B-cell acute lymphoblastic leukemia promotes an immune suppressive microenvironment that can be overcome by IL-12 Open Access

Abstract

Immunotherapies are a breakthrough in treatment for B-cell acute lymphoblastic leukemia (B-ALL), particularly in patients with relapsed or refractory disease. Thus, for my thesis project, we sought to identify mechanisms of immune suppression in high-risk B-ALL and strategies to overcome them. We first began by examining T- and myeloid cell responses when exposed to leukemia cell supernatant. We observed when T-cells were stimulated in ALL supernatant ex vivo there is a reduction in the surface expression of the T-cell activation marker, CD44, and CD107b, a degranulation marker. Macrophages co-cultured with leukemia cells were also unable to significantly upregulate activation markers, CD80 and CD86, compared to macrophages in unconditioned media.

Single-cell RNA-sequencing analysis of samples collected from patients with B-ALL with measurable residual disease (MRD) after induction chemotherapy revealed T-cell exhaustion. To investigate T cell exhaustion in vivo we used a mouse model of B-ALL and we observed reductions of T cell and dendritic cell numbers and activation similar to what is observed in MRD positive patients. Impressively, recombinant interleukin-12 (rIL-12) treatment of mice with B-ALL significantly increased the number of splenic and bone marrow resident T-cells and DCs. We also observed a shift to an immunostimulatory cytokine and chemokine bone marrow microenvironment in mice with B-ALL treated with rIL-12. Targeted RNA-sequencing of T-cells isolated from vehicle and rIL-12 treated mice with B-ALL provided mechanistic insight into how IL-12 treatment overcomes B-ALL induced immunosuppression. Genes associated with immune exhaustion, including Lag3 and Tigit, were suppressed with rIL-12 treatment, relative to levels observed in vehicle-treated mice. In addition to the beneficial effects of rIL-12 treatment in mice with B-ALL, the cytolytic capacity of the immunotherapy blinatumomab, a bispecific engager, was also enhanced in co-culture experiments with human T-cells and B-ALL cells. In the presence of B-ALL secretome, blinatumomab efficacy is reduced and this suppression can be overcome with IL-12 treatment.

Overall, this work provides mechanistic insight into how IL-12 overcomes B-ALL-mediated immune suppression. This suggests the potential for novel treatment strategies utilizing IL-12 for the treatment of B-ALL.

Table of Contents

Chapter 1: Introduction Pages 1-11

1.1 Cancer                                                                                                               

1.1.1 Cancer overview 2                                                                                                                   

1.1.2 Hallmarks of cancer 2-3                                                                                                 

1.1.3 Pediatric hematologic malignancies 3-4                                                                        

1.2 Overview of B-ALL: Prognosis and treatments                                           

1.2.1 B-cell acute lymphoblastic leukemia (ALL) 4-5                                                                    

1.2.2 Current standards of care 5-8                                                                                                      

1.2.3 Challenges to current standards of care 8                                                                 

1.2.4 Immunotherapies 8-11                                                                                               

Chapter 2: Overcoming mechanisms of immune evasion in B-ALL 14-27

2.1 Mechanisms of immune evasion                                                                       

2.1.1 Immune evasion mechanisms in cancer 15-16                                                        

2.1.2 Mechanisms of immune evasion in B-ALL 17-21                                                                                      

2.1.3 Current immunotherapies to overcome immune evasion in B-ALL 21-24              

2.2 IL-12

2.2.1 IL-12 overview 24-35                                                                                                                

2.2.2 Pre-clinical and clinical implications of IL-12 treatment 25-27                          

2.3 Rationale for studying IL-12 an immunotherapeutic in B-ALL 27-28

Chapter 3: IL-12 induced leukemia remission is dependent upon CD4 and CD8 T cells 33-49

3.1 Introduction 34                                                                                      

3.2 Background 34-36                                                                                                                  

3.3 Materials and Methods 36-38                                                                                     

3.4 Results 38-40                                                                                                                 

3.5 Discussion 40-42                                                                                                               

Figures 43-48                                                                                                                               

Chapter 4: B-cell acute lymphoblastic leukemia promotes an immune suppressive microenvironment that can be overcome by IL-12 49-76

4.1 Abstract 50                                                                                                                                                                                                                                                      

4.1 Introduction 50-52                                                                                                                    

4.2 Materials and Methods 52-58                                                                                              

4.3 Results 59-64                                                                                                                                 

4.4 Discussion 64-66                                                                                                                       

Figures 67-76                                                                                                                                    

Chapter 5: Discussion 77-85 

5.1 Summary and conclusions 78-79                                                                                            

5.2 Future directions 79-80                                                                                                           

5.3 Clinical implications for localized delivery of IL-12 in B-ALL 80-82                           

Figures 83-86                                                                                                                

References 87-101           

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Oncology Health Sciences, Immunology
Keyword	interleukin-12 blinatumomab immune suppression acute lymphoblastic leukemia T-cells
Committee Chair / Thesis Advisor	Porter, Christopher, Emory University
Committee Members	Selvaraj, Periasamy, Emory University Lesinski, Gregory, Emory University Henry, Curtis, Emory University Shanmugam, Mala, Emory University

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