Date on Master's Thesis/Doctoral Dissertation

5-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Shirwan, Haval

Committee Co-Chair (if applicable)

Yolcu, Esma

Committee Member

Suttles, Jill

Committee Member

Yan, Jun

Committee Member

Zhang, Huang-Ge

Abstract

SA-4-1BBL is a recombinant costimulatory molecule that is active in its soluble form and has pleiotropic effects on the functions of innate, adaptive, and regulatory immune cells. We have previously shown that SA-4-1BBL makes CD4+ T conventional cells (Tconvs) resistant to suppression by CD4+CD25+Foxp3+ T regulatory cells (Tregs). The mechanistic basis of this observation is the subject of this study. We demonstrate that Tconvs, but not Tregs, are the direct targets of SA-4-1BBL-mediated evasion of Treg suppression without contribution from antigen presenting cells (APCs). SA-4-1BBL engagement with its receptor, 4-1BB, on Tconvs resulted in the production of high levels of IL-2 cytokine that was necessary and sufficient in overcoming Treg suppression. Removal of IL-2 from culture supernatant restored Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcame suppression. In a series of studies our laboratory demonstrated the therapeutic efficacy of SA- 4-1BBL as an adjuvant component of subunit cancer vaccines in various preclinical rodent cancer models. Inasmuch as SA-4-1BBL contains streptavidin (SA) as a foreign bacterial protein, repeated vaccinations may generate anti-SA antibodies. Such antibodies may have positive or negative effect on the efficacy of the vaccines. Therefore, a series of studies were conducted to assess the potential impact of SA’s immunogenicity on the immuno-stimulatory function of SA-4-1BBL molecule. We demonstrated that repeated treatments with SA-4-1BBL generate both cellular and humoral immune responses against the SA portion of the molecule. Antibodies against SA neither blocked nor improved the costimulatory function of SA-4-1BBL on T cells. Importantly, preimmunization with SA-4-1BBL protein alone did not interfere with the therapeutic efficacy of a subunit vaccine consisting of the human papillomavirs (HPV) E7 tumor associated antigen (TAA) + SA-4-1BBL in the TC-1 cervical cancer tumor model. Surprisingly, pretreatment with SA-4-1BBL alone conferred protection regardless of the type of tumor tested. The robust anti-tumor function of SA-4-1BBL involved a communication bridge between CD4 and NK cells without significant contribution from CD8 cells or B cells. NK cells were found to be critical to the observed tumor suppression and required CD4+ T cell help for protective efficacy at both priming and effector stages. Depletion of either NK or CD4 cells negated SA-4-1BBL’s anti-tumor protection. Moreover, the tumor suppressive effect of SA-4-1BBL was shown to be a bona fide property of this molecule, as immunizing mice with an agonistic antibody to 4-1BB receptor did not confer any protection against TC-1 tumors. The significance of understating the mechanisms underlying SA-4-1BBL’s mode of action provides new tools in the evolving field of cancer immunotherapy and prevention. The implications also extend to transplantation settings as well as infectious diseases where tipping the balance between Tregs and T effector cells (Teffs) plays a critical role in achieving desired outcomes.

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