Engineering Immune Cells forTherapy

Author: Prof. Dr. Kathrin Schumann

Engineering Immune Cells for Therapy

Genetic manipulation of human primary cells has been largely impossible until recently, but advances in genome engineering methods offer new opportunities. Genome engineering of eukaryotic cells with CRISPR/Cas9 has been first described in 2012 and has since then revolutionized this research field (Jinek et al. 2013). It is widely accepted that T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV and autoimmune diseases, but genetic manipulation of human T cells has been challenging.

So far, our knowledge about this unique cell type is largely based on data generated in transgenic mice or with RNAi approaches in human T cells which exhibit significant off-target effects. We developed a robust CRISPR/Cas9 technology based on Cas9 ribonucleoproteins (Cas9 RNPs), recombinant Cas9 protein complexed with chemically synthesized gRNAs, that enables both knock-out and knock-in genome editing in primary human T cells to overcome this hurdle (Schumann and Lin et al. PNAS 2015).

Figure 1: Generation of knock-out and knock-in primary human T cells using Cas9 ribonukleoproteins (Cas9 RNPs).

We expanded the CRISPR/Cas9 technique to a variety of genes associate with HIV (Hultquist & Schumann et al. Cell Reports 2016), cancer (Rupp & Schumann et al. Scientific Reports 2017) and autoimmune diseases.

CRISPR screens in primary human T cells

We developed a medium-throughput platform to systematically ablate HIV host factors in primary human T cells in a rapid, arrayed fashion using Cas9 RNPs in a 96 well format, which has the potential to accelerate target validation for pharmaceutical and cellular therapies. We systematically screened for known and predicted HIV integrase interaction partners in T cells and could identify novel dependency and restriction factors (Hultquist & Schumann et al. Cell Reports 2016).

We apply this arrayed platform now to analyse the stability and the transcriptional patterns of different human T cell subset. We have a special interest in applying these technologies to human regulatory T cells.

Figure 2: Arrayed Cas9 RNP platform for screening HIV host factors in human T cells. A: Workflow for arrayed screens. B: Results of RNP screen targeting 45 known and predicted HIV integrase interaction partners.

Dissecting transcriptional regulation in human regulatory T cells

Regulatory T cells (Tregs) play a fundamental role in maintaining immune tolerance by suppressing autoreactive effector T cells. Tregs with pro-inflammatory features have been described in different context and with a variety of phenotypes. Murine “ex-Tregs” downregulate their master transcription factor Foxp3 and in humans and mice with autoimmune conditions Tregs with changed cytokine profile could be detected. However, the processes that lead to these heterogeneous phenotypes are incompletely understood. Methods to stabilize Tregs for the treatment of autoimmune diseases or actively destabilize Tregs to ablate tolerogenic effects on the tumor microenvironment have great therapeutic potential. To identify these potential targets for therapy we perform arrayed and pooled CRISPR screens to systematically ablate transcription factors in human Tregs.

We are interested in dissecting the gene networks regulated by individual transcription factors in human T cells by performing arrayed and pooled CRISRP screens. Besides that, we want to develop novel ex vivo models to functionally validate human CRISPR-engineered T cells.

With these insights we hope to engineer advanced cell therapies against autoimmunity or cancer in the future.


Publications

Lowe MM, Boothby I, Clancy S, Ahn RS, Liao W, Nguyen DN, Schumann K, Marson A, Mahuron KM, Kingsbury GA, Liu Z, Munoz Sandoval P, Rodriguez RS, Pauli ML, Taravati K, Arron ST, Neuhaus IM, Harris HW, Kim EA, Shin US, Krummel MF, Daud A, Scharschmidt TC, Rosenblum MD. Regulatory T cells use arginase 2 to enhance their metabolic fitness in tissues. JCI Insight., 4(24):129756 (2019).

Schober K, Müller TR, Gökmen F, Grassmann S, Effenberger M, Poltorak M, Stemberger C, Schumann K, Roth TL, Marson A, Busch DH. Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function. Nat Biomed Eng., 3(12):974-984 (2019).

Hultquist JF, Hiatt J, Schumann K, McGregor MJ, Roth TL, Haas P, Doudna JA, Marson A, Krogan NJ. CRISPR-Cas9 genome engineering of primary CD4(+) T cells for the interrogation of HIV-host factor interactions. Nat Protoc, 14:1-27 (2019)

Roth TL, Puig-Saus  C, Ruby Y, Shifrut E, Carnevale J, Hiatt J, Saco J, Li H, Li J, Tobin V, Nguyen D, Ferris AM, Chen J, Schickel JN, Pellerin L, Carmody D, Alkorta-Aranburu G, Del Gaudio D, Matsumoto H, Morell M, Mao Y, Quadros R, Gurumurthy C, Smith B, Haugwitz M, Hughes SH, Weissman J, Schumann K, May AP, Ashworth A, Kupfer G, Greeley S, Bacchetta R, Meffre E, Roncarolo MG, Romberg N, Herold KC, Ribas A, Leonetti MD, Marson A. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature, 559:405-409 (2018)

Simeonov DR, Gowen BG, Boontanrart M, Roth TL, Gagnon JD, Mumbach MR, Satpathy AT, Lee Y, Bray NL, Chan AY, Lituiev DS, Nguyen ML, Gate RE, Subramaniam M, Li Z, Woo JM, Mitros T, Ray GJ, Curie GL, Naddaf N, Chu JS, Ma H, Boyer E, Van Gool F, Huang H, Liu R, Tobin VR, Schumann K, Daly MJ, Farh KK, Ansel KM, Ye CJ, Greenleaf WJ, Anderson MS, Bluestone JA, Chang HY, Corn JE, Marson A. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature (2017), 7;549(7670):111-115.

Rupp LJ*, Schumann K*, Roybal KT, Gate RE, Ye JY, Lim WA, Marson A. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. (2017), 7(1):737.

Park RJ, Wang T, Koundakjian D, Hultquist JF, Lamothe-Molina P, Monel B, Schumann K, Yu H, Krupzcak KM, Garcia-Beltran W, Piechocka-Trocha A, Krogan NJ, Marson A, Sabatini DM, Lander ES, Hacohen N, Walker BD. A genome-wide CRISPRscreen identifies a restricted set of HIV host dependency factors. Nat Genet. (2017), 49(2):193-203.

Hultquist JF*, Schumann K*, Woo JM, Manganaro L, Doudna JA, Simon V, Krogan NJ, Marson A. A Cas9 ribonucleoprotein platform for functional genetic studies of HIV-Host interactions in primary human T Cells. Cell Reports (2016) 17(5):1438-1452

Schumann K*, Lin S*, Boyer E, Simeonov DR, Subramaniam M, Gate RE, Haliburton GDE, Ye CJ, Bluestone JA, Doudna JA, Marson A. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. PNAS (2015), 112(33):10437-42.

Wendland M, Willenzon S, Kocks J, Davalos-Misslitz AC, Hammerschmidt SI, Schumann K, Kremmer E, Sixt M, Hoffmeyer A, Pabst O, Förster R. Lymph node T cell homeostasis relies on steady state homing of dendritic cells. Immunity (2011) 35(6): 945-57.

Soriano SF*, Hons M*, Schumann K, Kumar V, Dennier TJ, Lyck R, Sixt M, Stein JV. In vivo analysis of uropod function during physiological T cell trafficking. J Immunol. (2011) 187(5): 2356-64.

Schumann K*, Lämmermann T*, Bruckner M, Legler DF, Polleux J, Spatz J, Schuler G, Förster R, Lutz MB, Sorokin L, Sixt M. Immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells. Immunity (2010) 32(5): 703-13.

Renkawitz J, Schumann K, Weber M, Lämmermann T, Pflicke H, Piel M, Polleux J, Spatz JP, Sixt M. Adaptive force transmission in amoeboid cell migration. Nat Cell Biol (2009) 11(12): 1438-43.

 

* equal contribution