Can genetic and clinical findings made in a single patient be considered sufficient to establish a causal relationship between genotype and phenotype? We report that up to 49 of the 232 monogenic etiologies (21%) of human primary immunodeficiencies (PIDs) were initially reported in single patients. The ability to incriminate single-gene inborn errors in immunodeficient patients results from the relative ease in validating the disease-causing role of the genotype by in-depth mechanistic studies demonstrating the structural and functional consequences of the mutations using blood samples. The candidate genotype can be causally connected to a clinical phenotype using cellular (leukocytes) or molecular (plasma) substrates. The recent advent of next generation sequencing (NGS), with whole exome and whole genome sequencing, induced pluripotent stem cell (iPSC) technology, and gene editing technologies—including in particular the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology—offer new and exciting possibilities for the genetic exploration of single patients not only in hematology and immunology but also in other fields. We propose three criteria for deciding if the clinical and experimental data suffice to establish a causal relationship based on only one case. The patient’s candidate genotype must not occur in individuals without the clinical phenotype. Experimental studies must indicate that the genetic variant impairs, destroys, or alters the expression or function of the gene product (or two genetic variants for compound heterozygosity). The causal relationship between the candidate genotype and the clinical phenotype must be confirmed via a relevant cellular phenotype, or by default via a relevant animal phenotype. When supported by satisfaction of rigorous criteria, the report of single patient–based discovery of Mendelian disorders should be encouraged, as it can provide the first step in the understanding of a group of human diseases, thereby revealing crucial pathways underlying physiological and pathological processes.
Cardiac macrophages (cM) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1+ cM diversifies into MHCII+ and MHCII– cells. Genetic fate mapping demonstrated that cM derived from CX3CR1+ embryonic progenitors persisted into adulthood but the initially high contribution to resident cM declined after birth. Consistent with this, the early significant proliferation rate of resident cM decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cM populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cM. Together, our observations indicate that in the heart, embryo-derived cM show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.
The (patho)physiological role of IgE in nonallergic inflammatory diseases is not well understood. Here, we explored the effect of IgE deficiency on the inflammatory response in FcRIIB-deficient mice as well as in mice carrying both a deletion of FcRIIB and the chromosomal translocation of Y-linked autoimmune acceleration (Yaa) that hastens and results in a more aggressive lupuslike disease in these mice. The findings show that deficiency of IgE delays disease development and severity as demonstrated by reduced autoantibody production and amelioration of organ pathologies. This was associated with decreased numbers of plasma cells and reduced levels of IgG2b and IgG3. Unexpectedly, the loss of IgE also caused a striking decrease of immune cell infiltration in secondary lymphoid organs with a marked effect on the presence of dendritic cells, monocytes, neutrophils, and eosinophils in these organs and decreased activation of basophils. The presence of autoreactive IgE in human systemic lupus erythematosus subjects was also associated with increased basophil activation and enhanced disease activity. These findings argue that IgE facilitates the amplification of autoimmune inflammation.
Activated B cells undergo immunoglobulin class-switch recombination (CSR) and differentiate into antibody-secreting plasma cells. The distinct transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors: those that maintain the B cell program, including BCL6 and PAX5, and plasma cell–promoting factors, such as IRF4 and BLIMP-1. We show that the complex of IRF8 and PU.1 controls the propensity of B cells to undergo CSR and plasma cell differentiation by concurrently promoting the expression of BCL6 and PAX5 and repressing AID and BLIMP-1. As the PU.1–IRF8 complex functions in a reciprocal manner to IRF4, we propose that concentration-dependent competition between these factors controls B cell terminal differentiation.
A single microRNA (miRNA) can regulate the expression of many genes, though the level of repression imparted on any given target is generally low. How then is the selective pressure for a single miRNA/target interaction maintained across long evolutionary distances? We addressed this problem by disrupting in vivo the interaction between miR-155 and PU.1 in mice. Remarkably, this interaction proved to be key to promoting optimal T cell–dependent B cell responses, a previously unrecognized role for PU.1. Mechanistically, miR-155 inhibits PU.1 expression, leading to Pax5 down-regulation and the initiation of the plasma cell differentiation pathway. Additional PU.1 targets include a network of genes whose products are involved in adhesion, with direct links to B–T cell interactions. We conclude that the evolutionary adaptive selection of the miR-155–PU.1 interaction is exercised through the effectiveness of terminal B cell differentiation.
The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte–induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell–specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.
The majority of patients with myeloproliferative neoplasms (MPNs) carry a somatic JAK2-V617F mutation. Because additional mutations can precede JAK2-V617F, it is questioned whether JAK2-V617F alone can initiate MPN. Several mouse models have demonstrated that JAK2-V617F can cause MPN; however, in all these models disease was polyclonal. Conversely, cancer initiates at the single cell level, but attempts to recapitulate single-cell disease initiation in mice have thus far failed. We demonstrate by limiting dilution and single-cell transplantations that MPN disease, manifesting either as erythrocytosis or thrombocytosis, can be initiated clonally from a single cell carrying JAK2-V617F. However, only a subset of mice reconstituted from single hematopoietic stem cells (HSCs) displayed MPN phenotype. Expression of JAK2-V617F in HSCs promoted cell division and increased DNA damage. Higher JAK2-V617F expression correlated with a short-term HSC signature and increased myeloid bias in single-cell gene expression analyses. Lower JAK2-V617F expression in progenitor and stem cells was associated with the capacity to stably engraft in secondary recipients. Furthermore, long-term repopulating capacity was also present in a compartment with intermediate expression levels of lineage markers. Our studies demonstrate that MPN can be initiated from a single HSC and illustrate that JAK2-V617F has complex effects on HSC biology.
The mutational repertoire of cancers creates the neoepitopes that make cancers immunogenic. Here, we introduce two novel tools that identify, with relatively high accuracy, the small proportion of neoepitopes (among the hundreds of potential neoepitopes) that protect the host through an antitumor T cell response. The two tools consist of (a) the numerical difference in NetMHC scores between the mutated sequences and their unmutated counterparts, termed the differential agretopic index, and (b) the conformational stability of the MHC I–peptide interaction. Mechanistically, these tools identify neoepitopes that are mutated to create new anchor residues for MHC binding, and render the overall peptide more rigid. Surprisingly, the protective neoepitopes identified here elicit CD8-dependent immunity, even though their affinity for Kd is orders of magnitude lower than the 500-nM threshold considered reasonable for such interactions. These results greatly expand the universe of target cancer antigens and identify new tools for human cancer immunotherapy.
Genetic studies have shown that the tuberous sclerosis complex (TSC) 1–TSC2–mammalian target of Rapamycin (mTOR) and the Hippo–Yes-associated protein 1 (YAP) pathways are master regulators of organ size, which are often involved in tumorigenesis. The crosstalk between these signal transduction pathways in coordinating environmental cues, such as nutritional status and mechanical constraints, is crucial for tissue growth. Whether and how mTOR regulates YAP remains elusive. Here we describe a novel mouse model of TSC which develops renal mesenchymal lesions recapitulating human perivascular epithelioid cell tumors (PEComas) from patients with TSC. We identify that YAP is up-regulated by mTOR in mouse and human PEComas. YAP inhibition blunts abnormal proliferation and induces apoptosis of TSC1–TSC2-deficient cells, both in culture and in mosaic Tsc1 mutant mice. We further delineate that YAP accumulation in TSC1/TSC2-deficient cells is due to impaired degradation of the protein by the autophagosome/lysosome system. Thus, the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth-permissive conditions. YAP may serve as a potential therapeutic target for TSC and other diseases with dysregulated mTOR activity.
Fibroblast-like cells of secondary lymphoid organs (SLO) are important for tissue architecture. In addition, they regulate lymphocyte compartmentalization through the secretion of chemokines, and participate in the orchestration of appropriate cell–cell interactions required for adaptive immunity. Here, we provide data demonstrating the functional importance of SLO fibroblasts during Notch-mediated lineage specification and immune response. Genetic ablation of the Notch ligand Delta-like (DL)1 identified splenic fibroblasts rather than hematopoietic or endothelial cells as niche cells, allowing Notch 2–driven differentiation of marginal zone B cells and of Esam+ dendritic cells. Moreover, conditional inactivation of DL4 in lymph node fibroblasts resulted in impaired follicular helper T cell differentiation and, consequently, in reduced numbers of germinal center B cells and absence of high-affinity antibodies. Our data demonstrate previously unknown roles for DL ligand-expressing fibroblasts in SLO niches as drivers of multiple Notch-mediated immune differentiation processes.
T cell receptor (TCR) signals can elicit full activation with acquisition of effector functions or a state of anergy. Here, we ask whether microRNAs affect the interpretation of TCR signaling. We find that Dicer-deficient CD4 T cells fail to correctly discriminate between activating and anergy-inducing stimuli and produce IL-2 in the absence of co-stimulation. Excess IL-2 production by Dicer-deficient CD4 T cells was sufficient to override anergy induction in WT T cells and to restore inducible Foxp3 expression in Il2-deficient CD4 T cells. Phosphorylation of Akt on S473 and of S6 ribosomal protein was increased and sustained in Dicer-deficient CD4 T cells, indicating elevated mTOR activity. The mTOR components Mtor and Rictor were posttranscriptionally deregulated, and the microRNAs Let-7 and miR-16 targeted the Mtor and Rictor mRNAs. Remarkably, returning Mtor and Rictor to normal levels by deleting one allele of Mtor and one allele of Rictor was sufficient to reduce Akt S473 phosphorylation and to reduce co-stimulation–independent IL-2 production in Dicer-deficient CD4 T cells. These results show that microRNAs regulate the expression of mTOR components in T cells, and that this regulation is critical for the modulation of mTOR activity. Hence, microRNAs contribute to the discrimination between T cell activation and anergy.
Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo–activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.
Dectin-1 functions as a pattern recognition receptor for sensing fungal infection. It has been well-established that Dectin-1 induces innate immune responses through caspase recruitment domain-containing protein 9 (CARD9)–mediated NF-B activation. In this study, we find that CARD9 is dispensable for NF-B activation induced by Dectin-1 ligands, such as curdlan or Candida albicans yeast. In contrast, we find that CARD9 regulates H-Ras activation by linking Ras-GRF1 to H-Ras, which mediates Dectin-1–induced extracellular signal-regulated protein kinase (ERK) activation and proinflammatory responses when stimulated by their ligands. Mechanistically, Dectin-1 engagement initiates spleen tyrosine kinase (Syk)–dependent Ras-GRF1 phosphorylation, and the phosphorylated Ras-GRF1 recruits and activates H-Ras through forming a complex with CARD9, which leads to activation of ERK downstream. Finally, we show that inhibiting ERK activation significantly accelerates the death of C. albicans–infected mice, and this inhibitory effect is dependent on CARD9. Together, our studies reveal a molecular mechanism by which Dectin-1 induces H-Ras activation that leads to ERK activation for host innate immune responses against fungal infection.