Cheating the Hunger Games; Mechanisms Controlling Clonal Diversity of CD8 Effector and Memory Populations

Effector and memory CD8 T cells have an intrinsic difference in the way they must approach antigen; effector cells need to address the pathogen at hand and therefore favor outgrowth of only high-affinity clones. In contrast, the memory pool benefits from greater clonal diversity to recognize and eliminate pathogens with mutations in their immunogenic epitopes. Effector and memory fates are ultimately the result of the same three signals that control T cell activation; T cell receptor (TCR) engagement together with co-stimulation and cytokines. Great progress has been made in our understanding of the transcriptional programs that drive effector or memory differentiation. However, how these two different programs result from the same initial cues is still a matter of debate. An emerging image is that not only the classical three signals determine T cell differentiation, but also the ability of cells to access these signals relative to that of other activated clones. Inter-clonal competition is therefore not only a selective force, but also a mediator of CD8 T cell fate. How this is regulated on a transcriptional level, especially in the context of a selective "hunger game" based on antigen-affinity in which only cells of high-affinity are supposed to survive, is still poorly defined. In this review, we discuss recent literature that illustrates how antigen-affinity dependent inter-clonal competition shapes effector and memory populations in an environment of antigen affinity-driven selection. We argue that fine-tuning of TCR signal intensity presents an attractive target for regulating the scope of CD8 T cell vaccines

Cheating the Hunger Games; Mechanisms Controlling Clonal Diversity of CD8 Effector and Memory Populations

Effector and memory CD8 T cells have an intrinsic difference in the way they must approach antigen; effector cells need to address the pathogen at hand and therefore favor outgrowth of only high-affinity clones. In contrast, the memory pool benefits from greater clonal diversity to recognize and eliminate pathogens with mutations in their immunogenic epitopes. Effector and memory fates are ultimately the result of the same three signals that control T cell activation; T cell receptor (TCR) engagement together with co-stimulation and cytokines. Great progress has been made in our understanding of the transcriptional programs that drive effector or memory differentiation. However, how these two different programs result from the same initial cues is still a matter of debate. An emerging image is that not only the classical three signals determine T cell differentiation, but also the ability of cells to access these signals relative to that of other activated clones. Inter-clonal competition is therefore not only a selective force, but also a mediator of CD8 T cell fate. How this is regulated on a transcriptional level, especially in the context of a selective “hunger game” based on antigen-affinity in which only cells of high-affinity are supposed to survive, is still poorly defined. In this review, we discuss recent literature that illustrates how antigen-affinity dependent inter-clonal competition shapes effector and memory populations in an environment of antigen affinity-driven selection. We argue that fine-tuning of TCR signal intensity presents an attractive target for regulating the scope of CD8 T cell vaccines

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling via deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45+Ly6G-CD3-B220-NK1.1- CD11b-/loCD115+). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced in vitro by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL ; macrophages by M-CSF ; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose E. coli particles, and osteoclasts to resorb bone and express tartrate- resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and Ctsk gene expression were upregulated in osteoclastogenic cultures from RBP-J+ mice, with the opposing results in NICD1+ mice. Notch modulation did not affect the number of in vitro differentiated macrophages and DCs. However, RBP-J deletion stimulated Il12b and Cd86 expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1+ and RBP-J+ strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases

NK cell receptor NKG2D sets activation threshold for the NCR1 receptor early in NK cell development

The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-γ. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3ζ. Reduced expression of CD3ζ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1, and CD3ζ deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell reactivity to cellular threats

ULOGA JAKOSTI AKTIVIRAJUĆEG SIGNALA NA T STANIČNI RECEPTOR U KONTROLI KLONALNE RAZNOLIKOSTI IMUNOLOŠKE MEMORIJE POSREDOVANE CD8 LIMFOCITIMA T

Objectives: CD8 T cell-mediated immunity plays a critical role in the protection from intracellular pathogens and tumors. To recognize the large number of potential threats, the naïve CD8 T-cell pool consists of millions of clones, each one unique based on its T-cell receptor (TCR). Upon infection, only a few of these clones are recruited to generate antigen-specific memory. Selection of memory clones is a trade-off between specificity and diversity; too much specificity restricts antigen-recognition, which precludes responsiveness against pathogens with small mutations. Too much diversity impairs efficiency of recall responses. Mechanisms controlling memory diversity are largely unknown. The proposed research aims to identify key molecular mechanisms that regulate the clonal diversity of the memory CD8 T-cell pool. Materials and Methods: To investigate how signal strength impacts memory differentiation, we set up an in vitro model in which we stimulated OT-1 T cells with SIINFEKL (N4 peptide) or altered peptide ligands (APLs). Using high-throughput transcriptome analysis, we identified potential master regulators of low-affinity memory formation (Eomes and Bcl-2). To confim these findings in vivo we used mCMV, LCMV or L. monocytogenes expressing N4 or APLs. To demonstrate a specific role for Eomes, we adoptively transferred cells from conditional knock-out mice (OT-1 Eomesfl/flCD4Cre; EomesCKO). Moreover, the inducible MXCre system was used to determine the timeframe in which Eomes mediates this pro-survival effect. To confirm this data in polyclonal system we generated mixed bone marrow chimeras (MBMCs) containing WT and EomesCKO cells. The chemical compound ABT-199 was used to specifically inhibit Bcl-2. To asses if Eomes binds to Bcl2 promoter region we performed Eomes ChIP-seq on activated OT-1 cells. To confirm that Eomes deficience results with reduced clonal diversity of memory CD8 T-cell pool we performed TCR sequencing of antigen-specific WT and EomesCKO memory CD8 T cells from MBMCs. Results: We find that memory precursors stimulated within a window of cumulative signal intensity maximally induce the transcription factor Eomes. Eomes directly drives expression of the pro-survival protein Bcl-2, providing a survival advantage for cells of submaximal affinity. Beyond this window, T-bet expression dominates, which inhibits Eomes-induced transcription of Bcl-2. The increased proliferative potential of clones activated with a TCR ligand of high-affinity causes them to dominate the memory pool, whereas the survival advantage of cells of submaximal affinity ensures their persistence into memory, despite an environment of antigen affinity-based selection. Conclusion: We demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection. These findings may be beneficial for the development of T cell-based vaccines with an increased scopeCilj istraživanja: Imunost posredovana CD8 limfocitima T igra ključnu ulogu u zaštiti od unutarstaničnih patogena i tumora. Da bi prepoznali veliki broj potencijalnih prijetnji, naivni CD8 limfociti T obuhvaćaju milijune različitih klonova, od kojih je svaki jedinstven na temelju T staničnog receptora. Nakon infekcije, samo nekolicina tih klonova ima sposobnost stvaranja dugoročne antigen-specifične imunološke memorije. Selekcija memorijskih klonova je bazirana na balansiranju specifičnosti i raznolikosti; prevelika specifičnost ograničava prepoznavanje antigena s mutacijama u imunodominantnim epitopima. Previše raznolikosti smanjuje učinkovitost specifičnog prepoznavanja. Mehanizmi koji kontroliraju raznolikost memorije još uvijek su nepoznati. Predloženo istraživanje ima za cilj identificirati ključne molekularne mehanizme koji reguliraju klonalnu raznolikost imunološke memorije posredovane CD8 limfocitima T. Materijali i metode: Kako bi razjasnili utjecaj jakosti aktivirajućeg signala na T- stanični receptor u kontekstu formiranja imunološke memorije uspostavili smo in vitro model - OT-1 CD8 limfociti T su stimulirani sa SIINFEKL (N4) peptidom ili izmijenjenim peptidnim ligandima (eng. altered peptide ligands - APL) s nižim afinitetoma za T stanični receptor. Koristeći visokoprotočnu analizu transkriptoma identificirali smo potencijalne ključne faktore za razvoj memorijskih stanica niskog afiniteta (Eomes i Bcl-2). Kako bi dobivene rezultate potvrdili in vivo koristili smo modele infekcija s mCMV, LCMV ili L. monocytogenes koji ispoljavaju N4 peptid ili APL. S ciljem dokazivanja ključne uloge transkripcijskog faktora Eomes u dugoročnom preživljavanju memorijskih stanica niskog afiniteta proveli smo pokuse adoptivnog transfera Eomes-deficijentnih stanica (OT-1 Eomesfl/flCD4Cre; EomesCKO). Osim toga, inducibilni MXCre sustav korišten je za određivanje vremenskog okvira u kojem Eomes posreduje navedeni učinak. Kako bi smo potvrdili dobivene rezultate u poliklonalnom sustavu stvorili smo mješovite kimerične životinje koristeći stanice koštane srži miševa divljeg tipa (WT) i EomesCKO miševa, dok je spoj ABT-199 korišten za specifično inhibiranje molekule Bcl-2. U svrhu ispitivanja vezanja faktora Eomes na promotorsku regiju Bcl2 gena, proveli smo Eomes ChIP-seq analizu na aktiviranim OT-1 stanicama. S ciljem određivanja molekularnog mehanizma koji je odgovoran za prednost u preživljavanju memorijskih stanica niskog afiniteta, koristili smo transgenične NIH3T3 i HEK293 stanične linije koje ispoljavaju Eomes i/ili T-bet. Navedeno ispitivanje smo proveli pomoću luciferaznog reporter eseja upotrebom specifičnih plazmida. Da bismo potvrdili da nedostatak transkripcijskog faktora Eomes rezultira smanjenom klonalnom raznolikošću proveli smo TCR sekvencioniranje antigen-specifičnih WT i EomesCKO CD8 limfocita T izoliranih iz mješovitih kimeričnih životinja 30 dana nakon infekcije LCMV-om. Rezultati: Stimulacija CD8 limfocita T submaksimalnim intenzitetom kumulativnog aktivirajućeg signala dovodi do najviše razine ispoljavanja transkripcijskog faktora Eomes. Eomes direktno na transkripcijskog razini potiče ispoljavanje Bcl-2 proteina što rezultira dugoročnim preživljavanjem memorijskih stanica submaksimalnog afiniteta. Ukoliko su stanice stimulirane jačim intenzitetom dolazi do dominacije transkripcijskog faktora T-bet koji inhibira Eomes-induciranu transkripciju Bcl-2. Povećani proliferativni potencijal klonova visokog afiniteta je odgovoran za njihovu dominaciju, dok prednost u preživljavanju stanica submaksimalnog afiniteta omogućuje njihovu perzistenciju u memorijskoj fazi imunološkog odgovora. Zaključak: Ovaj rad otkriva molekularni mehanizam koji je temelj za uspostavljanje potrebne raznolikost klonova memorijskih CD8 limfocita T. Dobiveni rezultati mogu se koristiti u svrhu unaprjeđenja imunoterapije posredovane limfocitima T

ULOGA JAKOSTI AKTIVIRAJUĆEG SIGNALA NA T STANIČNI RECEPTOR U KONTROLI KLONALNE RAZNOLIKOSTI IMUNOLOŠKE MEMORIJE POSREDOVANE CD8 LIMFOCITIMA T

Objectives: CD8 T cell-mediated immunity plays a critical role in the protection from intracellular pathogens and tumors. To recognize the large number of potential threats, the naïve CD8 T-cell pool consists of millions of clones, each one unique based on its T-cell receptor (TCR). Upon infection, only a few of these clones are recruited to generate antigen-specific memory. Selection of memory clones is a trade-off between specificity and diversity; too much specificity restricts antigen-recognition, which precludes responsiveness against pathogens with small mutations. Too much diversity impairs efficiency of recall responses. Mechanisms controlling memory diversity are largely unknown. The proposed research aims to identify key molecular mechanisms that regulate the clonal diversity of the memory CD8 T-cell pool. Materials and Methods: To investigate how signal strength impacts memory differentiation, we set up an in vitro model in which we stimulated OT-1 T cells with SIINFEKL (N4 peptide) or altered peptide ligands (APLs). Using high-throughput transcriptome analysis, we identified potential master regulators of low-affinity memory formation (Eomes and Bcl-2). To confim these findings in vivo we used mCMV, LCMV or L. monocytogenes expressing N4 or APLs. To demonstrate a specific role for Eomes, we adoptively transferred cells from conditional knock-out mice (OT-1 Eomesfl/flCD4Cre; EomesCKO). Moreover, the inducible MXCre system was used to determine the timeframe in which Eomes mediates this pro-survival effect. To confirm this data in polyclonal system we generated mixed bone marrow chimeras (MBMCs) containing WT and EomesCKO cells. The chemical compound ABT-199 was used to specifically inhibit Bcl-2. To asses if Eomes binds to Bcl2 promoter region we performed Eomes ChIP-seq on activated OT-1 cells. To confirm that Eomes deficience results with reduced clonal diversity of memory CD8 T-cell pool we performed TCR sequencing of antigen-specific WT and EomesCKO memory CD8 T cells from MBMCs. Results: We find that memory precursors stimulated within a window of cumulative signal intensity maximally induce the transcription factor Eomes. Eomes directly drives expression of the pro-survival protein Bcl-2, providing a survival advantage for cells of submaximal affinity. Beyond this window, T-bet expression dominates, which inhibits Eomes-induced transcription of Bcl-2. The increased proliferative potential of clones activated with a TCR ligand of high-affinity causes them to dominate the memory pool, whereas the survival advantage of cells of submaximal affinity ensures their persistence into memory, despite an environment of antigen affinity-based selection. Conclusion: We demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection. These findings may be beneficial for the development of T cell-based vaccines with an increased scopeCilj istraživanja: Imunost posredovana CD8 limfocitima T igra ključnu ulogu u zaštiti od unutarstaničnih patogena i tumora. Da bi prepoznali veliki broj potencijalnih prijetnji, naivni CD8 limfociti T obuhvaćaju milijune različitih klonova, od kojih je svaki jedinstven na temelju T staničnog receptora. Nakon infekcije, samo nekolicina tih klonova ima sposobnost stvaranja dugoročne antigen-specifične imunološke memorije. Selekcija memorijskih klonova je bazirana na balansiranju specifičnosti i raznolikosti; prevelika specifičnost ograničava prepoznavanje antigena s mutacijama u imunodominantnim epitopima. Previše raznolikosti smanjuje učinkovitost specifičnog prepoznavanja. Mehanizmi koji kontroliraju raznolikost memorije još uvijek su nepoznati. Predloženo istraživanje ima za cilj identificirati ključne molekularne mehanizme koji reguliraju klonalnu raznolikost imunološke memorije posredovane CD8 limfocitima T. Materijali i metode: Kako bi razjasnili utjecaj jakosti aktivirajućeg signala na T- stanični receptor u kontekstu formiranja imunološke memorije uspostavili smo in vitro model - OT-1 CD8 limfociti T su stimulirani sa SIINFEKL (N4) peptidom ili izmijenjenim peptidnim ligandima (eng. altered peptide ligands - APL) s nižim afinitetoma za T stanični receptor. Koristeći visokoprotočnu analizu transkriptoma identificirali smo potencijalne ključne faktore za razvoj memorijskih stanica niskog afiniteta (Eomes i Bcl-2). Kako bi dobivene rezultate potvrdili in vivo koristili smo modele infekcija s mCMV, LCMV ili L. monocytogenes koji ispoljavaju N4 peptid ili APL. S ciljem dokazivanja ključne uloge transkripcijskog faktora Eomes u dugoročnom preživljavanju memorijskih stanica niskog afiniteta proveli smo pokuse adoptivnog transfera Eomes-deficijentnih stanica (OT-1 Eomesfl/flCD4Cre; EomesCKO). Osim toga, inducibilni MXCre sustav korišten je za određivanje vremenskog okvira u kojem Eomes posreduje navedeni učinak. Kako bi smo potvrdili dobivene rezultate u poliklonalnom sustavu stvorili smo mješovite kimerične životinje koristeći stanice koštane srži miševa divljeg tipa (WT) i EomesCKO miševa, dok je spoj ABT-199 korišten za specifično inhibiranje molekule Bcl-2. U svrhu ispitivanja vezanja faktora Eomes na promotorsku regiju Bcl2 gena, proveli smo Eomes ChIP-seq analizu na aktiviranim OT-1 stanicama. S ciljem određivanja molekularnog mehanizma koji je odgovoran za prednost u preživljavanju memorijskih stanica niskog afiniteta, koristili smo transgenične NIH3T3 i HEK293 stanične linije koje ispoljavaju Eomes i/ili T-bet. Navedeno ispitivanje smo proveli pomoću luciferaznog reporter eseja upotrebom specifičnih plazmida. Da bismo potvrdili da nedostatak transkripcijskog faktora Eomes rezultira smanjenom klonalnom raznolikošću proveli smo TCR sekvencioniranje antigen-specifičnih WT i EomesCKO CD8 limfocita T izoliranih iz mješovitih kimeričnih životinja 30 dana nakon infekcije LCMV-om. Rezultati: Stimulacija CD8 limfocita T submaksimalnim intenzitetom kumulativnog aktivirajućeg signala dovodi do najviše razine ispoljavanja transkripcijskog faktora Eomes. Eomes direktno na transkripcijskog razini potiče ispoljavanje Bcl-2 proteina što rezultira dugoročnim preživljavanjem memorijskih stanica submaksimalnog afiniteta. Ukoliko su stanice stimulirane jačim intenzitetom dolazi do dominacije transkripcijskog faktora T-bet koji inhibira Eomes-induciranu transkripciju Bcl-2. Povećani proliferativni potencijal klonova visokog afiniteta je odgovoran za njihovu dominaciju, dok prednost u preživljavanju stanica submaksimalnog afiniteta omogućuje njihovu perzistenciju u memorijskoj fazi imunološkog odgovora. Zaključak: Ovaj rad otkriva molekularni mehanizam koji je temelj za uspostavljanje potrebne raznolikost klonova memorijskih CD8 limfocita T. Dobiveni rezultati mogu se koristiti u svrhu unaprjeđenja imunoterapije posredovane limfocitima T

ULOGA JAKOSTI AKTIVIRAJUĆEG SIGNALA NA T STANIČNI RECEPTOR U KONTROLI KLONALNE RAZNOLIKOSTI IMUNOLOŠKE MEMORIJE POSREDOVANE CD8 LIMFOCITIMA T

Objectives: CD8 T cell-mediated immunity plays a critical role in the protection from intracellular pathogens and tumors. To recognize the large number of potential threats, the naïve CD8 T-cell pool consists of millions of clones, each one unique based on its T-cell receptor (TCR). Upon infection, only a few of these clones are recruited to generate antigen-specific memory. Selection of memory clones is a trade-off between specificity and diversity; too much specificity restricts antigen-recognition, which precludes responsiveness against pathogens with small mutations. Too much diversity impairs efficiency of recall responses. Mechanisms controlling memory diversity are largely unknown. The proposed research aims to identify key molecular mechanisms that regulate the clonal diversity of the memory CD8 T-cell pool. Materials and Methods: To investigate how signal strength impacts memory differentiation, we set up an in vitro model in which we stimulated OT-1 T cells with SIINFEKL (N4 peptide) or altered peptide ligands (APLs). Using high-throughput transcriptome analysis, we identified potential master regulators of low-affinity memory formation (Eomes and Bcl-2). To confim these findings in vivo we used mCMV, LCMV or L. monocytogenes expressing N4 or APLs. To demonstrate a specific role for Eomes, we adoptively transferred cells from conditional knock-out mice (OT-1 Eomesfl/flCD4Cre; EomesCKO). Moreover, the inducible MXCre system was used to determine the timeframe in which Eomes mediates this pro-survival effect. To confirm this data in polyclonal system we generated mixed bone marrow chimeras (MBMCs) containing WT and EomesCKO cells. The chemical compound ABT-199 was used to specifically inhibit Bcl-2. To asses if Eomes binds to Bcl2 promoter region we performed Eomes ChIP-seq on activated OT-1 cells. To confirm that Eomes deficience results with reduced clonal diversity of memory CD8 T-cell pool we performed TCR sequencing of antigen-specific WT and EomesCKO memory CD8 T cells from MBMCs. Results: We find that memory precursors stimulated within a window of cumulative signal intensity maximally induce the transcription factor Eomes. Eomes directly drives expression of the pro-survival protein Bcl-2, providing a survival advantage for cells of submaximal affinity. Beyond this window, T-bet expression dominates, which inhibits Eomes-induced transcription of Bcl-2. The increased proliferative potential of clones activated with a TCR ligand of high-affinity causes them to dominate the memory pool, whereas the survival advantage of cells of submaximal affinity ensures their persistence into memory, despite an environment of antigen affinity-based selection. Conclusion: We demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection. These findings may be beneficial for the development of T cell-based vaccines with an increased scopeCilj istraživanja: Imunost posredovana CD8 limfocitima T igra ključnu ulogu u zaštiti od unutarstaničnih patogena i tumora. Da bi prepoznali veliki broj potencijalnih prijetnji, naivni CD8 limfociti T obuhvaćaju milijune različitih klonova, od kojih je svaki jedinstven na temelju T staničnog receptora. Nakon infekcije, samo nekolicina tih klonova ima sposobnost stvaranja dugoročne antigen-specifične imunološke memorije. Selekcija memorijskih klonova je bazirana na balansiranju specifičnosti i raznolikosti; prevelika specifičnost ograničava prepoznavanje antigena s mutacijama u imunodominantnim epitopima. Previše raznolikosti smanjuje učinkovitost specifičnog prepoznavanja. Mehanizmi koji kontroliraju raznolikost memorije još uvijek su nepoznati. Predloženo istraživanje ima za cilj identificirati ključne molekularne mehanizme koji reguliraju klonalnu raznolikost imunološke memorije posredovane CD8 limfocitima T. Materijali i metode: Kako bi razjasnili utjecaj jakosti aktivirajućeg signala na T- stanični receptor u kontekstu formiranja imunološke memorije uspostavili smo in vitro model - OT-1 CD8 limfociti T su stimulirani sa SIINFEKL (N4) peptidom ili izmijenjenim peptidnim ligandima (eng. altered peptide ligands - APL) s nižim afinitetoma za T stanični receptor. Koristeći visokoprotočnu analizu transkriptoma identificirali smo potencijalne ključne faktore za razvoj memorijskih stanica niskog afiniteta (Eomes i Bcl-2). Kako bi dobivene rezultate potvrdili in vivo koristili smo modele infekcija s mCMV, LCMV ili L. monocytogenes koji ispoljavaju N4 peptid ili APL. S ciljem dokazivanja ključne uloge transkripcijskog faktora Eomes u dugoročnom preživljavanju memorijskih stanica niskog afiniteta proveli smo pokuse adoptivnog transfera Eomes-deficijentnih stanica (OT-1 Eomesfl/flCD4Cre; EomesCKO). Osim toga, inducibilni MXCre sustav korišten je za određivanje vremenskog okvira u kojem Eomes posreduje navedeni učinak. Kako bi smo potvrdili dobivene rezultate u poliklonalnom sustavu stvorili smo mješovite kimerične životinje koristeći stanice koštane srži miševa divljeg tipa (WT) i EomesCKO miševa, dok je spoj ABT-199 korišten za specifično inhibiranje molekule Bcl-2. U svrhu ispitivanja vezanja faktora Eomes na promotorsku regiju Bcl2 gena, proveli smo Eomes ChIP-seq analizu na aktiviranim OT-1 stanicama. S ciljem određivanja molekularnog mehanizma koji je odgovoran za prednost u preživljavanju memorijskih stanica niskog afiniteta, koristili smo transgenične NIH3T3 i HEK293 stanične linije koje ispoljavaju Eomes i/ili T-bet. Navedeno ispitivanje smo proveli pomoću luciferaznog reporter eseja upotrebom specifičnih plazmida. Da bismo potvrdili da nedostatak transkripcijskog faktora Eomes rezultira smanjenom klonalnom raznolikošću proveli smo TCR sekvencioniranje antigen-specifičnih WT i EomesCKO CD8 limfocita T izoliranih iz mješovitih kimeričnih životinja 30 dana nakon infekcije LCMV-om. Rezultati: Stimulacija CD8 limfocita T submaksimalnim intenzitetom kumulativnog aktivirajućeg signala dovodi do najviše razine ispoljavanja transkripcijskog faktora Eomes. Eomes direktno na transkripcijskog razini potiče ispoljavanje Bcl-2 proteina što rezultira dugoročnim preživljavanjem memorijskih stanica submaksimalnog afiniteta. Ukoliko su stanice stimulirane jačim intenzitetom dolazi do dominacije transkripcijskog faktora T-bet koji inhibira Eomes-induciranu transkripciju Bcl-2. Povećani proliferativni potencijal klonova visokog afiniteta je odgovoran za njihovu dominaciju, dok prednost u preživljavanju stanica submaksimalnog afiniteta omogućuje njihovu perzistenciju u memorijskoj fazi imunološkog odgovora. Zaključak: Ovaj rad otkriva molekularni mehanizam koji je temelj za uspostavljanje potrebne raznolikost klonova memorijskih CD8 limfocita T. Dobiveni rezultati mogu se koristiti u svrhu unaprjeđenja imunoterapije posredovane limfocitima T

Immunological Mechanisms of Sickness Behavior in Viral Infection

Sickness behavior is the common denominator for a plethora of changes in normal behavioral routines and systemic metabolism during an infection. Typical symptoms include temperature, muscle weakness, and loss of appetite. Whereas we experience these changes as a pathology, in fact they are a carefully orchestrated response mediated by the immune system. Its purpose is to optimize immune cell functionality against pathogens whilst minimizing viral replication in infected cells. Sickness behavior is controlled at several levels, most notably by the central nervous system, but also by other organs that mediate systemic homeostasis, such as the liver and adipose tissue. Nevertheless, the changes mediated by these organs are ultimately initiated by immune cells, usually through local or systemic secretion of cytokines. The nature of infection determines which cytokine profile is induced by immune cells and therefore which sickness behavior ensues. In context of infection, sickness behavior is typically beneficial. However, inappropriate activation of the immune system may induce adverse aspects of sickness behavior. For example, tissue stress caused by obesity may result in chronic activation of the immune system, leading to lasting changes in systemic metabolism. Concurrently, metabolic disease prevents induction of appropriate sickness behavior following viral infection, thus impairing the normal immune response. In this article, we will revisit recent literature that elucidates both the benefits and the negative aspects of sickness behavior in context of viral infection

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45 Ly6G CD3 B220 NK1.1 CD11b CD115 ). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and gene expression were upregulated in osteoclastogenic cultures from RBP-J mice, with the opposing results in NICD1 mice. Notch modulation did not affect the number of differentiated macrophages and DCs. However, RBP-J deletion stimulated and expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1 and RBP-J strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling via deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45+Ly6G-CD3-B220-NK1.1-CD11b-/loCD115+). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced in vitro by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose E. coli particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and Ctsk gene expression were upregulated in osteoclastogenic cultures from RBP-J+ mice, with the opposing results in NICD1+ mice. Notch modulation did not affect the number of in vitro differentiated macrophages and DCs. However, RBP-J deletion stimulated Il12b and Cd86 expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1+ and RBP-J+ strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases