Efficient and reproducible generation of tumour-infiltrating lymphocytes for renal cell carcinoma

Abstract

Background: Tumour-infiltrating lymphocyte (TIL) therapy is showing great promise in the treatment of patients with advanced malignant melanoma. However, the translation of TIL therapy to non-melanoma tumours such as renal cell carcinoma has been less successful with a major constraint being the inability to reproducibly generate TILs from primary and metastatic tumour tissue.

Methods: Primary and metastatic renal cell carcinoma biopsies were subjected to differential tumour disaggregation methods and procedures that stimulate the specific expansion of TILs tested to determine which reliably generated TIL maintained antitumour specificity.

Results: Enzymatic or combined enzymatic/mechanical disaggregation resulted in equivalent numbers of TILs being liberated from renal cell carcinoma biopsies. Following mitogenic activation of the isolated TILs with anti-CD3/anti-CD28-coated paramagnetic beads, successful TIL expansion was achieved in 90% of initiated cultures. The frequency of T-cell recognition of autologous tumours was enhanced when tumours were disaggregated using the GentleMACS enzymatic/mechanical system.

Conclusion: TILs can be consistently produced from renal cell carcinoma biopsies maintaining autologous tumour recognition after expansion in vitro. While the method of disaggregation has little impact on the success of TIL growth, methods that preserve the cell surface architecture facilitate TIL recognition of an autologous tumour, which is important in terms of characterising the functionality of the expanded TIL population.

Figure 1

Isolation, expansion and phenotype of TILs from renal biopsies. (A) Number of isolated TILs after overnight (n=8) or GentleMACS (GM) (n=10) digestion from each biopsy sample. No statistically significant difference was observed between the digestion methods. NS, P>0.05, Mann–Whitney test. (B) Growth curves of TILs throughout the 15-day culture period in overnight-digested or (C) GM-processed samples. (D) Fold expansion of TILs after 15 days of culture after overnight or GM digests. (E, F) Phenotypic characterisation of TILs expanded from renal biopsies after 15 days in culture in overnight (o/n) digested and GM-processed samples. NS, P>0.05, *P<0.05, **P<0.01, Mann–Whitney test. Abbreviation: NS, not significant.

Figure 2

Functional activity of expanded TILs against their autologous tumour cells. (A) Interferon gamma (IFNγ) secreted after 24-h co-culture of TILs with overnight and (B) GentleMACS-disaggregated autologous tumour samples. (C) IFNγ release of TILs after co-culture with an autologous tumour generated with parallel processing of the same tumour with either overnight digestion or GentleMACS disaggregation. Mean±s.e.m. of three experimental replicates are shown. *P<0.05, ***P<0.001. Two-way ANOVA with Bonferroni post-test correction.

Figure 3

Surface expression of markers on TILs after overnight or GentleMACS disaggregation. (A) Flow cytometric analysis of CD4, CD8, EpCAM, HLA A,B,C and HLA DR,DP, DQ expression on isolated, uncultured TILs from four renal cell carcinoma biopsies after overnight (black empty line) or GentleMACS (grey-filled line) disaggregation. (B) Effect of MHC class I and class II blockade on the release of IFNγ after 24-h co-culture of TILs with an autologous tumour in two RCC biopsies. **P<0.01 Unpaired t-test.

Figure 4

Expansion and functional activity of TILs after undergoing a rapid expansion protocol. (A) Growth curves of TILs subjected to a rapid expansion protocol (REP). (B) IFNγ release of rapid-expanded TILs V6, V12, V14 and V15 after co-culture with an autologous tumour. (C) Alloreactivity of rapid-expanded TILs V6, V12, V14 and V15 as determined by IFNγ release after co-culture of TILs with renal (2220R), gastric (MKN45K), melanoma (Mel624) and neuroblastoma (LA15S and SK-N-SH) cell lines. ***P<0.001, two-way ANOVA with Bonferroni post-test correction.