Identification and Characterization of Antigen-Specific CD8+ T Cells Using Surface-Trapped TNF-α and Single-Cell Sequencing.

CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and ß-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.

Targeted molecular imaging of TLR4 in hepatocellular carcinoma using zwitterionic near-infrared fluorophores.

BACKGROUND: Tumor-associated macrophages (TAMs) are one of the most abundant immune cell types in solid tumors and implicated in tumor progression. Toll-like receptor 4 (TLR4) is expressed in TAMs and plays a key role in immune surveillance and tumor progression. Therefore, molecular imaging of TLR4 has potential not only for detection of TAM-enriched progressing tumors, but also evaluation of TLR4 expression in tumor microenvironment. METHODS: Here, we report that near-infrared (NIR) fluorescence imaging can provide a real-time imaging of a syngeneic model of murine hepatocellular carcinoma using targeted strategy against TLR4. We conjugated a zwitterionic NIR fluorophore ZW800-1C with minimal nonspecific tissue interactions to anti-TLR4 antibody and observed its targetability. The bioconjugates showed high affinity to murine macrophages in cell culture and in vivo. RESULTS: Interestingly, we observed predominant NIR signals in the tumor site, which persisted for more than 48 h after single intravenous administration of the bioconjugate. CONCLUSIONS: This result suggests that TLR4 targeting combined with NIR fluorescence imaging is a useful tool for cancer imaging. This imaging strategy could be used to detect cancerous tissue with the increased TAM content and evaluate the status of TLR4 signaling in solid tumors, ultimately impacting on the diagnostic and prognostic imaging of human cancers.

Novel Treatment of a Vaccinia Virus Infection from an Occupational Needlestick - San Diego, California, 2019.

Vaccinia virus (VACV) is an orthopoxvirus used in smallpox vaccines, as a vector for novel cancer treatments, and for experimental vaccine research (1). The Advisory Committee on Immunization Practices (ACIP) recommends smallpox vaccination for laboratory workers who handle replication-competent VACV (1). For bioterrorism preparedness, the U.S. government stockpiles tecovirimat, the first Food and Drug Administration-approved antiviral for treatment of smallpox (caused by variola virus and globally eradicated in 1980*,†) (2). Tecovirimat has activity against other orthopoxviruses and can be administered under a CDC investigational new drug protocol. CDC was notified about an unvaccinated laboratory worker with a needlestick exposure to VACV, who developed a lesion on her left index finger. CDC and partners performed laboratory confirmation, contacted the study sponsor to identify the VACV strain, and provided oversight for the first case of laboratory-acquired VACV treated with tecovirimat plus intravenous vaccinia immunoglobulin (VIGIV). This investigation highlights 1) the misconception among laboratory workers about the virulence of VACV strains; 2) the importance of providing laboratorians with pathogen information and postexposure procedures; and 3) that although tecovirimat can be used to treat VACV infections, its therapeutic benefit remains unclear.

Wound Botulism Outbreak Among Persons Who Use Black Tar Heroin - San Diego County, California, 2017-2018.

During September 29-October 6, 2017, the County of San Diego Public Health Services (COSD) was notified of two patients with suspected wound botulism and a history of using black tar heroin. On October 9, COSD, which had reported an average of one wound botulism case per year during 2001-2016, sent a health alert through the California Health Alert Network, notifying Southern California providers of these two patients, including their signs and symptoms and black tar heroin exposure. In collaboration with the California Department of Public Health, COSD conducted an investigation to identify additional cases, determine risk factors for illness, estimate cost of medical care, and develop recommendations to prevent further illness. By April 18, 2018, nine (eight confirmed and one probable) patients with wound botulism were identified, all of whom were hospitalized; one of the nine died. All nine were persons who inject drugs; seven specifically reported using black tar heroin and six practiced subcutaneous injection known as skin popping. Clinically compatible signs and symptoms included muscle weakness, difficulty swallowing, blurred vision, drooping eyelids, slurred speech, difficulty breathing, loss of facial expression, or descending paralysis. All patients were treated with heptavalent botulism antitoxin (BAT). Wound botulism is likely underrecognized because of its rarity and the overlapping signs and symptoms with opioid intoxication, overdose, and other neurologic syndromes including Guillain-Barré syndrome, the Miller Fisher variant of Guillain-Barré syndrome, and myasthenia gravis. Prompt diagnosis, administration of BAT, and provision of supportive care can help stop the progression of paralysis and be lifesaving.

Routine TBI screening following combat deployments.

A precise estimate of the rates of traumatic brain injury (TBI) in returning combat troops is difficult to establish given the challenges of screening large numbers of military personnel returning from combat deployments. The Brief Traumatic Brain Injury Screen (BTBIS) was implemented in the First Marine Expeditionary Force between 2004 and 2006. Nine percent of the 7909 marines who completed the BTBIS were considered having a positive screen; that is, they endorsed at least one injury mechanism and indicated a change in mental status at the time of injury. The majority of combat-related TBI's were due to multiple injury agents with the next largest group related to blast exposure only. Most importantly, of those who screened positive for TBI 70.5% (n=500) were first identified by the screen. Service members who endorsed items on the BTBIS were contacted for follow-up assessment of persistent symptoms related to TBI and clinical referrals were made as needed. Given the rate of positive TBI screens in this non-referred sample of military personnel returning from a combat deployment, routine TBI screening appears valuable in screening individuals who might not be identified otherwise. Furthermore, this study appears to refute the contention that routine TBI screening will result in an over-identification of TBI in this population.