromat 63X/1.4 oil immersion objective. For live-cell imaging, microscopes are covered by a full acrylic box to enable analysis at 37°C, 5% CO₂. For time-lapse fluorescence imaging, cells were resuspended in HBSS (Lonza), 2% FBS, 20 mM Hepes, and were plated onto PLL-coated 35-mm dishes (MatTek). Cells expressing GFP or GFP–Lamin-A were differentially stained with vibrant DiI and DiD cell labels (Invitrogen). Images were analyzed with Leica LASAF (Leica Microsystems), Metamorph (Molecular Devices) Imaris (Bitplane), or ImageJ (NIH) software. For immunofluorescence assays, cells were plated on slides coated with PLL (50 μg/ml) for 5 min at 37°C, fixed in 2% PFA-1% sucrose, incubated with the appropriate primary antibodies followed by species-matching secondary antibodies coupled to Alexa Fluor fluorochromes (Invitrogen), and then mounted in ProLong Gold antifade medium (Invitrogen). To detect intracellular proteins, cells were first permeabilized with 0.5% Triton X-100 for 5 min. F-actin accumulation at the immunological synapse was measured in three-dimensional (3D) confocal maximal projections with the Synapsemeasure plugin in ImageJ (75). In the charts, each dot corresponds to a T cell-B cell conjugate. The distance from MTOC to the contact area between a T cell and an APC was measured in 3D confocal maximal projections with ImageJ.

Quantitative RT-PCR analysis

Total RNA was isolated from mouse splenocytes or J77 cells with Qiazol Lysis Reagent (Qiagen) and isopropanol precipitation or with the RNeasy Mini kit (Qiagen), according to the manufacturer’s instructions. RNA concentration and purity were assessed from the ratio of absorbances at 260 and 280 nm, and RNA integrity was verified by separation on ethidium bromide-stained 1% agarose gels. Complementary DNA (cDNA) was generated from total RNA (0.1 to 1 μg per reaction) with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with random hexamers and RNase Inhibitor, according to the manufacturer’s protocol. Quantitative PCR was performed with the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) with the PCR Power SYBR Green PCR Master Mix (Applied Biosystems), and reactions were performed in technical triplicates. The sequences of the forward and reverse primers used for RT-PCR are as follows. Mouse genes: Cd69: 5′-ATCTCTCCGTGGACCACTTG-3′ and 5′-CACAGCCCAAGGGATAGAAA-3′; Cd25: 5′-AACAACTGCAATGACGGAGACAT-3′ and 5′-CAGCTGGCCACTGCTACCTT-3′; Hprt1: 5′-CCTAAGATGAGCGCAAGTTGAA-3′ and 5′-CCACAGGACTAGAACACCTGCTAA-3′. Human genes: IL-2: 5′-AAGTTTTACATGCCCAAGAAGG-3′ and 5′-AAGTGAAAGTTTTTGCTTTGAGC-3′; CD69: 5′-TCCGGAGAGTGGACAAGAAAAT-3′ and 5′-GACCCTTCATGACGTGTTGAGA-3′; CD25: 5′-ACGGGAAGACAAGGTGGAC-3′ and 5′-TGCCTGAGGCTTCTCTTCAC-3′; LAMIN-A: 5′-GACGAGGATGAGGATGGAGA-3′ and 5′-GACACTGGAGGCAGAAGAGC-3′; LAMIN-A/C (recognizing both LAMIN-A and LAMIN-C mRNAs): 5′-ATGGAGATGATCCCTTGCTG-3′ and 5′-CTTCTTCCCCAGTGGAGTTG-3′; LAMIN-C: 5′-CAACTCCACTGGGGAAGAAG-3′ and 5′-AACATTCTTTAATGAAAAGATTTTTGG-3′; HPRT1: 5′-TGACACTGGCAAAACAATGCA-3′ and 5′-GGTCCTTTTCACCAGCAAGCT-3′. The exent of expression of a gene of interest was analyzed by the comparative Ct method with Biogazelle qBasePLUS software using as an internal control the housekeeping gene HPRT1 (hypoxanthine phosphoribosyltransferase 1). Results are represented as fold change relative to control conditions (see details in figure legends).

Generation of chimeric mice, adoptive transfer experiments, and contact hypersensitivity assays

C57BL6 wild-type recipient mice received 9.5 Gy of total body irradiation administered in two treatments from a ¹³⁷Cs source. Bone marrow cells from CD45.2⁺ wild-type and CD45.2⁺ Lmna^−/− mice were transplanted into CD45.1⁺ wild-type recipients by tail-vein injections immediately after irradiation (ten mice per group). Eight weeks after transplantation, the chimeric condition of the mice was assessed by flow cytometric analysis of blood cells stained with a combination of fluorescently labeled anti-CD45.1 and anti-CD45.2 antibodies to detect CD45.2⁺ cells from donors and CD45.1⁺ cells from recipient mice, which confirmed that more than 90% of the cells analyzed were derived from the transplanted bone marrow cells (fig. S6). For the adoptive transfer experiments, CD4⁺ T cells from CD45.2⁺ Lmna^−/−, CD45.1⁺CD45.2⁺ wild-type, or CD45.2⁺ wild-type mice were isolated by negative selection from spleens with MACS separation columns (Miltenyi Biotec) after labeling the cells with a cocktail of biotinylated antibodies against B220, CD19, MHCII, CD11c, CD11b, CD44, and CD8α, as well as with a solution containing streptavidin-bound magnetic microbeads (Miltenyi Biotec). Adoptive transfer experiments were performed by inoculating CD45.1⁺ wild-type recipient mice with 2 × 10⁶ CD4⁺ T cells from the spleen of CD45.2⁺ Lmna^−/− or CD45.2⁺ wild-type mice or by inoculating CD45.1⁺ wild-type recipient mice with 4 × 10⁶ cells of a 1:1 mixture of CD4⁺ T cells from the spleens of CD45.2⁺ Lmna^−/− and CD45.1⁺CD45.2⁺ wild-type mice. In both cases, adoptive cell transfer was performed 24 hours before the first application of oxazolone. In mice transplanted with bone marrow cells as well as in adoptively transferred mice, contact hypersensitivity was induced by painting shaved mouse abdomen skin with 200 μl of 3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, Sigma) in ethanol. After 5 days, mice were challenged with 20 μl of 1% oxazolone on each side of the right ear. The left ear was painted with vehicle as a control. In transplanted mice, ear thickness was measured in a blinded manner every day for 7 days. Net ear swelling was calculated by subtracting the thickness of the vehicle-treated ear from the thickness of the oxazolone-challenged ear. Adoptively transferred mice were sacrificed 3 days after the second application of oxazolone, and the analysis of the numbers of transplanted CD4⁺ T cells was determined in the skin of the ears, the cervical draining lymph nodes, and the spleen. To analyze cells in the skin, the ears were removed from the mice and split into dorsal and ventral halves. The skin was placed in 2 ml of digestion buffer containing DNase I (0.1 mg/ml) and Liberase TL (0.2 mg/ml, Roche) diluted in 1% FBS in RPMI (GIBCO). After 1 hour of incubation at 37°C, ears were placed into a 70-mm cell strainer (BD Biosciences) and mashed through the mesh. The strainers were washed with 2 ml of 1% FBS, 2 mM EDTA in PBS (GIBCO).

TIRF microscopy

J77 cells were co-transfected with plasmids encoding CD3ζ-EGFP and either dsRED or dsRED-Lamin-A. Transfected cells were sorted on a FACSAriaI flow cytometer, cultured for 1 day, and then plated onto 35-mm dishes (MatTek) coated with purified anti-CD3 antibody (T3b, 10 μg/ml). Images were acquired for 10 min at 0.35-s intervals on a Leica AM TIRFM MC unit mounted on a Leica DMI6000B microscope fitted with a 100×/1.46 oil immersion objective, with 1.6× magnification and ~90-nm depth penetration. Images were analyzed with Imaris software (Bitplane) with a Brownian motion algorithm.

Statistical analysis

All statistical analyses were performed with Prism GraphPad or Microsoft Office Excel. Unless otherwise stated, statistical significance was calculated by two-tailed Student’s t-test. When specified, one-way ANOVA or two-way ANOVA with Bonferroni’s post-hoc multiple comparison test was used. Significance of differences was calculated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.