Browsing by Author "Bellamkonda, Ravi V"

Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities in the millions that suffer TBI every year. Important mediators of inflammation include macrophages and microglia and these cell types can possess a range of phenotypes. An anti-inflammatory, “M2-like” macrophage phenotype after TBI is associated with neurogenesis, axonal regeneration, and improved white matter integrity. To boost these subpopulations, a promising approach is the enrichment of two cytokines: Fractalkine (FKN, CX3CL1) or Interleukin-4 (IL-4). FKN is a chemokine and thus recruits non-classical monocytes which are precursors to M2-like macrophages. IL-4 polarizes and proliferates M2-like macrophages. However, delivering recombinant or purified cytokines is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we explore two strategies to enrich endogenous FKN or IL-4, obviating the need for delivery of exogenous proteins.

In the first study, we synthesize a biomaterial to elevate endogenous FKN at an injury site. Modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels (“aptagels”) that dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate local increases in non-classical monocytes and M2-like macrophages.

In our second approach, we augment mesenchymal stem/stromal cells (MSCs), to transiently express IL-4. As MSCs do not endogenously synthesize IL-4, we transfect them with synthetic IL-4 mRNA. We suggest that mRNA transfection is a better strategy than DNA transfection, viral transduction, and recombinant IL-4 delivery for TBI. Our studies first characterize the IL-4 expression. Then, in a TBI model of closed head injury, we observe that IL-4 expressing MSCs successfully induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression. Curiously, this does not translate to improvements in function, histology, or white matter integrity.

The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins. Both FKN-aptamer functionalized biomaterials and IL-4 expressing MSCs may be promising approaches to boost anti-inflammatory subpopulations at sites of injury. However, our studies also begin to question whether M2-like macrophages alone orchestrate the neurogenesis, axonal regeneration, and improved white matter integrity that has previously been observed.

Finally, both strategies could have important immunomodulatory roles outside of TBI. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic. IL-4 expressing MSCs could assist tissue regeneration in cavitary diseases or improve biomaterial integration into tissues.

Chimeric antigen receptor (CAR) T cell therapy for glioblastoma remains challenging due to insufficient CAR T cell abundance and antigen-negative tumor cells evading targeting. Unfortunately, most preclinical studies evaluating CAR T cells in glioblastoma use tumors expressing a single antigen, xenografts, and/or lymphodepletion. While lymphodepletion enhances CAR T cell efficacy, it diminishes the endogenous immune system, which has the potential for tumor eradication. Here, we orthotopically delivered IL7 and/or Flt3L expressing CAR T cells in 50% EGFRvIII-positive and -negative tumors pre-conditioned with non-lymphodepleting irradiation. IL7 and IL7 Flt3L CAR T cells increased intratumoral CD8 CAR T cell populations seven days after treatment. IL7 co-expression with Flt3L increased conventional dendritic cells (cDCs) as well as the CD103+XCR1+ population known for migration and antigen cross-presentation. Treatment with IL7 or IL7 Flt3L CAR T cells improved overall survival to 67% and 50%, respectively, compared to 9% in vCAR and vFL and no survivors in PBS treated. However, there was no significant difference in survival between IL7 and IL7 Flt3L CAR T cells. We conclude that CAR T cells modified to express IL7 demonstrate therapeutic potential in glioblastoma treated with non-lymphodepleting irradiation and co-expression with Flt3L modestly improved intratumoral dendritic cell populations.