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. 2011 Apr;34(3):236-50.
doi: 10.1097/CJI.0b013e318209e7ec.

Costimulation through the CD137/4-1BB pathway protects human melanoma tumor-infiltrating lymphocytes from activation-induced cell death and enhances antitumor effector function

Jessica Ann Hernandez-Chacon  1 , Yufeng Li, Richard C Wu, Chantale Bernatchez, Yijun Wang, Jeffrey S Weber, Patrick Hwu, Laszlo G Radvanyi
Affiliations

Costimulation through the CD137/4-1BB pathway protects human melanoma tumor-infiltrating lymphocytes from activation-induced cell death and enhances antitumor effector function

Jessica Ann Hernandez-Chacon et al. J Immunother. 2011 Apr.

Abstract

Adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) with high-dose interleukin-2 is a promising form of immunotherapy for stage IV melanoma having clinical response rates of 50% or more. One of the major problems preventing further success of this therapy is that the current protocols used to highly expand TIL for infusion drive CD8(+) T cells to differentiate into effector cells losing key costimulatory molecules such as CD28 and CD27. This has been associated with a lack of persistence in vivo for reasons not entirely clear. In this study, we demonstrate that while human melanoma CD8(+) TIL lost CD27 and CD28 expression during the rapid expansion for ACT, they gained expression of the alternative costimulatory molecule CD137/4-1BB, and to a lesser extent CD134/OX40. Postrapid expansion protocol (REP) TIL were found to be highly sensitive to activation-induced cell death when reactivated through the T-cell receptor with low levels of OKT3 antibody. However, coligation of 4-1BB using 2 different agonistic anti-4-1BB antibodies potently prevented activation-induced cell death of post-REP CD8(+) TIL, including those specific for melanoma antigen recognized by T cells, and facilitated even further cell expansion. This was correlated with increased levels of bcl-2 and bcl-xL together with decreased bim expression. 4-1BB costimulated post-REP TIL also expressed increased levels of the cytolytic granule proteins and exhibited enhanced cytotoxic T-cell activity against melanoma cells. Lastly, post-REP CD8(+) TIL were protected from cell death by anti-4-1BB ligation when exposed to human leukocyte antigen-matched melanoma cells. Our results indicate that 4-1BB costimulation may significantly improve TIL survival during melanoma ACT and boost antitumor cytolytic activity.

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Figures

Fig. 1
Fig. 1. Post-REP and re-activated TIL exhibit up-regulation of 4-1BB
TIL were stained for CD8, CD27, CD28, 4-1BB, or OX40 expression before and following the REP, or 24 h after TCR re-stimulation of post-REP TIL and post-REP TIL with OKT3. A, Staining of post-REP TIL from two representative patients showing changes in 4-1BB expression in the total CD8+ TIL population or in the CD8+CD27−, CD8+CD27+, CD8+CD28−, and CD8+CD28+ subsets. In each case, live cells were gated based on forward scatter and side scatter profiles. B, TIL from different patients were stained for CD8 and 4-1BB (n=26) or CD8 and OX40 (n=19) expression at the pre-REP, post-REP, and 24 h after re-stimulation of post-REP TIL with OKT3. The percentage of 4-1BB+ or OX40+ cells in the gated CD8+ population is shown for each TIL line. C, TIL from HLA-A0201+ patients were stained pre-REP, post-REP and after re-stimulation with CD8, 4-1BB, MART-1 or CD8, OX40 and MART-1. The live cells were gated on CD8+MART-1+ and analyzed for the different positive percentages of 4-1BB or OX40. The black bars indicate the average for all of the TIL lines for each marker. Statistical analysis was done using the Student’s t-test.
Fig. 2
Fig. 2. Induction of apoptosis and loss of viable cell recovery of post-REP CD8+ TIL after re-stimulation with OKT3 (OKT3)
Post-REP TIL were isolated and re-stimulated with plate-bound OKT3 and analyzed for apoptosis and viable cell recovery in the CD8+ TIL population. A, TIL were analyzed for apoptosis by staining with 7-AAD and Annexin V three days after re-stimulation with the indicated concentrations of plate-bound OKT3; this time interval was when maximal apoptosis was found in previous experiments (data not shown). OKT3 at 10 ng/ml was found to induce maximal apoptosis and was used in all subsequent experiments. B, Post-REP TIL (2 × 106 cells) from the indicated TIL lines were re-stimulated with 10 ng/ml OKT3 and recovery of viable CD8+ TIL determined after 7 days.
Fig. 3
Fig. 3. 4-1BB co-ligation during post-REP TIL re-stimulation with OKT3 inhibits induction of apoptosis
Multiple post-REP TIL lines were re-stimulated with coated OKT3 with or without anti-4-1BB antibody. On day 3 of the re-stimulation, apoptosis was measured using Annexin V and 7AAD staining in the CD8+ subset. A, Flow cytometry profiles of 7-AAD versus Annexin V of post-REP TIL lines re-stimulated for 3 days with or without the indicated concentrations of anti-4-1BB (4-1BB/1). Results with two representative TIL lines shown. Optimal protection from apoptosis was found at 30 ng/ml for 4-1BB/1. B, The experiment was repeated with the same TIL lines using 4-1BB/2 with optimal protection found at 10 ng/ml 4-1BB/2. C. Inhibition of OKT3-induced apoptosis by 4-1BB/1 (30 ng/ml) co-ligation in multiple patient post-REP TIL (n=13). D. Inhibition of OKT3-induced apoptosis by 4-1BB/2 (10 ng/ml) co-ligation in multiple patient post-REP TIL (n=7). The black bars indicate the average for all of the TIL lines for each marker. Statistical analysis was performed using the Student t-test.
Fig, 4
Fig, 4. Continued expansion of CD8+ post-REP TIL re-stimulated with 4-1BB co-stimulation
Post-REP TIL (3 × 106 per condition) were re-activated using OKT3 as before with or without anti-4-1BB antibody in 4 replicate wells. Viable cell numbers were determined on day 7 by hemocytometer counts after Trypan Blue staining of each well and flow cytometry analysis was performed to determine percentage of CD8+ T cells in the viable lymphocyte gate. CD8+ T-cell recovery was determined by multiplying the percentage of CD8+ T-cell found by flow cytometry by the viable cell count. CD8+ TIL recovery after re-stimulation with OKT3 together with different doses of 4-1BB/1 in TIL 2055A (A) or 4-1BB/2 in TIL 2060 (B) is shown. In each case an average of 2 cell counts per condition is shown. C and D, The experiment was repeated with 20 different patient TIL lines using 4-1BB/1 (C) and 10 different patient TIL lines using 4-1BB/2 (D). The black bars indicate the average for all of the TIL lines for each condition shown. Statistical analysis was performed using the Student t-test with biological relevance occuring when P<0.05.
Fig. 5
Fig. 5. Increased expansion and recovery of MART-1-specific CD8+ post-REP TIL following re-stimulation with 4-1BB co-stimulation
TIL from HLA-A0201+ patients that had a significant population of CD8+MART-1 tetramer+ T cells were subjected to the REP. The post-REP TIL were re-stimulated with or without OKT3, with or without anti-4-1BB (30 ng/ml 4-1BB/1) for 7 days in 4 replicate wells. Post-REP TIL re-stimulated with anti-CD28 was used as a control. On day 7 of the re-stimulation, viable cell counts were done using hemocytometer counts of each well and the recovered TIL were also stained and analyzed for MART-1 and CD8 using flow cytometry. The recovery of CD8+MART-1 tetramer+ T cells was then calculated for each of the re-stimulation conditions indicated. The results of two separate experiments with post-REP TIL lines from two patients is shown.
Fig. 6
Fig. 6. 4-1BB co-stimulation increases the relative expression of anti-apoptotic genes in post-REP TIL
Post-REP TIL lines from three separate patients were re-stimulated with OKT3 with or without anti-4-1BB antibody. On day 2, RNA was isolated and the expression of bcl-2, bcl-xL, and bim was determined using qRT-PCR. All qRT-PCR data for the different treatments indicated were normalized to the level of expression in post-REP TIL re-cultured with IL-2 alone (relative expression of 1). Each PCR reaction was ran in triplicate and the coefficient of variance (CV) was found to be <5%. A, Post-REP TIL from the indicated patients were re-stimulated with OKT3 (10 ng/ml) alone or with 30 ng/ml 4-1BB/1 (optimal concentration) or anti-CD28 (negative control). B, The same experiment was repeated with different doses of 4-1BB/2.
Fig. 7
Fig. 7. Increased antigen-specific CTL activity in post-REP TIL re-stimulated with anti-4-1BB co-stimulation without a change in antigen-specific T-cell frequency
Post-REP TIL from HLA-A0201+ patients were re-stimulated as before with or without 4-1BB/1 antibody or with anti-CD28 (negative control). After 7 days, CTL activity was monitored using a flow cytometry-based assay measuring the cleavage of caspase-3 in target cells as readout, as described in the Materials and Methods. A, CTL activity was determined in two separate patient TIL lines (#2199 and #2105) against HLA-A0201+ 624 melanoma targets (graphs on left hand side), or against an HLA unmatched melanoma cell line 938 as negative controls (graphs on right hand side). B, CTL activity from TIL line #2009 was assayed using MART-1 peptide-pulsed T2 target cells (graphs on left hand side), or T2 cells pulsed with HIV rev peptide as the negative control (graphs on right hand side). Fig. S2 (Supplementary Data online) is accompanying data to this figure showing that the frequency of CD8+ and CD8+MART-1 tetramer+ T cells isolated from all the treatment groups was similar.
Fig. 8
Fig. 8. GB and Perf expression in re-stimulated post-REP CD8+ TIL is increased by 4-1BB co-stimulation
Post-REP TIL were re-stimulated with OKT3 with or without anti-4-1BB antibody as before. On day 7 after the re-stimulation, phenotypic analysis of the live CD8+ T cells for Perf expression and GB expression was determined by flow cytometry analysis. Results of post-REP TIL from three different patients (#2199, #2198, and #2206) are shown. A, The frequency (left hand panels) and MFI (right hand panels) of GB+ expression in the TIL lines following re-stimulation with OKT3 with or without 4-1BB/1 (30 ng/ml). B, An analysis of Perf+ expression was similarly performed on the three TIL lines. In each case, 4-1BB co-stimulation markedly increased both the percentage of CD8+ TIL expressing Granzyme B and Perforin as well as the MFI for both proteins.
Fig. 9
Fig. 9. Increased IFN-γ production by post-REP TIL re-stimulated together with co-stimulation through 4-1BB
Post-REP TIL were re-activated with IL-2 alone, or with OKT3 with or without anti-4-1BB/1 antibody or 4-1BB/2. On day 7, the culture supernatants were collected and the concentration of IFN-γ determined using an ELISA. Results with post-REP TIL from 3 different patients (#2117, #2170 and #2228) are shown for co-stimulation with 4-1BB/1 at 30 ng/ml (A) or with the indicated concentrations of 4-1BB/2 (B). TILs re-stimulated with 4-1BB produced more IFN-γ than TILs stimulated with OKT3 (A). All assays were done in triplicate with averages and standard deviation shown.
Fig. 10
Fig. 10. Reduced apoptosis of antigen-specific CD8+ TIL co-incubated with melanoma cells expressing 4-1BB ligand
Post-REP TIL from two HLA-A0201+ patients containing MART-1-specific CD8+ T cells were co-incubated with vector control-transduced 624 melanoma cells (MART-1 expressing) or 624 melanoma cells transduced with 4-1BBL at a 1:1 ratio. After 3 days, the cells were stained and gated on CD8+ and MART-1+ tetramer, and apoptosis was measured using Annexin V plus 7-AAD staining. The same TIL were also co-incubated in parallel with vector-transduced 624 cells with 3 or 10 ng/ml of 4-1BB/2.
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