4-1BB-Enhanced Expansion of CD8+ TIL from Triple-Negative Breast Cancer Unveils Mutation-Specific CD8+ T Cells

Abstract

Triple-negative breast cancer (TNBC) highly infiltrated with CD8⁺ tumor-infiltrating lymphocytes (TIL) has been associated with improved prognosis. This observation led us to hypothesize that CD8⁺ TIL could be utilized in autologous adoptive cell therapy for TNBC, although this concept has proven to be challenging, given the difficulty in expanding CD8⁺ TILs in solid cancers other than in melanoma. To overcome this obstacle, we used an agonistic antibody (urelumab) to a TNFR family member, 4-1BB/CD137, which is expressed by recently activated CD8⁺ T cells. This approach was first utilized in melanoma and, in this study, led to advantageous growth of TILs for the majority of TNBC tumors tested. The agonistic antibody was only added in the initial setting of the culture and yet favored the propagation of CD8⁺ TILs from TNBC tumors. These expanded CD8⁺ TILs were capable of cytotoxic functions and were successfully utilized to demonstrate the presence of immunogenic mutations in autologous TNBC tumor tissue without recognition of the wild-type counterpart. Our findings open the way for a successful adoptive immunotherapy for TNBC. Cancer Immunol Res; 5(6); 439-45. ©2017 AACR.

Figure 1. Addition of agonistic anti-4-1BB antibody allows for the generation of CD8⁺ TILs from TNBC tumor fragments

(A) The total number of TILs expanded from 7 TNBC patients, reported as per single-fragment growth, because diverse number of fragments were put in culture depending on the tumor sample size obtained post-surgery. The absolute number is shown as determined by trypan blue exclusion. Growth per patient (left) and per culture condition (right) is shown. (B) The percentage of CD8⁺ TILs (left) and CD4⁺ TILs (right) obtained after 28 days of culture with IL2 alone or with IL2 + anti-4-1BB from 6 independent lines.

Figure 2. Enhanced cytolytic function of TNBC TILs generated with anti-4-1BB

TNBC TILs grown with IL2 alone or with IL2 + anti-4-1BB for 28 days from 5 independent lines were cocultured with target P815 (DDAO-SE labeled) cells pulsed with anti-CD3 (OKT3, diamonds). P815 cells not pulsed with OKT3 were used as a negative control (squares). Cells were stained for active caspase-3 by flow cytometry as a measure of tumor killing by TIL effectors. Gating was performed on DDAO-SE–positive target cells to determine the level of active caspase-3 detection. The graphs show the percentage of caspase-3 positive tumor targets at decreasing TIL: tumor ratios. Each experiment was independently performed per patient.

Figure 3. CD8⁺ TILs from patients BC7 and BC9 recognize class I–restricted mutated peptides

(A) IFNγ ELISPOT showing BC7 TIL recognition of the mutant version (H-RAREEIVAHIAL-OH) of RNA-binding protein MEX3B (mutation E→V at position 7) but not the wild-type form nor the negative control HIV-gag. The quantification of BC7 ELISPOT reactivity against mutant MEX3B is shown as number of spots/10⁶ TILs. A TIL-alone control is also included; n = 2 independent experiments (B) IFNγ ELISPOT demonstrating BC9 TIL recognition of the mutant version (H-FAFVTENTY-OH) of G2/mitotic-specific cyclin-B1 (mutation D→E at position 6) as compared to the wild-type form. Quantification of BC9 ELISPOT reactivity against mutant cyclin-B1 is shown as number of spots/5×10⁵ TIL; n = 3 independent experiments. For A) and B) the average number of spot is shown per condition as a label over the bar graphs. (C) IFNγ intracellular staining of BC9 TILs stimulated with either WT or mutant cyclin-B1 showing that mutated G2/mitotic-specific cyclin-B1–specific CD8⁺ T cells constituted over 50% of BC9 TILs. IFNγ expression is gated on live, CD3⁺CD4⁻CD8⁺ TILs.