Ileana S Mauldin 1,2,*, Jasmin Jo 3, Nolan A Wages 4, Lalanthica V Yogendran 5, Adela Mahmutovic 2, Samuel J Young 1, Maria Beatriz Lopes 6, Craig L Slingluff Jr 1, Loren D Erickson 7,8, Camilo E Fadul 5
Posted: Dec 1, 2021
Abstract
Background: tumor-infiltrating lymphocytes are prognostic in many human cancers. However, the prognostic value of lymphocytes infiltrating glioblastoma (GBM), and roles in tumor control or progression are unclear. We hypothesized that B and T cell density, and markers of their activity, proliferation, differentiation, or function, would have favorable prognostic significance for patients with GBM. Methods: initial resection specimens from 77 patients with IDH1/2 wild type GBM who received standard-of-care treatment were evaluated with multiplex immunofluorescence histology (mIFH), for the distribution, density, differentiation, and proliferation of T cells and B cells, as well as for the presence of tertiary lymphoid structures (TLS), and IFNγ expression. Immune infiltrates were evaluated for associations with overall survival (OS) by univariate and multivariate Cox proportional hazards modeling. Results: in univariate analyses, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.36, p = 0.001) and CD20+ cells (HR 0.51, p = 0.008), as well as CD8+Tbet+ cells (HR 0.46, p = 0.004), and RORγt+ cells (HR 0.56, p = 0.04). Conversely, IFNγ intensity was associated with diminished OS (HR 0.59, p = 0.036). In multivariable analyses, adjusting for clinical variables, including age, resection extent, Karnofsky Performance Status (KPS), and MGMT methylation status, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.15, p < 0.001), and higher ratios of CD8+ cells to CD4+ cells (HR 0.31, p = 0.005). Diminished OS was associated with increases in patient age (HR 1.21, p = 0.005) and higher mean intensities of IFNγ (HR 2.13, p = 0.027). Conclusions: intratumoral densities of proliferating CD8 T cells and higher CD8/CD4 ratios are independent predictors of OS in patients with GBM. Paradoxically, higher mean intensities of IFNγ in the tumors were associated with shorter OS. These findings suggest that survival may be enhanced by increasing proliferation of tumor-reactive CD8+ T cells and that approaches may be needed to promote CD8+ T cell dominance in GBM, and to interfere with the immunoregulatory effects of IFNγ in the tumor microenvironment.
NeoBiotechnologies’ products were used in this study:
In total, four micrometre thick sections were cut from formalin fixed paraffin embedded (FFPE) tissue specimens, and human lymph node and bowel were used as positive control tissues. Multispectral staining was performed according to the manufacturer’s protocol using the OPAL Multiplex Manual IHC kit, and antigen retrieval buffers (AR) 6 and 9 (Akoya Biosciences, Marlborough, MA, USA). Staining sequence, antibodies, and antigen retrieval buffers were performed on three serial sections as follows:
TIL Panel 1: AR9, CD4 (1:100, clone SP35) (Cell Marque, Rocklin, CA, USA) Opal520; AR9, CD8 (1:500, clone C8/144B) (Agilent, Santa Clara, CA, USA) Opal540; AR6, CD20 (1:4k, clone L26) (Agilent, Santa Clara, CA, USA) Opal570; DIVA, CD34 (1:200, clone QBEnd 10) (Agilent, Santa Clara, CA, USA) Opal620; DIVA, T-bet (1:50, clone 4B10) (Santa Cruz Biotechnology, Dallas, TX, USA) Opal650; and spectral DAPI (Akoya Biosciences, Marlborough, MA, USA).
TIL Panel 2: AR9, CD4 (1:100, clone SP35) (Cell Marque, Rocklin, CA, USA) Opal520; AR9, CD8 (1:500, clone C8/144B) (Agilent, Santa Clara, CA, USA) Opal540; AR6, CD20 (1:4k, clone L26) (Agilent, Santa Clara, CA, USA) Opal570; DIVA, IFNγ (1:1k, clone IFNG/466) (NeoBiotechnologies, Union City, CA, USA) Opal620; and Ki67 (1:20, clone SP6) (Abcam, Waltham, MA, USA) and spectral DAPI (Akoya Biosciences, Marlborough, MA, USA).
TLS Panel 3: DIVA, MAdCAM (1:200, clone355G8) (Invitrogen, Carlsbad, CA, USA) Opal620; AR9, CD8 (1:500, clone C8/144B) (Agilent, Santa Clara, CA, USA) Opal540; AR6, CD20 (1:1k, clone L26) (Agilent, Santa Clara, CA, USA) Opal570; AR6, PNAd (1:100, clone MECA-79) (BD Pharmingen, Franklin Lakes, NJ, USA) Opal650; DIVA, RORγt (1:1k, clone 6F3.1) (EMD Millipore, Burlington, MA, USA) Opal570; and spectral DAPI (Akoya Biosciences, Marlborough, MA, USA). Images of slides stained with these 3 mIFH panels are shown in Supplemental Figures S1–S3, as well as staining controls.
Keywords: immunology, tumor infiltrating lymphocytes, multiplex immunofluorescence histology, glioblastoma, human
Publication History:
Cells. 2021 Dec 1;10(12):3378. doi: 10.3390/cells10123378
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