Peggy A. Bulur

Immunosuppressive CD14+HLA-DRlow/− Monocytes in Prostate Cancer

To determine if the levels of circulating myeloid‐derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy.

Systemic immune suppression in glioblastoma: the interplay between CD14+HLA-DRlo/neg monocytes, tumor factors, and dexamethasone

Patients with glioblastoma (GBM) exhibit profound systemic immune defects that affect the success of conventional and immune-based treatments. A better understanding of the contribution of the tumor and/or therapy on systemic immune suppression is necessary for improved therapies, to monitor negative effects of novel treatments, to improve patient outcomes, and to increase understanding of this complex system. To characterize the immune profile of GBM patients, we phenotyped peripheral blood and compared these to normal donors. In doing so, we identified changes in systemic immunity associated with both the tumor and dexamethasone treated tumor bearing patients. In particular, dexamethasone exacerbated tumor associated lymphopenia primarily in the T cell compartment. We have also identified unique tumor and dexamethasone dependent altered monocyte phenotypes. The major population of altered monocytes (CD14(+)HLA-DR(lo/neg)) had a phenotype distinct from classical myeloid suppressor cells. These cells inhibited T cell proliferation, were unable to fully differentiate into mature dendritic cells, were associated with dexamethasone-mediated changes in CCL2 levels, and could be re-created in vitro using tumor supernatants. We provide evidence that tumors express high levels of CCL2, can contain high numbers of CD14(+) cells, that tumor supernatants can transform CD14(+)HLA-DR(+) cells into CD14(+)HLA-DR(lo/neg) immune suppressors, and that dexamethasone reduces CCL2 in vitro and is correlated with reduction of CCL2 in vivo. Consequently, we have developed a model for tumor mediated systemic immune suppression via recruitment and transformation of CD14(+) cells.

A novel source of viable peripheral blood mononuclear cells from leukoreduction system chambers.

BACKGROUND: Buffy coats are becoming less available as a source of research‐grade peripheral blood mononuclear cells (PBMNCs). Therefore, alternative sources of these cells were investigated.

Optimizing Preparation of Normal Dendritic Cells and bcr-abl+ Mature Dendritic Cells Derived from Immunomagnetically Purified CD14+ Cells

The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14+ mononuclear cells (MNC). We studied normal DC and bcr-abl+ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsorption and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14+ cells were indistinguishable. The percentage of bcr-abl+ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl+ the highest purity of bcr-abl+ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC.

Cancer Vaccines in the World of Immune Suppressive Monocytes (CD14+HLA-DRlo/neg Cells): The Gateway to Improved Responses

Dendritic cells are an important target in cancer immunotherapy based on their critical role in antigen presentation and response to tumor development. The capacity of dendritic cells to stimulate anti-tumor immunity has led investigators to use these cells to mediate anti-tumor responses in a number of clinical trials. However, these trials have had mixed results. The typical method for generation of ex vivo dendritic cells starts with the purification of CD14+ cells. Our studies identified a deficiency in the ability to generate mature dendritic cell using CD14+ cells from cancer patients that corresponded with an increased population of monocytes with altered surface marker expression (CD14+HLA-DRlo/neg). Further studies identified systemic immune suppression and increased concentrations of CD14+HLA-DRlo/neg monocytes capable of inhibiting T-cell proliferation and DC maturation. Together, these findings strongly suggest that protocols aimed at immune stimulation via monocytes/dendritic cells, if optimized on normal monocytes or in systems without these suppressive monocytes, are unlikely to engender effective DC maturation in vitro or efficiently trigger DC maturation in vivo. This highlights the importance of developing optimal protocols for stimulating DCs in the context of significantly altered monocyte phenotypes often seen in cancer patients.

Dendritic cell microvilli: a novel membrane structure associated with the multifocal synapse and T-cell clustering

Polarizing effects of productive dendritic cell (DC)-T-cell interactions on DC cytoskeleton have been known in some detail, but the effects on DC membrane have been studied to a lesser extent. We found that T-cell incubation led to DC elongation and segregation of characteristic DC veils to the broader pole of the cell. On the opposite DC pole, we observed a novel membrane feature in the form of bundled microvilli. Each villus was approximately 100 nm in diameter and 600 to 1200 nm long. Microvilli exhibited high density of antigen-presenting molecules and costimulatory molecules and provided the physical basis for the multifocal immune synapse we observed during human DC and T-cell interactions. T cells preferentially bound to this site in clusters often contained both CD4(+) and CD8(+) T cells.

Soluble B7-H1: Differences in production between dendritic cells and T cells

Tumor cells aberrantly express several T cell inhibitory molecules including members of the B7-H co-regulatory family. Presumably tumor-expressed B7-H1 and B7-H3 confer resistance to elimination by the immune system. In addition, elevated levels of soluble B7-H1 (sB7-H1) has been identified in the sera of cancer patients, including renal carcinoma patients and is associated with increased cancer related death. Here we report that sB7-H1 is produced and released by activated mature dendritic cells (mDC). Immature DC, macrophages, monocytes, or T cells are refractory to releasing sB7-H1. Exposure of CD4+ and CD8+ T cells to mDC-derived sB7-H1 molecules induced apoptosis. These data suggest that the immunobiology of B7-H1 is perhaps more complex than previously thought. sB7-H1 molecules may represent an unanticipated contributing factor to immune homeostasis. That both immune and tumor cells can be sources of sB7-H1 suggests that optimization of co-regulatory blockade immunotherapy for solid malignancies of necessity will require impact of targeting tumor and immune-derived B7-H1 molecules.

Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma

BackgroundDendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines.MethodsWe prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma.ResultsThree preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 ± 12 percent) in comparison to non-electroporated cells (82 ± 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections.ConclusionStandardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.

Maturation of dendritic cells infected by recombinant adenovirus can be delayed without impact on transgene expression

Adenovirus-mediated gene transfer to dendritic cells is highly efficient and often used, but the relationship among cell maturation, viral infection and expression of a transferred gene remains unclear. To study this relationship, we introduced a recombinant replication-defective adenovirus encoding the gene for green fluorescent protein to normal human immature myeloid dendritic cells. We induced maturation by the addition of TNF-α, IL-1β, IL-6 and prostaglandin E2 to the medium and assessed cell maturity by the levels of the secreted p40 subunit of IL-12 and of membrane-bound CD83. We quantified the efficiency of gene expression by GFP fluorescence and analyzed the data by a mixed-model analysis of variance; the model explained more than 97% of the effects. CD83 expression and p40 secretion depended solely on incubation time and maturation medium. The cells cultured in the absence of maturation medium remained immature and maintained the ability to respond to the later addition of the maturation irrespective of adenovirus infection and transferred gene expression. This expression was independent of cell maturation. In comparison with mature cells, the transferred gene was expressed in immature dendritic cells with a lag compatible with the less effective initial step (infection and/or gene transfer) in the absence of the maturation medium rather than less effective later GFP synthesis. Expression of CD83 and p40 were unaffected by adenovirus infection and transferred gene expression. Thus, immature dendritic cells infected with recombinant adenoviruses can be matured when desired after transferred gene expression.

Testing the safety of clinical-grade mature autologous myeloid DC in a phase I clinical immunotherapy trial of CML

BACKGROUND We conducted a phase I clinical immunotherapy trial of CML to evaluate the safety of a clinical-grade leukemic DC product standardized for purity and mature phenotype. METHODS We injected autologous DC into patients in late chronic or accelerated phases of CML. The patients received mature CD83+ and bcr-abl+ DC prepared from CD14+ cells. Two cohorts of three patients received four injections each of 3 x 10(6) DC and 15 x 10(6) DC/injection, respectively. The first patient was studied before imatinib mesylate (IM) was available, four patients were treated concurrently with IM therapy and one did not tolerate the IM and was off the drug at the time of DC therapy. IM effects on WBC counts precluded DC preparation in numbers sufficient for further dose escalation. The first patient received DC s.c. and all subsequent patients received DC into a cervical lymph node under ultrasound guidance. RESULTS DC injections were well tolerated. We observed no clinical responses. T cells drawn later in the course of therapy were more sensitive to stimulation by CML DC in vitro. DISCUSSION The increase in T-cell sensitivity to CML-specific stimulation that accompanied active immunization by CML DC justifies further clinical studies, possibly with modifications such as an increased frequency and number of DC injections.