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Supplementary Figures 1-16 and Legends from T-cell Responses in the Microenvironment of Primary Renal Cell Carcinoma—Implications for Adoptive Cell Therapy

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posted on 2023-04-03, 23:10 authored by Rikke Andersen, Marie Christine Wulff Westergaard, Julie Westerlin Kjeldsen, Anja Müller, Natasja Wulff Pedersen, Sine Reker Hadrup, Özcan Met, Barbara Seliger, Bjarne Kromann-Andersen, Thomas Hasselager, Marco Donia, Inge Marie Svane

S1.(A) The figure shows an image of cancer cells stained with May-Grünwald-Giemsa (MGG) after cytospin centrifugation of freshly detached cancer cells from a representative patient (RCC12), magnification x 200. The red arrow shows a mitotic figure. (B) The figure shows images of clotted cancer cells from RCC12 after formalin-fixation and paraffin-embedding followed by immunohistochemistry (IHC) staining for various RCC markers (as seen from the figure). S2. The FACS plots demonstrate CD107a mobilization (left panels) and cytokine production (TNF and IFNgamma right panels) from PBMCs alone (control) and after co-culture with autologous tumor cell line (TCL) in a representative RCC patient (RCC28). Plots are gated on CD3+ cells (left panels) and CD3+ CD8+ cells (right panels). S3. Changes in TIL reactivity during the rapid expansion protocol (REP). S4. The FACS plots demonstrate cytokine production (TNF and IFNgamma) after co-culture assay with autologous tumor cell lines from (A) unselected MM REP-TILs (gated on CD8+ T cells) and (B) CD8+CD107a+ sorted MM REP-TILs in a representative patient. S5. Massive expansion with alternative cytokine combinations S6. Cytolytic activity of RCC-TIL S7. Polyfunctional characterization of CD8+CD107a+ tumor-reactive T cells. S8. Differentiation/dysfunctional profile of CD8+ tumor-reactive Y-TIL S9. Differentiation/dysfunctional profile of CD8+ REP-TIL Up-regulation of HLA class I expression after exposure to IFNgamma S10. Up-regulation of HLA class I expression after exposure to IFNgamma.� S11. Constitutive expression of the major antigen processing machinery (APM) components in RCC and MM by qPCR. S12. IFNgamma-induced upregulation of the major antigen processing machinery (APM) components in RCC and MM by qPCR. S13. IFNgamma-induced fold induction of the major antigen processing machinery (APM) components in RCC and MM by qPCR. S14. Effects of IFNgamma on autologous tumor recognition by CD8+ T cells. S15. Up-regulation of HLA class II after exposure to IFNgamma in RCC and MM S16. Effects of IFNgamma on autologous tumor recognition by CD4+ T cells.

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Danish Cancer Society

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ARTICLE ABSTRACT

In vitro expansion of large numbers of highly potent tumor-reactive T cells appears a prerequisite for effective adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TIL) as shown in metastatic melanoma (MM). We therefore sought to determine whether renal cell carcinomas (RCC) are infiltrated with tumor-reactive T cells that could be efficiently employed for adoptive transfer immunotherapy. TILs and autologous tumor cell lines (TCL) were successfully generated from 22 (92%) and 17 (77%) of 24 consecutive primary RCC specimens and compared with those generated from metastatic melanoma. Immune recognition of autologous TCLs or fresh tumor digests was observed in CD8+ TILs from 82% of patients (18/22). Cytotoxicity assays confirmed the tumoricidal capacity of RCC-TILs. The overall expansion capacity of RCC-TILs was similar to MM-TILs. However, the magnitude, polyfunctionality, and ability to expand in classical expansion protocols of CD8+ T-cell responses was lower compared with MM-TILs. The RCC-TILs that did react to the tumor were functional, and antigen presentation and processing of RCC tumors was similar to MM-TILs. Direct recognition of tumors with cytokine-induced overexpression of human leukocyte antigen class II was observed from CD4+ T cells (6/12; 50%). Thus, TILs from primary RCC specimens could be isolated, expanded, and could recognize tumors. However, immune responses of expanded CD8+ RCC-TILs were typically weaker than MM-TILs and displayed a mono-/oligofunctional pattern. The ability to select, enrich, and expand tumor-reactive polyfunctional T cells may be critical in developing effective ACT with TILs for RCC. In summary, TILs isolated from primary RCC specimens could recognize tumors. However, their immune responses were weaker than MM-TILs and displayed a mono-/oligofunctional pattern. The ability to select and expand polyfunctional T cells may improve cell therapy for RCC. Cancer Immunol Res; 6(2); 222–35. ©2018 AACR.

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    Cancer Immunology Research

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    Biological Agents & TherapiesGene therapyGenitourinary CancersImmunologyImmune responses to cancerImmunotherapyCellular immunotherapy

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