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Innate Lymphoid Cell Dysregulation in Acute Myeloid Leukemia

Lordo, Matthew Robert
http://orcid.org/0000-0002-7108-9765
http://rave.ohiolink.edu/etdc/view?acc_num=osu1680775578447773

Abstract Details

2023, Doctor of Philosophy, Ohio State University, Biomedical Sciences.
Acute myeloid leukemia (AML) is an aggressive and relatively common form of leukemia with poor survival outcomes despite modern advances in targeted and cellular therapies. For patients able to tolerate induction therapy and go on to receive a bone marrow transplant, at least half will eventually relapse, underscoring the critical need to better understand the immune-related mechanisms likely underpinning this process. Natural killer (NK) cells are a type of innate lymphoid cell (ILC) with potent anti-leukemic functions, however AML patients often have hypomature and hypofunctional circulating NK cells which correlate with poor outcomes. Other ILCs lack the cytotoxic function of NK cells but possess more immunomodulatory functions. All these ILC populations share a common developmental precursor, and thus the overall goal and hypothesis of this research was that the hypomature NK cell phenotype observed in AML patients may be indicative of developmental rerouting away from the NK cell lineage and towards other ILCs. The results of these studies characterize the developmental reprogramming of ILC precursors (ILCPs) by AML-mediated activation of the aryl hydrocarbon receptor (AHR) pathway to skew development away from NK cells and towards both the ILC1 and ILC3 lineage. We utilize both patient samples and in vivo models to identify expansion of ILC populations and then use a combination of ex vivo, in vivo, and in vitro modeling approaches to demonstrate expansion of these populations is due at least in part to skewed development of ILCPs via an AHR-dependent process. First, by phenotyping the ILC compartment of a mouse model of AML that recapitulates human disease, we identified expansion of ILC1s in the bone marrow and liver. Functional testing of this population revealed that they produced levels of IFNγ lower than what would be produced by an NK cell. When we cultured human ILCPs in differentiating conditions with AML cell lines or AHR agonists, we observed increased production of TBET+EOMES- ILC1s and a concomitant decrease in TBET+EOMES+ NK cells. This observation was recapitulated in vivo. When the progeny of ILCPs derived in the presence of AML cells were stimulated with PMA/Ionomycin, we observed an overall hypofunctional phenotype as evidenced by decreased cytokine production. In contrast, when these same cells were exposed to a high-affinity AHR agonist such as FICZ, we observed increased production of IFNγ and TNFα, thus suggesting additional pathways to AHR may be responsible for both the developmental and functional defects observed in AML. To further support this, when we treated ILCPs with TGFβ, we noted a profound suppression of IFNγ and TNFα production in the resulting cells, suggesting crosstalk between these pathways may be at least partially responsible for our observations. We also found evidence of skewing of the CD56bright compartment in the bone marrow of newly diagnosed AML patients towards an ILC1-like phenotype (TBET+EOMESlo) and away from an NK cell. Second, we determined the contribution of a downstream mediator of AHR activity, the microRNA, miR-29b. Using both global and NKp46-conditional miR-29b knockout (KO) mouse models, we profiled the developmental status of splenic NK cells and observed a significant loss of terminally mature (CD27-CD11b+) NK cells and increased frequencies of intermediate (CD27+CD11b+) and immature (CD27+CD11b-) NK cell populations in the global miR-29b KO model. In the NKp46-conditional KO model, we observed increased frequencies of mature NK cells, suggesting miR-29b has both cell-intrinsic and -extrinsic roles in NK cell development. Within each of these populations, we also observed differential alterations in Tbet and Eomes expression depending on developmental stage, suggesting stage-specific effects of miR-29b regulation. We also compared expression of miR-29b between NK cells and the closely related ILC1 population. We observed that ILC1s express approximately double the level of miR-29b detected in NK cells. In global KO mice, we observed decreases in both NK cell and ILC1 numbers. Upon stimulation with PMA/Ionomycin, we observed a decreased ability of NK cells to produce IFNγ but no difference in ILC1’s ability to produce IFNγ, suggesting miR-29b regulates these populations differently. Because both populations play important roles in the response to cytomegalovirus (CMV), we also assessed how miR-29b levels were affected by murine CMV infection and observed temporal differences in expression between NK cells and ILC1s, again demonstrating that miR-29b regulates NK cells and ILC1s differently. We also found that both miR-29b KO models were potentially more susceptible to MCMV infection as evidenced by higher viral titers compared to littermate WT mice and failure of the ILC1s from KO mice to upregulate the checkpoint receptors Pd1 and Lag3 following infection. Because CMV reactivation is clinically relevant in the post-stem cell transplant setting and has implications for relapse risk, we assessed miR-29b expression within the CD56bright and CD56dim ILC populations of post-SCT patients and stratified them based on whether they experienced CMV reactivation. We observed significantly higher expression of miR-29b in the CD56bright, but not CD56dim, cells of CMV-reactivating patients. We are currently working on a study to determine how loss of miR-29b via gene editing at different stages of human ILC development impacts ILC development and function. Finally, we assessed the ability of AML cells to skew development of ILCPs towards the ILC3 lineage. We began by performing scRNA-seq on lineage-depleted bone marrow from an AML mouse model and identified clustering of an AML-specific population that resembled an ILC3 cluster, which we further validated by flow cytometry. This also informed our discovery of an increased frequency of Lin-EOMES-RORγT+ cells in the marrow of newly diagnosed AML patients compared to marrow from nonleukemic hip replacement patients. Mechanistic studies determined this expansion of ILC3s is also dependent on AML-mediated activation of the AHR pathway in ILCPs. Functional studies using both mouse and human cells revealed that ILC3s exposed to AML cells upregulate the pro-myeloid cytokine GM-CSF as well as the molecules TNFα and IL-2. We also found that ILC3s increase the resistance of AML cells to NK cell mediated cytotoxicity and increase leukemic colony formation in CFU assays. Overall, these results further dissect the ability of AML to skew ILC development away from cytotoxic NK cells and towards other ILC lineages. While some of these lineages appear to primarily be hypofunctional (i.e. ILC1s), allowing opportunities for immune escape, others (ILC3s) have direct pro-myeloid functions that make AML cells more resistant to NK cell-mediated killing while simultaneously increasing AML’s ability to form leukemic colonies, thus potentially promoting chemotherapy resistance and disease relapse. These studies establish a foundation and paradigm to elucidate why AML patients experience NK cell dysfunction and may not respond to some forms of immunotherapy targeting these populations. It is further hoped that the discoveries related to this work can be applied in translational research efforts that seek to optimize the role of the immune system in clinical therapies.
Bethany Mundy-Bosse (Advisor)
Aharon Freud (Committee Co-Chair)
Michael Caligiuri (Committee Member)
Susheela Tridandapani (Committee Member)
Eugene Oltz (Committee Member)
214 p.
Bioinformatics; Biology; Biomedical Research; Immunology; Molecular Biology
Innate Lymphoid Cells, ILC, NK cells, AML, AHR, Development, miR-29b, ILC3

Recommended Citations

Citations

  • Lordo, M. R. (2023). Innate Lymphoid Cell Dysregulation in Acute Myeloid Leukemia [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1680775578447773

    APA Style (7th edition)

  • Lordo, Matthew. Innate Lymphoid Cell Dysregulation in Acute Myeloid Leukemia. 2023. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1680775578447773.

    MLA Style (8th edition)

  • Lordo, Matthew. "Innate Lymphoid Cell Dysregulation in Acute Myeloid Leukemia." Doctoral dissertation, Ohio State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=osu1680775578447773

    Chicago Manual of Style (17th edition)

osu1680775578447773
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This open access ETD is published by The Ohio State University and OhioLINK.