Anti-CD3 Fab Fragments Enhance Tumor Killing by Human γδ T Cells Independent of Nck Recruitment to the γδ T Cell Antigen Receptor

Abstract

T lymphocytes expressing the γδ T cell receptor (γδ TCR) can recognize antigens expressed by tumor cells and subsequently kill these cells. γδ T cells are indeed used in cancer immunotherapy clinical trials. The anti-CD3ε antibody UCHT1 enhanced the in vitro tumor killing activity of human γδ T cells by an unknown molecular mechanism. Here, we demonstrate that Fab fragments of UCHT1, which only bind monovalently to the γδ TCR, also enhanced tumor killing by expanded human Vγ9Vδ2 γδ T cells or pan-γδ T cells of the peripheral blood. The Fab fragments induced Nck recruitment to the γδ TCR, suggesting that they stabilized the γδ TCR in an active CD3ε conformation. However, blocking the Nck-CD3ε interaction in γδ T cells using the small molecule inhibitor AX-024 neither reduced the γδ T cells' natural nor the Fab-enhanced tumor killing activity. Likewise, Nck recruitment to CD3ε was not required for intracellular signaling, CD69 and CD25 up-regulation, or cytokine secretion by γδ T cells. Thus, the Nck-CD3ε interaction seems to be dispensable in γδ T cells.

Keywords: AX-024; Fab fragments; Nck; T cell antigen receptor; activation; signaling; tumor; γδ T cells.

Figures

Figure 1

UCHT1 increased tumor killing by γδ T cells. 51Cr-labeled Daudi and Raji cells were incubated with zoledronate-expanded human γδ T cells without (−) or with the addition of 5 µg/ml UCHT1 using an effector to target (T cell to tumor cell) ratio of 12.5:1. After 5 h of co-culture, the amount of released 51Cr was measured in the supernatant by γ-ray spectroscopy. Data represent mean ± SD of triplicate wells (n = 3). Significance was determined by unpaired t-test, two-tailed between untreated γδ T cells and treated samples.

Figure 2

Using its SH3.1(Nck) domain Nck binds to the UCHT1-stimulated γδ T cell antigen receptor (TCR). (A) Schematic of the SH3.1(Nck) pull down assay. (B) Zoledronate-expanded γδ T cells were left untreated (−) or stimulated with 5 µg/ml UCHT1 at 37°C for 5 min. The cellular lysates were incubated with SH3.1(Nck)-coupled beads and bound proteins separated by SDS-PAGE along with aliquots of the cellular lysate. Western blotting was done using anti-CD3ζ and anti-GST antibodies. (C) The normalized ratio of the band intensities of CD3ζ to GST in the SH3.1(Nck) pull down is plotted. Data of three independent experiments were used to calculate the mean ± SD; statistics was done by two-tailed t-test. (D) Schematic of the in situ proximity ligation assay (PLA) using anti-CD3ε and anti-Nck antibodies. (E) Close proximity between the TCR and Nck was detected by in situ PLA. Zoledronate-expanded γδ T cells were either left untreated (−) or treated with 5 µg/ml UCHT1 in the absence or presence of 10 nM AX-024 at 37°C for 5 min. After fixation and permeabilization, PLA was performed using the primary antibodies goat anti-CD3ε (M20) and rabbit anti-Nck1, and the corresponding secondary antibodies. Nuclei were stained with DAPI. (F) The corresponding quantification of the red PLA dots and the mean ± SEM is displayed; statistics was done by two-tailed t-test. For each condition 500 cells were analyzed. Three independent experiments were performed (n = 3).

Figure 3

Preparation of UCHT1 Fab fragments. (A) The anti-CD3ε antibody UCHT1 was digested with papain using a kit from Thermo Fisher Scientific. The Fab fragments were purified with protein A coupled beads. The complete antibody (lane 1), the first and second digestions (lanes 2 and 3), and the purified Fab fragment (lane 4) were separated by reducing SDS-PAGE followed by Coomassie staining (n > 3). (B) Jurkat Vγ9Vδ2 cells were labeled with Fluo-3 AA and Fura Red AM and the baseline of cellular Ca2+ was measured by flow cytometry for 1 min. The indicated reagents were then added and Ca2+ levels were measured for additional 6 min (black lines). The gray lines represent baseline Ca2+ level without addition of any stimuli (n > 3). (C) Concanavalin A expanded γδ T cells were stained with UCHT1, Fab, Fabred, or left untreated, followed by an APC-labeled anti-mouse IgG antibody as a secondary reagent. Fluorescence intensities were quantified by flow cytometry (n > 3).

Figure 4

Fab and Fabred fragments enhanced tumor killing by γδ T cells. 51Cr-labeled Daudi and Raji cells were incubated with zoledronate (A) or concanavalin A (B) expanded γδ T cells in triplicates without (−) or with 5 µg/ml UCHT1, 3.33 µg/ml Fab, or 3.33 µg/ml Fabred (these concentrations were chosen to obtain equimolar amounts of the different reagents). The effector to target ratio was 12.5:1. After 5 h of co-culture, the amount of released 51Cr was measured in the supernatant by γ-ray spectroscopy (n = 3). (C) Concanavalin A expanded γδ T cells from four different donors (HD1, 2, 3, and 4) were used in a chromium-release assay using Daudi cells as target as in (A) (n = 1). Data represent mean ± SD of triplicate wells. Significance was determined by unpaired t-test, two-tailed between untreated γδ T cells and UCHT1, Fab or Fabred-treated samples.

Figure 5

UCHT1 Fab induced the recruitment of Nck to the γδ T cell antigen receptor. (A) The proximity ligation assay as in Figure 2 was performed using 5 µg/ml UCHT1, 3.33 µg/ml Fab, and 3.33 µg/ml Fabred in the absence or presence of 10 nM AX-024. (B) The data of (A) were analyzed as in Figure 2F; 700 cells were analyzed per condition. Two independent experiments were performed (n = 2).

Figure 6

Tumor killing by γδ T cells is independent of Nck recruitment to the γδ T cell antigen receptor. 51Cr-labeled Daudi cells were incubated with concanavalin A (A) or zoledronate (B) expanded γδ T cells without (−) or with 5 µg/ml UCHT1, 3.33 µg/ml Fab, or 3.33 µg/ml Fabred. In addition, 10 or 100 nM of AX-024 was included or not. The effector to target ratio was 12.5:1. After 5 h of co-culture, the amount of released 51Cr was measured in the supernatant by γ-ray spectroscopy. Data represent mean ± SD of triplicates (n = 3). Significance was determined by unpaired t-test, two-tailed between untreated samples and samples treated with AX-024.

Figure 7

The up-regulation of activation markers and cytokines is independent of Nck recruitment to the γδ T cell antigen receptor (TCR). Daudi and Raji cells were incubated with zoledronate-expanded γδ T cells without (−) or with 5 µg/ml UCHT1, 3.33 µg/ml Fab, or 3.33 µg/ml Fabred. In addition, 10 nM AX-024 was included or not. The γδ T cell to target cell ratio was 12.5:1. The cells were co-cultured at 37°C and 5% CO2 for 18 h. Subsequently, cells were either stained with anti-CD25 and anti-γ/δTCR (A,B) or with anti-CD69 and anti-γ/δTCR (C) antibodies. Fluorescence intensities were quantified by flow cytometry (A) and the mean fluorescence intensity is displayed (B,C). Data represent mean ± SD of triplicates (n = 3). Significance was determined by unpaired t-test, two-tailed between untreated samples and samples treated with AX-024. (D,E) The experiments were performed as in (A). After co-culturing of the cells for 18 h, the supernatants were harvested. TNFα (D) and IFNγ (E) concentrations were quantified by enzyme linked immunosorbent assay.

Figure 8

Calcium signaling is enhanced by Fab fragments and does not require the Nck-γδ T cell antigen receptor (TCR) interaction. (A) G8 γδ TCR-expressing Jurkat cells were stained with the γδ TCR-specific antibody GL3 and analyzed by flow cytometry. As controls, the GL3-stained parental Jurkat cells and unstained G8 γδ TCR-expressing cells are shown (n > 3). (B) Jurkat cells expressing the G8 γδ TCR were stimulated with the G8 ligand T22 tetramers (T22t), UCHT1 Fab, or a combination of both T22t and Fab. Intracellular calcium was measured using the dyes Fluo-3 and Fura-red by flow cytometry. Calcium influx is depicted as the median ratio of Fluo-3 to Fura-red fluorescence over time. As a negative control, steptavidin was added to the cells. The arrow indicates addition of the stimuli (n = 3). (C) The experiment was performed as in (C) with the difference that cells without and with 10 nM AX-024 were stimulated with the combination of T22t and Fab. As a control, PBS was added to the cells (n = 3).