1. Department of Hematology, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China 2. Department of Hematology, Henan Provincial People’s Hospital; Zhengzhou University People’s Hospital, Zhengzhou 450003, China 3. Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, China 4. Department of Hematology, Tongji Hospital of Tongji University, Shanghai 200065, China
Tumor-derived exosomes (TEXs) enriched in immune suppressive molecules predominantly drive T-cell dysfunction and impair antitumor immunity. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising treatment for refractory and relapsed hematological malignancies, but whether lymphoma TEXs have the same impact on CAR T-cell remains unclear. Here, we demonstrated that B-cell lymphoma-derived exosomes induce the initial activation of CD19–CAR T-cells upon stimulation with exosomal CD19. However, lymphoma TEXs might subsequently induce CAR T-cell apoptosis and impair the tumor cytotoxicity of the cells because of the upregulated expression of the inhibitory receptors PD-1, TIM3, and LAG3 upon prolonged exposure. Similar results were observed in the CAR T-cells exposed to plasma exosomes from patients with lymphoma. More importantly, single-cell RNA sequencing revealed that CAR T-cells typically showed differentiated phenotypes and regulatory T-cell (Treg) phenotype conversion. By blocking transforming growth factor β (TGF-β)–Smad3 signaling with TGF-β inhibitor LY2109761, the negative effects of TEXs on Treg conversion, terminal differentiation, and immune checkpoint expression were rescued. Collectively, although TEXs lead to the initial activation of CAR T-cells, the effect of TEXs suppressed CAR T-cells, which can be rescued by LY2109761. A treatment regimen combining CAR T-cell therapy and TGF-β inhibitors might be a novel therapeutic strategy for refractory and relapsed B-cell lymphoma.
EA Chong, M Ruella, SJ; Lymphoma Program Investigators at the University of Pennsylvania Schuster. Five-year outcomes for refractory b-cell lymphomas with car t-cell therapy. N Engl J Med 2021; 384(7): 673–674 https://doi.org/10.1056/NEJMc2030164
2
JN Kochenderfer, ME Dudley, SH Kassim, RP Somerville, RO Carpenter, M Stetler-Stevenson, JC Yang, GQ Phan, MS Hughes, RM Sherry, M Raffeld, S Feldman, L Lu, YF Li, LT Ngo, A Goy, T Feldman, DE Spaner, ML Wang, CC Chen, SM Kranick, A Nath, DA Nathan, KE Morton, MA Toomey, SA Rosenberg. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 2015; 33(6): 540–549 https://doi.org/10.1200/JCO.2014.56.2025
3
EJ Orlando, X Han, C Tribouley, PA Wood, RJ Leary, M Riester, JE Levine, M Qayed, SA Grupp, M Boyer, B De Moerloose, ER Nemecek, H Bittencourt, H Hiramatsu, J Buechner, SM Davies, MR Verneris, K Nguyen, JL Brogdon, H Bitter, M Morrissey, P Pierog, S Pantano, JA Engelman, W Winckler. Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia. Nat Med 2018; 24(10): 1504–1506 https://doi.org/10.1038/s41591-018-0146-z
4
E Sotillo, DM Barrett, KL Black, A Bagashev, D Oldridge, G Wu, R Sussman, C Lanauze, M Ruella, MR Gazzara, NM Martinez, CT Harrington, EY Chung, J Perazzelli, TJ Hofmann, SL Maude, P Raman, A Barrera, S Gill, SF Lacey, JJ Melenhorst, D Allman, E Jacoby, T Fry, C Mackall, Y Barash, KW Lynch, JM Maris, SA Grupp, A Thomas-Tikhonenko. Convergence of acquired mutations and alternative splicing of cd19 enables resistance to cart-19 immunotherapy. Cancer Discov 2015; 5(12): 1282–1295 https://doi.org/10.1158/2159-8290.CD-15-1020
5
C Tong, Y Zhang, Y Liu, X Ji, W Zhang, Y Guo, X Han, D Ti, H Dai, C Wang, Q Yang, W Liu, Y Wang, Z Wu, W Han. Optimized tandem CD19/CD20 CAR-engineered T cells in refractory/relapsed B-cell lymphoma. Blood 2020; 136(14): 1632–1644 https://doi.org/10.1182/blood.2020005278
6
W Deng, P Chen, W Lei, Y Xu, N Xu, JJ Pu, A Liang, W Qian. CD70-targeting CAR-T cells have potential activity against CD19-negative B-cell Lymphoma. Cancer Commun (Lond) 2021; 41(9): 925–929 https://doi.org/10.1002/cac2.12201
7
JY Spiegel, S Patel, L Muffly, NM Hossain, J Oak, JH Baird, MJ Frank, P Shiraz, B Sahaf, J Craig, M Iglesias, S Younes, Y Natkunam, MG Ozawa, E Yang, J Tamaresis, H Chinnasamy, Z Ehlinger, W Reynolds, R Lynn, MC Rotiroti, N Gkitsas, S Arai, L Johnston, R Lowsky, RG Majzner, E Meyer, RS Negrin, AR Rezvani, S Sidana, J Shizuru, WK Weng, C Mullins, A Jacob, I Kirsch, M Bazzano, J Zhou, S Mackay, SJ Bornheimer, L Schultz, S Ramakrishna, KL Davis, KA Kong, NN Shah, H Qin, T Fry, S Feldman, CL Mackall, DB Miklos. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nat Med 2021; 27(8): 1419–1431 https://doi.org/10.1038/s41591-021-01436-0
8
S Cordoba, S Onuoha, S Thomas, DS Pignataro, R Hough, S Ghorashian, A Vora, D Bonney, P Veys, K Rao, G Lucchini, R Chiesa, J Chu, L Clark, MM Fung, K Smith, C Peticone, M Al-Hajj, V Baldan, M Ferrari, S Srivastava, R Jha, F Arce Vargas, K Duffy, W Day, P Virgo, L Wheeler, J Hancock, F Farzaneh, S Domning, Y Zhang, NZ Khokhar, VGR Peddareddigari, R Wynn, M Pule, PJ Amrolia. CAR T cells with dual targeting of CD19 and CD22 in pediatric and young adult patients with relapsed or refractory B cell acute lymphoblastic leukemia: a phase 1 trial. Nat Med 2021; 27(10): 1797–1805 https://doi.org/10.1038/s41591-021-01497-1
9
H Liu, W Lei, C Zhang, C Yang, J Wei, Q Guo, X Guo, Z Chen, Y Lu, KH Young, Z Lu, W Qian. CD19-specific CAR T cells that express a PD-1/CD28 chimeric switch-receptor are effective in patients with PD-L1-positive B-cell lymphoma. Clin Cancer Res 2021; 27(2): 473–484 https://doi.org/10.1158/1078-0432.CCR-20-1457
10
X Yan, D Chen, Y Wang, Y Guo, C Tong, J Wei, Y Zhang, Z Wu, W Han. Identification of NOXA as a pivotal regulator of resistance to CAR T-cell therapy in B-cell malignancies. Signal Transduct Target Ther 2022; 7(1): 98 https://doi.org/10.1038/s41392-022-00915-1
11
L Milane, A Singh, G Mattheolabakis, M Suresh, MM Amiji. Exosome mediated communication within the tumor microenvironment. J Control Release 2015; 219: 278–294 https://doi.org/10.1016/j.jconrel.2015.06.029
12
L Mashouri, H Yousefi, AR Aref, AM Ahadi, F Molaei, SK Alahari. Exosomes: composition, biogenesis, and mechanisms in cancer metastasis and drug resistance. Mol Cancer 2019; 18(1): 75 https://doi.org/10.1186/s12943-019-0991-5
E Yang, X Wang, Z Gong, M Yu, H Wu, D Zhang. Exosome-mediated metabolic reprogramming: the emerging role in tumor microenvironment remodeling and its influence on cancer progression. Signal Transduct Target Ther 2020; 5(1): 242 https://doi.org/10.1038/s41392-020-00359-5
15
AK Ghosh, CR Secreto, TR Knox, W Ding, D Mukhopadhyay, NE Kay. Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression. Blood 2010; 115(9): 1755–1764 https://doi.org/10.1182/blood-2009-09-242719
16
F Ma, J Vayalil, G Lee, Y Wang, G Peng. Emerging role of tumor-derived extracellular vesicles in T cell suppression and dysfunction in the tumor microenvironment. J Immunother Cancer 2021; 9(10): e003217 https://doi.org/10.1136/jitc-2021-003217
17
MJ Szczepanski, M Szajnik, A Welsh, TL Whiteside, M Boyiadzis. Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-beta1. Haematologica 2011; 96(9): 1302–1309 https://doi.org/10.3324/haematol.2010.039743
18
EU Wieckowski, C Visus, M Szajnik, MJ Szczepanski, WJ Storkus, TL Whiteside. Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes. J Immunol 2009; 183(6): 3720–3730 https://doi.org/10.4049/jimmunol.0900970
19
Q Hao, Y Wu, Y Wu, P Wang, JV Vadgama. Tumor-derived exosomes in tumor-induced immune suppression. Int J Mol Sci 2022; 23(3): 1461 https://doi.org/10.3390/ijms23031461
20
Z Chen, L You, L Wang, X Huang, H Liu, JY Wei, L Zhu, W Qian. Dual effect of DLBCL-derived EXOs in lymphoma to improve DC vaccine efficacy in vitro while favor tumorgenesis in vivo. J Exp Clin Cancer Res 2018; 37(1): 190 https://doi.org/10.1186/s13046-018-0863-7
21
Y Hao, P Chen, X Zhang, Y Shao, Y Xu, W Qian. The effects of tumor-derived exosomes on T-cell function and efficacy of cancer immunotherapy. ImmunoMedicine 2021; 1(2): e1029 https://doi.org/10.1002/imed.1029
22
M Poggio, T Hu, CC Pai, B Chu, CD Belair, A Chang, E Montabana, UE Lang, Q Fu, L Fong, R Blelloch. Suppression of exosomal PD-L1 induces systemic anti-tumor immunity and memory. Cell 2019; 177(2): 414–427.e13 https://doi.org/10.1016/j.cell.2019.02.016
23
T Stüber, R Monjezi, L Wallstabe, J Kühnemundt, SL Nietzer, G Dandekar, A Wöckel, H Einsele, J Wischhusen, M Hudecek. Inhibition of TGF-β-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer. J Immunother Cancer 2020; 8(1): e000676 https://doi.org/10.1136/jitc-2020-000676
24
T Aung, B Chapuy, D Vogel, D Wenzel, M Oppermann, M Lahmann, T Weinhage, K Menck, T Hupfeld, R Koch, L Trümper, GG Wulf. Exosomal evasion of humoral immunotherapy in aggressive B-cell lymphoma modulated by ATP-binding cassette transporter A3. Proc Natl Acad Sci USA 2011; 108(37): 15336–15341 https://doi.org/10.1073/pnas.1102855108
25
M Aitamer, H Akil, C Vignoles, M Branchaud, J Abraham, N Gachard, J Feuillard, MO Jauberteau, H Shirvani, D Troutaud, H Bentayeb. CD20 expression, TrkB activation and functional activity of diffuse large B cell lymphoma-derived small extracellular vesicles. Br J Cancer 2021; 125(12): 1687–1698 https://doi.org/10.1038/s41416-021-01611-7
26
MJ Cox, F Lucien, R Sakemura, JC Boysen, Y Kim, P Horvei, C Manriquez Roman, MJ Hansen, EE Tapper, EL Siegler, C Forsman, SB Crotts, KJ Schick, M Hefazi, MW Ruff, I Can, M Adada, E Bezerra, LA Kankeu Fonkoua, WK Nevala, E Braggio, W Ding, SA Parikh, NE Kay, SS Kenderian. Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia. Mol Ther 2021; 29(4): 1529–1540 https://doi.org/10.1016/j.ymthe.2020.12.033
27
I Xhangolli, B Dura, G Lee, D Kim, Y Xiao, R Fan. Single-cell analysis of CAR-T cell activation reveals a mixed TH1/TH2 response independent of differentiation. Genom Proteom Bioinf 2019; 17(2): 129–139 https://doi.org/10.1016/j.gpb.2019.03.002
28
K Nakamura, A Kitani, W Strober. Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor beta. J Exp Med 2001; 194(5): 629–644 https://doi.org/10.1084/jem.194.5.629
29
S Huber, C Schramm, HA Lehr, A Mann, S Schmitt, C Becker, M Protschka, PR Galle, MF Neurath, M Blessing. TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells. J Immunol 2004; 173(11): 6526–6531 https://doi.org/10.4049/jimmunol.173.11.6526
30
N Tang, C Cheng, X Zhang, M Qiao, N Li, W Mu, XF Wei, W Han, H Wang. TGF-β inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight 2020; 5(4): e133977 https://doi.org/10.1172/jci.insight.133977
Y Tone, K Furuuchi, Y Kojima, ML Tykocinski, MI Greene, M Tone. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer. Nat Immunol 2008; 9(2): 194–202 https://doi.org/10.1038/ni1549
33
BV Park, ZT Freeman, A Ghasemzadeh, MA Chattergoon, A Rutebemberwa, J Steigner, ME Winter, TV Huynh, SM Sebald, SJ Lee, F Pan, DM Pardoll, AL Cox. TGFβ1-mediated smad3 enhances pd-1 expression on antigen-specific t cells in cancer. Cancer Discov 2016; 6(12): 1366–1381 https://doi.org/10.1158/2159-8290.CD-15-1347
34
MP Oksvold, A Kullmann, L Forfang, B Kierulf, M Li, A Brech, AV Vlassov, EB Smeland, A Neurauter, KW Pedersen. Expression of B-cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells. Clin Ther 2014; 36(6): 847–862.e1 https://doi.org/10.1016/j.clinthera.2014.05.010
35
S Ali, K Toews, S Schwiebert, A Klaus, A Winkler, L Grunewald, L Oevermann, HE Deubzer, A Tüns, MC Jensen, AG Henssen, A Eggert, JH Schulte, E Schwich, V Rebmann, A Schramm, A Künkele. Tumor-derived extracellular vesicles impair cd171-specific cd4+ car t cell efficacy. Front Immunol 2020; 11: 531 https://doi.org/10.3389/fimmu.2020.00531
36
SC Lin, K Haga, XL Zeng, MK Estes. Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning. Nat Protoc 2022; 17(4): 1004–1027 https://doi.org/10.1038/s41596-021-00669-0
37
DG Tantalo, AJ Oliver, B von Scheidt, AJ Harrison, SN Mueller, MH Kershaw, CY Slaney. Understanding T cell phenotype for the design of effective chimeric antigen receptor T cell therapies. J Immunother Cancer 2021; 9(5): e002555 https://doi.org/10.1136/jitc-2021-002555
38
JA Fraietta, SF Lacey, EJ Orlando, I Pruteanu-Malinici, M Gohil, S Lundh, AC Boesteanu, Y Wang, RS O’Connor, WT Hwang, E Pequignot, DE Ambrose, C Zhang, N Wilcox, F Bedoya, C Dorfmeier, F Chen, L Tian, H Parakandi, M Gupta, RM Young, FB Johnson, I Kulikovskaya, L Liu, J Xu, SH Kassim, MM Davis, BL Levine, NV Frey, DL Siegel, AC Huang, EJ Wherry, H Bitter, JL Brogdon, DL Porter, CH June, JJ Melenhorst. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 2018; 24(5): 563–571 https://doi.org/10.1038/s41591-018-0010-1
39
GM Chen, C Chen, RK Das, P Gao, CH Chen, S Bandyopadhyay, YY Ding, Y Uzun, W Yu, Q Zhu, RM Myers, SA Grupp, DM Barrett, K Tan. Integrative bulk and single-cell profiling of premanufacture t-cell populations reveals factors mediating long-term persistence of CAR T-cell therapy. Cancer Discov 2021; 11(9): 2186–2199 https://doi.org/10.1158/2159-8290.CD-20-1677
40
C Yang, SH Kim, NR Bianco, PD Robbins. Tumor-derived exosomes confer antigen-specific immunosuppression in a murine delayed-type hypersensitivity model. PLoS One 2011; 6(8): e22517 https://doi.org/10.1371/journal.pone.0022517
41
CC Kloss, J Lee, A Zhang, F Chen, JJ Melenhorst, SF Lacey, MV Maus, JA Fraietta, Y Zhao, CH June. Dominant-negative tgf-β receptor enhances psma-targeted human car t cell proliferation and augments prostate cancer eradication. Mol Ther 2018; 26(7): 1855–1866 https://doi.org/10.1016/j.ymthe.2018.05.003
42
X Chen, S Yang, S Li, Y Qu, HY Wang, J Liu, ZS Dunn, GE Cinay, MA MacMullan, F Hu, X Zhang, P Wang. Secretion of bispecific protein of anti-PD-1 fused with TGF-β trap enhances antitumor efficacy of CAR-T cell therapy. Mol Ther Oncolytics 2021; 21: 144–157 https://doi.org/10.1016/j.omto.2021.03.014
43
J Proff, CU Brey, A Ensser, W Holter, M Lehner. Turning the tables on cytomegalovirus: targeting viral Fc receptors by CARs containing mutated CH2-CH3 IgG spacer domains. J Transl Med 2018; 16(1): 26 https://doi.org/10.1186/s12967-018-1394-x
44
M Boyiadzis, TL Whiteside. Plasma-derived exosomes in acute myeloid leukemia for detection of minimal residual disease: are we ready?. Expert Rev Mol Diagn 2016; 16(6): 623–629 https://doi.org/10.1080/14737159.2016.1174578
