Daniela Zarcone

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

Systemic inflammation and neurodegeneration in a mouse model of multiple sulfatase deficiency

Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple sulfatase deficiency, mutations in the SUMF1 gene cause a reduction of sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in the Sumf1 gene. Sulfatase activities are completely absent in Sumf1−/− mice, indicating that Sumf1 is indispensable for sulfatase activation and that mammals, differently from bacteria, have a single sulfatase modification system. Similarly to multiple sulfatase deficiency patients, Sumf1−/− mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans. Sumf1−/− mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study sulfatase function and the mechanisms underlying lysosomal storage diseases.

Increased serum levels of soluble CD44 standard, but not of variant isoforms v5 and v6, in B cell chronic lymphocytic leukemia

The CD44 cell surface proteoglycan participates in a variety of functions including lymphohematopoiesis, lymphocyte homing and tumor metastasis. In addition to the standard form (CD44st), a large family of variant isoforms (CD44v) is generated by alternative splicing of a single gene. Certain CD44v (v5 and V6) are upregulated in the course of neoplastic progression and reflect the metastatic potential of tumor cells. CD44 v6 is expressed in high-grade non-Hodgkin’s lymphoma cells and is released in the serum, thus providing a soluble marker that reflects tumor burden, disease progression and treatment response. Here we show that serum CD44st is elevated in approximately half of B-CLL patients. In contrast, CD44v5 and v6 are detected at normal levels in the large majority of the cases. CD44st serum levels correlate significantly with the number of circulating leukemic B cells and with the levels of another soluble B-CLL marker, β2-microglobulin. Immunoprecipitation analyses of B-CLL sera allow detection of several high molecular weight bands and of a 78 kDa band that represents a soluble form of CD44st and is 4 kDa lower than a similar band (82 kDa) detected in B-CLL cell lysates. Elevated serum CD44st associates with a number of unfavorable prognostic factors such as high peripheral blood lymphocytosis, splenomegaly, advanced disease stage and therapy requirement. A follow-up study indicates that serum levels of CD44st are related to disease status, thus reinforcing our veiw that this molecule may represent a reliable tumor marker in B-CLL.

CD1d expression on B-precursor acute lymphoblastic leukemia subsets with poor prognosis.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Although therapeutical advances have been achieved, some ALL subgroups still fare poorly. CD1d is a monomorphic molecule that provides a suitable target for immunotherapy in view of the characterization of a glycolipid, α-galactosylceramide (α-GalCer), capable of being presented to CD1d-restricted T cells with cytotoxic potential. We investigated CD1d expression in 80 pediatric B-cell precursor (BCP) ALL cases defined according to immunophenotype, cytogenetic features and age at onset. CD1d was detected on ALL cells in 15% of the patients. CD1d+ ALLs were significantly associated with infant leukemia, pro-B phenotype and mixed-lineage leukemia (MLL)/AF4 gene rearrangement. Accordingly, overall survival of patients with CD1d+ ALL was significantly shorter. CD1d+ leukemic blasts were able to present α-GalCer via CD1d to cytotoxic CD1d-restricted T cells, which induced apoptosis of ALL cells that was inhibited by mAb to CD1d. CD1d+ blasts loaded with α-GalCer elicited cytokine secretion by CD1d-restricted T cells. Analysis of bone marrow (BM) cells derived from normal donors revealed that CD19+/CD1d+ cells were mostly mature B lymphocytes. However, a minority of BCPs expressed CD1d. Thus, expression of CD1d in ALL cases heralds an adverse prognosis but may provide a therapeutic tool.

CD85j (Leukocyte Ig-Like Receptor-1/Ig-Like Transcript 2) Inhibits Human Osteoclast-Associated Receptor-Mediated Activation of Human Dendritic Cells

Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated γ-chain, induced Ca2+ flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs.

Flow cytometry evaluation of cell-mediated cytotoxicity

A novel flow cytometry method for the evaluation of cell-mediated cytotoxicity is described. This method uses flow cytometry analysis to distinguish target cells from effector cells by differences in volume and light scatter characteristics. Non-viable target cells, following their interaction with effector cells, are determined via propidium iodide (PI) dye exclusion and then expressed as a percentage of the total target cell population. This assay is suitable both for analysis of systems which allow recycling of cytotoxic effector cells (total cell cytotoxicity assays, TCCA), and of systems in which recycling does not occur (single cell cytotoxicity assays, SCCA). Natural killer (NK) cell-mediated cytotoxicity evaluated by flow cytometry is significantly correlated with the standard 51Cr release assay. Flow cytometry can also be used to evaluate the competitive inhibition that certain cell types exert on the cell-mediated killing of NK-sensitive targets. A prerequisite for this assay is that competitor cells and target cells are distinguishable through their volume and light scatter characteristics. Advantages and pitfalls of the flow cytometry method are discussed, in comparison with the 51Cr-release assay.

Antibodies to adhesion molecules inhibit the lytic function of MHC-unrestricted cytotoxic cells by preventing their activation

We evaluated the effect of the antibodies to adhesion molecules CD2, CD11a/CD18 (LFA-1), and CD56 (N-CAM) on MHC-unrestricted cytotoxicity mediated by polyclonal NK cells and LAK cells or by CD3+ or CD3- cytolytic cell clones against a panel of tumor cell targets selected according to expression or absence of the corresponding ligands. We show that (i) antibodies to CD11a/CD18 and, to a lesser extent, antibodies to CD2 inhibit target cell lysis, whereas anti-CD56 antibodies exert little if any effect; (ii) in a model system using polyclonal NK/LAK cells as effectors and K562 or HL60-R (NK-resistant) cells as targets, inhibition of cytotoxicity occurs without a significant impairment of effector to target cell binding; (iii) the cytotoxic function of CD3+ or CD3- cytotoxic cell clones is inhibited differentially by antibodies to adhesion molecules; (iv) conjugates formed in the presence of antibodies which inhibit target cell lysis display a significant reduction of target to effector cell contact surface; and (v) this may lead to defective activation of effector cells, as indicated by lack of redistribution of the microtubular apparatus. We conclude that (i) MHC-unrestricted cytotoxicity is regulated by a number of molecular interactions that span far beyond our present knowledge and that it is strictly dependent on the surface phenotype of the effector cell and of the target cell; (ii) in certain types of effector/target cell interactions, antibodies to adhesion molecules do not prevent conjugate formation but reduce the extent of cell-to-cell surface contact which, in turn, leads to defective activation of the effector cell and, therefore, to inhibition of target cell lysis.

Adhesion Molecule Expression, Clinical Features and Therapy Outcome in Childhood Acute Lymphoblastic Leukemia

In view of the relevance of adhesion molecule expression for the mechanisms of homing, trafficking and spreading of malignant cells, we have investigated the expression of surface adhesion molecules in lymphoblasts from 57 acute lymphoblastic leukemia (ALL) cases and tried to correlate the adhesive phenotype with immunological typing, prognostic factors at diagnosis and clinical follow-up. Blasts from all cases expressed adhesion molecules at high rates. β1 integrin chain (CD18) was consistently found on blasts from most ALL cases; among integrins of the β2 family, LFA-1 was detected in 58% of cases, in the virtual absence of other α chains. CD54 and CD58 were expressed in variable proportions by ALL blasts and CD44 was detected in the majority of the malignant cells, whereas the CD62L selectin was only present in 24% of cases. B-lineage ALLs displayed similar adhesion molecule phenotypes irrespective of maturational stages of the leukemic cells. We found a significantly reduced expression of β2 αL integrins in the hybrid ALL cases (CD13 and/or CD33 positive). However, these cases did not show differences in clinical presentation and behaviour in comparison with patients of other groups. We did not find a significant correlation between adhesion molecule expression and well established risk factors (age, white blood cell count, central nervous system involvement, chromosomal abnormalities), with the exception of splenomegaly, that was significantly associated with CD18 expression. In the follow-up, no evidence of significant correlation between adhesive phenotype and adverse events such as leukemic relapse and death was found. In conclusion, although expression of adhesion molecules on lymphoblasts confirms the phenotypic heterogeneity of ALL, it appears that this is not relevant for the clinical aspects of the disease and for prognosis.

Adhesion Molecule Expression on B-Cells from Acute and Chronic Lymphoid Leukemias

Adhesion molecule expression on acute and chronic lymphoid leukemia cells of B lineage (B-ALL and B-CLL) may subserve several functions. Adhesion of leukemic cells to endothelial cells and to extracellular matrix components is relevant to homing, trafficking and spread of the malignant cells, and thus to clinical presentation, course and disease prognosis. Adhesive interactions between malignant cells and accessory cells, particularly stromal cells in the bone marrow environment, may support growth of the malignant cells via cytokine-delivered messages. They may also deliver signals that prevent or trigger programmed cell death of tumor cells. Here we review data on the adhesive phenotype of leukemic blasts from pro-B (CALLA +) ALL and of cells from B-CLL cases. We show that expression of certain adhesion molecules may help define disease subsets with distinctive clinical and prognostic features. One adhesion molecule, the lymphocyte homing receptor CD44, allows definition of two groups of B-CLL patients with significantly different survival.

X-linked codominant genes control all types of non-major histocompatibility complex-restricted cytotoxicity

In most individuals, natural killer (NK) activity is abolished after lymphocyte irradiation with 3,000 cGy, while lymphocytes from a minority of males retain 100% NK activity and lymphocytes from some females retain 50% NK activity after this dose. Radiation sensitivity of NK activity is controlled by X-linked codominant genes. The frequency of the allele that imparts resistance is 7%. We studied a unique family in which both parents have the resistant allele such that the father is completely resistant and the mother is partially resistant. The three offspring of this couple were one sensitive male, one partially resistant female, and one completely resistant female. The radiation sensitivity of nonspecific cytotoxic functions mediated by various types of effector cells from all five family members were evaluated in order to determine whether other cytotoxic functions were controlled by the same set of genes. The cytotoxic functions investigated were: NK and lymphokine-activated killing, anomalous killing and lectin-dependent cellular cytotoxicity, and antibody-dependent cell-mediated cytotoxicity. Our data indicate that the radiation sensitivity of all types of nonspecific cytotoxic cells is under the same genetic control.