Hygiene Hypothesis in Acute Lymphoblastic Leukemia Público

Abstract

The Hygiene Hypothesis (HH) posits that we live in a society that is becoming devoid of adequate pathogen exposure relative to our past ancestors without 21st-century medical care. Furthermore, this hypothesis suggests that our immune system becomes limited in identifying and responding to insults, including pathogens and cancer cells, which leads to higher mortality rates in these contexts. Cancer is driven by cell-intrinsic (e.g., mutations) and extrinsic (e.g., tumor microenvironment) parameters. 

The immune system governs the development or elimination of cancer cells, with T-cells (cell-mediated immunity) (TCs) playing a role in the destruction of malignant cells. Cell-mediated immunity relies on potent naive and memory TC responses to foreign antigens. As a result, research and clinical efforts are focused on optimizing their efficacy as new forms of cancer treatments, immunotherapy. In this study, we hypothesized that pathogenic exposure enhances the ability of TCs to eliminate cancer cells. We predict that this is due to components of the HH where memory TCs generated in response to pathogenic exposure, direct infections, or vaccinations eliminate cancer cells in a process called “cross protection.” This property relies on the potent killing capacity of memory TCs mediated by the death-receptor CD95 expressed on the surface of these cells and the inherent property of some cancer cells expressing high surface levels of the receptor for CD95 known as CD95 ligand (CD95L). This study will provide mechanistic insight into the extent of cross-protection in B-cell acute lymphoblastic leukemia (B-ALL) models and provide public health information regarding the potentially pleiotropic health benefits of vaccinating pediatric populations.  

Methods: To define the surface expression (SE) of CD95 on human T-cells and CD95L on human B-ALL cell lines, non-malignant primary B-cells, and malignant B-cells from pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL), we will fluorescently detect CD95/CD95L via flow cytometry (FC). The SE of the proteins will be determined using Flowjo software. To define if the “cross recognition/cross protection” of malignant B-cells by antigen-stimulated TCs is mediated through the CD95/CD95L pathway, FC, and live-cell imaging to determine malignant B-ALL death when co-cultured with unstimulated and stimulated TCs.

Results: We have confirmed human B-ALL SE of CD95L. We have also confirmed that TCs can cross-recognize and kill malignant B-ALL cells. Next, we will determine if cytotoxicity depends on the CD95/CD95L pathway and if primary B-ALL cells from patients are killed by TCs using this mechanism.

Table of Contents

I. Introduction 1

a. Cancer 2

i. Solid vs. Blood Cancer (Hematological Malignancies) 2

ii. High Risk Factors for Oncogenesis 3

iii. Immunosuppression 3

iv. Adiposity and Carcinogenesis 3

b. Vaccination 6

i. Vaccines and Cancer 6

c. Hygiene Hypothesis 7

d. The Death Receptor CD95 and its Natural Ligand CD95L 11

e. Experimental Goals and Significance 11

II. Methods 13

a. Cell Lines 13

b. Flow Cytometry Analysis of CD95L & CD95 Surface Stains 13

c. Cytotoxicity assay with Co-Co-coculture Experiments 13

d. Adipocyte Differentiation 14

III. Results 14

a. Surface Stain of CD95L on RCH-ACV B-ALL 14

b. Co-culture of Jurkat T-cells with B-ALL 16

IV. Discussion 17

V. Future Objectives and Conclusion 19

VI. Supplementary Data 21

VII. References 27

VIII. Appendix  34

a. Protocol 34

i. Micropropagation of Mesenchyme OP-9 and Generation of ACM/SCM 34

ii. Insulin Oleate Medium (IOM) Preparation 40

iii. Flow Cytometry Assay Set-Up Procedure 40

iv. T-cell Activation Protocol 42

v. Co-culture Experiment Protocol 42

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Biology, General Health Sciences, Immunology
Palabra Clave	B-ALL ALL Cancer Hygiene Hypothesis Immunology T-cell acute lymphoblastic leukemia Leukemia
Committee Chair / Thesis Advisor	Curtis J. Henry, Emory University
Committee Members	Leila Rieder, Emory University Michael McCormick, Emory University

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