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. 2014 Jan;99(1):131-9.
doi: 10.3324/haematol.2013.097311. Epub 2013 Oct 25.

Aminobisphosphonates prevent the inhibitory effects exerted by lymph node stromal cells on anti-tumor Vδ 2 T lymphocytes in non-Hodgkin lymphomas

Alessandra Musso  1 , Silvia Catellani, Paolo Canevali, Sara Tavella, Roberta Venè, Silvia Boero, Ivana Pierri, Marco Gobbi, Annalisa Kunkl, Jean-Louis Ravetti, Maria Raffaella Zocchi, Alessandro Poggi
Affiliations

Aminobisphosphonates prevent the inhibitory effects exerted by lymph node stromal cells on anti-tumor Vδ 2 T lymphocytes in non-Hodgkin lymphomas

Alessandra Musso et al. Haematologica. 2014 Jan.

Abstract

In this study, we analyzed the influence of mesenchymal stromal cells derived from lymph nodes of non-Hodgkin's lymphomas, on effector functions and differentiation of Vdelta (δ)2 T lymphocytes. We show that: i) lymph-node mesenchymal stromal cells of non-Hodgkin's lymphoma inhibit NKG2D-mediated lymphoid cell killing, but not rituximab-induced antibody-dependent cell-mediated cytotoxicity, exerted by Vδ2 T lymphocytes; ii) pre-treatment of mesenchymal stromal cells with the aminobisphosphonates pamidronate or zoledronate can rescue lymphoma cell killing via NKG2D; iii) this is due to inhibition of transforming growth factor-β and increase in interleukin-15 production by mesenchymal stromal cells; iv) aminobisphosphonate-treated mesenchymal stromal cells drive Vδ2 T-lymphocyte differentiation into effector memory T cells, expressing the Thelper1 cytokines tumor necrosis factor-α and interferon-γ. In non-Hodgkin's lymphoma lymph nodes, Vδ2 T cells were mostly naïve; upon co-culture with autologous lymph-node mesenchymal stromal cells exposed to zoledronate, the percentage of terminal differentiated effector memory Vδ2 T lymphocytes increased. In all non-Hodgkin's lymphomas, low or undetectable transcription of Thelper1 cytokines was found. In diffused large B-cell lymphomas and in a group of follicular lymphoma, transcription of transforming growth factor β and interleukin-10 was enhanced compared to non-neoplastic lymph nodes. Thus, in non-Hodgkin lymphomas mesenchymal stromal cells interfere with Vδ2 T-lymphocyte cytolytic function and differentiation to Thelper1 and/or effector memory cells, depending on the prominent in situ cytokine milieu. Aminobisphosphonates, acting on lymph-node mesenchymal stromal cells, can push the balance towards Thelper1/effector memory and rescue the recognition and killing of lymphoma cells through NKG2D, sparing rituximab-induced antibody-dependent cell-mediated cytotoxicity.

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Figures

Figure 1.
Figure 1.
N-BPs prevent the inhibition of NKG2D-mediated lymphoid cell killing and NKG2D downregulation induced by LNMSC on Vδ2 T cells. Vδ2 T cells isolated from PBMC were cultured with IL2 alone (white columns) or with IL2 and either untreated LNMSC or LNMSC pre-treated for 12 h with Pam (5 μg/mL) or Zol (1 μg/mL) at the 1:10 LNMSC:Vδ2 T ratio (gray columns). Some samples were treated for 48 h with mevastatin (Meva, 10 μM) and Zol added during the last 12 h. In some experiments, Vδ2 T cells were pre-treated with Pam or Zol in order to exclude any possible direct effect of NBPs on these cells. On Day 5, Vδ2 T cells were harvested and used in a redirected killing assay against the P815 cell line in the presence of the anti-NKG2D or the anti-CD16 mAb or an unrelated mAb (UnmAb) matched for the isotype (all at 5 μg/mL) (A), or in an ADCC assay against the C1R (B), or the C1R-MICA+ lymphoid cell lines (C), in the presence of the anti-CD20 therapeutic antibody rituximab (20 μg/mL, RTX). In some experiments, Vδ2 T cells were exposed to saturating amounts (5 μg/mL) of anti-NKG2D mAb before adding the C1R or the C1R-MICA+ targets The effector-target (E:T) ratio was 10:1. Target cells were labeled with 51Cr and used in a standard 4-h cytolytic assay. Results are shown as percentage of 51Cr specific release and are the mean±SD from 6 experiments. (A) *P<0.005 versus Vδ2 T cells without mAb or with UnmAb; **P<0.001 versus NKG2D-triggered Vδ2 T cells not co-cultured with LNMSC; ***P<0.001 versus Vδ2 T cells co-cultured with untreated LNMSC. (B and C) *P<0.001 versus Vδ2 T cells not co-cultured with LNMSC or in the absence of mAbs; **P<0.001 versus Vδ2 T cells after co-culture with LNMSC. (D) Transcription of NKG2D in Vδ2 T cells before (white column) or after (gray columns) co-culture with LNMSC either untreated or treated with Pam (5 μg/mL) or Zol (1 μg/mL) or Meva (10 μM), as indicated. Q-RT-PCR was performed with the fluorescent Taqman method. mRNAs were normalized to RPLP0 as a control gene and referred to a standard curve. After subtracting the threshold cycle (CT) value for RPLP0 from the CT values of the target genes, results were expressed as ΔCT. *P<0.001 versus Vδ2 T cells not co-cultured with LNMSC (white columns); **P<0.001 versus Vδ2 T cells co-cultured with LNMSC, with or without Meva (light gray columns). (E) FACS analysis of expression of NKG2D or CD16 by immunofluorescence using the specific mAbs followed by isotype specific PE-GAM before (dark gray) or after (gray) co-culture with LNMSC either untreated (medium) or treated with Pam (5 μg/mL) or Zol (1 μg/mL). Empty histograms show Vδ2 cells stained with an unrelated mAb (un-mAb) followed by PE-GAM (histograms of Vδ2 T cells from LNMSC co-cultures stained with un-mAb were exactly superimposable; data not shown). Results are expressed as log red fluorescence intensity (a.u.) versus number of cells and are from one representative experiment out of 6. (F) Vδ2 T cells harvested from the indicated culture conditions were analyzed for NKG2D expression. In some experiments cultures were performed with an anti-TGFβ mAb (5 μg/mL) as indicated. Results are shown as mean fluorescence intensity (MFI) of NKG2D expression and are the mean±SD from 6 experiments *P<0.005 versus Vδ2 T cells not co-cultured with LNMSC (white column); **P<0.001 versus Vδ2 T cells after co-culture with LNMSC. Statistical analysis: two-tailed Student’s t-test.
Figure 2.
Figure 2.
Effects of N-BPs on TGFβ or IL15 expression in LNMSC and IL10, TNFα, IFNγ production in Vδ2 T cells. Cultures of LNMSC or Vδ2 T cells alone or co-cultures of LNMSC and Vδ2 T cells (at 1:10 LNMSC: Vδ2 T-cell ratio were performed in medium with IL2. In some experiments, LNMSC were pre-treated for 12 h with Pam (5 μg/mL) or Zol (1 μg/mL), as indicated On Day 5, LNMSC (adherent cells) (A and B) or Vδ2 T cells (non-adherent cells) (E–G) were recovered and evaluation of mRNA transcription levels for TGFβ (A), IL15 (B), IL10 (E), TNFα (F) or IFNγ (G) was performed by Q-RT-PCR using specific probes with the fluorescent Taqman method. mRNAs were normalized to RPLP0 as a control gene and referred to a standard curve. After subtracting the threshold cycle (CT) value for RPLP0 from the CT values of the target genes, results were expressed as ΔCT. White columns: LNMSC (A and B) or Vδ2 T cells (E–G) cultured alone; gray columns LNMSC (A and B) or Vδ2 T cells (E–G) from LNMSC-Vδ2 T-cell co-cultures. Results are the mean±SD from 6 experiments. (A and B) *P<0.005 versus LNMSC alone; **P<0.001 versus LNMSC alone or versus untreated LNMSC co-cultured with Vδ2 T cells; ***P<0.005 versus untreated LNMSC. (E–G) *P<0.001 versus Vδ2 T cells alone. **P<0.001 versus LNMSC-Vδ2 T-cell co-cultures. TGFβ (C), IL-15 (D) measured by ELISA in the SN of LNMSC untreated (medium) or pre-treated with Pam (5 μg/mL) or Zol (1 μg/mL), as indicated. *P<0.001 versus untreated LNMSC (medium). TNFα (H) or IFNγ (I) measured by ELISA in the SN of Vδ2 T cells cultured alone (IL2) or co-cultured with untreated LNMSC (none) or LNMSC pre-treated with Pam or Zol. Results are expressed as pg/mL/106 cells and are the mean±SD from 6 experiments. *P<0.001 versus Vδ2 T cells alone (IL2); ** P<0.001 versus untreated LNMSC-Vδ2 T-cell co-cultures (none). Statistical analysis: two-tailed Student’s t-test.
Figure 3.
Figure 3.
N-BPs-treated LNMSC drive Vδ2 T lymphocyte to effector memory T cells. (A and B) Peripheral blood T lymphocytes (A) or CFSE-labeled purified Vδ2 T cells (B) were co-cultured with LNMSC, either untreated or pre-treated for 12 h with Pam (5 μM) or Zol (1 μM), as indicated. On Day 10, percentage of Vδ2 T cells was evaluated by immunofluorescence using the anti-Vδ2 mAb PE-BB3 and FACS analysis (A). Mean±SD from 4 experiments. *P<0.001 versus T or Vδ2 cells cultured on untreated LNMSC. (B) Proliferation was assessed on the basis of the decreased green (CFSEdull) fluorescence of dividing cells. Data were analyzed by the Modfit 3.1 computer program and expressed as cell number/generation of proliferating cells. (C and D) Immunophenotype of Vδ2 T cells cultured alone or co-cultured for 10 days with untreated or Pam or Zol pre-treated LNMSC, as indicated, stained with APC-anti-CD27 or PE-anti-CCR7 and FITC-anti-CD45RA. (C) Results are expressed as percentage of effector memory (EM) T cells, i.e. CD45RA-CD27- (white columns) or CD45RA-CCR7- cells (gray columns). Mean±SD from 6 experiments *P<0.001 versus Vδ2 T cells in the absence of LNMSC **P<0.001 versus Vδ2 T cells co-cultured with untreated LNMSC. (D) Vδ2 T cells alone (medium, left dot plots) or Vδ2 T cells co-cultured with untreated or treated LNMSC, as indicated, were stained as in (C). Results are expressed as log green fluorescence intensity (x-axis, MFI, a.u.) versus either log far red (first row) or red (second row) fluorescence intensity (y-axis, MFI, a.u.). One representative experiment out of 6 is shown. Percentages of positive cells in each quadrant show the naive (N, upper right, double positive), central memory (CM, upper left, CD45RA− and CD27+ or CCR7+), effector memory (EM, lower left, double negative) and terminally differentiated effector memory (TEMRA, lower right, CD45RA+ but CD27− or CCR7−) cells. Statistical analysis: two-tailed Student’s t-test.
Figure 4.
Figure 4.
Vδ2 T-lymphocyte phenotype in NHL in situ. (A and B) Cell suspensions obtained from 5 DLCL, 5 FL NHL and 3 non-neoplastic LN were analyzed for phenotype by FACS analysis. (A) Upper dot plots: cells stained with the VioBlue anti-CD3 and the FITC anti-Vδ2 mAbs. Lower dot plots: gate on Vδ2 T cells, double staining with PE-anti-CD45RA and APC anti-CD27 mAbs. One representative experiment; results are expressed as log violet (x-axis, MFI, a.u.) versus log green fluorescence intensity (first row) or log red (x-axis, MFI, a.u.) versus log far red (second row) fluorescence intensity (y-axis, MFI, a.u.). Percentages of positive cells in each quadrant of the lower row show the naive (N, upper right, double positive), central memory (CM, upper left, CD45RA−CD27+), effector memory (EM, lower left, double negative) and terminally differentiated effector memory (TEMRA, lower right, CD45RA+ CD27−) cells. (B) Results are expressed as percentage of naive (double positive) Vδ2 T cells; mean±SD from 3 non-neoplastic LN (dark gray columns), 5 FL (white columns) and 5 DLCL (gray columns) cell suspensions. *P<0.001 versus LN. (C) Cell suspensions from FL (white columns) or DLCL (gray columns) were co-cultured with autologous LNMSC, either untreated or pre-treated for 12 h with Zol (1 μM), as indicated. On Day 14, percentage of Vδ2 T cells was evaluated by immunofluorescence using the anti-Vδ2 mAb FITC-γδ123 and the VioBlue anti-CD3 and FACS analysis; mean±SD from 5 FL and 5 DLCL. *P<0.001 versus cell suspensions cultured with IL2 alone, or on untreated or Zol-treated LNMSC. (D and E) Immunophenotype of cells co-cultured for 14 days alone (IL2) or with untreated or Zol pre-treated LNMSC, stained with VioBlue anti-CD3, FITC anti-Vδ2 mAbs, APC-anti-CD27 and PE-anti-CD45RA. (D) One representative experiment, gate on Vδ2 T cells. Results are expressed as log red fluorescence intensity (x-axis, MFI, a.u.) versus log far red fluorescence intensity (y-axis, MFI, a.u.); percentage of CM, N, EM and TEMRA in each panel of the lower row. (E) Results are expressed as percentage of TEMRA Vδ2 T cells, i.e. CD45RA+CD27−. Mean±SD from 2 experiments with FL (white columns) and 2 with DLCL (gray columns) cell suspensions. *P<0.001 versus cells versus cells co-cultured with untreated LNMSC. Statistical analysis: two-tailed Student’s t-test.
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References

    1. Hayday AC. Gammadelta T cells and the lymphoid stress-surveillance response. Immunity. 2009;31(2):184–96 - PubMed
    1. Bonneville M, O’Brien RL, Born WK. Gammadelta T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol. 2010;10(7):467–78 - PubMed
    1. Das H, Wang L, Kamath A, Bukowski JF.V 9V 2 T-cell receptor-mediated recognition of aminobisphosphonates. Blood. 2001;98(5):1616–8 - PubMed
    1. Gober HJ, Kistowska M, Angman L, Jenö P, Mori L, De Libero G. Human T cell receptor gammadelta cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med. 2003;197(2):163–8 - PMC - PubMed
    1. Wang H, Sarikonda G, Puan K-J, Tanaka Y, Fenq J, Giner JL, et al. Indirect stimulation of human V 2V 2 T cells through alterations in isoprenoid metabolism. J Immunol. 2011;187(10):5099–113 - PMC - PubMed

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