Cell-based therapy with CD4 FOXP3 regulatory T cells (Tregs) is a promising strategy to limit organ rejection and graft-vs-host disease. Ongoing clinical applications have yet to consider how human Tregs could be modified to direct their migration to specific inflammation sites and/or tissues for more targeted immunosuppression. We show here that stable, homing-receptor-tailored human Tregs can be generated from thymic Tregs isolated from pediatric thymus or adult blood. To direct migration to Th1-inflammatory sites, addition of interferon-γ and IL-12 during Treg expansion produced suppressive, epigenetically stable CXCR3 TBET FOXP3 T helper (Th)1-Tregs. CXCR3 remained expressed after injection in vivo and Th1-Tregs migrated efficiently towards CXCL10 in vitro. To induce tissue-specific migration, addition of retinoic acid (RA) during Treg expansion induced expression of the gut-homing receptors α4β7-integrin and CCR9. FOXP3 RA-Tregs had elevated expression of the functional markers latency-associated peptide and glycoprotein A repetitions predominant, increased suppressive capacity in vitro and migrated efficiently to healthy and inflamed intestine after injection into mice. Homing-receptor-tailored Tregs were epigenetically stable even after long-term exposure to inflammatory conditions, suppressive in vivo and characterized by Th1- or gut-homing-specific transcriptomes. Tailoring human thymic Treg homing during in vitro expansion offers a new and clinically applicable approach to improving the potency and specificity of Treg therapy.

Cell-cycle progression and the acquisition of a migratory phenotype are hallmarks of human carcinoma cells that are perceived as independent processes but may be interconnected by molecular pathways that control microtubule nucleation at centrosomes. Here, cell-cycle progression dramatically impacts the engraftment kinetics of 4T1-luciferase2 breast cancer cells in immunocompetent BALB/c or immunocompromised NOD-SCID gamma (NSG) mice. Multiparameter imaging of wound closure assays was used to track cell-cycle progression, cell migration, and associated phenotypes in epithelial cells or carcinoma cells expressing a fluorescence ubiquitin cell-cycle indicator. Cell migration occurred with an elevated velocity and directionality during the S-G-phase of the cell cycle, and cells in this phase possess front-polarized centrosomes with augmented microtubule nucleation capacity. Inhibition of Aurora kinase-A (AURKA/Aurora-A) dampens these phenotypes without altering cell-cycle progression. During G-phase, the level of phosphorylated Aurora-A at centrosomes is reduced in hyaluronan-mediated motility receptor (HMMR)-silenced cells as is the nuclear transport of TPX2, an Aurora-A-activating protein. TPX2 nuclear transport depends upon HMMR-T703, which releases TPX2 from a complex with importin-α (KPNA2) at the nuclear envelope. Finally, the abundance of phosphorylated HMMR-T703, a substrate for Aurora-A, predicts breast cancer-specific survival and relapse-free survival in patients with estrogen receptor (ER)-negative ( = 941), triple-negative (TNBC) phenotype ( = 538), or basal-like subtype ( = 293) breast cancers, but not in those patients with ER-positive breast cancer ( = 2,218). Together, these data demonstrate an Aurora-A/TPX2/HMMR molecular axis that intersects cell-cycle progression and cell migration. Tumor cell engraftment, migration, and cell-cycle progression share common regulation of the microtubule cytoskeleton through the Aurora-A/TPX2/HMMR axis, which has the potential to influence the survival of patients with ER-negative breast tumors. .

Proteasome inhibitors have emerged as an effective therapy for the treatment of haematological malignancies; however, their efficacy can be limited by the development of tumour resistance mechanisms. Novel combination strategies including the addition of TLR adjuvants to increase cell death and augment immune responses may help enhance their effectiveness. Although generally thought to inhibit inflammatory responses and NF-κB activation, we found that under specific conditions proteasome inhibitors can promote inflammatory responses by mediating IL-1β maturation and secretion after TLR stimulation. This was dependent on the timing of proteasome inhibition relative to TLR stimulation where reversal of treatment order could alternatively increase or inhibit IL-1β secretion (P < 0.001). TLR stimulation combined with proteasome inhibition enhanced cell death in vitro and delayed tumour development in vivo in NOD SCID mice (P < 0.01). However, unlike IL-1β secretion, cell death occurred similarly regardless of treatment order and was only partially caspase dependent, possessing characteristics of both apoptosis and necrosis as indicated by activation of caspase-1, 3, 8 and RIP3 phosphorylation. Although stimulation of various TLRs was capable of driving IL-1β production, TLR4 stimulation was the most effective at increasing cell death in THP-1 and U937 cells. TLR4 stimulation and proteasome inhibition independently activated the RIP3 necroptotic pathway and ultimately reduced the effectiveness of caspase/necroptosis inhibitors in mitigating overall levels of cell death. This strategy of combining TLR stimulation with proteasome inhibition may improve the ability of proteasome inhibitors to generate immunogenic cell death and increase anti-tumour activity.