Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Physiology and molecular basis of thallium toxicity and accumulation in Arabidopsis thaliana.

Thallium (Tl) is a non-essential metal mobilized through industrial processes which can lead to it entering the environment and exerting toxic effects. Plants are fundamental components of all ecosystems. Therefore, understanding the impact of Tl on plant growth and development is of great importance for assessing the potential environmental risks of Tl. Here, the responses of Arabidopsis thaliana to Tl were elucidated using physiological, genetic, and transcriptome analyses. Thallium can be absorbed by plant roots and translocated to the aerial parts, accumulating at comparable concentrations throughout plant parts. Genetic evidence supported the regulation of Tl uptake and movement by different molecular compartments within plants. Thallium primarily caused growth inhibition, oxidative stress, leaf chlorosis, and the impairment of K homeostasis. The disturbance of redox balance toward oxidative stress was supported by significant differences in the expression of genes involved in oxidative stress and antioxidant defense under Tl exposure. Reduced GSH levels in cad2-1 mutant rendered plants highly sensitive to Tl, suggesting that GSH has a prominent role in alleviating Tl-triggered oxidative responses. Thallium down-regulation of the expression of LCHII-related genes is believed to be responsible for leaf chlorosis. These findings illuminate some of the mechanisms underlying Tl toxicity at the physiological and molecular levels in plants with an eye toward the future environment management of this heavy metal.

Maternal mood, anxiety and mental health functioning after combined myo-inositol, probiotics, micronutrient supplementation from preconception: Findings from the NiPPeR RCT.

Observational studies have reported associations between nutrition during pregnancy and mental wellbeing. As secondary outcomes, the NiPPeR double-blind randomized trial in women planning conception investigated whether a myo-inositol, probiotics and enriched micronutrients formulation (intervention) taken preconception and throughout pregnancy could improve mental wellbeing during pregnancy and post-delivery, compared with a standard micronutrient supplement (control). Mood and anxiety symptoms were ascertained (Edinburgh Postnatal Depression Scale (EPDS), State-Trait Anxiety Inventory (STAI-state)) at preconception (baseline), 7, 28 and 34 weeks gestation, 3-weeks and 6-months post-delivery. EPDS>=13 was categorised as low mood; STAI-state>=45 as high anxiety. Change in mental health functioning was assessed as difference between preconception baseline and 6-month post-delivery 12-item Short-Form Health Survey (SF-12v2) mental component scores. Adjusting for site, ethnicity and baseline scores, there were no robust differences in EPDS and STAI-state scores between intervention and control groups across pregnancy (n = 630) and post-delivery (n = 532). Compared to controls, intervention group women averaged a 1.21 (95 %CI 0.04,2.39) higher change in SF-12v2 mental component score from preconception to 6-months post-delivery. Taking a myo-inositol, micronutrient and probiotic supplement during preconception/pregnancy had no effect on mood and anxiety, but there was evidence of a modest improvement in mental health functioning from preconception to 6-months post-delivery.

Lytic granule exocytosis at immune synapses: lessons from neuronal synapses.

Regulated exocytosis is a central mechanism of cellular communication. It is not only the basis for neurotransmission and hormone release, but also plays an important role in the immune system for the release of cytokines and cytotoxic molecules. In cytotoxic T lymphocytes (CTLs), the formation of the immunological synapse is required for the delivery of the cytotoxic substances such as granzymes and perforin, which are stored in lytic granules and released via exocytosis. The molecular mechanisms of their fusion with the plasma membrane are only partially understood. In this review, we discuss the molecular players involved in the regulated exocytosis of CTL, highlighting the parallels and differences to neuronal synaptic transmission. Additionally, we examine the strengths and weaknesses of both systems to study exocytosis.

Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation.

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

Temporal transformation of indium speciation in rice paddy soils and spatial distribution of indium in rice rhizosphere.

Indium is a potentially toxic element that could enter human food chains, including soil-rice systems. The submerged environment in rice paddy soil results in temporal and spatial variations in the chemical properties of the rice rhizosphere and bulk soils, expected to cause changes in indium's chemical speciation and consequently affect its bioavailability. Therefore, this study aimed to investigate indium speciation and fractionation in soils at different periods of rice growth under continuous submergence using X-ray absorption spectroscopy and a sequential extraction method. The predominant indium species were identified as indium-associated Fe hydroxide, and indium hydroxide and phosphate precipitates. The reductive dissolution of indium-associated Fe hydroxides led to the release of indium into the soil solution under continuous submergence of soils, and the released indium concentration decreased with time due to re-sorption and re-precipitation. Meanwhile, indium hydroxide was found to be the predominant species in rice rhizosphere using µ-X-ray absorption spectroscopy. The relative depletion of indium-associated Fe hydroxides in the rice rhizosphere was attributed to the low mobility of indium from bulk soil to rice rhizosphere and the root uptake of indium associated with Fe hydroxide around rice roots. Consequently, indium uptake by rice roots was lower during the reproductive and grain-ripening stage of rice growth. Understanding the behavior of indium will help develop a strategy to minimize uptake into crops in indium-contaminated paddy soils.

Heterozygous OT-I mice reveal that antigen-specific CD8+ T cells shift from apoptotic to necrotic killers in the elderly.

Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8+ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT-I+/+ and OT-I+/- mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8+ T cells from adult OT-I+/- mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8+ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I+/- mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I+/- model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzyme-mediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation.

Study of Effector CD8+ T Cell Interactions with Cortical Neurons in Response to Inflammation in Mouse Brain Slices and Neuronal Cultures.

Cytotoxic CD8+ T cells contribute to neuronal damage in inflammatory and degenerative CNS disorders, such as multiple sclerosis (MS). The mechanism of cortical damage associated with CD8+ T cells is not well understood. We developed in vitro cell culture and ex vivo brain slice co-culture models of brain inflammation to study CD8+ T cell-neuron interactions. To induce inflammation, we applied T cell conditioned media, which contains a variety of cytokines, during CD8+ T cell polyclonal activation. Release of IFNγ and TNFα from co-cultures was verified by ELISA, confirming an inflammatory response. We also visualized the physical interactions between CD8+ T cells and cortical neurons using live-cell confocal imaging. The imaging revealed that T cells reduced their migration velocity and changed their migratory patterns under inflammatory conditions. CD8+ T cells increased their dwell time at neuronal soma and dendrites in response to added cytokines. These changes were seen in both the in vitro and ex vivo models. The results confirm that these in vitro and ex vivo models provide promising platforms for the study of the molecular details of neuron-immune cell interactions under inflammatory conditions, which allow high-resolution live microscopy and are readily amenable to experimental manipulation.

Time-to-conception and clinical pregnancy rate with a myo-inositol, probiotics, and micronutrient supplement: secondary outcomes of the NiPPeR randomized trial.

OBJECTIVE: To determine whether a combined myo-inositol, probiotics and micronutrient nutritional supplement impacts time-to-natural-conception and clinical pregnancy rates. DESIGN: Secondary outcomes of a double-blind randomized controlled trial. SETTING: Community recruitment. PATIENTS: Women aged 18 to 38 years planning to conceive in the United Kingdom, Singapore, and New Zealand, excluding those with diabetes mellitus or receiving fertility treatment. INTERVENTION: A standard (control) supplement (folic acid, iron, calcium, iodine, ß-carotene), compared with an intervention additionally containing myo-inositol, probiotics, and other micronutrients (vitamins B2, B6, B12, D, zinc). MAIN OUTCOME MEASURES: Number of days between randomization and estimated date of natural conception of a clinical pregnancy, as well as cumulative pregnancy rates at 3, 6, and 12 months. RESULTS: Of 1729 women randomized, 1437 (83%; intervention, n=736; control, n=701) provided data. Kaplan-Meier curves of conception were similar between intervention and control groups; the time at which 20% achieved natural conception was 90.5 days (95% confidence interval: 80.7, 103.5) in the intervention group compared with 92.0 days (76.0, 105.1) in the control group. Cox's proportional hazard ratios (HRs) comparing intervention against control for cumulative achievement of pregnancy (adjusted for site, ethnicity, age, body mass index, and gravidity) were similar at 3, 6, and 12 months. Among both study groups combined, overall time-to-conception lengthened with higher preconception body mass index, and was longer in non-White than in White women. Among women who were overweight the intervention shortened time-to-conception compared with control regardless of ethnicity (12-month HR=1.47 [1.07, 2.02], P=.016; 20% conceived by 84.5 vs. 117.0 days) and improved it to that comparable to nonoverweight/nonobese women (20% conceived by 82.1 days). In contrast, among women with obesity, time-to-conception was lengthened with intervention compared with control (12-month HR=0.69 [0.47, 1.00]; P=.053; 20% conceived by 132.7 vs. 108.5 days); an effect predominantly observed in non-White women with obesity. CONCLUSIONS: Time-to-natural-conception and clinical pregnancy rates within a year were overall similar in women receiving the intervention supplement compared with control. Overweight women had a longer time-to-conception but there was suggestion that the supplement may shorten their time-to-conception to that comparable to the nonoverweight/nonobese women. Further studies are required to confirm this. CLINICAL TRIAL REGISTRATION NUMBER: clinicaltrials.gov (NCT02509988).

Peripartum outcomes after combined myo-inositol, probiotics, and micronutrient supplementation from preconception: the NiPPeR randomized controlled trial.

BACKGROUND: Evidence that nutritional supplementation before and during pregnancy improves peripartum outcomes is sparse. In the Nutritional Intervention Preconception and During Pregnancy to Maintain Healthy Glucose Metabolism and Offspring Health (NiPPeR) trial, we previously reported that a combined myo-inositol, probiotics, and micronutrient supplement started at preconception showed no difference in the primary outcome of gestational glycemia, but did reduce the risk of preterm delivery, preterm prelabor rupture of membranes, and major postpartum hemorrhage. OBJECTIVE: This study aimed to examine the hypothesis that a reduction in major postpartum hemorrhage following a combined nutritional (myo-inositol, probiotics, and micronutrients) intervention is linked with promotion of labor progress and reduced operative delivery. STUDY DESIGN: This double-blind randomized controlled trial recruited 1729 women from the United Kingdom, Singapore, and New Zealand, aged 18 to 38 years, and planning conception between 2015 and 2017. The effects of the nutritional intervention compared with those of a standard micronutrient supplement (control), taken at preconception and throughout pregnancy, were examined for the secondary outcomes of peripartum events using multinomial, Poisson, and linear regression adjusting for site, ethnicity, and important covariates. RESULTS: Of the women who conceived and progressed beyond 24 weeks' gestation with a singleton pregnancy (n=589), 583 (99%) provided peripartum data. Between women in the intervention (n=293) and control (n=290) groups, there were no differences in rates of labor induction, oxytocin augmentation during labor, instrumental delivery, perineal trauma, and intrapartum cesarean delivery. Although duration of the first stage of labor was similar, the second-stage duration was 20% shorter in the intervention than in the control group (adjusted mean difference, -12.0 [95% confidence interval, -22.2 to -1.2] minutes; P=.029), accompanied by a reduction in operative delivery for delayed second-stage progress (adjusted risk ratio, 0.61 [0.48-0.95]; P=.022). Estimated blood loss was 10% lower in the intervention than in the control group (adjusted mean difference, -35.0 [-70.0 to -3.5] mL; P=.047), consistent with previous findings of reduced postpartum hemorrhage. CONCLUSION: Supplementation with a specific combination of myo-inositol, probiotics, and micronutrients started at preconception and continued in pregnancy reduced the duration of the second stage of labor, the risk of operative delivery for delay in the second stage, and blood loss at delivery.

Impaired bidirectional communication between interneurons and oligodendrocyte precursor cells affects social cognitive behavior.

Cortical neural circuits are complex but very precise networks of balanced excitation and inhibition. Yet, the molecular and cellular mechanisms that form the balance are just beginning to emerge. Here, using conditional γ-aminobutyric acid receptor B1- deficient mice we identify a γ-aminobutyric acid/tumor necrosis factor superfamily member 12-mediated bidirectional communication pathway between parvalbumin-positive fast spiking interneurons and oligodendrocyte precursor cells that determines the density and function of interneurons in the developing medial prefrontal cortex. Interruption of the GABAergic signaling to oligodendrocyte precursor cells results in reduced myelination and hypoactivity of interneurons, strong changes of cortical network activities and impaired social cognitive behavior. In conclusion, glial transmitter receptors are pivotal elements in finetuning distinct brain functions.

Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes.

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets.

The Effect of a 20-Hour Baby-Friendly Hospital Initiative Training Program on Nurses' Breastfeeding Knowledge, Attitudes and Confidence, in a Tertiary Hospital in Singapore.

OBJECTIVE: The Baby-Friendly Hospital Initiative (BFHI) enables maternity units to be centers of breastfeeding support to increase breastfeeding rates. This study evaluates the impact of the 20-hour BFHI training course on nurses' breastfeeding knowledge, attitude, and confidence in breastfeeding practice in a tertiary hospital in Singapore. STUDY DESIGN: Seventeen sessions of the 20-hour BFHI training course were conducted by lactation consultants from 2010 to 2013 at the National University Hospital, Singapore. An anonymous self-administered survey on knowledge, attitude, and confidence in breastfeeding practices were distributed to nurses before (2009) and after (2014) the training courses to assess effectiveness of training. RESULTS: One-hundred forty nurses and one hundred forty-eight nurses participated in the surveys in 2009 and 2014, respectively. Majority were registered nurses who worked in the postnatal wards and the neonatal intensive care unit. After training, there were significant improvements for five of eight items in infant feeding knowledge, including greater awareness of the International Code of Marketing of Breastmilk Substitutes and medical contraindication for breastfeeding. Participants reported more confidence in assisting mothers on breastfeeding, 77.1 to 88.5% (p = 0.019); advising hand expressing breast milk, 75.7 to 86.5% (p = 0.012); and advising attachment to the breast, 75.7 to 89.2% (p = 0.004) in 2014 compared with 2009. However, despite having high levels of confidence, only about half the nurses reported being able to assist mothers in breastfeeding, mainly due to time constraints. CONCLUSION: Implementation of the 20-hour BFHI training program positively influenced nurses' breastfeeding knowledge, attitude, and confidence in breastfeeding practices. Hospital procedures and manpower requirements should be re-examined to overcome nursing constraints in providing breastfeeding help to postpartum mothers. KEY POINTS: · Nurses have low breastfeeding knowledge pretraining.. · The 20-hour BFHI training course is effective.. · Nurses have inadequate time to support breastfeeding..

Soil gallium speciation and resulting gallium uptake by rice plants.

Gallium (Ga) is widely used in high-tech industries and is an emerging contaminant in the environment. This study aimed to determine Ga speciation in soils and Ga accumulation in rice plants (Oryza sativa L.) grown in three Ga-contaminated soils. The results showed that, among the soils, the acidic soil with a coarse texture had the highest soil Ga availability, which enhanced Ga uptake by rice roots. The Ga K-edge X-ray absorption near edge structure and sequential extraction results of the soils showed that the predominant species of Ga associated with iron hydroxides transformed to Ga(OH)3 precipitates, and the residue fraction increased with rice-growing time, resulting in lower Ga uptake by rice roots in the second half period of rice cultivation. A large fraction of Ga was accumulated in the rice roots, with only a small portion of Ga was transferred to the shoots and then to the rice grains. This study revealed that Ga speciation in soil-rice plant systems varied during rice cultivation and determined soil Ga availability to rice plants. Gallium accumulated in rice grains is distributed homogenously in the endosperm of the grains, suggesting a potential risk to public health via the intake of rice grains harvested from Ga-contaminated paddy fields.

Differential expression of hypoxia-inducible factors related to the invasiveness of epithelial ovarian cancer.

Ovarian cancer is the most lethal gynecological cancer, and it is frequently diagnosed at advanced stages, with recurrences after treatments. Treatment failure and resistance are due to hypoxia-inducible factors (HIFs) activated by cancer cells adapt to hypoxia. IGFBP3, which was previously identified as a growth/invasion/metastasis suppressor of ovarian cancer, plays a key role in inhibiting tumor angiogenesis. Although IGFBP3 can effectively downregulate tumor proliferation and vasculogenesis, its effects are only transient. Tumors enter a hypoxic state when they grow large and without blood vessels; then, the tumor cells activate HIFs to regulate cell metabolism, proliferation, and induce vasculogenesis to adapt to hypoxic stress. After IGFBP3 was transiently expressed in highly invasive ovarian cancer cell line and heterotransplant on mice, the xenograft tumors demonstrated a transient growth arrest with de-vascularization, causing tumor cell hypoxia. Tumor re-proliferation was associated with early HIF-1α and later HIF-2α activations. Both HIF-1α and HIF-2α were related to IGFBP3 expressions. In the down-expression of IGFBP3 in xenograft tumors and transfectants, HIF-2α was the major activated protein. This study suggests that HIF-2α presentation is crucial in the switching of epithelial ovarian cancer from dormancy to proliferation states. In highly invasive cells, the cancer hallmarks associated with aggressiveness could be activated to escape from the growth restriction state.

Characteristics Predicting a High Caregiver Burden in Patients with Vascular Cognitive Impairment: Using the Apriori Algorithm to Delineate the Caring Scenario.

PURPOSE: The aim of this study was to elucidate the factors and caring scenarios associated with a moderate to severe care burden in the caregivers of patients with vascular cognitive impairment (VCI). PATIENTS AND METHODS: This cross-sectional study included 158 patients with VCI and their caregivers who were managed by the dementia collaborative care team at Changhua Christian Hospital, Taiwan. Gender, age, clinical dementia rating, walking ability, behavioral symptoms, and psychological symptoms were the variables from the patients with VCI. Age, marital status, relation to the VCI patient, education, employment status, help of key activities, type of primary care, frequency of care, ZBI (Zarit burden interview) caregiving burden, and caregiver's mood were the evaluated variables for the caregivers. The Apriori algorithm was used to identify the attributes that resulted in different caregiving burdens from a comprehensive viewpoint of both VCI patients and their caregivers. RESULTS: A total of 1193 rules were identified with 1134 rules belonging to caregivers with a mild to moderate burden and 59 rules belonging to caregivers with a moderate to severe burden. Seven general rules were created based on a summary of these 59 rules. The results showed that an employed female caregiver who was taking care of her husband alone for ≥6 days per week, and who was helping with all key activities was likely to experience a moderate to severe burden when the patient had VCI. Moreover, if the caregiver had a relatively low education level and expressed an abnormal mood during the assessment, this increased the likelihood of the caregiver having a moderate to severe burden. CONCLUSION: The caregiver's gender, relation to the care recipient, education level, mood status, employment status, and care loading were associated with a higher burden of care for caregivers of patients with VCI. Therefore, a dementia care team should provide personalized training for caregivers about the disease, care skills for specific behaviors and psychological symptoms of dementia (BPSD), and strategies to cope with their own feelings. Caregivers should also be referred to appropriate social resources, such as support groups or respite care.

Indium Uptake and Accumulation by Rice and Wheat and Health Risk Associated with Their Consumption.

The increasing use of indium in high-tech industries has inevitably caused its release into the environment. However, knowledge of its environmental fate has been very limited so far. This study investigates the indium uptake and accumulation by two staple crops, rice (Oryza sativa L.) and wheat (Triticum aestivum L.), and evaluates potential risks associated with their consumption. Rice and wheat were grown on three kinds of soil, including acidic soils spiked with a high indium concentration (1.0 mmol kg-1), which is considered the worst-case scenario, because high soil acidity promotes indium bioavailability. The results revealed that a large portion of soil indium was associated with iron hydroxides, even in acidic soils. Indium precipitates in soils resulted in relatively low availability at the plant root site. Most absorbed indium accumulated at the roots, with only a tiny portion reaching the grains. The corresponding Hazard Quotient indicated no adverse effects on human health. Due to the low translocation of indium from soil to grain, the consumption of rice and wheat grains harvested from indium-contaminated soils may pose an insignificant risk to human health. Further field studies are necessary to better elucidate the risks associated with consuming crops grown in indium-contaminated soils.

Studying the biology of cytotoxic T lymphocytes in vivo with a fluorescent granzyme B-mTFP knock-in mouse.

Understanding T cell function in vivo is of key importance for basic and translational immunology alike. To study T cells in vivo, we developed a new knock-in mouse line, which expresses a fusion protein of granzyme B, a key component of cytotoxic granules involved in T cell-mediated target cell-killing, and monomeric teal fluorescent protein from the endogenous Gzmb locus. Homozygous knock-ins, which are viable and fertile, have cytotoxic T lymphocytes with endogeneously fluorescent cytotoxic granules but wild-type-like killing capacity. Expression of the fluorescent fusion protein allows quantitative analyses of cytotoxic granule maturation, transport and fusion in vitro with super-resolution imaging techniques, and two-photon microscopy in living knock-ins enables the visualization of tissue rejection through individual target cell-killing events in vivo. Thus, the new mouse line is an ideal tool to study cytotoxic T lymphocyte biology and to optimize personalized immunotherapy in cancer treatment.

Cytotoxic, or killer, T cells are a key part of the immune system. They carry a lethal mixture of toxic chemicals, stored in packages called cytotoxic granules. Killer T cells inject the contents of these granules into infected, cancerous or otherwise foreign cells, forcing them to safely self-destruct. In test tubes, T cells are highly efficient serial killers, moving from one infected cell to the next at high speed. But, inside the body, their killing rate slows down. Researchers think that this has something to do with how killer T cells interact with other immune cells, but the details remain unclear. To get to grips with how killer T cells work in their natural environment, researchers need a way to follow them inside the body. One approach could be to use genetic engineering to attach a fluorescent tag to a protein found inside killer T cells. That tag then acts as a beacon, lighting the cells up and allowing researchers to track their movements. Tagging a protein inside the cytotoxic granules would allow close monitoring of T cells as they encounter, recognize and kill their targets. But fluorescent tags are bulky, and they can stop certain proteins from working as they should. To find out whether it is possible to track killer T cells with fluorescent tags, Chitirala, Chang et al. developed a new type of genetically modified mouse. The modification added a teal-colored tag to a protein inside the granules of the killer T cells. Chitirala, Chang et al. then used a combination of microscopy techniques inside and outside of the body to find out if the T cells still worked. This analysis showed that, not only were the tagged T cells able to kill diseased cells as normal, the tags made it possible to watch it happening in real time. Super-resolution microscopy outside of the body allowed Chitirala, Chang et al. to watch the killer T cells release their toxic granule content. It was also possible to follow individual T cells as they moved into, and destroyed, foreign tissue that had been transplanted inside the mice. These new mice provide a tool to understand how killer T cells really work. They could allow study not only of the cells themselves, but also their interactions with other immune cells inside the body. This could help to answer open questions in T cell research, such as why T cells seem to be so much more efficient at killing in test tubes than they are inside the body. Understanding this better could support the development of new treatments for viruses and cancer.

Investigation of Cytotoxic T Lymphocyte Function during Allorejection in the Anterior Chamber of the Eye.

Cytotoxic T lymphocytes (CTL) are an essential part of our immune system by killing infected and malignant cells. To fully understand this process, it is necessary to study CTL function in the physiological setting of a living organism to account for their interplay with other immune cells like CD4+ T helper cells and macrophages. The anterior chamber of the eye (ACE), originally developed for diabetes research, is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, particularly allorejection of islets of Langerhans cells by CTLs. We follow the onset of the rejection after vascularization on islets until the end of the rejection process for about a month by repetitive two-photon microscopy. We find that CTLs show reduced migration on allogeneic islets in vivo compared to in vitro data, indicating CTL activation. Interestingly, the temporal infiltration pattern of T cells during rejection is precisely regulated, showing enrichment of CD4+ T helper cells on the islets before arrival of CD8+ CTLs. The adaptation of the ACE to immune responses enables the examination of the mechanism and regulation of CTL-mediated killing in vivo and to further investigate the killing in gene-deficient mice that resemble severe human immune diseases.