Our laboratory studies the basic biology and clinical utilization of NK cells. The following are the major areas of our focus:

Transcriptional Regulation of Human NK cell development.

GATA2 is an essential transcription factor for NK cell development that contains two zinc finger (ZF1 and ZF2) domains, two transactivation domains, and one negative regulatory domain. The specific loss of the CD56bright NK cell population is a salient feature in GATA2T354M patients, with or without reduced CD56dim NK cells. Using a combination of scRNA-seq, ATAC-seq, and CUT&Tag of NK cells from GATA2T354M patients and healthy controls and in vitro expression of GATA2 and GATA2T354M constructs and western blot analyses, we uncovered a novel GATA2-TGF-b1-TAL1 transcriptional network. We are currently testing a central hypothesis that an interplay between the GATA2, TGF-b1, and TAL1 is obligatory for the development and functions of human NK cells. GATA2, along with TAL1, forms a heptameric transcriptional complex. Using scRNA-seq data from 10 patients with GATA2, we demonstrated that TAL1 target genes, including GIMAPs, are upregulated, while TGF-b1 and its downstream targets are downregulated. Ectopic expression of the clinical GATA2T354M variant upregulated TAL1 and its target genes. Decreasing TAL1 levels in GATA2T354M-expressing cells attenuated the expression of GIMAPs. GATA2 occupied the TGF-b1 promoter and elevated TGF-b1 expression, while GATA2T354M had reduced binding activity. A reduction in TGF-b1 decreased chromatin accessibility at AP-1 binding regions, leading to defective NK cell activation. In addition, TGF-b1 promoted TAL1 degradation. Thus, GATA2-TAL1-TGF-b1 constitutes a vital axis that controls human NK cell development. This work is supported by an NIH-NIAID R01 grant (December 27th, 2024-November 30th, 2029). Role: Contact PI.

Transcriptional regulation of memory NK cells.

The developmental origins and molecular mechanisms involved in the maintenance and persistence of long-lived Memory NKG2C+ NK cells remain largely unknown. Managing human cytomegalovirus (HCMV) infections relies on a subset of NKG2C+ Natural Killer (NK) cells. The NKG2C/CD94 receptor complex recognizes and responds to HCMV-infected cells expressing HLA-E loaded with viral gpUL40 peptide. Upon activation, NKG2C+ NK cells mount a response akin to adaptive immunity and establish a reservoir of long-lived memory cells in HCMV+ individuals. The origins by which memory NKG2C+ NK cells are maintained remain unknown. Here, we used human spleens to investigate the transcriptional signatures and the developmental origins of memory NKG2C+ NK cells. We discover that the human spleen houses a distinct subset of memory NKG2CHi NK cells that express pro-survival genes and high levels of IL-7R and intracellular CD3e. Upon re-exposure to the gpUL40 viral peptide, memory NKG2CHi NK cells expand and increase their effector functions. Developmental trajectory analyses reveal memory NKG2CHi NK cells develop from dynamic NKG2C+NKG2A+ NK cells. We show that this subset becomes NKG2C+ or NKG2A+ single positive, depending on the external stimuli. Together, these findings suggest that a distinct subset of long-lived memory NKG2CHi NK cells exist within the human spleen, and their maintenance is dependent on key pro-survival genes and transcriptional regulators.

Genomic and Immunological Predisposition to CD20/CD19 Bi-specific CAR T cell Therapy.

Bi-specific anti-CD20/CD19 Chimeric Antigen Receptor (CAR, 4-1BB-CD3zLV20.19)-transduced T cell therapy provides a revolutionary approach to treat relapsed and refractory B cell malignancies. Yet a significant number of patients relapse or have only partial remission. In this project, leveraging an ongoing Phase I/II clinical trial, we performed scRNA and bulk RNA sequencing on pre-PBMCs, CAR+ T cell infusion product, recovered CAR+ T cells during peak and late in vivo response from six patients with complete remission (CR) and six patients with progressive disease (PD) or partial remission (PR). CAR+ T cell therapies achieve durable remissions in 40-50% of patients with DLBCL, the most common type of non-Hodgkin’s lymphoma (NHL). We find that some patients may be genetically predisposed to poor CAR T cell responses. First, the IEGs, a set of cellular activation markers, were significantly less expressed in the non-responders. The expression of IEGs is also regulated by the phosphorylated cAMP response element-binding protein (CREB), which requires histone lysine acetyltransferase CBP. Thus, a significant reduction of IEGs strongly supports that a pre-existing mutation can lead to a failure of T cell activation. Secondly, a pioneer transcription factor, GATA3, and its target genes were not optimally expressed in the non-responder T cells. It is important to note that complexes, including CBP/p300 and RNA polymerase II facilitate the binding of GATA3 to its GATA3 response element (CGRE). Thirdly, T cells from the non-responders harbored higher levels of genes and regulon activity that represented increased cell cycle and DNA repair pathways. Our findings provide mechanistic explanations and set the stage to identify them prognostically with a panel of potential biomarkers.

• Nicholas Family Foundation, "NK cell-based Immunotherapies," Role: PI (10/01/2014 - 09/30/2019)
• High Risk Hematological Malignancies Program - MACC-Fund Initiatives, "Novel CAR-based Immunotherapeutic Approaches to Pediatric Cancer," Role: PI (07/01/2014 - 06/30/2019)
• NCI, 1R01 CA179363, "Molecular signature of inflammation," Role: PI (03/10/2014 - 02/28/2019)
• NIH-NIAID, R01 AI102893, "Molecular mechanisms of signaling co-ordination in innate lymphocytes," Role: PI (08/01/2013 - 07/31/2017)
• Alex's Lemonade Stand Foundation, "Control of inflammation in genetically-modified lymphocytes," Role: PI (09/01/2013-08/30/2014)
• Clinical and Translational Science Institute, "Cellular and adoptive immunotherapy using hematopoietic cell transplantation and NK cell infusion for the treatment of high risk pediatric and adult solid tumors: a Phase I/II Study," Role: Co-I (07/01/2012 - 06/30/2014)
• American Cancer Society Pilot Research Grant, "NK cell immunotherapy in relapsed and refractory solid tumors," Role: Co-I (07/01/2012 - 06/30/2014)
• MACC-Fund Novel Initiatives, "Cellular therapy using haploidentical donor NK cells," Role: PI (07/01/2012 - 06/30/2014)

Subramaniam Malarkannan, PhD
PI, Molecular Immunology and Immunotherapy
Professor of Medicine - Hematology and Oncology & Microbiology and Molecular Genetics
Senior Investigator, Blood Research Institute

Research Interests: We are interested in the basic biology and clinical utilization of NK cells.  In particular, we study signaling cascades in muring and human NK cells.  This helps us to correlate the role of individual signaling proteins with the development, terminal maturation and effector functions on NK cells.  Using our findings, we are also exploring the translational relevance of NK and T cells.  

Email: SMalarkannan@Versiti.org

Dandan Wang, PhD
Postodoctoral Fellow

Email: dwang2@Versiti.org

Ao Mei
Graduate Student

Email: amei@versiti.org

Fangfei Zhang
Graduate Student

Email: fzhang@mcw.edu

1. IQGAP1-based Ras-MEK1/2-ERK1/2-based signalosome is essential for light chain recombination and early B cell development. Lella RK and Malarkannan S. Cell Mol Life Sci. 2024 Nov 25;81(1):462. doi: 10.1007/s00018-024-05509-4. PMID: 39585462
2. Expansion and characterization of immune suppressive CD56^BrightPerforin- regulatory natural killer cells in chronic graft-versus-host disease Corresponding Author: Madeline Patricia Lauener; Erin Tanaka; Ao Mei; Sayeh Abdossamadi; Elena Ostroumov; Ramon Klein Geltink; Subramaniam Malarkannan, Kirk Schultz. Cytotherapy, (2024) DOI:https://doi.org/10.1016/j.jcyt.2024.07.013.
3. Novel PI(3)K-p85α/p110δ-ITK-LAT-PLC-γ2 and Fyn-ADAP-Carma1-TAK1 Pathways Define Reverse Signaling via FasL. Kumar P, Rajasekaran K, Malarkannan S. (Original article) Crit Rev Immunol. 2024;44(1):55-77. doi: 10.1615/CritRevImmunol.2023049638.PMID: 37947072.
4. Transcriptomic Diversity of Innate Lymphoid Cells in Human Lymph nodes. Elaheh Hashemi, Colleen McCarthy, Sridhar Rao, and Subramaniam Malarkannan. Commun Biol 7, 769 (2024). https://doi.org/10.1038/s42003-024-06450-9 
5. CD36 Restricts Lipid-Associated Macrophages Accumulation in White Adipose Tissues During Atherogenesis. Jue Zhang, Jackie Chang, Vaya Chen, Mirza Beg, Lance Vick, Dandan Wang, Ankan Gupta, Yaxin Wang, Ziyu Zhang, Wen Dai, Mindy Kim, Shan Song, Duane Pereira, Ze Zheng, Komal Sodhi, Joseph I Shapiro, Roy L Silverstein, S. Malarkannan and Yiliang Chen. Frontiers in Cardiovascular Medicine, Volume 11 - 2024 | https://doi.org/10.3389/fcvm.2024.1436865
6. Transcriptomic-Based Microenvironment Classification Reveals Precision Medicine Strategies for PDAC (2024). George B, Kudryashova O, Thalji S, Malarkannan S, Kurzrock R, Kravets A, Chernyavskaya E, Gusakova M, Kravchenko D, Tychinin D, Savin E, Alekseeva L, Butusova A, Bagaev A, Shin N, Brown J, Sethi I, Wang D, Taylor B, McFall T, Kamgar M, Hall W, Erickson B, Christians K, Evans D, and Tsai S. Gastroenterology. 2024 May;166(5):859-871.e3. doi: 10.1053/j.gastro.2024.01.028. Epub 2024 Jan 25.PMID: 38280684.
7. Successes and Challenges in Taming the Beast: Cytotoxic Immune Effectors in Amyotrophic Lateral Sclerosis. Kaur K, Chen PC, Ko MW, Mei A, Huerta-Yepez S, Maharaj D, Malarkannan S, Jewett A. Crit Rev Immunol. 2023;43(1):1-11. doi: 10.1615/CritRevImmunol.2023047235.PMID: 37522557
8. Sequential therapy with supercharged NK cells with either chemotherapy drug cisplatin or anti-PD-1 antibody decreases the tumor size and significantly enhances the NK function in Hu-BLT mice. Kaur K, Chen PC, Ko MW, Mei A, Senjor E, Malarkannan S, Kos J, Jewett A. Front Immunol. 2023 May 1;14:1132807. doi: 10.3389/fimmu.2023.1132807.
9. The potential role of cytotoxic immune effectors in induction, progression, and pathogenesis of Amyotrophic Lateral Sclerosis (ALS). Kaur K, Chen PC, Ko MW, Mei A, Chovatiya N, Huerta-Yepez S, Ni W, Mackay S, Zhou J, Maharaj D, Malarkannan S, Jewett A. Cells. 2022 Oct 31;11(21):3431. doi: 10.3390/cells11213431.
10. Innatus Immunis: Evolving paradigm of Adaptive NK cells (2022). Khalil M and  Malarkannan S. Journal of Experimental Medicine 219(11):e20221254. doi: 10.1084/jem.20221254. Epub 2022.PMID: 36066493.
11. Transcriptomic perspectives of memory-like NK cells during aging (2022). Wang D and Malarkannan S. Genome Medicine May 25;14(1):57. doi: 10.1186/s13073-022-01059-1.PMID: 35610660.
12. Methods for isolating and defining single-cell of tissue-resident NK cells. (2022) Hashemi E, Khalil M, Mei A, Wang D, Malarkannan S. Methods Mol Biol. (2022;2463:103-116. doi: 10.1007/978-1-0716-2160-8_8). PMID: 35344170.
13. Methods to infect and define the single-cell transcriptomes of MCMV-specific murine NK cells. (2022) Khalil M, Wang D, Mei A, Hashemi E, Terhune S, Malarkannan S. Methods Mol Biol. (2022;2463:195-204. doi: 10.1007/978-1-0716-2160-8_14). PMID: 35344176.
14. Methods to analyze the developmental stages of Murine and Human Primary NK cells using Monocle and SCENIC analyses. (2022) Wang D, Khalil M, Mei A, Hashemi E, Malarkannan S. Methods Mol Biol. (2022;2463:81-102. doi: 10.1007/978-1-0716-2160-8_7). PMID: 35344169.
15. Impaired NK cell Development and functions in patients with GATA2 mutation (2021). Wang D, Hashemi E, Thakar MS, and Malarkannan S. Critical Reviews in Immunology (DOI: 10.1615/CritRevImmunol.2021037643).
16. Developmental and functional defects of NK cells in Fanconi Anemia patients (2021). Hashemi E, Wang D, Thakar MS, and Malarkannan S. Critical Reviews in Immunology (DOI: 10.1615/CritRevImmunol.2021037644).
17. NK cell-mediated immunotherapy: The exquisite role of PGC-1a in metabolic reprogramming (2021) Gerbec Z and Malarkannan S. NK Immunotherapy: Breaking Tolerance to Cancer Resistance. Ed: Ben Bonavida and Anahid Jewett (ISBN 978-0-12-824375-6).
18. Isolation of Innate Lymphoid Cells from Murine Intestinal Lamina Propria. (2022) Mei A, Hashemi E, Khalil M, Wang D, Malarkannan S. Methods Mol Biol. (2022;2463:3-9. doi: 10.1007/978-1-0716-2160-8_1). PMID: 35344163.
19. Role of microRNAs in NK cell development and functions (2021). Nanbakhsh A and Malarkannan S. Cells 10(8):2020 (doi: 10.3390/cells10082020).
20. MyD88 is an essential regulator of Ly49H-mediated signaling and proliferation in NK cells (2021). Dixon KJ, Siebert JR, Abel AM, Johnson KE, Riese MJ, Terhune SS, Tarakanova VL, Thakar MS, and Malarkannan S. Molecular Immunology 137:94-104 (doi: 10.1016/j.molimm.2021.07.001. Epub 2021 Jul 6.PMID: 34242922).
21. Implications of a ‘third signal’ in NK cells (2021). Khalil M, Wang D, Hashemi E, Terhune SS, Malarkannan S. Cells 10(8):1955 (doi: 10.3390/cells10081955).
22. Transcriptional regulation of NK cell development by mTOR complexes (2020). Yang C and Malarkannan S. Frontiers in Cell and Dev Biology (https://doi.org/10.3389/fcell.2020.566090).
23. Conditional Deletion of PGC-1α Results in Energetic and Functional Defects in NK Cells. (2020) Gerbec ZJ, Hashemi E, Nanbakhsh A, Holzhauer S, Yang C, Mei A, Tsaih SW, Lemke A, Flister MJ, Riese MJ, Thakar MS, and Malarkannan S. iScience (https://doi.org/10.1016/j.isci.2020.101454). 
24. NKG7 makes a better killer (2020). Malarkannan S. News and Views. Nature Immunology. doi: 10.1038/s41590-020-0767-5.
25. Reverse signaling mediated by FasL (2020). Malarkannan S. Molecular Immunology (127:31-37. doi: 10.1016/j.molimm.2020.08.010).
26. Single-cell transcriptome reveals the novel role of T-bet in suppressing the immature NK gene signature (2020). Yang C, Siebert JR, Burns R, Zheng Y, Mei A, Bonacci B, Wang D, Urrutia RA, Riese MJ, Rao S, Carlson K-S, Thakar MS, and Malarkannan S. eLife 2020;9:e51339 DOI: 10.7554/eLife.51339.
27. Transcriptional regulation of NK cell development and function (2020). Wang D and Malarkannan S. Cancers 12(6), 1591; https://doi.org/10.3390/cancers12061591.
28. Tissue-resident NK cells: Development, functions, and clinical relevance (2020). Hashemi E and Malarkannan S. Cancers 12(6), 1553; https://doi.org/10.3390/cancers12061553
29. Entinostat Activates Human NK cells Through a Novel IFIT1-STING-IRF1-STAT4 Signaling Axis (2020). Idso J, Lao S, Schloemer N, Knipstein J, Burns R, Thakar MS, and Malarkannan S. Oncotarget: 11(20):1799-1815. doi: 10.18632/oncotarget.27546. 
30. Containing Cytokine-Release Syndrome to harness the potentials of CAR therapy. Thakar MS, Kearl T, and Malarkannan S (2020). Frontiers in Oncology 9: https://doi.org/10.3389/fonc.2019.01529.
31. In Vivo Assessment of NK Cell-Mediated Cytotoxicity by Adoptively Transferred Splenocyte Rejection. (2020) Schloemer NJ, Abel AM, Thakar MS, Malarkannan S. Methods Mol Biol. 2097:115-123. doi: 10.1007/978-1-0716-0203-4-8. (PMID:31776923). 
32. Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells. (2020) Nanbakhsh A, Malarkannan S. Methods Mol Biol. 2097:107-113. doi: 10.1007/978-1-0716-0203-4-7. (PMID: 31776922). 
33. Beyond the Cell Surface: Targeting Intracellular Negative Regulators to Enhance T cell Anti-tumor Activity (2019). Sitaram P, Uyemora B, Malarkannan S, and Riese MJ. Int J Mol Sci.  Nov 20;20 (23). pii: E5821. doi: 10.3390/ijms20235821. (PMID: 31756921).
34. Oxidized LDL re-purpose macrophage mitochondria functions for immune-activation through CD36-mediated fatty acid trafficking. (2019) Chen Y, Yang M, Huang W, Chen W, Zhao Y, Schulte M, Gerbec Z, Zhang J, Smith B, 1, Malarkannan S, Xie Z, Silverstein RL Circulation Research. Oct 18. doi: 10.1161/CIRCRESAHA.119.315833. (PMID: 31625810).
35. Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression. Reimer M Jr, Pulakanti K, Shi L, Abel A, Liang M, Malarkannan S, and Rao S (2019). BMC Developmental Biology (PMID: 31286885). 
36. The development and heterogeneity of human NK cells defined by single-cell transcriptome (2019). Yang C Siebert J, Burns R, Gerbec ZJ, Bonacci B, Rymaszewski A, Rau M, Riese MJ, Rao S, Carlson K-S, Routes JM, Verbsky JW, Thakar MS, and Malarkannan S. Nature Communications (DOI: 10.1038/s41467-019-11947-7).
37. Long-Term Single Center Donor Lymphocyte Infusion (DLI) Experience Suggests a Role for Durable Response in Children with High-Risk Lymphoid Malignancies (2019). Liberio N, Robinson H, Nugent M, Simpson P, Margolis DA, Malarkannan S, Keever-Taylor C, Thakar MS Pediatric Blood, and Cancer (PMID: 31368194, Doi: 10.1002/pbc.27950)
38. MicroRNA Mirc11 optimizes the inflammatory responses by silencing ubiquitin modifiers and altering K63 and K48 ubiquitylation of TRAF6 (2019). Nanbakhsh A, Shirng-Wern, Flister M, Thakar MS, and Malarkannan S. (2019) Cancer Immunology Research 7(10):1647-1662. PMID: 31515257. doi: 10.1158/2326-6066.CIR-18-0934.
39. Immune checkpoint VISTA controls TLR-mediated anti-tumor immunity via regulating TRAF6 protein turnover and activation in myeloid cells. Xu W, Zheng Y, Zhou J, Yuan Y, Rajasekaran K, Miller H, Olson M, Dong J, Ernstoff MS, Wang D, Malarkannan S, Wang L (2019) Cancer Immunology Research PMID: 31340983 DOI:10.1158/2326-6066.CIR-18-0489
40. IL-27 regulates NK cell effector functions via MafF-Nrf2 pathway during influenza infection. Kumar P, Rajasekaran K, Thakar M, and Malarkannan S. (2019). Scientific Reports 9(1):4984. doi: 10.1038/s41598-019-41478-6. PMCID: PMC6428861
41. Platelet gene therapy provokes targeted peripheral tolerance by clonal deletion and induction of antigen-specific regulatory T cells. Luo X, Chen J, Schroeder JA, Allen KP, Baumgartner CK, Malarkannan S, Hu J, Williams CB, and Shi Q. (2018). Frontiers in Immunology Sep 6;9:1950. doi: 10.3389/fimmu.2018.01950. PMCID: PMC6136275
42. mTORC1 and mTORC2 differentially regulate NK cell development. Yang C, Thakar M, and Malarkannan S (2018) eLife. doi: 10.7554/eLife.35619. PMCID: PMC5976438
43. NK cells: Development, Maturation, and Clinical Utilization. Abel AM, Yang C, Thakar MS, Malarkannan S (2018). Frontiers in Immunology. Aug 13;9:1869. https://doi.org/10.3389/fimmu.2018.01869. PMCID: PMC6099181