Quantitative phosphoproteomics of cytotoxic T cells to reveal Protein Kinase D 2 regulated networks

The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages

Generation of virus-specific cytotoxic T cells in vitro I. Induction conditions of primary and secondary Sendai virus-specific cytotoxic T cells

H-2-restricted cytotoxic T cells specific for Sendai virus were generated in vitro in a primary response from normal mouse lymphocytes cultured in the presence of infective as well as inactivated Sendai virus. Antigen-presenting cells of different origin, including T cells, were found to be effective stimulators. Antibodies to Sendai virus were shown to inhibit the activation of specific precursor killer cells when added to cultures before, but not after, the addition of viral antigen. Data obtained by Lyt phenotyping, revealed that precursor killer cells specific for Sendai virus reside in the Lyt-2,3+ T cell population and that Lyt-l,2,3+ T cells are not required for the generation of cytotoxic lymphocytes. Different activation kinetics were demonstrated for primary and secondary antiviral cytotoxic responses, and the analysis of the proliferation and stimulation requirements suggests qualitative differences

Cytotoxic T cells and mycobacteria

How the immune system kills Mycobacterium tuberculosis is still a puzzle. the classical picture of killing due to phagocytosis by activated macrophages may be only partly correct. Based on recent evidence, we express here the view that cytotoxic T lymphocytes also make an important contribution and suggest that DNA vaccines might be a good way to enhance this. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Univ São Paulo, Sch Med Ribeirao Preto, Dept Biochem & Immunol, BR-14049900 Ribeirao Preto, SP, BrazilUniv São Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Clin Anal Bromatol & Toxicol, BR-14049 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol & Immunol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol & Immunol, São Paulo, BrazilWeb of Scienc

Lack of evidence for MHC-unrestricted (atypical) recognition of mucin by mucinous pancreatic tumour-reactive T-cells

Cytotoxic T-cells generated against heterologous, mucinous pancreatic tumour cells were shown to recognize mucin in a major histocombatibility complex (MHC)-unrestricted fashion. In contrast, the present study demonstrates a typical allogeneic response of heterologous cytotoxic T-cells established against mucin-expressing pancreatic tumour cells. Heterologous cytotoxic T cells lysed targets that were used as stimulators and other targets that shared human leucocyte antigen (HLA) with the stimulator. These cytotoxic T-cells lysed mucin-expressing stimulator cells but not autologous tumour cells in spite of expressing mucin on their surface. Likewise, tumour-infiltrating CD4+T-cells proliferated against its own tumour cell target, while such T-cells did not respond to heterologous, mucin-expressing pancreatic tumour cells. Culturing heterologous tumour-specific cytotoxic T-cells with purified pancreatic tumour cell-mucin rendered them unresponsive to their target cells. Furthermore, purified mucin did not produce a mucin-specific response in mucinous pancreatic tumour patients' primary T-cells even in the presence of antigen-presenting cells. Our study finds no evidence for MHC-unrestricted recognition of mucin by pancreatic cancer patients' T-cells. © 2000 Cancer Research Campaig

Immunologic recognition of influenza virus-infected cells. II. Expression of influenza A matrix protein on the infected cell surface and its role in recognition by cross-reactive cytotoxic T cells

Two distinct subpopulations of cytotoxic T cells are generated in the primary or secondary response of mice to type A influenza viruses. One subpopulation is specific for the immunizing virus strain. The other subpopulation shows a high degree of cross-reactivity for heterologous type A virus of a different subtype. This report examines the possibility that distinct influenza virus antigens, expressed on the surface of the infected cell, are recognized by the different subpopulations of influenza-specific cytotoxic T cells. Data are presented which demonstrate that influenza A matrix protein, an internal virion antigen, is detectable on the surface of target cells infected with influenza A viruses of different subtypes. Since this viral antigen is type specific, i.e., serologically cross-reactive among all type A influenza viruses, it could serve as the target for cross-reactive cytotoxic T cells. To further examine the specificity of the two cytotoxic T-cell subpopulations, experiments were carried out by using the inhibitor of glycoprotein synthesis - 2-Deoxy-D-Glucose 2-DG. These experiments examine first the effect of 2-DG on the expression of influenza matrix protein and viral glycoprotein on the infected cell surface and second, the susceptibility of 2-DG-treated target cells to lysis by cytotoxic T cells. 2-DG inhibits the expression of the viral hemagglutinin glycoprotein on the cell surface but does not inhibit the expression of the nonglycosylated matrix protein. Furthermore, inhibition of glycoprotein synthesis in infected target cells abrogates the reactivity of infected target cells to lysis by virus strain-specific but not cross- reactive cytotoxic T cells. These findings suggest that the influenza glycoproteins (hemagglutinin and/or neuraminidase) and the nonglycosylated matrix protein are the targets for the virus strain- specific and cross-reactive cytotoxic T cells, respectively. These results are discussed in the light of available information on influenza virus structure and the biology of influenza infection and in terms of current models for cytotoxic T-cell recognition of virus-infected cells

T-cell cytotoxicity in the absence of viral protein synthesis in target cells

CYTOTOXIC T cells lyse only those virus infected target cells in vitro which express, in addition to the viral antigen(s), those K or D region products of the major histocompati-bility complex (MHC) which were present during anti-viral sensitisation in vivo. This 'associative recogniton' by cytotoxic T cells could reflect the interaction of two T-cell receptors with specificity for target K or D gene products and independently for the viral antigen, or one receptor with specificity for virally altered K or D region products (see ref. 1 and refs therein). There are various ways that the MHC antigens could be altered, including 'modification from within', where the virus modifies host protein synthesis by interfering with transcription2, translation or post-translational glycosylation; or 'modification from without' where enzymic or chemical alteration of cell membrane proteins are induced by virus activity at the cell surface. In this report we show that inactivated Sendai virus or isolated Sendai virus envelopes can serve to modify a cell and make it a specific target for Sendai-immune T-cell killing, thus excluding the possibility of 'modification from within' in this system

Involvement of fusion activity of ultraviolet light-inactivated sendai virus in formation of target antigens recognized by cytotoxic T cells

Mice inoculated with ultraviolet light-inactivated Sendai virus mount a cell- mediated immune response to the virus. Cytotoxic T cells specific for Sendai virus can be obtained by in vitro secondary stimulation of primed spleen cells with syngeneic stimulator cells coated with UV-inactivated Sendai virus. Neither in vivo nor in vitro stimulation alone is sufficient to generate specific cytotoxic T cells. Sharing of the H-2 haplotype between cytotoxic T cells and target cells is required for the Sendai virus-specific lysis to occur. The fusion (F) glycoprotein of Sendai virus has been implicated in target antigen formation (20). Ethanol treatment of Sendai virus causes complete inactivation of the cell-fusion and hemolytic activities of the envelope, but does not affect the antigenicity of the F glycoprotein; furthermore, hemagglutinin and neuraminidase activities of the envelope HANA glycoprotein are also left intact after ethanol treatment. Target cells can be prepared by coating them with various numbers of UV-inactivated Sendai virus that have been treated with ethanol or, as a control, phosphate-buffered saline (PBS). The amount of virus adsorbed to target cells during the cytotoxicity reaction time using either ethanol-treated or untreated (PBS "treated") virions is essentially identical, but target cells coated with ethanol-treated Sendai virus fail to serve as targets for cytotoxic T cells. These results indicate that fusion activity of the Sendai virus envelope is essential to the formation of the target antigen and that virus adsorption to cell surfaces without fusion of the envelope with cell membranes is not sufficient to allow killing by virus-specific cytotoxic T cells