Limited Effects of Class II Transactivator-Based Immunotherapy in Murine and Human Glioblastoma

Abstract

Background: The major histocompatibility complex type II is downregulated in glioblastoma (GB) due to the silencing of the major transcriptional regulator class II transactivator (CIITA). We investigated the pro-immunogenic potential of CIITA overexpression in mouse and human GB.

Methods: The intracerebral growth of wildtype GL261-WT cells was assessed following contralateral injection of GL261-CIITA cells or flank injections with GL261-WT or GL261-CIITA cells. Splenocytes obtained from mice implanted intracerebrally with GL261-WT, GL261-CIITA cells or phosphate buffered saline (PBS) were transferred to other mice and subsequently implanted intracerebrally with GL261-WT. Human GB cells and (syngeneic) GB-infiltrating immune cells were isolated from surgical samples and co-cultured with GB cells expressing CIITA or not, followed by RT-qPCR assessment of the expression of key immune regulators.

Results: Intracerebral vaccination of GL261-CIITA significantly reduced the subsequent growth of GL261-WT cells implanted contralaterally. Vaccination with GL261-WT or -CIITA subcutaneously, however, equivalently retarded the intracerebral growth of GL261 cells. Adoptive cell transfer experiments showed a similar antitumor potential of lymphocytes harvested from mice implanted intracerebrally with GL261-WT or -CIITA. Human GB-infiltrating myeloid cells and lymphocytes were not activated when cultured with CIITA-expressing GB cells. Tumor-infiltrating NK cells remained mostly inactivated when in co-culture with GB cells, regardless of CIITA.

Conclusion: these results question the therapeutic potential of CIITA-mediated immunotherapy in glioblastoma.

Keywords: class II transactivator; glioblastoma; natural killer cells; tumor microenvironment; tumor-associated macrophages; tumor-infiltrating lymphocytes.

Figure 1

(a) Set up of experiment. (b) Coronal brain slices of mice after the experiment. (c) Direct intracranial injection of GL261-wildtype or -CIITA cells in C57Bl/6 mice. The bargraph represents the mean ± SD. (d) Survival curve of mice after direct i.c. injection with GL261-WT or -CIITA. Survival was significantly longer after injection with GL261-CIITA. (e) Set up of intracerebral contralateral vaccination experiment. (f) Coronal brain slices of mice after the vaccination experiment. The dashed line depicts the tumor engraftment after (*) PBS or (**) GL261-CIITA vaccination in the contralateral hemisphere. (g) The bargraph represents the mean ± SD after GL261-CIITA vaccination PBS vaccination. Abbreviations: I.c. = intracranial. WT = wildtype. ACT = Adoptive Cell Transfer. PBS = Phosphate Buffered Saline. * p < 0.05. *** p < 0.0001.

Figure 2

(a) Technical set up of peripheral vaccination experiment. (b) Representative images of coronal brain slices. (c) Bargraph, representing mean, SD and separate datapoints (orange). ANOVA p = 0.045. (d) Set-up of peripheral vaccination experiment. (e) Survival curve, representing survival of mice injected intracerebrally with GL261-WT and subsequently injected with splenocytes that were primed with either GL261-WT, -CIITA or PBS (sham-vaccinated). Abbreviations: S.c. = subcutanetous (injection). I.c. = intracranial (injection). WT = wildtype. PBS = Phosphate Buffered Saline. m = micrometer. * p < 0.05.

Figure 3

(a) Phenotypic changes after co-culturing tumor-associated macrophages with U87-CIITA or -wildtype cells versus U87 monocultures. Scale bar in white: 100 micrometer, the same scale for all images. (b–d) Gene expression of the indicated genes was determined by qPCR. Each bar represents the mean ± SD relative gene expression. * p < 0.05, ** p = 0.01. (b) Gene expression in tumor-associated macrotphages after co-culture with U87 glioblastoma cells and in monoculture. (c) Gene expression in tumor-infiltrating NK-cells after co-culture with U87 (c1) and GM2 (c2). (d) Gene expression of genes expressed in tumor-infiltrating T lymphocytes (d1) and of CD155 expressed in tumor cells (d2). Abbreviations: TAM = tumor-associated myeloid cells. NK = Natural Killer cell. TIL = Tumor-infiltrating T lymphocytes. WT = wildtype. CC = co-cultured.

Figure 4

(a) Survival curve of patients with glioblastoma with low (<25%) or high (>24%) CIITA or (b) HLA-DRa expression. (c) Two representative images of TMA with glioblastoma cells with high or no CIITA-expression. (d) Correlation between HLA-DRa expression and CIITA expression in glioblastoma. Using Gepia.cancer.pku online software, we calculated Kaplan meier curves for CIITA (e) and HLA-DRa (f) expression in patients with glioblastoma of TGCA cancer atlas. (g) Correlation between HLA-DRa and CIITA expression in glioblastoma.