Expansion and Retroviral Transduction of Primary Murine T Cells for CAR T-Cell Therapy.

The development of chimeric antigen receptor (CAR) T cells has been a revolutionary technology for the treatment of relapsed and refractory leukemias and lymphomas. The synthetic CAR molecule redirects T cell function toward tumor surface-expressed antigens through a single-chain variable fragment (scFv) fused to CD3z and intracellular costimulatory domains. Here, we describe a protocol for the generation of CAR T cells using primary mouse T cells and a gammaretroviral vector encoding a CAR transgene. This protocol outlines several transduction and expansion methods based on the use of two transduction enhancers, RetroNectin® and Vectofusin®-1, and cell culture systems such as conventional plates or G-Rex® devices.

Differentiation of Bone Marrow Monocytes into Alveolar Macrophages-like Cells through Co-culture with Lung Epithelial Cells and Group 2 Innate Lymphoid Cells.

During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes. Key features • Ex vivo co-culture model of the alveolar niche. • Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.

Dampening type 2 properties of group 2 innate lymphoid cells by a gammaherpesvirus infection reprograms alveolar macrophages.

Immunological dysregulation in asthma is associated with changes in exposure to microorganisms early in life. Gammaherpesviruses (γHVs), such as Epstein-Barr virus, are widespread human viruses that establish lifelong infection and profoundly shape host immunity. Using murid herpesvirus 4 (MuHV-4), a mouse γHV, we show that after infection, lung-resident and recruited group 2 innate lymphoid cells (ILC2s) exhibit a reduced ability to expand and produce type 2 cytokines in response to house dust mites, thereby contributing to protection against asthma. In contrast, MuHV-4 infection triggers GM-CSF production by those lung ILC2s, which orders the differentiation of monocytes (Mos) into alveolar macrophages (AMs) without promoting their type 2 functions. In the context of γHV infection, ILC2s are therefore essential cells within the pulmonary niche that imprint the tissue-specific identity of Mo-derived AMs and shape their function well beyond the initial acute infection.

Ly6Chi monocytes balance regulatory and cytotoxic CD4 T cell responses to control virus-induced immunopathology.

Gammaherpesviruses (γHVs) have coevolved with their host, leading to a remarkably high infection prevalence and establishment of latency. The lifelong persistence of γHVs in hosts appears to broadly shape host immunity, and we show here that pulmonary infection with Murid herpesvirus 4 (MuHV-4), a mouse γHV, drives the recruitment of Ly6Chi monocytes (MOs) into the airway, thereby modulating the host immune response. The absence of Ly6Chi MOs is associated with severe virus-induced immunopathology and the systemic release of inflammatory mediators. Mechanistically, MuHV-4-imprinted MOs recruit CD4 T cells to the airways and trigger immunosuppressive signaling pathways through the PD-L1/PD-1 axis, thereby dampening the deleterious activation of cytotoxic CD4 T cells. These results uncover a role for Ly6Chi MOs in modulating CD4 T cell functions and reveal pathways that could be targeted therapeutically to reduce detrimental immunopathological responses associated with respiratory viral infections.

Chylous ascites associated with abdominal trauma and intestinal resection-anastomosis in a pet ferret (Mustela putorius furo).

CASE DESCRIPTION A 10-week-old 0.73-kg (1.6-lb) castrated male domestic ferret (Mustela putorius furo) was referred for exploratory laparotomy because of pneumoperitoneum and possible septic peritonitis after being bitten by the owner's dog. CLINICAL FINDINGS Abdominal exploration revealed a large laceration of the duodenum, tears of the jejunal mesentery, and 2 small tears in the abdominal wall. Chylous abdominal effusion developed 48 hours after surgery. TREATMENT AND OUTCOME Postoperative care included supportive treatment, analgesia, and antimicrobials. An abdominal drain was placed during the laparotomy and enabled monitoring of abdominal fluid production. Enteral feeding was provided through an esophagostomy tube. The chylous fluid production rapidly decreased after treatment with octreotide was initiated, and the ferret improved. Chyloabdomen resolved after 8 days of hospitalization and medical treatment. CLINICAL RELEVANCE Findings suggested that chylous ascites can potentially develop secondary to blunt abdominal trauma in ferrets. In this ferret, chyloabdomen was successfully treated with octreotide administration and abdominal drainage.

Bite injuries of grey seals (Halichoerus grypus) on harbour porpoises (Phocoena phocoena).

Bite-like skin lesions on harbour porpoises (Phocoena phocoena) have been suspected to be caused by grey seals (Halichoerus grypus), and a few field observations have been reported. Bite-like skin lesions observed on stranded animals were characterized by two main components: large flaps of loose or missing skin and blubber with frayed edges and puncture lesions. Definitive demonstration of predation by a grey seal was not reported so far in those stranded animals. In this study, five stranded porpoises with bite-like skin lesions were swabbed for genetic investigations. In addition, the head of a recently dead grey seal was used to mimic bite-like skin injuries on a porpoise carcass. Subsequently, the artificial skin injuries were swabbed, along with the gum of the seal used for inflicting them (positive controls). Total DNA was extracted from the swabs and was used to retrieve a fragment of mitochondrial DNA by PCR. Primers were designed to amplify a specific stretch of mitochondrial DNA known to differ between grey seals and porpoises. The amplicon targeted was successfully amplified from the positive control and from two of the stranded porpoises, and grey seal-specific mitochondrial DNA was retrieved from all those samples. We conclude that (1) it is possible to detect grey seal DNA from dead porpoises even after several days in seawater and (2) bite-like skin lesions found on dead porpoises definitively result from grey seals attacks. The attacks are most likely linked with predation although, in a number of cases, scavenging and aggressive behaviour cannot be excluded.