TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.

Background: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. Methods: We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. Results: We show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. Conclusion: These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

Longitudinal study of total body potassium in healthy men.

OBJECTIVES: Total body potassium (TBK) is an index of fat-free mass and an accurate measure of body cell mass (BCM). To date, however, no longitudinal studies have evaluated body composition using TBK in aging healthy adults. The purpose of this study was to assess TBK and BCM in healthy men over a 21-year period, and to evaluate changes with age. DESIGN: A 21-year longitudinal, prospective, observational study evaluating TBK and BCM and their relationship to changes with aging and body mass index (BMI). SUBJECTS: Body composition of 133 healthy Italian men, 20 to 66 years of age, was measured by whole-body counting of (40)K. TBK was calculated by using this formula: (40)K × 8.474. BCM was calculated from TBK by using this formula: BCM (kg) = 0.00833 × TBK (mmol). RESULTS: The prevalence of overweight and obesity increased significantly from baseline to end of study (p < 0.01), with no change in TBK observed over the 21-year period. BCM summaries were tested to determine if any was a good predictor of BMI after age 30. Participants with a low maximum BCM (<27 kg) at an early age tended to exhibit decreased BMI as they aged, whereas those with a high maximum BCM (>27 kg) at an early age tended to show increased BMI as they aged. CONCLUSION: Despite an overall increase in the incidence of overweight and obesity over the 21-year period, a high maximum BCM at an early age was a predictor of an increase in BMI as men got older.