Cytotoxic T lymphocytes (CTLs) are the primary component of the adaptive immune system responsible for clearance of virally infected and tumorigenic cells. In cancer however, this tumor-specific immune response is often impaired. The impairment is multifactorial; some cancers utilize mechanisms to evade the immune system through downregulation of Major Histocompatability Complex I or lack of tumor-specific antigens, while others use methods to actively inhibit local function of tumor-induced immune responses via production of immunosuppressive cytokines, Fas-mediated apoptosis, or recruitment of T regulatory cells (Tregs). These Tregs function to further immune regulate and inhibit CTLs, using methods such as suppressive cytokines, and cytotoxic killing. All of these components lead to an “on/off” phenotype, where CTL effector function is shut down within the Tumor Immunosuppressive Microenvironment (TIM), but can be recovered quickly upon removal of CTLs from the TIM. The transient impairment of Tumor Infiltrating Lymphocytes (TIL) has been described in mouse models, but is poorly characterized in humans.
In this dissertation, we examined infiltration of CTLs across several types of human pediatric cancers, taken from patients who had not undergone prior treatment. We found tumors associated with favorable prognoses, including Wilms’ Tumor and Neuroblastoma (NB), had higher levels of CTL infiltration than those with less favorable prognoses, e.g. Ependymoma, which possessed no observable infiltration. Additionally, we demonstrate the TIL “on/off” phenotype in a case of Pilocytic Astrocytoma, demonstrating significant recovery of TIL effector function.
We proposed that the poor infiltration and impaired effector function in these pediatric tumors was a direct result of the TIM, and sought to improve this immune response by developing an attenuated live cell vaccine, utilizing a murine NB model, Neuro2a, to create a NB line with knock down (KD) of Inhibitor of Differentiation 2 (Id2), which impaired their ability to form tumors in vivo. In prophylactic and therapeutic models, introduction of Id2-KD cells in combination with the immune checkpoint blockade inhibitor anti-CTLA-4, induced an increase in CTLs capable of homing to the tumor, that were also able to employ effector function within the TIM, resulting in clearance of wild-type Neuro2a tumors.
A separate emerging immunotherapeutic approach is to express a Chimeric Antigen Receptor (CAR) on CTLs that allows them to be activated to kill cells expressing the CAR-specific protein, bypassing MHC presentation. Using a murine Rhabdomyosarcoma model, we demonstrate that tumor infiltrating Tregs express lytic molecules, encouraging us to develop a method of successfully transducing Tregs with a CAR (DC101), rather than CTLs, thereby exploiting characteristics of the Treg in the TIM, specifically their cytotoxic capability and their unique recruitment and ability to thrive in that environment. We demonstrate in vitro CAR-mediated redirection of lytic effector function using DC101-expressing CTLs against tumor cell lines, though attempting to increase Treg cytotoxicity in vitro via known inducers of CTL cytotoxicity (IFNα or IL-12) or known inducers of Tregs within the TIM (TGF-β1) showed no increase in Treg cytotoxicity.
|Advisor:||Sandler, Anthony D.|
|Commitee:||Cruz, Conrad Russell Y., Leitenberg, David, Nagaraju, Kanneboyina, Vukmanovic, Stanislav|
|School:||The George Washington University|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 76/10(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Immunology, Oncology|
|Keywords:||Chimeric antigen receptor, Cytotoxic T lymphocytes, Neuroblastoma, T cells, T regulatory cells, Tumor infiltrating lymphocytes|
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