Approximately 20% of persons living in areas hyperendemic for tuberculosis (TB) display persistent lack of tuberculin skin test (TST) reactivity and appear to be naturally resistant to infection by Mycobacterium tuberculosis. Among those with a positive response, the intensity of TST reactivity varies greatly. The genetic basis of TST reactivity is not known. We report on a genome-wide linkage search for loci that have an impact on TST reactivity, which is defined either as zero versus nonzero (TST-BINa) or as extent of TST in millimeters (TST–quantitative trait locus [QTL]) in a panel of 128 families, including 350 siblings, from an area of South Africa hyperendemic for TB. We detected a major locus (TST1) on chromosomal region 11p14 (P = 1.4 x 10^–5), which controls TST-BINa, with a lack of responsiveness indicating T cell–independent resistance to M. tuberculosis. We also detected a second major locus (TST2) on chromosomal region 5p15 (P < 10^–5), which controls TST-QTL or the intensity of T cell–mediated delayed type hypersensitivity (DTH) to tuberculin. Fine mapping of this region identified SLC6A3, encoding the dopamine transporter DAT1, as a promising gene for further studies. Our results pave the way for the understanding of the molecular mechanisms involved in resistance to M. tuberculosis infection in endemic areas (TST1) and for the identification of critical regulators of T cell–dependent DTH to tuberculin (TST2).

Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro–differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

During somatic hypermutation (SHM), B cells introduce mutations into their immunoglobulin genes to generate high affinity antibodies. Current models suggest a separation in the generation of G/C transversions by the Ung2-dependent pathway and the generation of A/T mutations by the Msh2/ubiquitinated proliferating cell nuclear antigen (PCNA-Ub)–dependent pathway. It is currently unknown whether these pathways compete to initiate mutagenesis and whether PCNA-Ub functions downstream of Ung2. Furthermore, these models do not explain why mice lacking Msh2 have a more than twofold reduction in the total mutation frequency. Our data indicate that PCNA-Ub is required for A/T mutagenesis downstream of both Msh2 and Ung2. Furthermore, we provide evidence that both pathways are noncompetitive to initiate mutagenesis and even collaborate to generate half of all G/C transversions. These findings significantly add to our understanding of SHM and necessitate an update of present SHM models.

Both class switch recombination (CSR) and somatic hypermutation (SHM) require transcription and the trans-acting factor activation-induced cytidine deaminase (AID), and must be up-regulated during antigen-dependent differentiation of B lymphocytes. To test the role of the heavy chain 3' enhancers in both CSR and SHM, we used a BAC transgene of the entire heavy chain constant region locus. Using Cre-loxP recombination to delete a 28-kb region that contains the four known 3' heavy chain enhancers, we isolated lines of BAC transgenic mice with an intact heavy chain locus and paired lines in the same chromosomal insertion site lacking the 3' enhancers. Intact heavy chain transgenes undergo CSR to all heavy chain genes and mutate their transgenic VDJ exon. In paired transgenes lacking the 3' enhancer region, CSR to most heavy chain genes is reduced to ~1% of the levels for intact heavy chain loci; SHM is also reduced. Finally, we find that in B cells with a transgene lacking the 3' enhancers, interchromosomal recombination between the transgenic VDJ exon and the endogenous heavy chain C genes is more easily detected than CSR within the transgene.

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre–B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Ig DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-/ locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.

B lymphocyte–intrinsic Toll-like receptor (TLR) signals amplify humoral immunity and can exacerbate autoimmune diseases. We identify a new mechanism by which TLR signals may contribute to autoimmunity and chronic inflammation. We show that TLR4 signaling enhances B lymphocyte trafficking into lymph nodes (LNs), induces B lymphocyte clustering and interactions within LN follicles, leads to sustained in vivo B cell proliferation, overcomes the restriction that limits the access of nonantigen-activated B cells to germinal center dark zones, and enhances the generation of memory and plasma cells. Intravital microscopy and in vivo tracking studies of B cells transferred to recipient mice revealed that TLR4-activated, but not nonstimulated, B cells accumulated within the dark zones of preexisting germinal centers even when transferred with antigen-specific B cells. The TLR4-activated cells persist much better than nonstimulated cells, expanding both within the memory and plasma cell compartments. TLR-mediated activation of B cells may help to feed and stabilize the spontaneous and ectopic germinal centers that are so commonly found in autoimmune individuals and that accompany chronic inflammation.

The origin of IgM⁺CD27⁺ B lymphocytes with mutated IgV genes, which account for ~20% of human peripheral blood (PB) B cells, is controversially discussed. A generation in a primary diversification pathway, in T cell–independent immune responses, or in T cell–dependent germinal center (GC) reactions has been proposed. We show here that IgM⁺IgD⁺CD27⁺ and IgM⁺IgD^–/lowCD27⁺ B cell subsets carry, like class-switched memory B cells, mutations in the Bcl6 gene as a genetic trait of a GC experience. Moreover, the identification of PB IgM⁺IgD⁺CD27⁺ B cells clonally related to GC-derived IgG⁺ memory B cells with shared and distinct IgV gene mutations demonstrates the GC origin also of the former subset. These findings provide genetic evidence for a GC derivation of somatically mutated IgM⁺ B cells and indicate that adult humans harbor a large population of IgM⁺IgD⁺ post-GC memory B cells. Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction. This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.

B cell activation factor of the TNF family (BAFF) activates noncanonical nuclear factor B (NF-B) heterodimers that promote B cell survival. We show that although MALT1 is largely dispensable for canonical NF-B signaling downstream of the B cell receptor, the absence of MALT1 results in impaired BAFF-induced phosphorylation of NF-B2 (p100), p100 degradation, and RelB nuclear translocation in B220⁺ B cells. This corresponds with impaired survival of MALT1^–/– marginal zone (MZ) but not follicular B cells in response to BAFF stimulation in vitro. MALT1^–/– MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor–mediated signaling, and TRAF3 was found to interact with MALT1. Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell–intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction.

The transcription factor Ets1 contributes to the differentiation of CD8 lineage cells in the thymus, but how it does so is not understood. In this study, we demonstrate that Ets1 is required for the proper termination of CD4 expression during the differentiation of major histocompatability class 1 (MHC I)–restricted thymocytes, but not for other events associated with their positive selection, including the initiation of cytotoxic gene expression, corticomedullary migration, or thymus exit. We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression. Enforced Runx3 expression in Ets1-deficient MHC I–restricted thymocytes largely rescued their impaired Cd4 silencing, indicating that Ets1 is not required for Runx3 function. Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription. These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.

Forkhead box P3 (FOXP3)⁺CD4⁺CD25⁺ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4⁺ T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4⁺ T cells into Foxp3⁺ iT reg cells was significantly decreased in adoptively transferred Cbfb^F/F CD4-cre naive T cells into Rag2^–/– mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and Cbfb^F/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4⁺ CD25^high CD127^– T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4⁺CD25^– cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.

Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (~6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

West Nile virus (WNV) infection causes a life-threatening meningoencephalitis that becomes increasingly more prevalent over the age of 50 and is 40–50x more prevalent in people over the age of 70, compared with adults under the age of 40. In a mouse model of age-related vulnerability to WNV, we demonstrate that death correlates with increased viral titers in the brain and that this loss of virus control with age was the result of defects in the CD4 and CD8 T cell response against WNV. Specific age-related defects in T cell responses against dominant WNV epitopes were detected at the level of cytokine and lytic granule production, each of which are essential for resistance against WNV, and in the ability to generate multifunctional anti-WNV effector T cells, which are believed to be critical for robust antiviral immunity. In contrast, at the peak of the response, old and adult T cells exhibited superimposable peptide sensitivity. Most importantly, although the adult CD4 or CD8 T cells readily protected immunodeficient mice upon adoptive transfer, old T cells of either subset were unable to provide WNV-specific protection. Consistent with a profound qualitative and quantitative defect in T cell immunity, old brains contained at least 12x fewer total effector CD8 T cells compared with adult mice at the peak of brain infection. These findings identify potential targets for immunomodulation and treatment to combat lethal WNV infection in the elderly.

Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of G subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gβ is unknown. We now show that impairment of Gβ function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gβ1, the predominant Gβ isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gβ1 from RACK1, resulting in Gβ1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gβ1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gβ1-induced barrier recovery indicating RACK1 negatively regulates Gβ1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn^–/– mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gβ1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcµR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcµR in human B-lineage cDNA libraries. FcµR is defined as a transmembrane sialoglycoprotein of ~60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fc/µR) but exhibits an exclusive Fcµ-binding specificity. The cytoplasmic tail of FcµR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcµR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcµR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcµR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcµR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

Leukocyte migration across endothelial cell borders (paracellular) and through endothelial cells (transcellular) appear to be distinct processes. During paracellular migration, membrane from a parajunctional reticulum of interconnected vesicles, the endothelial lateral border recycling compartment (LBRC), moves to surround the leukocyte in a kinesin-mediated, microtubule-dependent manner. We show that transcellular migration likewise requires targeted trafficking of LBRC membrane. We show that in addition to platelet/endothelial cell adhesion molecule (PECAM; CD31), CD99 and junctional adhesion molecule A (JAM-A), but apparently not vascular endothelial cell–specific cadherin (cadherin 5, CD144), are components of the LBRC. During transcellular migration, LBRC membrane invests the transmigrating leukocyte. Intracellular adhesion molecule 1 (ICAM-1) on the apical endothelial surface is enriched around adherent leukocytes. Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%. Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration. We conclude that similar molecular mechanisms promote both para- and transcellular migration.

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT₁ receptor agonistic autoantibodies (AT₁-AAs) that contribute to the disease features. However, the exact role of AT₁-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT₁ receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT₁-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT₁-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT₁-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT₁-AA–induced placental damage. Our findings highlight AT₁-AAs as important therapeutic targets.

The bloodstream is an important route of dissemination of invading pathogens. Most of the small bloodborne pathogens, like bacteria or viruses, are filtered by the spleen or liver sinusoids and presented to the immune system by dendritic cells (DCs) that probe these filters for the presence of foreign antigen (Ag). However, larger pathogens, like helminths or infectious emboli, that exceed 20 µm are mostly trapped in the vasculature of the lung. To determine if Ag trapped here can be presented to cells of the immune system, we used a model of venous embolism of large particulate Ag (in the form of ovalbumin [OVA]-coated Sepharose beads) in the lung vascular bed. We found that large Ags were presented and cross-presented to CD4 and CD8 T cells in the mediastinal lymph nodes (LNs) but not in the spleen or liver-draining LNs. Dividing T cells returned to the lungs, and a short-lived infiltrate consisting of T cells and DCs formed around trapped Ag. This infiltrate was increased when the Toll-like receptor 4 was stimulated and full DC maturation was induced by CD40 triggering. Under these conditions, OVA-specific cytotoxic T lymphocyte responses, as well as humoral immunity, were induced. The T cell response to embolic Ag was severely reduced in mice depleted of CD11c^hi cells or Ly6C/G⁺ cells but restored upon adoptive transfer of Ly6C^hi monocytes. We conclude that the lung vascular filter represents a largely unexplored site of immune induction that traps large bloodborne Ags for presentation by monocyte-derived DCs.

Hematopoietic stem cells (HSCs) are thought to reside in discrete niches through stable adhesion, yet previous studies have suggested that host HSCs can be replaced by transplanted donor HSCs, even in the absence of cytoreductive conditioning. To explain this apparent paradox, we calculated, through cell surface phenotyping and transplantation of unfractionated blood, that ~1–5% of the total pool of HSCs enters into the circulation each day. Bromodeoxyuridine (BrdU) feeding experiments demonstrated that HSCs in the peripheral blood incorporate BrdU at the same rate as do HSCs in the bone marrow, suggesting that egress from the bone marrow to the blood can occur without cell division and can leave behind vacant HSC niches. Consistent with this, repetitive daily transplantations of small numbers of HSCs administered as new niches became available over the course of 7 d led to significantly higher levels of engraftment than did large, single-bolus transplantations of the same total number of HSCs. These data provide insight as to how HSC replacement can occur despite the residence of endogenous HSCs in niches, and suggest therapeutic interventions that capitalize upon physiological HSC egress.