Silvia Catellani

Vδ1 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 Vδ1 T lymphocytes were significantly increased (100 to 300 cells/μL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/μL). We studied these Vδ1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor α or IFN-γ 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 γδ T cells and activates their cytolytic function. The Vδ1 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 Vδ1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vδ1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vδ1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vδ1 T lymphocytes may play a role in limiting the progression of B-CLL.

Vδ1 T lymphocytes producing IFN-γ 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.

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(+)αβT 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(+)αβT and γδT cells strongly reduced their cytolytic activity against NKG2D-L(+) targets; this seems to be the result of TGF-β, 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(+)αβT and γδT cells from the lymph nodes of cHL patients, cocultured in vitro with LN MSCs, underwent TGF-β-mediated down regulation of NKG2D. Thus, in cHL the tumor microenvironment is prone to inhibit the development of an efficient antitumor response.

Lack of the leukocyte-associated Ig-like receptor-1 expression in high-risk chronic lymphocytic leukaemia results in the absence of a negative signal regulating kinase activation and cell division

In this study, we analysed 30 patients with B-cell chronic lymphocytic leukaemia (CLL), compared with 10 healthy donors, for the expression and function of the leukocyte-associated Ig-like receptor-1 (LAIR-1). LAIR-1 is an inhibitory receptor containing a cytoplasmic tyrosine-based inhibitory motif (ITIM) that binds to the SH2 domain of phosphatases, leading to dephosphorylation of different kinases. Constitutive activation of c-Jun aminoterminal kinase (JNK), p38 mitogen-activated protein kinase and extracellular signal-regulated kinase, has been reported in CLL. We show that LAIR-1 is absent in high–risk (HR) CLL and differently expressed on intermediate- and low-risk CLL and the intensity of expression, which is always significantly lower than in healthy donors, correlates with disease stage and progression. Interestingly, both constitutive and sIgM-induced phosphorylation of p38 and JNK is inhibited by LAIR-1 through an ITIM-dependent signal, as demonstrated by the use of specific ITIM-binding peptides; importantly, this inhibitory signal is missing when LAIR-1 is not expressed as occurs in HR CLL. Moreover, engagement of LAIR-1 blocks constitutive and sIgM-induced Akt phosphorylation, besides nuclear factor κ-B nuclear translocation, and prevents CLL division. These results suggest that CLL lacking LAIR-1 may miss one of the molecular mechanisms controlling B-cell activation and proliferation.

Gammadelta T Lymphocytes Producing IFNγ and IL-17 in Response to Candida Albicans or Mycobacterial Antigens: Possible Implications for Acute and Chronic Inflammation

T lymphocytes bearing the gammadelta T cell receptor are known to play an important role in the first-line defense against viral, bacterial and fungal pathogens. Two main subsets of gammadelta T cells are known, showing distinct functional behaviour: Vdelta2 T lymphocytes, circulating in the peripheral blood, are involved in the response to mycobacterial infections and certain viruses, including coxsakie virus B3 and herpes simplex virus type 2. Vdelta1 T cells are resident in the mucosal-associated lymphoid tissue and are reported to participate in the immunity against Listeria monocytogenes and cytomegalovirus. Vdelta2 T lymphocytes recognize non-peptidic phosphorylated metabolites of isoprenoid biosynthesis, expressed by mycobacteria, while Vdelta1 T cells mainly interact with MHC-related antigens (MIC-A and MIC-B) and with receptors, called UL-16 binding proteins, for the UL-16 protein produced by cytomegalovirus-infected cells. Both Vdelta1 and Vdelta2 T cells can produce interferon-gamma in response to MIC-A(+) cells or non-peptide antigens, respectively. Moreover, production of TNF-alpha by human Vgamma9/Vdelta2 T cells has been demonstrated in response to bacterial products and non-peptidic molecules. Recently, it has been reported that gammadelta T lymphocytes can produce IL-17 during Escherichia coli or Mycobacterium tuberculosis infections in mice. This is of interest as IL-17 is emerging as a cytokine crucial in the control of intracellular pathogens and fungi. In this review, we will discuss the possible role of IL-17 producing gammadelta T cells in the regulation of acute and chronic inflammation, focusing on the different response of the two subsets to mycobacterial, viral or fungal antigens.

Regulation of γδ T cell survival by soluble HLA-I: Involvement of CD8 and activating killer Ig-like receptors

We show that human Vδ1 or Vδ2 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 α3 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 γδ 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‐α. Noteworthy, sHLA‐I molecules synergize with antigen‐mediated activation of Vδ2 T cells: Indeed, Vδ2 T lymphocytes produced TNF‐α when stimulated with isopentenyl pyrophosphate, and this effect was enhanced by sHLA‐I. These findings suggest that sHLA‐I can regulate γδ T cell survival and that activating KIR may amplify antigen‐specific Vδ2 T cell responses.

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 responeìder 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.

Migratory Pathways of γδ T Cells and Response to CXCR3 and CXCR4 Ligands

Abstract:  Gammadelta T lymphocytes are thought to be involved in multiple sclerosis (MS) pathogenesis. In this work, we discuss the characteristics of these cells and possible implications in the pathogenesis of MS, focusing on the mechanism(s) underlying extravasation and tissue localization. Phenotype and transendothelial migration of γδ T cells from healthy donors and patients with relapsing–remitting MS were studied. In MS patients the Vδ2 T cell subset, expressing NKRP1A/CD161 adhesion molecule, is expanded and capable of transendothelial migration. Vδ1/Vδ2 subsets use distinct signal transduction pathways: Vδ1 cells lack NKRP1A and express PECAM‐1/CD31, which drives transmigration, while Vδ2 cells are PECAM‐1 negative and use NKRP1A. Vδ2 migration is coupled with CAMKII, whereas Vδ1 depend on PI‐3K. NKRP1A and PECAM‐1 selectively activate the two pathways: indeed, oligomerization of NKRP1A on Vδ2 T cells leads to CAMKII activation, occupancy of PECAM‐1 on Vδ1 cells triggers the PI‐3K‐dependent Akt/PKB pathway. Moreover, Vδ2 T cells are CXCR3brightCXCR4dull, while Vδ1 are mostly CXCR4+. Vδ1 and Vδ2 cells transmigrate in response to IP‐10/CXCL10 and SDF‐1/CXCL12 according to the expression of their specific receptors. In a fraction of Vδ1 T cells coexpressing CXCR3 and CXCR4, the homeostatic chemokine 6Ckine/SLC/CCL21 is more effective. IP‐10/CXCL10 or 6Ckine/SLC/CCL21 and SDF‐1/CXCL12‐induced transmigration is coupled to PI‐3K/Akt/PKB, but only CXCR3 is capable of inducing CAMKII activation. We suggest that both subsets of γδ T lymphocytes may migrate to the site of lesion in MS using two different signaling pathways to extravasate and responding to different chemokines.

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-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.

Adhesion Molecules and Kinases Involved in γ δ T Cells Migratory Pathways:Implications for Viral and Autoimmune Diseases

γ δ 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 Vδ1 and Vδ2 γ δT 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-γ-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 Vδ1 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 Vδ2 γ δ T cells and this is related to interleukin-12-mediated upregulation of NKRP1A. Finally, the possible role of γ δ T lymphocytes in post-transplantation immune reconstitution is discussed.