Meeting report by Luciani Cécilia who was awarded a travel grant by the CFCD
Luciani Cécilia is a PhD student of the Aix Marseille University, working at the CIML in Marseille, France
Turning up the heat: reversing immunoediting by activating innate immunity in the tumor microenvironment – Judy Lieberman:
After an infection or a danger, cytosolic pattern recognition receptors (PRRs) are able to recognize molecular patterns carried by pathogens. These PRRs can form multiprotein complexes called inflammasomes. One of the main roles of the inflammasome is to induce the activation of inflammatory caspases (caspases 1, 4, 5, and 11) in order to generate the maturation, and therefore, the activation of inflammatory cytokines (IL-1 and IL-18). Inflammatory caspases also trigger pyroptosis by inducing the cleavage of gasdermin D (GSMD) which is known to induce pore structures in the membrane (Kuang et al,114 (40) 10642-10647, PNAS, biophysic and computational biology, 2017).
The mechanism that induces pyroptosis after cleavage of gasdermin D by inflammatory caspases has been study by Judy Liebermann. The active form of gasdermin D (GSDMD-NT) can trigger pore in the cell membrane inducing cell death. Using flag-tagged mouse GSDMD-NT, her team observed that GSDMD-NT oligomerizes in membranes producing pores visible by electron microscopy. Moreover, the oligomerization and the pyroptosis can be blocked after the induction of mutations on some residues of GSDMD-NT. Her team also showed that GSDMD-NT can bind to compounds that are present in cell membrane inner leaflet (phosphatidylserine, phosphatidylinositol) or in the outer and inner leaflets of bacterial membranes (cardiolipin). Regarding the binding of GSDMD-NT to cardiolipin, they demonstrated the role of GSDMD-NT in bactericidal effect, as in the absence of GSDMD-NT, there is an inhibition of bacterial killing.
Her team has also been interested in other gasdermin family members like gasdermine E (GSDME). GSDME is activated by caspase-3 and its expression in cells converts apoptosis to pyroptosis. Indeed, overexpression of GSDME in B16 cells treated with a rapid activator of capase-3 (raptinal) triggers pyroptosis compared to control B16 cells that become apoptotic. Several studies showed that GSDME expression is suppressed in many cancers and suggested a tumor suppressor role for GSDME. Liebermann’s team was interested in the impact of GSDME expression on tumor growth and tumor-infiltrating immune cells. They observed a more rapid tumor growth in Gsdme-deficient tumors (knock out) compared to tumors expressing endogenous Gsdme. Moreover, the tumor microenvironment (TME) in tumors deleted for Gsdme (Gsdme-/- EMT6) had fewer CD8+ T cells, aNK cells and tumor-associated macrophages (TAM). These tumors also expressed less granzyme B and perforin and produced less IFNand TNF after PMA and ionomycin stimulation. They also observed that in absence of CD8+ T cells and NK cells there was a significant reduction of GSDME-mediated tumor suppression. As they noticed that NK cells can induced pyroptosis in GSDME-expressing cells they hypothesised that granzyme protease might cleave GSDME. Indeed, they showed that granzyme B can directly and indirectly (by activating caspase-3) cleave GSDME.
Bibliography:
- Kuang et al,114 (40) 10642-10647, PNAS, biophysic and computational biology, 2017).
- Liu et al, 535(7610):153-158, Nature, 2016, doi : 10.1038/nature18629
- Zhang et al, 579(7799) :415-420, Nature, 2020. Doi : 10.1038/s41586-020-2071-9
Bcl6/Blimp-1 axis in DCs determines CD4+ T cell fate decisions– Dirk Baumjohann:
To prevent harmful actions of antigens, innate and adaptive immunity responses are put in place to defend the organism. Innate immunity is mainly based on phagocytes that excel in the presentation of antigens to the actors of the adaptative immunity that are T and B cells. After priming, T cells differentiate into several effectors including T follicular helper (Tfh) cells thanks to the expression of the transcription factor Bcl6. Tfh cells provide help to promote the formation of germinal centers. The germinal center is essential to produce high-affinity antibodies against pathogens. However, excessive number of Tfh cells can promote auto-immune diseases. In order to avoid overproduction of Tfh, an antagonist of Bcl6, Blimp-1, is expressed. Indeed, expression of Blimp-1 prevents the expression of Bcl6 and the generation of Tfh. Expression of Blimp-1 is linked to the proliferation and differentiation of non-Tfh CD4+ cells. Recently, it has been described that Bcl6 is expressed in cDC1 and to a lower extent in cDC2 but the role and function of this expression remain unknown. Therefore, Baumjohann’s team dissected how the Bcl6/Blimp-1 axis in DCs impacts their ability to induce Tfh cells and to drive T helper cell fate decisions.
Baumjohann et al have previously shown that Bcl6 deficiency mainly induces the loss of CD11b- DC (cDC1). In order to fully understand the impact of the Bcl6 deficiency on cDC1, they used transgenic mice models in which Bcl6 is depleted only in cDC1 (XCR1.Bcl6ko) or in CD11c+ cells (CD11c.Bcl6ko). In the spleen, they observed a decrease of XCR1+ cDC1 in both Bcl6-deficient mice models. Moreover XCR1- CD11b+ cDC2 were also diminished in CD11c.Bcl6ko.
To better characterize the impact of Bcl6 deficiency in cDC1, they compared the genes expressed in XCR1.Bcl6ko and wt mice. They observed a deregulation of genes involved in cytokine signaling and cell adhesion in cDC1. Despite these alterations, cDC1 maintain their ability to cross-present antigens in Bcl6-deficient mice. Indeed, the proliferation of OT-I that strictly depends on cDC1 cross presentation of ovalbumin was identical in XCR1.Bcl6ko or wt mice injected with heat-killed ovalbumin-expressing mouse embryonic fibroblasts.
In addition, they noticed that Bcl6 depletion has also an impact on spleen cDC2. In the spleen, the majority of cDC2 expresses endothelial cell specific adhesion marker (ESAM). Their development is supported by Notch2-signaling pathway. Phenotyping of cDC2 in the spleen of CD11c.Bcl6ko combined with confocal microscopy showed the loss of ESAM+ cDC2. As these cells have an essential role in the activation of CD4+ T cells and in the formation of germinal centers, they transferred wt and CD11c.Bcl6ko mice with OT-II T cells and immunized the mice intraperitoneally with ovalbumin + poly I:C and looked at the generation of antigen-specific helper T cells three days later. They observed that even if OT-II cells expanded normally after immunization in CD11c.Bcl6ko there was a diminution in the generation of early antigen-specific Tfh cells.
In conclusion, Baumjohann’s team showed that in the spleen deletion of Bcl6 induces an alteration of genes expressed by cDC1 that does not impact their ability to cross-present antigens. Morever, Bcl6 is an essential transcription factor for ESAM+ cDC2 in the spleen. Indeed, the absence of Bcl6 induces a loss of ESAM+cDC2 and therefore a diminution of early antigen-specific Tfh cells in response to immunization.
Bibliography:
- Bcl6 Promotes Follicular Helper T Cell Differentiation and PD-1 Expression in a Blimp1-independent Manner; Markus M. Xie et al; Eur J Immunol; 2018; doi: 10.1002/eji.201747034
- Dependence on Bcl6 and Blimp1 drive distinct differentiation of murine memory and follicular helper CD4+ T cells; Thomas Ciucci et al; J Exp Med. 2022; doi: 10.1084/jem.20202343
- Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation; Crotty et al; Nat.Immunol; 2010
- Transcriptional and functional profiling of human intestinal dendritic cells reveals conserved specialization and a role for Bcl-6 and Blimp-1 in terminal subset differentiation; Watchmarker et al, Nat.Immunol; 2014; doi: 10.1038/ni.2768
- Loss of Bcl6 transcriptionally alters classical dendritic cells and diminish T follicular helper cell-inducing cDC2 in mouse spleen; Xiao et al Manuscript BioRxiv; 2022; doi: https://doi.org/10.1101/2022.08.10.503322
Luciani Cécilia is a PhD student of the Aix Marseille University, working at the CIML in Marseille, France
Turning up the heat: reversing immunoediting by activating innate immunity in the tumor microenvironment – Judy Lieberman:
After an infection or a danger, cytosolic pattern recognition receptors (PRRs) are able to recognize molecular patterns carried by pathogens. These PRRs can form multiprotein complexes called inflammasomes. One of the main roles of the inflammasome is to induce the activation of inflammatory caspases (caspases 1, 4, 5, and 11) in order to generate the maturation, and therefore, the activation of inflammatory cytokines (IL-1 and IL-18). Inflammatory caspases also trigger pyroptosis by inducing the cleavage of gasdermin D (GSMD) which is known to induce pore structures in the membrane (Kuang et al,114 (40) 10642-10647, PNAS, biophysic and computational biology, 2017).
The mechanism that induces pyroptosis after cleavage of gasdermin D by inflammatory caspases has been study by Judy Liebermann. The active form of gasdermin D (GSDMD-NT) can trigger pore in the cell membrane inducing cell death. Using flag-tagged mouse GSDMD-NT, her team observed that GSDMD-NT oligomerizes in membranes producing pores visible by electron microscopy. Moreover, the oligomerization and the pyroptosis can be blocked after the induction of mutations on some residues of GSDMD-NT. Her team also showed that GSDMD-NT can bind to compounds that are present in cell membrane inner leaflet (phosphatidylserine, phosphatidylinositol) or in the outer and inner leaflets of bacterial membranes (cardiolipin). Regarding the binding of GSDMD-NT to cardiolipin, they demonstrated the role of GSDMD-NT in bactericidal effect, as in the absence of GSDMD-NT, there is an inhibition of bacterial killing.
Her team has also been interested in other gasdermin family members like gasdermine E (GSDME). GSDME is activated by caspase-3 and its expression in cells converts apoptosis to pyroptosis. Indeed, overexpression of GSDME in B16 cells treated with a rapid activator of capase-3 (raptinal) triggers pyroptosis compared to control B16 cells that become apoptotic. Several studies showed that GSDME expression is suppressed in many cancers and suggested a tumor suppressor role for GSDME. Liebermann’s team was interested in the impact of GSDME expression on tumor growth and tumor-infiltrating immune cells. They observed a more rapid tumor growth in Gsdme-deficient tumors (knock out) compared to tumors expressing endogenous Gsdme. Moreover, the tumor microenvironment (TME) in tumors deleted for Gsdme (Gsdme-/- EMT6) had fewer CD8+ T cells, aNK cells and tumor-associated macrophages (TAM). These tumors also expressed less granzyme B and perforin and produced less IFNand TNF after PMA and ionomycin stimulation. They also observed that in absence of CD8+ T cells and NK cells there was a significant reduction of GSDME-mediated tumor suppression. As they noticed that NK cells can induced pyroptosis in GSDME-expressing cells they hypothesised that granzyme protease might cleave GSDME. Indeed, they showed that granzyme B can directly and indirectly (by activating caspase-3) cleave GSDME.
Bibliography:
- Kuang et al,114 (40) 10642-10647, PNAS, biophysic and computational biology, 2017).
- Liu et al, 535(7610):153-158, Nature, 2016, doi : 10.1038/nature18629
- Zhang et al, 579(7799) :415-420, Nature, 2020. Doi : 10.1038/s41586-020-2071-9
Bcl6/Blimp-1 axis in DCs determines CD4+ T cell fate decisions– Dirk Baumjohann:
To prevent harmful actions of antigens, innate and adaptive immunity responses are put in place to defend the organism. Innate immunity is mainly based on phagocytes that excel in the presentation of antigens to the actors of the adaptative immunity that are T and B cells. After priming, T cells differentiate into several effectors including T follicular helper (Tfh) cells thanks to the expression of the transcription factor Bcl6. Tfh cells provide help to promote the formation of germinal centers. The germinal center is essential to produce high-affinity antibodies against pathogens. However, excessive number of Tfh cells can promote auto-immune diseases. In order to avoid overproduction of Tfh, an antagonist of Bcl6, Blimp-1, is expressed. Indeed, expression of Blimp-1 prevents the expression of Bcl6 and the generation of Tfh. Expression of Blimp-1 is linked to the proliferation and differentiation of non-Tfh CD4+ cells. Recently, it has been described that Bcl6 is expressed in cDC1 and to a lower extent in cDC2 but the role and function of this expression remain unknown. Therefore, Baumjohann’s team dissected how the Bcl6/Blimp-1 axis in DCs impacts their ability to induce Tfh cells and to drive T helper cell fate decisions.
Baumjohann et al have previously shown that Bcl6 deficiency mainly induces the loss of CD11b- DC (cDC1). In order to fully understand the impact of the Bcl6 deficiency on cDC1, they used transgenic mice models in which Bcl6 is depleted only in cDC1 (XCR1.Bcl6ko) or in CD11c+ cells (CD11c.Bcl6ko). In the spleen, they observed a decrease of XCR1+ cDC1 in both Bcl6-deficient mice models. Moreover XCR1- CD11b+ cDC2 were also diminished in CD11c.Bcl6ko.
To better characterize the impact of Bcl6 deficiency in cDC1, they compared the genes expressed in XCR1.Bcl6ko and wt mice. They observed a deregulation of genes involved in cytokine signaling and cell adhesion in cDC1. Despite these alterations, cDC1 maintain their ability to cross-present antigens in Bcl6-deficient mice. Indeed, the proliferation of OT-I that strictly depends on cDC1 cross presentation of ovalbumin was identical in XCR1.Bcl6ko or wt mice injected with heat-killed ovalbumin-expressing mouse embryonic fibroblasts.
In addition, they noticed that Bcl6 depletion has also an impact on spleen cDC2. In the spleen, the majority of cDC2 expresses endothelial cell specific adhesion marker (ESAM). Their development is supported by Notch2-signaling pathway. Phenotyping of cDC2 in the spleen of CD11c.Bcl6ko combined with confocal microscopy showed the loss of ESAM+ cDC2. As these cells have an essential role in the activation of CD4+ T cells and in the formation of germinal centers, they transferred wt and CD11c.Bcl6ko mice with OT-II T cells and immunized the mice intraperitoneally with ovalbumin + poly I:C and looked at the generation of antigen-specific helper T cells three days later. They observed that even if OT-II cells expanded normally after immunization in CD11c.Bcl6ko there was a diminution in the generation of early antigen-specific Tfh cells.
In conclusion, Baumjohann’s team showed that in the spleen deletion of Bcl6 induces an alteration of genes expressed by cDC1 that does not impact their ability to cross-present antigens. Morever, Bcl6 is an essential transcription factor for ESAM+ cDC2 in the spleen. Indeed, the absence of Bcl6 induces a loss of ESAM+cDC2 and therefore a diminution of early antigen-specific Tfh cells in response to immunization.
Bibliography:
- Bcl6 Promotes Follicular Helper T Cell Differentiation and PD-1 Expression in a Blimp1-independent Manner; Markus M. Xie et al; Eur J Immunol; 2018; doi: 10.1002/eji.201747034
- Dependence on Bcl6 and Blimp1 drive distinct differentiation of murine memory and follicular helper CD4+ T cells; Thomas Ciucci et al; J Exp Med. 2022; doi: 10.1084/jem.20202343
- Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation; Crotty et al; Nat.Immunol; 2010
- Transcriptional and functional profiling of human intestinal dendritic cells reveals conserved specialization and a role for Bcl-6 and Blimp-1 in terminal subset differentiation; Watchmarker et al, Nat.Immunol; 2014; doi: 10.1038/ni.2768
- Loss of Bcl6 transcriptionally alters classical dendritic cells and diminish T follicular helper cell-inducing cDC2 in mouse spleen; Xiao et al Manuscript BioRxiv; 2022; doi: https://doi.org/10.1101/2022.08.10.503322