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Adoptive immunotherapy with Tregs is a promising approach for preventing or treating type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR redirected NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Cotransfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In WT NOD mice, InsB-g7 CAR Tregs prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising therapeutic approach for the prevention of autoimmune diabetes.

The notion that T cell insulitis increases as type 1 diabetes (T1D) develops is unsurprising, however, the quantitative analysis of CD4 + and CD8 + T cells within the islet mass is complex and limited with standard approaches. Optical microscopy is an important and widely used method to evaluate immune cell infiltration into pancreatic islets of Langerhans for the study of disease progression or therapeutic efficacy in murine T1D. However, the accuracy of this approach is often limited by subjective and potentially biased qualitative assessment of immune cell subsets. In addition, attempts at quantitative measurements require significant time for manual analysis and often involve sophisticated and expensive imaging software. In this study, we developed and illustrate here a streamlined analytical strategy for the rapid, automated and unbiased investigation of islet area and immune cell infiltration within (insulitis) and around (peri-insulitis) pancreatic islets. To this end, we demonstrate swift and accurate detection of islet borders by modeling cross-sectional islet areas with convex polygons (convex hulls) surrounding islet-associated insulin-producing β cell and glucagon-producing α cell fluorescent signals. To accomplish this, we used a macro produced with the freeware software ImageJ equipped with the Fiji Is Just ImageJ (FIJI) image processing package. Our image analysis procedure allows for direct quantification and statistical determination of islet area and infiltration in a reproducible manner, with location-specific data that more accurately reflect islet areas as insulitis proceeds throughout T1D. Using this approach, we quantified the islet area infiltrated with CD4 + and CD8 + T cells allowing statistical comparison between different age groups of non-obese diabetic (NOD) mice progressing towards T1D. We found significantly more CD4 + and CD8 + T cells infiltrating the convex hull-defined islet mass of 13-week-old non-diabetic and 17-week-old diabetic NOD mice compared to 4-week-old NOD mice. We also determined a significant and measurable loss of islet mass in mice that developed T1D. This approach will be helpful for the location-dependent quantitative calculation of islet mass and cellular infiltration during T1D pathogenesis and can be combined with other markers of inflammation or activation in future studies.

CD8 + T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8 + resident memory T cells (T RM cells) within the reproductive tracts of live female mice. We found that mucosal T RM cells were highly motile, but paused and underwent in situ division after local antigen challenge. T RM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent T RM cell differentiation in situ. However, the proliferation of pre-existing T RM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary T RM cell population. We observed similar results in skin. Thus, T RM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue. Masopust and colleagues show that mucosal tissue-resident memory T cells proliferate in situ in response to local antigen and dominate the local recall response.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, which showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks of age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks of age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

We investigate how myeloid subsets differentially shape immunity to pancreatic ductal adenocarcinoma (PDA). We show that tumor antigenicity sculpts myeloid cell composition and functionality. Antigenicity promotes accumulation of type 1 dendritic cells (cDC1), which is driven by Xcr1 signaling, and overcomes macrophage-mediated suppression. The therapeutic activity of adoptive T cell therapy or programmed cell death ligand 1 blockade required cDC1s, which sustained splenic Klrg1+ cytotoxic antitumor T cells and functional intratumoral T cells. KLRG1 and cDC1 genes correlated in human tumors, and PDA patients with high intratumoral KLRG1 survived longer than patients with low intratumoral KLRG1. The immunotherapy CD40 agonist also required host cDC1s for maximal therapeutic benefit. However, CD40 agonist exhibited partial therapeutic benefit in cDC1-deficient hosts and resulted in priming of tumor-specific yet atypical CD8+ T cells with a regulatory phenotype and that failed to participate in tumor control. Monocyte/macrophage depletion using clodronate liposomes abrogated T cell priming yet enhanced the antitumor activity of CD40 agonist in cDC1-deficient hosts via engagement of innate immunity. In sum, our study supports that cDC1s are essential for sustaining effective antitumor T cells and supports differential roles for cDC1s and monocytes/macrophages in instructing T cell fate and immunotherapy response.

Type 1 diabetes is caused by autoreactive T cell-mediated β cell destruction. Even though co-inhibitory receptor programmed death-1 (PD-1) restrains autoimmunity, the expression and regulation of its cognate ligands on β cell remains unknown. Here, we interrogated β cell-intrinsic programmed death ligand-1 (PD-L1) expression in mouse and human islets. We measured a significant increase in the level of PD-L1 surface expression and the frequency of PD-L1 + β cells as non-obese diabetic (NOD) mice aged and developed diabetes. Increased β cell PD-L1 expression was dependent on T cell infiltration, as β cells from Rag1-deficient mice lacked PD-L1. Using Rag1-deficient NOD mouse islets, we determined that IFN-γ promotes β cell PD-L1 expression. We performed analogous experiments using human samples, and found a significant increase in β cell PD-L1 expression in type 1 diabetic samples compared to type 2 diabetic, autoantibody positive, and non-diabetic samples. Among type 1 diabetic samples, β cell PD-L1 expression correlated with insulitis. In vitro experiments with human islets from non-diabetic individuals showed that IFN-γ promoted β cell PD-L1 expression. These results suggest that insulin-producing β cells respond to pancreatic inflammation and IFN-γ production by upregulating PD-L1 expression to limit self-reactive T cells.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.

Here we present evidence of the first field observation of the nonnative Ictalurus furcatus (Blue Catfish) occurring on the Satilla River, GA, in May 2011, and additional collections since then. This is the second large, non-native riverine catfish to be found in the Satilla River basin. Pylodictis olivaris (Flathead catfish) was first collected from the Satilla River in May 1996. The ecological effects of Blue Catfish on native mussel and fish species in the Satilla River are currently unknown, but competition with native catfishes is likely.

CD8.sup.+ T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8.sup.+ resident memory T cells (T.sub.RM cells) within the reproductive tracts of live female mice. We found that mucosal T.sub.RM cells were highly motile, but paused and underwent in situ division after local antigen challenge. T.sub.RM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent T.sub.RM cell differentiation in situ. However, the proliferation of pre-existing T.sub.RM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary T.sub.RM cell population. We observed similar results in skin. Thus, T.sub.RM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue.

Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T‐cell functions. The aggregation of specific GM1 lipid rafts can control many T‐cell activation events, including their novel association with T‐cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow‐dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F‐actin, the resistance to shear‐induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin ‘inside‐out’ signaling mechanisms. This was seen through increased integrin–cytoskeleton associations and enhanced soluble binding of FN and VCAM‐1, suggesting the induction of high‐affinity integrin conformations. The activation of these adhesion‐strengthening characteristics appears to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft‐associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion‐strengthening processes.