Role of caspase-8 and/or RIPK3 expression in target cells during In vivo antigen-specific by CD8+ T lymphocytes

• Grant number: 16/24912-7
• Support Opportunities: Regular Research Grants
• Start date: April 01, 2017
• End date: March 31, 2019
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: João Gustavo Pessini Amarante Mendes
• Grantee: João Gustavo Pessini Amarante Mendes
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Ricardo Weinlich

Abstract

The direct elimination of target cells by cytotoxic T lymphocytes (CTL) plays an important role in protective immunity against pathogens and tumor cells, and can be triggered by the combined action of perforin and granzymes, or by FasL-Fas interaction. The FasL-Fas interaction promotes apoptosis through activation of caspase-8 and subsequent cleavage of effector caspases-3, -6 and -7, or Bid, a pro-apoptotic member of the Bcl-2 family. Recently, it was shown that caspase-8 is an inhibitor of Necroptosis, another form of programmed cell death, coordinated by RIPK1, RIPK3 and MLKL. Interestingly, nothing is know about the role of necroptosis in in vivo target elimination by CTL. In addition, a correlation between CASP8 gene mutations in tumor cells from patients with high cytolytic activity has been established, suggesting that caspase-8 deficiency may represent a mechanism by which tumor cells evade the apoptotic signal triggered by CTLs. Interestingly, the role of the central components of necroptosis, another form of programmed cell death, in resistance/susceptibility to CTL attack remains obscure. Therefore, the objective of this project is to investigate the relative contribution of Casp8 or RIPK3 expression in target cells, to their in vivo elimination by antigen-specific T CD8 lymphocytes. To this end, our group has been using recombinant human adenovirus type 5 (hAd5) to study in vivo CTL responses. Immunized or non-immunized mice receive syngeneic splenocytes labeled with different concentrations of CFSE, or a combination of CFSE and Cell Trace, and pulsed (target) or not-pulsed (control) with cognate peptide, mixed at 1:1 ratio. Specific elimination of target cells is assessed by flow cytometry of splenocytes after 18-24h. A deviation of the 1:1 ratio correlates with specific target elimination. To investigate on the role of caspase-8 and/or RIPK3 expression, splenocytes from wild type, Ripk3-/- and Casp-8-/-Ripk3-/- C57BL/6 mice will be used as targets in the system. Furthermore, C57BL/6 mice deficient in perforin (Prf-/-) and FasL (gld) will be used to evaluate the relative contribution of these two apoptotic pathways. In vitro experiments will be performed in addition to the in vivo studies. Therefore, this project will contribute to understanding the role of caspase-8 and RIPK3 (necroptose) in the resistance of tumor cells to eliminate in vivo by cytotoxic T lymphocytes. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

HERNANDEZ-TORRES, MARCELA; DO NASCIMENTO, SILVA ROGERIO; REBOUCAS, MONICA CARDOZO; CASSADO, ALEXANDRA; MATTEUCCI, KELY CATARINE; D'IMPERIO-LIMA, MARIA REGINA; VASCONCELOS, JOSE RONNIE C.; BORTOLUCI, KARINA R.; ALVAREZ, JOSE MARIA; AMARANTE-MENDES, GUSTAVO P.. Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection (vol 12, 817, 2021). CELL DEATH & DISEASE, v. 12, n. 9, p. 1-pg., 2021-08-30. (15/20432-8, 15/09568-5, 18/15607-1, 17/25942-0, 18/25984-7, 16/24912-7, 12/24737-0)