Epithelial organoid supports resident memory CD8 T cell differentiation.

Resident Memory T cells (TRM) play a vital role in regional immune defense in barrier organs. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency, sampling bias and low cell survival rates have limited our ability to conduct TRM-focused high-throughput assays. Here, we engineered a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation in vitro. The three-dimensional VEOs established from murine adult stem cells resembled stratified squamous vaginal epithelium and induced gradual differentiation of activated CD8 T cells into epithelial TRM. These in vitro generated TRM were phenotypically and transcriptionally similar to in vivo TRM, and key tissue residency features were reinforced with a second cognate-antigen exposure during co-culture. TRM differentiation was not affected even when VEOs and CD8 T cells were separated by a semipermeable barrier, indicating soluble factors' involvement. Pharmacological and genetic approaches showed that TGF-ß signaling played a crucial role in their differentiation. We found that the VEOs in our model remained susceptible to viral infections and the CD8 T cells were amenable to genetic manipulation; both of which will allow detailed interrogation of antiviral CD8 T cell biology in a reductionist setting. In summary, we established a robust model which captures bonafide TRM differentiation that is scalable, open to iterative sampling, and can be subjected to high throughput assays that will rapidly add to our understanding of TRM.

Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease.

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5'-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (αIIbß3) integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

Visualization of Hyperconjugation and Subsequent Structural Distortions through 3D Printing of Crystal Structures.

BACKGROUND: Structural distortions due to hyperconjugation in organic molecules, like norbornenes, are well captured through X-ray crystallographic data, but are sometimes difficult to visualize especially for those applying chemical knowledge and are not chemists. METHODS: Crystal structure from the Cambridge database were downloaded and converted to .stl format. The structures were then printed at the desired scale using a 3D printer. RESULTS: Replicas of the crystal structures were accurately reproduced in scale and any resulting distortions were clearly visible from the macroscale models. Through space interactions or effect of through space hyperconjugation was illustrated through loss of symmetry or distortions thereof. CONCLUSION: The norbornene structures exhibits distortion that cannot be observed through conventional ball and stick modelling kits. We show that 3D printed models derived from crystallographic data capture even subtle distortions in molecules. We translate such crystallographic data into scaled-up models through 3D printing.