Putative regulation of macrophage-mediated inflammation by catestatin.

Catestatin (CST) is a bioactive cleavage product of the neuroendocrine prohormone chromogranin A (CgA). Recent findings show that CST can exert anti-inflammatory and antiadrenergic effects by suppressing the inflammatory actions of mammalian macrophages. However, recent findings also suggest that macrophages themselves are major CST producers. Here, we hypothesize that macrophages produce CST in an inflammation-dependent manner and thereby might self-regulate inflammation in an autocrine fashion. CST is associated with pathological conditions hallmarked by chronic inflammation, including autoimmune, cardiovascular, and metabolic disorders. Since intraperitoneal injection of CST in mouse models of diabetes and inflammatory bowel disease has been reported to be beneficial for mitigating disease, we posit that CST should be further investigated as a candidate target for treating certain inflammatory diseases.

Non-invasive imaging of sympathetic innervation of the pancreas in individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Compromised pancreatic sympathetic innervation has been suggested as a factor involved in both immune-mediated beta cell destruction and endocrine dysregulation of pancreatic islets. To further explore these intriguing findings, new techniques for in vivo assessment of pancreatic innervation are required. This is a retrospective study that aimed to investigate whether the noradrenaline (norepinephrine) analogue 11C-hydroxy ephedrine (11C-HED) could be used for quantitative positron emission tomography (PET) imaging of the sympathetic innervation of the human pancreas. METHODS: In 25 individuals with type 2 diabetes and 64 individuals without diabetes, all of whom had previously undergone 11C-HED-PET/CT because of pheochromocytoma or paraganglioma (or suspicion thereof), the 11C-HED standardised uptake value (SUVmean), 11C-HED specific binding index (SBI), pancreatic functional volume (FV, in ml), functional neuronal volume (FNV, calculated as SUVmean × FV), specific binding index with functional volume (SBI FV, calculated as SBI × FV) and attenuation on CT (HU) were investigated in the entire pancreas, and additionally in six separate anatomical pancreatic regions. RESULTS: Generally, 11C-HED uptake in the pancreas was high, with marked individual variation, suggesting variability in sympathetic innervation. Moreover, pancreatic CT attenuation (HU) (p<0.001), 11C-HED SBI (p=0.0049) and SBI FV (p=0.0142) were lower in individuals with type 2 diabetes than in individuals without diabetes, whereas 11C-HED SUVmean (p=0.15), FV (p=0.73) and FNV (p=0.30) were similar. CONCLUSIONS/INTERPRETATION: We demonstrate the feasibility of using 11C-HED-PET for non-invasive assessment of pancreatic sympathetic innervation in humans. These findings warrant further prospective evaluation, especially in individuals with theoretical defects in pancreatic sympathetic innervation, such as those with type 1 diabetes.

Perivascular Macrophages Regulate Blood Flow Following Tissue Damage.

[Figure: see text].

Regulatory Immune Mechanisms beyond Regulatory T Cells.

In autoimmunity, aggressive immune responses are counteracted by suppressive rejoinders. For instance, FOXP3-expressing regulatory T cells (Tregs), have shown remarkable effects in limiting autoimmunity in preclinical models. However, early results from human Treg trials have not been as positive. Here, we highlight questions surrounding Treg transfers as putative treatments for autoimmunity. We discuss whether lack of antigenic recognition might be key to shifting cells from contributing to an aggressive autoresponse, to being part of a regulatory network. Moreover, we argue that identifying the physiological range of immunosuppression of Tregs might help potentiate their efficacy. We propose widening the view on immunoregulation by considering the participation of CD8+ Tregs in this process, which could have major implications in autoimmunity.

The effect of Toll-like receptor stimulation on the motility of regulatory T cells.

Regulatory T cells (Tregs) have suppressive functions and play an important role in controlling inflammation and autoimmunity. The migratory capacity of Tregs determines their location and their location determines whether they inhibit the priming of naïve lymphocytes in lymphoid tissues or the effector phase of immune responses at inflamed sites. Tregs generated or expanded in vitro are currently being tested in clinics for the treatment of autoimmune disorders, however, little is known about the factors controlling their migration towards therapeutically relevant locations. In this study, we have modulated Treg dynamics using Toll-like receptor (TLR) agonists. Dynamic imaging with confocal and two-photon microscopy revealed that Tregs generated in vitro and stimulated with P3C (a TLR2 agonist) but not with R848 (a TLR7 agonist) or LPS (a TLR4 agonist) showed enhanced cell migration within splenic white pulp or draining lymph node when transferred into mice intravenously or into the footpad, respectively. In summary, our data demonstrate that Tregs are more motile in response to direct TLR stimulation in particular towards TLR2 signals. This may have implications for efficient clinical Treg induction protocols.

The neutrophil: one cell on many missions or many cells with different agendas?

The unique role of neutrophils in host defense is not only based on their abilities to kill bacteria but is also due to their abundance in circulation and their ability to quickly migrate and accumulate in great numbers at afflicted sites. The high number of circulating neutrophils is the result of regulated release of new neutrophils from bone marrow as well as from marginated pools to balance their recruitment to tissue. Marginated pools, such as the spleen and lung, have previously been attributed to passively delay neutrophil transit time due to their large capillary network, but recent reports demonstrate that they are comprised of neutrophils with specific functions. The spleen, for instance, holds neutrophil subpopulations at different anatomical locations with distinct functions important for, e.g., bacterial eradication, and the lung was recently shown to re-educate neutrophils that had trafficked from a site of sterile injury to home back to bone marrow for elimination. Further, recent reports demonstrate subpopulations of neutrophils with different actions during homeostasis, infection, tissue restitution and cancer. It is becoming increasingly clear that this cannot be due to different stages of neutrophil activation during their life span but instead points towards distinct subpopulations of neutrophils with different effector functions. Whether these cellular distinctions are due to different education or origin is, however, not yet known. Together, the accumulating information about the heterogeneous neutrophils presents important insights into their role in development of pathologies, as well as revealing novel targets in the form of certain subpopulations to treat disease.

Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans.

Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.

Matrix metalloproteinase-9 is essential for physiological Beta cell function and islet vascularization in adult mice.

The availability of paracrine factors in the islets of Langerhans, and the constitution of the beta cell basement membrane can both be affected by proteolytic enzymes. This study aimed to investigate the effects of the extracellular matrix-degrading enzyme gelatinase B/matrix metalloproteinase-9 (Mmp-9) on islet function in mice. Islet function of Mmp9-deficient (Mmp9(-/-)) mice and their wild-type littermates was evaluated both in vivo and in vitro. The pancreata of Mmp9(-/-) mice did not differ from wild type in islet mass or distribution. However, Mmp9(-/-) mice had an impaired response to a glucose load in vivo, with lower serum insulin levels. The glucose-stimulated insulin secretion was reduced also in vitro in isolated Mmp9(-/-) islets. The vascular density of Mmp9(-/-) islets was lower, and the capillaries had fewer fenestrations, whereas the islet blood flow was threefold higher. These alterations could partly be explained by compensatory changes in the expression of matrix-related proteins. This in-depth investigation of the effects of the loss of MMP-9 function on pancreatic islets uncovers a deteriorated beta cell function that is primarily due to a shift in the beta cell phenotype, but also due to islet vascular aberrations. This likely reflects the importance of a normal islet matrix turnover exerted by MMP-9, and concomitant release of paracrine factors sequestered on the matrix.

VEGF-A recruits a proangiogenic MMP-9-delivering neutrophil subset that induces angiogenesis in transplanted hypoxic tissue.

Recruitment and retention of leukocytes at a site of blood vessel growth are crucial for proper angiogenesis and subsequent tissue perfusion. Although critical for many aspects of regenerative medicine, the mechanisms of leukocyte recruitment to and actions at sites of angiogenesis are not fully understood. In this study, we investigated the signals attracting leukocytes to avascular transplanted pancreatic islets and leukocyte actions at the engraftment site. Expression of the angiogenic stimulus VEGF-A by mouse pancreatic islets was elevated shortly after syngeneic transplantation to muscle. High levels of leukocytes, predominantly CD11b(+)/Gr-1(+)/CXCR4(hi) neutrophils, were observed at the site of engraftment, whereas VEGF-A-deficient islets recruited only half of the amount of leukocytes when transplanted. Acute VEGF-A exposure of muscle increased leukocyte extravasation but not the levels of SDF-1α. VEGF-A-recruited neutrophils expressed 10 times higher amounts of MMP-9 than neutrophils recruited to an inflammatory stimulus. Revascularization of islets transplanted to MMP-9-deficient mice was impaired because blood vessels initially failed to penetrate grafts, and after 2 weeks vascularity was still disturbed. This study demonstrates that VEGF-A recruits a proangiogenic circulating subset of CD11b(+)/Gr-1(+) neutrophils that are CXCR4(hi) and deliver large amounts of the effector protein MMP-9, required for islet revascularization and functional integration after transplantation.

Acute sleep deprivation in healthy young men: impact on population diversity and function of circulating neutrophils.

Lack of sleep greatly affects our immune system. The present study investigates the acute effects of total sleep deprivation on blood neutrophils, the most abundant immune cell in our circulation and the first cell type recruited to sites of infection. Thus, the population diversity and function of circulating neutrophils were compared in healthy young men following one night of total sleep deprivation (TSD) or after 8h regular sleep. We found that neutrophil counts were elevated after nocturnal wakefulness (2.0 ± 0.2 × 10(9)/l vs. 2.6 ± 0.2 × 10(9)/l, sleep vs. TSD, respectively) and the population contained more immature CD16(dim)/CD62L(bright) cells (0.11 ± 0.040 × 10(9)/l [5.5 ± 1.1%] vs. 0.26 ± 0.020 × 10(9)/l [9.9 ± 1.4%]). As the rise in numbers of circulating mature CD16(bright)/CD62L(bright) neutrophils was less pronounced, the fraction of this subpopulation showed a significant decrease (1.8 ± 0.15 × 10(9)/l [88 ± 1.8%] vs. 2.1 ± 0.12 × 10(9)/l [82 ± 2.8%]). The surface expression of receptors regulating mobilization of neutrophils from bone marrow was decreased (CXCR4 and CD49d on immature neutrophils; CXCR2 on mature neutrophils). The receptor CXCR2 is also involved in the production of reactive oxygen species (ROS), and in line with this, total neutrophils produced less ROS. In addition, following sleep loss, circulating neutrophils exhibited enhanced surface levels of CD11b, which indicates enhanced granular fusion and concomitant protein translocation to the membrane. Our findings demonstrate that sleep loss exerts significant effects on population diversity and function of circulating neutrophils in healthy men. To which extent these changes could explain as to why people with poor sleep patterns are more susceptible to infections warrants further investigation.

Boundary cap neural crest stem cells promote angiogenesis after transplantation to avulsed dorsal roots in mice and induce migration of endothelial cells in 3D printed scaffolds.

Dorsal root avulsion injuries lead to loss of sensation and to reorganization of blood vessels (BVs) in the injured area. The inability of injured sensory axons to re-enter the spinal cord results in permanent loss of sensation, and often also leads to the development of neuropathic pain. Approaches that restore connection between peripheral sensory axons and their CNS targets are thus urgently need. Previous research has shown that sensory axons from peripherally grafted human sensory neurons are able to enter the spinal cord by growing along BVs which penetrate the CNS from the spinal cord surface. In this study we analysed the distribution of BVs after avulsion injury and how their pattern is affected by implantation at the injury site of boundary cap neural crest stem cells (bNCSCs), a transient cluster of cells, which are located at the boundary between the spinal cord and peripheral nervous system and assist the growth of sensory axons from periphery into the spinal cord during development. The superficial dorsal spinal cord vasculature was examined using intravital microscopy and intravascular BV labelling. bNCSC transplantation increased vascular volume in a non-dose responsive manner, whereas dorsal root avulsion alone did not decrease the vascular volume. To determine whether bNCSC are endowed with angiogenic properties we prepared 3D printed scaffolds, containing bNCSCs together with rings prepared from mouse aorta. We show that bNCSC do induce migration and assembly of endothelial cells in this system. These findings suggest that bNCSC transplant can promote vascularization in vivo and contribute to BV formation in 3D printed scaffolds.

GABA-mediated inhibition of human CD4+ T cell functions is enhanced by insulin but impaired by high glucose levels.

BACKGROUND: γ-aminobutyric acid (GABA), known as the main inhibitory neurotransmitter in the brain, exerts immunomodulatory functions by interaction with immune cells, including T cells. Metabolic programs of T cells are closely linked to their effector functions including proliferation, differentiation, and cytokine production. The physiological molecules glucose and insulin may provide environmental cues and guidance, but whether they coordinate to regulate GABA-mediated T cell immunomodulation is still being examined. METHODS: CD4+ T cells that were isolated from blood samples from healthy individuals and from patients with type 1 diabetes (T1D) were activated in vitro. We carried out metabolic assays, multiple proximity extension assay (PEA), ELISA, qPCR, immunoblotting, immunofluorescence staining, flow cytometry analysis, MS-based proteomics, as well as electrophysiology and live-cell Ca2+ imaging. FINDINGS: We demonstrate that GABA-mediated reduction of metabolic activity and the release of inflammatory proteins, including IFNγ and IL-10, were abolished in human CD4+ T cells from healthy individuals and patients with T1D when the glucose concentration was elevated above levels typically observed in healthy people. Insulin increased GABAA receptor-subunit ρ2 expression, enhanced the GABAA receptors-mediated currents and Ca2+ influx. GABA decreased, whereas insulin sustained, hexokinase activity and glycolysis in a glucose concentration-dependent manner. INTERPRETATION: These findings support that metabolic factors, such as glucose and insulin, influence the GABA-mediated immunomodulation of human primary T cells effector functions. FUNDING: The Swedish Children's Diabetes Foundation, The Swedish Diabetes Foundation, The Swedish Research Council 2018-02952, EXODIAB, The Ernfors Foundation, The Thurings Foundation and the Science for Life Laboratory.

Three-dimensional Co-culture Model for Live Imaging of Pancreatic Islets, Immune Cells, and Neurons in Agarose Gel.

During the onset of autoimmune diabetes, nerve-immune cell interactions seem to play an important role; however, there are currently no models to follow and interfere with these interactions over time in vivo or in vitro. Two-dimensional in vitro models provide insufficient information and microfluidics or organs on a chip are usually challenging to work with. We present here what we believe to be the first simple model that provides the opportunity to co-culture pancreatic islets with sympathetic nerves and immune cells. This model is based on our stamping device that can be 3D printed (STL file provided). Due to the imprint in the agarose gel, sympathetic neurons, pancreatic islets, and macrophages can be seeded in specific locations at a level that allows for confocal live-cell imaging. In this protocol, we provide the instructions to construct and perform live cell imaging experiments in our co-culture model, including: 1) design for the stamping device to make the imprint in the gel, 2) isolation of sympathetic neurons, pancreatic islets, and macrophages, 3) co-culture conditions, 4) how this can be used for live cell imaging, and 5) possibilities for wider use of the model. In summary, we developed an easy-to-use co-culture model that allows manipulation and imaging of interactions between sympathetic nerves, pancreatic islets, and macrophages. This new co-culture model is useful to study nerve-immune cell-islet interactions and will help to identify the functional relevance of neuro-immune interactions in the pancreas. Key features • A novel device that allows for 3D co-culture of sympathetic neurons, pancreatic islets, and immune cells • The device allows the capture of live interactions between mouse sympathetic neurons, pancreatic islets, and immune cells in a controlled environment after six days of co-culturing. • This protocol uses cultured sympathetic neurons isolated from the superior cervical ganglia using a previously established method (Jackson and Tourtellotte, 2014) in a 3D co-culture. • This method requires 3D printing of our own designed gel-stamping device (STL print file provided on SciLifeLab FigShare DOI: 10.17044/scilifelab.24073062).

Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency.

Vascular endothelial growth factor (VEGF)-A regulates angiogenesis, vascular morphology and permeability by signaling through its receptor VEGFR-2. The Shb adapter protein has previously been found to relay certain VEGFR-2 dependent signals and consequently vascular physiology and structure was assessed in Shb knockout mice. X-ray computed tomography of vessels larger than 24 µm diameter (micro-CT) after contrast injection revealed an increased frequency of 48-96 µm arterioles in the hindlimb calf muscle in Shb knockout mice. Intravital microscopy of the cremaster muscle demonstrated a less regular vasculature with fewer branch points and increased vessel tortuosity, changes that led to an increased blood flow velocity. Reduced in vivo angiogenesis was observed in Shb knockout Matrigel™ plugs. Unlike the wild-type situation, VEGF-A did not provoke a dissociation of VE-cadherin from adherens junctions in Shb knockout venules. The reduced angiogenesis and altered properties of junctions had consequences for two patho-physiological responses to arterial occlusion: vascular permeability was reduced in the Shb knockout cremaster muscle after ligation of one supplying artery and heat-induced blood flow determined by Laser-Doppler measurements was decreased in the hindlimb after ligation of the femoral artery. Consequently, the Shb knockout mouse exhibited structural and functional (angiogenesis and vascular permeability) vascular abnormalities that have implications for understanding the function of VEGF-A under physiological conditions.

A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils.

During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site.

The anti-inflammatory peptide Catestatin blocks chemotaxis.

Increased levels of the anti-inflammatory peptide Catestatin (CST), a cleavage product of the pro-hormone chromogranin A, correlate with less severe outcomes in hypertension, colitis, and diabetes. However, it is unknown how CST reduces the infiltration of monocytes and macrophages (Mϕs) in inflamed tissues. Here, it is reported that CST blocks leukocyte migration toward inflammatory chemokines. By in vitro and in vivo migration assays, it is shown that although CST itself is chemotactic, it blocks migration of monocytes and neutrophils to inflammatory attracting factor CC-chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, it directs CX3 CR1+ Mϕs away from pancreatic islets. These findings suggest that the anti-inflammatory actions of CST are partly caused by its regulation of chemotaxis.

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology.

BACKGROUND: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. METHODS: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFNγ) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. RESULTS: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFNγ and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFNγ in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. CONCLUSION: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.

Phagocytosis and Efferocytosis by Resident Macrophages in the Mouse Pancreas.

The tissue microenvironment in the mouse pancreas has been shown to promote very different polarizations of resident macrophages with islet-resident macrophages displaying an inflammatory "M1" profile and macrophages in the exocrine tissue mostly displaying an alternatively activated "M2" profile. The impact of this polarization on tissue homeostasis and diabetes development is unclear. In this study, the ability of pancreas-resident macrophages to phagocyte bacterial and endogenous debris was investigated. Mouse endocrine and exocrine tissues were separated, and tissue-resident macrophages were isolated by magnetic immunolabeling. Isolated macrophages were subjected to flow cytometry for polarization markers and qPCR for phagocytosis-related genes. Functional in vitro investigations included phagocytosis and efferocytosis assays using pH-sensitive fluorescent bacterial particles and dead fluorescent neutrophils, respectively. Intravital confocal imaging of in situ phagocytosis and efferocytosis in the pancreas was used to confirm findings in vivo. Gene expression analysis revealed no significant overall difference in expression of most phagocytosis-related genes in islet-resident vs. exocrine-resident macrophages included in the analysis. In this study, pancreas-resident macrophages were shown to differ in their ability to phagocyte bacterial and endogenous debris depending on their microenvironment. This difference in abilities may be one of the factors polarizing islet-resident macrophages to an inflammatory state since phagocytosis has been found to imprint macrophage heterogeneity. It remains unclear if this difference has any implications in the development of islet dysfunction or autoimmunity.

Means, Motive, and Opportunity: Do Non-Islet-Reactive Infiltrating T Cells Contribute to Autoimmunity in Type 1 Diabetes?

In human type 1 diabetes and animal models of the disease, a diverse assortment of immune cells infiltrates the pancreatic islets. CD8+ T cells are well represented within infiltrates and HLA multimer staining of pancreas sections provides clear evidence that islet epitope reactive T cells are present within autoimmune lesions. These bona fide effectors have been a key research focus because these cells represent an intellectually attractive culprit for ß cell destruction. However, T cell receptors are highly diverse in human insulitis. This suggests correspondingly broad antigen specificity, which includes a majority of T cells for which there is no evidence of islet-specific reactivity. The presence of "non-cognate" T cells in insulitis raises suspicion that their role could be beyond that of an innocent bystander. In this perspective, we consider the potential pathogenic contribution of non-islet-reactive T cells. Our intellectual framework will be that of a criminal investigation. Having arraigned islet-specific CD8+ T cells for the murder of pancreatic ß cells, we then turn our attention to the non-target immune cells present in human insulitis and consider the possible regulatory, benign, or effector roles that they may play in disease. Considering available evidence, we overview the case that can be made that non-islet-reactive infiltrating T cells should be suspected as co-conspirators or accessories to the crime and suggest some possible routes forward for reaching a better understanding of their role in disease.

An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses.

Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.