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. 2017 Oct;7(10):1154-1167.
doi: 10.1158/2159-8290.CD-16-0850. Epub 2017 Jun 2.

Overcoming the Immunosuppressive Tumor Microenvironment of Hodgkin Lymphoma Using Chimeric Antigen Receptor T Cells

Marco Ruella  1   2   3 , Michael Klichinsky  1   3   4 , Saad S Kenderian  1   5 , Olga Shestova  1   2   3 , Amy Ziober  2   3 , Daniel O Kraft  1 , Michael Feldman  2   3 , Mariusz A Wasik  2   3 , Carl H June  1   2   3   5 , Saar Gill  6   3   5
Affiliations

Overcoming the Immunosuppressive Tumor Microenvironment of Hodgkin Lymphoma Using Chimeric Antigen Receptor T Cells

Marco Ruella et al. Cancer Discov. 2017 Oct.

Abstract

Patients with otherwise treatment-resistant Hodgkin lymphoma could benefit from chimeric antigen receptor T-cell (CART) therapy. However, Hodgkin lymphoma lacks CD19 and contains a highly immunosuppressive tumor microenvironment (TME). We hypothesized that in Hodgkin lymphoma, CART should target both malignant cells and the TME. We demonstrated CD123 on both Hodgkin lymphoma cells and TME, including tumor-associated macrophages (TAM). In vitro, Hodgkin lymphoma cells convert macrophages toward immunosuppressive TAMs that inhibit T-cell proliferation. In contrast, anti-CD123 CART recognized and killed TAMs, thus overcoming immunosuppression. Finally, we showed in immunodeficient mouse models that CART123 eradicated Hodgkin lymphoma and established long-term immune memory. A novel platform that targets malignant cells and the microenvironment may be needed to successfully treat malignancies with an immunosuppressive milieu.Significance: Anti-CD123 chimeric antigen receptor T cells target both the malignant cells and TAMs in Hodgkin lymphoma, thereby eliminating an important immunosuppressive component of the tumor microenvironment. Cancer Discov; 7(10); 1154-67. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

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Figures

Figure 1
Figure 1. The IL-3 receptor α, CD123, is expressed in Hodgkin Lymphoma cells and in tumor-associated macrophages
A. Gene expression analysis of microdissected HRS (GSE-39133) reveals high expression of CD123 as compared to controls (microdissected germinal center B cells) B. Primary samples of Hodgkin lymphoma were stained for CD30 and CD123 by immunohistochemistry (IHC). Expression of CD123 was found of the HL Reed-Sternberg cells but also in the tumor microenvironment, as opposed to CD30 that was only positive on HRS. C. CD123 was expressed on tumor-associated macrophages by IHC (left), dual IHC (right, top; brown CD68 and red CD123) and immune fluorescence (right, bottom; red CD123 and green CD68. D. RNA expression of CD123 and CD68 (left) and CD11b (right) in 130 whole HL biopsies (GSE17920): a strong correlation between macrophage markers (CD68, CD11b) and CD123 expression was observed. E. High CD123 protein expression on 4 standard HL cell lines (HDLM-2, KM-H2, SUP-HD1 and L428) by flow cytometry.
Figure 2
Figure 2. HL cells polarize normal macrophages to a M2-like phenotype
A. Human normal donor macrophages differentiated from peripheral blood monocytes were cultured with a control acute lymphoblastic leukemia cell line (NALM-6), IL-4 (M2 positive control), or HL cell line HDLM-2 conditioned supernatant. HDLM-2 cells can polarize macrophages toward an M2 phenotype (CD163+CD206+) after a 24-hour culture. B. HDLM2 supernatants trigger phosphorylation of STAT6 on macrophages, similarly to IL-4. C. M0, M2-polarized (IL-4 or IL-13), and HL polarized macrophages express CD123 by flow cytometry. D. M2-polarized (IL-4) and, to a greater extent, HL-polarized macrophages can reduce anti-CD19 chimeric antigen receptor T cell proliferation in response to irradiated CD19+ NALM-6 target cells, as shown by CFSE (Carboxyfluorescein succinimidyl ester - a fluorescent cell stained dye that is diluted with cell proliferation) dilution assay. E. HL-polarized macrophages reduce CART19 proliferation in response to irradiated NALM-6 target cells, as shown by absolute T cell numbers at day 5 F. Heatmap demonstrating that HL cell lines HDLM2, KMH2, SUPHD1, and L428 secreted significantly more myeloid cell recruiting chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10), M2 polarizing cytokines (IL13, IL10, IL6) and macrophage supporting factor GM-CSF than control non-HL cell lines K562 and NALM6 or media alone. G. RNA expression analysis of 29 microdissected HRS cells (dataset GSE-39133) confirmed the expression of M2 polarizing cytokines (IL13, IL10, IL6), macrophage supporting factors (GM-CSF, M-CSF), and the immunosuppressive checkpoint ligand PDL1 in primary samples.
Figure 3
Figure 3. Anti-CD123 chimeric antigen receptor T cells exert potent effector function against Hodgkin lymphoma in vitro
A. HL cells (HDLM-2) were co-cultured with CART123 for 4–6 hours. CAR+ but not CAR- T cells expressed high levels of the degranulation marker CD107A and produced intra-cellular cytokines like IFNγ, IL-2 and TNFα. B. CART123, but not UTD, exert potent cytotoxicity (luciferase-based killing assay) against HL cells in a dose-dependent manner. C. HL cells (HDLM-2) were co-cultured at long term with CART123 or control UTD. At day 20, CART123 but not UTD killed HL cells and proliferated. D. CART123 or UTD were co-cultured with media, PMA/Ionomycin (positive control) or two HL cell lines (HDLM-2 and KM-H2) for 5 days; CART123 but not UTD controls showed significant proliferation as absolute number and CFSE dilution (E). F. HL cells stimulated CART123 but not UTD cells to release multiple cytokines including GM-CSF, IFNγ, MIP1β and TNFα. E:T= effector : target ratio.
Figure 4
Figure 4. Anti-CD123 chimeric antigen receptor T cells exert potent effector function against Hodgkin lymphoma in vivo
A Experiment schema: 2 ×106 Luciferase-positive HDLM-2 cells were injected i.v. in NSG mice and tumor engraftment was monitored by bioluminescence imaging. At day 42 mice were randomized to receive no treatment, 2 ×106 control untransduced T cells (UTD) or 2 ×106 CART123. B. Mice receiving CART123, but not controls, experienced complete response with long term remission of disease (>250 days), representative experiment. C. CART123-treated mice have a significantly longer overall survival as compared to controls (3 experiments combined). D. CAR123 T cells engraft, expand and disappear from the peripheral blood after clearing the tumor. E. T cells in the PB of CART123-treated mice were both CD8 and CD4 with high expression of the CAR.
Figure 5
Figure 5. CART123 establish long-term immunological memory in mice with HL
A. Experiment schema: mice previously treated with CART123 and experiencing a long-term remission were rechallenged at day 250 with HL cells (HDLM-2). As a control a tumor-naïve group of mice were also injected with the same HL cells. B. HL cells only engrafted and grew in tumor-naïve mice while long-term surviving mice (post-CART123) were able to control disease growth. C. A re-expansion of CART123 cells was observed in mice previously treated with CART123. D. An improved overall survival was observed in mice with previous exposure to CART123.
Figure 6
Figure 6. CART123 are resistant to inhibition by M2-macrophages
A. In a 5-day CFSE proliferation assay, CART123 are completely resistant to HL-polarized macrophage mediated inhibition. B. CART123 cells rapidly (day 1) recognize M2-macrophages, forming clusters around macrophages and clearing them by day 5 as shown by phase contrast microscopy (20X) and flow cytometry, respectively. C. CART19 but not CART123 cytokine secretion was significantly reduced in the presence of M2 (IL-4 polarized) macrophages in vitro.
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