Hem1 inborn errors of immunity: waving goodbye to coordinated immunity in mice and humans.

Inborn errors of immunity (IEI) are a group of diseases in humans that typically present as increased susceptibility to infections, autoimmunity, hyperinflammation, allergy, and in some cases malignancy. Among newly identified genes linked to IEIs include 3 independent reports of 9 individuals from 7 independent kindreds with severe primary immunodeficiency disease (PID) and autoimmunity due to loss-of-function mutations in the NCKAP1L gene encoding Hematopoietic protein 1 (HEM1). HEM1 is a hematopoietic cell specific component of the WASp family verprolin homologous (WAVE) regulatory complex (WRC), which acts downstream of multiple immune receptors to stimulate actin nucleation and polymerization of filamentous actin (F-actin). The polymerization and branching of F-actin is critical for creating force-generating cytoskeletal structures which drive most active cellular processes including migration, adhesion, immune synapse formation, and phagocytosis. Branched actin networks at the cell cortex have also been implicated in acting as a barrier to regulate inappropriate vesicle (e.g. cytokine) secretion and spontaneous antigen receptor crosslinking. Given the importance of the actin cytoskeleton in most or all hematopoietic cells, it is not surprising that HEM1 deficient children present with a complex clinical picture that involves overlapping features of immunodeficiency and autoimmunity. In this review, we will provide an overview of what is known about the molecular and cellular functions of HEM1 and the WRC in immune and other cells. We will describe the common clinicopathological features and immunophenotypes of HEM1 deficiency in humans and provide detailed comparative descriptions of what has been learned about Hem1 disruption using constitutive and immune cell-specific mouse knockout models. Finally, we discuss future perspectives and important areas for investigation regarding HEM1 and the WRC.

Hem-1 regulates protective humoral immunity and limits autoantibody production in a B cell-specific manner.

Hematopoietic protein-1 (Hem-1) is a member of the actin-regulatory WASp family verprolin homolog (WAVE) complex. Loss-of-function variants in the NCKAP1L gene encoding Hem-1 were recently discovered to result in primary immunodeficiency disease (PID) in children, characterized by poor specific Ab responses, increased autoantibodies, and high mortality. However, the mechanisms of how Hem-1 deficiency results in PID are unclear. In this study, we utilized constitutive and B cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in B cell development and functions. B cell-specific disruption of Hem-1 resulted in reduced numbers of recirculating follicular (FO), marginal zone (MZ), and B1 B cells. B cell migration in response to CXCL12 and -13 were reduced. T-independent Ab responses were nearly abolished, resulting in failed protective immunity to Streptococcus pneumoniae challenge. In contrast, T-dependent IgM and IgG2c, memory B cell, and plasma cell responses were more robust relative to WT control mice. B cell-specific Hem-1-deficient mice had increased autoantibodies against multiple autoantigens, and this correlated with hyperresponsive BCR signaling and increased representation of CD11c+T-bet+ age-associated B cell (ABC cells) - alterations associated with autoimmune diseases. These results suggest that dysfunctional B cells may be part of a mechanism explaining why loss-of-function Hem-1 variants result in recurring infections and autoimmunity.

The actin-regulatory protein Hem-1 is essential for alveolar macrophage development.

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell-specific actin-regulatory protein. Loss-of-function (LOF) variants in the NCKAP1L gene encoding Hem-1 have recently been found to result in primary immunodeficiency disease (PID) in humans, characterized by recurring respiratory infections, asthma, and high mortality. However, the mechanisms of how Hem-1 variants result in PID are not known. In this study, we generated constitutive and myeloid cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in lung immunity. We found that Hem-1-deficient mice accumulated excessive surfactant and cell debris in airways (pulmonary alveolar proteinosis) due to impaired development of alveolar macrophages (AMs) and reduced expression of the AM differentiation factor Pparg. Residual Hem-1-deficient AMs shifted to a proinflammatory phenotype, and Hem-1-deficient neutrophils and monocytes failed to migrate normally. Myeloid cell-specific Hem-1-deficient mice exhibited increased morbidity following influenza A virus or Streptococcus pneumoniae challenge. These results provide potential mechanisms for how LOF variants in Hem-1 result in recurring respiratory diseases.

Folliculin Interacting Protein 1 Maintains Metabolic Homeostasis during B Cell Development by Modulating AMPK, mTORC1, and TFE3.

Folliculin interacting protein 1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with the master metabolic sensor 5' AMP-activated protein kinase (AMPK) and Folliculin, a protein mutated in individuals with Birt-Hogg-Dubé Syndrome. In response to low energy, AMPK stimulates catabolic pathways such as autophagy to enhance energy production while inhibiting anabolic pathways regulated by the mechanistic target of rapamycin complex 1 (mTORC1). We previously found that constitutive disruption of Fnip1 in mice resulted in a lack of peripheral B cells because of a block in B cell development at the pre-B cell stage. Both AMPK and mTORC1 were activated in Fnip1-deficient B cell progenitors. In this study, we found inappropriate mTOR localization at the lysosome under nutrient-depleted conditions. Ex vivo lysine or arginine depletion resulted in increased apoptosis. Genetic inhibition of AMPK, inhibition of mTORC1, or restoration of cell viability with a Bcl-xL transgene failed to rescue B cell development in Fnip1-deficient mice. Fnip1-deficient B cell progenitors exhibited increased nuclear localization of transcription factor binding to IgHM enhancer 3 (TFE3) in developing B cells, which correlated with an increased expression of TFE3-target genes, increased lysosome numbers and function, and increased autophagic flux. These results indicate that Fnip1 modulates autophagy and energy response pathways in part through the regulation of AMPK, mTORC1, and TFE3 in B cell progenitors.

Loss of Fnip1 alters kidney developmental transcriptional program and synergizes with TSC1 loss to promote mTORC1 activation and renal cyst formation.

Birt-Hogg-Dube' Syndrome (BHDS) is a rare genetic disorder in humans characterized by skin hamartomas, lung cysts, pneumothorax, and increased risk of renal tumors. BHDS is caused by mutations in the BHD gene, which encodes for Folliculin, a cytoplasmic adapter protein that binds to Folliculin interacting proteins-1 and -2 (Fnip1, Fnip2) as well as the master energy sensor AMP kinase (AMPK). Whereas kidney-specific deletion of the Bhd gene in mice is known to result in polycystic kidney disease (PKD) and renal cell carcinoma, the roles of Fnip1 in renal cell development and function are unclear. In this study, we utilized mice with constitutive deletion of the Fnip1 gene to show that the loss of Fnip1 is sufficient to result in renal cyst formation, which was characterized by decreased AMPK activation, increased mTOR activation, and metabolic hyperactivation. Using RNAseq, we found that Fnip1 disruption resulted in many cellular and molecular changes previously implicated in the development of PKD in humans, including alterations in the expression of ion and amino acid transporters, increased cell adhesion, and increased inflammation. Loss of Fnip1 synergized with Tsc1 loss to hyperactivate mTOR, increase Erk activation, and greatly accelerate the development of PKD. Our results collectively define roles for Fnip1 in regulating kidney development and function, and provide a model for how loss of Fnip1 contributes to PKD and perhaps renal cell carcinoma.

Murine norovirus inhibits B cell development in the bone marrow of STAT1-deficient mice.

Noroviruses are a leading cause of gastroenteritis in humans and it was recently revealed that noroviruses can infect B cells. We demonstrate that murine norovirus (MNV) infection can significantly impair B cell development in the bone marrow in a signal transducer and activator of transcription 1 (STAT1) dependent, but interferon signaling independent manner. We also show that MNV replication is more pronounced in the absence of STAT1 in ex vivo cultured B cells. Interestingly, using bone marrow transplantation studies, we found that impaired B cell development requires Stat1-/- hematopoietic cells and Stat1-/- stromal cells, and that the presence of wild-type hematopoietic or stromal cells was sufficient to restore normal development of Stat1-/- B cells. These results suggest that B cells normally restrain norovirus replication in a cell autonomous manner, and that wild-type STAT1 is required to protect B cell development during infection.

A functional genomics predictive network model identifies regulators of inflammatory bowel disease.

A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.

Control of B lymphocyte development and functions by the mTOR signaling pathways.

Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase originally discovered as the molecular target of the immunosuppressant rapamycin. mTOR forms two compositionally and functionally distinct complexes, mTORC1 and mTORC2, which are crucial for coordinating nutrient, energy, oxygen, and growth factor availability with cellular growth, proliferation, and survival. Recent studies have identified critical, non-redundant roles for mTORC1 and mTORC2 in controlling B cell development, differentiation, and functions, and have highlighted emerging roles of the Folliculin-Fnip protein complex in regulating mTOR and B cell development. In this review, we summarize the basic mechanisms of mTOR signaling; describe what is known about the roles of mTORC1, mTORC2, and the Folliculin/Fnip1 pathway in B cell development and functions; and briefly outline current clinical approaches for targeting mTOR in B cell neoplasms. We conclude by highlighting a few salient questions and future perspectives regarding mTOR in B lineage cells.

Distinct mechanisms of B and T lymphocyte accumulation generate tumor-draining lymph node hypertrophy.

Tumor-draining lymph nodes (TDLNs) often enlarge in human cancer patients and in murine tumor models, due to lymphocyte accumulation and lymphatic sinus growth. B lymphocytes within TDLNs can drive lymph node hypertrophy in response to tumor growth, however little is known about the mechanisms directing the preferential accumulation of B lymphocytes relative to T cells in enlarging TDLNs. To define why B and T lymphocytes accumulate in TDLNs, we quantified lymphocyte proliferation, apoptosis, entry, and exit in TDLNs versus contralateral non-TDLNs (NTDLNs) in a footpad B16-F10 melanoma mouse model. B and T lymphocyte proliferation and apoptosis were increased as the TDLNs enlarged, although relative rates were similar to those of NTDLNs. TDLN entry of B and T lymphocytes via high endothelial venules was also modestly increased in enlarged TDLNs. Strikingly, the egress of B cells was strongly reduced in TDLNs versus NTDLNs, while T cell egress was modestly decreased, indicating that regulation of lymphocyte exit from TDLNs is a major mechanism of preferential B lymphocyte accumulation. Surface sphingosine-1-phosphate receptor 1 (S1PR1) which binds S1P and signals lymphocyte egress, exhibited greater downregulation in B relative to T lymphocytes, consistent with preferential retention of B lymphocytes in TDLNs. TDLN lymphocytes did not activate surface CD69 expression, indicating a CD69-independent mechanism of downregulation of S1PR1. B and T cell trafficking via afferent lymphatics to enter TDLNs also increased, suggesting a pathway for accumulation of tumor-educated lymphocytes in TDLNs. These mechanisms regulating TDLN hypertrophy could provide new targets to manipulate lymphocyte responses to cancer.

Conditional Disruption of Raptor Reveals an Essential Role for mTORC1 in B Cell Development, Survival, and Metabolism.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.

Increased Susceptibility to Skin Carcinogenesis Associated with a Spontaneous Mouse Mutation in the Palmitoyl Transferase Zdhhc13 Gene.

Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis, and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared with wild-type (WT) epidermis in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than WT littermates. To our knowledge, this is the first report of a protective role for PAT in skin carcinogenesis.

Regulatory B cells preferentially accumulate in tumor-draining lymph nodes and promote tumor growth.

Our previous studies found that B16-F10 melanoma growth in the rear footpad of immunocompetent mice induces marked B cell accumulation within tumor-draining popliteal lymph nodes (TDLN). This B cell accumulation drives TDLN remodeling that precedes and promotes metastasis, indicating a tumor-promoting role for TDLN B cells. Here we show that phenotypic characterization of lymphocytes in mice bearing B16-F10 melanomas identifies preferential accumulation of T2-MZP B cells in the TDLN. Comparison of non-draining LNs and spleens of tumor-bearing mice with LNs and spleens from naïve mice determined that this pattern of B cell accumulation was restricted to the TDLN. B cell-deficient and immunocompetent mice reconstituted with T2-MZP B cells but not with other B cell subsets displayed accelerated tumor growth, demonstrating that T2-MZP B cells possess regulatory activity in tumor-bearing mice. Unlike splenic regulatory B cells, however, these TDLN B cells did not exhibit increased IL-10 production, nor did they promote Treg generation in the TDLN. These findings demonstrate that tumors initially signal via the lymphatic drainage to stimulate the preferential accumulation of T2-MZP regulatory B cells. This local response may be an early and critical step in generating an immunosuppressive environment to permit tumor growth and metastasis.

Fnip1 regulates skeletal muscle fiber type specification, fatigue resistance, and susceptibility to muscular dystrophy.

Mammalian skeletal muscle is broadly characterized by the presence of two distinct categories of muscle fibers called type I "red" slow twitch and type II "white" fast twitch, which display marked differences in contraction strength, metabolic strategies, and susceptibility to fatigue. The relative representation of each fiber type can have major influences on susceptibility to obesity, diabetes, and muscular dystrophies. However, the molecular factors controlling fiber type specification remain incompletely defined. In this study, we describe the control of fiber type specification and susceptibility to metabolic disease by folliculin interacting protein-1 (Fnip1). Using Fnip1 null mice, we found that loss of Fnip1 increased the representation of type I fibers characterized by increased myoglobin, slow twitch markers [myosin heavy chain 7 (MyH7), succinate dehydrogenase, troponin I 1, troponin C1, troponin T1], capillary density, and mitochondria number. Cultured Fnip1-null muscle fibers had higher oxidative capacity, and isolated Fnip1-null skeletal muscles were more resistant to postcontraction fatigue relative to WT skeletal muscles. Biochemical analyses revealed increased activation of the metabolic sensor AMP kinase (AMPK), and increased expression of the AMPK-target and transcriptional coactivator PGC1α in Fnip1 null skeletal muscle. Genetic disruption of PGC1α rescued normal levels of type I fiber markers MyH7 and myoglobin in Fnip1-null mice. Remarkably, loss of Fnip1 profoundly mitigated muscle damage in a murine model of Duchenne muscular dystrophy. These results indicate that Fnip1 controls skeletal muscle fiber type specification and warrant further study to determine whether inhibition of Fnip1 has therapeutic potential in muscular dystrophy diseases.

Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development.

Folliculin-interacting protein 1 (Fnip1) is an adaptor protein that physically interacts with AMPK, an energy-sensing kinase that stimulates mitochondrial biogenesis and autophagy in response to low ATP, while turning off energy consumption mediated by mammalian target of rapamycin. Previous studies with Fnip1-null mice revealed that Fnip1 is essential for pre-B-cell development. Here we report a critical role of Fnip1 in invariant natural killer T (iNKT) cell development. Thymic iNKT development in Fnip1(-/-) mice was arrested at stage 2 (NK1.1(-)CD44(+)) but development of CD4, CD8, γδ T-cell, and NK cell lineages proceeded normally. Enforced expression of a Vα14Jα18 iNKT TCR transgene or loss of the proapoptotic protein Bim did not rescue iNKT cell maturation in Fnip1(-/-) mice. Whereas most known essential transcription factors for iNKT cell development were represented normally, Fnip1(-/-) iNKT cells failed to down-regulate Promyelocytic leukemia zinc finger compared with their WT counterparts. Moreover, Fnip1(-/-) iNKT cells contained hyperactive mTOR and reduced mitochondrial number despite lower ATP levels, resulting in increased sensitivity to apoptosis. These results indicate that Fnip1 is vital for iNKT cell development by maintaining metabolic homeostasis in response to metabolic stress.

Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes.

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and ß- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1⁻/⁻ erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1⁻/⁻ erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

Interleukin-7 receptor blockade suppresses adaptive and innate inflammatory responses in experimental colitis.

BACKGROUND: Interleukin-7 (IL-7) acts primarily on T cells to promote their differentiation, survival, and homeostasis. Under disease conditions, IL-7 mediates inflammation through several mechanisms and cell types. In humans, IL-7 and its receptor (IL-7R) are increased in diseases characterized by inflammation such as atherosclerosis, rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel disease. In mice, overexpression of IL-7 results in chronic colitis, and T-cell adoptive transfer studies suggest that memory T cells expressing high amounts of IL-7R drive colitis and are maintained and expanded with IL-7. The studies presented here were undertaken to better understand the contribution of IL-7R in inflammatory bowel disease in which colitis was induced with a bacterial trigger rather than with adoptive transfer. METHODS: We examined the contribution of IL-7R on inflammation and disease development in two models of experimental colitis: Helicobacter bilis (Hb)-induced colitis in immune-sufficient Mdr1a-/- mice and in T- and B-cell-deficient Rag2-/- mice. We used pharmacological blockade of IL-7R to understand the mechanisms involved in IL-7R-mediated inflammatory bowel disease by analyzing immune cell profiles, circulating and colon proteins, and colon gene expression. RESULTS: Treatment of mice with an anti-IL-7R antibody was effective in reducing colitis in Hb-infected Mdr1a-/- mice by reducing T-cell numbers as well as T-cell function. Down regulation of the innate immune response was also detected in Hb-infected Mdr1a-/- mice treated with an anti-IL-7R antibody. In Rag2-/- mice where colitis was triggered by Hb-infection, treatment with an anti-IL-7R antibody controlled innate inflammatory responses by reducing macrophage and dendritic cell numbers and their activity. CONCLUSIONS: Results from our studies showed that inhibition of IL-7R successfully ameliorated inflammation and disease development in Hb-infected mice by controlling the expansion of multiple leukocyte populations, as well as the activity of these immune cells. Our findings demonstrate an important function of IL-7R-driven immunity in experimental colitis and indicate that the therapeutic efficacy of IL-7R blockade involves affecting both adaptive and innate immunity.

Evaluation of reference genes for quantitative PCR analysis of mouse lymphocytes.

Normalization to a reference gene is the method of choice for quantitative PCR analysis. The stability of reference genes is critical for accurate gene expression analysis, as significant variations in reference gene expression can alter experimental results and conclusions. In this study, we evaluated the expression stability of five commonly used reference genes found in mouse lymphocytes. Using NormFinder and BestKeeper algorithms, we consistently show that ubiquitin C (Ubc) is the optimal reference gene for normalizing qPCR data obtained from mouse lymphocytes, whereas beta-actin (Actb) is not a suitable reference gene due to its extensive variability in expression. Our findings emphasize the importance of validating reference genes for qPCR analyses. We provide a shortlist of reference genes to use for normalization and recommend freely available software programs as a rapid approach to validate potential reference genes.

Disruption of Fnip1 reveals a metabolic checkpoint controlling B lymphocyte development.

The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By using a chemical mutagenesis strategy in mice, we identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.

B lymphocytes promote lymphogenous metastasis of lymphoma and melanoma.

The prognosis of patients with many types of cancers correlates with the degree of metastasis to regional lymph nodes (LNs) and vital organs. However, the mechanisms and route of cancer cell metastasis are still unclear. Previous studies determined that B-cell accumulation in tumor-draining LNs (TDLNs) induces lymphatic sinus growth (lymphangiogenesis) and increases lymph flow, which could actively promote tumor dissemination through the lymphatic system. Using young Eµ-c-Myc mice that feature LN B-cell expansion as hosts for tumor transplants, we show that subcutaneously implanted lymphomas or melanomas preferentially spread to TDLNs over non-TDLNs, thus demonstrating that these tumors initially metastasize through lymphatic rather than through hematogenous routes. In addition, the rate and amount of tumor dissemination is greater in Eµ-c-Myc mice versus wild-type hosts, which correlates with LN B-cell accumulation and lymphangiogenesis in Eµ-c-Myc hosts. The increased lymphatic dissemination in Eµ-c-Myc hosts is further associated with rapid hematogenous tumor spread of subcutaneously implanted lymphomas, suggesting that TDLN metastasis secondarily drives lymphoma spread to distant organs. In contrast, after intravenous tumor cell injection, spleen metastasis of lymphoma cells or lung metastasis of melanoma cells is similar in Eµ-c-Myc and wild-type hosts. These studies demonstrate that the effect of Eµ-c-Myc hosts to promote metastasis is limited to the lymphatic route of dissemination. TDLN B-cell accumulation, in association with lymphangiogenesis and increased lymph flow, thus significantly contributes to dissemination of lymphomas and solid tumors, providing new targets for therapeutic intervention to block metastasis.