Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans

CD4+ T cells are one of the key immune cells contributing to the immunopathogenesis of type 1 diabetes (T1D). Previous studies have reported that platelet-derived mitochondria suppress the proliferation of peripheral blood mononuclear cells (PBMC). To further characterize the immune modulation of platelet-derived mitochondria, the purified CD4+ T cells were treated, respectively, with platelet-derived mitochondria. The data demonstrated that MitoTracker Deep Red-labeled platelet-derived mitochondria could directly target CD4+ T cells through C-X-C motif chemokine receptor 4 (CXCR4) and its ligand stromal cell-derived factor-1 (SDF-1), regulating the anti-CD3/CD28 bead-activated CD4+ T cells. The result was an up-regulation of Naïve and central memory (TCM) CD4+ T cells, the down-regulation of effector memory (TEM) CD4+ T cells, and modulations of cytokine productions and gene expressions. Thus, platelet-derived mitochondria have a translational potential as novel immune modulators to treat T1D and other autoimmune diseases.

Existence of Circulating Mitochondria in Human and Animal Peripheral Blood

Mitochondria are usually located in the cytoplasm of cells where they generate adenosine triphosphate (ATP) to empower cellular functions. However, we found circulating mitochondria in human and animal blood. Electron microscopy confirmed the presence of mitochondria in adult human blood plasma. Flow cytometry analyses demonstrated that circulating mitochondria from the plasma of human cord blood and adult peripheral blood displayed the immune tolerance-associated membrane molecules such as CD270 and PD-L1 (programmed cell death-ligand 1). Similar data were obtained from fetal bovine serum (FBS) and horse serum of different vendors. Mitochondria remained detectable even after 56 °C heat inactivation. A real-time PCR array revealed purified mitochondria from animal sera expressed several genes that contribute to human T- and B-cell activation. Transwell experiments confirmed the migration capability of mitochondria through their expression of the chemokine receptor CXCR4 in responses to its ligand stromal-derived factor-1α (SDF-1α). Functional analysis established that human plasma mitochondria stimulated the proliferation of anti-CD3/CD28 bead-activated PBMC, up-regulated the percentage of activated CD4+ T and CD8+ T cells, and reduced the production of inflammatory cytokines. These findings suggested that the existence of circulating mitochondria in blood may function as a novel mediator for cell-cell communications and maintenance of homeostasis. Plasma-related products should be cautiously utilized in cell cultures due to the mitochondrial contamination.

Generation of Treg-Like Cells from CD4+CD25- T Cells Occurs Via Both Foxp3 Dependent and Independent Pathways

Regulatory T cells (Treg) contribute to the maintenance of self-tolerance and have been demonstrated to both suppress autoimmune diseases and mitigate GvHD in mouse models. Major obstacles for their routine use in human clinical trials to reduce autoimmunity or GvHD include the low number of Treg found in the peripheral blood in the resting state (5–10% of CD4+ T cells), low yields and efficiencies of purification and severe limitations maintaining suppressor function after ex vivo expansion. The master gene responsible for the normal development and suppressor function of Treg is Foxp3 which is exclusively expressed in Treg. Recent identification of demethylated CpG islands within the Foxp3 locus only in Treg lead us to investigate whether an FDA-approved demethylating agent, decitabine, could be used to enhance expression of Foxp3 via an epigenetic effect and functionally convert CD4+CD25- T cells into Treg. Treatment of human CD4+CD25-FOXP3- T cells with decitabine in the presence of anti-CD3/CD28 beads and hIL-2 (50u/ml) induced FOXP3 expression. Optimal expression of FOXP3 was seen in those cells incubated with 1–5 uM decitabine. Real time RT-PCR demonstrated levels of mRNA for FOXP3 that were comparable to that seen in bead-activated natural Treg (10–12 fold increase above baseline). This resulted in increased expression at the protein level in 60% of decitabine-treated cells (dcT) (5% of PBS-treated cells (pbsT)). Decitabine also induced Foxp3 expression in 80% of anti-CD3/CD28 bead-activated murine CD4+CD25- T cells. To determine the duration of Foxp3 expression in dcT, we performed intracellular staining for Foxp3 each day for seven days and found that about 50% of dcT were Foxp3+ at day 7. Consistent with these results, decitabine also induced marked increase GFP expression (from undetectable levels) in anti-CD3/CD28 bead-activated CD4+CD25- T cells from Foxp3-ires-GFP KI mice (kindly provided by Tim Ley). In order to identify regulatory elements which mediate decitabine induced expression of Foxp3, we cloned both the 6kb 5′ promoter and the 6kb intron 1 of the human FOXP3 locus upstream of luciferase and tested expression of luciferase in stable Jurkat transfectants +/− decitabine by BLI. Decitabine induced 5–7 fold increase luciferase only in those 5′ transfectants. Of note is that dcT could potently suppress proliferation of CD4+CD25- T cells in vitro (ratio 1:1) in response to both anti-CD3/CD28 bead activation and to allo-APC in mixed lymphocyte cultures. Transwell experiments demonstrated that the suppressor function of dcT is cell-contact dependent. Surprisingly, we found that their suppressive properties are independent of Foxp3 expression in that dcT from Foxp3 KO mice were equally suppressive in bead-based proliferation assays as WT dcT. These data strongly suggest that decitabine may allow for the reexpression of genes such as Foxp3 and critical genes downstream of Foxp3 which mediate the suppressor phenotype in vitro. In murine T-cell depleted BMT model (B6→Balb/c), conventional T-cells (Tconv) (10×106) incubated with anti-CD3/CD28 beads and decitabine were found to promote enhanced engraftment with reduced GvHD, compared to mice receiving PBS-treated Tconv. In addition, addition of dcT with Tconv (1:1 ratio) resulted in decreased GvHD and improved survival, compared to Balb/c recipients receiving B6 Tconv and pbsT. In summary, decitabine-treatment enhanced Foxp3 expression in CD4+CD25- non-Treg cells. These dcT efficiently suppressed the proliferation of allo-reactive T cells in vitro and mitigated GvHD in vivo. The suppressor function of dcT was cell-contact dependent but unexpectedly Foxp3-independent.

Comparison of the Division Rate and Proliferative Capacity of Naive and Ex Vivo Activated T Cells after Allogeneic Bone Marrow Transplantation.

Maintaining T cell function after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously showed that murine T cells activated ex vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) retain greater GVHD-inducing potential than cells activated with either soluble anti-CD3 antibody alone or plate bound anti-CD3 and anti-CD28 antibodies after allogeneic BMT. However, CD3/CD28 bead activated T cells still exhibit reduced GVHD-inducing potential compared to naïve T cells. In this study, we used CFSE and congenic mice to monitor the proliferative kinetics of naïve and CD3/CD28 bead activated, transduced, and selected T cells in the same mouse after allogeneic and syngeneic BMT. High efficiency (>50%) gene transfer of our chimeric suicide gene, CD34/TK, into C57BL/6 (B6) murine T cells was achieved 24 h after CD3/CD28 bead activation and gene-modified cells were purified to >98% by CD34 immunomagnetic selection 48 h later. CD34/TK+ T cells were then rested for 3 days in medium containing 10 U/mL IL-2. Purified CD34/TK+ (CD45.1+) and naïve (CD45.2+) T cells from B6 mice were then labeled with CFSE, mixed 1/1 (3e6 total T cells), and injected, along with T cell depleted B6 (CD45.1+) BM, into lethally irradiated allogeneic (BALB/c, CD45.2+) or syngeneic (B6, CD45.2+) recipients. Mice were sacrificed daily up to 6 days after BMT to assess donor T cell engraftment, division, and phenotype by five color flow cytometry. The CD34/TK+ donor T cells (both CD4+ and CD8+ T cells) underwent 1–2 rounds of division within 24 h after infusion. In contrast, <5% of the naïve T cells divided during the first 24 h after infusion. Thereafter, the CD34/TK+ and naïve CD4+ and CD8+ T cells exhibited similar division kinetics between days 1 and 4 after BMT. At 3 days after BMT, both CD34/TK+ and naïve CD4+ and CD8+ T cells were detected in the 8 cell divisions discernible by CFSE, with approximately equal percentages (5%–15%) of cells in each division cycle. However, virtually all of the CD34/TK+ and naïve CD4+ and CD8+ T cells had divided more than 7 times by day 4 after allogeneic BMT. In contrast, <10% of the CD34/TK+ or naïve CD4+ and CD8+ T cells had undergone more than 7 cell divisions in the syngeneic recipients. Interestingly, the CD34/TK+ T cells exhibited a dramatic decrease in expansion compared with the naïve T cells between days 4 and 6 after allogeneic BMT. Although ~ equal percentages of CD34/TK+ and naïve T cells were observed 4 days after infusion, >6-fold and 10-fold more naïve CD4+ and CD8+ T cells, respectively, were detected in the allogeneic recipients at day 6 after BMT. This effect was specific to the allogeneic response, because we observed no difference in expansion between the CD34/TK+ and naïve CD4+ and CD8+ T cells in the syngeneic recipients. Phenotypically, both the CD34/TK+ and naïve CD4+ and CD8+ cells upregulated CD25 expression after 4 divisions, upregulated CD69 and CD44 expression after 1 to 2 divisions, and downregulated CD62L expression. In summary, CD3/CD28 bead activated, transduced, and selected T cells exhibit decreased expansion compared to naïve T cells after injection into allogeneic recipients. Ongoing studies are evaluating whether this decrease in expansion is caused by activation induced cell death, altered trafficking, or a decrease in the proliferative capacity of the ex vivo manipulated cells.