Thomas F. Conway

Long-Term Engraftment and Expansion of Tumor-Derived Memory T Cells Following the Implantation of Non-Disrupted Pieces of Human Lung Tumor into NOD-scid IL2Rγnull Mice

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rγnull mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45+ tumor-associated leukocytes within the xenograft are predominantly CD3+ T cells with fewer CD138+ plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-γ in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-γ and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45+ cells. The majority of CD45+ cells were CD3+ and expressed a phenotype consistent with an effector memory T cell, consisting of CD4+ or CD8+ T cells that were CD45RO+, CD44+, CD62L−, and CD25−. Following adoptive transfer into non-tumor bearing NOD-scid IL2Rγnull mice, these human T cells were found to expand in the spleen, produce IFN-γ, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rγnull tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.

Controlled-release Particulate Cytokine Adjuvants for Cancer Therapy

Cytokine therapy can induce tumor regression in cancer patients but systemic administration of cytokines is accompanied with severe toxicity. Loco-regional delivery represents an effective and less toxic alternative to systemic injection. However; the requirement for frequent repeated injections of recombinant cytokine or the logistical difficulties associated with gene-modification have limited wide-spread use of loco-regional therapy. A simpler alternative local delivery strategy involves the use of controlled-release cytokine depot formulations. These formulations provide the advantage that physiological doses of cytokines are directly released to the tumor microenvironment in a sustained manner. Anti-tumor efficacy of IL-2; IL-12; GM-CSF or TNFalpha-encapsulated polymer microspheres has been evaluated in syngeneic murine and human tumor /SCID mouse xenograft models. A single intra-tumoral injection of these formulations; particularly that of IL-12 in combination with GM-CSF or TNFalpha; promoted the regression of established primary tumors; induced systemic anti-tumor T- and NK-cell responses and achieved complete eradication of disseminated disease. Cellular and molecular analysis of post-therapy tumor microenvironment demonstrated that treatment promoted the activation of tumor-associated T-effector/memory cells; the elimination of CD4+ CD25+ Foxp3+ T-suppressors and the de novo priming of tumor-specific CD8+ T-effector cells. Long-term monitoring of post-therapy tumors revealed that reversal of intra-tumoral immune suppression was transient and that T-suppressor cells rapidly re-infiltrated tumors. Repeated treatment resurrected anti-tumor activity; however, therapeutic efficacy declined with each treatment cycle. The observed loss of therapeutic efficacy was associated with a progressive intensification of the post-treatment T-suppressor cell rebound. In contrast; depletion of T-suppressor cells with low dose chemotherapy prior to each cycle of treatment resulted in a dramatic enhancement of long-term therapeutic efficacy leading to complete remissions. Clinical implications of these findings are discussed herein.

Monoclonal antibodies directed against the T-cell activation molecule CD137 (interleukin-A or 4-1BB) block human lymphocyte-mediated suppression of tumor xenografts in severe combined immunodeficient mice.

A monoclonal antibody specific for the human analog of the murine T-cell activation molecule 4-1BB was generated and is shown here to react selectively with activated human CD4+ and CD8+ T lymphocytes. Treatment of these T cells in a one-way mixed lymphocyte culture with the anti–h4-1BB antibody enhanced the cell proliferation of the allostimulated lymphocytes. Previous studies in the mouse have shown that treatment of tumor-bearing mice with antibodies to 4-1BB augments anti-tumor immunity that is mediated by both CD4+ and CD8+ T cells. The authors consider the possibility that a similar approach may be efficacious for human cancer immunotherapy. This question was addressed by evaluating the effect of an anti–h4-1BB monoclonal antibody on human lymphocyte-mediated suppression of a human tumor xenograft in SCID mice. Mice treated with a control antibody and co-injected with the tumor and peripheral blood lymphocytes exhibited a lymphocyte dose-dependent suppression of tumor growth. In mice treated with the anti–h4-1BB antibody, the lymphocyte-mediated tumor suppression was completely eliminated and tumors grew progressively (as was observed in mice inoculated with tumors without lymphocytes). This monoclonal antibody specific for anti–h4-1BB, which augments the proliferation of allostimulated cells in vitro, blocks T-cell anti-tumor activity in vivo. These results suggest that although 4-1BB plays a role in the human peripheral blood lymphocyte–mediated suppression of tumor growth, antibodies to this molecule on human cells fail to stimulate anti-tumor activity, as was observed in tumor-bearing mice treated with an antibody to murine 4-1BB.

Growth of human tumor xenografts in SCID mice quantified using an immunoassay for tumor marker protein in serum

The accurate measurement of the response of a tumor to a given treatment is critical to evaluating novel therapeutic modalities. An experimental design is reported here that can be generally applied to monitoring human tumor xenografts growing in immunodeficient mice. A human non-small cell lung tumor cell line was transfected with a mammalian expression vector containing the gene encoding human prostate specific antigen (PSA) and has been shown to grow progressively following the subcutaneous, intraperitoneal and intravenous inoculation of the tumor into severe combined immunodeficient (SCID) mice. The transfected human tumor cells produce PSA that accumulates in the sera of all tumor inoculated SCID mice. An enzyme-linked immunoassay using a rabbit polyclonal and a mouse monoclonal antibody specific for PSA was designed and tested for the detection and quantification of serum PSA in tumor-bearing mice. Over a 5-week period, the serum levels of PSA of mice inoculated subcutaneously with the tumor increased progressively, and the estimated tumor volumes correlated with the amount of PSA detected in the serum. Serum PSA levels correlated even better with total tumor mass following the intraperitoneal inoculation of tumor cells into SCID mice. Serum PSA levels fell rapidly following the surgical debulking of tumor xenograft, reaching background levels of PSA in the serum 1 week after tumor removal. Serum PSA levels were also observed in SCID mice inoculated intravenously with a PSA transfected human lung tumor cell line adapted to grow orthotopically in the lung. The transfection of human tumors with a tumor marker and the use of an immunoassay to detect this marker establish an experimental design that provides a reliable, non-invasive, accurate and simple approach to monitor and quantify the growth of human tumor xenografts in SCID mice.

Oral interleukin-10 alleviates polyposis via neutralization of pathogenic T-regulatory cells

Immune dysregulation drives the pathogenesis of chronic inflammatory, autoimmune, and dysplastic disorders. While often intended to address localized pathology, most immune modulatory therapies are administered systemically and carry inherent risk of multiorgan toxicities. Here, we demonstrate, in a murine model of spontaneous gastrointestinal polyposis, that site-specific uptake of orally administered IL10 microparticles ameliorates local and systemic disease to enhance survival. Mechanistic investigations showed that the therapeutic benefit of this treatment derived from neutralization of disease-promoting FoxP3(+)RoRγt(+)IL17(+) pathogenic T-regulatory cells (pgTreg), with a concomitant restoration of FoxP3(+)RoRγt(-)IL17(-) conventional T-regulatory cells (Treg). These findings provide a proof-of-principle for the ability of an oral biologic to restore immune homeostasis at the intestinal surface. Furthermore, they implicate local manipulation of IL10 as a tractable therapeutic strategy to address the inflammatory sequelae associated with mucosal premalignancy.

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patient’s T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patient’s PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.

Oral Delivery of Particulate Transforming Growth Factor Beta 1 and All-Trans Retinoic Acid Reduces Gut Inflammation in Murine Models of Inflammatory Bowel Disease

BACKGROUND AND AIMS We investigated oral delivery of transforming growth factor beta 1 [TGFβ]- and all-trans retinoic acid [ATRA]-loaded microspheres as therapy for gut inflammation in murine models of inflammatory bowel disease [IBD]. METHODS ATRA and TGFβ were separately encapsulated in poly [lactic-co-glycolic] acid or polylactic acid microspheres [respectively]. TGFβ was encapsulated using proprietary phase-inversion nanoencapsulation [PIN] technology. RESULTS PIN particles provided sustained release of bioactive protein for at least 4 days and were stable for up to 52 weeks when stored at either 4(0)C or -20(0)C. In the SCID mouse CD4 + CD25- T cell transfer model of IBD, oral treatment starting at disease onset prevented weight loss, significantly reduced average disease score [~ 50%], serum amyloid A levels [~ 5-fold], colon weight-to-length ratio [~ 50%], and histological score [~ 5-fold]. CONCLUSIONS Both agents given together outperformed either separately. Highest TGFβ doses and most frequent dose schedule were most effective. Activity was associated with a significant increase [45%] in Foxp3 expression by colonic lamina propria CD4+ CD25+ T-cells. Activity was also demonstrated in dextran sulphate sodium-induced colitis. The data support development of the combination product as a novel, targeted immune based therapy for treatment for IBD.

Memory T Cells in the Chronic Inflammatory Microenvironment of Nasal Polyposis are Hyporesponsive to Signaling Through the T Cell Receptor

A majority of T cells from chronic inflammatory tissues derived from patients with nasal polyposis were found to express an effector memory phenotype. We report here that these memory T cells failed to activate NF-κB in response to TCR stimulation but responded normally when the proximal TCR signaling molecules were bypassed with PMA and ionomycin. The dysfunction of these cells was associated with a decrease in the phosphorylation of several TCR proximal signaling molecules including ZAP70, Lck and SLP-76. In addition to the disruption in the TCR signaling pathway, the nasal polyp-associated T cells were shown to have a defect in their ability to translocate LAMP-1 to the cell surface. The results presented here establish that the phenotype and anergy of the T cells in the nasal polyp are similar to those which is seen in memory T cells derived from human tumors and other sites of chronic inflammation.