- Email: [email protected]
Qa-2 associated lipid rafts are indispensable in the final maturation of CD4+CD8− thymocytes
Immunology Letters 148 (2012) 163–171
Contents lists available at SciVerse ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Qa-2 associated lipid rafts are indispensable in the final maturation of CD4+ CD8− thymocytes Juan Li a , Hai-Dong Li b , Yu Zhang c , Jun Zhang c,∗ a Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Educational Ministry, Tianjin Medical University, 22 Qixiangtai Road, Tianjin 300070, China b Department of Biochemistry, Tianjin Medical University, Tianjin, China c Department of Immunology, School of Basic Sciences, Peking University Health Science Center, Beijing, China
a r t i c l e
i n f o
Article history: Received 24 April 2012 Received in revised form 21 September 2012 Accepted 9 October 2012 Available online 17 October 2012 Keywords: Thymocyte Qa-2 Lipid rafts CD4SP SP4
a b s t r a c t Lipid rafts have been shown to play significant roles in thymocyte development. However, the exact role of lipid rafts in single positive (SP) thymocyte differentiation is poorly characterized. We previously defined a developmental program (SP1 → SP4) for CD4SP thymocytes. In this study, we found that lipid raft components were up-regulated during CD4SP maturation. Qa-2, a unique marker for the most mature SP4 subset, was localized to lipid rafts and heterogeneously expressed in SP4 cells. Functional assays showed that the proliferation capacity of SP4 cells correlated with the expression of Qa-2. Raft-disruption on both CD4SP and epithelial cells by cholesterol extraction or cholesterol oxidation in a medullary thymic epithelial cell (mTEC)-supported co-culture system impaired the transition from SP3 to SP4. This result was further confirmed in fetal thymic organ culture system. Collectively, these studies suggest that raftassociated signaling between mTECs and thymocytes drives the differentiation of CD4SP thymocytes and lipid rafts are involved in the final maturation of SP4 thymocytes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lipid rafts are specialized detergent-resistant microdomains of the eukaryotic plasma membrane that are mainly composed of sphingomyelins, glycosphingolipids and cholesterol. Accumulating evidence has suggested that lipid rafts play a key role in vital cellular processes, including signaling, apoptosis and cell adhesion. Lipid rafts are proposed to serve as signaling platforms to facilitate the propagation of signaling cascades from various membranebound receptors, especially during T cell activation [1]. This role has been emphasized by the finding that TCR is recruited to lipid rafts upon receptor stimulation. Several acylated proteins involved in TCR signaling, such as Lck and Fyn, reside constitutively in lipid rafts. Many signaling proteins become concentrated in lipid rafts upon TCR activation, including ZAP-70, PKC and PKB [2,3]. Lipid rafts also contribute to the cascade of events leading to T cell apoptosis after CD95/Fas ligation [4]. Notably, several studies have demonstrated that lipid rafts may play a crucial role in thymocyte development. Differentiating thymocytes are subdivided into four major subpopulations: CD4− CD8− (double-negative, DN), CD4+ CD8+ (double-positive, DP) and CD4+ CD8− and CD4− CD8+ SP. The roles
∗ Corresponding author. Tel.: +86 10 82805649. E-mail address: [email protected] (J. Zhang). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2012.10.006
of lipid rafts in thymocyte development are well documented from the DN to SP stage. The transition from the DN to DP stage requires the palmitoylation of the pre-TCR␣ chain and its recruitment to the lipid raft [5]. The selection of DP cells to become SP cells occurs after CD3 associates with TCR in the raft regions [6,7]. Moreover, a recent study suggests that the CD4 lineage commitment from DP is directed by the raft-associated presentation of MHC class II molecules [8]. It has also been suggested that raft-associated Lck and Fyn may be involved in nearly all stages, from DN to SP [9]. However, the exact functions of lipid rafts in SP thymocyte development remain poorly characterized. Recently, more and more studies indicate that SP thymocytes are heterogeneous and undergo an ordered maturational program in the thymic medulla. Several important events occur in this process, including negative selection and functional maturation. We previously resolved CD4SP thymocytes into four subsets: SP1 (6C10+ CD69+ Qa-2− ), SP2 (6C10− CD69+ Qa2− ), SP3 (6C10− CD69− Qa-2− ) and SP4 (6C10− CD69− Qa-2+ ) cells [10,11]. In vivo and in vitro assays demonstrated that the differentiation of CD4SP thymocytes followed a strict order from SP1 to SP4 and exhibited increased maturity from SP1 to SP4. Notably, our study identified the SP3/SP4 transition as a critical checkpoint in CD4SP development [10]. The major difference between SP3 and SP4 stage phenotype is the surface expression of Qa-2 in SP4 cells. Qa-2 is the product of the Ped (preimplantation embryonic development) gene. Qa-2+ embryos have an increased rate of
164
J. Li et al. / Immunology Letters 148 (2012) 163–171
preimplantation development and of subsequent embryonic survival than Qa-2− embryos [12]. Qa-2, the murine homolog of HLA-G, which belongs to the family of non-classical MHC proteins, has a glycosylphosphatidylinositol (GPI) linkage to the cell membrane [13,14]. GPI-anchored proteins preferentially localize in lipid rafts in the cell membrane, and Qa-2 has indeed been reported to localize to lipid rafts in T cells [15]. Cross-linking Qa-2 induces resting T cells to proliferate. The Qa-2-mediated signaling pathway requires Fyn, the PI-3 kinase and Akt [14]. In general, Qa-2 has only been used as a thymocyte maturation marker [16], and its exact role in thymocytes remains elusive. In the present study, we demonstrate that the expression level of Qa-2 in lipid rafts of SP4 correlates with cell functionality and that raft disruption impairs CD4SP thymocyte differentiation in vitro and in fetal thymic organ culture system, suggesting a potential role for Qa-2 associated lipid rafts in the functional maturation of SP4 cells and a key role of lipid rafts in CD4SP thymocyte development. 2. Materials and methods
After extensive rinsing, cells were cultured in vitro or labeled with filipin. 2.5. Flow cytometric assay of differential detergent resistance (FCDR) FCDR was performed as described [18]. Briefly, the fluorescence intensities of unlabeled, labeled or MCD-pretreated cells were recorded on FACS before and 5 min after mixing the cells with 0.1% (v/v) Triton X-100 (Sigma–Aldrich) dissolved in PBS. The extent of detergent resistance was estimated using the FCDR index, which is calculated according to the following equation: FCDR =
(FLdet − FLBg det ) (FLmax − FLBg )
FLmax , fluorescence of labeled untreated cells; FLdet , fluorescence of the cells treated with detergent for 5 min; FLBg , autofluorescence of the untreated cells; FLBg det , autofluorescence of the detergenttreated cells.
2.1. Mice C57BL/6 mice were purchased from Beijing Vitalriver Co. and bred in the Animal Breeding Facilities of Tianjin Medical University. A ␥-irradiated sterile diet and autoclaved distilled water were used. The animal studies were approved by the ethics committee of Tianjin Medical University. 2.2. Flow cytometry and cell sorting Freshly isolated thymocytes were stained with fluorochromelabeled mAbs, sorted using a FACSCalibur instrument (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Tree Star, USA) software. The following mAbs were used: anti-Qa-2 and anti-CD8 fluorescein isothiocyanate (FITC)-conjugated mAbs; anti-CD69 phycoerythrin (PE)-conjugated mAb; anti-CD4 PerCPcy5.5-conjugated mAb; and allophycocyanin (APC)-conjugated streptavidin (eBioscience, San Diego, CA). Cholera toxin B (CTB)conjugated biotin was obtained from Sigma–Aldrich (St. Louis, MO). For filipin staining, the cells were treated with 250 g/ml fresh filipin III (Sigma–Aldrich) in balanced salt solution (BSS) at 4 ◦ C for 2 h, and then acquired by LSRII (BD Biosciences) with the krypton ion laser tuned to UV (350.7–356.4 nm) and emission detected at 420–460 nm. The isolation of different subsets of CD4SP thymocytes was performed as described [10]. The resulting viable cells were then stained for CD4, CD69 and Qa-2 and sorted into various subsets with FACSAria (BD Bioscience). The purity of cells harvested was >98% when reanalyzed on FACS. 2.3. Proliferation assay SP4 thymocytes were sorted into Qa-2lo , Qa-2int and Qa-2hi subsets, seeded at 1 × 105 per well into 96-well tissue plates and cultured in the presence of 2.5 g/ml Con A (Amersham Pharmacia, Piscataway, NJ) for 72 h. Then, 0.5 Ci per well [3 H] thymidine was added to the culture for the last 12 h. Cells were harvested and their thymidine incorporation was measured. 2.4. Depletion of plasma membrane cholesterol by methylcyclodextrin (MCD) Thymocytes and mTEC1 cell line (106 /ml) were treated with 10 mM MCD (Sigma–Aldrich) at 37 ◦ C in BSS containing 0.1% BSA for 30 min or 15 min, respectively. This treatment removes approximately 40–50% of the plasma membrane cholesterol [17].
2.6. Immunofluorescence CD4SP thymocytes were stained with purified anti-Qa-2 mAb and biotin-CTB, followed by IF488-anti-mouse IgG and PEstreptavidin (Sungene Biotech, Tianjin, China). Then the cell suspensions were layered onto poly(l-lysine)-coated coverslips for 1 h at room temperature. Images were acquired using PerkinElmer Ultraview VoX spinning disk confocal microscope (PerkinElmer, Waltham, MA). 2.7. Sucrose gradient centrifugation and Western blotting CD4SP thymocytes (1 × 108 cells) were lysed in 1 ml ice-cold lysis buffer containing 20 mM Tris–HCl (pH 7.2), 150 mM KCl, 0.05% Triton X-100, and protease inhibitors. The cells lysates were homogenized with 10 strokes of a glass dounce homogenizer, then mixed with 1 ml of 85% sucrose in lysis buffer, and transferred to SW41 centrifuge tubes. The samples were then overlaid with 6 ml of 35% sucrose and 3.5 ml of 5% sucrose and ultracentrifuged (Beckman, Palo Alto, CA) at 200,000 × g for 16 h. Following centrifugation, 10 fractions of 1.2 ml each were collected. Aliquots of each fraction were boiled in SDS-PAGE sample buffer, loaded onto SDSPAGE, and transferred to polyvinylidene fluoride membrane. Qa-2 and p38 MAPK were detected by specific mAbs (eBioscience,Cell Signaling Technology, respectively) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma–Aldrich). GM1 was detected by HRP-conjugated CTB (Sigma–Aldrich). Signal detection was performed by an enhanced chemiluminescence system (Bio-Rad, Hersules, CA). 2.8. In vitro SP thymocyte culture MTEC1 cells were cultured in 48-well plates at 2 × 104 /well. After growing to 80–90% confluence, mTEC1 cells were treated with mitomycin C (MMC, 50 g/ml; Kyowa, Tokyo, Japan) at 37 ◦ C for 1 h and then washed twice with BSS. MTEC1 cells were treated with or without 10 mM MCD for 15 min at 37 ◦ C, and washed in BSS. CD69+ Qa-2− CD4SP thymocytes isolated from 6-week-old mice were treated with BSS containing 10 mM MCD and 0.1% BSA and incubated for 30 min at 37 ◦ C. After extensively rinsing, MCDtreated or -untreated thymocytes were cultured at 2 × 105 per well with MCD-treated or -untreated mTEC1 cells in AIM V serum-free medium (Invitrogen, Carlsbad, CA) containing 20 ng/ml IL-7 (PeproTech, Rock Hill, NJ) with or without 10% FBS. On day 3 of culture,
J. Li et al. / Immunology Letters 148 (2012) 163–171
165
the expression of CD69 and Qa-2 in the thymocytes was analyzed using flow cytometry. In some experiments, CD69+ Qa-2− CD4SP thymocytes and mTEC1 were both pre-treated with cholesterol oxidase from Streptomyces sp. (Sigma–Aldrich) at the concentration of 0.5 U/ml for 60 min and co-cultured as mentioned above. After 48 h, the thymocytes were harvested. 2.9. FTOC Fetal thymic lobes were prepared from C57BL/6 mice on embryonic day 15.5 and treated with DMEM containing 10 mM MCD and incubated for 30 min at 37 ◦ C. The lobes were then rinsed extensively in serum-free DMEM. MCD-treated or -untreated lobes were placed on 0.8-m pore size polycarbonate filters (Merck Millipore, Billerica, MA) floating on AIM V serum-free medium with or without 10% FBS, and cultured at 37 ◦ C at 5% CO2 . On day 5 of culture, the culture was replenished with the same fresh medium. On day 9 of culture, the thymic lobes were stained with anti-CD4, anti-CD8, anti-CD69 and anti-Qa-2 mAbs and analyzed on FACS. 3. Theory The final maturation of CD4SP thymocytes may be dependent on the integrity of Qa-2 associated lipid rafts. 4. Results 4.1. The heterogeneity of Qa-2 expression in SP4 correlates with functionality SP4 represents the most mature CD4SP cells before emigration to the periphery. This subset has the most potent functionality, but is still less responsive than splenic CD4+ T cells after antigenic stimulation [10]. The continued maturation of CD4SP cells in the periphery has also been confirmed by other groups [19]. Given that Qa-2 is a unique marker for SP4, it is pertinent to assess Qa-2 at the single cell level in SP4 and analyze its relationship with the functional capacity of these cells. We first compared Qa-2 expression in SP4 cells with that in splenic CD4+ T cells. The expression level of Qa-2 in SP4 cells was approximately 2–3-fold lower than that in splenic CD4+ T cells, as measured by mean fluorescence intensity (MFI) (Fig. 1A). Then, SP4 thymocytes were sorted into three fractions based on Qa-2 expression level: Qa-2lo , Qa-2int and Qa-2hi , as shown in Fig. 1B. Their proliferative capacity in response to Con A stimulation was subsequently tested, and a progressive increase in proliferation was observed with the increase of Qa-2 expression (Fig. 1C). These results indicate that the functionality of SP4 thymocytes correlates with Qa-2 expression level, and suggest that this subset is not static but rather features a progressive up-regulation of Qa-2. The continuous maturation of SP4 is due to the increase in functionality on a per cell basis, not in cell number. 4.2. Qa-2 localization in lipid raft microdomains of SP4 thymocytes Qa-2 has been well documented to be localized in lipid rafts of peripheral resting T cells. Here, we attempted to determine whether Qa-2 is localized in lipid rafts in SP4 thymocytes. We first adopted the FCDR developed by Gombos et al. [18]. This assay allows the rapid differentiation of membrane proteins based on their distinct detergent solubility properties and on the mechanisms that cause detergent resistance (e.g., raft localization and/or cytoskeletal connections). The increased detergent solubility of selected membrane proteins after cholesterol depletion by MCD
Fig. 1. The heterogeneity of Qa-2 expression in SP4 correlates with functionality (A) The mean fluorescence intensity (MFI) of Qa-2 expression in SP4 cells and splenic CD4+ T cells. The dashed line, thin line and bold line represent the isotype control, SP4 cells and splenic CD4+ T cells, respectively. The experiments were repeated three times, and similar results were obtained for each set of experiments. (B, C) Proliferation capacity of SP4 cells with distinct Qa-2 expression levels. SP4 cells were sorted into Qa-2lo , Qa-2int and Qa-2hi subsets as indicated and stimulated with Con A. Cell proliferation was measured using a thymidine incorporation assay. The data shown are the mean ± SD of three repeated experiments. The MFIs of the three subsets are indicated.
can be interpreted as a sign of their association with lipid rafts; MCD is known to extract cholesterol from the plasma membrane and thereby disrupt rafts [20]. As shown in Fig. 2A, Qa-2 in CD4SP thymocytes displayed significantly increased detergent solubility after MCD treatment, suggesting its association with rafts. The raft marker GM1, which was detected by CTB, also showed a cholesterol-sensitive resistance to Triton X-100. Fig. 2B shows the FCDR values calculated for Qa-2 and GM1 before and after cholesterol depletion by MCD. The high FCDR values for Qa-2 and GM1 (0.97 and 0.83, respectively) before MCD treatment indicated their detergent resistance. However, after MCD treatment, the FCDR values for both proteins decreased significantly. This suggested that the observed detergent resistance was mainly due to the localization in raft microdomains. Moreover, immunofluorescent staining analysis by confocal microscopy revealed Qa-2 co-stained with the raft marker GM1 in CD4SP
166
J. Li et al. / Immunology Letters 148 (2012) 163–171
Fig. 2. Qa-2 localization in lipid raft microdomains of SP4 thymocytes. (A) The effect of cholesterol depletion by MCD on the detergent solubility of Qa-2 and GM1. The fluorescence of CD4SP thymocytes labeled with anti-Qa-2 or CTB before detergent treatment (red lines) and after 5 min of treatment with 0.1% cold Triton X-100 (blue lines) are shown. MCD untreated cells are regarded as control cells. (B) FCDR values for Qa-2 and GM1 in CD4SP thymocytes. The FCDR values are the means of three independent measurements. (C) Co-localization of Qa-2 with lipid raft microdomains in CD4SP thymocytes by confocal microscopy analysis. Qa-2 (IF488) and GM1 (PE) are detected with appropriate Abs and co-localization is shown in the merged panel. (D) CD4SP thymocytes were solubilized with 0.05% Triton X-100, subjected to sucrose gradient centrifugation, and analyzed by western blotting for the indicated molecules. The numbers indicate sucrose gradient fractions. Fractions 2–4 are low-density fractions containing lipid rafts.
thymocytes and suggested that the GM1-containing domains co-localized with Qa-2 (Fig. 2C). The localization of Qa-2 in GM1enriched lipid rafts was further confirmed by ultracentrifugation of cell lysates in a sucrose gradient, followed by western blotting of the resulting gradient fractions with anti-Qa-2 or anti-p38 MAPK. Fig. 2D shows the enrichment of Qa-2 in fractions containing lipid raft marker GM1 in CD4SP thymocytes. Thus, the integrated data from three different assays demonstrated the localization of Qa-2 in lipid rafts of SP4 thymocytes. 4.3. Quantitative analysis of lipid raft components in CD4SP subsets To investigate whether lipid rafts play a role in CD4SP thymocyte development, we analyzed the quantitative levels of specific lipid raft components GM1 in CD4SP subsets. By flow cytometric analysis, we found that almost 100% of CD4SP thymocytes expressed GM1, which is consistent with previous observations [21,22]. We further dissected the GM1 expression levels in distinct CD4SP subsets and observed a hierarchy: CD69+ Qa2− (SP1 + SP2) < CD69− Qa-2− (SP3) < CD69− Qa-2+ (SP4) (Fig. 3A). Meanwhile, a significant increase in GM1 expression was observed following the increase of Qa-2 levels in SP4 (Fig. 3A). After exit from the thymus, CD4SP cells undergo further functional maturation and acquire the highest expression of Qa-2 in periphery. We compared the GM1 expression levels between thymocytes and splenocytes and observed that the GM1 membrane content of splenic CD4+ T cells was more than five-fold higher than that of SP4 thymocytes (Fig. 3B). Taken together, differences in lipid raft components by thymocyte subsets may contribute to the process of T cell differentiation or functional maturation. 4.4. Lipid raft disruption impairs CD4SP thymocyte differentiation in vitro The quantitative differences in lipid raft components of CD4SP subsets prompted us to investigate whether lipid raft integrity is required for CD4SP development. We previously established an
mTEC1-supported culture system that is capable of supporting CD4SP thymocyte differentiation in vitro [10]. Here, we adopted this system to investigate the role of membrane rafts in CD4SP thymocyte differentiation. As mentioned above, the cholesterol depleting reagent MCD is commonly used to disrupt lipid rafts. Because the effect of MCD treatment is temporary and probably followed by a rapid up-regulation of cholesterol synthesis within the cell [23], serum-free AIM V medium was used. The concentration and duration of MCD treatment were carefully determined for thymocytes and mTEC1 because these cell types cannot survive inappropriate MCD treatment. We first confirmed that MCD indeed removed cholesterol from the membranes of thymocytes and mTEC1. Filipin III was used to stain cholesterol on thymocytes. Filipin is a fluorescent polyene antibiotic from Saccharomyces filipinensis, and it forms a multimeric globular complex with cholesterol in the cell membrane [24]. Flow cytometric analysis showed that treatment of thymocytes with MCD reduced the staining with filipin to approximately 50% of that in control cells (Fig. 4A). The effect of MCD on mTEC1 was also confirmed (data not shown). When MCD-treated CD69+ Qa-2− CD4SP cells were co-cultured with MCDtreated mTEC1 cells in AIM V medium, the resulting CD69− Qa-2+ (SP4) cells comprised 36.6% of the culture at 72 h, compared to 53.3% of the co-culture of untreated thymocytes and mTEC1. The SP4 population in the MCD-treated group was recovered by the addition of FBS to the culture. The proportion of SP4 cells in the MCD + FBS treated culture was 50.9%, very close to that of the culture without MCD treatment (Fig. 4B). This effect was also observed in the actual numbers of SP3 and SP4 cells developed (Fig. 4C). To further support the notion that cholesterol-rich rafts are involved in CD4SP thymocyte development, we also treated the cells with cholesterol oxidase (COase) in the same co-culture system as mentioned above. COase converts cholesterol into cholestenone and can disrupt the raft integrity. In a 48 h co-culture system, our data demonstrated that similar to MCD treatment, COase-treatment hindered CD4SP thymocyte development. The percentage and absolute cell number of SP4 subset were both significantly decreased (Fig. 4D and E).
J. Li et al. / Immunology Letters 148 (2012) 163–171
167
Fig. 3. Quantitative analysis of lipid raft components in CD4SP subsets and splenic CD4+ T cells (A) GM1 expression in CD4SP thymocyte subsets. CD4SP thymocytes were analyzed by flow cytometry after staining with anti-Qa-2, anti-CD69 and CTB. A significant difference in the MFI of GM1 was observed among CD69+ Qa-2− , CD69− Qa-2− and CD69− Qa-2+ CD4SP cells and among Qa-2lo , Qa-2int and Qa-2hi SP4 cells. The experiments were repeated three times, and similar results were obtained for each set of experiments. (B) GM1 expression in SP4 thymocytes was compared with that in splenic CD4+ T cells.
4.5. Effect of raft disruption on CD4SP thymocyte differentiation in fetal thymic organ culture To investigate the impact of cholesterol depletion on SP thymocyte development in a more physiological system, fetal thymic organ cultures (FTOC) were established with and without MCD treatment. When fetal thymi were treated with MCD and cultured in serum-free medium, compared to the control group, the percentage of CD4SP cells only decreased a little bit. However, further analysis of the profile of CD69 and Qa-2 revealed that the differentiation of CD4SP cells were significantly blocked after MCD treatment and the percentage of SP4 subset among CD4SP cells reduced to 4.2%, compared to 19.8% of that without MCD treatment. Similar to the results in the co-culture system, cholesterol replenishment by the addition of FBS recovered the differentiation of CD4SP cells and the percentage of SP4 subset increased to 17.6% (Fig. 5A). This effect was also reflected in the absolute cell numbers of distinct developing subsets of CD4SP (Fig. 5B).
Both data from in vitro co-culture system and FTOC suggest that when the raft integrity is disrupted, the differentiation of CD4SP thymocytes is significantly hindered, especially the transition from SP3 to SP4 (Fig. 6). 5. Discussion Recently, many efforts have underlined the importance of CD4SP thymocyte differentiation in the medulla. Here, we demonstrated that CD4SP exhibited heterogeneity in expression of lipid raft components GM1. Qa-2, a unique marker for SP4, localizes to lipid rafts. It is heterogeneously expressed in SP4 cells, and its expression level correlates with cell functionality. Raft disruption impairs the expression of Qa-2, which is an important event during the transition from SP3 to SP4. SP4 represents the most mature subset of CD4SP cells. The data from intrathymic adoptive transfer assays showed that some SP4 cells stayed in the thymus longer than others [11]. The consequences of this extended retention in the medulla
168
J. Li et al. / Immunology Letters 148 (2012) 163–171
Fig. 4. Effect of raft disruption on CD4SP thymocyte differentiation in vitro. (A) Cholesterol depletion by MCD treatment. Thymocytes were treated with 10 mM MCD or left untreated, and the cholesterol in the plasma membrane was stained with filipin III and analyzed by flow cytometry. (B) In vitro differentiation of CD69+ Qa-2− CD4SP thymocytes after cholesterol depletion with MCD treatment. MCD treated or untreated CD69+ Qa-2− CD4SP thymocytes were cultured in AIM V medium in the presence of MCD treated mTEC1. The effect of FBS on cholesterol reconstitution was determined simultaneously. Cells were harvested at day 3 and analyzed for surface expression of CD69 and Qa-2. The number of cells recovered from different cultures is shown in brackets, and the number in each quadrant indicates the percentage of total cells in the corresponding subset. The results are representative of three independent experiments. (C) The numbers of SP3 and SP4 cells from different cultures were calculated. The SD is shown as error bars. (D) In vitro differentiation of CD69+ Qa-2− CD4SP thymocytes after cholesterol oxidation with cholesterol oxidase (COase). COase treated CD69+ Qa-2− CD4SP thymocytes were cultured with COase treated mTEC1. Co-cultures without COase treatment are regarded as control. Cells were harvested at day 2 and analyzed for surface expression of CD69 and Qa-2. (E) The cell numbers of CD4SP subsets from cultures with or without COase treatment were calculated. The SD is shown as error bars.
aroused our interests. In analysis of proliferation response upon mitogen stimulation in the SP4 subpopulations with different Qa-2 expression level, we observed a significant hierarchy: Qa-2lo < Qa2int < Qa-2hi . This phenomenon reveals that the phenotype of SP4 cells is not uniform but rather increases with Qa-2 expression level, and SP4 cells undergo continuous functional maturation concomitant with the up-regulation of Qa-2 expression. These results may explain the extended retention of some SP4 cells in thymic medulla. The correlation between Qa-2 expression and cell functionality is further supported by the comparison of GM1 expression between splenic CD4+ T cells and thymocytes. The analysis of the genes expressed by various CD4SP subsets showed that a variety of molecules were up-regulated along with the CD4SP development, including MHCs, costimulatory molecules, cytokines and their receptors [25]. Among them, several molecules such as MHC II and IL-2R are associated with lipid rafts of T cells [26,27]. Considering the role of lipid rafts as signal platforms that facilitate efficient and specific signal transduction, the clustering of these molecules into lipid rafts may be responsible for the functional maturation of T cells. Our finding that Qa-2 localizes to lipid rafts in SP4, together with the fact that Qa-2 expression correlates with cell functionality, further supports the role of lipid rafts in the functional maturation of SP4 cells. By analyzing the expression of lipid raft components in SP4 cells, we
observed that GM1 expression increased with the up-regulation of Qa-2. Thus, the difference in lipid raft contents may influence the intensity of the signals required for cell maturation. To extend the studies of the roles of the lipid raft in SP thymocyte development, we conducted further experiments to show that the expression of GM1 is up-regulated during CD4SP thymocyte development. This finding suggests that the development of CD4SP thymocytes in medulla follows the formation of lipid rafts. So far, there are various gangliosides (GM1, GM2, GM3) that have been identified. GM1 is widely studied for the adoption of CTB and is generally considered to be a reliable marker for lipid rafts. It is well-documented that GM1 is very important for T cell activation. In recent years, GM2 and GM3 have received more attention. A recent report shows that CD4+ and CD8+ T cells require different ganglioside subsets for activation [28]. The activation of CD4+ T cells from GM3 synthase null mice, deficient in GM3derived gangliosides, is severely compromised, suggesting that GM3 is also very important for CD4+ T cell activation. Another interesting phenomenon is that with the differentiation from thymocytes to mature T cell subsets, selective glycosphingolipids are up-regulated. For example, GM3S, GM2/GD2S expressions are markedly increased from thymocytes to mature CD4+ T cells. In the present study, we demonstrate Qa-2 is localized in lipid rafts. Besides GM1, whether Qa-2 is also enriched in GM2 or GM3 related
J. Li et al. / Immunology Letters 148 (2012) 163–171
169
Fig. 5. Effect of raft disruption on CD4SP thymocyte differentiation in FTOC. MCD treated or untreated fetal thymic lobes were cultured in AIM V medium, with or without FBS. The thymocytes were analyzed on day 9 of incubation by flow cytometry with anti-CD4, CD8, CD69 and Qa-2 mAbs. (A) Profiles of thymocyte subpopulations from thymic lobes cultured are shown. The cell number of each lobe from different cultures is shown in brackets. CD4/CD8 expression profiles are in the top row and the number in each quadrant indicates the percentage of subset among all cells. CD69/Qa-2 expression profiles in CD4SP subsets are shown in the bottom row and the number in individual quadrant represents the percentage of subsets among CD4SP cells. The results are representative of three independent experiments. (B) The cell number in different CD4SP subsets of each lobe from cultures was calculated. The SD is shown as error bars.
Fig. 6. Schematic representation of the effect of raft disruption on CD4SP thymocyte development.
170
J. Li et al. / Immunology Letters 148 (2012) 163–171
lipid rafts needs further studies. Future studies focusing on the functions of GM2 or GM3 in CD4SP development, dissecting the developmental program of CD4SP in GM3 synthase null mice or GM2/GD2 synthase null mice will be very helpful to have a deeper understanding of the functions of lipid rafts in thymocyte development. The appropriate development of thymocytes in the thymus requires the interaction between thymocytes and thymic stromal cells. During the transition from the DN to the SP stage, different signaling molecules are involved in the lympho-stromal communication to ensure that key events happen at the right stages. In addition to their involvement in central tolerance, mTECs play essential roles in SP thymocyte development. Defects in mTECs (such as in Relb−/− or Aire−/− mice) cause the arrest of CD4SP cells at SP3. Furthermore, the efficient generation of SP4 cells in our in vitro studies requires the presence of a special type of mTECs [10]. The potential role of soluble factors produced by mTECs in this process was excluded by in vitro experiments in a co-culture system (data not shown). These results indicate that signaling between mTECs and thymocytes drives the differentiation of SP thymocytes, although the details of this signaling remain to be further defined. It has been demonstrated that the presentation of MHC II molecules on epithelial cells induces raft-associated signaling in thymocytes, thereby inducing CD4+ T cell development [8]. Given the heterogeneity of lipid rafts among various CD4SP subsets, we speculate that raft-associated signaling is essential for the supporting role of mTECs in CD4SP thymocyte development, and lipid rafts acting as signal platforms for crosstalk between mTECs and thymocytes may be a prerequisite for CD4SP thymocyte maturation. Our finding of a significant reduction in the transition from SP3 to SP4 in the mTEC1-supported culture system after raft disruption by cholesterol extraction or cholesterol oxidation supports this hypothesis. Besides, raft-disruption either on mTEC1 or on thymocytes also hindered the differentiation of CD4SP thymocyte to SP4 stage (data not shown), and these data further emphasize the indispensability of crosstalk between mTECs and thymocytes in the final maturation of SP thymocytes. It seems that raft-associated signaling in the final maturation of CD4SP is not mediated by TCR-MHC II interaction because the expansion and differentiation of SP cells still occur in co-cultures in which epithelial cells express no or mismatched MHC molecules [[29] and unpublished data]. Recent studies have demonstrated that the Lck unique domain is important for both the DN-DP transition and the selection of SP cells, and Fyn plays a minor compensatory role during thymocyte development [9,30]. Therefore, we propose that raft-associated signaling via Lck and Fyn may be responsible for CD4SP thymocyte development. In our study, Qa-2 is regarded as a clue which correlates the differentiation and functional maturation of SP thymocytes with lipid rafts, whereas its exact role in this process is still unknown. Further studies to analyze the developmental program of CD4SP in Ped low strain mice may provide more suggestive and convincing data to explain the exact roles of Qa-2 in the development of CD4SP thymocytes. To our knowledge, this is the first study to demonstrate the critical role of lipid rafts in the process of medullary CD4SP thymocyte differentiation and in the final maturation of SP4 cells. However, the specific raft-associated molecules required for CD4SP thymocyte development require further investigation.
6. Conclusions Findings reported here demonstrate that Qa-2 associated lipid rafts are indispensable for the final maturation of CD4+ CD8− thymocytes. Qa-2, the unique marker for mature CD4SP cells, may also be involved in the functional maturation of CD4SP cells. The exact
mechanism for Qa-2 and lipid raft-associated signaling in thymocyte development is under study now.
Acknowledgements This work was supported by National Natural Science Foundation of China (30900735 and 81070271), the Key Project of Chinese Ministry of Education (211012) and the National Basic Research Program of China (2011CB946103).
References [1] Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev 2000;1:31–41. [2] Kabouridis PS. Lipid rafts in T cell receptor signaling. Mol Membr Biol 2006;23:49–57. [3] Kabouridis PS, Juryb EC. Lipid rafts and T-lymphocyte function: Implications for autoimmunity. FEBS Lett 2008;582:3711–8. [4] Malorni W, Giammarioli AM, Garofalo T, Sorice M. Dynamics of lipid raft components during lymphocyte apoptosis: the paradigmatic role of GD3. Apoptosis 2007;12:941–9. [5] Werlen G, Hausmann B, Palmer E. A motif in the alpha-beta T cell receptor controls positive selection by modulating ERK activity. Nature 2000;406:422–6. [6] Wong P, Barton GM, Forbush KA, Rudensky AY. Dynamic tuning of T cell reactivity by self-peptide-major histocompatibility complex ligands. J Exp Med 2001;193:1179–87. [7] Ebert PJ, Baker JF, Punt JA. Immature CD4+ CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals. J Immunol 2000;165:5435–42. [8] Komaniwa S, Hayashi H, Kawamoto H, Sato SB, Ikawa T, Katsura Y, et al. Lipidmediated presentation of MHC class II molecules guides thymocytes to the CD4 lineage. Eur J Immunol 2009;39:96–112. [9] Palacios EH, Weiss A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 2004;23:7990–8000. [10] Li J, Li Y, Yao JY, Jin R, Zhu MZ, Qian XP, et al. Developmental pathway of CD4+ CD8− medullary thymocytes during mouse ontogeny and its defect in Aire−/− mice. Proc Natl Acad Sci USA 2007;104:18175–80. [11] Jin R, Wang W, Yao JY, Zhou YB, Qian XP, Zhang J, et al. Characterization of the in vivo dynamics of medullary CD4+ CD8− thymocyte development. J Immunol 2008;180:2256–63. [12] McElhinny AS, Exley GE, Warner CM. Painting Qa-2 onto Ped slow preimplantation embryos increases the rate of cleavage. Am J Reprod Immunol 2000;44:52–8. [13] Comiskey M, Domino KE, Warner CM. HLA-G is found in lipid rafts and can act as a signaling molecule. Hum Immunol 2007;68:1–11. [14] De Fazio SR, Warner CM. Activation of T cells by cross-linking Qa-2, the Ped gene product, requires Fyn. Am J Reprod Immunol 2007;58:315–24. [15] Suzuki K, Sheetz MP. Binding of cross-linked glycosylphosphatidylinositolanchored proteins to discrete actin-associated sites and cholesterol-dependent domains. Biophys J 2001;81:2181–9. [16] Vernachio J, Li M, Donnenberg AD, Soloski MJ. Qa-2 expression in the adult murine thymus. A unique marker for a mature thymic subset. J Immunol 1989;142:48–56. [17] Matkó J, Bodnár A, Vereb G, Bene L, Vámosi G, Szentesi G, et al. GPI-microdomains (membrane rafts) and signaling of the multi-chain interleukin-2 receptor in human lymphoma/leukemia T-cell lines. Eur J Biochem 2002;269:1199–208. [18] Gombos I, Bacsó Z, Detre C, Nagy H, Goda K, Andrásfalvy M, et al. Cholesterol sensitivity of detergent resistance: a rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins. Cytometry A 2004;61:117–26. [19] Boursalian TE, Golob J, Soper DM, Cooper CJ, Fink PJ. Continued maturation of thymic emigrants in the periphery. Nat Immunol 2004;5:418–25. [20] Cherukuri A, Dykstra M, Pierce SK. Floating the raft hypothesis: lipid rafts play a role in immune cell activation. Immunity 2001;14:657–60. [21] De Mello Coelho V, Nguyen D, Giri B, Bunbury A, Schaffer E, Taub DD. Quantitative differences in lipid raft components between murine CD4+ and CD8+ T cells. BMC Immunol 2004;5:2. [22] Haks MC, Belkowski SM, Ciofani M, Rhodes M, Lefebvre JM, Trop S, et al. Low activation threshold as a mechanism for ligand-independent signaling in pre-T cells. J Immunol 2003;170:2853–61. [23] Hampton R, Dimster-Denk D, Rine J. The biology of HMG-CoA reductase: the pros of contra-regulation. Trends Biochem Sci 1996;21:140–5. [24] Hassall DG, Graham A. Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages. Cytometry 1995;21:352–62. [25] Teng F, Zhou YB, Jin R, Chen Y, Pei X, Liu Y, et al. The molecular signature underlying the thymic migration and maturation of TCR␣+ CD4+ CD8− thymocytes. PloS ONE 2011;6:e25567. [26] Li Q, Wang M, Tan L, Wang C, Ma J, Li N, et al. Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts. J Lipid Res 2005;46:1904–13.
J. Li et al. / Immunology Letters 148 (2012) 163–171 [27] Karacsonyi C, Knorr R, Fülbier A, Lindner R. Association of major histocompatibility complex II with cholesterol- and sphingolipid-rich membranes precedes peptide loading. J Biol Chem 2004;279:34818–26. [28] Nagafuku M, Okuyama K, Onimaru Y, Suzuki A, Odagiri Y, Yamashita T, et al. CD4 and CD8 T cells require different membrane gangliosides for activation. Proc Natl Acad Sci USA 2012;109:E336–42.
171
[29] Hare KJ, Wilkinson RW, Jenkinson EJ, Anderson G. Identification of a developmentally regulated phase of postselection expansion driven by thymic epithelium. J Immunol 1998;160:3666–72. [30] Salmond RJ, Filby A, Pirinen N, Magee AI, Zamoyska R. Mislocalization of Lck impairs thymocyte differentiation and can promote development of thymomas. Blood 2011;117:108–17.
Contents lists available at SciVerse ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Qa-2 associated lipid rafts are indispensable in the final maturation of CD4+ CD8− thymocytes Juan Li a , Hai-Dong Li b , Yu Zhang c , Jun Zhang c,∗ a Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Educational Ministry, Tianjin Medical University, 22 Qixiangtai Road, Tianjin 300070, China b Department of Biochemistry, Tianjin Medical University, Tianjin, China c Department of Immunology, School of Basic Sciences, Peking University Health Science Center, Beijing, China
a r t i c l e
i n f o
Article history: Received 24 April 2012 Received in revised form 21 September 2012 Accepted 9 October 2012 Available online 17 October 2012 Keywords: Thymocyte Qa-2 Lipid rafts CD4SP SP4
a b s t r a c t Lipid rafts have been shown to play significant roles in thymocyte development. However, the exact role of lipid rafts in single positive (SP) thymocyte differentiation is poorly characterized. We previously defined a developmental program (SP1 → SP4) for CD4SP thymocytes. In this study, we found that lipid raft components were up-regulated during CD4SP maturation. Qa-2, a unique marker for the most mature SP4 subset, was localized to lipid rafts and heterogeneously expressed in SP4 cells. Functional assays showed that the proliferation capacity of SP4 cells correlated with the expression of Qa-2. Raft-disruption on both CD4SP and epithelial cells by cholesterol extraction or cholesterol oxidation in a medullary thymic epithelial cell (mTEC)-supported co-culture system impaired the transition from SP3 to SP4. This result was further confirmed in fetal thymic organ culture system. Collectively, these studies suggest that raftassociated signaling between mTECs and thymocytes drives the differentiation of CD4SP thymocytes and lipid rafts are involved in the final maturation of SP4 thymocytes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lipid rafts are specialized detergent-resistant microdomains of the eukaryotic plasma membrane that are mainly composed of sphingomyelins, glycosphingolipids and cholesterol. Accumulating evidence has suggested that lipid rafts play a key role in vital cellular processes, including signaling, apoptosis and cell adhesion. Lipid rafts are proposed to serve as signaling platforms to facilitate the propagation of signaling cascades from various membranebound receptors, especially during T cell activation [1]. This role has been emphasized by the finding that TCR is recruited to lipid rafts upon receptor stimulation. Several acylated proteins involved in TCR signaling, such as Lck and Fyn, reside constitutively in lipid rafts. Many signaling proteins become concentrated in lipid rafts upon TCR activation, including ZAP-70, PKC and PKB [2,3]. Lipid rafts also contribute to the cascade of events leading to T cell apoptosis after CD95/Fas ligation [4]. Notably, several studies have demonstrated that lipid rafts may play a crucial role in thymocyte development. Differentiating thymocytes are subdivided into four major subpopulations: CD4− CD8− (double-negative, DN), CD4+ CD8+ (double-positive, DP) and CD4+ CD8− and CD4− CD8+ SP. The roles
∗ Corresponding author. Tel.: +86 10 82805649. E-mail address: [email protected] (J. Zhang). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.imlet.2012.10.006
of lipid rafts in thymocyte development are well documented from the DN to SP stage. The transition from the DN to DP stage requires the palmitoylation of the pre-TCR␣ chain and its recruitment to the lipid raft [5]. The selection of DP cells to become SP cells occurs after CD3 associates with TCR in the raft regions [6,7]. Moreover, a recent study suggests that the CD4 lineage commitment from DP is directed by the raft-associated presentation of MHC class II molecules [8]. It has also been suggested that raft-associated Lck and Fyn may be involved in nearly all stages, from DN to SP [9]. However, the exact functions of lipid rafts in SP thymocyte development remain poorly characterized. Recently, more and more studies indicate that SP thymocytes are heterogeneous and undergo an ordered maturational program in the thymic medulla. Several important events occur in this process, including negative selection and functional maturation. We previously resolved CD4SP thymocytes into four subsets: SP1 (6C10+ CD69+ Qa-2− ), SP2 (6C10− CD69+ Qa2− ), SP3 (6C10− CD69− Qa-2− ) and SP4 (6C10− CD69− Qa-2+ ) cells [10,11]. In vivo and in vitro assays demonstrated that the differentiation of CD4SP thymocytes followed a strict order from SP1 to SP4 and exhibited increased maturity from SP1 to SP4. Notably, our study identified the SP3/SP4 transition as a critical checkpoint in CD4SP development [10]. The major difference between SP3 and SP4 stage phenotype is the surface expression of Qa-2 in SP4 cells. Qa-2 is the product of the Ped (preimplantation embryonic development) gene. Qa-2+ embryos have an increased rate of
164
J. Li et al. / Immunology Letters 148 (2012) 163–171
preimplantation development and of subsequent embryonic survival than Qa-2− embryos [12]. Qa-2, the murine homolog of HLA-G, which belongs to the family of non-classical MHC proteins, has a glycosylphosphatidylinositol (GPI) linkage to the cell membrane [13,14]. GPI-anchored proteins preferentially localize in lipid rafts in the cell membrane, and Qa-2 has indeed been reported to localize to lipid rafts in T cells [15]. Cross-linking Qa-2 induces resting T cells to proliferate. The Qa-2-mediated signaling pathway requires Fyn, the PI-3 kinase and Akt [14]. In general, Qa-2 has only been used as a thymocyte maturation marker [16], and its exact role in thymocytes remains elusive. In the present study, we demonstrate that the expression level of Qa-2 in lipid rafts of SP4 correlates with cell functionality and that raft disruption impairs CD4SP thymocyte differentiation in vitro and in fetal thymic organ culture system, suggesting a potential role for Qa-2 associated lipid rafts in the functional maturation of SP4 cells and a key role of lipid rafts in CD4SP thymocyte development. 2. Materials and methods
After extensive rinsing, cells were cultured in vitro or labeled with filipin. 2.5. Flow cytometric assay of differential detergent resistance (FCDR) FCDR was performed as described [18]. Briefly, the fluorescence intensities of unlabeled, labeled or MCD-pretreated cells were recorded on FACS before and 5 min after mixing the cells with 0.1% (v/v) Triton X-100 (Sigma–Aldrich) dissolved in PBS. The extent of detergent resistance was estimated using the FCDR index, which is calculated according to the following equation: FCDR =
(FLdet − FLBg det ) (FLmax − FLBg )
FLmax , fluorescence of labeled untreated cells; FLdet , fluorescence of the cells treated with detergent for 5 min; FLBg , autofluorescence of the untreated cells; FLBg det , autofluorescence of the detergenttreated cells.
2.1. Mice C57BL/6 mice were purchased from Beijing Vitalriver Co. and bred in the Animal Breeding Facilities of Tianjin Medical University. A ␥-irradiated sterile diet and autoclaved distilled water were used. The animal studies were approved by the ethics committee of Tianjin Medical University. 2.2. Flow cytometry and cell sorting Freshly isolated thymocytes were stained with fluorochromelabeled mAbs, sorted using a FACSCalibur instrument (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Tree Star, USA) software. The following mAbs were used: anti-Qa-2 and anti-CD8 fluorescein isothiocyanate (FITC)-conjugated mAbs; anti-CD69 phycoerythrin (PE)-conjugated mAb; anti-CD4 PerCPcy5.5-conjugated mAb; and allophycocyanin (APC)-conjugated streptavidin (eBioscience, San Diego, CA). Cholera toxin B (CTB)conjugated biotin was obtained from Sigma–Aldrich (St. Louis, MO). For filipin staining, the cells were treated with 250 g/ml fresh filipin III (Sigma–Aldrich) in balanced salt solution (BSS) at 4 ◦ C for 2 h, and then acquired by LSRII (BD Biosciences) with the krypton ion laser tuned to UV (350.7–356.4 nm) and emission detected at 420–460 nm. The isolation of different subsets of CD4SP thymocytes was performed as described [10]. The resulting viable cells were then stained for CD4, CD69 and Qa-2 and sorted into various subsets with FACSAria (BD Bioscience). The purity of cells harvested was >98% when reanalyzed on FACS. 2.3. Proliferation assay SP4 thymocytes were sorted into Qa-2lo , Qa-2int and Qa-2hi subsets, seeded at 1 × 105 per well into 96-well tissue plates and cultured in the presence of 2.5 g/ml Con A (Amersham Pharmacia, Piscataway, NJ) for 72 h. Then, 0.5 Ci per well [3 H] thymidine was added to the culture for the last 12 h. Cells were harvested and their thymidine incorporation was measured. 2.4. Depletion of plasma membrane cholesterol by methylcyclodextrin (MCD) Thymocytes and mTEC1 cell line (106 /ml) were treated with 10 mM MCD (Sigma–Aldrich) at 37 ◦ C in BSS containing 0.1% BSA for 30 min or 15 min, respectively. This treatment removes approximately 40–50% of the plasma membrane cholesterol [17].
2.6. Immunofluorescence CD4SP thymocytes were stained with purified anti-Qa-2 mAb and biotin-CTB, followed by IF488-anti-mouse IgG and PEstreptavidin (Sungene Biotech, Tianjin, China). Then the cell suspensions were layered onto poly(l-lysine)-coated coverslips for 1 h at room temperature. Images were acquired using PerkinElmer Ultraview VoX spinning disk confocal microscope (PerkinElmer, Waltham, MA). 2.7. Sucrose gradient centrifugation and Western blotting CD4SP thymocytes (1 × 108 cells) were lysed in 1 ml ice-cold lysis buffer containing 20 mM Tris–HCl (pH 7.2), 150 mM KCl, 0.05% Triton X-100, and protease inhibitors. The cells lysates were homogenized with 10 strokes of a glass dounce homogenizer, then mixed with 1 ml of 85% sucrose in lysis buffer, and transferred to SW41 centrifuge tubes. The samples were then overlaid with 6 ml of 35% sucrose and 3.5 ml of 5% sucrose and ultracentrifuged (Beckman, Palo Alto, CA) at 200,000 × g for 16 h. Following centrifugation, 10 fractions of 1.2 ml each were collected. Aliquots of each fraction were boiled in SDS-PAGE sample buffer, loaded onto SDSPAGE, and transferred to polyvinylidene fluoride membrane. Qa-2 and p38 MAPK were detected by specific mAbs (eBioscience,Cell Signaling Technology, respectively) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma–Aldrich). GM1 was detected by HRP-conjugated CTB (Sigma–Aldrich). Signal detection was performed by an enhanced chemiluminescence system (Bio-Rad, Hersules, CA). 2.8. In vitro SP thymocyte culture MTEC1 cells were cultured in 48-well plates at 2 × 104 /well. After growing to 80–90% confluence, mTEC1 cells were treated with mitomycin C (MMC, 50 g/ml; Kyowa, Tokyo, Japan) at 37 ◦ C for 1 h and then washed twice with BSS. MTEC1 cells were treated with or without 10 mM MCD for 15 min at 37 ◦ C, and washed in BSS. CD69+ Qa-2− CD4SP thymocytes isolated from 6-week-old mice were treated with BSS containing 10 mM MCD and 0.1% BSA and incubated for 30 min at 37 ◦ C. After extensively rinsing, MCDtreated or -untreated thymocytes were cultured at 2 × 105 per well with MCD-treated or -untreated mTEC1 cells in AIM V serum-free medium (Invitrogen, Carlsbad, CA) containing 20 ng/ml IL-7 (PeproTech, Rock Hill, NJ) with or without 10% FBS. On day 3 of culture,
J. Li et al. / Immunology Letters 148 (2012) 163–171
165
the expression of CD69 and Qa-2 in the thymocytes was analyzed using flow cytometry. In some experiments, CD69+ Qa-2− CD4SP thymocytes and mTEC1 were both pre-treated with cholesterol oxidase from Streptomyces sp. (Sigma–Aldrich) at the concentration of 0.5 U/ml for 60 min and co-cultured as mentioned above. After 48 h, the thymocytes were harvested. 2.9. FTOC Fetal thymic lobes were prepared from C57BL/6 mice on embryonic day 15.5 and treated with DMEM containing 10 mM MCD and incubated for 30 min at 37 ◦ C. The lobes were then rinsed extensively in serum-free DMEM. MCD-treated or -untreated lobes were placed on 0.8-m pore size polycarbonate filters (Merck Millipore, Billerica, MA) floating on AIM V serum-free medium with or without 10% FBS, and cultured at 37 ◦ C at 5% CO2 . On day 5 of culture, the culture was replenished with the same fresh medium. On day 9 of culture, the thymic lobes were stained with anti-CD4, anti-CD8, anti-CD69 and anti-Qa-2 mAbs and analyzed on FACS. 3. Theory The final maturation of CD4SP thymocytes may be dependent on the integrity of Qa-2 associated lipid rafts. 4. Results 4.1. The heterogeneity of Qa-2 expression in SP4 correlates with functionality SP4 represents the most mature CD4SP cells before emigration to the periphery. This subset has the most potent functionality, but is still less responsive than splenic CD4+ T cells after antigenic stimulation [10]. The continued maturation of CD4SP cells in the periphery has also been confirmed by other groups [19]. Given that Qa-2 is a unique marker for SP4, it is pertinent to assess Qa-2 at the single cell level in SP4 and analyze its relationship with the functional capacity of these cells. We first compared Qa-2 expression in SP4 cells with that in splenic CD4+ T cells. The expression level of Qa-2 in SP4 cells was approximately 2–3-fold lower than that in splenic CD4+ T cells, as measured by mean fluorescence intensity (MFI) (Fig. 1A). Then, SP4 thymocytes were sorted into three fractions based on Qa-2 expression level: Qa-2lo , Qa-2int and Qa-2hi , as shown in Fig. 1B. Their proliferative capacity in response to Con A stimulation was subsequently tested, and a progressive increase in proliferation was observed with the increase of Qa-2 expression (Fig. 1C). These results indicate that the functionality of SP4 thymocytes correlates with Qa-2 expression level, and suggest that this subset is not static but rather features a progressive up-regulation of Qa-2. The continuous maturation of SP4 is due to the increase in functionality on a per cell basis, not in cell number. 4.2. Qa-2 localization in lipid raft microdomains of SP4 thymocytes Qa-2 has been well documented to be localized in lipid rafts of peripheral resting T cells. Here, we attempted to determine whether Qa-2 is localized in lipid rafts in SP4 thymocytes. We first adopted the FCDR developed by Gombos et al. [18]. This assay allows the rapid differentiation of membrane proteins based on their distinct detergent solubility properties and on the mechanisms that cause detergent resistance (e.g., raft localization and/or cytoskeletal connections). The increased detergent solubility of selected membrane proteins after cholesterol depletion by MCD
Fig. 1. The heterogeneity of Qa-2 expression in SP4 correlates with functionality (A) The mean fluorescence intensity (MFI) of Qa-2 expression in SP4 cells and splenic CD4+ T cells. The dashed line, thin line and bold line represent the isotype control, SP4 cells and splenic CD4+ T cells, respectively. The experiments were repeated three times, and similar results were obtained for each set of experiments. (B, C) Proliferation capacity of SP4 cells with distinct Qa-2 expression levels. SP4 cells were sorted into Qa-2lo , Qa-2int and Qa-2hi subsets as indicated and stimulated with Con A. Cell proliferation was measured using a thymidine incorporation assay. The data shown are the mean ± SD of three repeated experiments. The MFIs of the three subsets are indicated.
can be interpreted as a sign of their association with lipid rafts; MCD is known to extract cholesterol from the plasma membrane and thereby disrupt rafts [20]. As shown in Fig. 2A, Qa-2 in CD4SP thymocytes displayed significantly increased detergent solubility after MCD treatment, suggesting its association with rafts. The raft marker GM1, which was detected by CTB, also showed a cholesterol-sensitive resistance to Triton X-100. Fig. 2B shows the FCDR values calculated for Qa-2 and GM1 before and after cholesterol depletion by MCD. The high FCDR values for Qa-2 and GM1 (0.97 and 0.83, respectively) before MCD treatment indicated their detergent resistance. However, after MCD treatment, the FCDR values for both proteins decreased significantly. This suggested that the observed detergent resistance was mainly due to the localization in raft microdomains. Moreover, immunofluorescent staining analysis by confocal microscopy revealed Qa-2 co-stained with the raft marker GM1 in CD4SP
166
J. Li et al. / Immunology Letters 148 (2012) 163–171
Fig. 2. Qa-2 localization in lipid raft microdomains of SP4 thymocytes. (A) The effect of cholesterol depletion by MCD on the detergent solubility of Qa-2 and GM1. The fluorescence of CD4SP thymocytes labeled with anti-Qa-2 or CTB before detergent treatment (red lines) and after 5 min of treatment with 0.1% cold Triton X-100 (blue lines) are shown. MCD untreated cells are regarded as control cells. (B) FCDR values for Qa-2 and GM1 in CD4SP thymocytes. The FCDR values are the means of three independent measurements. (C) Co-localization of Qa-2 with lipid raft microdomains in CD4SP thymocytes by confocal microscopy analysis. Qa-2 (IF488) and GM1 (PE) are detected with appropriate Abs and co-localization is shown in the merged panel. (D) CD4SP thymocytes were solubilized with 0.05% Triton X-100, subjected to sucrose gradient centrifugation, and analyzed by western blotting for the indicated molecules. The numbers indicate sucrose gradient fractions. Fractions 2–4 are low-density fractions containing lipid rafts.
thymocytes and suggested that the GM1-containing domains co-localized with Qa-2 (Fig. 2C). The localization of Qa-2 in GM1enriched lipid rafts was further confirmed by ultracentrifugation of cell lysates in a sucrose gradient, followed by western blotting of the resulting gradient fractions with anti-Qa-2 or anti-p38 MAPK. Fig. 2D shows the enrichment of Qa-2 in fractions containing lipid raft marker GM1 in CD4SP thymocytes. Thus, the integrated data from three different assays demonstrated the localization of Qa-2 in lipid rafts of SP4 thymocytes. 4.3. Quantitative analysis of lipid raft components in CD4SP subsets To investigate whether lipid rafts play a role in CD4SP thymocyte development, we analyzed the quantitative levels of specific lipid raft components GM1 in CD4SP subsets. By flow cytometric analysis, we found that almost 100% of CD4SP thymocytes expressed GM1, which is consistent with previous observations [21,22]. We further dissected the GM1 expression levels in distinct CD4SP subsets and observed a hierarchy: CD69+ Qa2− (SP1 + SP2) < CD69− Qa-2− (SP3) < CD69− Qa-2+ (SP4) (Fig. 3A). Meanwhile, a significant increase in GM1 expression was observed following the increase of Qa-2 levels in SP4 (Fig. 3A). After exit from the thymus, CD4SP cells undergo further functional maturation and acquire the highest expression of Qa-2 in periphery. We compared the GM1 expression levels between thymocytes and splenocytes and observed that the GM1 membrane content of splenic CD4+ T cells was more than five-fold higher than that of SP4 thymocytes (Fig. 3B). Taken together, differences in lipid raft components by thymocyte subsets may contribute to the process of T cell differentiation or functional maturation. 4.4. Lipid raft disruption impairs CD4SP thymocyte differentiation in vitro The quantitative differences in lipid raft components of CD4SP subsets prompted us to investigate whether lipid raft integrity is required for CD4SP development. We previously established an
mTEC1-supported culture system that is capable of supporting CD4SP thymocyte differentiation in vitro [10]. Here, we adopted this system to investigate the role of membrane rafts in CD4SP thymocyte differentiation. As mentioned above, the cholesterol depleting reagent MCD is commonly used to disrupt lipid rafts. Because the effect of MCD treatment is temporary and probably followed by a rapid up-regulation of cholesterol synthesis within the cell [23], serum-free AIM V medium was used. The concentration and duration of MCD treatment were carefully determined for thymocytes and mTEC1 because these cell types cannot survive inappropriate MCD treatment. We first confirmed that MCD indeed removed cholesterol from the membranes of thymocytes and mTEC1. Filipin III was used to stain cholesterol on thymocytes. Filipin is a fluorescent polyene antibiotic from Saccharomyces filipinensis, and it forms a multimeric globular complex with cholesterol in the cell membrane [24]. Flow cytometric analysis showed that treatment of thymocytes with MCD reduced the staining with filipin to approximately 50% of that in control cells (Fig. 4A). The effect of MCD on mTEC1 was also confirmed (data not shown). When MCD-treated CD69+ Qa-2− CD4SP cells were co-cultured with MCDtreated mTEC1 cells in AIM V medium, the resulting CD69− Qa-2+ (SP4) cells comprised 36.6% of the culture at 72 h, compared to 53.3% of the co-culture of untreated thymocytes and mTEC1. The SP4 population in the MCD-treated group was recovered by the addition of FBS to the culture. The proportion of SP4 cells in the MCD + FBS treated culture was 50.9%, very close to that of the culture without MCD treatment (Fig. 4B). This effect was also observed in the actual numbers of SP3 and SP4 cells developed (Fig. 4C). To further support the notion that cholesterol-rich rafts are involved in CD4SP thymocyte development, we also treated the cells with cholesterol oxidase (COase) in the same co-culture system as mentioned above. COase converts cholesterol into cholestenone and can disrupt the raft integrity. In a 48 h co-culture system, our data demonstrated that similar to MCD treatment, COase-treatment hindered CD4SP thymocyte development. The percentage and absolute cell number of SP4 subset were both significantly decreased (Fig. 4D and E).
J. Li et al. / Immunology Letters 148 (2012) 163–171
167
Fig. 3. Quantitative analysis of lipid raft components in CD4SP subsets and splenic CD4+ T cells (A) GM1 expression in CD4SP thymocyte subsets. CD4SP thymocytes were analyzed by flow cytometry after staining with anti-Qa-2, anti-CD69 and CTB. A significant difference in the MFI of GM1 was observed among CD69+ Qa-2− , CD69− Qa-2− and CD69− Qa-2+ CD4SP cells and among Qa-2lo , Qa-2int and Qa-2hi SP4 cells. The experiments were repeated three times, and similar results were obtained for each set of experiments. (B) GM1 expression in SP4 thymocytes was compared with that in splenic CD4+ T cells.
4.5. Effect of raft disruption on CD4SP thymocyte differentiation in fetal thymic organ culture To investigate the impact of cholesterol depletion on SP thymocyte development in a more physiological system, fetal thymic organ cultures (FTOC) were established with and without MCD treatment. When fetal thymi were treated with MCD and cultured in serum-free medium, compared to the control group, the percentage of CD4SP cells only decreased a little bit. However, further analysis of the profile of CD69 and Qa-2 revealed that the differentiation of CD4SP cells were significantly blocked after MCD treatment and the percentage of SP4 subset among CD4SP cells reduced to 4.2%, compared to 19.8% of that without MCD treatment. Similar to the results in the co-culture system, cholesterol replenishment by the addition of FBS recovered the differentiation of CD4SP cells and the percentage of SP4 subset increased to 17.6% (Fig. 5A). This effect was also reflected in the absolute cell numbers of distinct developing subsets of CD4SP (Fig. 5B).
Both data from in vitro co-culture system and FTOC suggest that when the raft integrity is disrupted, the differentiation of CD4SP thymocytes is significantly hindered, especially the transition from SP3 to SP4 (Fig. 6). 5. Discussion Recently, many efforts have underlined the importance of CD4SP thymocyte differentiation in the medulla. Here, we demonstrated that CD4SP exhibited heterogeneity in expression of lipid raft components GM1. Qa-2, a unique marker for SP4, localizes to lipid rafts. It is heterogeneously expressed in SP4 cells, and its expression level correlates with cell functionality. Raft disruption impairs the expression of Qa-2, which is an important event during the transition from SP3 to SP4. SP4 represents the most mature subset of CD4SP cells. The data from intrathymic adoptive transfer assays showed that some SP4 cells stayed in the thymus longer than others [11]. The consequences of this extended retention in the medulla
168
J. Li et al. / Immunology Letters 148 (2012) 163–171
Fig. 4. Effect of raft disruption on CD4SP thymocyte differentiation in vitro. (A) Cholesterol depletion by MCD treatment. Thymocytes were treated with 10 mM MCD or left untreated, and the cholesterol in the plasma membrane was stained with filipin III and analyzed by flow cytometry. (B) In vitro differentiation of CD69+ Qa-2− CD4SP thymocytes after cholesterol depletion with MCD treatment. MCD treated or untreated CD69+ Qa-2− CD4SP thymocytes were cultured in AIM V medium in the presence of MCD treated mTEC1. The effect of FBS on cholesterol reconstitution was determined simultaneously. Cells were harvested at day 3 and analyzed for surface expression of CD69 and Qa-2. The number of cells recovered from different cultures is shown in brackets, and the number in each quadrant indicates the percentage of total cells in the corresponding subset. The results are representative of three independent experiments. (C) The numbers of SP3 and SP4 cells from different cultures were calculated. The SD is shown as error bars. (D) In vitro differentiation of CD69+ Qa-2− CD4SP thymocytes after cholesterol oxidation with cholesterol oxidase (COase). COase treated CD69+ Qa-2− CD4SP thymocytes were cultured with COase treated mTEC1. Co-cultures without COase treatment are regarded as control. Cells were harvested at day 2 and analyzed for surface expression of CD69 and Qa-2. (E) The cell numbers of CD4SP subsets from cultures with or without COase treatment were calculated. The SD is shown as error bars.
aroused our interests. In analysis of proliferation response upon mitogen stimulation in the SP4 subpopulations with different Qa-2 expression level, we observed a significant hierarchy: Qa-2lo < Qa2int < Qa-2hi . This phenomenon reveals that the phenotype of SP4 cells is not uniform but rather increases with Qa-2 expression level, and SP4 cells undergo continuous functional maturation concomitant with the up-regulation of Qa-2 expression. These results may explain the extended retention of some SP4 cells in thymic medulla. The correlation between Qa-2 expression and cell functionality is further supported by the comparison of GM1 expression between splenic CD4+ T cells and thymocytes. The analysis of the genes expressed by various CD4SP subsets showed that a variety of molecules were up-regulated along with the CD4SP development, including MHCs, costimulatory molecules, cytokines and their receptors [25]. Among them, several molecules such as MHC II and IL-2R are associated with lipid rafts of T cells [26,27]. Considering the role of lipid rafts as signal platforms that facilitate efficient and specific signal transduction, the clustering of these molecules into lipid rafts may be responsible for the functional maturation of T cells. Our finding that Qa-2 localizes to lipid rafts in SP4, together with the fact that Qa-2 expression correlates with cell functionality, further supports the role of lipid rafts in the functional maturation of SP4 cells. By analyzing the expression of lipid raft components in SP4 cells, we
observed that GM1 expression increased with the up-regulation of Qa-2. Thus, the difference in lipid raft contents may influence the intensity of the signals required for cell maturation. To extend the studies of the roles of the lipid raft in SP thymocyte development, we conducted further experiments to show that the expression of GM1 is up-regulated during CD4SP thymocyte development. This finding suggests that the development of CD4SP thymocytes in medulla follows the formation of lipid rafts. So far, there are various gangliosides (GM1, GM2, GM3) that have been identified. GM1 is widely studied for the adoption of CTB and is generally considered to be a reliable marker for lipid rafts. It is well-documented that GM1 is very important for T cell activation. In recent years, GM2 and GM3 have received more attention. A recent report shows that CD4+ and CD8+ T cells require different ganglioside subsets for activation [28]. The activation of CD4+ T cells from GM3 synthase null mice, deficient in GM3derived gangliosides, is severely compromised, suggesting that GM3 is also very important for CD4+ T cell activation. Another interesting phenomenon is that with the differentiation from thymocytes to mature T cell subsets, selective glycosphingolipids are up-regulated. For example, GM3S, GM2/GD2S expressions are markedly increased from thymocytes to mature CD4+ T cells. In the present study, we demonstrate Qa-2 is localized in lipid rafts. Besides GM1, whether Qa-2 is also enriched in GM2 or GM3 related
J. Li et al. / Immunology Letters 148 (2012) 163–171
169
Fig. 5. Effect of raft disruption on CD4SP thymocyte differentiation in FTOC. MCD treated or untreated fetal thymic lobes were cultured in AIM V medium, with or without FBS. The thymocytes were analyzed on day 9 of incubation by flow cytometry with anti-CD4, CD8, CD69 and Qa-2 mAbs. (A) Profiles of thymocyte subpopulations from thymic lobes cultured are shown. The cell number of each lobe from different cultures is shown in brackets. CD4/CD8 expression profiles are in the top row and the number in each quadrant indicates the percentage of subset among all cells. CD69/Qa-2 expression profiles in CD4SP subsets are shown in the bottom row and the number in individual quadrant represents the percentage of subsets among CD4SP cells. The results are representative of three independent experiments. (B) The cell number in different CD4SP subsets of each lobe from cultures was calculated. The SD is shown as error bars.
Fig. 6. Schematic representation of the effect of raft disruption on CD4SP thymocyte development.
170
J. Li et al. / Immunology Letters 148 (2012) 163–171
lipid rafts needs further studies. Future studies focusing on the functions of GM2 or GM3 in CD4SP development, dissecting the developmental program of CD4SP in GM3 synthase null mice or GM2/GD2 synthase null mice will be very helpful to have a deeper understanding of the functions of lipid rafts in thymocyte development. The appropriate development of thymocytes in the thymus requires the interaction between thymocytes and thymic stromal cells. During the transition from the DN to the SP stage, different signaling molecules are involved in the lympho-stromal communication to ensure that key events happen at the right stages. In addition to their involvement in central tolerance, mTECs play essential roles in SP thymocyte development. Defects in mTECs (such as in Relb−/− or Aire−/− mice) cause the arrest of CD4SP cells at SP3. Furthermore, the efficient generation of SP4 cells in our in vitro studies requires the presence of a special type of mTECs [10]. The potential role of soluble factors produced by mTECs in this process was excluded by in vitro experiments in a co-culture system (data not shown). These results indicate that signaling between mTECs and thymocytes drives the differentiation of SP thymocytes, although the details of this signaling remain to be further defined. It has been demonstrated that the presentation of MHC II molecules on epithelial cells induces raft-associated signaling in thymocytes, thereby inducing CD4+ T cell development [8]. Given the heterogeneity of lipid rafts among various CD4SP subsets, we speculate that raft-associated signaling is essential for the supporting role of mTECs in CD4SP thymocyte development, and lipid rafts acting as signal platforms for crosstalk between mTECs and thymocytes may be a prerequisite for CD4SP thymocyte maturation. Our finding of a significant reduction in the transition from SP3 to SP4 in the mTEC1-supported culture system after raft disruption by cholesterol extraction or cholesterol oxidation supports this hypothesis. Besides, raft-disruption either on mTEC1 or on thymocytes also hindered the differentiation of CD4SP thymocyte to SP4 stage (data not shown), and these data further emphasize the indispensability of crosstalk between mTECs and thymocytes in the final maturation of SP thymocytes. It seems that raft-associated signaling in the final maturation of CD4SP is not mediated by TCR-MHC II interaction because the expansion and differentiation of SP cells still occur in co-cultures in which epithelial cells express no or mismatched MHC molecules [[29] and unpublished data]. Recent studies have demonstrated that the Lck unique domain is important for both the DN-DP transition and the selection of SP cells, and Fyn plays a minor compensatory role during thymocyte development [9,30]. Therefore, we propose that raft-associated signaling via Lck and Fyn may be responsible for CD4SP thymocyte development. In our study, Qa-2 is regarded as a clue which correlates the differentiation and functional maturation of SP thymocytes with lipid rafts, whereas its exact role in this process is still unknown. Further studies to analyze the developmental program of CD4SP in Ped low strain mice may provide more suggestive and convincing data to explain the exact roles of Qa-2 in the development of CD4SP thymocytes. To our knowledge, this is the first study to demonstrate the critical role of lipid rafts in the process of medullary CD4SP thymocyte differentiation and in the final maturation of SP4 cells. However, the specific raft-associated molecules required for CD4SP thymocyte development require further investigation.
6. Conclusions Findings reported here demonstrate that Qa-2 associated lipid rafts are indispensable for the final maturation of CD4+ CD8− thymocytes. Qa-2, the unique marker for mature CD4SP cells, may also be involved in the functional maturation of CD4SP cells. The exact
mechanism for Qa-2 and lipid raft-associated signaling in thymocyte development is under study now.
Acknowledgements This work was supported by National Natural Science Foundation of China (30900735 and 81070271), the Key Project of Chinese Ministry of Education (211012) and the National Basic Research Program of China (2011CB946103).
References [1] Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev 2000;1:31–41. [2] Kabouridis PS. Lipid rafts in T cell receptor signaling. Mol Membr Biol 2006;23:49–57. [3] Kabouridis PS, Juryb EC. Lipid rafts and T-lymphocyte function: Implications for autoimmunity. FEBS Lett 2008;582:3711–8. [4] Malorni W, Giammarioli AM, Garofalo T, Sorice M. Dynamics of lipid raft components during lymphocyte apoptosis: the paradigmatic role of GD3. Apoptosis 2007;12:941–9. [5] Werlen G, Hausmann B, Palmer E. A motif in the alpha-beta T cell receptor controls positive selection by modulating ERK activity. Nature 2000;406:422–6. [6] Wong P, Barton GM, Forbush KA, Rudensky AY. Dynamic tuning of T cell reactivity by self-peptide-major histocompatibility complex ligands. J Exp Med 2001;193:1179–87. [7] Ebert PJ, Baker JF, Punt JA. Immature CD4+ CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals. J Immunol 2000;165:5435–42. [8] Komaniwa S, Hayashi H, Kawamoto H, Sato SB, Ikawa T, Katsura Y, et al. Lipidmediated presentation of MHC class II molecules guides thymocytes to the CD4 lineage. Eur J Immunol 2009;39:96–112. [9] Palacios EH, Weiss A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 2004;23:7990–8000. [10] Li J, Li Y, Yao JY, Jin R, Zhu MZ, Qian XP, et al. Developmental pathway of CD4+ CD8− medullary thymocytes during mouse ontogeny and its defect in Aire−/− mice. Proc Natl Acad Sci USA 2007;104:18175–80. [11] Jin R, Wang W, Yao JY, Zhou YB, Qian XP, Zhang J, et al. Characterization of the in vivo dynamics of medullary CD4+ CD8− thymocyte development. J Immunol 2008;180:2256–63. [12] McElhinny AS, Exley GE, Warner CM. Painting Qa-2 onto Ped slow preimplantation embryos increases the rate of cleavage. Am J Reprod Immunol 2000;44:52–8. [13] Comiskey M, Domino KE, Warner CM. HLA-G is found in lipid rafts and can act as a signaling molecule. Hum Immunol 2007;68:1–11. [14] De Fazio SR, Warner CM. Activation of T cells by cross-linking Qa-2, the Ped gene product, requires Fyn. Am J Reprod Immunol 2007;58:315–24. [15] Suzuki K, Sheetz MP. Binding of cross-linked glycosylphosphatidylinositolanchored proteins to discrete actin-associated sites and cholesterol-dependent domains. Biophys J 2001;81:2181–9. [16] Vernachio J, Li M, Donnenberg AD, Soloski MJ. Qa-2 expression in the adult murine thymus. A unique marker for a mature thymic subset. J Immunol 1989;142:48–56. [17] Matkó J, Bodnár A, Vereb G, Bene L, Vámosi G, Szentesi G, et al. GPI-microdomains (membrane rafts) and signaling of the multi-chain interleukin-2 receptor in human lymphoma/leukemia T-cell lines. Eur J Biochem 2002;269:1199–208. [18] Gombos I, Bacsó Z, Detre C, Nagy H, Goda K, Andrásfalvy M, et al. Cholesterol sensitivity of detergent resistance: a rapid flow cytometric test for detecting constitutive or induced raft association of membrane proteins. Cytometry A 2004;61:117–26. [19] Boursalian TE, Golob J, Soper DM, Cooper CJ, Fink PJ. Continued maturation of thymic emigrants in the periphery. Nat Immunol 2004;5:418–25. [20] Cherukuri A, Dykstra M, Pierce SK. Floating the raft hypothesis: lipid rafts play a role in immune cell activation. Immunity 2001;14:657–60. [21] De Mello Coelho V, Nguyen D, Giri B, Bunbury A, Schaffer E, Taub DD. Quantitative differences in lipid raft components between murine CD4+ and CD8+ T cells. BMC Immunol 2004;5:2. [22] Haks MC, Belkowski SM, Ciofani M, Rhodes M, Lefebvre JM, Trop S, et al. Low activation threshold as a mechanism for ligand-independent signaling in pre-T cells. J Immunol 2003;170:2853–61. [23] Hampton R, Dimster-Denk D, Rine J. The biology of HMG-CoA reductase: the pros of contra-regulation. Trends Biochem Sci 1996;21:140–5. [24] Hassall DG, Graham A. Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP-1 macrophages. Cytometry 1995;21:352–62. [25] Teng F, Zhou YB, Jin R, Chen Y, Pei X, Liu Y, et al. The molecular signature underlying the thymic migration and maturation of TCR␣+ CD4+ CD8− thymocytes. PloS ONE 2011;6:e25567. [26] Li Q, Wang M, Tan L, Wang C, Ma J, Li N, et al. Docosahexaenoic acid changes lipid composition and interleukin-2 receptor signaling in membrane rafts. J Lipid Res 2005;46:1904–13.
J. Li et al. / Immunology Letters 148 (2012) 163–171 [27] Karacsonyi C, Knorr R, Fülbier A, Lindner R. Association of major histocompatibility complex II with cholesterol- and sphingolipid-rich membranes precedes peptide loading. J Biol Chem 2004;279:34818–26. [28] Nagafuku M, Okuyama K, Onimaru Y, Suzuki A, Odagiri Y, Yamashita T, et al. CD4 and CD8 T cells require different membrane gangliosides for activation. Proc Natl Acad Sci USA 2012;109:E336–42.
171
[29] Hare KJ, Wilkinson RW, Jenkinson EJ, Anderson G. Identification of a developmentally regulated phase of postselection expansion driven by thymic epithelium. J Immunol 1998;160:3666–72. [30] Salmond RJ, Filby A, Pirinen N, Magee AI, Zamoyska R. Mislocalization of Lck impairs thymocyte differentiation and can promote development of thymomas. Blood 2011;117:108–17.