Investigation of mitochondrial fusion-mediated T cell expansion and persistence for cancer immunotherapy applications Pubblico
Gupta, Pulkit (Spring 2023)
Abstract
Chimeric antigen receptor (CAR)-T cell therapy targeting the surface antigen CD19 is an exciting novel therapy for B-chronic lymphocytic leukemia (B-CLL), but CAR-T cell exhaustion remains a challenge for the therapy’s efficacy. It is known that mitochondrial biogenesis and metabolism are necessary for robust T cell immune responses and T cell memory and that increased numbers of CD8+ memory T cells are associated with higher responses to CD19 CAR-T therapy. Here, I test the hypothesis that increased mitochondrial fusion improves the survival/expansion of T cells and overcomes T cell exhaustion. In the present study, I utilize a newly-identified mitofusin (MFN) agonist, MASM7, as a tool compound to examine this hypothesis. T cells were isolated from normal donor and B-CLL patient donor peripheral blood mononuclear cells and activated with CD3/CD28 beads. Activated cells were then stimulated with IL-2 treatment and expanded over the course of eight days, with and without MASM7 treatment. Endpoint analysis was performed via fluorochrome-conjugated antibody staining and flow cytometry analysis. Additionally, Seahorse cellular energetics assays, mitochondrial membrane potential staining, and mitochondrial morphology assays were conducted to verify the activity of the drug. Though MASM7 was shown to increase mitochondrial membrane potential and mitochondrial remodeling in multiple different cell types, there were no significant differences in expansion, immune inhibitory checkpoint molecule expression, and activation marker expression between MASM7-treated T cells and vehicle-treated T cells. The current findings suggest that activation of mitochondrial fusion with MASM7 does not improve T cell expansion and persistence. However, further work is required to assess the effects of MASM7 on sustained maintenance of mitochondrial fusion and use of an optimal extrinsic environment for expansion and development.
Table of Contents
1. Introduction (p. 1-9)
1.1: B-CLL and CD19 CAR-T therapy
1.2: PI3K dual-inhibition therapy for T cell persistence
1.3: Mitochondrial dynamics
1.4: Mechanisms of mitochondrial fusion and fission
1.5: MASM7 is a small-molecule mitochondrial fusion activator
1.6: MASM7 increases T cell expansion
1.7: MASM7 decreases expression of T cell exhaustion markers
1.8: MASM7 increases mitochondrial volume in patient-derived B-CLL T cells
1.9: Research objectives and hypotheses
2. Methods (p. 10-12)
2.1: T cell isolation and expansion
2.2: T cell marker staining and flow cytometry analysis
2.3: Cellular energetics assays
2.4: Mitochondrial membrane potential and mitochondrial visualization assays
3. Results and Analysis (p. 13-24)
3.1: MASM7 does not induce expansion of healthy donor-derived T cells
3.2: B-CLL patient-derived T cells are not able to replicate the MASM7 expansion phenotype
3.3: Time course experiment reveals no significant differences with MASM7 treatment
3.4: Seahorse experiment provides evidence for inactive MASM7 drug
3.5: New MASM7 stock is effective at increasing mitochondrial membrane potential and fusion
3.6: Final MASM7 T cell experiment yields no appreciable differences in expansion
4. Discussion and Conclusions (p. 25-26)
4.1: Treatment with MASM7 is not sufficient to induce MFN-mediated T cell expansion
4.2: Future experimentation
5. References (p. 27-34)
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