OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism.

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

Nasopharyngeal Exposure to Streptococcus pneumoniae Induces Extended Age-Dependent Protection against Pulmonary Infection Mediated by Antibodies and CD138+ Cells.

Streptococcus pneumoniae commonly resides asymptomatically in the nasopharyngeal (NP) cavity of healthy individuals but can cause life-threatening pulmonary and systemic infections, particularly in the elderly. NP colonization results in a robust immune response that protects against invasive infections. However, the duration, mechanism, and cellular component of such responses are poorly understood. In this study, we found that repeated NP exposure of mice to S. pneumoniae TIGR4 strain results in pneumococcal-specific Ab responses that protect against lethal lung challenge. Abs were necessary and sufficient for protection because Ab-deficient µMT mice did not develop postexposure protection, only becoming resistant to lung infection after transfer of immune sera from NP-exposed mice. T cells contributed to immunity at the time of NP exposure, but neither CD4+ nor CD8+ T cells were required. The protective activity was detectable 20 wk after exposure and was maintained in irradiated mice, suggesting involvement of long-lived Ab-secreting cells (ASC), which are radioresistant and secrete Abs for extended periods of time in the absence of T cells or persistent Ag. CD138+ bone marrow cells, likely corresponding to long-lived ASC, were sufficient to confer protection. NP exposure of aged mice failed to protect against subsequent lung infection despite eliciting a robust Ab response. Furthermore, transfer of CD138+ bone marrow cells or sera from NP-exposed old mice failed to protect naive young mice. These findings suggest that NP exposure elicits extended protection against pneumococcal lung infection by generating long-lived CD138+ ASC and that the protective efficacy of these responses declines with age.

Alcohol-induced adipose tissue macrophage phenotypic switching is independent of myeloid Toll-like receptor 4 expression.

Alcoholic liver disease results from a combination of immune and metabolic pathogenic events. In addition to liver injury, chronic alcohol consumption also causes adipose tissue inflammation. The specific immune mechanisms that drive this process are unknown. Here, we sought to determine the role of the innate immune receptor Toll-like receptor 4 (TLR4) in alcohol-induced adipose tissue inflammation. Using a model of chronic, multiple-binge alcohol exposure, we showed that alcohol-mediated accumulation of proinflammatory adipose tissue macrophages was absent in global TLR4 knockout mice. Proinflammatory macrophage accumulation did not depend on macrophage TLR4 expression; LysMCre-driven deletion of Tlr4 from myeloid cells did not affect circulating endotoxin or the accumulation of M1 macrophages in adipose tissue following alcohol exposure. Proinflammatory cytokine/chemokine production in the adipose stromal vascular fraction also occurred independently of TLR4. Finally, the levels of other adipose immune cells, such as dendritic cells, neutrophils, B cells, and T cells, were modulated by chronic, multiple-binge alcohol and the presence of TLR4. Together, these data indicate that TLR4 expression on cells, other than myeloid cells, is important for the alcohol-induced increase in proinflammatory adipose tissue macrophages.

YY1 Regulates the Germinal Center Reaction by Inhibiting Apoptosis.

The germinal center (GC) reaction produces high-affinity Abs for a robust adaptive immune response. When dysregulated, the same processes cause GC B cells to become susceptible to lymphomagenesis. It is important to understand how the GC reaction is regulated. In this study, we show that transcription factor YY1 is required to maintain a robust GC reaction in mice. Selective ablation of YY1 significantly decreased in the frequency and number of GC B cells during the GC reaction. This decrease of GC B cells was accompanied by increased apoptosis in these cells. Furthermore, we found that loss of YY1 disrupted the balance between dark zones and light zones, leading to a preferential decrease in dark zone cells. Collectively, these results indicate that YY1 plays an important role in regulating the balance between dark zone and light zone cells in GCs and between survival and death of GC B cells.

Localized DNA demethylation at recombination intermediates during immunoglobulin heavy chain gene assembly.

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D(H)) and joining (J(H)) gene segments were methylated, DJ(H) junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eµ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.

Enhancing T lineage production in aged mice: a novel function of Foxn1 in the bone marrow niche.

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. Whereas reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with early T cell progenitors (ETP), derived from multipotent progenitors (MPP) in the bone marrow (BM). A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1 transgenic (Tg); whether the effect is initiated in the BM with MPP is not known. In this study, we report that Foxn1 is expressed in wild-type BM and overexpressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of hematopoietic stem cells. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, common lymphoid progenitors and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared with wild type. We identified a novel population of lineage(neg/low), CD45(pos) EpCAM(pos), SCA1(pos), CD117(neg), CD138(neg), MHCII(neg) cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, and the Foxn1 BM niche may function in skewing MPP development toward T lineage progenitors.

Emerging Cerebrospinal Fluid Biomarkers of Disease Activity and Progression in Multiple Sclerosis.

Importance: Biomarkers distinguishing nonrelapsing progressive disease biology from relapsing biology in multiple sclerosis (MS) are lacking. Cerebrospinal fluid (CSF) is an accessible fluid that most closely reflects central nervous system biology. Objective: To identify CSF biological measures associated with progressive MS pathobiology. Design, Setting, and Participants: This cohort study assessed data from 2 prospective MS cohorts: a test cohort provided serial CSF, clinical, and imaging assessments in a multicenter study of patients with relapsing MS (RMS) or primary progressive MS (PPMS) who were initiating anti-CD20 treatment (recruitment: 2016-2018; analysis: 2020-2023). A single-site confirmation cohort was used to assess CSF at baseline and long-term (>10 year) clinical follow-up (analysis: 2022-2023). Exposures: Test-cohort participants initiated standard-of-care ocrelizumab treatment. Confirmation-cohort participants were untreated or received standard-of-care disease-modifying MS therapies. Main Outcomes and Measures: Twenty-five CSF markers, including neurofilament light chain, neurofilament heavy chain, and glial fibrillary acid protein (GFAP); 24-week confirmed disability progression (CDP24); and brain magnetic resonance imaging measures reflecting focal injury, tissue loss, and progressive biology (slowly expanding lesions [SELs]). Results: The test cohort (n = 131) included 100 patients with RMS (mean [SD] age, 36.6 [10.4] years; 68 [68%] female and 32 [32%] male; Expanded Disability Status Scale [EDSS] score, 0-5.5), and 31 patients with PPMS (mean [SD] age, 44.9 [7.4] years; 15 [48%] female and 16 [52%] male; EDSS score, 3.0-6.5). The confirmation cohort (n = 68) included 41 patients with RMS and 27 with PPMS enrolled at diagnosis (age, 40 years [range, 20-61 years]; 47 [69%] female and 21 [31%] male). In the test cohort, GFAP was correlated with SEL count (r = 0.33), greater proportion of T2 lesion volume from SELs (r = 0.24), and lower T1-weighted intensity within SELs (r = -0.33) but not with acute inflammatory measures. Neurofilament heavy chain was correlated with SEL count (r = 0.25) and lower T1-weighted intensity within SELs (r = -0.28). Immune markers correlated with measures of acute inflammation and, unlike GFAP, were impacted by anti-CD20. In the confirmation cohort, higher baseline CSF GFAP levels were associated with long-term CDP24 (hazard ratio, 2.1; 95% CI, 1.3-3.4; P = .002). Conclusions and Relevance: In this study, activated glial markers (in particular GFAP) and neurofilament heavy chain were associated specifically with nonrelapsing progressive disease outcomes (independent of acute inflammatory activity). Elevated CSF GFAP was associated with long-term MS disease progression.

Notch1 inhibition targets the leukemia-initiating cells in a Tal1/Lmo2 mouse model of T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy largely caused by aberrant activation of the TAL1/SCL, LMO1/2, and NOTCH1 oncogenes. Approximately 30% of T-ALL patients relapse, and evidence is emerging that relapse may result from a failure to eliminate leukemia-initiating cells (LICs). Thymic expression of the Tal1 and Lmo2 oncogenes in mice results in rapid development of T-ALL; and similar to T-ALL patients, more than half the leukemic mice develop spontaneous mutations in Notch1. Using this mouse model, we demonstrate that mouse T-ALLs are immunophenotypically and functionally heterogeneous with approximately 1 of 10,000 leukemic cells capable of initiating disease on transplantation. Our preleukemic studies reveal expansion of Notch-active double-negative thymic progenitors, and we find the leukemic DN3 population enriched in disease potential. To examine the role of Notch1 in LIC function, we measured LIC activity in leukemic mice treated with vehicle or with a γ-secretase inhibitor. In 4 of 5 leukemias examined, Notch inhibition significantly reduced or eliminated LICs and extended survival. Remarkably, in 2 mice, γ-secretase inhibitor treatment reduced LIC frequency below the limits of detection of this assay, and all transplanted mice failed to develop disease. These data support the continued development of Notch1 therapeutics as antileukemia agents.

Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge.

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.

Endemic Burkitt lymphoma avatar mouse models for exploring inter-patient tumor variation and testing targeted therapies.

Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children.

Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue.

Wnt5a is a member of the Wnt family of secreted glycoproteins that play essential organizing roles in development. Similar to other Wnt members, Wnt5a can upregulate cell proliferation and has been proposed to have oncogenic function. Here we report that Wnt5a signals through the noncanonical Wnt/Ca++ pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation in a cell-autonomous manner. Wnt5a hemizygous mice develop myeloid leukemias and B cell lymphomas that are clonal in origin and display loss of Wnt5a function in tumor tissues. Furthermore, analysis of human primary leukemias reveals deletion of the WNT5A gene and/or loss of WNT5A expression in a majority of the patient samples. These results demonstrate that Wnt5a suppresses hematopoietic malignancies.

E47 is required for V(D)J recombinase activity in common lymphoid progenitors.

Common lymphoid progenitors (CLPs) are the first bone marrow precursors in which V(D)J recombinase activity is up-regulated. Here, we show that loss of the transcription factor E47 produces a reduced CLP population that lacks V(D)J recombinase activity and D-J(H) rearrangements in vivo. Apart from a profound arrest before the pro-B cell stage, other downstream lymphoid progeny of CLPs are still intact in these mice albeit at reduced numbers. In contrast to the inhibition of recombinase activity in early B lineage precursors in E47-deficient animals, loss of either E47 or its cis-acting target Erag (enhancer of rag transcription) has little effect on recombinase activity in thymic T lineage precursors. Taken together, this work defines a role for E47 in regulating lineage progression at the CLP stage in vivo and describes the first transcription factor required for lineage-specific recombinase activity.

B cell receptor-mediated sustained c-Rel activation facilitates late transitional B cell survival through control of B cell activating factor receptor and NF-kappaB2.

Signaling from the BCR and B cell activating factor receptor (BAFF-R or BR3) differentially regulates apoptosis within early transitional (T1) and late transitional (T2; CD21(int)-T2) B cells during selection processes to generate mature B lymphocytes. However, molecular mechanisms underlying the differential sensitivity of transitional B cells to apoptosis remain unclear. In this study, we demonstrate that BCR signaling induced more long-term c-Rel activation in T2 and mature than in T1 B cells leading to increased expression of anti-apoptotic genes as well as prosurvival BAFF-R and its downstream substrate p100 (NF-kappaB2). Sustained c-Rel activation required de novo c-Rel gene transcription and translation via Btk-dependent mechanisms. Like T1 cells, mature B cells from Btk- and c-Rel-deficient mice also failed to activate these genes. These findings suggest that the gain of survival potential within transitional B cells is dependent on the ability to produce a long-term c-Rel response, which plays a critical role in T2 B cell survival and differentiation in vivo by inducing anti-apoptotic genes, BAFF-R and NF-kappaB2, an essential component for BAFF-R survival signaling. Thus, acquisition of resistance to apoptosis during transitional B cell maturation is achieved by integration of BCR and BAFF-R signals.

Developmental separation of V(D)J recombinase expression and initiation of IgH recombination in B lineage progenitors in vivo.

In B lineage progenitors, V(D)J recombination occurs only during distinct stages of development and is restricted to immunoglobulin loci. This process is thought to be controlled by both regulated expression of the V(D)J recombinase and by limited accessibility of target loci to the recombinase complex. However, it is unknown whether these two processes occur concomitantly in developing B lineage progenitors or whether these events are temporally distinct and, therefore, potentially independently regulated. To distinguish between these possibilities, we developed a transgenic V(D)J recombination substrate that is not governed by the same chromatin remodeling constraints as endogenous immunoglobulin heavy chain (IgH) loci and examined the requirements for V(D)J recombination to initiate in early B lineage progenitors. We find that single B lineage precursors express an active V(D)J recombinase in vivo before the stage when IgH rearrangements are frequently detectable. Our results indicate that the onset of recombinase activity and the initiation of IgH recombination are developmentally distinct events in the B lineage.

The earliest step in B lineage differentiation from common lymphoid progenitors is critically dependent upon interleukin 7.

Little is known about the signals that promote early B lineage differentiation from common lymphoid progenitors (CLPs). Using a stromal-free culture system, we show that interleukin (IL)-7 is sufficient to promote the in vitro differentiation of CLPs into B220(+) CD19(+) B lineage progenitors. Consistent with current models of early B cell development, surface expression of B220 was initiated before CD19 and was accompanied by the loss of T lineage potential. To address whether IL-7 receptor (R) activity is essential for early B lineage development in vivo, we examined the frequencies of CLPs and downstream pre-pro- and pro-B cells in adult mice lacking either the alpha chain or the common gamma chain (gamma(c)) of the IL-7R. The data indicate that although gamma(c)(-/-) mice have normal frequencies of CLPs, both gamma(c)(-/-) and IL-7R(alpha)(-/-) mice lack detectable numbers of all downstream early B lineage precursors, including pre-pro-B cells. These findings challenge previous notions regarding the point in B cell development affected by the loss of IL-7R signaling and suggest that IL-7 plays a key and requisite role during the earliest phases of B cell development.

Bone marrow microenvironmental changes underlie reduced RAG-mediated recombination and B cell generation in aged mice.

During aging, adaptive immunity is severely compromised, due in part to decreased production of B lymphocytes and loss of immunoglobulin (Ig) diversity. However, the molecular mechanisms that underlie age-associated diminished B cell production remain unclear. Using in vivo labeling, we find that this reduction in marrow pre-B cells reflects increased attrition during passage from the pro-B to pre-B cell pool. Analyses of reciprocal bone marrow chimeras reveal that the magnitude and production rates of pre-B cells are controlled primarily by microenvironmental factors, rather than intrinsic events. To understand changes in pro-B cells that could diminish production of pre-B cells, we evaluated rag2 expression and V(D)J recombinase activity in pro-B cells at the single cell level. The percentage of pro-B cells that express rag2 is reduced in aged mice and is correlated with both a loss of V(D)J recombinase activity in pro-B cells and reduced numbers of pre-B cells. Reciprocal bone marrow chimeras revealed that the aged microenvironment also determines rag2 expression and recombinase activity in pro-B cells. Together, these observations suggest that extrinsic factors in the bone marrow that decline with age are largely responsible for less efficient V(D)J recombination in pro-B cells and diminished progression to the pre-B cell stage.

B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors.

Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals.

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.