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Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Baylor College of Medicine

We consider statistics of the disruption and Lyapunov times in an hierarchical restricted three-body problem. We show that at the edge of disruption the orbital periods and the size of the orbit of the escaping body exhibit Lévy flights. Due to them, the time decay of the survival probability is heavy-tailed with the power-law index equal to -2/3, while the relation between the Lyapunov and disruption times is quasilinear. Applicability of these results in an "hierarchical resonant scattering" setting for a three-body interaction is discussed.

Hamiltonian intermittency and Lévy flights in the three-body problem.

Comment on "Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b(+)Gr1(+) cells".

Modern theoretical methods for estimating the width of the chaotic layer in the presence of prominent marginal resonances are considered in the perturbed pendulum model of nonlinear resonance. The fields of applicability of these methods are explicitly and precisely formulated. The comparative accuracy is investigated in massive and long-run numerical experiments. It is shown that the methods are naturally subdivided in classes applicable for adiabatic and nonadiabatic cases of perturbation. It is explicitly shown that the pendulum approximation of marginal resonance works well in the nonadiabatic case. In this case, the role of marginal resonances in determining the total layer width is demonstrated to diminish with increasing main parameter λ (equal to the ratio of the perturbation frequency to the frequency of small-amplitude phase oscillations on the resonance). Solely the "bending effect" is important in determining the total amplitude of the energy deviations of the near-separatrix motion at λ≳7. In the adiabatic case, it is demonstrated that the geometrical form of the separatrix cell can be described analytically quite easily by means of using a specific representation of the separatrix map. It is shown that the nonadiabatic (and, to some extent, intermediary) case is most actual, in comparison with the adiabatic one, for the physical or technical applications that concern the energy jumps in the near-separatrix chaotic motion.

Width of the chaotic layer: maxima due to marginal resonances.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human neoplasms with extremely poor prognosis and a low survival rate. Immunosuppressive cell populations, e.g. regulatory T cells (Treg), appear to be important in PDAC, contributing to patient's poor prognosis. Therefore, we investigated the PDAC microenvironment with a focus on conventional and regulatory T cells in view of their potential therapeutic importance. We found that tumors from the murine Panc02 orthotopic model of PDAC were infiltrated with high numbers of Treg. Remarkably, these cells exhibited the effector/memory phenotype, suggesting their enhanced suppressive activity and higher proliferation capacity. Although we observed a steady increase in transforming growth factor-β (TGF-β) levels in the tumors, treatment with a specific inhibitor of TGF-β receptor I kinase failed to abrogate Treg accumulation. A CCR4 antagonist did not affect Treg percentage in the tumor either. However, intense Treg cell division in the tumor microenvironment was demonstrated, suggesting local proliferation as a major mechanism of Treg accumulation in PDAC. Notably, this accumulation was reduced by low-dose gemcitabine administration, resulting in a modestly increased survival of PDAC mice. Our results provide an insight into mechanisms of immunosuppression in PDAC, suggesting an important role for proliferative expansion of effector/memory Treg. Low-dose gemcitabine therapy selectively depletes Treg, providing a basis for new modalities of PDAC therapy.

Low-dose gemcitabine depletes regulatory T cells and improves survival in the orthotopic Panc02 model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human neoplasms, having extremely poor prognosis with a 5-year survival rate of <1 % and a median survival of 6 months. In contrast to other malignancies, pancreatic cancer is highly resistant to chemotherapy and targeted therapy. Therefore, new treatment options are urgently needed to improve the survival of patients with PDAC. Based on our data showing that patients with higher CD8+ T cell tumour infiltration exhibited prolonged overall and disease-free survival compared to patients with lower or without CD8+ T cell tumour infiltration, we suggested that immunotherapy could be a promising treatment option for PDAC. However, clinical data from the chemoradioimmunotherapy with interferon-α (IFN) trial did not point to an improved efficiency of chemoradiation combined with IFN as compared to chemoradiotherapy alone, suggesting an important role of the immune suppression induced by PDAC and/or unspecific immune stimulation. In support of this hypothesis, we found that the PDAC patients and experimental mice had an increased number of regulatory T cells and myeloid-derived suppressor cells. These results allowed us to conclude that PDAC provokes not only an anti-tumour immune response, but also strong immune suppression. Thus, we supposed that new immunotherapeutical strategies should involve not only stimulation of the immune system of PDAC patients, but also exert control over the tumour immune suppressive milieu.

Two immune faces of pancreatic adenocarcinoma: possible implication for immunotherapy.

Malignant melanoma is characterized by the development of chronic inflammation in the tumor microenvironment, which leads to a strong immunosuppression associated with a rapid tumor progression. Adenosine is considered as one of the main immunosuppressive factors in the tumor environment. It is produced via enzymatic hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 localized on cell surface. Using the ret transgenic mouse melanoma model that closely mimics human melanoma, we demonstrated an increased frequency of ectonucleotidase-positive myeloid-derived suppressor cells (MDSCs) in melanoma lesions and lymphoid organs. Furthermore, we observed that conventional CD4(+)FoxP3(-) and CD8(+) T cells infiltrating melanoma lesions of ret transgenic mice were distinctly enriched in the CD39(+)CD73(+) subpopulation that co-expressed also PD-1. Ectonucleotidase expression was also up-regulated in CD4(+) and CD8(+) T cells upon activation. In addition, these ectoenzymes were largely found to be expressed on memory T cell compartment (in particular, on effector memory cells). Our data suggest that extracellular adenosine produced by regulatory T cells (Tregs) and MDSCs can suppress T cell effector functions through paracrine signaling. Another mechanism involves its production also by effector T cells and an inhibition of their anti-tumor reactivity via autocrine signaling as a part of the negative feedback loop. This mode of adenosine signaling could be also used by Tregs and MDSCs to enhance their immunosuppressive activity.

Extracellular adenosine metabolism in immune cells in melanoma.

We hypothesized that the combination of oncolytic virotherapy with immune checkpoint modulators would reduce tumor burden by direct cell lysis and stimulate antitumor immunity. In this study, we have generated attenuated Measles virus (MV) vectors encoding antibodies against CTLA-4 and PD-L1 (MV-aCTLA-4 and MV-aPD-L1). We characterized the vectors in terms of growth kinetics, antibody expression, and cytotoxicity in vitro. Immunotherapeutic effects were assessed in a newly established, fully immunocompetent murine model of malignant melanoma, B16-CD20. Analyses of tumor-infiltrating lymphocytes and restimulation experiments indicated a favorable immune profile after MV-mediated checkpoint modulation. Therapeutic benefits in terms of delayed tumor progression and prolonged median overall survival were observed for animals treated with vectors encoding anti-CTLA-4 and anti-PD-L1, respectively. Combining systemic administration of antibodies with MV treatment also improved therapeutic outcome. In vivo oncolytic efficacy against human tumors was studied in melanoma xenografts. MV-aCTLA-4 and MV-aPD-L1 were equally efficient as parental MV in this model, with high rates of complete tumor remission (> 80%). Furthermore, we could demonstrate lysis of tumor cells and transgene expression in primary tissue from melanoma patients. The current results suggest rapid translation of combining immune checkpoint modulation with oncolytic viruses into clinical application.

CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers in the world. PDAC cells activate tumor-specific immune responses but simultaneously trigger a strong immunosuppression. We showed that PDAC cells produce high amount of chronic inflammatory mediators and PDAC tumors build an immunosuppressive cytokine milieu, which correlates with tumor progression. We observed a low frequency of dendritic cells (DC) and a pronounced accumulation of macrophages and myeloid-derived suppressor cells (MDSC) in murine PDAC tumors. A strong accumulation of MDSC has also been demonstrated in the peripheral blood of resected PDAC patients. While DC and macrophages seem not to play a significant role in this PDAC model in the context of immunosuppression, MDSC are highly suppressive, and their accumulation is associated with an increase in intratumoral VEGF concentration during the PDAC progression. Application of the phosphodiesterase-5 inhibitor sildenafil led to a prolonged survival of PDAC-bearing female mice, which was due to the decrease in MDSC frequencies and in the systemic VEGF level. This led to a restoration of anticancer immune responses, manifested in the recovery of T lymphocyte functions and in an increase in the frequency of conventional CD4(+) T cells in tumors and IFNγ level in serum of PDAC-bearing mice. Thus, MDSC are strongly involved in the PDAC-associated immunosuppression and that their depletion could create new approaches for therapy of PDAC.

Ivan Shevchenko

Department of Pediatrics

Baylor College of Medicine

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Ivan Shevchenko

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics

Baylor College of Medicine

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Department of Pediatrics