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Both natural influenza infection and current seasonal influenza vaccines primarily induce neutralizing antibody responses against highly diverse epitopes within the 'head' of the viral hemagglutinin (HA) protein. There is increasing interest in redirecting immunity toward the more conserved HA stem or stalk as a means of broadening protective antibody responses. Here we examined HA stem-specific B cell and T follicular helper (Tfh) cell responses in the context of influenza infection and immunization in mouse and monkey models. We found that during infection, the stem domain was immunologically subdominant to the head in terms of serum antibody production and antigen-specific B and Tfh cell responses. Similarly, we found that HA stem immunogens were poorly immunogenic compared with the full-length HA with abolished sialic acid binding activity, with limiting Tfh cell elicitation a potential constraint to the induction or boosting of anti-stem immunity by vaccination. Finally, we confirm that currently licensed seasonal influenza vaccines can boost preexisting memory responses against the HA stem in humans. An increased understanding of the immune dynamics surrounding the HA stem is essential to inform the design of next-generation influenza vaccines for broad and durable protection., Introduction Influenza viruses cause significant global morbidity and mortality through seasonal epidemics and periodic pandemics. The effectiveness of influenza vaccination is limited by the focusing of humoral immunity on a [...]

Author(s): Devin Sok [1, 2, 3, 4]; Khoa M. Le [1, 2, 3, 4]; Melissa Vadnais [5]; Karen L. Saye-Francisco [1, 2, 3]; Joseph G. Jardine [1, 2, 3]; Jonathan [...]

The ability to predict how different individuals will respond to vaccination and to understand what best protects individuals from infection greatly facilitates developing next-generation vaccines. It facilitates both the rapid [...]

Forkhead box P3 [(Foxp3).sup.+] T regulatory ([T.sub.reg]) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors (1), (2). [Foxp3.sup.+] [T.sub.reg] [...]

The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and [...]

Adaptation of tumor cells to the host is a major cause of cancer progression, failure of therapy, and ultimately death. Immune selection drives this adaptation in human cancer by enriching tumor cells with a cancer stem cell-like (CSC-like) phenotype that makes them resistant to CTL-mediated apoptosis; however, the mechanisms that mediate CSC maintenance and proliferation are largely unknown. Here, we report that CTL-mediated immune selection drives the evolution of tumor cells toward a CSC-like phenotype and that the CSC-like phenotype arises through the Akt signaling pathway via transcriptional induction of Tcl1a by Nanog. Furthermore, we found that hyperactivation of the Nanog/Tcl1a/Akt signaling axis was conserved across multiple types of human cancer. Inhibition of Nanog in a murine model of colon cancer rendered tumor cells susceptible to immune-mediated clearance and led to successful, long-term control of the disease. Our findings establish a firm link among immune selection, disease progression, and the development of a stem-like tumor phenotype in human cancer and implicate the Nanog/Tcl1a/Akt pathway as a central molecular target in this process., Introduction Despite heroic efforts by humankind to combat cancer over millennia, this disease remains the second leading cause of death in the United States, afflicting approximately 50% of all men [...]

Ig class-switch recombination (CSR) is a region-specific process that exchanges the constant Ig heavy-chain region and thus modifies an antibody's effector function. DNA lesions in switch (S) regions are induced by activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase 2 (UNG2), subsequently processed to DNA breaks, and resolved by either the classical nonhomologous end-joining pathway or the alternative end-joining pathway (XRCC4/ DNA ligase 4-and/or Ku70/Ku80-independent and prone to increased microhomology usage). We examined whether the induction of DNA lesions influences DNA end-joining during CSR by analyzing S[mu]-S[alpha] recombination junctions in various human Ig CSR defects of DNA lesion induction. We observed a progressive trend toward the usage of microhomology in S[mu]-S[alpha] recombination junctions from AID-heterozygous to AID--autosomal dominant to UNG2-deficient B lymphocytes. We thus hypothesize that impaired induction of DNA lesions in S regions during CSR leads to unusual end-processing of the DNA breaks, resulting in microhomology-mediated end-joining, which could be an indication for preferential processing by alternative end-joining rather than by classical nonhomologous end-joining. antibody maturation | switch junction | DNA repair doi/ 10.1073/pnas.1012591108

Pathogens use specialized secretion systems and targeting signals to translocate effector proteins inside host cells, a process that is essential for promoting disease and parasitism. However, the amino acid sequences that determine host delivery of eukaryotic pathogen effectors remain mostly unknown. The Crinkler (CRN) proteins of oomycete plant pathogens, such as the Irish potato famine organism Phytophthora infestans, are modular proteins with predicted secretion signals and conserved N-terminal sequence motifs. Here, we provide direct evidence that CRN N termini mediate protein transport into plant cells. CRN host translocation requires a conserved motif that is present in all examined plant pathogenic oomycetes, including the phylogenetically divergent species Aphanomyces euteiches that does not form haustoria, specialized infection structures that have been implicated previously in delivery of effectors. Several distinct CRN C termini localized to plant nuclei and, in the case of CRN8, required nuclear accumulation to induce plant cell death. These results reveal a large family of ubiquitous oomycete effector proteins that target the host nucleus. Oomycetes appear to have acquired the ability to translocate effector proteins inside plant cells relatively early in their evolution and before the emergence of haustoria. Finally, this work further implicates the host nucleus as an important cellular compartment where the fate of plant-microbe interactions is determined. plant immunity | Phytophthora | Crinklers | translocation | nuclear localization doi/ 10.1073/pnas.1008491107

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness. hemagglutinin | protein design | bacterial expression doi/ 10.1073/pnas.1007465107

Specificity of the Tn4430 target immunity signal was examined by fusing the transposase TnpA to the LacI repressor of Escherichia coli. The resulting chimeric proteins failed to impose immunity to DNA targets carrying copies of the lacO operator, though they were proficient in lacO binding in vivo and remained responsive to wild-type immunity conferred by the Tn4430 inverted repeat end. Intriguingly, the presence of lacO repeats within the target was found to strongly influence target site selection by Tn4430, but in a LacI-independent manner. doi: 10.1128/JB.00477-10

A study was conducted to evaluate the frequency of vaccine-induced seropositivity/reactivity (VISP) in the case of noninfected HIV vaccine trial recipients. Results indicated that the induction of VISP is common in the case of such recipients and the development and detection of VISP seems to be associated with immunogenicity of the vaccine and the EIA assay used.

[alpha][beta] T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR--pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I. MHC restriction | T cell recognition | structural immunology | immunogenetics doi/ 10.1073/pnas.1004926107

NK cell-mediated murine cytomegalovirus (MCMV) resistance ([Cmv.sup.1]) is under H-[2.sup.k] control in MA/My mice, but the underlying gene(s) is unclear. Prior genetic analysis mapped [Cmv.sup.r] to the MHC class I (MHC-I) [D.sup.k] gene interval. Because NK cell receptors are licensed by and responsive to MHC class I molecules, [D.sup.k] itself is a candidate gene. A 10-kb genomic [D.sup.k] fragment was subcloned and microinjected into MCMV-susceptible ([Cmv.sup.5]) (MA/My.L-[H2.sup.b] x C57L)[F.sub.1] or (B6 x DBA/2)[F.sub.2] embryos. Transgenic founders, which are competent for [D.sup.k] expression and germline transgene transmission, were identified and further backcrossed to MA/My. L-[H2.sup.b] or C57L mice. Remarkably, [D.sup.k] expression delivered NK-mediated resistance in either genetic background. Further, NK cells with cognate inhibitory Ly49G receptors for self-MHC-I [D.sup.k] were licensed and critical in protection against MCMV infection. In radiation bone marrow chimeras, NK resistance was significantly diminished when MHC-I [D.sup.k] expression was restricted to only hematopoietic or nonhematopoietic cells. Thus, MHC-I [D.sup.k] is the H-[2.sup.k]-linked [Cmv.sup.r] locus; these findings suggest a role for NK cell interaction with [D.sup.k]-bearing hematopoietic and nonhematopoietic cells to shape NK-mediated virus immunity. NK cell | virus immunity | Ly49G | MCMV | H-2D transgenic doi/10.1073/pnas.0913126107

Chemokines are known to regulate the steady-state and inflammatory migration of cutaneous dendritic cells (DCs). The [beta]-chemokine CCL17, a ligand of CCR4, is inducibly expressed in a subset of DCs and is strongly up-regulated in atopic diseases. Using an atopic dermatitis model, we show that CCL17-deficient mice develop acanthosis as WT mice, whereas dermal inflammation, T helper 2-type cytokine production, and the allergen-specific humoral immune response are significantly decreased. Notably, CCL17-deficient mice retained Langerhans cells (LCs) in the lesional skin after chronic allergen exposure, whereas most LCs emigrated from the epidermis of allergen-treated WT controls into draining lymph nodes (LNs). Moreover, CCL17-deficient LCs showed impaired emigration from the skin after exposure to a contact sensitizer. In contrast, the absence of CCR4 had no effect on cutaneous DC migration and development of atopic dermatitis symptoms. As an explanation for the major migratory defect of CCL17-deficient DCs in vivo, we demonstrate impaired mobility of CCL17-deficient DCs to CCL19/21 in 3D in vitro migration assays and a blockade of intracellular calcium release in response to CCR7 ligands. In addition, responsiveness of CCL17-deficient DCs to CXCL12 was impaired as well. We demonstrate that the inducible chemokine CCL17 sensitizes DCs for CCR7- and CXCR4-dependent migration to LN-associated homeostatic chemokines under inflammatory conditions and thus plays an important role in cutaneous DC migration. atopic dermatitis | CCL22 | CCR4 | GPCR | Langerhans cells doi/10.1073/pnas.0906126107

We have studied folding and complex formation of the yeast Mediator head-module protein subunits Med8, Med18, and Med20. Using a combination of immunoprecipitation, far-UV circular dichroism, and fluorescence measurements on recombinantly expressed and denatured proteins that were allowed to renature separately or in different combinations, we found that Meal8, Med18, and Med20 can fold in different ways to form both soluble monomeric proteins and different distinct subcomplexes. However, the concurrent presence of all three protein subunits during the renaturation process is required for proper folding and trimer complex formation. assembly | protein folding | transcription doi/10.1073/pnas.0907645106

Due to their low immunogenicity in patients, humanized or fully human mAbs are becoming increasingly important for the treatment of a growing number of diseases, including cancer, infections, and immune disorders. Here, we describe a technology allowing for the rapid isolation of fully human mAbs. In contrast to previously described methods, B cells specific for an antigen of interest are directly isolated from peripheral blood mononuclear cells (PBMC) of human donors. Recombinant, antigen-specific single-chain Fv (scFv) libraries are generated from this pool of B cells and screened by mammalian cell surface display by using a Sindbis virus expression system. This method allows isolating antigen-specific antibodies by a single round of FACS. The variable regions (VRs) of the heavy chains (HCs) and light chains (LCs) are isolated from positive clones and recombinant fully human antibodies produced as whole IgG or Fab fragments. In this manner, several hypermutated high-affinity antibodies binding the Q[beta] virus like particle (VLP), a model viral antigen, as well as antibodies specific for nicotine were isolated. All antibodies showed high expression levels in cell culture. The human nicotine-specific mAbs were validated preclinically in a mouse model. Thus, the technology presented here allows for rapid isolation of high-affinity, fully human antibodies with therapeutic potential from human volunteers.

BAFF-R-dependent activation of the alternative NF-KB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-KB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal plO0 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-KB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked. IKB kinase | NF-KB | Hyperplasia | p100 processing | knockin

Natural killer (NK) cells constitute a subpopulation of lymphocytes that develop from precursors in the bone marrow (BM), but the transcriptional regulation of their development and maturation is only beginning to be understood, in part due to their relatively rare abundance, especially of developmental subsets. Using a mouse model in which NK cells are arrested at an immature stage of development, and a gene expression profiling approach, we uncovered transient normal NK cell expression of a homeobox transcription factor (TF) family, called Distal-less (DIx), which had been primarily implicated in murine CNS, craniofacial, limb, and skin development. Our studies demonstrate that Dlx1, DIx2, and DIx3 are transiently expressed in immature Mac-1(10) NK cells within the BM, with DIx3 being the predominantly expressed member. These genes are expressed in a temporally regulated pattern with overlapping waves of expression, and they display functional redundancy. Expression is extinguished in fully mature splenic NK cells, and persistent expression of DIx genes leads to functionally immature NK cells arrested at the Mac-1 stage. Whereas conventional splenic NK cells develop but are arrested at an immature stage, there appears to be a complete failure to develop CD127+ thymic NK cells when DIx genes are persistently expressed. We also observed that T and B cells fail to develop in the context of persistent DIx1 expression. Thus, these studies indicate that DIx TFs play a functional role in lymphocyte development. Dlxl NK | lymphocyte gene expression

UL18 is a human cytomegalovirus class I MHC (MHCI) homolog that binds the host inhibitory receptor LIR-1 and the only known viral MHC homolog that presents peptides. The 2.2-[Angstrom] structure of a LIR-1/UL18/peptide complex reveals increased contacts and optimal surface complementarity in the LIR-1/UL18 interface compared with LIR/MHCI interfaces, resulting in a > 1,000-fold higher affinity. Despite sharing only [approximately equal to] 25% sequence identity, UL18's structure and peptide binding are surprisingly similar to host MHCI. The crystal structure suggests that most of the UL18 surface, except where LIR-1 and the host-derived light chain bind, is covered by carbohydrates attached to 13 potential N-glycosylation sites, thereby preventing access to bound peptide and association with most MHCI-binding proteins. The LIR-1/UL18 structure demonstrates how a viral protein evolves from its host ancestor to impede unwanted interactions while preserving and improving its receptor-binding site. cytomegalovirus | immune evasion | LIR-1 | x-ray crystallography

Regulatory T (Treg) cells safeguard against autoimmunity and immune pathology. Because determinants of the Treg cell fate are not completely understood, we have delineated signaling events that control the de novo expression of Foxp3 in naive peripheral CD4 T cells and in thymocytes. We report that premature termination of TCR signaling and inibition of phosphatidyl inositol 3-kinase (PI3K) p110[alpha], p110[delta], protein kinase B (Akt), or mammalian target of rapamycin (mTOR) conferred Foxp3 expression and Treg-like gene expression profiles. Conversely, continued TCR signaling and constitutive PI3K/Akt/mTOR activity antagonised Foxp3 induction. At the chromatin level, di- and trimethylation of lysine 4 of histone H3 (H3K4me2 and -3) near the Foxp3 transcription start site (TSS) and within the 5' untranslated region (UTR) preceded active Foxp3 expression and, like Foxp3 inducibility, was lost upon continued TCR stimulation. These data demonstrate that the PI3K/Akt/mTOR signaling network regulates Foxp3 expression.

Bombyx mori densovirus type 2 (BmDNV-2), a parvo-like virus, replicates only in midgut columnar cells and causes fatal disease. The resistance expressed in some silkworm strains against the virus is determined by a single gene, nsd-2, which is characterized as nonsusceptibility irrespective of the viral dose. However, the responsible gene has been unknown. We isolated the nsd-2 gene by positional cloning. The virus resistance is caused by a 6-kb deletion in the ORF of a gene encoding a 12-pass transmembrane protein, a member of an amino acid transporter family, and expressed only in midgut. Germ-line transformation with a wild-type transgene expressed in the midgut restores susceptibility, showing that the defective membrane protein is responsible for resistance. Cumulatively, our data show that the membrane protein is a functional receptor for BmDNV-2. This is a previously undescribed report of positional cloning of a mutant gene in Bombyx and isolation of an absolute virus resistance gene in insects. virus resistance | positional cloning | transgenesis