Mixed Bone Marrow Chimeras Research Articles

Cell-intrinsic regulation of phagocyte function by interferon lambda during pulmonary viral, bacterial super-infection.

Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1-/- mice with influenza A/PR/8/34 followed by S. aureus USA300. We demonstrated that global IFNLR1-/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX3CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells.

RIPK1 protects naive and regulatory T cells from TNFR1-induced apoptosis

The T cell population size is stringently controlled before, during, and after immune responses, as improper cell death regulation can result in autoimmunity and immunodeficiency. RIPK1 is an important regulator of peripheral T cell survival and homeostasis. However, whether different peripheral T cell subsets show a differential requirement for RIPK1 and which programmed cell death pathway they engage in vivo remains unclear. In this study, we demonstrate that conditional ablation of Ripk1 in conventional T cells (Ripk1ΔCD4) causes peripheral T cell lymphopenia, as witnessed by a profound loss of naive CD4+, naive CD8+, and FoxP3+ regulatory T cells. Interestingly, peripheral naive CD8+ T cells in Ripk1ΔCD4 mice appear to undergo a selective pressure to retain RIPK1 expression following activation. Mixed bone marrow chimeras revealed a competitive survival disadvantage for naive, effector, and memory T cells lacking RIPK1. Additionally, tamoxifen-induced deletion of RIPK1 in CD4-expressing cells in adult life confirmed the importance of RIPK1 in post-thymic survival of CD4+ T cells. Ripk1K45A mice showed no change in peripheral T cell subsets, demonstrating that the T cell lymphopenia was due to the scaffold function of RIPK1 rather than to its kinase activity. Enhanced numbers of Ripk1ΔCD4 naive T cells expressed the proliferation marker Ki-67+ despite the peripheral lymphopenia and single-cell RNA sequencing revealed T cell-specific transcriptomic alterations that were reverted by additional caspase-8 deficiency. Furthermore, Ripk1ΔCD4Casp8 ΔCD4 and Ripk1ΔCD4Tnfr1−/− double-knockout mice rescued the peripheral T cell lymphopenia, revealing that RIPK1-deficient naive CD4+ and CD8+ cells and FoxP3+ regulatory T cells specifically die from TNF- and caspase-8-mediated apoptosis in vivo. Altogether, our findings emphasize the essential role of RIPK1 as a scaffold in maintaining the peripheral T cell compartment and preventing TNFR1-induced apoptosis.

Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5′-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

Glycan-Reactive Innate-like B Cells and Developmental Checkpoints.

Using an Ig H chain conferring specificity for N-acetyl-d-glucosamine (GlcNAc), we developed transgenic (VHHGAC39 TG) mice to study the role of self-antigens in GlcNAc-reactive B-1 B cell development. In VHHGAC39 TG mice, GlcNAc-reactive B-1 B cell development during ontogeny and in adult bone marrow was normal. However, adult TG mice exhibited a block at transitional-2 immature B cell stages, resulting in impaired allelic exclusion and accumulation of a B cell subset coexpressing endogenous Ig gene rearrangements. Similarly, VHHGAC39 B cell fitness was impeded compared with non-self-reactive VHJ558 B TG cells in competitive mixed bone marrow chimeras. Nonetheless, adult VHHGAC39 mice immunized with Streptococcus pyogenes produce anti-GlcNAc Abs. Peritoneal cavity B cells transferred from VHHGAC39 TG mice into RAG-/- mice also exhibited robust expansion and anti-GlcNAc Ab production. However, chronic treatment of young VHHGAC39 mice with GlcNAc-specific mAbs leads to lower GlcNAc-binding B cell frequencies while increasing the proportion of GlcNAc-binding B1-a cells, suggesting that Ag masking or clearance of GlcNAc Ags impedes maturation of newly formed GlcNAc-reactive B cells. Finally, BCR H chain editing promotes expression of endogenous nontransgenic BCR alleles, allowing potentially self-reactive TG B cells to escape anergy or deletion at the transitional stage of precursor B cell development. Collectively, these observations indicate that GlcNAc-reactive B cell development is sensitive to the access of autologous Ags.

DCIR expression on dendritic cells decelerates early T-cell activation in a murine mixed bone marrow chimera model of neurotropic virus infections

DCIR expression on dendritic cells decelerates early T-cell activation in a murine mixed bone marrow chimera model of neurotropic virus infections

Co-adjuvanting DDA/TDB liposomes with a TLR7 agonist allows for IgG2a/c class-switching in the absence of Th1 cells

Class-switching to IgG2a/c in mice is a hallmark response to intracellular pathogens. T cells can promote class-switching and the predominant pathway for induction of IgG2a/c antibody responses has been suggested to be via stimulation from Th1 cells. We previously formulated CAF®01 (cationic liposomes containing dimethyldioctadecylammonium bromide (DDA) and Trehalose-6,6-dibehenate (TDB)) with the lipidated TLR7/8 agonist 3M-052 (DDA/TDB/3M-052), which promoted robust Th1 immunity in newborn mice. When testing this adjuvant in adult mice using the recombinant Chlamydia trachomatis (C.t.) vaccine antigen CTH522, it similarly enhanced IgG2a/c responses compared to DDA/TDB, but surprisingly reduced the magnitude of the IFN-γ+Th1 response in a TLR7 agonist dose-dependent manner. Single-cell RNA-sequencing revealed that DDA/TDB/3M-052 liposomes initiated early transcription of class-switch regulating genes directly in pre-germinal center B cells. Mixed bone marrow chimeras further demonstrated that this adjuvant did not require Th1 cells for IgG2a/c switching, but rather facilitated TLR7-dependent T-bet programming directly in B cells. This study underlines that adjuvant-directed IgG2a/c class-switching in vivo can occur in the absence of T-cell help, via direct activation of TLR7 on B cells and positions DDA/TDB/3M-052 as a powerful adjuvant capable of eliciting type I-like immunity in B cells without strong induction of Th1 responses.

Conditional NKT Cell Depletion in Mice Reveals a Negative Feedback Loop That Regulates CTL Cross-Priming.

NKT cells are unconventional T cells whose biological role is incompletely understood. Similar to TH cells, activated NKT cells can cause dendritic cell (DC) maturation, which is required for effective CTL responses. However, it is unclear whether and how NKT cells affect CTLs downstream of the DC maturation phase. This is partially due to the lack of techniques to conditionally deplete NKT cells invivo. To overcome this problem, we have developed two approaches for this purpose in mice: the first is based on mixed bone marrow chimeras where Jα18 knockout and depletable CD90 congenic bone marrow is combined, and the second used PLZFCre × iDTR bone marrow chimeras, which target innate-like T cells. Using these tools, we found that NKT cell depletion at 20 h, that is, after initial DC activation, did not render CTLs helpless, as CD40L signaling by non-NKT cells sufficed. Instead, NKT cell depletion even augmented CD8 T cell expansion and cytotoxicity by mechanisms distinct from reduced STAT6 signaling. These findings revealed a negative feedback loop by which NKT cells control CTL cross-priming downstream of DC maturation. The techniques described in this study expand the toolbox to study NKT cells and other unconventional T cell subsets invivo and uncovered a hidden immunoregulatory mechanism.

Immunological Phenotyping of Mice with a Point Mutation in Cdk4.

Cyclin-dependent kinases (CDKs) play a crucial role in regulation of the mammalian cell cycle. CDK4 and CDK6 control the G1/S restriction checkpoint through their ability to associate with cyclin D proteins in response to growth factor signals. CDK4 deficiency in mice gives rise to a range of endocrine-specific phenotypes including diabetes, infertility, dwarfism, and atrophy of the anterior pituitary. Although CDK6 deficiency can cause thymic atrophy due to a block in the double-negative (DN) to double-positive (DP) stage of T cell development, there are no overt defects in immune cell development reported for CDK4-deficient mice. Here, we examined the impact of a novel N-ethyl-N-nitrosourea-induced point mutation in the gene encoding CDK4 on immune cell development. Mutant mice (Cdk4wnch/wnch) showed normal development and differentiation of major immune cell subsets in the thymus and spleen. Moreover, T cells from Cdk4wnch/wnch mice exhibited normal cytokine production in response to in vitro stimulation. However, analysis of the mixed bone marrow chimeras revealed that Cdk4wnch/wnch-derived T cell subsets and NK cells are at a competitive disadvantage compared to Cdk4+/+-derived cells in the thymus and periphery of recipients. These results suggest a possible role for the CDK4wnch mutation in the development of some immune cells, which only becomes apparent when the Cdk4wnch/wnch mutant cells are in direct competition with wild-type immune cells in the mixed bone marrow chimera.

Impaired tissue homing by the Ikzf3N159S variant is mediated by interfering with Ikaros function.

AIOLOS, encoded by IKZF3, is a member of the IKZF family of proteins that plays an important role in regulating late B-cell differentiation. Human individuals heterozygous for the AIOLOS p.N160S variant displayed impaired humoral immune responses as well as impaired B and T cell development. We have previously reported that a mouse strain harboring an Ikzf3N159S allele that corresponds to human IKZF3N160S recapitulated immune-deficient phenotypes, such as impaired B cell development and loss of CD23 expression. In this study, we investigated the effect of the Ikzf3N159S variant and found that B1a cell development was impaired in Ikzf3N159S/N159S mice. In addition, CD62L expression was severely decreased in both B and T lymphocytes by the Ikzf3N159S mutation, in a dose-dependent manner. Mixed bone marrow chimera experiments have revealed that most immunodeficient phenotypes, including low CD62L expression, occur in intrinsic cells. Interestingly, while Ikzf3N159S/N159S lymphocytes were still present in the spleen, they were completely outcompeted by control cells in the lymph nodes, suggesting that the capacity for homing or retention in the lymph nodes was lost due to the Ikzf3N159S mutation. The homing assay confirmed severely decreased homing abilities to lymph nodes of Ikzf3N159S/N159S B and T lymphocytes but selective enrichment of CD62L expressing Ikzf3N159S/N159S lymphocytes in lymph nodes. This finding suggests that impaired CD62L expression is the major reason for the impaired homing capacity caused by the Ikzf3N159S mutation. Interestingly, an excess amount of Ikaros, but not Aiolos, restored CD62L expression in Ikzf3N159S/N159S B cells. Together with the loss of CD62L expression due to Ikaros deficiency, the AiolosN159S mutant protein likely interferes with Ikaros function through heterodimerization, at least in activating the Sell gene encoding CD62L expression. Thus, our results revealed that AiolosN159S causes some immunodeficient phenotypes via the pathogenesis referred to as the heterodimeric interference as observed for AiolosG158R variant.

Interleukin-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration in allergic airway inflammation

Allergic asthma is a chronic lung disease characterized by airway hyperresponsiveness and cellular infiltration that is exacerbated by immunoglobulin E-dependent mast cell (MC) activation. Interleukin-9 (IL-9) promotes MC expansion during allergic inflammation but precisely how IL-9 expands tissue MCs and promotes MC function is unclear. In this report, using multiple models of allergic airway inflammation, we show that both mature MCs (mMCs) and MC progenitors (MCp) express IL-9R and respond to IL-9 during allergic inflammation. IL-9 acts on MCp in the bone marrow and lungs to enhance proliferative capacity. Furthermore, IL-9 in the lung stimulates the mobilization of CCR2+ mMC from the bone marrow and recruitment to the allergic lung. Mixed bone marrow chimeras demonstrate that these are intrinsic effects in the MCp and mMC populations. IL-9-producing T cells are both necessary and sufficient to increase MC numbers in the lung in the context of allergic inflammation. Importantly, T cell IL-9-mediated MC expansion is required for the development of antigen-induced and MC-dependent airway hyperreactivity. Collectively, these data demonstrate that T cell IL-9 induces lung MC expansion and migration by direct effects on the proliferation of MCp and the migration of mMC to mediate airway hyperreactivity.

The adaptor protein TRAF3 is an immune checkpoint that inhibits myeloid-derived suppressor cell expansion.

Myeloid-derived suppressor cells (MDSCs) are aberrantly expanded in cancer patients and under other pathological conditions. These cells orchestrate the immunosuppressive and inflammatory network to facilitate cancer metastasis and mediate patient resistance to therapies, and thus are recognized as a prime therapeutic target of human cancers. Here we report the identification of the adaptor protein TRAF3 as a novel immune checkpoint that critically restrains MDSC expansion. We found that myeloid cell-specific Traf3-deficient (M-Traf3 -/-) mice exhibited MDSC hyperexpansion during chronic inflammation. Interestingly, MDSC hyperexpansion in M-Traf3 -/- mice led to accelerated growth and metastasis of transplanted tumors associated with an altered phenotype of T cells and NK cells. Using mixed bone marrow chimeras, we demonstrated that TRAF3 inhibited MDSC expansion via both cell-intrinsic and cell-extrinsic mechanisms. Furthermore, we elucidated a GM-CSF-STAT3-TRAF3-PTP1B signaling axis in MDSCs and a novel TLR4-TRAF3-CCL22-CCR4-G-CSF axis acting in inflammatory macrophages and monocytes that coordinately control MDSC expansion during chronic inflammation. Taken together, our findings provide novel insights into the complex regulatory mechanisms of MDSC expansion and open up unique perspectives for the design of new therapeutic strategies that aim to target MDSCs in cancer patients.

TLR3 Signaling is Required for Robust Extrafollicular B Cell Response (EF) in Influenza A/PR/8 Infection

Abstract Following influenza A virus (IAV) infection B cells rapidly proliferate and differentiate into plasma cells (PCs) in extrafollicular foci (EF) within mediastinal lymph nodes (medLN), resulting in protective antibody secretion. We had shown that co-expression of both TLR adaptor proteins, TRIF and MyD88, is required for robust EF responses. Only two TLRs, TLR3 and 4, signal using TRIF. As shown previously TLR3 and 7, but not TLR4, expression were upregulated by medLN B cells within 2 days post infection (dpi) in a Type-I IFN-dependent manner, suggesting that TLR3 might utilize TRIF in B cells during IAV infection. To test this we generated MyD88 × TLR3 double-deficient (DKO) mice. Flow cytometry on MedLN at 7 dpi showed significantly reduced EF B cells (CD19 loB220 loCD38 loCD24 hi) and CD138 +plasma cells (PCs), including IAV-specific PCs, as well as reduced serum IAV-specific IgG and IgG 2Cin DKO compared to C57BL/6 or single- MyD88 and TLR3 deficient mice. IAV infection of mixed bone marrow chimeras indicated that the TLR3-TRIF axis contributes to EF formation at least in part in a B cell-intrinsic manner. In vitro stimulation of B cells with the TLR3 agonist, Poly: IC, caused expression of several activation/costimulatory molecules but did not induce proliferation. Together, the data suggest that TLR3-mediated B cell is a non-redundant driver of rapid protective antibody responses. Supported by NIH/NIAID R01AI117890 (NB) and NIH T32 HL007013-40 (JHL and EJK).

Identifying the expression and function of Blimp-1 in ILC2s in allergic asthma

Abstract Allergic asthma is driven by type 2 inflammation from Th2 cell and Type 2 innate lymphoid cells (ILC2s). We recently demonstrated a critical role for Blimp-1, a transcriptional repressor, in Th2 cells to promote allergic asthma. However, the expression and function of Blimp-1 in ILC2s remains undefined. Using genetically modified mouse strains that report Blimp-1 expression via YFP, we surveyed Blimp-1 expression in allergic asthma and other type 2 immune models across various tissues. We found that Blimp-1 is induced in ILC2s in the lung in response to the typical alarmins IL-33 and IL-25, as well as house dust mite (HDM). However, ILC2 activation did not upregulate Blimp-1 in the skin or gut in response to peanut allergen, suggesting a tissue-specific requirement for Blimp-1 in ILC2s. IL-33, as known to be a potent activator of ILC2s, was not required for Blimp-1 expression upon HDM stimulation. Interestingly, IL-10, a potent driver of Blimp-1 in Th2 cells, induced Blimp-1 expression in ILC2s, suggesting IL-10 but not IL-33 may be important for a Blimp-1+ ILC2 state. Intriguingly, Blimp-1 expression did not correlate with cytokine production, potentially representing a unique functional state of ILC2 cells that are high for Blimp-1. To evaluate Blimp-1 function, we deleted Blimp-1 in ILC populations using IL-7Ra Cre crossed to Blimp-1 floxed mice and generated mixed bone marrow chimeras with IL-7Ra Cre control donors. Blimp-1 knockout ILC2s in response to HDM exhibited an alteration in markers of ILC2 activation including ST2, KLRG1 and GATA3, suggesting Blimp-1 plays a role in promoting ILC2 differentiation. Collectively, our study has identified a lung-specific state of ILC2s expressing Blimp-1 that are found in a model of allergic asthma.

The ubiquitin ligase Itch skews light zone selection in germinal centers

Abstract Immunoglobulin diversification occurs in peripheral lymphoid organs, after the central tolerance checkpoints during B cell development. High levels of somatic hypermutation (SHM) of Ig genes occurs in germinal center (GC) B cells, followed by selection of mutated clones. This process is essential for generation of high affinity antibody to pathogens, but can also produce autoreactive Ig receptors. Selection mechanisms that govern antigen-driven Ig diversification are still incompletely understood. The E3 ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop debilitating, multi-faceted, potentially fatal, autoimmune disease; yet how Itch regulates GC B cell fate or function is not well understood. By studying Itch deficient mice, we have recently shown that Itch directly limits GC B cell activity to shape antibody responses. Proteomic profiling of GC B cells uncovered that Itch deficient cells exhibit high mTORC1 and Myc activity, hallmarks of positive selection. Mixed bone marrow chimeras revealed that Itch acts within B cells to limit proportions of light zone GC cells, and Itch deficient light zone GC cells were mainly in G1/G0 rather than in cell cycle. These results support a novel role for Itch in skewing selection of GC B cells to restrict LZ accumulation and shape GC-derived humoral immunity. Determining how B cells integrate cues within GCs to navigate through LZs and DZs will aid in understanding how autoreactive clones emerge from GCs in those suffering from autoimmune disease. Supported by a grant from NIH (K22AI148597)

Absence of Toll-like receptor 5 confers survival in mice bearing ovarian tumors treated with anti-PD-L1

Abstract Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system. Patients bearing ovarian tumors infiltrated with high frequencies of T cells associate with a greater survival probability. However, therapies targeting T cells in ovarian cancers are ineffective. We observed in models of late-stage murine ovarian cancer that in absence of Toll-Like Receptor 5 (TLR5) signaling, anti-PD-L1 promotes significant survival and protection against tumor rechallenge. Ovarian tumors from rechallenged TLR5KO mice exhibited significantly higher frequency/number of CD8 T cells expressing functional and activation markers. Thus, in the absence of TLR5 signaling, T cells mount/maintain a stronger response to ovarian tumors after inhibitory receptor blockade. Furthermore, protection against tumor progression in TLR5KO mice corresponds with a significant increase in tumor infiltrating cross presenting and IL-12 producing Dendritic cells (DCs). Additionally, in CD11c Cre x TLR5 fl/fl mice, whose DCs lack TLR5 expression, PD-L1-blockade mediated extended survival in ovarian tumor-bearing mice. Using a mixed bone marrow chimera approach, we demonstrate that direct TLR5 signaling functionally alters DCs to become more suppressive in the tumor microenvironment, impacting T cell differentiation and anti-tumor activity, thereby reducing survival during aPD-L1 therapy. Clinically, roughly 7.5% of the general population harbor a TLR5 SNP that diminishes TLR5 signaling and is associated with increased long-term survival for ovarian cancer patients. Therefore, patients who express the TLR5 SNP may immediately benefit from anti-PDL1 therapy and those without the SNP, TLR5 antagonism. NIH/NCI 1R01CA253285 UVA Farrow Fellowship

Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1

Liver X receptor (LXR) is a critical regulator of cholesterol homeostasis that inhibits T cell receptor (TCR)-induced proliferation by altering intracellular sterol metabolism. However, the mechanisms by which LXR regulates helper T cell subset differentiation remain unclear. Here, we demonstrate that LXR is a crucial negative regulator of follicular helper T (Tfh) cells invivo. Both mixed bone marrow chimera and antigen-specific T cell adoptive cotransfer studies show a specific increase in Tfh cells among LXRβ-deficient CD4+ T cell population in response to immunization and lymphocytic choriomeningitis mammarenavirus (LCMV) infection. Mechanistically, LXRβ-deficient Tfh cells express augmented levels of T cell factor 1 (TCF-1) but comparable levels of Bcl6, CXCR5, and PD-1 in comparison with those of LXRβ-sufficient Tfh cells. Loss of LXRβ confers inactivation of GSK3β induced by either AKT/Extracellular signal-regulated kinase (ERK) activation or Wnt/β-catenin pathway, leading to elevated TCF-1 expression in CD4+ Tcells. Conversely, ligation of LXR represses TCF-1 expression and Tfh cell differentiation in both murine and human CD4+ Tcells. LXR agonist significantly diminishes Tfh cells and the levels of antigen-specific IgG upon immunization. These findings unveil a cell-intrinsic regulatory function of LXR in Tfh cell differentiation via the GSK3β-TCF1 pathway, which may serve as a promising target for pharmacological intervention in Tfh-mediated diseases.

Abstract TMP74: HIF-1a And MTORC2 Co-regulate Macrophage Inflammation And Metabolism In Intracerebral Hemorrhage

Introduction: Recruited macrophages are known to spur secondary inflammation-induced damage in intracerebral hemorrhage (ICH), and targeted metabolic modulation of these macrophages could limit inflammation. Recent macrophage transcriptomic work has associated increased expression of HIF-1a, master regulator of the hypoxia response, and mTORC2, the more recently-discovered mTOR kinase complex, with good patient outcomes. The goal of this work was to identify the inflammatory and metabolic effects of these factors, providing insight into their putatively beneficial role in acute brain injury. Methods: Human monocyte-derived macrophages were stimulated either with ICH-associated danger signals (DAMP) (S100A9 (2 ug/mL, R&D) + IL-1β (10 ng/mL, Biolegend)), or 1 ng/mL LPS (Invivogen). Echinomycin (Sigma Aldrich), NR1 (Aobious), JR-AB2-011 (Aobious), and Torin-1 (LC Labs) were used to inhibit HIF-1, mTORC1, mTORC2, and mTOR signaling respectively. Cytokine levels were quantified using cytometric bead array (BD) and prostaglandin-E2 (PGE 2 ) levels were quantified using ELISA (Cayman). Metabolic flux was measured using the Seahorse platform (Agilent). Results/Conclusions: HIF-1a was necessary for the production of inflammatory and reparative cytokines, consistent with previous literature. Inhibition of mTORC1 or mTORC2 had little effect on cytokine levels, while broad inhibition of mTOR resulted in a step-wise decrease in chemokine secretion. mTORC2 was required for the production of PGE 2 (p<0.01; Figure 1A), a lipid mediator highly expressed in ICH patients with good outcomes. This effect was dependent on HIF-1a signaling (p<0.01; Figure 1A) and resulted in an increase in glycolytic flux (p<0.001; Figure 1B), suggesting a neuroprotective mTORC2-HIF-PGE 2 axis. Future research will explore the role of HIF-1a and mTORC2 in murine ICH models using pharmacological inhibition and LysM-Cre HIF-1a-floxed mixed bone marrow chimeras.

Targeting ATR in TP53-Mutant AML/MDS

Mutations of TP53 are found in approximately 10% of cases of de novo AML/MDS and approximately 30% of therapy related AML/MDS. These patients have an exceptionally poor prognosis, with median survival of 6-9 months. Many patients with TP53-mutant AML/MDS respond to hypomethylating agents, although the response is not durable, similar to standard induction therapy. The mechanisms that contribute to the relative sensitivity of TP53-mutated AML/MDS to hypomethylating agents is unknown. Hypomethylating agents work, at least in part, by inhibiting DNMT1, the major DNA methyltransferase responsible for maintaining methylation of genomic DNA. Loss of DNMT1 and DNA hypomethylation has been previously shown to induce DNA replication stress and activate the ATM-Rad3-related (ATR) pathway. TP53 has multiple roles in the replication stress response, including control of the G1/S checkpoint and regulation of DNA replication progression and restart. Based on these observations, we hypothesize that ATR inhibition selectively sensitizes TP53-mutated MDS/AML to decitabine-induced replication stress. To test this hypothesis, initial studies focused on MOLM13 cells, a TP53-wildtype human AML cell line. Treatment with decitabine for 4 days induced global hypomethylation, and increased phosphorylation of CHK1, which is indicative of ATR activation. We next performed single-molecule DNA fiber assays to assess replication fork progression after exposure to decitabine. We used CRISPR-Cas9 gene editing to generate TP53-/- (or control) MOLM13 cells. In untreated cells, no difference in replication fork progression rate was observed between wildtype and TP53-/- MOLM13 cells (Figure 1). However, after decitabine treatment, a significant decrease in replication fork length was observed in TP53-/- MOLM13, but not wildtype MOLM13 cells. These data show that p53 is required for the efficient resolution of replication stress induced by decitabine. Since ATR is a major regulator of replication stress, we next examined the response of TP53-/- MOLM13 cells to decitabine alone, AZD6738 (an ATR inhibitor) alone, or the combination. Compared to controls, TP53-/- MOLM13 cells showed a modest increased sensitivity to decitabine, but not AZD6738. However, strong synergistic killing was observed with the combination in TP53-/-, but not control, MOLM13 cells (Zip synergy score of 10.1 vs 5.8). Similar results were observed in a second cell line, murine AML1. Cell cycle analysis showed that combined treatment results in a selective increase in polyploid TP53-/- MOLM13 cells, which is suggestive of mitotic catastrophe. We next developed a xenotransplantation model of TP53-mutated AML by transplanting TP53-/- or control MOLM13 cells into NSG mice. After 4 days to allow for engraftment, mice were treated with vehicle alone, decitabine alone (0.1mg/kg), AZD6738 alone (50mg/kg), or the combination for 14 days. In mice engrafted with wildtype MOLM13 cells, no significant benefit of single agent or combination therapy was observed. In contrast, treatment with decitabine alone modestly, but significantly, prolonged survival in mice engrafted with TP53-/- MOLM13 cells (Figure 2). Importantly, the survival of mice treated with combined AZD6738 and decitabine had a significantly prolonged survival compared to decitabine alone. These data show that even a single cycle of AZD6738 and decitabine is sufficient to prolong survival in the xenograft model of human AML. Finally, to assess the impact of combination treatment on TP53-mutant hematopoietic stem cells (HSCs), we established mixed bone marrow chimeras transplanted with both wildtype and R172H TP53 bone marrow. Mice were treated with either cis-platinum or decitabine ± AZD6738. Whereas cis-platinum treatment resulted in a significant expansion of TP53-mutant HSCs, treatment with decitabine plus AZD6738 selectively inhibited TP53-mutant HSCs. Collectively, these data support the hypothesis that TP53-mutated AML/MDS is selectively sensitive to ATR inhibition in combination with decitabine-induced replicative stress. A clinical trial of this drug combination in patients with TP53-mutated AML/MDS is being developed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Adoptive Transfer and Bone Marrow Chimera Models to Analyze Treg Function and Differentiation.

Cellular adoptive transfer and mixed bone marrow chimera are cornerstone experimental tools for immuno-biology. Here we describe protocols for adoptive transfer and bone marrow chimera to address the effect of a specific mutation on T regulatory cell (Treg) function and differentiation, respectively. Treg function can be quantitatively measured by analyzing the expansion of conventional CD4 T cells and their differentiation into helper cells. The quantitative measure of Treg differentiation is addressed by analyzing the number and phenotype of Foxp3-expressing cells. The use of congenic markers is instrumental for these approaches.

IL-4/IL-13-producing ILC2s are required for timely control of intestinal helminth infection in mice.

Infection of mice with Nippostrongylus brasiliensis (Nb) serves as a model for human hookworm infection affecting about 600 million people world-wide. Expulsion of Nb from the intestine requires IL-13-mediated mucus secretion from goblet cells and activation of smooth muscles cells. Type 2 innate lymphoid cells (ILC2s) are a major cellular source of IL-13 but it remains unclear whether IL-13 secretion from ILC2s is required for Nb expulsion. Here, we compared the immune response to Nb infection in mixed bone marrow chimeras with wild-type or IL-4/IL-13-deficient ILC2s. ILC2-derived IL-4/IL-13 was required for recruitment of eosinophils to the lung but had no influence of systemic eosinophil levels. In the small intestine, goblet cell hyperplasia and tuft cell accumulation was largely dependent on IL-4/IL-13 secretion from ILC2s. This further translated to higher eggs counts and impaired worm expulsion in mice with IL-4/IL-13-deficient ILC2s. Overall, we demonstrate that ILC2s constitute a non-redundant source of IL-4/IL-13 required for protective immunity against primary Nb infection.