CD4 and CD8 T cells require different membrane for activation

Masakazu Nagafukua,b, Kaori Okuyamac, Yuri Onimarud, Akemi Suzukie, Yuta Odagiria, Tadashi Yamashitaf, Katsunori Iwasakid,g, Michihiro Fujiwarad, Motoaki Takayanagic, Isao Ohnoc, and Jin-ichi Inokuchia,b,g,1 aDivision of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, and cDepartment of Pathophysiology, Tohoku Pharmaceutical University, Sendai 981-8558, Japan; bCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan; dDepartment of Neuropharmacology, Faculty of Pharmaceutical Sciences, and gAcademic, Industrial and Governmental Institute for Aging and Brain Sciences, Fukuoka University, Fukuoka 814-0180, Japan; eInstitute of Glycoscience, Tokai University, Kanagawa 259-1292, Japan; and fDivision of Integrated Life Science, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan AUTHOR SUMMARY

T cells compose a crucial part of the im- of mice carried a disrupted enzyme, mune system and require activation. The A known as GM3 synthase (GM3S), which first step of T cell activation requires the is required for synthesis and movement of one of their surface mole- therefore lacked several gangliosides cules, known as the receptor, into cell- (GM3, GD3, GM2, and GM1a of a-series membrane regions known as rafts. gangliosides) but expressed several others Molecules called gangliosides are known (GA1, GM1b, GalNAcGM1b, and ex- to be major components of lipid rafts, tended GM1b of o-series gangliosides). but their role in T-cell activation has not B The other mice had an altered form of been established. Here, we show that another enzyme, GM2/GD2 synthase different types of T cells require different (GM2/GD2S), and thus lacked all of the and distinct ganglioside types for activa- above-mentioned gangliosides, except tion. We also describe a role for gan- GM3 and GD3 (Fig. P1A). gliosides in asthma, a disorder involving CD4+ T cells from wild-type (WT) the immune system. mice, in which the synthases were not T cells are generally designated as ei- mutated, mainly expressed GM1a and − − ther CD4+CD8 or CD4 CD8+, labels GD1b. The cells from the mice lacking that refer to molecules on the cells’ sur- GM3 synthase (GM3-null mice) exhibited faces. These molecules are involved in severe impairments in TCR-mediated the T cells’ main function: recognizing immune responses. These responses in- foreign invaders presented by other cells cluded the production of cytokines in the form of small fragments called (molecules that participate in immune- “antigens.” Research has shown that the Fig. P1. Selection of T cells to be produced cell signaling) and the multiplication of T-cell receptor (TCR), in conjunction (repertoire selection) by ganglioside selection. (A) specific T cells in response to specific with the CD4 and CD8 molecules, inter- The core biosynthetic pathway of gangliosides. invaders, a process known as clonal ex- acts with antigen-presenting cells through Staining by CTx-B detects not only GM1a gan- pansion. However, the defect was rescued glioside but also extended-GM1b. (B) Distinct a unique “immunological synapse” (IS) by preincubation of the cells with GM3 “ ” difference of lipid rafts in individual T-cell subsets. + (1). Lipid rafts have long been pro- The repertoire selection from immature thymo- and GM1a. CD8 T cells from WT mice posed to play a role (2) in this interaction. cytes to mature T-cell subpopulations is accom- expressed GA1, GM1b, GalNAc-GM1b, Polarization of the ganglioside, known as panied by selective ganglioside expression. and extended-GM1b. The cells from GM1, occurs only in CD4+ T cells after Analysis confirms that CD4+ T cells dominantly GM2/GD2S-null mice also exhibited se- TCR clustering (3). In addition, GM1 express a-series gangliosides due to up-regulation vere impairments in TCR-mediated cyto- fi (increased expression) of GM3S genes. Meanwhile, and GM3 gangliosides de ne different + kine production and clonal expansion. types of raft membrane domains that CD8 T cells carry o-series gangliosides due to Correspondingly, they were rescued by down-regulation (decreased expression) of GM3S preincubation with GA1 and GM1b. segregate either to the leading pole or to genes and up-regulation of GM2/GD2S expression. the trailing uropod, or extension, of the This result suggests that each T-cell subset has These results indicate that for T cell polarized human T cell (4). Expression of unique rafts composed of different ganglioside activation when their TCR binds an GM1 (GM1a) differs by cell type and types and that these rafts provide distinct antigen, different T cells require different + developmental stage, and staining with functions following stimulation of T cells via their gangliosides. CD4 T cells require a- cholera toxin B subunit (CTx-B) is com- receptors (TCRs). This ganglioside selection process series gangliosides, such as GM3 and monly used to visualize rafts. However, may be indispensable in the formation of distinct GM1a. Correspondingly, CD8+ T cells this toxin also reacts with another type of and functional lipid rafts in mature T cells. require o-series gangliosides, such as ganglioside known as extended GM1b (Fig. P1A). Thus, to understand the role of rafts in the differ- + entiation, maturation, and activation processes of CD4 T and Author contributions: M.N., A.S., T.Y., K.I., M.F., M.T., I.O., and J.-i.I. designed research; CD8+ T cells in vivo, it is critical to define the ganglioside M.N., K.O., Y. Onimaru, A.S., Y. Odagiri, I.O., and J.-i.I. performed research; M.N., K.O., A.S., composition in each respective T-cell subset. I.O., and J.-i.I. analyzed data; and M.N., A.S., I.O., and J.-i.I. wrote the paper. In this study, we determined which gangliosides are expressed The authors declare no conflict of interest. on thymocytes (the cells that develop into T cells) and CD4+ This Direct Submission article had a prearranged editor. T and CD8+ T cells and whether the activation of individual 1To whom correspondence should be addressed. E-mail: [email protected]. T-cell subsets requires distinct species of gangliosides. We used See full research article on page E336 of www.pnas.org. two kinds of mice with specific genetic characteristics. One type Cite this Author Summary as: PNAS 10.1073/pnas.1114965109.

1832–1833 | PNAS | February 7, 2012 | vol. 109 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1114965109 Downloaded by guest on September 25, 2021 GM1b and extended-GM1b. (Fig. P1B). We confirmed the ex- We also studied the role of gangliosides in allergic responses. PNAS PLUS pression of the GM3S and GM2/GD2S genes in thymocytes and Allergic airway inflammation is tightly regulated by some im- peripheral CD4+ T and CD8+ T cells from WT mice. GM3S mune processes, in which CD4+ T cells play a crucial role. We expression increased to 180% in CD4+ T cells and decreased to examined allergic airway responses induced by inhalation of 30% in CD8+ T cells compared with expression in thymocytes. ovalbumin (OVA) (the protein from the white of eggs) in OVA- GM2/GD2S expression was markedly increased in both CD4+ T sensitized mice (a common method of experimentally inducing and CD8+ T cells compared with thymocytes. Thus, these gene asthma). We found that these allergic responses were less severe expression patterns, together with the results of ganglioside in GM3S-null mice. These mice lack GM1a, which in turn results + analysis, demonstrate that CD4 T cells dominantly express a- in selective dysfunction of CD4+ T cells. This finding suggests series gangliosides due to increased expression of the GM3S that inhibition of GM3S would be a powerful treatment for + gene. On the other hand, CD8 T cells carry o-series ganglio- allergic airway diseases. sides due to decreased expression of the GM3S gene and con- We propose that the selection of specific T cells from the comitant up-regulation of the GM2/GD2S gene. Taken together, thymocyte stage (repertoire selection) is accompanied by selec- these results strongly suggest that each T-cell subset has unique tive ganglioside expression in individual T-cell subsets. This rafts consisting of different gangliosides and that these rafts ganglioside selection process may be indispensable for the for- provide distinct functions in different events following stimula- mation of distinct and functional lipid rafts in mature T cells tion via the TCR. B fi + + (Fig. P1 ). These ndings may open up a strategy for targeting Why are CD4 T cells and CD8 T cells dependent on ex- helper T cells to treat forms of allergic inflammation, including pression of distinct gangliosides for proper immune function? asthma, by controlling ganglioside expression in lipid rafts. Although they share the common mechanisms of TCR-mediated

signaling, the two subsets do have different cellular and molec- 1. Fooksman DR, et al. (2010) Functional anatomy of T cell activation and synapse ular modifications. CD4 and CD8 localize to lipid rafts by pal- formation. Annu Rev Immunol 28:79–105. mitoylation (a method of attaching to other molecules), yet 2. Dykstra M, Cherukuri A, Sohn HW, Tzeng SJ, Pierce SK (2003) Location is everything: raft localization seems not to be determined by that process Lipid rafts and immune cell signaling. Annu Rev Immunol 21:457–481. alone. To ensure that CD4 and CD8 are moved to a proper place 3. Kovacs B, et al. (2002) Human CD8+ T cells do not require the polarization of lipid rafts for activation and proliferation. Proc Natl Acad Sci USA 99:15006–15011. in the cell and on the membrane, it might be vital for CD4/CD8 4. Gómez-Móuton C, et al. (2001) Segregation of leading-edge and uropod components to interact with rafts carrying a specific ganglioside. into specific lipid rafts during T cell polarization. Proc Natl Acad Sci USA 98:9642–9647. IMMUNOLOGY

Nagafuku et al. PNAS | February 7, 2012 | vol. 109 | no. 6 | 1833 Downloaded by guest on September 25, 2021