Fig 2: CAR and BDNF Tregs decrease inflammation in the spinal cord of mSOD1-NSG mice. Spinal cord tissue from G93A-hSOD1 transgenic NSG mice at 13 weeks of age was evaluated for mRNA expression of inflammatory mediators. Human Tregs expressing CAR and BDNF (green) or vehicle (gray) were injected at 8 weeks of age. RNA from spinal cord was isolated. Human CD52 mRNA, an indicator of human Tregs, was found in 8 of 12 mice injected with CAR and BDNF Tregs at 13 weeks. Mouse NOX-2, TNF-α, IL-1β, CCL2, and CCL4 mRNAs were decreased in CAR + BDNF Treg-injected mice relative to vehicle-injected mice. Circles represent individual mice (n = 12 CAR + BDNF and n = 16 vehicle) combined from three experiments. Mouse mRNA values are expressed as fold-change over values from age-matched non-transgenic littermate controls (NTLs, n = 12). p values determined by unpaired t-test with Welch’s correction. * p < 0.05; ** p < 0.01.

Fig 3: Characteristics of human Tregs transduced with a retroviral vector expressing both a CAR and BDNF. (A) Representative flow cytometry plots of the isolation of Tregs from human PBMCs and sorted for CD4+CD25hiCD127lo cells. Q1, Q2, Q3, and Q4 refer to quadrants. (B) Treg expansion over 17 days in vitro. Values are mean fold-change ± SEM relative to day 0 from twelve independent Tregs sorts from ten different human donors. (C) Representative flow cytometry plots of CAR- and BDNF-transduced Tregs after 17 days of expansion for different cell markers and CAR expression. Red is specific staining, and blue is unstained control. Orange is isotype control for intracellular FoxP3 staining.

Fig 4: Human Tregs expressing both CAR and BDNF are similar to Tregs expressing CAR alone other than constitutive secretion of BDNF. (A) CAR + BDNF Tregs secreted BDNF into culture media in wells coated with G93A hSOD1 or media only. Little BDNF was measured in media of CAR Tregs (n = 3 mean ± SEM, p value determined by unpaired t-test with Welch’s correction). (B) CAR + BDNF Tregs and CAR Tregs secreted IL-10 upon culture on G93A hSOD1-coated wells, whereas no IL-10 was measured in the absence of CAR antigen (n = 3 mean ± SEM, p value determined by unpaired t-test with Welch’s correction). (C) CAR + BDNF Tregs (green) and CAR Tregs (blue) have similar levels of cell expansion, expression of FoxP3, CD127, CD39, and CAR as measured by flow cytometry (Each circle is an individual experiment, for cell expansion, %FoxP3, and %CAR n = 5 for CAR Treg and n = 11 for CAR and BDNF Tregs, for %CD127 and %CD39 n = 4 for CAR Treg and n = 10 for CAR and BDNF Tregs, black horizontal lines are means, and colored bars are SEMs, p value determined by unpaired t-test with Welch’s correction). * p < 0.05; *** p < 0.001.

Fig 5: Human Tregs expanded in vitro for 17 days and transduced with a BDNF retroviral vector secrete BDNF and are neuroprotective. (A) BDNF Tregs secreted BDNF into culture media, whereas little BDNF was measured in media of mock Tregs (n = 6, mean ± SEM, p value determined by unpaired t-test with Welch’s correction). (B). The viability of human neuronal cells co-cultured with Tregs in the presence of H2O2. Data are shown as % viability compared to untreated Luc-SH-SY5Y neuronal cells. Tregs transduced with BDNF or catalase inhibited H2O2-induced Luc-SH-SY5Y loss relative to mock-transduced Tregs (n = 4, mean ± SEM, p value determined by Dunnett’s multiple comparisons test versus mock Treg). *** indicates p < 0.001.