Author ORCID Identifier

http://www.orcid.org/0000-0003-3114-7665

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Immunology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Tomasz Zal, Ph.D.

Committee Member

Kimberly Schluns, Ph.D.

Committee Member

Amy Heimberger, M.D.

Committee Member

Joya Chandra, Ph.D.

Committee Member

Robert Dantzer, DVM, Ph.D.

Abstract

Visualizing the dynamics of immune surveillance in brain tumors by intravital multiphoton microscopy

By

Felix I. Nwajei, MD

Supervisory Professor: Tomasz Zal, Ph.D.

Brain tumors (BTs) generally have a bad prognosis despite conventional treatment strategies. Immunotherapy is a relatively novel treatment approach that has shown benefit for durable treatment of melanoma, and is a promising candidate for different tumor types including BTs. Immunotherapeutic strategies work by exploiting and/or enhancing natural anti-tumor immune response, a process that is critically dependent on adaptive immunity, T cell infiltration and surveillance of tumor. However, little is known about the dynamics and regulation of T cell surveillance in BTs. Resident immune cells of the myeloid lineage known as microglia are ubiquitous in the brain parenchyma while tissue-resident myeloid dendritic cells (DCs) known to activate T cells are relatively rare in the brain compared to DCs in other organs. Accumulating evidence indicates that myeloid cells infiltrate and create an immune suppressive microenvironment in BTs, but the identity of these myeloid cells and their role in the adaptive immune surveillance of BTs by T cells is unclear. Based on the predominance of microglia in the brain tissue, studies focused on understanding how BT immune surveillance is regulated, have been skewed toward microglia. Many conclusions regarding microglia function have been deduced from in vitro experiments. Nonetheless, in vivo studies in parallel models such as EAE indicate that DCs are superior to microglia in antigen presentation to T cells in the brain and to date, there is no direct in vivo evidence to suggest otherwise. In addition, DCs are well-established cellular regulators of T cell surveillance in extracranial tumors. Therefore, I hypothesized that DCs, rather than microglia, play a major role in regulating T cell surveillance in BTs. To address this hypothesis, I have developed experimental imaging systems for longitudinal intravital multiphoton microscopy of immune cell dynamics in BTs in living mice and used this approach to interrogate T cell behavior in orthotopic glioma and in experimental intracranial metastases in vivo. I found that the myeloid infiltration of BTs was dominated by CD11c+ DC cells rather than microglia. Quantitative in situ tissue cell image cytometry further revealed that myeloid-derived CCR2+ monocytes accumulated in the BT core, CD11c+ DCs at the tumor margin, and CX3CR1+ microglia outside the tumor. T cells formed clusters around CD11c+ DCs, but not the microglia. Within these clusters, T cells vigorously interacted directly with CD11c+ DCs. CD11c+ DCs retained T cells and controlled their motility patterns, indicating that CD11c+ DCs play a major role in regulating T cell retention and motility in BT. Corresponding to the preferential distribution of CD11c+ DCs at BT margins was expression of the neuronal chemokine Fractalkine (CX3CL1). Deficiency of the Fractalkine receptor CX3CR1 resulted in decreased CD11c+ DC recruitment. In addition, decreased CD11c+ DC recruitment was accompanied by decreased T cell recruitment, an increase in the spatial diffusion of the few BT-infiltrating T cells, and subsequent outgrowth of a fibrosarcoma BT, which spontaneously regresses in the brain of control wild type mice in a CD8 T cell dependent manner.

In summary, by using novel intravital imaging systems for longitudinal visualization of BT immune surveillance across several types of cancer, I showed that the recruitment, migration and retention of tumor infiltrating T cells in the brain is mediated by incoming CD11c+ DCs rather than by the brain-resident CX3CR1 microglia, and identified the neuronal chemokine Fractalkine as a key molecule that promotes T cell surveillance in BTs by recruiting CD11c+ DCs.

These findings suggest that the non-microglial tumor-associating CD11c+ myeloid cells and the fractalkine/CX3CR1 chemokine pathway control T cell surveillance in BT and represent attractive immunotherapeutic targets that could be modulated for guiding endogenous or adoptive transfer of T cells to BT sites and for therapeutic modulation to enhance immunity against BTs.

Keywords

Brain tumors, Glioma, Brain metastases, Immune surveillance, Microglia, CD11c dendritic cells, Chemokines, Fractalkine, Longitudinal intravital multiphoton microscopy, T cells

Available for download on Thursday, April 12, 2018

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