Quantitative differences in lipid raft components between murine CD4+ and CD8+ T cells
- Valeria de Mello Coelho†1,
- Dzung Nguyen†1,
- Banabihari Giri1,
- Allyson Bunbury1,
- Eric Schaffer1 and
- Dennis D Taub1Email author
© de Mello Coelho et al; licensee BioMed Central Ltd. 2004
Received: 10 September 2003
Accepted: 30 January 2004
Published: 30 January 2004
Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4+ helper and CD8+ cytotoxic T cells. However, the differential expression of lipid raft components between CD4+ and CD8+ T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4+ and CD8+ T cell subpopulations.
We found that CD4+CD8- and CD8+CD4- thymocytes at different stages of maturation display distinct GM1 surface expression. This phenomenon did not change with progressive aging, as these findings were consistent over the lifespan of the mouse. In the periphery, CD8+ T cells express significantly higher levels of GM1 than CD4+ T cells. In addition, we observed that GM1 levels increase over aging on CD8+ T cells but not in CD4+ T cells. We also verified that naïve (CD44lo) and memory (CD44hi) CD8+ T cells as well as naïve and memory CD4+ T cells express similar levels of GM1 on their surface. Furthermore, we found that CD8+ T cells express higher levels of the GPI-anchored cell surface protein Thy-1 associated with lipid raft domains as compared to CD4+ T cells. Finally, we observed higher levels of total cellular cholesterol in CD8+ T cells than CD4+ T cells.
These results demonstrate heterogeneity of lipid raft components between CD4+ and CD8+ T cells in young and aged mice. Such differences in lipid raft composition may contribute to the differential CD4 and CD8 molecule signaling pathways as well as possibly to the effector responses mediated by these T cell subsets following TCR activation.
Lipid rafts are characterized as organized plasma membrane domains enriched in sphingolipids and cholesterol, originally identified by their resistance to non-ionic detergent lysis at 4°C [1, 2]. These microdomains are enriched in GPI-linked proteins on the extracellular surface, such as Thy-1 and CD59, and acylated signaling proteins on the cytoplasmic surface, including Src kinases, Ras proteins, G proteins, Vav, PKC, and LAT [1–4]. Lipid rafts play an integral role in synapse formation between antigen presenting cells and T cells due to their ability to serve as platforms for the recruitment of TCR and signaling molecules. To identify lipid rafts on the surface of cells, GM1, a monosialoganglioside and glycosphingolipid, is a commonly used marker, which is detected using bacterial-derived cholera toxin B subunit (CTB) [5, 6]. Other markers to lipid rafts include the GPI-linked proteins, which associate with sphingolipids, glycolipids and cholesterol in the cell membrane and with several cytoplasmic proteins possibly facilitating raft domains downstream signaling [reviewed in ].
Cholesterol is also essential to the formation and function of lipid rafts. Studies involving the extraction of membrane cholesterol by β-cyclodextrins, as well as membrane cholesterol sequestering by filipin and nystatin, implicate a critical role for cholesterol in lipid raft formation [reviewed in ]. The cholesterol molecule is believed to pack more tightly in the membrane with unsaturated fatty acid chains, increasing membrane order and conferring detergent resistance in these regions at low temperatures . Thus, the overall concentration of cholesterol in cell membranes is believed to impact on cell function. Evidence from aging human immune cells suggests that an excess of membrane cholesterol may affect TCR signaling pathways, although the specific mechanisms involved are not completely understood [10, 11].
During the process of T cell maturation in the thymus, the expression of CD4 and CD8 molecules changes on thymocyte subsets. Immature CD4-CD8- T cell progenitors, originating from the bone marrow, enter the thymus and undergo differentiation and selection to become immunocompetent mature T cells capable of emigrating to the peripheral lymphoid organs . During this process, CD4-CD8- T cells become CD4+CD8+ and then differentiate into mature CD4+CD8- or CD8+CD4- T cells [12, 13]. Interestingly, CD4 and CD8 molecules on fully differentiated mature T cells are palmitoylated and are constitutively associated with lipid raft microdomains .
During antigen presentation, the CD8 and CD4 molecules in combination with the TCR bind to the peptide-MHC class I or II components, respectively, on antigen-presenting cells. This interaction favors the formation of immunological synapses where signaling, adhesion and cytoskeleton molecules are concentrated within lipid raft microdomains following TCR co-aggregation [15–18]. Although lipid rafts are important in all of these processes, an association between these effects and the quantitative levels of specific lipid rafts components, namely GM1, GPI-linked proteins and cholesterol, have not been described in thymic or peripheral T cell subsets. In addition, deficiencies in T cell signaling identified in aging human and murine cells could be potentially be explained by differences in lipid raft composition between cells from young versus old subjects. In the present work, we have examined the GM1 expression on distinct CD4+ and CD8+ thymic and peripheral T cell subsets from young and aged mice as well as the levels of the GPI-anchored protein Thy-1 and cholesterol contents in these same cell populations.
Results and discussion
GM1 expression levels on distinct thymocyte subsets do not change with aging1
CD4 - CD8 -
797.8 ± 141.4
874.8 ± 45.1
1242.5 ± 343.0
1144.4 ± 426.3
947.5 ± 12.0
CD4 + CD8 +
593.0 ± 71.4
622.14 ± 14.0
729.1 ± 129.1
672.6 ± 81.0
671.6 ± 63.9
CD4 + CD8 -
302.3 ± 7.6
329.8 ± 18.3
360.7 ± 25.2
350.7 ± 6.6
387.1 ± 7.3
CD8 + CD4 -
1534.7 ± 507.7
1316.6 ± 184.2
1554.0 ± 39.5
1472.3 ± 279.2
1561.4 ± 217.7
Previous studies have demonstrated that lipid rafts may play a role in thymic T cell differentiation. In this context, the development of CD4-CD8- immature thymocytes to the CD4+CD8+ stage requires the pre-TCR α chain palmitoylation and recruitment to lipid raft domains . Furthermore, the process of thymic selection of CD4+CD8+ T cells to become CD4+CD8- or CD8+CD4- mature thymocytes occurs after association of CD3 molecules to the TCR in the raft regions and interaction with the complex self peptide-MHC in the thymus [22–24]. Moreover, additional studies have demonstrated the polarization of lipid rafts to the sites of TCR-activation on mature CD4+ and CD8+ T cells while CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals . Additionally, the commitment of thymocytes to the CD4+CD8- lineage requires a significantly stronger stimulus and a prolonged MAPK signal compared to what was required for a CD8+CD4- lineage commitment . Thus, it seems possible that differences in lipid raft components by thymic subsets may contribute to the process of T cell selection and differentiation. Given that CD4+ and CD8+ T cells are derived from CD4+CD8+ precursors, it may be possible that highly GM1-expressing CD4+CD8+ cells are positively selected to become CD8+ T cells, while lower GM1-expressing cells are selected to become CD4+ T cells. This selection would not likely be occurring as a direct result of GM1 expression, but rather, through the effects of glycosphingolipid levels on TCR avidity and signaling during positive and negative selection in the thymus. Accordingly, Drake and Baciale have demonstrated that MHC class I tetramer binding to functional CD8+ T cells requires lipid raft integrity . It may be that the optimal levels of GM1 for CD4+ and CD8+ T cells to survive negative and positive selection require distinct windows. Further studies will be necessary to answer this question.
Expression of GM1 on peripheral T cell subsets of young and aged mice
CD4 + CD8 -
997.15 ± 25.50
1298 ± 196.43
CD8 + CD4 -
2114.0 ± 292.92
3449.12 ± 54.17*
Next, we examined whether there are any alterations in the expression of GM1 in peripheral CD44lo (naïve) and CD44hi (memory) CD4+ and CD8+ T cells , due to observations suggesting that these cell subsets have differential requirements for stimulation [28, 29]. As shown in figure 2B, we failed to observe any significant differences in the expression of GM1 between CD44loCD4+ and CD44hiCD4+ cells or CD44loCD8+ and CD44hiCD8+ cells isolated from Balb/c mice spleens. These results are in contrast to studies utilizing total human T cells isolated from peripheral blood where the levels of GM1 were shown to be higher on memory T cells compared to naïve T cells .
While GM1 is commonly utilized as a marker for lipid rafts on cellular surfaces, this marker may not be absolutely indicative of total lipid raft expression on cells as GPI-associated proteins and other glycolipids are also involved in raft formation. Thy-1, a GPI-linked protein, and cholesterol are enriched in lipid rafts and contribute to the formation of lipid raft membrane domains through the interaction with phospho- and sphingolipids, including GM1 [31, 32]. In light of our GM1 findings, we next examined the expression of Thy-1 in lipid raft fractions isolated from CD4+ and CD8+ T cells (Figure 3B). Previously, we verified that the CD8+ cell line, RF3370, expressed higher levels of GM1 than the CD4+ T cell line, D0-11.10 (data not shown), at similar ratios and patterns as observed for primary peripheral CD4+ and CD8+ T cells (Fig. 2A). Utilizing immunoprecipitation and immunoblot analyses, we analyzed the expression of Thy-1 in lipid raft fractions isolated from these cell lines. In accordance with our GM1 results, we found that the expression of Thy-1 was approximately 50 % higher in CD8+ T cells as compared to CD4+ T cells (Fig. 3B and 3C).
These results support the concept that CD8+ T cells do express greater numbers of lipid rafts and/or have a greater surface area of lipid rafts than CD4+ T cells. Such differences may influence cellular activation and functional responses mediated by these cells following TCR activation. In fact, it has been demonstrated that changes in the cholesterol levels influence the interactive molecular stabilization and activity of CD4 among other molecules present in raft regions of the plasma membrane of T cells [20, 32–34]. Polarization of membrane receptor molecules in lipid raft platforms is critical to immunological synapse formation. Differences in lipid raft content and/or numbers between T cell subsets may influence the intensity or threshold of signals required for T cell activation.
It is not unreasonable to propose that CD4+ and CD8+ T cells require differing levels of cell surface lipid rafts for optimal signaling. It is generally accepted that rafts are essential for function in both cell types, but the experimental approaches used often test for an "all-or-none" phenotype regarding lipid rafts. Our results suggest that the levels of lipid rafts in CD4+ and CD8+ T cells require some degree of fine tuning, seen in the relatively consistent GM1 expression in both CD4+ and CD8+ T cells (Fig. 1A and ref. ). The recruitment of signaling molecules and lipid rafts to the immunological synapse is a hallmark of CD4+ T cell activation. In contrast, it is interesting to note that CD8+ T cells that do require lipid rafts for signaling do not polarize lipid rafts during signaling and activation . Could this be due to the elevated levels of lipid rafts already present on these cells? If raft concentrations in CD8+ T cells are maintained at a high level, no further capping may be necessary to mediate signaling at the site of cell-cell contact, whereas CD4+ cells would require capping due to the relatively low lipid raft concentrations in these regions.
In summary, our results suggest that differences in lipid raft composition may contribute to the differential CD4 and CD8 molecule signaling pathways as well as possibly to the effector responses mediated by these T cell subsets following TCR activation.
Our results demonstrate heterogeneity of lipid raft components between CD4+ and CD8+ T cells, which might influence in distinct effector response of these cells. Based on these results, it would seem appropriate to investigate the activity of molecules associated with lipid rafts in T lymphocytes using purified CD4+ and CD8+ T cell subpopulations rather than total T cells to avoid variable and/or biased results.
Thymocyte and T cell purification
Pooled thymi were homogenized in a glass potter within RPMI supplemented with 0.5% bovine serum albumin (BSA). Supernatant was collected, centrifuged and the cells were counted for subsequent analysis. Splenocytes were derived from the pooled spleens of albino Swiss or Balb/c mice and subsequently treated with ACK lysing buffer (150 mM NH4Cl, 1 mM KHCO3 and 0.001 mM EDTA) to remove erythrocytes. CD4+CD8- or CD8+CD4- splenic T cells were purified via a negative selection technique utilizing mouse T cell subset columns (R&D system, Minneapolis, MN) following the manufacturer's instructions. Following isolation, T cells were ultracentrifuged at room temperature for 5 minutes and resuspended in PBS containing 0.5% BSA. Typically, fresh T cell subset selection yielded greater than 95% purity for CD3+CD4+ or CD3+CD8+ T cells as assessed by flow cytometric analysis using anti-CD3-FITC (clone 145-2C11) and anti-CD8-PE (clone OKT8) and/or anti-CD4-PE (clone OKT4) antibodies (PharMingen/BD, San Diego, CA).
T cell lines
Subclones of the T cell hybridoma cell lines, D0-11.10 (CD4+, H2d restricted, OVA-specific) and RF3370 (CD8+, H2Kb restricted, OVA specific) were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% heat inactivated bovine calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin.
Flow Cytometry analysis
Purified T cell subsets or T Cell lines were stained with FITC-conjugated Cholera Toxin B (CTB) (Calbiochem, San Diego, CA) in combination with either anti-CD4 or anti-CD8 and anti-CD44 antibodies conjugated with fluorochrome on ice for 15 minutes and subsequently analyzed on a FACScan flow cytometer (Becton Dickinson, Sand Diego, CA).
Lipid raft isolation
Murine primary CD4+ and CD8+ (5 × 106) T cells or T cell lines (1 × 108 cells) were lysed in 0.4 ml of ice-cold MNE buffer (25 mM MES Ph 6.5, 150 mM NaCl, 2 Mm EDTA) containing 1% Triton X-100, the E-64 protease inhibitors, and 1 mM sodium orthovanadate. These cell lysates were brought to 1 ml using 40% sucrose solution and then overlaid with 2 ml of sucrose 35% and 1 ml of sucrose 5% in MNE buffer. Lysates were then ultracentrifuged in an SW55Ti rotor (Beckman, Palo Alto, CA) at 100,000 g for 16 hours to separate lipid rafts from cytosol. Aliquots of 0.4 ml of gradient fractions were then collected to yield a total of 9 fractions, which were analyzed for low-density lipid raft components and high-density detergent soluble cytosolic and plasma membrane components by immunodot.
One hundred μl of each fraction obtained from the ultracentrifugation for lipid raft isolation were blotted on nitrocellulose membranes, after which the membrane was blocked for 15 minutes using a 3% milk solution blocking buffer. After incubation, the membranes were incubated at 4°C overnight with HRP-conjugated CTB diluted to 2 μg/ml. Membranes were subsequently washed twice in PBS and signal detection was performed by Hyperfilm ECL according to the manufacturer's protocol (Amersham Biosciences, Piscataway, NJ).
Immunoprecipitation and western blot analysis
Lipid raft fractions 2, 3 & 4 and the non-raft fractions 7, 8 & 9 were prepared from CD4+ and CD8+ T cell lines lysates were pooled and proteins were quantitated by Bradford reagents (Bio-Rad, Hercules, CA). Both pooled fractions were mixed with immunoprecipitation buffer (IP buffer) (10 mM Tris-Hcl pH 7.5, 150 mM NaCl, 10 mM MgCl2 0.5% NP40, 1 mM Sodiumorthovanadate, leupeptine and pepstatin) with anti-thy1 mAb (Abcam, Cambridge, UK) along with protein agarose G beads (Calbiochem, San Diego, CA) overnight at 4°C. Immunocomplexes with agarose-bound protein G were pelleted down by centrifugation at 10,000 rpm for 5 min. The pellets were washed three times with IP buffer and then subjected to SDS-PAGE, followed by immunoblotting. Pellets were solubilized in IP buffer containing 2-mercaptoethanol. After heating for 5 min at boiling water bath, proteins were separated by SDS-PAGE on 12% polyacrylamide gel and transferred onto 0.22 μm polyvinylidine (difluoride) membranes (Invitrogen). Immunoblot analysis was performed using anti-Thy-1 mAb at a dilution of 1:500. Signal detection was performed by Hyperfilm ECL according to the manufacturer's protocol (Amersham Biosciences).
Purified T cells were extensively washed with PBS prior to use and subsequently lysed in a buffer containing SDS 0.1%, Na2EDTA 1 mM and Tris-HCL 0.1 M, pH 7.4. These cells were subsequently examined for their cholesterol content using a sensitive cholesterol oxidase-based assay using the Amplex Red cholesterol kit (Molecular Probes, Eugene, OR).
Significant statistical differences between groups were conducted using Student's t test and indicated as *p ≤ 0.05 or **p ≤ 0.02.
List of Abbreviations
T cell receptor
Cholera Toxin B
We would like to thank Drs. Paritosh Ghosh and Ashani Weeraratna for critical review of this manuscript.
- Simons K, Ikonen E: Functional rafts in cell membranes. Nature. 1997, 387: 569-72. 10.1038/42408.View ArticlePubMedGoogle Scholar
- Brown DA, London E: Functions of lipid rafts in biological membranes. Ann Rev Cell Dev Bio. 1998, 14: 111-36. 10.1146/annurev.cellbio.14.1.111.View ArticleGoogle Scholar
- Alonso MA, Millan J: The role of lipid rafts in signalling and membrane trafficking in T lymphocytes. J Cell Sci. 2001, 114: 3957-65.PubMedGoogle Scholar
- Zhang W, Trible RP, Samelson LP: LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation. Immunity. 1998, 9: 239-246. 10.1016/S1074-7613(00)80606-8.View ArticlePubMedGoogle Scholar
- Nichols BJ, Kenworthy AK, Polishchuk RS, Lodge R, Roberts TH, Hirschberg K, Phair RD, Lippincott-Schwartz J: Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J Cell Biol. 2001, 153: 529-41. 10.1083/jcb.153.3.529.PubMed CentralView ArticlePubMedGoogle Scholar
- Merritt EA, Sarfaty S, van den Akker SF, L'Hoir C, Martial JA, Hol WG: Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Sci. 1994, 3: 166-75.PubMed CentralView ArticlePubMedGoogle Scholar
- Magee T, Pirinen N, Adler J, Pagakis SN, Parmryd I: Lipid rafts: cell surface platforms for T cell signaling. Biol Res. 2002, 35: 127-31.View ArticlePubMedGoogle Scholar
- Silvius JR: Role of cholesterol in lipid raft formation: lessons from lipid model systems. Biochim Biophys Acta. 2003, 1610: 174-83. 10.1016/S0005-2736(03)00016-6.View ArticlePubMedGoogle Scholar
- London E, Brown DA: Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts). Biochim Biophys Acta. 2000, 1508: 182-95. 10.1016/S0304-4157(00)00007-1.View ArticlePubMedGoogle Scholar
- Stulnig TM, Buhler E, Bock G, Kirchebner C, Schonitzer D, Wick G: Altered switch in lipid composition during T-cell blast transformation in the healthy elderly. J Gerontol A Biol Sci Med Sci. 1995, 50: B383-90.View ArticlePubMedGoogle Scholar
- Fulop T, Douziech N, Goulet AC, Desgeorges S, Linteau A, Lacombe G, Dupuis G: Cyclodextrin modulation of T lymphocyte signal transduction with aging. Mech Ageing Dev. 2001, 122: 1413-30. 10.1016/S0047-6374(01)00274-3.View ArticlePubMedGoogle Scholar
- Shortman K, Egerton M, Spangrude GJ, Scollay R: The generation and fate of thymocytes. Semin Immunol. 1990, 2: 3-12.PubMedGoogle Scholar
- Ellmeier W, Sawada S, Littman DR: The regulation of CD4 and CD8 coreceptor gene expression during T cell development. Annu Rev Immun. 1999, 17: 523-54. 10.1146/annurev.immunol.17.1.523.View ArticlePubMedGoogle Scholar
- Bosselut R, Zhang J, Ashe JM, Kopacz JL, Samelson LE, Singer A: Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction. J Exp Med. 1999, 190: 1517-26. 10.1084/jem.190.10.1517.PubMed CentralView ArticlePubMedGoogle Scholar
- Kovacs B, Maus MV, Riley JL, Derimanov GS, Koretzky GA, June CH, Finkel TH: Human CD8+ T cells do not require the polarization of lipid rafts for activation and proliferation. Proc Natl Acad Sci USA. 2002, 99: 15006-11. 10.1073/pnas.232058599.PubMed CentralView ArticlePubMedGoogle Scholar
- Morrison WJ, Offner H, Vandenbark AA: Enhanced T-helper cell function following CD4 modulation. Cell Immunol. 1994, 153: 392-400. 10.1006/cimm.1994.1037.View ArticlePubMedGoogle Scholar
- Gao GF, Rao Z, Bell JI: Molecular coordination of alpha-beta T-cell receptors and coreceptors CD8 and CD4 in their recognition of peptide-MHC ligands. Trends Immunol. 2002, 23: 408-13. 10.1016/S1471-4906(02)02282-2.View ArticlePubMedGoogle Scholar
- Dykstra M, Cherukuri A, Pierce SK: Rafts and synapses in the spatial organization of immune cell signaling receptors. J Leukoc Biol. 2001, 70: 699-707.PubMedGoogle Scholar
- Haks MC, Belkowski SM, Ciofani M, Rhodes M, Lefebvre JM, Trop S, Hugo P, Zuniga-Pflucker JC, Wiest DL: Low activation threshold as a mechanism for ligand-independent signaling in pre-T cells. J Immunol. 2003, 170: 2853-61.View ArticlePubMedGoogle Scholar
- Garcia GG, Miller RA: Single-cell analyses reveal two defects in peptide-specific activation of naive T cells from aged mice. J Immunol. 2001, 166: 3151-57.View ArticlePubMedGoogle Scholar
- 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. 10.1038/35019094.View ArticlePubMedGoogle Scholar
- Delgado P, Fernandez E, Dave V, Kappes D, Alarcon B: CD3delta couples T-cell receptor signaling to ERK activation and thymocyte positive selection. Nature. 2000, 406: 426-30. 10.1038/35019102.View ArticlePubMedGoogle Scholar
- 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. 10.1084/jem.193.10.1179.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- Wilkinson B, Kaye J: Requirement for sustained MAPK signaling in both CD4 and CD8 lineage commitment: a threshold model. Cell Immunol. 2000, 211: 86-95. 10.1006/cimm.2001.1827.View ArticleGoogle Scholar
- Drake DR, Braciale TJ: Cutting edge: lipid raft integrity affects the efficiency of MHC class I tetramer binding and cell surface TCR arrangement on CD8+ T cells. J Immunol. 2001, 166: 7009-13.View ArticlePubMedGoogle Scholar
- Budd RC, Cerottini JC, Horvath C, Bron C, Pedrazzini T, Howe RC, MacDonald HR: Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation. J Immunol. 1987, 138: 3120-9.PubMedGoogle Scholar
- Berard M, Tough DF: Qualitative differences between naive and memory T cells. Immunology. 2002, 106: 127-38. 10.1046/j.1365-2567.2002.01447.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Seder RA, Ahmed R: Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat Immunol. 2003, 4: 835-42. 10.1038/ni969.View ArticlePubMedGoogle Scholar
- Tuosto L, Parolini I, Schroder S, Sargiacomo M, Lanzavecchia A, Viola A: Organization of plasma membrane functional rafts upon T cell activation. Eur J Immunol. 2001, 31: 345-9. 10.1002/1521-4141(200102)31:2<345::AID-IMMU345>3.3.CO;2-C.View ArticlePubMedGoogle Scholar
- Harder T, Scheiffele P, Verkade P, Simons K: Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol. 1998, 141: 929-42. 10.1083/jcb.141.4.929.PubMed CentralView ArticlePubMedGoogle Scholar
- Gimpl G, Burger K, Fahrenholz F: Cholesterol as modulator of receptor function. Biochemistry. 1997, 36: 10959-74. 10.1021/bi963138w.View ArticlePubMedGoogle Scholar
- Nguyen DH, Taub D: Cholesterol is essential for macrophage inflammatory protein 1 beta binding and conformational integrity of CC chemokine receptor 5. Blood. 2002, 99: 4298-306. 10.1182/blood-2001-11-0087.View ArticlePubMedGoogle Scholar
- Nguyen DH, Taub D: CXCR4 function requires membrane cholesterol: implications for HIV infection. J Immunol. 2002, 168: 4121-26.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.