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T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G0/G1 phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/β and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34+/CD4−/CD7−), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL. [ABSTRACT FROM AUTHOR]

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4+ T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34+/CD7−/CD4−) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL. [ABSTRACT FROM AUTHOR]

T-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting. [ABSTRACT FROM AUTHOR]

Background: The presence of antibodies to CTLA-4, a negative regulator of T-cell activation, was investigated in multiply transfused patients with malignant and non- malignant hematologic diseases. A previous study showed that, in multiply transfused patients, an immune response against nuclear matrix proteins can be induced by WBCs undergoing apoptosis during RBC unit storage. This study evaluated whether the same phenomenon could be involved in the induction of CTLA-4 antibodies in the patients analyzed.Study Design and Methods: Patient sera were tested for binding to the recombinant full-length CTLA-4 beta-galactosidase fusion protein by an ELISA. Immuno-fluorescence stainings were performed to analyze the CTLA-4 epitopes recognized by the antibodies and to detect such epitopes in the apoptotic cells present in the RBC units.Results: CTLA-4 antibodies were found in multiply transfused patients with beta-thalassemia (40%) and with other hemolytic diseases (33%) including leukemias (42%). A higher incidence of CTLA-4 antibodies was found in patients receiving non-WBC-reduced blood (88%) than in those receiving WBC-reduced blood (26%). Immunofluorescence staining showed that WBCs undergoing apoptosis in the RBC unit expressed CTLA-4 epitopes.Conclusions: The apoptotic WBCs present in the RBC units, after cold storage, express CTLA-4 epitopes. These epitopes can be released and induce formation of CTLA-4 antibodies with profound implications in the development of autoimmune disorders and in facilitating tumor dissemination and metastasis. [ABSTRACT FROM AUTHOR]

Ten patients with high-grade non-Hodgkin's lymphoma (HG-NHL) entered a subcutaneous (s.c.) recombinant interleukin 2 (rIL2) trial within 2 months of undergoing autologous bone marrow transplantation (ABMT). Immunological studies, consisting in T- and natural killer (NK)-cell subset assessment, together with functional assays, such as NK activity and CD16-mediated redirected killing assay, were performed before therapy, after 2 weeks, and then monthly. Phenotypic analysis showed a significant increase (p = 0.01) of CD16 and CD56 NK cells, from 12% to 28% and from 17% to 37%, respectively. In particular, the CD56bright NK cell population showed a tenfold increase, while CD56dim NK cells remained unmodified compared with pretreatment values. The expression of IL2 receptors was also studied and a significant increase (p = 0.01) of CD122 (p75)-positive cells from 8% to 30% was found, while no significant increase was observed in CD25 (p55)-positive cells. Furthermore, rIL2 administration led to an increase of NK activity even at the lowest effectors:target ratio and to an increase of CD16-mediated redirected killing assay. These phenotypic and functional modifications lasted throughout the duration of rIL2 therapy and remained after completion of therapy. In addition, none of the ten patients relapsed, and two of them who started IL2 treatment while still showing residual disease experienced a complete disappearance of the disease after 10 and 7 months of therapy, respectively. Our data suggest that infusion of rIL2 s.c. after ABMT is safe, can selectively increase NK cell number and function, and may have a beneficial effect on the minimal residual disease. [ABSTRACT FROM AUTHOR]

A phenotypic and functional analysis of the circulating natural killer (NK) cell population was carried out in a series of patients with hairy ceil leukaemia (HCL). The overall mean NK activity of both the mononuclear and T cell fractions was reduced compared to that of normal controls (466 lytic units (lu) v 573 lu and 226 lu v 381 lu, respectively), though this difference did not reach statistical significance (P < 0.05), individual analysis of the data showed that in five out of 15 and in seven out of 16 cases the K562 killing by the mononuclear and T cells respectively was below the lowest s.d. limit for normal subjects. This reduced NK function was associated with a decreased ability of the effector cells to bind the target. The NK response lo exogenous human leucocyte interferon was also generally depressed in cases with a low basal NK activity. The functional studies were complemented with the evaluation of the membrane expression of NK associated antigens. The percentage of circulating T cells recognized by the monoclonal antibody (MoAb) Leu-7 was significant higher (P <0.001) in HCL than in normal blood (25.2% ± 10.2 v 11.9 ± 5.9 s.d.). However, the reactivity with two other NK-related MoAb, Leu-11 and AB8.28, was significantly lower (7.1% ± 6.9 and 9.8% ± 8.5; P <0.002) than with Leu-7 and moderately reduced compared with that of normal circulating T cells(11.7% +-6.l & 12%,+ 5.5). These findings suggest that in a proportion of patients with HCL there is an Impairment of the NK compartment, which may contribute towards the occurrence of the infective complications which are the primary cause of death in this disease. [ABSTRACT FROM AUTHOR]

Peripheral blood B lymphocytes, depleted of adherent cells, from 10 patients with multiple myeloma were cultured in the presence of PWM with autologous or donor T lymphocytes. The results show that: (1) co-cultures with allogeneic T lymphocytes produced more plasma cells than those with autologous ones; (2) the k/λ ratio overlapped the values obtained in normal controls, irrespective of the light chain produced by the neoplastic plasma cells and (3) the immunological phenotype of plasma cells obtained from PWM stimulated peripheral B cells (RFA2+, RFA3+, A10+) was clearly different from that one of myelomatous plasma cells (RFA2-, RFA3-, A10+). These data confirm the T cell imbalance already seen in myeloma patients; moreover they show that PWM responsive B cell are functionally normal and phenotypically different from bone marrow myeloma cells. These results support the view that most of the peripheral B lymphocytes, previously identified as monoclonal arc in fact normal cells bearing adherent monoclonal Ig molecules. [ABSTRACT FROM AUTHOR]

The distribution of I lymphocyte subsets was assessed using monoclonal antibodies (MoAbs) in 44 untreated patients with multiple myeloma (MM) subdivided according to the clinical stage of the disease. A significant reduction (P<0.001) of T lymphocytes was observed only in stage II and III patients. The proportion and absolute number of OKT4 positive cells (helper/inducer phenotype) were significantly reduced in all stages of the disease; this quantitative abnormality was more pronounced in advanced disease. While the proportion of OKT8 positive cells (suppressor/cytotoxic phenotype) was increased above normal in all stages, the absolute number (of 0K18 positive cells) was high only in stage I patients; on the contrary in stage Il-Ill patients the total OKT8 count was reduced compared with normal controls. A significantly reduced OKT4/OKT8 ratio was found in both groups of patients (P < 0005). Functional studies, carried out on the unfractionated T cells of patients with MM, demonstrated a consistent helper defect in the ability to induce the differentiation of normal B lymphocytes into antibody producing cells in a pokeweed mitogen driven system. However, the removal of OKT8 positive cells produces a significant increase in helper capacity, suggesting that the reduced helper function of I lymphocytes in toto is probably due to excessive suppressor activity. The possible immunoregulatory role of MM T cell disease is discussed. [ABSTRACT FROM AUTHOR]