Identifying a novel γδ T-cell stress ligand: Potential for cancer immunotherapy

Background: γδ T-cells can kill multiple cancer types and virally infected cells independent of human leukocyte antigen (HLA). Targeting is believed to occur via recognition of generic, unidentified, stress signals at the target cell surface via the γδ T-cell receptor (TCR). Consequently, γδ TCR and its ligands offer potential for pan-population, off-the-shelf immunotherapies for a range of pathologies including cancer. I aimed to identify pan-cancer targeting γδ T-cells, describe their TCRs and discover the ligands they use to recognise such a wide range of targets. Results: I used herpesvirus infected cells to prime and isolate rare, tumour reactive γδ T-cell lines from three healthy donors. An interesting T-cell clone from each individual was subjected to novel technologies to find the TCR ligand. A whole genome CRISPR/Cas9 library screen determined that tumour recognition by one clone required that transformed cells express SCNN1A, a key component of the major epithelial sodium transporter. Surprisingly, CRIPSR gene editing and lentiviral transduction technologies demonstrated that the two other clones recognised allogeneic HLA class II alleles expressed on tumour cells. Conclusions: My results offer opportunities for broad-ranging cancer immunotherapies that are HLA-independent and could be applied in all individuals. My discovery that γδ T-cells can recognise allogeneic HLA alleles also serves as a cautionary note to those wishing to apply therapeutic approaches via allogeneic γδ T-cells. My results blur the boundaries between conventional αβ T-cell recognition of peptide-HLA and the ‘unconventional’ γδ T-cell subset that are often currently believed to be blind to HLA. These findings will be of major interest to the γδ T-cell field and should serve as important milestone to future studies of HLA-restricted and non-HLA restricted γδ T-cells

Punishment in Prison: Constituting the Normal and the Atypical in Solitary and Other Forms of Confinement

What aspects of human liberty does incarceration impinge? A remarkable group of Black and white prisoners, most of whom had little formal education and no resources, raised that question in the 1960s and 1970s. Incarcerated individuals asked judges for relief from corporal punishment; radical food deprivations; strip cells; solitary confinement in dark cells; prohibitions on bringing these claims to courts, on religious observance, and on receiving reading materials; and from transfers to long- term isolation and to higher security levels. Judges concluded that some facets of prison that were once ordinary features of incarceration, such as racial segregation, rampant violence, and filth, violated the Constitution. Today, even as implementation is erratic and at times abysmal, correctional departments no longer claim they have unfettered authority to do what they want inside prisons walls. And, even as the courts have continued to tolerate the punishment of solitary confinement in the last decade, a few lower courts have held unconstitutional the profound sensory deprivations such isolation has entailed. Prisoners have also sought procedural protections to constrain arbitrary decision-making about placements in solitary confinement and transfers to adverse settings. In response, the Supreme Court has required that, to state a Fourteenth Amendment claim that their liberty had been infringed, prisoners have to demonstrate that a specific practice imposed an “atypical” and “significant hardship.” What is typical in prisons? What are the sources of knowledge and the baselines used by Justices to decide? How did isolation come to be seen as an ordinary incident of prison life? We answer these questions through analyzing debates in both the U.S. Supreme Court and lower courts about what deprivations in prison are “normal.” After excavating the conflicts within the Court about the kinds of liberty interests prisoners retained, we mined hundreds of lower court opinions to learn how judges determine when constrictions on human movement meet the test of atypicality and hardship. By documenting the high tolerance many federal judges have for periods of isolation lasting months, years, and decades, we demonstrate the central role judges play in constructing the “normal” of prisons

Optimized peptide-MHC multimer protocols for detection and isolation of autoimmune T-cells

<p>Peptide–MHC (pMHC) multimers have become the “gold standard” for the detection and isolation of antigen-specific T-cells but recent evidence shows that normal use of these reagents can miss fully functional T-cells that bear T-cell receptors (TCRs) with low affinity for cognate antigen. This issue is particularly pronounced for anticancer and autoimmune T-cells as self-reactive T-cell populations are enriched for low-affinity TCRs due to the removal of cells with higher affinity receptors by immune tolerance mechanisms. Here, we stained a wide variety of self-reactive human T-cells using regular pMHC staining and an optimized technique that included: (i) protein kinase inhibitor (PKI), to prevent TCR triggering and internalization, and (ii) anti-fluorochrome antibody, to reduce reagent dissociation during washing steps. Lymphocytes derived from the peripheral blood of type 1 diabetes patients were stained with pMHC multimers made with epitopes from preproinsulin (PPI), insulin-β chain, glutamic acid decarboxylase 65 (GAD65), or glucose-6-phospate catalytic subunit-related protein (IGRP) presented by disease-risk allelles HLA A*02:01 or HLA*24:02. Samples from ankylosing spondylitis patients were stained with a multimerized epitope from vasoactive intestinal polypeptide receptor 1 (VIPR1) presented by HLA B*27:05. Optimized procedures stained an average of 40.5-fold (p = 0.01, range between 1.4 and 198) more cells than could be detected without the inclusion of PKI and cross-linking anti-fluorochrome antibody. Higher order pMHC dextramers recovered more cells than pMHC tetramers in parallel assays, and standard staining protocols with pMHC tetramers routinely recovered less cells than functional assays. HLA A*02:01-restricted PPI-specific and HLA B*27:05-restricted VIPR1-specific T-cell clones generated using the optimized procedure could not be stained by standard pMHC tetramer staining. However, these clones responded well to exogenously supplied peptide and endogenously processed and presented epitopes. We also showed that anti-fluorochrome antibody-conjugated magnetic beads enhanced staining of self-reactive T-cells that could not be stained using standard protocols, thus enabling rapid ex vivo isolation of autoimmune T-cells. We, therefore, conclude that regular pMHC tetramer staining is generally unsuitable for recovering self-reactive T-cells from clinical samples and recommend the use of the optimized protocols described herein.</p

Recognition memory, self-other source memory, and theory-of-mind in children with autism spectrum disorder.

This study investigated semantic and episodic memory in autism spectrum disorder (ASD), using a task which assessed recognition and self-other source memory. Children with ASD showed undiminished recognition memory but significantly diminished source memory, relative to age- and verbal ability-matched comparison children. Both children with and without ASD showed an “enactment effect”, demonstrating significantly better recognition and source memory for self-performed actions than other-person-performed actions. Within the comparison group, theory-of-mind (ToM) task performance was significantly correlated with source memory, specifically for other-person-performed actions (after statistically controlling for verbal ability). Within the ASD group, ToM task performance was not significantly correlated with source memory (after controlling for verbal ability). Possible explanations for these relations between source memory and ToM are considered

Dye Tracing to Camp Coldwater Spring, Minneapolis, MN

Camp Coldwater Spring is a ~6.3 liter/sec spring that emerges from a Platteville Limestone ledge at the top of the west side of the Mississippi River gorge. It was the original water supply for Ft. Snelling in the early-mid 1800’s and is a registered Minnesota State Landmark. Potential impacts from nearby highway construction led to two successful dye traces to help define the groundwater basin feeding the spring. These traces are the first traces through the Platteville in the Twin Cities. Dye input trenches were dug with a backhoe to the top of the water table. Input B reached the top of the Platteville and the water table was a few inches above the bedrock surface. Eosin dye input into the trench reached Camp Coldwater Spring, 125 meters away in less than 1.5 hours. The minimum flow velocity in the fractured Platteville Limestone was 83 m/hr. Input C reached the water table while still in glaciofluvial sediments and was 305 meters from the spring. Fluorescein dye from Input C reached the spring in 16 days. The slower flow velocity is a combination of flow through the glaciofluvial sediments and through the fractured Platteville Limestone. These two positive traces demonstrate that Inputs B and C are inside the ground-watershed that supplies the Spring and support concerns about the potential impact of dewatering and construction activities on the Spring. The trace is ongoing.Minnehaha Creek Watershed Distric

Delayed self-recognition in children with autism spectrum disorder.

This study aimed to investigate temporally extended self-awareness (awareness of one’s place in and continued existence through time) in autism spectrum disorder (ASD), using the delayed self-recognition (DSR) paradigm (Povinelli et al., Child Development 67:1540–1554, 1996). Relative to age and verbal ability matched comparison children, children with ASD showed unattenuated performance on the DSR task, despite showing significant impairments in theory-of-mind task performance, and a reduced propensity to use personal pronouns to refer to themselves. The results may indicate intact temporally extended self-awareness in ASD. However, it may be that the DSR task is not an unambiguous measure of temporally extended self-awareness and it can be passed through strategies which do not require the possession of a temporally extended self-concept

Effect of an audiovisual message for tetanus booster vaccination broadcast in the waiting room

<p>Abstract</p> <p>Background</p> <p>General practitioners (GPs) often lack time and resources to invest in health education; audiovisual messages broadcast in the waiting room may be a useful educational tool. This work was designed to assess the effect of a message inviting patients to ask for a tetanus booster vaccination.</p> <p>Methods</p> <p>A quasi experimental study was conducted in a Belgian medical practice consisting of 6 GPs and 4 waiting rooms (total: 20,000 contacts/year). A tetanus booster vaccination audiovisual message was continuously broadcast for 6 months in 2 randomly selected waiting rooms (intervention group - 3 GPs) while the other 2 waiting rooms remained unequipped (control group - 3 GPs). At the end of the 6-month period, the number of vaccine adult-doses delivered by local pharmacies in response to GPs' prescriptions was recorded. As a reference, the same data were also collected retrospectively for the general practice during the same 6-month period of the previous year.</p> <p>Results</p> <p>During the 6-month reference period where no audiovisual message was broadcast in the 4 waiting rooms, the number of prescriptions presented for tetanus vaccines was respectively 52 (0.44%) in the intervention group and 33 (0.38%) in the control group (p = 0.50). By contrast, during the 6-month study period, the number of prescriptions differed between the two groups (p < 0.0001), rising significantly to 91 (0.79%) in the intervention group (p = 0.0005) while remaining constant in the control group (0.38% vs 0.39%; p = 0.90).</p> <p>Conclusions</p> <p>Broadcasting an audiovisual health education message in the GPs' waiting room was associated with a significant increase in the number of adult tetanus booster vaccination prescriptions delivered by local pharmacies.</p