Inducible deletion of CDK4 and CDK6 – deciphering CDK4/6 inhibitor effects in the hematopoietic system

Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors are considered a breakthrough in cancer therapy. Currently approved for breast cancer treatment, CDK4/6 inhibitors are extensively tested in other cancer subtypes. Frequently observed side effects include hematological abnormalities such as reduced numbers of neutrophils, erythroid cells and platelets that are associated with anemia, bleeding and a higher risk of infections. In order to understand whether the adverse effects within the hematopoietic system are related to CDK4 or CDK6 we generated transgenic mice that lack either CDK4 or CDK6 in adult hematopoiesis. Anemia and perturbed erythroid differentiation are associated with the absence of CDK6 but did not manifest in CDK4- deficient mice. Total CDK6 knockout mice accumulate the most dormant fraction of hematopoietic stem cells due to an impaired exit of the quiescent state. We recapitulated this finding by deleting CDK6 in adult hematopoiesis. In addition, unlike total CDK6 knockout, all stem cell fractions were affected and increased in numbers. The deletion of CDK6 was also accompanied by neutropenia which is frequently seen in patients receiving CDK4/6 inhibitors. This was not the case in the absence of CDK4; CDK4 deficiency resulted in elevated numbers of myeloid progenitors without translating into numeric changes of differentiated myeloid cells. By using Cdk4fl/fl and Cdk6fl/fl mice we assign side effects of CDK4/6 inhibitors predominantly to the absence of CDK6. These mice represent a novel and powerful tool that will enable to study the distinct functions of CDK4 and CDK6 in a tissue-dependent manner

Myeloid STAT3 promotes formation of colitis-associated colorectal cancer in mice

Myeloid cells lacking STAT3 promote antitumor responses of NK and T cells but it is unknown if this crosstalk affects development of autochthonous tumors. We deleted STAT3 in murine myeloid cells (STAT3(Δm)) and examined the effect on the development of autochthonous colorectal cancers (CRCs). Formation of Azoxymethane/Dextransulfate (AOM/DSS)-induced CRCs was strongly suppressed in STAT3(Δm) mice. Gene expression profiling showed strong activation of T cells in the stroma of STAT3(Δm) CRCs. Moreover, STAT3(Δm) host mice were better able to control the growth of transplanted MC38 colorectal tumor cells which are known to be killed in a T cell-dependent manner. These data suggest that myeloid cells lacking STAT3 control formation of CRCs mainly via cross activation of T cells. Interestingly, the few CRCs that formed in STAT3(Δm) mice displayed enhanced stromalization but appeared normal in size indicating that they have acquired ways to escape enhanced tumor surveillance. We found that CRCs in STAT3(Δm) mice consistently activate STAT3 signaling which is implicated in immune evasion and might be a target to prevent tumor relapse

Myeloid Cells Restrict MCMV and Drive Stress-Induced Extramedullary Hematopoiesis through STAT1

Cytomegalovirus (CMV) has a high prevalence worldwide, is often fatal for immunocompromised patients, and causes bone marrow suppression. Deficiency of signal transducer and activator of transcription 1 (STAT1) results in severely impaired antiviral immunity. We have used cell-type restricted deletion of Stat1 to determine the importance of myeloid cell activity for the defense against murine CMV (MCMV). We show that myeloid STAT1 limits MCMV burden and infection-associated pathology in the spleen but does not affect ultimate clearance of infection. Unexpectedly, we found an essential role of myeloid STAT1 in the induction of extramedullary hematopoiesis (EMH). The EMH-promoting function of STAT1 was not restricted to MCMV infection but was also observed during CpG oligodeoxynucleotide-induced sterile inflammation. Collectively, we provide genetic evidence that signaling through STAT1 in myeloid cells is required to restrict MCMV at early time points post-infection and to induce compensatory hematopoiesis in the spleen

TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors

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

Dendritic Cell-Secreted Lipocalin2 Induces CD8⁺ T-Cell Apoptosis, Contributes to T-Cell Priming and Leads to a T_H1 Phenotype

<div><p>Lipocalin 2 (LCN2), which is highly expressed by dendritic cells (DCs) when treated with dexamethasone (Dex) and lipopolysaccharide (LPS), plays a key role in the defence against bacteria and is also involved in the autocrine apoptosis of T-cells. However, the function of LCN2 when secreted by DCs is unknown: this is a critical gap in our understanding of the regulation of innate and adaptive immune systems. Tolerance, stimulation and suppression are functions of DCs that facilitate the fine-tuning of the immune responses and which are possibly influenced by LCN2 secretion. We therefore examined the role of LCN2 in DC/T-cell interaction. WT or Lcn2^−/− bone marrow-derived DCs were stimulated with LPS or LPS+IFN-γ with and without Dex and subsequently co-cultured with T-cells from ovalbumin-specific TCR transgenic (OT-I and OT-II) mice. We found that CD8⁺ T-cell apoptosis was highly reduced when Lcn2^−/− DCs were compared with WT. An <i>in vivo</i> CTL assay, using LPS-treated DCs, showed diminished killing ability in mice that had received Lcn2^−/− DCs compared with WT DCs. As a consequence, we analysed T-cell proliferation and found that LCN2 participates in T-cell-priming in a dose-dependent manner and promotes a T_H1 microenvironment. DC-secreted LCN2, whose function has previously been unknown, may in fact have an important role in regulating the balance between T_H1 and T_H2. Our results yield insights into DC-secreted LCN2 activity, which could play a pivotal role in cellular immune therapy and in regulating immune responses.</p></div

CTL assay with DC and peptide immunisation.

<p>(A) Gating strategy of lymph nodes, splenocytes were selected for CFSE staining and analysed. On the right, CD8⁺ and CD4⁺ T-cells in lymph nodes. (B) Analysis of the splenocytes in immunised WT or Lcn2^−/− recipient mice. Cells were selected based on the CFSE staining. On the left, the control group received just PBS, in the middle mice immunised with 6 h-LPS-treated Lcn2^−/− DCs and on the right, mice immunised with 6 h-LPS-treated WT DCs. Histograms show the specific killing effects using Lcn2^−/− or WT DC immunisation. These data show the most representative of pooled mice (5 mice/group) performed 3 independent times and shown as mean ± SEM, p value is **p<0.01. (C) FACS analysis of perforin and granzyme B intracellular staining in OT-I cells after 72 h of WT or Lcn2^−/− DC/T-cell co-culture. DCs were pre-treated for 6 h with LPS. Histograms show the percentage of positive cells in the co-culture. (D) Comparison of <i>in vivo</i> CTL assays in WT and Lcn2^−/− recipient mice immunized with SIINFEKL_257–264 and CpG peptides for 7 days. These data show the most representative experiment of pooled mice (5 mice/group) performed 3 independent times.</p

T-cell priming in DC/T-cell co-culture. WT and Lcn2^−/− DCs were pre-treated for 6 h with LPS and then co-cultured with T-cells.

<p>The extent of proliferation was calculated after 72-culture from the decrease in fluorescence per cell of the cell proliferation dye (CPD). (A) Histograms represent CD8⁺ T-cell divisions. Activated T-cells were stained and gated on cells positive for CD8⁺CD25⁺. Generation 0 was the undivided population initially labelled with CPD and all other generations were the result of the sequentially halved CPD fluorescence. The results were analysed by the proliferation tool of FlowJo. (B) Analysis of the T-cell apoptosis at 1∶1 (10⁵∶10⁵), 1∶5 (2×10⁴∶10⁵) and 1∶10 (10⁴∶10⁵) DC/T-cell ratios. T-cells (OT-I) were co-cultured with 6 h-LPS-treated Lcn2^−/− DCs (black bars) or 6 h-LPS-treated WT DCs (grey bars). Five independent experiments were performed in triplicate. Mean ± SEM, *p<0.05, ****p<0.0001. (C) Analysis of T-cell apoptosis after adding Recombinant Lcn2 (Rec Lcn2) in DC/T-cell co-culture. Lcn2^−/− DCs, pre-treated with LPS for 6 h, were co-cultured 72 h with T-cells in medium supplemented with Rec Lcn2. Five independent experiments were performed in triplicate. Mean ± SEM, *p<0.05, ***p<0.001. (D) Proliferation and apoptosis of T-cells (OT-I) was determined after co-culture in the absence or presence of desferrioxamine (1∶1, 1∶5 and 1∶10 ratio) for 72 h with DCs that had been previously incubated for 6 h with LPS. The two groups were analysed by ANOVA and shown as mean ± SEM, *p<0.05, ***p<0.001.</p

WT and Lcn2^−/− DC/T-cell co-culture and analysis of CD8⁺ T-cell apoptosis.

<p>T-cells were analysed after DC/T-cell co-culture by flow cytometry. WT and Lcn2^−/− DCs were treated for 6 h with Dex, Dex+LPS (DL), Dex+LPS+IFN-γ (DLI), LPS+IFN-γ (LI), LPS alone (L) or on the left without any treatment (Co), and co-cultured with OT-I or OT-II T-cells for 48 and 72 h. (A) Gating strategy, FACS analysis was done with cells positive for CD3⁺CD8⁺ or CD3⁺CD4⁺ fluorescence and the output data were divided in 3 main groups: alive, Annexin V and DAPI to distinguish early and late apoptosis. (B) Early and late apoptosis of OT-I or OT-II T-cells after 48 and 72 h. OT-I T-cells, 1240, underwent early apoptosis in DL-treated Lcn2^−/− DCs compared with WT DCs co-culture, 6920, p value <0.05. In the same condition, the comparison of 4829 and 6958 OT-II T-cells was not statistically relevant. Absolute cell numbers were determined with Trucount (BD). The two groups were analysed by ANOVA followed by Bonferroni correction for multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). P value refers to the comparison at 48 or 72 h. (C) LCN2 protein expression measured by ELISA in supernatant from treated WT DC/OT-I or treated WT DC/OT-II T-cell co-cultures after 48 and 72 h incubation. These data were pooled from 3 independent experiments performed in triplicate per OT condition and are shown as mean ± SEM. (D) Quantitative PCR (TaqMan) analysis of LCN2 receptor, Lcn2R (Slc22A17) expression in OT-I and OT-II T-cells after 72 h DC/T-cell co-cultures at 1∶1, 1∶5, 1∶10 ratio. Black bars represent T-cells co-cultured with 6 h-LPS-treated Lcn2^−/− DCs, grey bars represent the co-culture with 6 h-LPS-treated WT DCs. The two groups were analysed by ANOVA. P value is *p<0.05, **p<0.01, ***p<0.001.</p

Characterisation of WT and Lcn2^−/− DC maturation.

<p>(A) Bone marrow-derived DCs were stained for major histocompatibility complex MHC class I and II, co-stimulatory molecules CD80/B7.1, CD86/B7.2 and the maturation marker CD83 after LPS treatment for 6 and 24 h. Negative control (Co). (B) Comparison of WT and Lcn2^−/− bone marrow-derived DCs treated for 24 h with Dexamethasone (Dex) added 20 min prior LPS (DL) and LPS+IFN-γ (DLI), or LPS (L), LPS+IFN-γ (LI), and analysed by FACS after CD86/B7.2 and MHC Class II staining. Negative control (Co). (C) Expression of Lcn2 mRNA and protein in DCs treated for 6 and 24 h with Dex, Dex+LPS (DL), Dex+LPS+IFN-γ (DLI), LPS+IFN-γ (LI), LPS alone (L) or on the left without any treatment (Co). The kinetic of LCN2 secretion was performed up to 72 h in DCs stimulated with DL and L and analysed by ELISA. The difference in the amount was due to the distinct amount of plated DCs. The two groups were analysed by ANOVA. P value is *p<0.05, ***p<0.001. (D) Proinflammatory cytokines IL-1α, IL-6, TNF-α, IFN-γ and IL-12p70 were measured in supernatant of DCs treated for 6 h with Dex, DL, DLI, LI, and L or on the left without any treatment (Co) as control, and quantified by Cytomix. IFN-γ identified what was previously added. These data are pooled from 6 independent experiments performed in triplicates and shown as mean ± SEM.</p