Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3343A allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

For effective adaptive immunity to foreign antigens (Ag), secondary lymphoid organs (SLO) provide the confined environment in which Ag-restricted lymphocytes, with very low precursor frequencies, interact with Ag on Ag-presenting cells (APC). The spleen is the primordial SLO, arising in conjunction with adaptive immunity in early jawed vertebrates. The spleen, especially the spleen’s lymphoid compartment, the white pulp (WP), has undergone numerous modifications over evolutionary time. We describe the progressive advancement of splenic WP complexity, which evolved in parallel with the increasing functionality of adaptive immunity. The Ag-presenting function of follicular dendritic cells (FDC) also likely emerged at the inception of adaptive immunity, and we propose that a single type of hematopoietically derived APC displayed Ag to both T and B cells. A dedicated FDC, derived from a vascular precursor, is a recent evolutionary innovation that likely permitted the robust affinity maturation found in mammals.

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC-dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that in Xenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence of bona fide FDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.

Studies using inbred strains of mice have been invaluable for identifying alleles that adversely affect hearing. However, the efficacy of those studies is limited by the phenotypes that these strains express and the alleles that they segregate. Here, by selectively breeding phenotypically and genetically heterogeneous NIH Swiss mice, we generated two lines—the all-frequency hearing loss (AFHL) line and the high-frequency hearing loss (HFHL) line—with differential hearing loss. The AFHL line exhibited characteristics typical of severe, early-onset, sensorineural hearing impairment. In contrast, the HFHL line expressed a novel early-onset, mildly progressive, and frequency-specific sensorineural hearing loss. By quantitative trait loci (QTLs) analyses in these two lines, we identified QTLs on chromosomes 7, 8, and 10 that significantly affected hearing function. The loci on chromosomes 7 and 8 (Hfhl1 and Hfhl2, respectively) are novel and appear to adversely affect only high frequencies (≥30 kHz). Mice homozygous for NIH Swiss alleles at either Hfhl1 or Hfhl2 have 32-kHz auditory-evoked brain stem response thresholds that are 8–14 dB SPL higher than the corresponding heterozygotes. DNA sequence analyses suggest that both the Cdh23 ahl and Gipc3 ahl5 variants contribute to the chromosome 10 QTL detected in the AFHL line. The frequency-specific hearing loss indicates that the Hfhl1 and Hfhl2 alleles may affect tonotopic development. In addition, dissecting the underlying complex genetics of high-frequency hearing loss may prove relevant in identifying less severe and common forms of hearing impairment in the human population.

Progressive sensorineural hearing loss is the most common form of acquired hearing impairment in the human population. It is also highly prevalent in inbred strains of mice, providing an experimental avenue to systematically map genetic risk factors and to dissect the molecular pathways that orchestrate hearing in peripheral sensory hair cells. Therefore, we ascertained hearing function in the inbred long sleep (ILS) and inbred short sleep (ISS) strains. Using auditory-evoked brain stem response (ABR) and distortion product otoacoustic emission (DPOAE) measurements, we found that ISS mice developed a high-frequency hearing loss at twelve weeks of age that progressed to lower frequencies by 26 weeks of age in the presence of normal endocochlear potentials and unremarkable inner ear histology. ILS mice exhibited milder hearing loss, showing elevated thresholds and reduced DPOAEs at the higher frequencies by 26 weeks of age. To map the genetic variants that underlie this hearing loss we computed ABR thresholds of 63 recombinant inbred stains derived from the ISS and ILS founder strains. A single locus was linked to markers associated with ISS alleles on chromosome 10 with a highly significant logarithm of odds (LOD) score of 15.8. The 2-LOD confidence interval spans ∼4 Megabases located at position 54–60 Mb. This locus, termed sensorineural hearing loss 1 (Snhl1), accounts for approximately 82% of the phenotypic variation. In summary, this study identifies a novel hearing loss locus on chromosome 10 and attests to the prevalence and genetic heterogeneity of progressive hearing loss in common mouse strains.

Five-year survival rates for patients diagnosed with metastatic melanoma are less than 5%. Adoptive cell transfer (ACT) has achieved an objective response of 50% by Response Evaluation Criteria in Solid Tumors (RECIST) in this patient population. For ACT to be maximally effective, the host must first be lymphodepleted. It is hypothesized that lymphodepletion may remove regulatory elements and cytokine sinks, or increase the activation and availability of antigen presenting cells (APCs). We use an in vivo model to study the ACT of tumor-associated antigen (TAA)-specific CD4+ T cells (TRP-1 cells). We have discovered that depletion of NK1.1+ cells enhances the rejection of established melanoma tumors by adoptively transferred TRP-1 CD4+ T cells. NK1.1+ cell depletion increases the number of CD4+ T cells, the serum concentration of pro-inflammatory cytokines, autoimmune vitiligo, host survival and prevented recurrence after ACT. Because multiple cells express NK1.1, we targeted different NK1.1+ cell populations using antibodies specific for NK cells, pre-mNK cells, and innate lymphoid cells (ILCs). Our data suggests that NK1.1+B220+ pre-mNK cells (also known as interferon-producing killer dendritic cells; IKDCs) are an important inhibitor of the CD4+ T cell response to melanoma. Understanding this mechanism may help design new immunotherapies to modulate the activity of pre-mNKs in the face of an antitumor immune response and inhibit their suppression of adoptively transferred T cells.

Secondary lymphoid organs (SLO) provide the structural framework for co-concentration of antigen and antigen-specific lymphocytes required for an efficient adaptive immune system. The spleen is the primordial SLO, and evolved concurrently with Ig/TCR:pMHC-based adaptive immunity. The earliest cellular/histological event in the ontogeny of the spleen’s lymphoid architecture, the white pulp (WP), is the accumulation of B cells around splenic vasculature, an evolutionarily conserved feature since the spleen’s emergence in early jawed vertebrates such as sharks. In mammals, B cells are indispensable for both formation and maintenance of SLO microarchitecture; their expression of lymphotoxin α1β2 (LTα1β2) is required for the LTα1β2:CXCL13 positive feedback loop without which SLO cannot properly form. Despite the spleen’s central role in the evolution of adaptive immunity, neither the initiating event nor the B cell subset necessary for WP formation has been identified. We therefore sought to identify both in mouse. We detected CXCL13 protein in late embryonic splenic vasculature, and its expression was TNFα- and RAG-2-independent. A substantial influx of CXCR5+ transitional B cells into the spleen occurred 18 hours before birth. However, these late embryonic B cells were unresponsive to CXCL13 (though responsive to CXCL12) and phenotypically indistinguishable from blood-derived B cells. Only after birth did B cells acquire CXCL13 responsiveness, accumulate around splenic vasculature, and establish the uniquely splenic B cell compartment, enriched for CXCL13-responsive late transitional cells. Thus, CXCL13 is the initiating component of the CXCL13:LTα1β2 positive feedback loop required for WP ontogeny, and CXCL13-responsive late transitional B cells are the initiating subset.

The Escherichia coli DNA polymerase III and ␥ subunits are single-strand DNA-dependent ATPases (the latter requires the ␦ and ␦ subunits for significant ATPase activity) involved in loading processivity clamp ␤. They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic /␥ homologs and 1 eukaryotic /␥ homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.The dnaX polymerization gene of Escherichia coli encodes two DNA polymerase III components, and ␥. is the fulllength translational product of the DnaX reading frame. The shorter ␥ is identical to the first 430 residues of , but its C terminus is generated by a programmed Ϫ1 ribosomal frameshift which results in the incorporation of a glutamate as the 431st amino acid followed by a stop codon (3,19,63).functions as the replisome organizer, dimerizing the core polymerase (30,33,43,58) and interacting with and stimulating the replicative DnaB helicase and primase (31,73). also contributes to processivity by stabilizing the processivity clamp (32) and the holoenzyme (73) on the leading strand.␥ functions in a five-subunit complex (␥ 2-4 -␦-␦Ј--) (12,20,42,49,51,62) to load and unload the processivity clamp ␤ (2,5,25,26,45,47,59,67). The binding of two or three ATP molecules by the ␥ subunit of the complex alters the conformation of the complex, allowing ␦ to bind directly to and open the clamp and allowing assembly of a primed DNA-open clamp-␥ complex structure (25,26,45). Hydrolysis of the ATP, required for closing the clamp around primed DNA, occurs in two sequential steps. The first might release ␤ from the ␥ complex; the second might then release DNA (enclosed within the clamp). Alternatively, the first hydrolysis might release DNA from the ␥ complex into the open clamp and the second would then release ␤ (encircling the DNA). Another possibility for the second hydrolysis might be resetting of the ␥ complex for the next cycle (26).The N-terminal region of is identical to ␥, except for the 431st residue, and is capable of all the known activities of ␥ in vitro, including loading ␤ (with ␦ or ␦-␦Ј) (59) and assembly in vitro or in vivo (from an artificial -complex operon) into clamp-loading complexes ( 2-4 -␦-␦Ј--) (13, 49, 54). The ␥ complex is often thought to be the principal clam...