Early expression of mature αß TCR in CD4-CD8- T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations.

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Slow angled-descent forepaw grasping (SLAG): an innate behavioral task for identification of individual experimental mice possessing functional vision.

BACKGROUND: There is significant interest in the generation of improved assays to clearly identify experimental mice possessing functional vision, a property that could qualify mice for inclusion in behavioral and neuroscience studies. Widely employed current methods rely on mouse responses to visual cues in assays of reflexes, depth perception, or cognitive memory. However, commonly assessed mouse reflexes can sometimes be ambiguous in their expression, while depth perception assays are sometimes confounded by variation in anxiety responses and exploratory conduct. Furthermore, in situations where experimental groups vary in their cognitive memory capacity, memory assays may not be ideal for assessing differences in vision. RESULTS: We have optimized a non-invasive behavioral assay that relies on an untrained, innate response to identify individual experimental mice possessing functional vision: slow angled-descent forepaw grasping (SLAG). First, we verified that SLAG performance depends on vision and not olfaction. Next, all members of an age-ranged cohort of 158 C57BL/6 mice (57 wild-type, 101 knockout, age range 44-241 days) were assessed for functional vision using the SLAG test without training or conditioning. Subjecting the population to a second innate behavioral test, Dark Chamber preference, corroborated that the functional vision assessment of SLAG was valid. CONCLUSIONS: We propose that the SLAG assay is immediately useful to quickly and clearly identify experimental mice possessing functional vision. SLAG is based on a behavioral readout with a significant innate component with no requirement for training. This will facilitate the selection of mice of known sighted status in vision-dependent experiments that focus on other types of behavior, neuroscience, and/or cognitive memory.

Oncolytic Urabe mumps virus: A promising virotherapy for triple-negative breast cancer.

Historically, the clinical utility of oncolytic virotherapy as a treatment for a wide range of cancer types was first demonstrated by three pilot human clinical trials conducted in Japan in the 1970s and 1980s using a wild-type Urabe mumps virus (MuV) clinical isolate. Using a sample of the actual original oncolytic Urabe MuV clinical trial virus stock (MuV-U-Japan) used in these Japanese clinical trials, we found that MuV-U-Japan consisted of a wide variety of very closely related Urabe MuVs that differed by an average of only three amino acids. Two MuV-U-Japan isolates, MuV-UA and MuV-UC, potently killed a panel of established human breast cancer cell lines in vitro, significantly extended survival of nude mice with human triple-negative breast cancer (TNBC) MDA-MB-231 tumor xenografts in vivo, and demonstrated significant killing activity against breast cancer patient-derived xenograft (PDX) cell lines grown as 3D organoids, including PDXs from patients resistant to anthracycline- and taxane-based chemotherapy. We also report success in developing a large-scale MuV-U production and purification process suitable for supporting Investigational New Drug applications for clinical trials. This study demonstrates the suitability of the MuV-UC virus for translation to modern clinical trials for treating patients with TNBC.

The early proximal αß TCR signalosome specifies thymic selection outcome through a quantitative protein interaction network.

During αß T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αß T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ0 mice. We found that CD3δ0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αß T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αß T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome.

A co-housing strategy to improve fecundity of mice in timed matings.

Timed matings of mice are often carried out to obtain offspring of a precise age when required for a study. Timed matings involve housing male and female mice together for a limited time period, typically overnight. A limitation of this practice is that many mouse pairs fail to mate during the brief co-housing period. The authors co-housed each breeding pair in the same cage but separated by a transparent partition for 3 d before carrying out timed matings. This co-housing strategy resulted in increased copulation during the timed mating period and also significantly increased the average number of pups produced per breeding pair. The authors suggest that co-housing likely permits male urine-borne pheromones to induce female estrus and also enables the expression of male and female mating behaviors.