Maria Raffaella Zocchi

Human γδ T cells: A nonredundant system in the immune-surveillance against cancer

Abstract Down-regulation of expression of MHC alleles, as well as tumor-specific antigens, is observed frequently during tumor progression, resulting in an impairment of MHC-restricted, αβ-T-cell-mediated, tumor-specific immunity. Given the unique set of antigens recognized and the lack of requirement for classical antigen-presenting molecules, γδ T cells might, therefore, represent a nonredundant system in anticancer surveillance, as proposed for the immune response against pathogens. Evidence that γδ and αβ T cells make distinct contributions to anticancer surveillance has been provided recently in mice. Here, we discuss the potential role played by resident Vδ1 + and circulating Vδ2 + T cells in the defense against solid tumors and hematological malignancies.

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.

Interaction between Human NK Cells and Bone Marrow Stromal Cells Induces NK Cell Triggering: Role of NKp30 and NKG2D Receptors

In this study we have analyzed the interaction between in vitro cultured bone marrow stromal cells (BMSC) and NK cells. Ex vivo-isolated NK cells neoexpressed the activation Ag CD69 and released IFN-γ and TNF-α upon binding with BMSC. Production of these proinflammatory cytokines was dependent on ligation of ICAM1 expressed on BMSC and its receptor LFA1 on NK cells. Furthermore, the NKp30, among natural cytotoxicity receptors, appeared to be primarily involved in triggering NK cells upon interaction with BMSC. Unexpectedly, autologous IL-2-activated NK cells killed BMSC. Again, LFA1/ICAM1 interaction plays a key role in NK/BMSC interaction; this interaction is followed by a strong intracellular calcium increase in NK cells. More importantly, NKG2D/MHC-I-related stress-inducible molecule A and/or NKG2D/UL-16 binding protein 3 engagement is responsible for the delivery of a lethal hit. It appears that HLA-I molecules do not protect BMSC from NK cell-mediated injury. Thus, NK cells, activated upon binding with BMSC, may regulate BMSC survival.

NK cell-mediated lysis of autologous antigen-presenting cells is triggered by the engagement of the phosphatidylinositol 3-kinase upon ligation of the natural cytotoxicity receptors NKp30 and NKp46.

Interleukin‐2 (IL‐2)‐activated polyclonal or clonal NK cells lysed autologous antigen presenting cells (APC) through the engagement of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. NK cell‐mediated cytolysis of APC correlated with the surface density of these NCR. Indeed, NK cell clones bearing low amounts of NKp30 and NKp46 did not lyse autologous APC, whereas NK cell clones with bright expression of these NCR efficiently killed autologous APC. Upon masking of NKp30 or NKp46 by specific monoclonal antibodies a strong reduction (by 50%) of APC lysis could be detected and the complete inhibition was achieved by the simultaneous masking of these NCR. Interestingly, NK cell‐mediated APC lysis was impaired by the phosphatidylinositol 3‐kinase (PI‐3 K) inhibitors LY294002 or wortmannin. Similarly, these drugs strongly reduced NK cell activation triggered by NKp30 or NKp46 in a re‐directed killing assay as well as the activation of Akt/PKB, substrate of PI‐3 K, induced by the engagement of these receptors. Altogether, these findings strongly suggest that NCR are responsible for the killing of autologous APC through the activation of PI‐3 K.

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.

Functional Association of Platelet Endothelial Cell Adhesion Molecule-1 and Phosphoinositide 3-Kinase in Human Neutrophils

In this paper we show that the engagement of the platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) up-regulates the adhesion of human neutrophils to the EA.hy926 endothelial cell line through a phosphoinositide 3-kinase (PI3K)-dependent pathway. Indeed, LY294002 and wortmannin prevented the effect of PECAM-1/CD31 cross-linking on cell adhesion, at concentrations known to inhibit PI3K without affecting other kinases. Both compounds blocked neutrophil binding to murine fibroblasts transfected with human ICAM-1, to purified ICAM-1 protein, or to fibronectin, suggesting that PECAM-1/CD31-mediated up-regulation of β2 and β1 integrin-mediated adhesion is PI3K-sensitive. We also provide evidence for the association of PECAM-1/CD31 to PI3K, because PI3K was detectable in neutrophil lysates after PECAM-1/CD31 cross-linking and immunoprecipitation. PECAM-1/CD31-dependent recruitment of PI3K was suggested by the finding that the serine/threonine kinase p70 S6 kinase (S6K), a signaling protein downstream of PI3K, is activated in neutrophils upon PECAM-1/CD31 cross-linking, based on the appearance of serine phosphorylation in S6K immunoprecipitates. In turn, S6K is not directly involved in the up-regulation of integrin function because rapamycin, which can inhibit S6K independent of PI3K, did not block PECAM-1/CD31-induced adhesion of neutrophils to β1 and β2 integrin substrates. In conclusion, PECAM-1/CD31 appears to be one of the molecules functionally coupled to PI3K, suggesting that this enzyme may represent a common pathway of integrin and adhesiveness regulation in leukocytes.

p40/LAIR-1 regulates the differentiation of peripheral blood precursors to dendritic cells induced by granulocyte-monocyte colony-stimulating factor

p40/LAIR‐1, a member of the immunoglobulin superfamily, is a surface molecule broadly distributed among leukocytes which has been shown to down‐regulate T and NK cell activation. In this study, we show that p40/LAIR‐1 is highly expressed in CD14+ peripheral blood mononuclear cells (PBMC). When cultured in the presence of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) for 10 – 14 days, CD14+ cells acquired morphologic and phenotypic features ( i.e. loss of CD14 and expression of CD80bright and CD86bright ) typical of dendritic cells (DC) and lost the expression of p40/LAIR‐1. Engagement of p40/LAIR‐1 (but not of CD58) by specific monoclonal antibodies prevented CD14+ PBMC differentiation into DC; when cultured in the presence of GM‐ CSF upon p40/LAIR‐1 cross‐linking, the resulting cells were CD14+ CD80dull CD86dull and displayed a macrophage‐like morphology. We have recently demonstrated that peripheral blood CD14+ cells co‐expressing the CD34 progenitor marker represent the circulating precursors of CD83+ DC. Herein we show that cross‐linking of p40/LAIR‐1 prevented the maturation of CD14+ CD34+ cells into CD83+ DC. This effect appears to be consequent to the impairment of GM‐CSF receptor‐mediated activation signaling. Indeed, triggering of GM‐CSF receptors in both CD14+ and CD14+ CD34+ cells led to increases in the intracellular free calcium concentrations which were inhibited by p40/LAIR‐1 engagement. Taken together, these data suggest a possible regulating role played by p40/LAIR‐1 in the process of differentiation from peripheral blood precursors into DC induced by GM‐CSF.

The platelet endothelial cell adhesion molecule-1 (PECAM1) contributes to endothelial barrier function

In this study we have analyzed the role of the platelet‐endothelial cell adhesion molecule‐1 (PECAM1) in vascular barrier function. PECAM1 is an immunoglobulin gene superfamily member expressed by endothelial cells at the cell boundaries. Macromolecule permeability assays performed on cell monolayers that express native or transfected PECAM1, indicated that the molecule participates in the establishment and maintenance of vascular barrier function in vitro. This hypothesis was confirmed by the finding that in vivo injection of the specific monoclonal antibody directed against the murine vascular PECAM1 led to a detectable leakage of hepatic and renal blood vessels.

ZAP-70 is expressed by normal and malignant human B-cell subsets of different maturational stage

ZAP-70 tyrosine kinase is involved in signalling pathways following T-cell receptor stimulation and was originally described only in T cells and natural killer cells. ZAP-70 expression has been reported in normal mouse B lineage cells and in human malignant B lymphocytes, mainly in chronic lymphocytic leukemia (CLL) where it correlates with clinical outcome. We analyzed several B-cell lines and ex vivo malignant B cells, ranging from acute lymphoblastic leukemia to multiple myeloma and reflecting different stages of B-cell differentiation, and they showed ZAP-70 expression regardless their maturation stage. We then analyzed by Western blot and flow cytometry different human normal B-lymphocyte subpopulations: naïve, germinal center and memory B cells from tonsils, CD19+ CD5+ cells from cord blood and CD19+ lymphocytes from peripheral blood. All expressed ZAP-70 protein, though at different levels depending on their differentiation, activation and tissue localization. In addition, ZAP-70 expression levels could be modulated following stimulation via the B-cell receptor. These findings implicate a potential role of ZAP-70 in the signalling pathway of B lymphocytes at different maturational stages, indicate that ZAP-70 expression is not a CLL-specific feature among B-cell malignancies and suggest that the absence of ZAP-70 rather than its presence should be considered abnormal for malignant B lymphocytes.

Escape of monocyte-derived dendritic cells of HIV-1 infected individuals from natural killer cell-mediated lysis

Objective: To verify whether the in vitro sensitivity of immature dendritic cells (iDC) to lysis by autologous natural killer (NK) cells from HIV-infected individuals might be correlated with HIV disease progression. Design: Both dendritic cells (DC) and interlekin (IL)-2 activated NK cells were obtained from 13 HIV-infected individuals early after seroconversion and not receiving highly active antiretroviral therapy (HAART) and from 14 individuals with chronic HIV infection under HAART. The rate of NK cell-mediated killing of autologous iDC was correlated with classical parameters of HIV evolution. Methods: Peripheral blood monocytes obtained from the Ficoll-derived leukocyte fraction after adherence to plastic were stimulated with granulocyte–macrophage colony stimulating factor plus IL-4 to induce their differentiation into iDC to be used as target cells in a standard 4-h cytotoxicity assay. A fraction of autologous leukocytes was stimulated with IL-2 to induce activation of NK cells to be used as effector cells. Results: During early HIV infection the extent of ex vivo lysis of monocyte-derived DC by activated autologous NK cells was inversely and directly correlated with the levels of viraemia and with the percentage of circulating CD4 T cells, respectively. In contrast, the capacity of NK cells to kill iDC was lost independently of the levels of plasma viraemia or the concurrence of HAART in chronically infected individuals. Addition of exogenous HIV Tat during the cytotoxicity assay inhibited NK cell-mediated lysis of DC. Conclusions: NK cell-mediated immune surveillance against infected DC may be effective only during early HIV infection and may not be restored by HAART.