Tumor infiltrating lymphocytes expanded from pediatric neuroblastoma display heterogeneity of phenotype and function

Abstract

Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs) has led to clinical benefit in some patients with melanoma but has not demonstrated convincing efficacy in other solid cancers. Whilst the presence of TILs in many types of cancer is often associated with better clinical prognosis, their function has not been systematically evaluated across cancer types. Responses to immunological checkpoint inhibitors in a wide range of cancers, including those for which adoptive transfer of expanded TILs has not shown clinical benefit, has clearly delineated a number of tumor type associated with tumor-reactive lymphocytes capable of effecting tumor remissions. Neuroblastoma is an aggressive childhood solid cancer in which immunotherapy with GD2-directed antibodies confers a proven survival advantage through incompletely understood mechanisms. We therefore evaluated the feasibility of ex vivo expansion of TILs from freshly resected neuroblastoma tumors and the potential therapeutic utility of TIL expansions. TILs were successfully expanded from both tumor biopsies or resections. Significant numbers of NKT and γδT cells were identified alongside the mixed population of cytotoxic (CD8+) and helper (CD4+) T cells of both effector and central memory phenotypes. Isolated TILs were broadly non-reactive against autologous tumor and neuroblastoma cell lines, so enhancement of neuroblastoma killing was attained by transducing TILs with a second-generation chimeric antigen receptor (CAR) targeting GD2. CAR-TILs demonstrated antigen-specific cytotoxicity against tumor cell lines. This study is the first to show reproducible expansion of TILs from pediatric neuroblastoma, the high proportion of innate-like lymphocytes, and the feasibility to use CAR-TILs therapeutically.

Fig 1. Large numbers of TILs can be expanded from neuroblastoma samples.

(A) Overview of TILs expansion protocol. (B) Representative microscope images of TILs at day 14 of initial expansion (left), and TILs at the end of REP (right). (C) Cell count per well at the end of initial expansion (left) and cell count per 75cm² flask at the end of REP (right). D) Fold change after REP. Data represented as mean ± Standard Error of Mean (SEM). Each case comprised a mean of 7.3 fragments (range 1–30 n = 19) for initial expansion and mean of 3 (range 1–8 n = 17) independent cultures per case for REP. Note case 1224 is the average of 4 independent tumors from the same patient. Accurate cell counts were missing from 4 further cases that went on to REP.

Fig 2. Phenotypic analysis of TILs after initial and REPs.

(A) T-cell subsets shown as % of CD3⁺ cells (n = 75). Statistical comparison by one-way Analysis of Variance (ANOVA) with Sidak’s correction for multiple comparisons. (B) Memory phenotype of T-cells after initial expansion and after the REP (n = 63) using the following criteria: Naïve CD62L⁺/CD45RA⁺/CD27⁺, CM CD62L⁺/CD45RA^-/CD27⁺, EM CD62L^-/CD45RA^-/CD27^-, TEMRA CD62L^-/CD45RA⁺/CD27^-. There was a significant reduction in the proportion of CM cells (p = 0.007) and a significant increase in the proportion of TEMRA cells (p = <0.0001). Statistical comparison by one-way ANOVA with Sidak’s correction for multiple comparisons. (C) Memory phenotype for paired TIL samples (n = 12) after initial expansion and then following REP. Proportions of CM cells reduced significantly after REP (p = 0.038). Statistical comparison by paired one-way ANOVA with Sidak’s correction for multiple comparisons. (D) PD1 status of paired CD4⁺. CD8⁺ and CD4^-/CD8^- T-cells after initial expansion and then following REP (n = 7). The percentage of CD4⁺ cells expressing PD1 fell significantly after REP (p = 0.015). Statistical comparison by paired one-way ANOVA with Sidak’s correction for multiple comparisons.

Fig 3. Innate lymphocyte subtype analysis within TILs population.

(A) Gating strategy for identification of γδT cells subtypes after gating on CD3 and γδTCR positive cells. This representative example is from a TIL sample following initial 14-day expansion. (B) γδT cell numbers expressed as % of CD3⁺ cells in TILs following initial expansion (n = 31) and then after REP (n = 41): statistical analysis by unpaired t-test. (C) γδT cell numbers expressed as % of CD3⁺ cells in 6 paired TIL samples following initial expansion and then after REP. (D) γδT cell subset distributions in the TILs population defined as in (A) following initial expansion (n = 14) and then after REP (n = 12). Vδ1⁺ cell proportions fell significantly (p = 0.006) and Vδ1^-/Vδ2^- proportions rose significantly after REP (p = <0.0001): statistical comparison by one-way ANOVA with Sidak’s correction for multiple comparisons. (E) γδT cell subset distributions in 5 paired TILs populations defined as in (A) following initial expansion and then after REP.

Fig 4. NKT subtype analysis within TILs population.

Multiple TIL cultures were analyzed for each case after the 14 days of initial expansion (n = 31) and after REP (n = 41). (A) Gating strategy for NKT cell analysis. NKT cells are defined as CD3⁺/γδTCR^-/CD56⁺. Invariant NKT (iNKT) are defined as CD3⁺/γδTCR^-/CD56⁺/Vα24Jα18⁺ and non-classical NKT cells are defined as CD3⁺/γδTCR^-/CD56⁺/Vα24Jα18^-. Data shown from a representative TIL sample following initial expansion (14 days). (B) NKT cell numbers in TILs expressed as proportion on CD3⁺ cells following initial expansion (n = 31) and following REP (n = 41). NKT cell proportions rose significantly following REP (p = 0.0006 by unpaired t-test). (C) NKT cell numbers in TILs expressed as proportion on CD3⁺ cells following initial expansion and following REP in 5 paired samples. (D) NKT subsets within the TILs following initial expansion (n = 14) and following rapid expansion (n = 12). The majority of NKT cells fell in the CD3⁺/γδTCR^-/CD56⁺/Vα24Jα18^- non-classical NKT cell gate, and the distribution of NKT cell subsets was not affected by the REP.

Fig 5. Functional potential of neuroblastoma expanded TILs.

(A) LAN1 neuroblastoma cells were co-cultured overnight with TILs and cell viability was assessed using an MTT assay. Presence of TILs significantly reduced viability of LAN1 cells in a manner which could not be blocked using TCR or Natural Killer Group 2D (NKG2D) blocking antibodies. (B) Example of a donor whose TILs were able to mount an IFNγ response against 1/6 autologous tumor fragments. In this case, IFNγ production by TILs was significantly increased in the presence of a 1-2mm tumor fragment (p = < 0.0001 by paired 2-way ANOVA with Sidak’s correction). Error bars are ±SEM of technical replicates (n = 1–3) for each tumor fragment/TIL co-culture. (C) IFNγ secreted by TILs in the absence or presence of autologous neuroblastoma tumors after initial expansion (n = 24) or after REP (n = 6). Stimulation with PMA is used as a positive control to demonstrate the capacity to mount an IFNγ response. (D) Migration of T-cells and TILs towards neuroblastoma cell lines (Lan1, SKNDZ and IMR32) at two different time points (4 and 24 h). T-cells or TILs were placed in the top chamber of a trans-well and target cells were placed in the bottom. At 4h, TILs: n = 3 and T cells: n = 2. At 24h, TILs: n = 8 and T cells: n = 3. Data represented as mean ± SEM.

Fig 6. Functional analysis of chimeric antigen receptor expressing TILs.

TILs were transduced to express an anti GD2-28ζ CAR with a mean transduction efficiency of 16.2±2.8%. (A) Killing of GD2^- SupT1, GD2⁺ SupT1-GD2 and GD2⁺ LAN1 neuroblastoma cells by transduced and non-transduced (NT) TILs (n = 16). Killing of SupT1-GD2 and LAN1 was significantly enhanced by the presence of GD2-28ζ (p = <0.0001). Statistical analysis by 2-way ANOVA with Sidak’s correction for multiple comparisons. (B) IFNγ release by non-transduced and GD2-28ζ transduced TILs in response to SupT1 or GD2-SupT1 (n = 8). Presence of the GD2-28ζ CAR increases background IFNγ production (p = 0.011) but the presence of GD2⁺ SupT1-GD2 leads to much greater IFNγ release (p = <0.0001). Statistical analysis by one-way ANOVA with Sidak’s correction for multiple comparisons. (C) Transduction with GD2-28ζ does not reduce the migratory capacity of TILs towards neuroblastoma cell lines, as determined using a 24h trans-well assay with target cells in the bottom chamber and effectors in the top chamber.