Targeting the Epidermal Growth Factor Receptor Can Counteract the Inhibition of Natural Killer Cell Function Exerted by Colorectal Tumor-Associated Fibroblasts.

Mesenchymal stromal cells (MSC) present in the tumor microenvironment [usually named tumor-associated fibroblasts (TAF)] can exert immunosuppressive effects on T and natural killer (NK) lymphocytes, favoring tumor immune escape. We have analyzed this mechanism in colorectal carcinoma (CRC) and found that co-culture of NK cells with TAF can prevent the IL-2-mediated NKG2D upregulation. This leads to the impairment of NKG2D-mediated recognition of CRC cells, sparing the NK cell activation through DNAM1 or FcγRIIIA (CD16). In situ, TAF express detectable levels of epidermal growth factor receptor (EGFR); thus, the therapeutic anti-EGFR humanized antibody cetuximab can trigger the antibody-dependent cellular cytotoxicity of TAF, through the engagement of FcγRIIIA on NK cells. Importantly, in the tumor, we found a lymphoid infiltrate containing NKp46+CD3- NK cells, enriched in CD16+ cells. This population, sorted and cultured with IL-2, could be triggered via CD16 and via NKG2D. Of note, ex vivo NKp46+CD3- cells were able to kill autologous TAF; in vivo, this might represent a control mechanism to reduce TAF-mediated regulatory effect on NK cell function. Altogether, these findings suggest that MSC from the neoplastic mucosa (TAF) of CRC patients can downregulate the immune cell recognition of CRC tumor cells. This immunosuppression can be relieved by the anti-EGFR antibody used in CRC immunotherapy.

Zoledronate can induce colorectal cancer microenvironment expressing BTN3A1 to stimulate effector γδ T cells with antitumor activity.

Amino-bis-phosphonates (N-BPs) such as zoledronate (Zol) have been used in anticancer clinical trials due to their ability to upregulate pyrophosphate accumulation promoting antitumor Vγ9Vδ2 T cells. The butyrophilin 3A (BTN3A, CD277) family, mainly the BTN3A1 isoform, has emerged as an important structure contributing to Vγ9Vδ2 T cells stimulation. It has been demonstrated that the B30.2 domain of BTN3A1 can bind phosphoantigens (PAg) and drive the activation of Vγ9Vδ2 T cells through conformational changes of the extracellular domains. Moreover, BTN3A1 binding to the cytoskeleton, and its consequent membrane stabilization, is crucial to stimulate the PAg-induced tumor cell reactivity by human Vγ9Vδ2 T cells. Aim of this study was to investigate the relevance of BTN3A1 in N-BPs-induced antitumor response in colorectal cancer (CRC) and the cell types involved in the tumor microenvironment. In this paper, we show that (i) CRC, exposed to Zol, stimulates the expansion of Vδ2 T lymphocytes with effector memory phenotype and antitumor cytotoxic activity, besides sensitizing cancer cells to γδ T cell-mediated cytotoxicity; (ii) this effect is partially related to BTN3A1 expression and in particular with its cellular re-distribution in the membrane and cytoskeleton-associated fraction; (iii) BTN3A1 is detected in CRC at the tumor site, both on epithelial cells and on tumor-associated fibroblasts (TAF), close to areas infiltrated by Vδ2 T lymphocytes; (iv) Zol is effective in stimulating antitumor effector Vδ2 T cells from ex-vivo CRC cell suspensions; and (v) both CRC cells and TAF can be primed by Zol to trigger Vδ2 T cells.

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

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-ß 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 ß 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.

High ERp5/ADAM10 expression in lymph node microenvironment and impaired NKG2D ligands recognition in Hodgkin lymphomas.

Herein we describe that in classic Hodgkin lymphomas (cHL, n = 25) the lymph node (LN) stroma displayed in situ high levels of transcription and expression of the disulfide-isomerase ERp5 and of the disintegrin-metalloproteinase ADAM10, able to shed the ligands for NKG2D (NKG2D-L) from the cell membrane. These enzymes were detected both in LN mesenchymal stromal cells (MSCs) and in Reed-Sternberg (RS) cells; in addition, MIC-A and ULBP3 were present in culture supernatants of LN MSCs or RS cells. NKG2D-L-negative RS cells could not be killed by CD8(+)αßT or γδT cells; tumor cell killing was partially restored by treating RS cells with valproic acid, which enhanced NKG2D-L surface expression. Upon coculture with LN MSCs, CD8(+)αßT and γδT cells strongly reduced their cytolytic activity against NKG2D-L(+) targets; this seems to be the result of TGF-ß, present at the tumor site, produced in vitro by LN MSCs and able to down-regulate the expression of NKG2D on T lymphocytes. In addition, CD8(+)αßT and γδT cells from the lymph nodes of cHL patients, cocultured in vitro with LN MSCs, underwent TGF-ß-mediated down regulation of NKG2D. Thus, in cHL the tumor microenvironment is prone to inhibit the development of an efficient antitumor response.

Imatinib treatment induces CD5+ B lymphocytes and IgM natural antibodies with anti-leukemic reactivity in patients with chronic myelogenous leukemia.

Imatinib mesylate is a first line treatment of Chronic Myelogenous Leukemia and of a rare form of gastrointestinal stromal cancer, where the response to the drug is also linked to the immune system activation with production of antineoplastic cytokines. In this study, forty patients in the chronic phase of disease, treated with imatinib mesylate, were analyzed. Bone marrow aspirates were drawn at diagnosis, after 3, 6, 12, 18 months for haematological, cytofluorimetric, cytogenetic, biomolecular evaluation and cytokine measurement. Responder and non responder patients were defined according to the European LeukemiaNet recommendations. In responder patients (n = 32), the percentage of bone marrow CD20(+)CD5(+)sIgM(+) lymphocytes, and the plasma levels of IgM, were significantly higher, at 3 months and up to 9 months, than in non responders. These IgM reacted with O-linked sugars expressed by leukemic cells and could induce tumor cell apoptosis. In responder patients the stromal-derived factor-1 and the B-lymphocyte-activating factor of the tumor necrosis factor family significantly raised in the bone marrow after imatinib administration, together with the bone morphogenetic proteins-2 and -7. All patients with high number of CD20(+)CD5(+)sIgM(+) cells and high stromal-derived factor-1 and B lymphocyte activating factor levels, underwent complete cytogenetic and/or molecular remission by 12 months. We propose that CD20(+)CD5(+)sIgM(+) lymphocytes producing anti-carbohydrate antibodies with anti-tumor activity, might contribute to the response to imatinib treatment. As in multivariate analysis bone marrow CD20(+)CD5(+)sIgM(+) cells and stromal-derived factor-1 and B-lymphocyte-activating factor levels were significantly related to cytogenetical and molecular changes, they might contribute to the definition of the pharmacological response.

Differential survival of γδT cells, αßT cells and NK cells upon engagement of NKG2D by NKG2DL-expressing leukemic cells.

Herein, we show that γδT, CD8(+) αßT lymphocytes and natural killer (NK) cells display a different sensitivity to survival signals delivered via NKG2D surface receptor. All the three effector cell populations activate Akt1/PKBalpha through the engagement of this molecule. Upon binding to leukemic cells expressing NKG2D ligands (NKG2DL), including chronic lymphocytic leukemias treated with transretinoic acid, most γδT (>60%) and half CD8(+) αßT cells (about 50%) received a survival signal, at variance with the majority of NK cells (>80%) that underwent apoptosis by day 5. Interestingly, oligomerization of NKG2D in γδT or CD8(+) αßT cells, led to a significant rise in nuclear/cytoplasmic ratio of both NF-kBp52 and RelB, the two NF-kB subunits mainly involved in the transcription of antiapoptotic proteins of the Bcl family. Indeed, the ratio between the antiapoptotic protein Bcl-2 or Bcl-x(L) and the proapoptotic protein Bax raised in γδT or CD8(+) αßT cells following NKG2D engagement by specific monoclonal antibodies or by NKG2DL expressing leukemic cells. Conversely, nuclear translocation of NF-kBp52 or RelB did not increase, nor the Bcl-2/Bax or the Bcl-x(L) /Bax ratios changed significantly, in NK cells upon oligomerizaton of NKG2D. Of note, transcripts for α5 importin, responsible for nuclear translocation of NF-kBp52/Rel B heterodimer, are significantly higher in γδT and CD8(+) αßT cells than in NK cells. These biochemical data may explain, at least in part, why γδT and CD8(+) αßT cells are cytolytic effector cells more resistant to target-induced apoptosis than NK cells.

Engagement of CD31 delivers an activating signal that contributes to the survival of chronic lymphocytic leukaemia cells.

The present study showed that engagement of CD31 delivers a survival signal in chronic lymphocytic leukaemia (CLL) cells. We describe two groups of CLL, showing different kinetics of apoptosis in vitro and distinct ratios between anti-apoptotic and pro-apoptotic proteins: CLL-I displayed low Bcl-x(L) /Bax and Bcl-2/Bax ratio and underwent rapid apoptosis in vitro; CLL-II had high Bcl-x(L) /Bax and Bcl-2/Bax ratio and were resistant to apoptosis for several days. Nurse-like cells, expressing vimentin, CD68 and CD31 were detected mainly in CLL-II cultures. Of note, CD31 cross-linking, obtained with a specific monoclonal antibody (mAb), induced phosphatidylinositol-3-kinase-dependent Akt phosphorylation and nuclear translocation of the nuclear factor-kBp65 and p52 subunits in both CLL groups, leading to upregulation of Bcl-2 and Bcl-x(L) transcription and increased cell survival. Binding to CD31(+) stable transfectants, could also deliver an anti-apoptotic signal in B cells of both CLL-I and CLL-II, increasing the Bcl-2 and Bcl-x(L) protein content, regardless the expression of CD38. On the other hand, the addition of the F(ab')2 (that is unable to oligomerize the target molecule) of the anti-CD31 mAb prevented these effects. These data suggest that the CD31 adhesion system may play a role also in vivo in maintaining CLL survival.

Vdelta1 T lymphocytes producing IFN-gamma and IL-17 are expanded in HIV-1-infected patients and respond to Candida albicans.

In early HIV-1 infection, Vdelta1 T lymphocytes are increased in peripheral blood and this is related to chemokine receptor expression, chemokine response, and recirculation. Herein we show that, at variance with healthy donors, in HIV-1-infected patients ex vivo-isolated Vdelta1 T cells display cytoplasmic interferon-gamma (IFN-gamma). Interestingly, these cells coexpress cytoplasmic interleukin-17 (IL-17), and bear the CD27 surface marker of the memory T-cell subset. Vdelta1 T cells, isolated from either patients or healthy donors, can proliferate and produce IFN-gamma and IL-17 in response to Candida albicans in vitro, whereas Vdelta2 T cells respond with proliferation and IFN-gamma/IL-17 production to mycobacterial or phosphate antigens. These IFN-gamma/IL-17 double-producer gammadelta T cells express the Th17 RORC and the Th1 TXB21 transcription factors and bear the CCR7 homing receptor and the CD161 molecule that are involved in gammadelta T-cell transendothelial migration. Moreover, Vdelta1 T cells responding to C albicans express the chemokine receptors CCR4 and CCR6. This specifically equipped circulating memory gammadelta T-cell population might play an important role in the control of HIV-1 spreading and in the defense against opportunistic infections, possibly contributing to compensate for the impairment of CD4(+) T cells.

Adhesion molecules and kinases involved in gammadelta T cells migratory pathways: implications for viral and autoimmune diseases.

gammadelta T lymphocytes are involved in the defence from viral and mycobacterial infections; however they are also responsible for autoimmune reactions. Herein, we discuss the characteristics of these cells, focusing on the mechanism(s) underlying extravasation and tissue localization. We show that Vdelta1 and Vdelta2 gammadeltaT cells display differential expression of adhesion molecules and chemokine receptors, the former being preferentially PECAM-1(+)CXCR4(+), the latter expressing NKRP1A and CXCR3. The two cell populations transmigrate across endothelial cells by activation of distinct kinase pathways and in response to interferon-gamma-inducing protein-10 (IP-10/CXCL10) or stromal-derived factor-1 (SDF-1/CXCL12) according to the expression of the specific receptors CXCR3 and CXCR4. IP-10/CXCL10 and SDF-1/CXCL12-induced transmigration are phosphoinositide-3 kinase (PI-3K) and Akt/PKB-dependent. In addition, occupancy of CXCR3, but not of CXCR4, leads to CAMKII activation; blocking of CAMKII decreases IP-10/CXCL10 and 6Ckine/SLC/CCL21-driven transmigration. We report that HIV-1-infected patients have an increased number of circulating Vdelta1 T cells possibly due to the interference of Tat protein on the function of chemokine receptors. In turn, patients with relapsing-remitting multiple sclerosis (MS), display an increase in peripheral Vdelta2 gammadelta T cells and this is related to interleukin-12-mediated upregulation of NKRP1A. Finally, the possible role of gammadelta T lymphocytes in post-transplantation immune reconstitution is discussed.

Expansion of Vdelta1 T lymphocytes producing IL-4 in low-grade non-Hodgkin lymphomas expressing UL-16-binding proteins.

Data on 23 patients with low-grade non-Hodgkin lymphomas (NHLs), 4 mantle (MT), 4 marginal zone (MZ), and 15 follicular (FL), were analyzed and compared with 10 high-risk (HR) B-cell chronic lymphocytic leukemias (B-CLLs) with lymph node involvement and 4 diffuse large-cell lymphomas (DLCLs). A significant increase in circulating Vdelta1 T lymphocytes producing interleukin-4 (IL-4) was found in patients with FL, MT, and MZ NHL, at variance with DLCL and HR B-CLL. IL-4 was also detectable in the sera and lymph nodes of the same patients. In 19 of the 23 patients with NHL with increased circulating Vdelta1 T lymphocytes, B cells expressing the UL-16-binding proteins (ULBPs) ULBP2 or ULBP3 or both were found in peripheral blood, bone marrow, or lymph nodes. Of note, in HR B-CLL or in DLCL, where leukemic cells were negative for ULBPs, no Vdelta1 T-cell increase was found. Moreover, Vdelta1 T lymphocytes from patients with FL NHL proliferate in response to ULBP2+ and ULBP3+ lymphoma cells. Finally, patients with high expression of ULBPs, increased circulating Vdelta1 T lymphocytes, and high levels of serum IL-4 showed stable disease in a 1-year follow-up in contrast to patients with low circulating Vdelta1 T cells and undetectable IL-4 or ULBPs.

Regulation of gammadelta T cell survival by soluble HLA-I: involvement of CD8 and activating killer Ig-like receptors.

We show that human Vdelta1 or Vdelta2 T lymphocytes secrete FasL and undergo apoptosis upon incubation with soluble HLA (sHLA)-I or after cross-linking of CD8, with a kinetics different from that observed following ligation of TCR. sHLA-I-induced apoptosis was blocked by anti-CD8 mAb; on the other hand, sHLA-I was not effective in CD8- clones, while an HLA-I mutated in the alpha3 domain, responsible for CD8 binding, was not functional on CD8+ clones. Purified sHLA-Cw3 or -Cw4 alleles, isolated from the Cw3- or Cw4-transfected 721.221 lymphoblastoid cell line, triggered gammadelta T cell apoptosis, interacting with the specific receptors CD158j/KIR2DS2 or CD158 h/KIR2DS1, respectively, also known as activating isoforms of killer Ig-like receptors (KIR). Again, this effect was dependent on FasL secretion and it was blocked by specific mAb to KIR2DS2 or KIR2DS1. The engagement of CD8 or activating KIR also triggered the production of TNF-alpha. Noteworthy, sHLA-I molecules synergize with antigen-mediated activation of Vdelta2 T cells: Indeed, Vdelta2 T lymphocytes produced TNF-alpha when stimulated with isopentenyl pyrophosphate, and this effect was enhanced by sHLA-I. These findings suggest that sHLA-I can regulate gammadelta T cell survival and that activating KIR may amplify antigen-specific Vdelta2 T cell responses.

Vdelta1 T lymphocytes from B-CLL patients recognize ULBP3 expressed on leukemic B cells and up-regulated by trans-retinoic acid.

We analyzed 38 untreated patients with chronic lymphocytic leukemia of B-cell type (B-CLL): 24 low-, 8 intermediate-, and 6 high-risk stage. In 15 patients (13 low risk and 2 intermediate risk), circulating Vdelta1 T lymphocytes were significantly increased (100 to 300 cells/muL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/muL). We studied these Vdelta1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor alpha or IFN-gamma in response to autologous leukemic B cells but not to normal lymphocytes. However, they were unable to kill resting autologous B cells, which lack the MHC-related MIC-A antigen and express low levels of the UL16-binding protein (ULBP) 3 and undetectable levels of ULBP1, ULBP2, and ULBP4. All these molecules are reported ligands for the NKG2D receptor, which is expressed by gammadelta T cells and activates their cytolytic function. The Vdelta1 T lymphocytes studied were able to lyse the ULBP3(+) C1R B-cell line upon transfection with MIC-A. More importantly, they also lysed autologous B-CLL cells when transcription and expression of MIC-A or up-regulation of ULBP3 were achieved either by activation or by exposure to trans-retinoic acid. The NKG2D receptor expressed on Vdelta1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vdelta1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vdelta1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vdelta1 T lymphocytes may play a role in limiting the progression of B-CLL.