Lipid raft-based membrane order is important for antigen-specific clonal expansion of CD4(+) T lymphocytes.

BACKGROUND: Lipid rafts are cholesterol and saturated lipid-rich, nanometer sized membrane domains that are hypothesized to play an important role in compartmentalization and spatiotemporal regulation of cellular signaling. Lipid rafts contribute to the plasma membrane order and to its spatial asymmetry, as well. The raft nanodomains on the surface of CD4(+) T lymphocytes coalesce during their interaction with antigen presenting cells (APCs). Sensing of foreign antigen by the antigen receptor on CD4(+) T cells occurs during these cell-cell interactions. In response to foreign antigen the CD4(+) T cells proliferate, allowing the expansion of few antigen-specific primary CD4(+) T cell clones. Proliferating CD4(+) T cells specialize in their function by undergoing differentiation into appropriate effectors tailored to mount an effective adaptive immune response against the invading pathogen. RESULTS: To investigate the role of lipid raft-based membrane order in the clonal expansion phase of primary CD4(+) T cells, we have disrupted membrane order by incorporating an oxysterol, 7-ketocholesterol (7-KC), into the plasma membrane of primary CD4(+) T cells expressing a T cell receptor specific to chicken ovalbumin323-339 peptide sequence and tested their antigen-specific response. We report that 7-KC, at concentrations that disrupt lipid rafts, significantly diminish the c-Ovalbumin323-339 peptide-specific clonal expansion of primary CD4(+) T cells. CONCLUSIONS: Our findings suggest that lipid raft-based membrane order is important for clonal expansion of CD4(+) T cells in response to a model peptide.

Ly-6A-Induced Growth Inhibition and Cell Death in a Transformed CD4+ T Cell Line: Role of Tumor Necrosis Factor-α.

Ly-6A, a member of the Ly-6/uPAR supergene family of proteins, is a cell adhesion and cell signaling protein. Signaling through Ly-6A activates the cell-intrinsic apoptotic cell death pathway in CD4+ T cell lines, as indicated by the release of cytochrome C, and activation of caspases 9 and 3. In addition, Ly-6A induces cytokine production and growth inhibition. The mechanism underlying the distinct cellular responses that are triggered by engaging Ly-6A protein has remained unknown. To examine the relatedness of these distinct responses, we have quantified the production of pro-apoptotic, growth inhibitory and tumor suppressive cytokines, such as TNF-α, TGF-ß and a related protein GDF-10, in response to Ly-6A signaling. Anti-Ly-6A monoclonal antibody-induced activation of YH16.33 CD4+ T cell line generated low levels of TGF-ß and GDF-10 but elevated levels of TNF-α. Blocking the biological activity of TNF-α resulted in reduced Ly-6A-induced apoptosis in T cells. The Ly-6A-induced response in the T cell line was distinct, as signaling through the antigen receptor complex did not cause growth inhibition and apoptosis despite high levels of TGF-ß and GDF-10 that were detected in these cultures. Additionally, in response to antigen receptor complex signaling, lower amount of TNF-α was detected. These results indicate the contribution of TNF-α in the observed Ly-6A-induced growth inhibition and apoptosis and provide a mechanistic explanation for the biologically distinct responses observed in CD4+ T cells after engaging Ly-6A protein. Additionally, the findings reported here will aid in the understanding of inhibitory signaling initiated by Ly-6A protein, especially in the context of its potential immune checkpoint inhibitory role in T cells.

Human LY6 gene family: potential tumor-associated antigens and biomarkers of prognosis in uterine corpus endometrial carcinoma.

The human Lymphocyte antigen-6 (LY6) gene family has recently gained interest for its possible role in tumor progression. We have carried out in silico analyses of all known LY6 gene expression and amplification in different cancers using TNMplot and cBioportal. We also have analyzed patient survival by Kaplan-Meier plotter after mining the TCGA database. We report that upregulated expression of many LY6 genes is associated with poor survival in uterine corpus endometrial carcinoma (UCEC) cancer patients. Importantly, the expression of several LY6 genes is elevated in UCEC when compared to the expression in normal uterine tissue. For example, LY6K expression is 8.25× higher in UCEC compared to normal uterine tissue, and this high expression is associated with poor survival with a hazard ratio of 2.42 (p-value = 0.0032). Therefore, some LY6 gene products may serve as tumor-associated antigens in UCEC, biomarkers for UCEC detection, and possibly targets for directing UCEC patient therapy. Further analysis of tumor-specific expression of LY6 gene family members and LY6-triggered signaling pathways is needed to uncover the function of LY6 proteins and their ability to endow tumor survival and poor prognosis in UCEC patients.

Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts.

BACKGROUND: Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. RESULTS: Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. CONCLUSIONS: Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

Cholesterol-dependent plasma membrane order (Lo) is critical for antigen-specific clonal expansion of CD4+ T cells.

Early "T cell activation" events are initiated within the lipid microenvironment of the plasma membrane. Role of lipid membrane order (Lo) in spatiotemporal signaling through the antigen receptor in T cells is posited but remains unclear. We have examined the role of membrane order (Lo)/disorder (Ld) in antigen specific CD4+ T cell activation and clonal expansion by first creating membrane disorder, and then reconstituting membrane order by inserting cholesterol into the disordered plasma membrane. Significant revival of antigen specific CD4+ T cell proliferative response was observed after reconstituting the disrupted membrane order with cholesterol. These reconstitution experiments illustrate Koch's postulate by demonstrating that cholesterol-dependent membrane order (Lo) is critical for responses generated by CD4+ T cells and point to the importance of membrane order and lipid microenvironment in signaling through T cell membrane antigen receptors.

Engaging Ly-6A/Sca-1 triggers lipid raft-dependent and -independent responses in CD4+ T-cell lines.

INTRODUCTION: The lymphocyte antigen 6 (Ly-6) supergene family encodes proteins of 12-14 kda in molecular mass that are either secreted or anchored to the plasma membrane through a glycosyl-phosphatidylinisotol (GPI) lipid anchor at the carboxy-terminus. The lipidated GPI-anchor allows localization of Ly-6 proteins to the 10-100 nm cholesterol-rich nano-domains on the membrane, also known as lipid rafts. Ly-6A/Sca-1, a member of Ly-6 gene family is known to transduce signals despite the absence of transmembrane and cytoplasmic domains. It is hypothesized that the localization of Ly-6A/Sca-1 with in lipid rafts allows this protein to transduce signals to the cell interior. METHODS AND RESULTS: In this study, we found that cross-linking mouse Ly-6A/Sca-1 protein with a monoclonal antibody results in functionally distinct responses that occur simultaneously. Ly-6A/Sca-1 triggered a cell stimulatory response as gauged by cytokine production with a concurrent inhibitory response as indicated by growth inhibition and apoptosis. While production of interleukin 2 (IL-2) cytokine by CD4+ T cell line in response to cross-linking Ly-6A/Sca-1 was dependent on the integrity of lipid rafts, the observed cell death occurred independently of it. Growth inhibited CD4+ T cells showed up-regulated expression of the inhibitory cell cycle protein p27kip but not of p53. In addition, Ly-6A/Sca-1 induced translocation of cytochrome C to the cytoplasm along with activated caspase 3 and caspase 9, thereby suggesting an intrinsic apoptotic cell death mechanism. CONCLUSIONS: We conclude that opposing responses with differential dependence on the integrity of lipid rafts are triggered by engaging Ly-6A/Sca-1 protein on the membrane of transformed CD4+ T cells.

Investigating B Cell Development, Natural and Primary Antibody Responses in Ly-6A/Sca-1 Deficient Mice.

Ly-6A/Stem cell antigen-1 (Ly-6A/Sca-1) is a glycosylphosphatidylinositol-anchored protein expressed on many cell types including hematopoietic stem cells (HSCs) and early lymphoid-specific progenitors. Ly-6A/Sca-1 is expressed on CD4+ T cells and plays a role in regulating cellular responses to foreign antigens. The role of Ly-6A/Sca-1 in primary antibody responses has not been defined. To investigate whether Ly-6A/Sca-1 functions in humoral immunity, we first injected Ly-6A/Sca-1-deficient and wild-type control mice with chicken ovalbumin (c-Ova) protein mixed with an adjuvant. We then assessed the ability of the mice to generate a primary antibody response against cOva. We further examined the development of B cells and circulating antibody isotypes in non-immunized Ly-6A/Sca-1deficient mice to determine if Ly6A/Sca-1 functions in development irrespective of antigen-specific immune activation. Ly-6A/Sca-1/Sca-1-deficient mice did not show any significant changes in the number of B lymphocytes in the bone marrow and peripheral lymphoid tissues. Interestingly, Ly-6A/Sca-1/Sca-1-/- mice have significantly elevated serum levels of IgA with λ light chains compared to wild type controls. B cell clusters with high reactivity to anti-IgA λ monoclonal antibody were detected in the lamina propria of the gut, though this was not observed in the bone marrow and peripheral lymphoid tissues. Despite these differences, the Ly-6A/Sca-1deficient mice generated a similar primary antibody response when compared to the wild-type mice. In summary, we conclude that the primary antibody response to cOva antigen is similar in Ly-6A/Sca-1deficient and sufficient mice. In addition, we report significantly higher expression of the immunoglobulin λ light chain by B cells in lamina propria of Ly-6A/Sca-1deficient mice when compared to the wild-type control.

In vitro derivation of interferon-γ producing, IL-4 and IL-7 responsive memory-like CD4(+) T cells.

CD4(+) memory is critical for successful protection against pathogenic challenge. As such, understanding the heterogeneity of cells that arise and survive after initial stimulation of naïve CD4(+) T cells will aid in the design of more successful vaccines. In previous studies, in vivo experimental systems have been extensively used to generate functional memory responses by lymphocytes. Here, we have attempted to develop an in vitro experimental system to generate memory CD4(+) T lymphocytes. CD4(+) T cells stimulated through the antigen receptor complex were examined for their memory-like characteristics after 3 weeks of cell culture. A subset of surviving cells expressed high levels of CD44 and low levels of CD45RB (CD44(hi)CD45(lo)), a phenotype that is similar to bonafide memory CD4(+) T cells. In vitro generated memory-like CD4(+) T cells secreted higher levels of IFN-γ, with rapid kinetics, upon re-stimulation than their naïve counterparts. In addition, these memory-like CD4(+) T cells did not produce either IL-2 or IL-4 but readily proliferated when cultured in the presence of IL-7 and IL-4. These observations suggest that CD4(+) cells surviving the expansion phase of immune response produce a Th1-signature cytokine and retain responsiveness to IL-4, a Th-2 cytokine, as well as to a well described survival factor, interleukin-7.