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

Hunter, Alisha Desiree "Rae" (Summer 2022)

Permanent URL: https://etd.library.emory.edu/concern/etds/kp78gh60r?locale=en


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           

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