Cell-Extrinsic Defective Lymphocyte Development in Lmna-/- Mice

Background Mutations in the LMNA gene, which encodes all A-type lamins, result in a variety of human diseases termed laminopathies. Lmna-/- mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna-/- mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development. Principal Findings Lmna-/- mice displayed severe age-dependent defects in T and B cell development which coincided with runting. Lmna-/- bone marrow reconstituted normal T and B cell development in irradiated wild-type recipients, driving generation of functional and self-MHC restricted CD4+ and CD8+ T cells. Transplantation of Lmna-/- neonatal thymus lobes into syngeneic wild-type recipients resulted in good engraftment of thymic tissue and normal thymocyte development. Conclusions Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna-/- mice do not result directly from the loss of A-type lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna -/- mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice.


Introduction
Nuclear lamins are intermediate filament proteins and have roles in various nuclear processes, including DNA replication, chromatin organization, and gene transcription [1][2][3][4]. One gene, LMNA, encodes the A-type nuclear lamins, which produce the predominant A-type lamin proteins A and C. Mutations in LMNA are associated with more than 13 different tissue-specific diseases, collectively termed laminopathies, which include muscular dystrophies, cardiomyopathies, and more recently, a series of progeroid diseases that resemble some aspects of premature aging (for review, see Cohen et al.) [5]. Lmna -/mice are characterized by a variety of tissue-specific defects consistent with those observed in human laminopathies, but additionally display growth defects as early as 2 weeks of age, reduced thymus and spleen size, defective spermatogenesis, and death by 6-8 weeks of age [6,7].
A-type lamins have limited expression in the hematopoietic system [8]. Previous studies describing the presence of lamin A/C proteins in lymphocyte lineages are mixed, but largely suggest limited to no presence of A-type lamin proteins in early stages of B and T cell development, with increasing protein abundance in mature B and T cells and stimulated lymphocytes [9][10][11][12][13]. Furthermore, A-type lamin proteins are found only in a minority of cells from the bone marrow, thymus, spleen, and lymph nodes, which may represent cell types of the stroma such as epithelial cells, pericytes, inflammatory cells, fibroblasts, endothelial cells, and smooth muscle cells [8,[13][14][15].
Although no immune disease to date has been linked to mutations in LMNA, Lmna -/mice display reduced thymus and spleen size, suggesting a potential role for A-type lamins in the postnatal development and/or homeostasis of lymphocyte lineages. Here, we investigate the reduced thymus and spleen size in Lmna -/mice and identify a progressive, age-dependent impairment in T and B cell development. These defects are not cell-autonomous, because transplanted Lmna -/bone marrow can reconstitute irradiated recipient Lmna +/+ immune tissues. Transplanted Lmna -/thymus lobes show good engraftment in wild-type hosts and drive normal thymocyte development. Therefore, the altered immune cell development is not specific to loss of Lmna expression in developing lymphocytes or thymic epithelial cells, but likely an indirect effect of loss of lamin expression in other non-lymphoid tissues.

Mice
Lmna +/mice were obtained from Colin Stewart (Institute of Medical Biology, Immunos Singapore) [7] and sibling mating was used to generate Lmna +/+ and Lmna -/mice. This line was also backcrossed to C57BL/6J (B6) mice for 9 additional generations and the resulting mice, B6.129S1(Cg)-Lmna tm1Stw /BkknJ, were used for thymus transplantation studies. B6.SJL (B6.SJL-Ptprc a -Pepc b /BoyJ) Ly5.1 + and C57BL/6J Ly5.2 + mice were crossed to generate F1 Ly5.1 + Ly5.2 + mice used as recipients in bone marrow chimera experiments. Mice were bred and maintained under specific-pathogen free conditions. All experiments were conducted in accordance with the review board of the University of Washington Institutional Animal Care and Use Committee, who approved this study.

Bone marrow chimeras
Single-cell suspensions of hind leg bone marrow from Lmna +/+ and Lmna -/mice were T cell-depleted by staining with PEconjugated anti-CD4 and anti-CD8 followed by removal of PElabeled cells using the EasySep PE selection separation kit (Stem Cell Technologies; Vancouver, Canada). Alternatively, T cells were depleted using anti-Thy1 (13.4.6), anti-CD4 (RL172), and anti-CD8 (3.168.8) followed by lysis using rabbit complement (Cedarlane Laboratories Limited; Hornby, Canada). Sex-matched recipient B6 Ly5.1 + Ly5.2 + mice were irradiated with 1000 rads 6-8 hours prior to bone marrow injection, and maintained on antibiotic water (polymixin B sulfate and neomycin sulfate from Invitrogen), for 2 days prior to 2 weeks following irradiation. Recipient mice were injected with 325610 6 T cell-depleted bone marrow cells into the lateral tail vein.

LCMV infection
Mice were infected by intraperitoneal injection with 2610 5 PFU of LCMV Armstrong (provided by M.K. Kaja; University of Washington). Spleens were harvested 8 days later for analysis of LCMV-specific CD4 + and CD8 + T cells by intracellular cytokine staining and flow cytometry.

Thymic lobe transplantation
Thymic lobes from 1-day-old Lmna +/+ and Lmna -/neonates were transplanted into 10-to 16-week-old female Lmna +/+ host mice by engraftment underneath the kidney capsule. Briefly, 2 days prior through 3 days after surgery, host mice were given medicated water (ibuprofen 0.2 mg/mL). Neonatal thymic lobes were placed on sterile nitrocellulose pads and suspended at the culture medium interface to ensure high oxygen transfer and cell survival. Thymuses were cultured at 37uC, 5% CO 2 for less than 24 hours prior to transplantation while genotypes of extracted neonatal thymuses were being determined. The culture medium consisted of DMEM, 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.1 mM non-essential amino acids, 0.3% sodium bicarbonate, 25 mM HEPES, and 50 mM b-mercaptoethanol. Host mice were anesthetized with ketamine and xylazine (130 mg/kg and 8.8 mg/kg respectively) and a laparotomy was performed after hair removal and sterilization of the surgical area. Thymic lobes were transferred underneath the left kidney capsule. Host mice were then sutured and monitored until full recovery from anesthesia. Transplanted thymuses were allowed to engraft for 6 weeks prior to analysis.

Runting of Lmna -/mice is accompanied by progressive thymic and splenic atrophy
It has been previously reported that Lmna -/have mice small lymphoid organs, including the spleen and thymus [7]. We analyzed lymphoid organs from Lmna -/mice to better understand the effects of Lmna deficiency on the immune system. Neonatal Lmna -/mice were indistinguishable from Lmna +/+ littermates 1 week after birth. By 4 weeks of age and older, Lmna -/mice were severely runted compared to Lmna +/+ mice ( Figure 1A). The thymus and spleen from 6-week old Lmna -/mice were reduced in size ( Figure 1B). We observed that while thymic cellularity in neonatal Lmna +/+ and Lmna -/mice was similar, 4-week old Lmna -/mice displayed decreased thymic cellularity compared to Lmna +/+ littermates that became more striking with age ( Figure 1C). Similarly, splenic cellularity was unaffected in neonatal Lmna -/mice, but was severely reduced at 4 and 9 weeks of age ( Figure 1D). Thymic and splenic cellularity were reduced even when normalized to the reduced body weight of Lmna -/mice ( Figure  S1).

Defective thymocyte maturation in the atrophic Lmna -/thymus
Given the striking decrease in thymic cellularity in Lmna -/mice, we predicted a decrease in the proportion of the predominant CD4 + CD8 + double-positive (DP) thymocyte population. Surprisingly, flow cytometric analysis revealed no perturbation in the frequency of DP thymocytes (Figure 2A and 2B). Slight reductions were noted in the percent of CD4 single-positive (SP) and CD8 SP thymocytes, starting at 4 weeks of age. There was also a significant increase in the proportion of double-negative (DN) thymocytes at 9 weeks of age in Lmna -/mice, possibly indicating that cells are partially blocked in their developmental progress at one of the DN stages. To further explore this possibility, we analyzed DN thymocytes, and found that the percent of DN cells that were DN1 (least mature), DN2, DN3, and DN4 (most mature) remained unaltered in Lmna -/mice, indicating that the relative increase in the percent of DN thymocytes at 9 weeks does not result from a developmental block at one of the DN stages ( Figure S2).
After completion of TCRb rearrangement in the thymus, cells proliferate at the DN3 to DN4 stage and upregulate CD4 and CD8 coreceptors to become CD4 + CD8 + DP thymocytes. At the DP thymocyte stage, cells undergo TCRa rearrangement, and test the newly generated TCRa chain paired with the already available TCRb chain for the ability to interact with self-MHC class I and class II molecules. Useful TCR interactions will result in positive selection of the cell, upregulation of TCR and CD69 surface expression, and lineage commitment of CD4 SP and CD8 SP thymocytes. Failure to be selected results in additional TCRa rearrangement, and eventually, if no positive selection takes place, the cell undergoes apoptosis (for review, see Starr et al.) [17]. We observed that the DPdull (CD4 low CD8 low ) thymocytes, a sub-population of DP thymocytes that are normally enriched for selected TCRb + cells, lacked positively-selected TCRb high CD69 + cells in 9-week-old Lmna -/mice ( Figure 2C and 2D). Although there was not a significant decrease in the percent of CD4 SP and CD8 SP gated thymocytes from 9-week-old Lmna -/mice, these cells displayed clear signs of altered development, including decreased cell surface TCRb expression for both CD4 SP and CD8 SP thymocytes, and decreased frequency of post-positive selection TCRb + mature CD8 SP thymocytes ( Figure 2E and 2F). Thus, the development of TCRb + mature SP thymocytes is defective in Lmna -/mice, suggesting that positive selection is impaired.

Age-dependent progressive T and B cell lymphopenia in
Lmna -/mice The apparent defect in positive selection in the thymus, compounded with the reduced thymic size in Lmna -/mice, suggested the size of the peripheral T cell compartment in these mice would be diminished. To investigate this possibility, we analyzed splenic CD4 + and CD8 + T cells. While Lmna -/mice exhibit an increased percent of splenic CD4 + and CD8 + T cells  Lmna -/mice display an age-dependent defect in thymic T cell development. Thymocytes were stained for CD4, CD8, CD69, and panTCRb surface expression. A. Representative flow cytometry plots show CD4/CD8 analysis of live-gated thymocytes. B. Charts show the percent of thymocytes that are DN, DP, CD4 SP, and CD8 SP for each indicated age group compiled from all mice analyzed. C. CD69/TCRb analysis of CD4 low CD8 low gated DP dull cells from 9-week-old mice with a gate indicating the percent of post-selection TCRb high CD69 + cells. D. The percent of CD4 low CD8 low DP dull cells that are TCRb high CD69 + for 4-and 9-week-old mice. E. Histograms of gated CD4 SP and CD8 SP cells showing TCRb expression. Colored numbers (Blue: Lmna +/+ ; Red: Lmna -/-) in histogram indicate the percent of SP cells that are TCRb + . F. CD4 SP and CD8 SP gated thymocytes were analyzed for the percent that are post-positive selection TCRb+ mature SP thymocytes. Charts show the mean percent with error bars indicating the standard deviation (Lmna +/+ N = 3; Lmna -/-N = 3 for each indicated age group). P values were calculated using an unpaired twotailed Student's t test (* p,0.05, ** p,0.001, *** p,0.0001). doi:10.1371/journal.pone.0010127.g002 compared to Lmna +/+ mice ( Figure 3A), the absolute number of CD4 + and CD8 + T cells is dramatically decreased in Lmna -/mice at both 4 and 9 weeks of age ( Figure 3B). These reduced T cell numbers are a reflection of the loss in absolute numbers of total splenocytes in the Lmna -/mice at these ages ( Figure 1D). Given this severe T cell lymphopenia, we predicted that the remaining T cells in Lmna -/mice would be driven to an activated/memory CD62L low CD44 high phenotype through lymphopenia-induced homeostatic proliferation. Surprisingly, there was no change in the percent of CD4 + and CD8 + cells that bear an activated/ memory phenotype in 4-week-old (not shown) and 9-week-old Lmna -/spleens ( Figure 3C). Although there were subtle increases in the proportion of T cells that are CD44 high and either CD62 low or CD62L high in the lymph nodes, these remained dramatically lower than other mouse systems characterized by T cell lymphopenia [18,19]. In 9-week-old Lmna -/spleens, we also observed an alteration in the percent of B cells ( Figure 3D). This alteration coincided with a severe reduction in B cell numbers in the spleen ( Figure 3E). Taken together, these data indicate that the development and/or survival of both B and T lymphocytes is defective in Lmna -/mice.

B cell development is interrupted in Lmna -/mice
The cellularity of bone marrow from Lmna -/mice was decreased in 4-week-old mice ( Figure 4A). Because splenic B cell numbers were dramatically reduced in 4-and 9-week-old Lmna -/mice compared to Lmna +/+ mice, we analyzed bone marrow to determine whether B cell development was altered. Indeed, the population of developing B220 + cells committed to the B cell lineage was severely reduced in Lmna -/mice, and composed primarily of a B220 high subpopulation ( Figure 4B). This reduction occurred as early as 4 weeks of age in Lmna -/mice ( Figure 4C and 4D). Of the remaining B220 + cells in the bone marrow of Lmna -/mice, there was a reduced percent and number of IgM -IgDcells and IgM + IgDimmature B cells, and an increased percent but normal number of IgM + IgD + mature B cells ( Figure 4E and 4F). Because the developing B220 low IgM -IgDcells and IgM + IgDimmature B cells are so severely affected in Lmna -/mice, it is likely that these B220 high IgM + IgD + mature B cells reached maturity before these mice experienced the precipitous decline in B cell development. Therefore, B cell development is impaired in Lmna -/mice with age.

Lmna -/-B and T cell defects are not cell-autonomous
The alteration in lymphocyte development and homeostasis in Lmna -/mice could result from cell-intrinsic defects caused by Lmna gene deficiency, from the contribution of A-type lamin function in non-hematopoietic cells that impact lymphocyte development, or instead, from the severe pathology and poor health of the Lmna -/mouse. We generated bone marrow chimeras by reconstituting lethally irradiated Lmna +/+ mice with Lmna +/+ or Lmna -/bone marrow to determine whether the defects in T and B cell development and homeostasis were cell-autonomous. Because thymocyte and splenocyte defects in Lmna -/mice were agedependent, we waited .40 weeks after irradiation and reconstitution to analyze the number of Lmna +/+ and Lmna -/populations in bone marrow, thymus, and spleen of chimeras. Bone marrow chimeric mice reconstituted with either Lmna -/or Lmna +/+ bone marrow had similar frequencies and numbers of total bone marrow cells and B220 + bone marrow cells ( Figure 5A). Bone marrow chimeras that received either Lmna -/or Lmna +/+ bone marrow had identical relative and absolute numbers of donorderived DN, DP, and SP thymocyte populations ( Figure 5B). Additionally, the relative and absolute number of donor-derived B and T cells in the spleen were the same in chimeric mice reconstituted with either Lmna -/or Lmna +/+ bone marrow ( Figure 5C).
To assess the immunocompetence of Lmna -/-CD4 + and CD8 + T cells that appear to develop normally in Lmna +/+ bone marrow chimeric hosts, we generated Lmna +/+ /Lmna -/mixed bone marrow chimeras by reconstituting lethally-irradiated Lmna +/+ mice with a mix of Lmna +/+ and Lmna -/bone marrow. Chimeras were infected 18 weeks post-reconstitution with the acute virus LCMV, known to generate a strong virus-specific response from both CD4 + and CD8 + T cells in B6 mice. Eight days later (at the peak of the T cell response), we analyzed Lmna +/+ and Lmna -/-CD4 + and CD8 + T cells for the presence of antigen-specific effector T cells. IFNc + , IL-2 + , and TNFa + virus-specific effector cells were present at the same frequencies among both Lmna +/+ and Lmna -/-CD4 + T cells within the same mixed bone marrow chimera ( Figure 6A and 6B). Similarly, virus-specific cytokineproducing effectors were present at the same frequencies among Lmna +/+ and Lmna -/-CD8 + T cells ( Figure 6C and 6D). These data show that Lmna -/-CD4 + and CD8 + T cells that develop in an Atype-lamin-sufficient environment can recognize foreign antigen in the context of self MHC, and proliferate and produce cytokine following antigen encounter. Therefore, in a Lmna +/+ host, Lmna -/-T cells are appropriately selected and functional, even when in direct competition with Lmna +/+ T cells.
Thus, the splenic and thymic cellularity defects and the apparent deficiencies in lymphocyte development observed in Lmna -/mice are not cell-autonomous for T or B lymphocytes. This implies that A-type lamin function/expression is not required in these cell types for normal development and homeostasis. Further, it implies the possibility that A-type lamin expression in non-hematopoietic cells influences the function and development of these lymphocytes.

Lmna -/thymus supports normal T cell development
We examined the possibility that defective T cell development in Lmna -/mice might result from specific defects in thymic stromal cells, which are required for positive selection of developing DP thymocytes. To determine whether Lmna -/thymic lobes could foster proper thymocyte development, we transplanted Lmna -/neonatal thymic lobes into adult syngeneic recipients and analyzed thymic grafts 6 weeks later, after seeding of the lobes with hostderived Lmna +/+ stem cells. Grafted Lmna -/and Lmna +/+ thymuses were similar in appearance and contained similar numbers of thymocytes ( Figure 7A and 7B). Engraftment of both Lmna +/+ and Lmna -/thymic lobes appeared normal, as both supported a normal distribution of DN, DP, and CD4 SP and CD8 SP developing thymocytes ( Figure 7C). Unlike the CD4 SP and CD8 SP thymocytes from Lmna -/mice (see Figure 2E), CD4 SP and CD8 SP thymocytes within Lmna -/thymuses grafted into Lmna +/+ hosts had normal TCRb expression and a similar percent of TCRb+ mature SP thymocytes ( Figure 7D). Therefore, Lmna -/thymic stromal cells support normal T cell development when they are in a healthy, A-type-lamin-sufficient host.

Discussion
In this study, we sought to determine the nature of lymphoid organ atrophy and lymphocyte population deficiencies in Lmna -/mice. Young adult Lmna -/mice exhibit striking decreases in splenic T and B lymphocyte numbers that correlate with reduced populations of developing thymocytes and B220 + B cells in bone marrow. The apparent selective block in T cell positive selection and early B cell development in Lmna -/mice hinted at specific developmental checkpoints at which A-type lamin function was required. However, global reduction in both developing and mature lymphocytes coincided with the severe runting, as well as cardiac and skeletal muscle disease onset that occurs in Lmna -/mice [7,20]. In addition, 1-week-old neonatal Lmna -/mice are healthy and indistinguishable from wild-type littermates, with comparable thymocyte numbers and splenic T and B cell numbers. These latter observations suggest a more general, perhaps indirect, impact on lymphocyte development resulting from the rapidly degrading health of older Lmna -/mice.
Bone marrow chimeras were generated to test the possibility that lamin A/C expression is required in developing lymphocytes for normal T and B cell maturation. We did not find any evidence for this, as Lmna -/bone marrow completely reconstituted wildtype recipient mice with normal T and B cell development and peripheral lymphocyte populations for more than 40 weeks following bone marrow transfer. Lmna -/-CD4 + and CD8 + T cell populations in mixed bone marrow chimeras were able to generate self-MHC class II and class I restricted T cell responses to LCMV infection. These antigen-specific and differentiated CD4 + IFNc + and CD8 + IFNc + effector cells were generated at the same frequency and demonstrated the same cytokine-producing potential from Lmna -/and Lmna +/+ cells within the same mixed bone marrow chimeras. These data show that even in differentiated T cell effector lineages, Lmna gene deficiency has no cell-autonomous consequence. Together, these data show that intrinsic lamin A/C expression is not required either for B cell development or for T cell development and function.
A second possible explanation for the lymphocyte developmental defects observed in Lmna -/mice is that stromal elements within primary lymphoid organs require lamin A/C expression for their function in promoting normal T and B cell development. Thymic epithelial cells play a critical role in the positive selection of developing thymocytes as rearranged TCRa and TCRb gene products are tested for useful and safe TCR interactions with self-MHC:peptide complexes. We tested whether there were thymic epithelial cell-specific defects in the Lmna -/thymus by transplanting mutant thymic lobes into wild-type hosts, and allowing them to engraft for sufficient time to be seeded by wild-type hematopoietic stem cells. Within these mice, Lmna -/thymic lobes maintained normal size and thymocyte cellularity compared to wild-type thymic grafts. Thus, Lmna -/thymic stroma displayed no cellintrinsic defects that can explain the reduced cellularity and altered thymocyte development observed in Lmna -/mice.
However, a formal possibility still exists that defective stromal elements may influence B cell development in the Lmna -/bone marrow. The stromal and hematopoietic microenvironments in the bone marrow are different than in the thymus and perhaps represent more complex interactions with diverse cell types. Of note, mesenchymal stem cells also inhabit the bone marrow and, in addition to their role in repopulating tissues of mesenchymal origin, have a regulatory role stimulating hematopoietic stem cell growth and differentiation (reviewed in Uccelli, et al.) [21,22]. Because most tissues affected in laminopathy syndromes are of mesenchymal origin and because recent findings have suggested that altered A-type lamin function affects the maintenance of mesenchymal stem cell identity and ability to differentiate [23], defective communication between these cell types could also lead to altered lymphocyte development. Unfortunately, bone marrow architecture is difficult to preserve in transplantation studies, and future studies to address these possibilities will require conditional deletion of Lmna within select cell types of the bone marrow.
A third possible explanation for the lymphocyte developmental defects observed in Lmna -/mice is an indirect effect of laminopathies at distal sites. Lmna -/mice suffer from stunted growth and previously characterized cardiac and skeletal muscle defects contribute to their decline in health and premature death at around 6-8 weeks of age [7,20,24,25]. Given that A-type lamins influence a variety of cell types, it is difficult to distinguish between cell-autonomous defects in specific tissues of Lmna deficiency and possible indirect effects rendered by so many dystrophic tissues. Thus, the atrophic lymphoid organs and accompanying reduced lymphocyte development and maintenance in the Lmna -/mice may result from the overall poor health of the mice. These defects may be driven by altered stress hormone levels, cytokines from a chronic inflammatory response to striated muscle dysfunction that is present in Lmna -/mice [7,20,26,Frock et al. manuscript in preparation], or decreased metabolic function resulting in nutritional deprivation of lymphocytes. Possibly supporting the argument for a role in nutritional defects, homeostasis of peripheral T cells appears to be impaired in Lmna -/mice. Despite severe decreases in splenic T cell numbers, there was no upregulation of CD44 or downregulation of CD62L in spleen ( Figure 3C), changes that characterize T cells that have undergone lymphopenia-induced homeostatic proliferation.
In summary, we have shown that Lmna -/mice undergo a progressive decline in the size of their thymus and spleen, a decline of developing B220 + B cells in the bone marrow, a decline of total developing thymocytes, and striking decreases in peripheral B and T cells. All of these changes occurred after 1 week of age, and coincided with runting of Lmna -/mice and onset of multi-tissue dystrophies. These immune defects were completely rescued when lymphocyte development was uncoupled from Lmna -/animals through bone marrow chimera and thymic grafting experiments. We conclude that these effects on lymphocyte development in Lmna -/mice are indirect, and most likely result from an unhealthy animal that is unable to support normal immune system development and homeostasis. Our data indicate that even apparently selective developmental blocks can arise from lymphocyte-extrinsic, indirect effects and underscore the need to analyze lymphocyte development in animals that provide a healthy environment before concluding that a particular gene or cell type of interest impacts development of a functional immune system. Figure S1 Progressive cellularity defects in spleen and thymus occur even when organ cellularity is normalized to body weight of Lmna -/mice. A. Thymic and B. splenic cellularity was normalized to the body weight of each individual mouse of each of the indicated ages. Charts show the mean normalized value with error bars indicating the standard deviation (Lmna +/+ N = 3; Lmna -/-N = 3 for each indicated age group). P values were calculated using an unpaired two-tailed Student's t test (* p,0.05, ** p,0.001). Found at: doi:10.1371/journal.pone.0010127.s001 (0.25 MB TIF) Figure S2 Normal composition of double-negative DN1-DN4 thymocytes in 9-week old Lmna -/mice. Thymocytes from 9-week old mice were stained for CD4, CD8, CD25, and CD44 surface expression. DN thymocytes were analyzed for the percent that are DN1 (CD44 + CD25 2 ), DN2 (CD44 + CD25 + ), DN3 (CD44 2 CD25 + ), and DN4 (CD44 2 CD25 2 ). Chart shows the mean percent with error bars indicating the standard deviation (Lmna +/+ N = 3, Lmna -/-N = 3). P values were calculated using an unpaired two-tailed Student's t test and none reached significance. Found at: doi:10.1371/journal.pone.0010127.s002 (0.10 MB TIF)