THEME I: Defining the influence of apoptotic and autophagic cell death on immunity
This past year has been devoted in large part to assay development. We established a novel method using multispectral imaging flow cytometry and unique imaging analysis scripts that permits simultaneous assessment of both apoptosis and autophagic pathways at the single cell level. The multispectral imaging flow cytometer (ImageStreamX) captures high-content images of cells in flow (up to 1000 cells / second). Typical protocols rely on the manual counting of cells containing microtubule associated protein light chain-3 (LC3) punctae as a measure of its aggregation within and decoration of autophagosomes. While prior methods have automated this approach using multispectral imaging and scanning array systems, our studies suggested that the heterogeneity of LC3 punctae in their size, shape and intensity make it difficult to accurately quantitate. In addition, several pathologic stressors result in the inhibition of autophagosome / lysosome fusion resulting in massive LC3 accumulation and aggregation, further complicating enumeration of punctae as they are no longer appearing as distinct sub-cellular structures. To overcome these limitations, we established a stream-lined image analysis strategy based on the cell’s texture features. This work was recently published in Autophagy and has served as a basis for current work on dissecting the interplay between cell death and autophagy.
In addition to fundamental mechanisms of antigen delivery and cross-priming, we have also taken steps to identify strategies for optimizing vaccination protocols. This work has concerned the delivery of cell- associated antigen, an important strategy for tumor antigen vaccination. While many experimental models have been developed in order to define the critical parameters for efficient cross-priming, few have utilized quantitative methods that permit the study of the endogenous repertoire. Comparing different strategies of immunization, we report that local delivery of cell-associated antigen results in delayed T cell cross-priming due to the increased time required for antigen capture and presentation. In comparison, delivery of disseminated antigen resulted in rapid T cell priming. Surprisingly, local injection of cell- associated antigen, while slower, resulted in the differentiation of a more robust, polyfunctional, effector response. We also evaluated the combination of cell-associated antigen with poly I:C delivery and observed an immunization route-specific effect regarding the optimal timing of innate immune stimulation. These studies highlight the importance of considering the timing and persistence of antigen presentation, and suggest that intradermal injection with delayed adjuvant delivery is the optimal strategy for achieving CD8+ T cell cross-priming. This study has been recently published in Frontiers in Immunology.
THEME II: To identify mechanisms of tumor immunity in patients with superficial transitional cell carcinoma of the bladder
After several years of investment, we are beginning to see important results coming from our work on bladder cancer. Briefly, our long term interest in bladder cancer is based on the fact that treatment for non-muscle invasive carcinoma of the bladder represents one of the few examples of successful tumor immunity. Six weekly intravesical instillations of Bacillus Calmette-Guerin (BCG), often followed by maintenance schedule, result in up to 50–70% clinical response. Prior models suggested that the mechanism of action involves the non-specific activation of innate effector cells, which may be capable of acting in the absence of an antigen-specific response. For example, recent evidence suggests that BCG-activated neutrophils, as well as other innate effector cells are not selective in their targeting— thus surrounding cells may be influenced by degranulation and/or cytokine production. To establish if these observed conditions could account for clinically effective tumor immunity, we built a mathematical model reflecting the early events and tissue conditioning in patients undergoing BCG therapy. The model incorporates key features of tumor growth, BCG instillations and the observed prime/boost pattern of the innate immune response. Model calibration established that each innate effector cell must kill 90–95 bystander cells for achieving the expected 50–70% clinical response. This prediction was evaluated both empirically and experimentally and found to vastly exceed the capacity of the innate immune system. We therefore conclude that the innate immune system alone is unable to eliminate the tumor cells. We infer that other aspects of the immune response (e.g., antigen- specific lymphocytes) decisively contribute to the success of BCG immunotherapy. This work was recently published in OncoImmunology.
We have also established and validated an experimental mouse model, thus allowing us to demonstrate that while BCG dissemination to bladder-draining lymph nodes and priming of interferon-γ producing T cells may occur following a single instillation, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. What was unexpected is that parenteral exposure to BCG prior to instillation overcame that requirement, triggering a more robust acute inflammatory process at the first intravesical instillation and accelerating the recruitment of T cells to the bladder. Moreover, exposure to BCG prior to orthotopic tumor challenge dramatically improved response to BCG therapy. Retrospective analysis of clinical data showed a significantly better response to therapy in patients with pre-existing immunity to BCG. Together these data suggest that monitoring response to purified protein derivative (PPD) may serve as a predictor of treatment outcome; and that boosting BCG responses by parenteral exposure may be a safe and effective means of improving intravesical BCG-induced clinical responses. This work has been submitted for publication.
THEME III: To characterize the complex role of type I IFNs and interferon stimulated genes (ISGs) in HCV disease pathogenesis and related emergent viral infections
In the past year we have successully completed a 5yr effort, which provided the first in vivo evidence for CXCL10 antagonism in humans. These studies were conducted in patients with chronic HCV but are highly applicable to therapeutic vaccination and tumor immunity. The application of our observations for such purposes is currently being explored in pre-clinical models.
Our efforts on chronic infection with hepatitis C virus (HCV) are based on its being a major public health problem (with nearly 170 million infected individuals worldwide) and the infection being the primary cause of liver cancer. Treatment for chronic infection has been the combination of pegylated IFN-α2 and ribavirin (RBV); however, this treatment is effective in fewer than 50% of patients infected with HCV geno- type 1 or 4. Recent studies identified the chemokine CXCL10 (also known as IP-10) as an important negative prognostic biomarker. Given that CXCL10 mediates chemoattraction of activated lymphocytes, it is counter- intuitive that this chemokine correlates with therapeutic nonresponsiveness. We offered new insight into this paradox and provided evidence that CXCL10 in the plasma of patients chronically infected with HCV exists in an antagonist form, due to in situ amino-terminal truncation of the protein. We further demonstrated that dipeptidyl peptidase IV (DPP4; also known as CD26), possibly in combination with other proteases, mediates the generation of the antagonist form(s) of CXCL10. These data offer what we believe to be the first evidence for CXCL10 antagonism in human disease and identify a possible factor contributing to the inability of patients to clear HCV. The work was published in Journal of Clinical Investigation in early 2011.
In addition, we have made efforts to validate our assays for CXCL10 agonist and antagonist forms. This work provided a detailed description of the assay systems that has been established to discriminate the agonist form of CXCL10 from the NH2-terminal truncated form of the molecule generated by dipeptidylpeptidase IV (DPP4) cleavage. In addition, we demonstrated the utility of this assay for monitoring agonist and antagonist forms of CXCL10 in culture supernatant and urine samples from bladder cancer patients. Given the important role of CXCL10 in chronic inflammatory diseases and its’ suggested role as a predictive marker in managing patients with chronic hepatitis C, asthma, atopic dermatitis, transplantation, tuberculosis, kidney injury, cancer and others diseases, we believe that our results will be of general interest to the research and medical community. This study was recently accepted for publication in Clinical and Experimental Immunology.
Regarding our studies on Chikungunya virus (ChikV) infection, we have continued our strong involvement as part of the Institut Pasteur ChikV taskforce that formed following the outbreak in 2005 / 2006. We have demonstrated that ChikV is controlled by type I IFN produced by non-hematopoetic cells. The principle project concerns the role of type I IFN in chikungunya disease pathogenesis. This work has provided an additional viral model for use to dissect the role of type I IFNs in immune regulation and anti-viral responses. Specifically, we have worked with Drs. Olivier Schwartz, Marc Lecuit (both Institut Pasteur) and Deborah Lenschow (Washington University, St Louis) to define the tropism of CHIKV infection, to develop an in vivo mouse model, and to establish the effort pathways responsible for inhibiting viral replication. Using these approaches, we have demonstrated a critical role for type I IFNs in the control of CHIKV replication and disease. In addition, we have identified RIG-I as the host sensor responsible for producing IFNα/β in CHIKV infected cells. Finally, we have characterizing the disease pathogenesis in neonates and identified interferon stimulated gene-15 (ISG-15) as an important antiviral factor responsible for restricting CHIKV replication.
Image of dendritic cells engulfing apoptotic cells—the first step for antigen cross-presentation