Umbilical Cord Tissue-Derived Mesenchymal Stem Cells Inhibit T Cell Response to Peptide

Mesenchymal stem cells (MSC) have been shown to possess immunomodulatory properties that highlight their potential as a cellular therapy for autoimmune disease. We propose to examine the in vitro potential of stem cells derived from umbilical cord tissue to suppress the effector functions of human auto-reactive T cells. While the mechanism(s) of suppression of T cell function are not fully understood, it has been hypothesized that MSC-derived immunosuppressive soluble factors and cell-to-cell contact are important. We developed an in vitro culture assay to assess the effects of umbilical cord derived MSC (TC-MSC) on T cell function. Various doses of low-passage TC-MSCs were adhered to collagen-coated 96 well plates or in the lower chamber wells of transwell plates. HLA-matched EBV transformed B cells were pulsed +/- with appropriate autoantigenic peptide and cultured with adherent MSC or in the upper transwell chambers with the appropriate T cell clone. After 48 hours, cells were stained for CD4 and stained intracellularly for IFN-γ and analyzed by flow cytometry. We observed decreased T cell effector function with MSC co-culture and this was partially restored by separation of MSC and T cell+B cell+peptide in the transwell. We examined if prostaglandin E2 derived from the MSC also contributed to decreased T cell effector function. The inclusion of a COX-2 inhibitor in the culture system led to partially restored T cell effector function. We conclude that TC-MSC-derived soluble factor(s) and TC-MSC:T cell contact both contribute to the TC-MSC’s immunosuppressive effects. Primary TC-MSC isolates (with no prior cell culture) will also be tested in this system to determine if they possess similar immunosuppressive effects as adherent, cultured TC-MSC. These studies will pinpoint the functional mechanisms of the TC-MSC immunomodulatory properties on T cell effector function and may suggest avenues of enhancing MSC function in the treatment of autoimmune disease

Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study

Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG-GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG-GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG-GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG-GCSF and 20kPEG-GCSF. It was observed that even 5kPEG-GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG-GCSF. Biological activity measurements demonstrated that both 5kPEG-GCSF and 20kPEG-GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface