ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

Diagnosis of Pregnancy Complications Using Blind Ultrasound Sweeps Performed by Individuals Without Prior Formal Ultrasound Training.

OBJECTIVE: To estimate the diagnostic accuracy of blind ultrasound sweeps performed with a low-cost, portable ultrasound system by individuals with no prior formal ultrasound training to diagnose common pregnancy complications. METHODS: This is a single-center, prospective cohort study conducted from October 2020 to January 2022 among people with second- and third-trimester pregnancies. Nonspecialists with no prior formal ultrasound training underwent a brief training on a simple eight-step approach to performing a limited obstetric ultrasound examination that uses blind sweeps of a portable ultrasound probe based on external body landmarks. The sweeps were interpreted by five blinded maternal-fetal medicine subspecialists. Sensitivity, specificity, and positive and negative predictive values for blinded ultrasound sweep identification of pregnancy complications (fetal malpresentation, multiple gestations, placenta previa, and abnormal amniotic fluid volume) were compared with a reference standard ultrasonogram as the primary analysis. Kappa for agreement was also assessed. RESULTS: Trainees performed 194 blinded ultrasound examinations on 168 unique pregnant people (248 fetuses) at a mean of 28±5.85 weeks of gestation for a total of 1,552 blinded sweep cine clips. There were 49 ultrasonograms with normal results (control group) and 145 ultrasonograms with abnormal results with known pregnancy complications. In this cohort, the sensitivity for detecting a prespecified pregnancy complication was 91.7% (95% CI 87.2-96.2%) overall, with the highest detection rate for multiple gestations (100%, 95% CI 100-100%) and noncephalic presentation (91.8%, 95% CI 86.4-97.3%). There was high negative predictive value for placenta previa (96.1%, 95% CI 93.5-98.8%) and abnormal amniotic fluid volume (89.5%, 95% CI 85.3-93.6%). There was also substantial to perfect mean agreement for these same outcomes (range 87-99.6% agreement, Cohen κ range 0.59-0.91, P<.001 for all). CONCLUSION: Blind ultrasound sweeps of the gravid abdomen guided by an eight-step protocol using only external anatomic landmarks and performed by previously untrained operators with a low-cost, portable, battery-powered device had excellent sensitivity and specificity for high-risk pregnancy complications such as malpresentation, placenta previa, multiple gestations, and abnormal amniotic fluid volume, similar to results of a diagnostic ultrasound examination using a trained ultrasonographer and standard-of-care ultrasound machine. This approach has the potential to improve access to obstetric ultrasonography globally.

Breast Ultrasound Volume Sweep Imaging: A New Horizon in Expanding Imaging Access for Breast Cancer Detection.

OBJECTIVE: The majority of people in the world lack basic access to breast diagnostic imaging resulting in delay to diagnosis of breast cancer. In this study, we tested a volume sweep imaging (VSI) ultrasound protocol for evaluation of palpable breast lumps that can be performed by operators after minimal training without prior ultrasound experience as a means to increase accessibility to breast ultrasound. METHODS: Medical students without prior ultrasound experience were trained for less than 2 hours on the VSI breast ultrasound protocol. Patients presenting with palpable breast lumps for standard of care ultrasound examination were scanned by a trained medical student with the VSI protocol using a Butterfly iQ handheld ultrasound probe. Video clips of the VSI scan imaging were later interpreted by an attending breast imager. Results of VSI scan interpretation were compared to the same-day standard of care ultrasound examination. RESULTS: Medical students scanned 170 palpable lumps with the VSI protocol. There was 97% sensitivity and 100% specificity for a breast mass on VSI corresponding to 97.6% agreement with standard of care (Cohen's κ = 0.95, P < .0001). There was a detection rate of 100% for all cancer presenting as a sonographic mass. High agreement for mass characteristics between VSI and standard of care was observed, including 87% agreement on Breast Imaging-Reporting and Data System assessments (Cohen's κ = 0.82, P < .0001). CONCLUSIONS: Breast ultrasound VSI for palpable lumps offers a promising means to increase access to diagnostic imaging in underserved areas. This approach could decrease delay to diagnosis for breast cancer, potentially improving morbidity and mortality.

Opposing T cell responses in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

Transcript-indexed ATAC-seq for precision immune profiling.

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.

Activated ß2 Integrins Restrict Neutrophil Recruitment during Murine Acute Pseudomonal Pneumonia.

Rapid neutrophil recruitment is critical for the efficient clearance of bacterial pathogens from the lungs. Although ß2 integrins and their activation are required for neutrophil recruitment from postcapillary venules of the systemic circulation into inflamed tissues, the involvement of integrins in neutrophil recruitment in response to respiratory infection varies among bacterial pathogens. For stimuli eliciting ß2 integrin-dependent neutrophil influx, including Pseudomonas aeruginosa, it remains unclear whether the activation of ß2 integrins is an essential step in this process. In the current study, we analyze neutrophil trafficking within the lungs of mice infected with Pseudomonas aeruginosa and evaluate the role of ß2 integrin activation through genetic deletion of talin-1 or Kindlin-3 or by pharmacological inhibition of high-affinity ß2 integrins using a small molecule allosteric antagonist. We observe that attenuation of high-affinity ß2 integrins leads to an enhancement of neutrophil emigration into lung interstitium and airspaces. Neutrophil effector functions, including the production of reactive oxygen species and the phagocytosis of bacteria, are only partially dependent on high-affinity ß2 integrins. These results reveal a mechanism by which activated ß2 integrins limit neutrophil entry into the lung tissue and airspaces during acute pseudomonal pneumonia and suggest potential strategies for modulating neutrophil-mediated host defense.