Tumour-reactive T cell subsets in the microenvironment of ovarian cancer

Abstract

Background: Solid malignancies are frequently infiltrated with T cells. The success of adoptive cell transfer (ACT) with expanded tumour-infiltrating lymphocytes (TILs) in melanoma warrants its testing in other cancer types. In this preclinical study, we investigated whether clinical-grade TILs could be manufactured from ovarian cancer (OC) tumour specimens.

Methods: Thirty-four tumour specimens were obtained from 33 individual patients with OC. TILs were analysed for phenotype, antigen specificity and functionality.

Results: Minimally expanded TILs (Young TILs) were successfully established from all patients. Young TILs contained a high frequency of CD3+ cells with a variable CD4/CD8 ratio. TILs could be expanded to clinical numbers. Importantly, recognition of autologous tumour cells was demonstrated in TILs in >50% of the patients. We confirmed with mass spectrometry the presentation of multiple tumour antigens, including peptides derived from the cancer-testis antigen GAGE, which could be recognised by antigen-specific TILs. Antigen-specific TILs could be isolated and further expanded in vitro.

Conclusion: These findings support the hypothesis that patients with OC can benefit from ACT with TILs and led to the initiation of a pilot clinical trial at our institution .

Trial registration: clinicaltrials.gov: NCT02482090.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Ovarian tumour-infiltrating lymphocytes (TILs). Young TILs were further expanded using a small scale Rapid Expansion Protocol (REP). a Fold expansion of TILs from 24 ovarian cancer (OC) patients (purple lines) and from 11 metastatic melanoma (MM) patients (blue lines) following a 14 days REP performed in parallel were compared. Median fold expansion for all OC patients (n = 34) was 1660 (range [440–5544]). Median fold expansion for MM (n = 11) was 2842 (range [816–6900]). b Scatterplot showing the final fold expansion. No significant difference was seen when comparing the final fold expansion of OC-TILs (n = 34) and MM-TILs (n = 11). The two groups were compared using the Mann–Whitney test. Data are presented with the median

Fig. 2

Phenotypic characterisation of ovarian cancer TILs. Young TILs and REP-TILs were analysed with flow cytometry for phenotypic markers. a Scatterplot showing percentages of CD3+ T cells in Young TIL (n = 34) and REP TIL (n = 33) populations. Data are presented with the median. b Scatterplot showing the percentage of NK cells in the Young TIL population (n = 34). Data are presented with the median. c Scatterplot showing percentages of γδ T cells in Young TIL (n = 34) and REP TIL (n = 33) populations. Data are presented with the median. d Scatterplot showing the CD4/CD8 ratios in Young TILs (n = 34) and REP-TILs (n = 33). The median CD4/CD8 ratio in Young TILs was 7.2 (range [0.01–306]) and 10.0 (range [0.02–1067]) in REP TIL population. Data were log-transformed before the two groups were compared. A significant increase in the ratio was observed (p = 0.0253). Data are presented with the median. e The pie charts show the phenotypic distribution of CD8+, CD4+, CD4+CD8+ and CD4−CD8− of CD3+ TILs in the Young TIL (n = 34) and REP TIL (n = 33) populations. Data are presented with mean values. f, g Scatterplots illustrating the percentage of naïve T cells, central memory T cells and effector memory T cells and h, i Scatterplots illustrating the percentage of Exhaustion markers: LAG-3, BTLA, PD-1 and TIM-3 in f, h the CD8+ T cell population and g, i the CD4+ T cell population in Young TILs (n = 34) and REP-TILs (n = 33). Data are presented with the median with interquartile range. Panel f shows a significant increase of CD8+ Tem cells p = 0.0004 during the REP and a significant decrease in the Naïve T cell subpopulation, p < 0.0001. g The CD4-expressing population had a significant increase in CD4+ Tem cells during the REP, p = 0.0003, and a significant decrease in the Naïve T cell subpopulation during the REP, p < 0.0001. Panel h shows significant increase of CD8+ LAG-3+ T cells p = 0.0263 during the REP and a significant decrease in the PD-1+ T cells, p = 0.0002, and a significant increase in the TIM-3+ T cells, p = 0.0379. i The CD4-expressing population had a significant decrease in CD4+ BTLA+ cells during the REP, p = 0.013, and a significant decrease in the PD-1+ T cell subpopulation during the REP, p < 0.0001. Young TILs and REP-TILs were compared using the Wilcoxon signed rank test. Statistical significant differences is indicated with *, **, *** or **** for p values less than 0.05, 0.01, 0.001, or 0.0001, respectively

Fig. 3

In vitro antitumour activity of CD8+ Young TILs. The antitumour activity of the in vitro-expanded TILs was evaluated by defining the frequency of T cells expressing at least one of the following T cell functions: TNF-α, IFN-γ or CD107a, upon stimulation with autologous Fresh tumour digest (FTD) or tumour cell line (TCL) treated with low-dose IFN-γ (100 IU/ml) or left untreated. A specific antitumour response was defined as the presence of minimum 0.5% responding cells, with a minimum number of 50 positive events. The frequency of tumour-reactive cells in stimulated samples was subtracted from the unstimulated samples. In all, 0.5% was used as a threshold for detection of tumour reactivity. a Antitumour responses of CD8+ T cells were detected in 13 of 31 patients analysed. *: OC.TIL.03 is not tested with FTD. ϕ: TILs generated from OC.TIL.04 2nd was tested for reactivity against FTD from OC.TIL.04. b FACS plot showing cytokine production from TIL alone (unstimulated, serving as a negative control) and TIL stimulated with autologous TCL, from a representative patient (OC.TIL.11). c FACS plot showing CD107a mobilisation of TIL upon co-culture with an autologous TCL. Unstimulated TIL (TIL alone) serves as a negative control. An example of the gating strategy is showed in Supplementary Figure 7A

Fig. 4

In vitro antitumour activity of CD4+ Young TILs. The antitumour activity of the in vitro-expanded TILs was evaluated by defining the frequency of T cells expressing at least one of the following T cell functions: TNF-α, IFN-γ or CD107a, upon stimulation with autologous fresh tumour digest (FTD) or tumour cell line (TCL) treated with low-dose IFN-γ (100 IU/ml) or left untreated. A specific antitumour response was defined as the presence of minimum 0.5% responding cells, with a minimum number of 50 positive events. The frequency of tumour-reactive cells in stimulated samples was subtracted from unstimulated samples; 0.5% was used as a threshold for detection of tumour reactivity. a Antitumour responses of CD4+ T cells were detected in 16 of 31 patients. *OC.TIL.03 is not tested with FTD. ϕ: TILs generated from OC.TIL.04 2nd was tested for reactivity against FTD from OC.TIL.04. b FACS plot showing cytokine production from TIL alone (unstimulated, serving as a negative control) and TIL stimulated with autologous TCL, from a representative patient (OC.TIL.15). An example of the gating strategy is showed in Supplementary Figure 7A

Fig. 5

IFN-γ ELISPOT assay: Selected peptides identified by MS-based immunopeptidomics tested for the ability to induce an IFN-γ response in REP-TILs from patient OC.TIL.11. Background (spots in wells without added peptides) was subtracted. Example of IFN-γ ELISPOT response against GAGE peptides

Fig. 6

GAGE-specific TILs. a 1600 GAGE12-specific Young TILs from patient OC.TIL.11 were sorted using tetramers HLA-A3/STYYWPRPR APC/PE. b The sorted culture was tested for specificity with tetramers HLA-A3/STYYWPRPR APC/PE after two times REP. c FACS plot illustrating cytokine production (upper panel) and CD107a mobilisation (lower panel) in TIL alone (unstimulated) as a negative control, TILs stimulated with autologous tumour cell line (TCL), TILs stimulated with autologous TCL pre-stimulated with IFN-γ for 3 days prior to experiment and TILs stimulated with the GAGE peptide. An example of the gating strategy is showed in Supplementary Figure 7A. d xCELLigence killing assay showing the killing of autologous tumour cells with GAGE-specific TILs from patients OC.TIL.11 (ratio 0.2:1). HLA class I antibody, HLA class II antibody and a combination were added as controls