Abstract 4061: Systemic autologous lymphocyte transfer can enhance tumor-infiltrating lymphocyte infiltration in glioblastoma and license co-stimulatory immunotherapy
Authors: Singh Kirit , Hotchkiss Kelly M. , Moelker Eliese , Archer Gary E. , Sampson John H. , Khasraw Mustafa 
Published: April 2023

Abstract
Glioblastoma is an immunologically ‘cold’ malignancy where few tumor infiltrating lymphocytes (TILs) are present. We sought to overcome this by ‘seeding’ the tumor with TILs via administration of autologous lymphocytes (ALT). We hypothesized that prior administration of ALT will enhance subsequent immunomodulatory therapies (e.g. immune checkpoint blockade (ICB) or co-stimulation). To evaluate this, we initially generated a pro-infiltrative TIL phenotype. CD45.1 splenocytes (C57/Bl6 background) were co-cultured with either IL2 and Concanavalin A (Con A) or IL7 and Con A for 5 days. Flow cytometric analysis found that both conditions resulted in similar CD4:CD8 ratios (20:80%) but IL7 co-culture upregulated expression of VLA-4, a pro migratory integrin. We then compared the entry of TILs into tumor bearing CNS when co-cultured with either IL2 or IL7. 8–10-week-old C57/Bl6 mice (n=5-6 per group) were implanted orthotopically with 30,000 CT2AvIII cells (syngeneic glioma) which established for 14 days. Mice received (1) CD45.1 lymphocytes activated with IL-7 and Con A (single intravenous (IV) injection, 1 × 107 cells) or (2) CD45.1 lymphocytes activated with IL2 and serial Con-A stimulation. Mice were sacrificed 3- and 48-hours following ALT and brains analysed for CD45.1+CD3+ populations. Groups were compared using a Mann-Whitney U test. Mice in Group 1 demonstrated significantly enhanced entry of CD8+ effector and memory T cells into tumor bearing hemispheres at both time points following administration compared to Group 2 (p = 0.005, p = 0.0159 respectively). By 48 hours post ALT, IL7 co-culture resulted in an average 810.1% expansion of CD8 T cell numbers in tumor bearing CNS compared to counts at Day 0. We then evaluated if mice with a TIL enriched CNS might be more responsive to ICB or co-stimulatory therapy. 8–10-week-old C57/Bl6 mice (n=5-7 per group) were implanted with 30,000 CT2AvIII cells that established over 10 days. On day 10, mice received IV ALT of either IL7 T cells only, or IL-7 and treatment courses of IP ICB (anti-PD1, anti-CTLA 4 or anti-41BB). Mice given ICB following IL7 ALT demonstrated a median survival (aPD1: 27 days, aCTLA-4: 25 days) similar to mice given IL7 ALT alone (25 days). However, 4/7 mice who received IL7 ALT and anti-41BB were still alive 45 days post tumor implantation (p=0.0004). Taken together, we demonstrated that certain ex vivo autologous lymphocyte co-culture conditions can generate a T cell population which infiltrates tumor bearing CNS in significant numbers. Further, we found that seeding a tumor with TILs prior to stimulatory therapy yields long term survival in vivo. Future work will focus on optimizing ex vivo culturing approaches for ALT and determine whether therapeutic efficacy is due to expansion of resident TILs at the tumor site, or ongoing recruitment of endogenous T cells from the periphery.
Citation Format: Kirit Singh, Kelly M. Hotchkiss, Eliese Moelker, Gary E. Archer, John H. Sampson, Mustafa Khasraw. Systemic autologous lymphocyte transfer can enhance tumor-infiltrating lymphocyte infiltration in glioblastoma and license co-stimulatory immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4061.