Northwestern University Feinberg School of Medicine

Wainwright Laboratory of Brain Aging, Cancer Immunology, and Immunotherapy

Examples of Our Work


  1. Adult glioblastoma (GBM; grade IV glioma) is the most common, aggressive, primary brain cancer with a median age of diagnosis at 65 years of age[1]. De novo wild-type isocitrate dehydrogenase (wtIDH) GBM reflects 90% of all GBM diagnoses and has a median age of onset at ~68-70 years old. It arises without any previous diagnosis of cancer and is therefore not the result of a lower grade glioma transitioning into GBM. While the median overall survival (mOS) of patients with GBM following surgery, radiotherapy, chemotherapy, and tumor treating fields is 18-21 months according to a recent phase III clinical trial[2], the mOS is less than 6 months for those GBM patients that are ≥65 years old[3]; highlighting an underappreciated aspect of this central nervous system age-related disease and raising the question of why GBM has a dramatically poorer prognosis among older adults?
  2. Advanced age is associated with a higher mortality rate (Fig. 1), as well as decreased mOS and long-term survival (LTS) of subjects with GBM (Fig. 2). Although GBM can arise at any age, there is a significant enrichment of diagnoses among subjects ≥65 years of age. Coincident among this enriched population is a decreased mOS and fewer long-term survivors that are consistent with the hypothesis: advanced age increases factors in the brain that contribute to the onset, progression, and/or failure of immune system control of tumor burden.

  3. Similar to older human subjects with GBM, older mice engrafted with intracranial malignant glioma have fewer long-term survivors and are more sensitive to lymphopenia (Fig. 3). To determine whether the negative effects of advanced age observed in human GBM patients can be recapitulated in an experimental model, we analyzed the mOS and LTS of C57BL/6 mice intracranially-engrafted with 500 syngeneic CT-2A glioma cells at 6-, 23-, 47-, and 75-weeks of age that were administered IgG control antibodies (Fig. 3A) or anti-CD4 and anti-CD8 T cell-depleting antibodies (Fig. 3B). There were different levels of mOS and LTS depending on the age of subject engrafted with glioma cells such that, there were higher levels of LTS among middle aged subjects with T cells, whereas older adults had no LTS and their mOS was decreased by the absence of T cells. These data collectively suggest three primary conclusions: (i) T cells provide different levels of protection from glioma that depend on the subject’s age; (ii) older adult T cells are critical for preventing a catastrophic decrease of mOS from glioma; (iii) older adults with glioma have poorer LTS outcomes as compared to younger counterparts.

  4. Older adult human patients with GBM have a decreased mOS after treatment with immune checkpoint blockade (ICB). Because we found a lower mOS among older human adults with GBM (Fig. 2), we next questioned whether this effect extended to dw4.jpgGBM patients treated with ICB such as anti-PD-1, anti-PD-L1 or anti-CTLA-4 mAbs. Information was extracted from medical records including age, sex, MGMT promoter methylation status, and IDH status for patients with recurrent GBM (rGBM) that were administered ICB in the adjuvant setting (Fig. 4A). Ninety-two patients were dichotomized into groups defined as <65 years of age (n=65) and ≥65 years of age (n=27; Fig. 4B). rGBM patients <65 years of age treated with ICB had a higher mOS of 278 days as compared to patients ≥65 years of age that had a mOS of only 154 days (Fig. 4A; p=0.022). 

  5. Older adult mice with intracranial glioma have decreased survival as compared to younger counterparts following treatment with ICB. dw5.jpgThe negative association between advanced age and decreased mOS in human GBM patients (Fig. 2A-D), combined with the negative association between advanced age and decreased mOS in human rGBM patients treated with adjuvant ICB (Fig. 4A), prompted us to generate a table of major analogous age groups between wild-type C57BL/6 mice and humans (Fig 5A). Based on our previous work determining the promising survival benefit of anti-CTLA-4 mAb/anti-PD-L1 mAb ICB treatment in young syngeneic mice[4], we next questioned how advanced age would affect the preclinical survival benefit. Similar to the clinical setting, older adult mice with glioma had a significantly decreased mOS of 25.5 days and 27% LTS as compared to younger subjects that had an undefined mOS and 67% LTS (Fig 5B). The overlapping negative outcomes of advanced age in mouse and human subjects with malignant glioma suggests that factors exist in older adults, but not in younger subjects that, decrease the survival benefit of therapy.
     
  6. Advanced age decreases therapeutic efficacy through a non-tumor, non-hematopoietic cell-dependent mechanism. To explore the basis for how advanced age decreases therapeutic efficacy in subjects with glioma, we next investigated bone marrow chimeric mice with different ages of host non-tumor stroma and hematopoietic cells. Given our previous work validating the combination of radiation (RT), anti-PD-1 mAb, and an indoleamine 2,3 dioxygenase 1 enzyme inhibitor (IDOi) to improve the survival of young mice with well-established glioma36, which serves as the basis for the Phase I clinical trial in newly-diagnosed GBM patients [NCT04047706] led by Dr. Rimas Lukas, MD (see included letter of support), we next evaluated this clinically-relevant therapeutic strategy while assessing the major potential sources of immune suppression during advanced age. When comparing either the young WT mouse group or the old WT mouse group with glioma, no differences in mOS were found. (Fig. 6). In contrast, there was a significant increase in both the mOS and the LTS of young mice with glioma that were reconstituted with either young or older adult bone marrow, as compared to older adult mice that were reconstituted with either young or older adult bone marrow (p<0.0001). These data collectively suggest that advanced age suppresses therapeutic efficacy against glioma through a non-hematopoietic-, non-tumor stromal-cell dependent mechanism.dw6.jpg
     
  7. Immunosuppressive IDO and dendritic cells (DCs) coincidently increase in the brain during advanced age. We and others have demonstrated substantial immunological changes in the brain during advanced age. Notably, these changes occur independent of tumor burden and include: (i) increased human[5] (Fig. 7A) and murine[6](Fig. 7B) IDO expression, as well as an increased presence of DCs in the human (Fig. 7A) and mouse brain parenchyma (Fig. 7C)[7, 8]. DCs are specialized antigen-presenting cells capable of activating and regulating T cell-mediated anti-GBM immune responses[9-11]. However, when DCs express IDO, they potently inhibit T cell activation and effector functions[12]. Taken together, these data suggest the possibility that advanced age increases DCs expressing IDO in the brain, which in-turn, suppresses the anti-glioma immune response.

  8. IDO decreases the mOS of older adult mice with glioma despite the treatment with an IDO enzyme inhibitor (IDOi). IDO is an inducible enzyme that converts tryptophan (Trp) into kynurenine (Kyn)[13, 14] (Fig. 8) and the depletion of tryptophan and/or accumulation of kynurenine has been the canonical mechanism attributed to how IDO suppresses T cell effector functions[12, 15, 16]dw7.jpgnotably, this mechanism has primarily been supported by in vitro observations. We therefore aimed to confirm the mechanism by IDO participated in older adults with glioma. In-line with our previous observations[6, 17], we found higher IDO expression in the older adult brain as compared to younger counterparts (Fig. 9A; next page). Strikingly, the advanced age-increased IDO levels were markedly higher in the contralateral side of the brain without tumor as compared to the ipsilateral brain hemisphere with glioma. dw8.jpgTo determine the significance of the increased IDO expression, we next evaluated the effect of IDO enzyme inhibitor (IDOi) pre-treatment in young and older adult WT mice. In young WT mice with glioma, IDOi increased the mOS as compared to the vehicle-treated group (Fig. 9B). Similarly, young IDOKO mice with glioma had an improved mOS as compared to WT counterparts treated with the vehicle. In contrast, older adult IDOKO mice had an improved mOS from glioma as compared to both the older adult WT mice pre-treated with vehicle or with IDOi (Fig. 9C). These data confirm that pre-treatment with a pharmacologic IDO enzyme inhibitor marginally improves the mOS of young subjects but not older adults with glioma, suggesting that advanced age changes the targetability of IDO.dw9.jpg

  9. IDO decreases therapeutic efficacy in older adults with glioma and its effects are not reversed by treatment with a metabolic inhibitor. Our finding of higher IDO levels in the older adult brain (Figs. 7A,B and 9A), combined with the survival decreasing effects of IDO in older adults (Fig. 9C), prompted us to next evaluate the effects of advanced age-increased IDO in a therapeutic setting. Young and old, WT and IDOKO mice with intracranial glioma were treated with RT, anti-PD-1 mAb, and IDOi at 14 days after glioma cell engraftment (Fig. 10) – a strategy that we recently translated into a clinical trial [NCT04047706] and for which I serve as a Project Leader of the Brain SPORE [P50CA221747]. In support of our previous findings36, there was a significant decrease of mOS and LTS in older adult WT mice as compared to younger counterparts (p<0.0001). Also in-line with our previous report, there was no difference of mOS or LTS in young WT and IDOKO mice with glioma treated with the triple combination therapy. In marked contrast, older adult IDOKO mice treated with the triple combination showed an impressive improvement in both mOS and LTS as compared to the older adult WT counterparts (p<0.01). The data collectively suggest that advanced age increases IDO expression in the brain (Fig. 9A) and suppresses therapeutic efficacy independent of IDO enzyme activity.

  10. A working hypothesis for how IDO suppresses the anti-glioma immune response and/or response to glioma immunotherapy (Fig. 1). Greater than 90% of human patient-resected dw10.jpgGBM expresses IDO[18] and intratumoral IDO expression levels are directly increased by tumor-infiltrating T cells[18, 19] but unchanged by the effects of aging (Fig. 11A)[20]. Glioma cell IDO increases intratumoral immunosuppressive regulatory T cells (Tregs; CD4+CD25+FoxP3+)[21] and decreases overall survival regardless of treatment with a pharmacologic IDO enzyme inhibitor[22] or IDO metabolism by glioma cells (Fig. 11B)[23, 24]. Advanced age is associated with increased non-metabolic immunosuppressive IDO activity in the brain (Figs. 9C, 10)[6, 20, 22] and is mediated by DCs (Fig. 7A-C), which inhibit T cell effector functions and decrease therapeutic efficacy against glioma. Senescent cells increase in the brain during advanced age and express the SASP, which in-turn increases inflammation-inducible immunosuppressive IDO levels45,56 due to IDO promoter sensitivity to pro-inflammatory stimuli[25].

 

dw11.jpg

 

 

 

 

 

 


REFERENCES

  1. Ostrom, Q.T., et al., CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014. Neuro Oncol, 2017. 19(suppl_5): p. v1-v88.
  2. Stupp, R., et al., Effect of Tumor-Treating Fields Plus Maintenance Temozolomide vs Maintenance Temozolomide Alone on Survival in Patients With Glioblastoma: A Randomized Clinical Trial. JAMA, 2017. 318(23): p. 2306-2316.
  3. Arvold, N.D. and D.A. Reardon, Treatment options and outcomes for glioblastoma in the elderly patient. Clin Interv Aging, 2014. 9: p. 357-67.
  4. Wainwright, D.A., et al., Durable therapeutic efficacy utilizing combinatorial blockade against IDO, CTLA-4 and PD-L1 in mice with brain tumors. Clinical Cancer Research, 2014.
  5. Ladomersky, E., Scholtens, D.M., Kocherginsky, M., Hibler, E.A., Bartom, E.T., Otto-Meyer, S., Zhai, L., Lauing, K.L., Choi, J., Sosman, J.A., Wu, J.D., Zhang, B., Lukas, R.V., Wainwright, D.A., The glioblastoma patient mortality rate and immunosuppression coincidently increase during advanced age. Frontiers in Pharmacology, 2019.
  6. Ladomersky, E., et al., Advanced age negatively impacts survival in an experimental brain tumor model. Neurosci Lett, 2016. 630: p. 203-8.
  7. Kaunzner, U.W., et al., Accumulation of resident and peripheral dendritic cells in the aging CNS. Neurobiol Aging, 2012. 33(4): p. 681-693 e1.
  8. Bulloch, K., et al., CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain. J Comp Neurol, 2008. 508(5): p. 687-710.
  9. Prins, R.M., et al., Gene expression profile correlates with T-cell infiltration and relative survival in glioblastoma patients vaccinated with dendritic cell immunotherapy. Clin Cancer Res, 2011. 17(6): p. 1603-15.
  10. Prins, R.M., et al., Comparison of glioma-associated antigen peptide-loaded versus autologous tumor lysate-loaded dendritic cell vaccination in malignant glioma patients. J Immunother, 2013. 36(2): p. 152-7.
  11. Lasky, J.L., 3rd, et al., Autologous tumor lysate-pulsed dendritic cell immunotherapy for pediatric patients with newly diagnosed or recurrent high-grade gliomas. Anticancer Res, 2013. 33(5): p. 2047-56.
  12. Munn, D.H., et al., Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science, 2002. 297(5588): p. 1867-70.
  13. Fujiwara, M., et al., Indoleamine 2,3-dioxygenase. Formation of L-kynurenine from L-tryptophan in cultured rabbit fineal gland. J Biol Chem, 1978. 253(17): p. 6081-5.
  14. Wainwright, D.A., et al., Targeting Tregs in Malignant Brain Cancer: Overcoming IDO. Front Immunol, 2013. 4: p. 116.
  15. Hwu, P., et al., Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J Immunol, 2000. 164(7): p. 3596-9.
  16. Munn, D.H., et al., Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med, 1999. 189(9): p. 1363-72.
  17. Ladomersky, E., et al., IDO1 Inhibition Synergizes with Radiation and PD-1 Blockade to Durably Increase Survival Against Advanced Glioblastoma. Clin Cancer Res, 2018.
  18. Zhai, L., et al., Infiltrating T Cells Increase IDO1 Expression in Glioblastoma and Contribute to Decreased Patient Survival. Clin Cancer Res, 2017. 23(21): p. 6650-6660.
  19. O'Rourke, D.M., et al., A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med, 2017. 9(399).
  20. Ladomersky, E., et al., The Coincidence Between Increasing Age, Immunosuppression, and the Incidence of Patients With Glioblastoma. Front Pharmacol, 2019. 10: p. 200.
  21. Wainwright, D.A., et al., IDO expression in brain tumors increases the recruitment of regulatory T cells and negatively impacts survival. Clin Cancer Res, 2012. 18(22): p. 6110-21.
  22. Ladomersky, E., et al., IDO1 Inhibition Synergizes with Radiation and PD-1 Blockade to Durably Increase Survival Against Advanced Glioblastoma. Clin Cancer Res, 2018. 24(11): p. 2559-2573.
  23. Zhai, L., et al., Non-tumor cell IDO1 predominantly contributes to enzyme activity and response to CTLA-4/PD-L1 inhibition in mouse glioblastoma. Brain Behav Immun, 2017. 62: p. 24-29.
  24. Zhai, L., et al., Tumor cell IDO1 increases intratumoral immunosuppressive Tregs independent of enzyme activity. The Journal of Immunology, 2019. 202(1 Supplement): p. 137.2-137.2.
  25. Zhai, L., et al., Molecular Pathways: Targeting IDO1 and Other Tryptophan Dioxygenases for Cancer Immunotherapy. Clin Cancer Res, 2015.