γδ T Cells Differentially Regulate Bone Loss in Periodontitis Models.

γδ T cells are nonclassical T lymphocytes representing the major T-cell population at epithelial barriers. In the gingiva, γδ T cells are enriched in epithelial regions adjacent to the biofilm and are considered to regulate local immunity to maintain host-biofilm homeostatic interactions. This delicate balance is often disrupted resulting in the development of periodontitis. Previous studies in mice lacking γδ T cells from birth (Tcrd-/- mice) examined the impact of these cells on ligature-induced periodontitis. Data obtained from those studies proposed either a protective effect or no impact to γδ T cells in this setting. Here, we addressed the role of γδ T cells in periodontitis using the recently developed Tcrd-GDL mice, enabling temporal ablation of γδ T cells. Specifically, the impact of γδ T cells during periodontitis was examined in 2 modalities: the ligature model and the oral infection model in which the pathogen Porphyromonas gingivalis was administrated via successive oral gavages. Ablation of γδ T cells during ligature-induced periodontitis had no impact on innate immune cell recruitment to the ligated gingiva. In addition, the number of osteoclasts and subsequent alveolar bone loss were unaffected. However, γδ T cells play a pathologic role during P. gingivalis infection, and their absence prevented alveolar bone loss. Further analysis revealed that γδ T cells were responsible for the recruitment of neutrophils and monocytes to the gingiva following the exposure to P. gingivalis. γδ T-cell ablation also downregulated osteoclastogenesis and dysregulated long-term immune responses in the gingiva. Collectively, this study demonstrates that whereas γδ T cells are dispensable to periodontitis induced by the ligature model, they play a deleterious role in the oral infection model by facilitating pathogen-induced bone-destructive immune responses. On a broader aspect, this study highlights the complex immunopathologic mechanisms involved in periodontal bone loss.

γδT Cells Are Essential for Orthodontic Tooth Movement.

Sustained mechanical forces applied to tissue are known to shape local immunity. In the oral mucosa, mechanical stress, either naturally induced by masticatory forces or externally via mechanical loading during orthodontic tooth movement (OTM), is translated, in part, by T cells to alveolar bone resorption. Nevertheless, despite being considered critical for OTM, depletion of CD4+ and CD8+ T cells is reported to have no impact on tooth movement, thus questioning the function of αßT cells in OTM-associated bone resorption. To further address the role of T cells in OTM, we first characterized the leukocytes residing in the periodontal ligament (PDL), the tissue of interest during OTM, and compared it to the neighboring gingiva. Unlike the gingiva, monocytes and neutrophils represent the major leukocytes of the PDL. These myeloid cells were also the main leukocytes in the PDL of germ-free mice, although at lower levels than SPF mice. T lymphocytes were more enriched in the gingiva than the PDL, yet in both tissues, the relative fraction of the γδT cells was higher than the αß T cells. We thus sought to examine the role of γδT cells in OTM. γδT cells residing in the PDL were mainly Vγ6+ and produced interleukin (IL)-17A but not interferon-γ. Using Tcrd-GDL mice allowing conditional ablation of γδT cells in vivo, we demonstrate that OTM was greatly diminished in the absence of γδT cells. Further analysis revealed that ablation of γδT cells decreased early IL-17A expression, monocyte and neutrophil recruitment, and the expression of the osteoclastogenic molecule receptor activator of nuclear factor-κß ligand. This, eventually, resulted in reduced numbers of osteoclasts in the pressure site during OTM. Collectively, our data suggest that γδT cells are essential in OTM for translating orthodontic mechanical forces to bone resorption, required for relocating the tooth in the alveolar bone.

PD-L1 Checkpoint Inhibition Narrows the Antigen-Specific T Cell Receptor Repertoire in Chronic Lymphocytic Choriomeningitis Virus Infection.

Checkpoint inhibitors are effective in restoring exhausted CD8+ T cell responses in persistent viral infections or tumors. Several compounds are in clinical use for different malignancies, but trials in patients with chronic viral infections have also been conducted. In a mouse model of persistent lymphocytic choriomeningitis virus (LCMV) infection, it was shown that checkpoint inhibitor treatment increased T cell proliferation and functionality, but its influence on the antigen-specific T cell receptor (TCR) repertoire is unknown. NP396-specific CD8+ T cells dominate during acute LCMV infection and are predominantly exhausted during chronic infection. Next-generation sequencing of NP396-specific TCRs showed that exhaustion corresponds with a significantly reduced NP396-specific TCR repertoire diversity: Shannon indices of 4 in immunized mice to 2.6 in persistently infected mice. Anti-PD-L1 treatment during persistent LCMV infection restored NP396-specific T cell responses and reduced viral titers. Nevertheless, anti-PD-L1-treated mice showed an even more narrowed TCR repertoire, with reduced TCR diversity compared to that of persistently infected control mice (Shannon indices of 2.1 and 2.6, respectively). Interestingly, anti-PD-L1 treatment-induced narrowing of the TCR repertoire negatively correlates with functional and physical restoration of the antigen-specific T cell response. Further, we found that private, hyperexpanded TCR clonotypes dominated the T cell response after anti-PD-L1 treatment. Although being private, these top clonotypes from anti-PD-L1-treated mice revealed a more closely related CDR3 motif than those of top clonotypes from persistently infected control mice. In conclusion, although targeting the PD-1/PD-L1 pathway reinvigorates exhausted CD8+ T cells, it fails to restore T cell repertoire diversity.IMPORTANCE Checkpoint inhibitors are effective immunotherapeutics to restore cancer- and virus-induced exhausted CD8+ T cells, by enhancing the quality and survival of immune responses. Although checkpoint inhibitors are already used as therapy against various cancers, not much is known about their multifaceted impact on the exhausted CD8+ T cell receptor (TCR) repertoire. This report describes for the first time the evolvement of an exhausted antigen-specific CD8+ TCR repertoire under checkpoint inhibitor treatment. By using a well-established virus model, we were able to show major shifts toward oligoclonality of the CD8+ TCR repertoire response against a massively exhausted lymphocytic choriomeningitis virus (LCMV) epitope. While supporting viral control in the LCMV model, oligoclonality and more private of TCR repertoires may impact future pathogenic challenges and may promote viral escape. Our results may explain the ongoing problems of viral escapes, unpredictable autoimmunity, and heterogeneous responses appearing as adverse effects of checkpoint inhibitor treatments.

Development and Function of γδT Cells in the Oral Mucosa.

To successfully withstand a wide variety of microbial and mechanical challenges, the immune system of the oral mucosa is composed of tissue-resident and specially recruited leukocytes. These leukocytes facilitate the establishment and maintenance of local homeostasis but are also capable to cause oral pathologies when are unrestrained. γδT cells represent an important tissue-resident innate T-cell population in various mucosal and nonmucosal barrier tissues, in which they are ideally located to assist in immunosurveillance, tissue repair, and homeostasis. Whereas most works studying γδT cells were focused on tissues such as the skin and intestine, these cells in the oral mucosa were only recently thoroughly studied. The findings obtained by those studies appear to be both complementary and contradicting, likely reflecting differences in the experimental settings and the type of transgenic mouse modalities employed by each study. Nevertheless, oral γδT cells were shown to consist of developmentally distinct tissue-resident Vγ6 cells and circulating Vγ1 and Vγ4 subsets that are independently maintained in the oral mucosa. In the gingiva, a particularly challenging barrier tissue due to its proximity to the dental plaque, γδT cells are strategically positioned close to the plaque and represent the major source of IL-17. While this suggests that γδT cells might be involved in controlling the dental biofilm, conflicting data were reported in this regard. In vivo studies have shown that γδT cells either play a protective role during age-associated bone loss or, alternatively, have no impact in this process. Also, recent reports suggested opposing data concerning the impact of γδT cells in experimental periodontitis based on the ligature model. This review summarizes and discusses the most up-to-date literature on oral γδT cells, providing a balanced perspective regarding our current understanding on the development of oral γδT cells and their role under physiologic conditions and certain oral pathologies.

Germ Line Deletion Reveals a Nonessential Role of Atypical Mitogen-Activated Protein Kinase 6/Extracellular Signal-Regulated Kinase 3.

Mitogen-activated protein kinase 6/extracellular signal-regulated kinase 3 (MAPK6/ERK3) is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary dysfunction and by defects in function, activation, and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon 3 flanked by loxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing the first evidence for the existence of the ERK3/MK5 signaling complex in vivo In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile and do not display pulmonary hypoplasia, acute respiratory failure, abnormal T-cell development, reduction of thymocyte numbers, or altered T-cell selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality and lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin resistance cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal, suggesting redundant functions of both protein kinases.

Neutrophilic NLRP3 inflammasome-dependent IL-1ß secretion regulates the γδT17 cell response in respiratory bacterial infections.

Traditionally regarded as simple foot soldiers of the innate immune response limited to the eradication of pathogens, neutrophils recently emerged as more complex cells endowed with a set of immunoregulatory functions. Using a model of invasive pneumococcal disease, we highlighted an unexpected key role for neutrophils as accessory cells in innate interleukin (IL)-17A production by lung resident Vγ6Vδ1+ T cells via nucleotide-binding oligomerization domain receptor, pyrin-containing 3 (NLRP3) inflammasome-dependent IL-1ß secretion. In vivo activation of the NLRP3 inflammasome in neutrophils required both host-derived and bacterial-derived signals. Elaborately, it relies on (i) alveolar macrophage-secreted TNF-α for priming and (ii) subsequent exposure to bacterial pneumolysin for activation. Interestingly, this mechanism can be translated to human neutrophils. Our work revealed the cellular and molecular dynamic events leading to γδT17 cell activation, and highlighted for the first time the existence of a fully functional NLRP3 inflammasome in lung neutrophils. This immune axis thus regulates the development of a protective host response to respiratory bacterial infections.

Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation.

Foxp3 (forkhead box P3 transcription factor)-expressing regulatory T cells (Tregs) are essential for immunological tolerance, best illustrated by uncontrolled effector T-cell responses and autoimmunity upon loss of Foxp3 expression. Tregs can adopt specific effector phenotypes upon activation, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3(+)CD4(+) T cells coexpressing retinoic acid-related orphan receptor-γt (RORγt), the master transcription factor for T helper type 17 (Th17) cells, represent a stable effector Treg lineage. Transcriptomic and epigenetic profiling revealed that Foxp3(+)RORγt(+) T cells display signatures of both Tregs and Th17 cells, although the degree of similarity was higher to Foxp3(+)RORγt(-) Tregs than to Foxp3(-)RORγt(+) T cells. Importantly, Foxp3(+)RORγt(+) T cells were significantly demethylated at Treg-specific epigenetic signature genes such as Foxp3, Ctla-4, Gitr, Eos, and Helios, suggesting that these cells have a stable regulatory rather than inflammatory function. Indeed, adoptive transfer of Foxp3(+)RORγt(+) T cells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and revealed an enhanced suppressive capacity as compared with Foxp3(+)RORγt(-) Tregs. Thus, our data suggest that RORγt expression in Tregs contributes to an optimal suppressive capacity during gut-specific immune responses, rendering Foxp3(+)RORγt(+) T cells as an important effector Treg subset in the intestinal system.

[Digital vs. analog hearing aids for children. Is there a method for making an objective comparison possible?]. / Digitale vs. analoge Hörgeräte bei Kindern. Haben wir eine Methode, die uns einen objektiven Vergleich ermöglicht?

Until now, the assumed benefits of digital hearing aids are reflected only in subjective descriptions by patients with hearing aids, but cannot be documented adequately by routine diagnostic methods. Seventeen schoolchildren with moderate severe bilateral symmetrical sensorineural hearing loss were examined in a double-blinded crossover study. Differences in performance between a fully digital hearing aid (DigiFocus compact/Oticon) and an analogous digitally programmable two-channel hearing aid were evaluated. Of the 17 children, 13 choose the digital and 4 the analogous hearing aid. In contrast to the clear subjective preferences for the fully digital hearing aid, we could not obtain any significant results with routine diagnostic methods. Using the "virtual hearing aid," a subjective comparison and speech recognition performance task yielded significant differences. The virtual hearing aid proved to be suitable for a direct comparison of different hearing aids and can be used for double-blind testing in a pediatric population.

A "virtual hearing aid" for comparing hearing aids in children: a double-blind crossover study.

In this study, a "virtual hearing aid" was used to compare different types of hearing aids. A digital hearing aid (Oticon DigiFocus Compact) and an analogue, automatic reference hearing aid were compared in a group of 17 schoolchildren (median age: 10 years) with moderate to severe, symmetrical, sensorineural hearing loss. Differences in performance were assessed using routine diagnostic methods (speech recognition performance tests, loudness scaling), subjective assessments (questionnaires) and the "virtual hearing aid". Guaranteeing double-blind testing conditions, the "virtual hearing aid" offers the possibility to directly compare individual in-situ recordings of different hearing aids. In contrast to the clear subjective preferences for the digital hearing aid, we could not obtain any significant results with routine diagnostic methods. Using the "virtual hearing aid", the subjective comparison and speech recognition performance tasks yielded significant differences. The "virtual hearing aid" proved to be suitable for directly comparing different hearing aids under double-blind testing conditions.

Differential processing of cytosolic and mitochondrial caspases.

The mitochondria have been shown to play a key role in the initiation of caspase activation during apoptosis. Recently, some caspases have been shown to be associated with mitochondria. In this study, we used Jurkat T-lymphoblasts to show that caspases -2 and -3 are located in the mitochondrial intermembrane space, associated with the inner membrane. Caspase-9 is associated with the outer membrane and is exposed to the cytosolic compartment. Caspase activation took place predominantly in the cytosol in response to Fas ligation, but staurosporine treatment led to caspase activation in both cytosol and mitochondria. In response to both Fas and staurosporine treatment, caspase processing could be detected earlier in cytosol than in mitochondria, but this could reflect the limits of sensitive detection by immunoblotting. Only trace amounts of Apaf-1 were found in association with the mitochondria. However, staurosporine treatment led to preferential auto-processing of caspase-9 associated with mitochondria. These findings suggest that mitochondrial caspases are regulated independently of the cytosolic pool of caspases. The data are also consistent with the notion of a caspase nucleation site associated with mitochondria. Using a stable transfected CEM cell line, we show that Bcl-2 suppressed caspase processing in both cytosolic and mitochondrial compartments in response to both staurosporine and Fas ligation.

Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells.

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.