Advanced Age Increases Immunosuppression in the Brain and Decreases Immunotherapeutic Efficacy in Subjects with Glioblastoma

Abstract

Purpose: Wild-type isocitrate dehydrogenase-expressing glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median age at diagnosis of ≥65 years. It accounts for approximately 90% of all GBMs and has a median overall survival (OS) of <15 months. Although immune checkpoint blockade (ICB) therapy has achieved remarkable survival benefits in a variety of aggressive malignancies, similar success has yet to be achieved for GBM among phase III clinical trials to date. Our study aimed to understand the relationship between subject age and immunotherapeutic efficacy as it relates to survival from glioma.

Experimental design: (i) Clinical data: GBM patient datasets from The Cancer Genome Atlas, Northwestern Medicine Enterprise Data Warehouse, and clinical studies evaluating ICB were stratified by age and compared for OS. (ii) Animal models: young, middle-aged, and older adult wild-type and indoleamine 2,3 dioxygenase (IDO)-knockout syngeneic mice were intracranially engrafted with CT-2A or GL261 glioma cell lines and treated with or without CTLA-4/PD-L1 mAbs, or radiation, anti-PD-1 mAb, and/or a pharmacologic IDO enzyme inhibitor.

Results: Advanced age was associated with decreased GBM patient survival regardless of treatment with ICB. The advanced age-associated increase of brain IDO expression was linked to the suppression of immunotherapeutic efficacy and was not reversed by IDO enzyme inhibitor treatment.

Conclusions: Immunosuppression increases in the brain during advanced age and inhibits antiglioma immunity in older adults. Going forward, it will be important to fully understand the factors and mechanisms in the elderly brain that contribute to the decreased survival of older patients with GBM during treatment with ICB.

Figure 1.. Advanced aging is associated with decreased overall survival (OS) among glioblastoma (GBM) patients, but not in human subjects with lung-, breast-, kidney-, or colon- metastases in the brain.

Human subject OS analyzed from the medical records (A,B) for the wild-type isocitrate dehydrogenase (wtIDH) cohort of GBM patients in the cancer genome atlas (TCGA) and (C,D) GBM patients in the Northwestern Enterprise Data Warehouse (NW EDW) and analyzed as described in Supp. Fig. 1. Subjects were stratified by (A,C) <20–34 (blue)-, 35–44 (red)-, 45–54 (green)-, 55–64 (purple)-, 65–74 (orange)-, and 75–84+ (black)-years of age, or by (B,D) <65 (blue)- or ≥65 (red)-years of age. The NW EDW database was also evaluated for the OS of human subjects with brain metastases originating from the (E) lung, (F) breast, (G) kidney, and (H) colon and stratified by <65 (blue)- or ≥65 (red)-years of age. Overall survival estimated using the method of Kaplan-Meier (A, C, E-H). Age groups compared using Cox proportional hazards models; pairwise groups comparisons adjusted using Scheffe’s method (A, C). Median OS with 95%CI (B, D). Age groups compared using logrank test (E-H). *p<0.05; **p<0.01 ***p<0.001; ****p<0.0001

Figure 2.. Advanced aging limits sporadic long-term survivor (LTS) generation in syngeneic mice with intracranial CT-2A glioma.

Timeline and overall survival analysis of 6-, 23-, 47-, and 75-week old C57BL/6 wild-type (WT) mice intracranially-engrafted with 500 CT-2A cells and administered (A) non-specific IgG Ab or (B) CD4⁺ and CD8⁺ T cell-depleting Abs. Individual age groups treated with IgG Ab or T cell-depleting Abs were further stratified by (C) 6-, (D) 23-, (E) 47-, and (F) 75-week old subjects, respectively. Overall survival estimated using the method of Kaplan-Meier; age groups compared using Cox proportional hazards models (A-F).

Figure 3.. Advanced aging is associated with decreased overall survival (OS) of recurrent glioblastoma (rGBM) patients and syngeneic mice treated with immune checkpoint blockade therapy.

(A) OS and (B) patient demographics of rGBM patients treated with adjuvant nivolumab (anti-PD-1 mAb), pembrolizumab (anti-PD-1 mAb), libtayo (anti-PD-1 mAb), durvalumab (anti-PD-L1 mAb), tremelimumab (anti-CTLA-4 mAb), or a combination of durvalumab and tremelimumab, and ultimately stratified by <65 (blue)- or ≥65 (red)-years of age. (C) Schematic illustration (created with biorender.com) of lifespan milestones and comparative ages between humans and C57BL/6 mice at the time of birth, weaning, puberty, sexual maturity, reproductive senescence, and life expectancy. The first dotted red line denotes the analogous mouse and human age range for common syngeneic mouse glioma modeling at 6–12 weeks old, which corresponds to the age of 11.5–16 year old human. The second dotted red line denotes the analogous mouse and human age ranges for the median age of a human GBM patient diagnosis at 64 years old in humans, which corresponds to 87 weeks old in mice. (D) Treatment timeline and OS of C57BL/6 WT mice intracranially-engrafted with 5×10⁴ GL261 cells and simultaneously treated with anti-CTLA-4 and anti-PD-L1 mAbs beginning at day 14 after tumor cell injection. Overall survival estimated using the method of Kaplan-Meier; age groups compared using Cox proportional hazards models (A, D); *p<0.05; **p<0.01

Figure 4.. Advanced aging licenses non-tumor-, non-hematopoietic-stroma to suppress immunotherapeutic efficacy in subjects with malignant glioma.

(A) Timeline and overall survival (OS) analysis of 8–12 week old C57BL/6 wild-type (WT) mice treated with or without intraperitoneally (i.p.)-administered vancomycin, metronidazole, and neomycin, as well as ampicillin via the water supply, beginning at day -35, followed by intracranial-engraftment with 2.0×10⁵ GL261 cells at day 0, and treatment with 2 Gy whole brain radiation (RT) × 5 days total, one 500 μg loading dose followed by three 100 μg maintenance doses of anti-PD-1 mAb (clone J43), and the IDO enzyme inhibitor (IDOi; BGB-7204), beginning at day 14 after tumor cell injection and as previously described. (B) Timeline for the mixed age bone marrow chimeric mouse experiment. Briefly, 4–6 week old and 62–64 week old C57BL/6 WT mice were sublethally irradiated with 9 Gy whole body radiation (WBRT) and reconstituted with the bone marrow from either 4–6 week old or 62–64 week old C57BL/6 WT mice during the same day. Mice received treatment with neomycin between days -84 to -56. Approximately three months later, all groups were intracranially-engrafted with 5.0×10⁴ GL261 cells, and treated with RT, anti-PD-1 mAb, and IDO1 enzyme inhibitor, beginning at day 14 after tumor cell injection and as described in (A). The resulting OS analysis is presented as Kaplan Meier survival curves. Overall survival estimated using the method of Kaplan-Meier (A, B). Age groups compared using Cox proportional hazards models; pairwise groups comparisons adjusted using Scheffe’s method (A, B). **p≤0.01; ***p≤0.001

Figure 5.. Advanced aging licenses non-tumor cell IDO to decrease overall survival (OS) despite the treatment with a pharmacologic IDO enzyme inhibitor in subjects with malignant glioma.

(A) Timeline and OS analysis of 8 week old wild-type (WT) C57BL/6 mice intracranially-engrafted with 2.0×10⁵ GL261 cells and treated with IgG Abs, or with 2 Gy whole brain radiation (RT) × 5 days total, one 500 μg loading dose followed by three 100 μg maintenance doses of anti-PD-1 mAb (clone J43) and combined with either BGB-5777 (tool compound IDO enzyme inhibitor which we previously published with) or BGB-7204 (clinical grade IDO enzyme inhibitor) beginning at day 14 after tumor cell injection. (B-F) IDO expression- and OS-analysis of 8–12 week old and 72–78 week old, WT and IDOKO (C57BL/6 background) mice, pre-treated with vehicle alone or with BGB-7204 (IDOi), followed by the intracranial-engraftment with 5.0×10⁴ GL261.fl cells modified to express luciferase. (B) IDO mRNA expression reflects the levels in the contralateral brain without tumor, as well as the syngeneic brain tumor. (E) Bioluminescence of GL261.fl brain tumors expressed as a percentage of the final radiance for each mouse. Overall survival estimated using the method of Kaplan-Meier; age groups compared using Cox proportional hazards models; pairwise groups comparisons unadjusted (A) or adjusted using Scheffe’s (C, D). Mean ± SEM; groups compared using linear regression model; pairwise groups comparisons adjusted using Scheffe’s method. *p<0.05; **p<0.01; ****p<0.0001

Figure 6.. Advanced aging enhances the immunosuppressive effects of IDO, which is not reversed during the treatment with a pharmacologic enzyme inhibitor in subjects with malignant glioma.

(A) Timeline and overall survival (OS) analysis of 8–12 week old- and 78–86 week old-C57BL/6 wild-type (WT) and IDOKO (C57BL/6 background) mice intracranially-engrafted with 5.0×10⁴ GL261 cells and treated with 2 Gy whole brain radiation (RT) × 5 days total, one 500 μg loading dose followed by three 100 μg maintenance doses of anti-PD-1 mAb (clone J43), and IDO enzyme inhibitor (IDOi; BGB-7204) beginning at day 14 after tumor cell injection. (B) Composition and numbers of young and older adult male and female WT mice that are represented in (A) and their association with median and long-term OS. (C) A working hypothesis for how IDO suppresses: (i) the anti-glioma immune response; (ii) responsiveness to glioma immunotherapy, in subjects with malignant glioma. Greater than 90% of human patient-resected glioblastoma (GBM) is IDO positive and for which, intratumoral IDO expression levels increase with higher tumor-infiltrating T cell levels but are not changed with aging. Glioma cell IDO increases intratumoral immunosuppressive regulatory T cells (Tregs; CD4⁺FoxP3⁺) regardless of the treatment with an IDO enzyme inhibitor and the absence of IDO metabolism by glioma cells^,. In contrast, IDO metabolism is detectable in the brain tumor-draining cervical lymph nodes (cLN). Advanced age increases IDO expression in the brain (Fig. 5B)^,, and decreases immunotherapeutic efficacy against glioma despite IDO enzyme inhibitor treatment. Given their association with mediating inflammation, senescent cells with the senescence-associated secretory phenotype (SASP) indirectly upregulate immunosuppressive IDO expression in the brain during advanced aging^,, due to the high sensitivity for IDO expression by pro-inflammatory molecule expression. Overall survival estimated using the method of Kaplan-Meier (A). Age groups compared using Cox proportional hazards models; pairwise groups comparisons adjusted using Scheffe’s method (A). *p<0.05; ****p<0.0001