4-1BB-4-1BBL cis-interaction contributes to the survival of self-reactive CD8+ T cell

Abstract

4-1BB is an inducible receptor expressed on activated T cells, while its ligand, 4-1BBL, is mainly expressed in antigen-presenting cells and macrophages [ 1 , 2 ]. To the best of our knowledge, ligand-mediated transactivation of 4-1BB is responsible for the survival and immune effector functions of T cells. However, there have been reports of 4-1BBL also being expressed in T cells [ 3 , 4 ]. As 4-1BB has only one known ligand, 4-1BBL, this coexpression of 4-1BB and 4-1BBL on T cells raises questions about cis -interactions among the proteins and their potential role in T-cell activation and immune function. Therefore, to investigate whether 4-1BB and 4-1BBL signals are necessary to suppress tumor growth, we designed an experiment as shown in Fig. 1a . In vivo administration of the 3H3 clone strongly triggers 4-1BB signals along with no or minor 4-1BBL signals, TKS-1 induces a mild agonistic effect on 4-1BBL with blockade of 4-1BB signals, 17B5 blocks both 4-1BB and 4-1BBL signals, and coadministration of 3H3 and TKS-1 enhances both 4-1BB and 4-1BBL signals [ 5 , 6 , 7 ]. C57BL/6 mice received mAbs intraperitoneally every 5 days starting from 9 days after MC38 tumor injection (Fig. 1a ). Compared to rat IgG treatment, 17B5 treatment did not affect the tumor growth rate, but it did increase mortality (Fig. 1b ). While TKS-1 treatment alone was ineffective, the combination treatment of 3H3 and TKS-1 effectively suppressed tumor growth (Fig. 1b ). The percentage and absolute number of tumor-specific pGP70 + CD8 + T cells were significantly higher in the inguinal tumor-draining lymph nodes (iTDLNs) of mice treated with 3H3 alone or with TSK-1 in combination with 3H3 than in those treated with rat IgG, TKS-1, or 17B5 alone (Fig. 1c, d ). The results suggest that the combination of 3H3 and TKS-1 enhances tumor-specific CD8 + T-cell responses in MC38 tumor-bearing mice. Fig. 1 Anti-4-1BBL enhances 4-1BB-stimulated antitumor responses: ( a ) C57BL/6 mice were s.c. injected with MC38 tumor cells and i.p. administered 100 μg of mAbs on the indicated days. b Tumor growth rate. c iTDLN cells were counted and stained with pGP70-PE and anti-CD8β-PE-Cy5. d The absolute numbers of total and pGP70 + CD8 + T cells in the inguinal TDLN were calculated. 4-1BBL promotes LN migration and tumor infiltration of activated pmel-1 CD8 + T cells: ( e ) Schematic diagram of the experiment. f Single-cell suspensions of inguinal LNs at Day 7 were counted and stained as described above. Gated CD8 + cells were plotted CFSE vs. Thy1.1. g Absolute numbers of inguinal LN cells and percentages and absolute numbers of pmel-1 Thy1.1 + CD8 + T cells in inguinal LNs ( n = 4 mice per experiment from three independent experiments). h Single-cell suspensions of tumor tissues at Day 12 were counted and stained with anti-CD8-PE, anti-Thy1.1-PE-Cy5, and anti-CD45-APC antibodies, and the percentages of CD45 + TILs and Thy1.1 + and Thy1.1 − cells in CD45 + TILs and tumor tissues were calculated ( i ). Peripheral 4-1BBL is required to induce migration of activated pmel-1 CD8 + T cells: ( j ) WT, 4-1BB −/− , and 4-1BBL −/− C57BL/6 mice were i.v. injected with CFSE-labeled pme-1 Thy1.1 + CD8 + T cells, s.c. immunized with 20 μg mgp100 peptide in IFA and i.p. administered 100 μg of rat IgG, 3H3, and 3H3 plus TKS-1 on Days 0 and 2. On Day 7, iTDLN cells were counted and stained with anti-CD8-PE and anti-Thy1.1-PE-Cy5 antibodies. Gated CD8 + cells were plotted CFSE vs. Thy1.1. k Absolute numbers of iTDLN cells. Percentages and absolute numbers of pmel-1 Thy1.1 + CD8 + T cells in iTDLNs. ( n = 6 mice per experiment from two independent experiments). Cellular division and migration of WT, 4-1BB −/− , and 4-1BBL −/− pmel-1 CD8 + T cells: ( l ) C57BL/6 mice were i.v. injected with CFSE-labeled WT, 4-1BB −/− , and 4-1BBL −/− pme-1 Thy1.1 + CD8 + T cells and further immunized and administered with Abs as described. m Single-cell suspensions of iTDLNs at Day 7 were counted and stained with anti-CD8-PE and anti-Thy1.1-PE-Cy5 antibodies. Gated CD8 + cells were plotted CFSE vs. Thy1.1. n Absolute numbers of iTDLN cells and percentages and absolute numbers of pmel-1 Thy1.1 + CD8 + T cells in the TDLN were calculated. ( n = 4 mice per experiment from three independent experiments). Data are presented as the mean ± SD. p values were calculated using Student’s t test (* p < 0.05; ** p < 0.01; *** p < 0.005) Full size image

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