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MLL-rearranged acute lymphoblastic leukaemia (ALL) represents an aggressive malignancy in infants (<1 year of age), associated with poor outcome. Current treatment intensification is not further possible, and novel therapy strategies are needed. Notably, MLL-rearranged ALL is characterised by a strongly deregulated epigenome and shows sensitivity to epigenetic perturbators. Here we demonstrate the in vivo efficacy of the histone deacetylase inhibitor panobinostat (LBH589) using xenograft mouse models of MLL-rearranged ALL. Panobinostat monotherapy showed strong anti-leukaemic effects, extending survival and reducing overall disease burden. Comprehensive molecular analyses in vitro showed that this anti-leukaemic activity involves depletion of H2B ubiquitination via suppression of the RNF20/RNF40/WAC E3 ligase complex; a pivotal pathway for MLL-rearranged leukaemic maintenance. Knockdown of WAC phenocopied loss of H2B ubiquitination and concomitant cell death induction. These combined data demonstrate that panobinostat cross-inhibits multiple epigenetic pathways, ultimately contributing to its highly efficacious targeting of MLL-rearranged ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult–to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Protein kinase CK2 sustains acute myeloid leukemia cell growth, but its role in leukemia stem cells is largely unknown. Here, we discovered that the CK2 catalytic α and regulatory β subunits are consistently expressed in leukemia stem cells isolated from acute myeloid leukemia patients and cell lines. CK2 inactivation with the selective inhibitor CX-4945 or RNA interference induced an accumulation of leukemia stem cells in the late S–G2–M phases of the cell cycle and triggered late-onset apoptosis. As a result, leukemia stem cells displayed an increased sensitivity to the chemotherapeutic agent doxorubicin. From a molecular standpoint, CK2 blockade was associated with a downmodulation of the stem cell-regulating protein BMI-1 and a marked impairment of AKT, nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) activation, whereas FOXO3a nuclear activity was induced. Notably, combined CK2 and either NF-κB or STAT3 inhibition resulted in a superior cytotoxic effect on leukemia stem cells. This study suggests that CK2 blockade could be a rational approach to minimize the persistence of residual leukemia cells.

The recently cloned cytokine interleukin-15 (IL-15) shares several functional activities with IL-2 in different cell systems. Although IL-15 does not show sequence homology with IL-2, it uses components of the IL-2 receptor (IL-2R) for binding and signal transduction, namely, p75 (ß) and the p64 (?) chains of IL-2R. To evaluate whether IL-15 is involved in the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), we evaluated the ability of IL-15 to stimulate GL proliferation, cytotoxic function, and the role of IL-2R ß and ? molecules on relevant cells. Our results show that IL-15 stimulates cell proliferation and cytotoxic activity of GL in LDGL patients. Reverse-transcriptase polymerase chain reaction (RT-PCR) and phenotypic analyses using the anti–IL-2R ?-chain–specific TUGh4 monoclonal antibody (MoAb) indicate that both CD3+ and CD3- GL express the p64 IL-2R, a result previously unknown. IL-15 activity was inhibited by antibodies against p75 and p64 IL-2R chains, while no inhibitory effects are detectable with anti-p55 IL-2R antibody. The association of anti-p75 and anti-p64 IL-2R MoAbs resulted in a nearly complete (95%) inhibition of IL-15–induced GL proliferation. Using RT-PCR analysis, we demonstrated that highly purified CD3+ and CD3- GL did not express mRNA for IL-15 or IL-2. By contrast, a clear-cut IL-15 mRNA signal was detected by RT-PCR in patients' peripheral blood mononuclear cells, with monocytes likely accounting for the source of IL-15 in LDGL patients. However, even in concentrated supernatants from enriched monocyte populations, we could not demonstrate the presence of IL-15 protein. Using anti–IL-15 specific MoAbs, a membrane-bound form of this cytokine was demonstrated both on CD3+ and CD3- LDGL cells. By RT-PCR analysis, purified GL from these patients were found to express the message for IL-15 receptor a chain. Taken together, these results indicate that both CD3+ and CD3- GL are stimulated by IL-15 and that this cytokine mediates its activity through the ß and ? chains of the IL-2R, providing further suggestions for the interpretation of the mechanisms that lead to cell expansion in patients with LDGL.

The cell membrane expression and functional role of the interleukin-2 receptor (IL-2R) was analyzed in nine patients with lymphoproliferative disease of granular lymphocytes (LDGL) using monoclonal antibodies (MoAbs) specific for the p75 (TU27) and the p55 (anti-Tac) subunits of IL-2R. Four patients were characterized by the proliferation of CD3+CD8+ granular lymphocytes (GL) expressing the alpha/beta T-cell receptor (T alpha beta) and one case by the proliferation of CD3+CD4- CD8- GL expressing the gamma/delta T-cell receptor (T gamma delta); in four additional cases proliferating cells were CD3 negative GL. Consistent with data observed on normal GL, phenotypic analysis demonstrated that patients' GL lack the expression of the p55 IL-2R, whereas the p75 subunit is constitutionally expressed by expanding GL of both T-cell (either T alpha beta and T gamma delta) and natural killer (NK) origin in variable proportions (11% to 94% of cells). The analysis of the cytotoxic and proliferative activity demonstrated that the anti-p55 MoAb failed to inhibit IL-2-mediated activation, whereas a marked inhibition of both cytotoxicity and proliferation were obtained using the anti-p75 chain specific MoAb. These data indicate that the p75 chain of IL-2R is responsible for IL-2 signal transduction in both CD3+ and CD3- LDGL patients' GL.

Natural killer (NK) cell activity is severely impaired in untreated patients with hairy cell leukemia (HCL). In an attempt to investigate whether this impairment is related to a defect at the target cell binding and/or at the post target cell binding level, we evaluated the peripheral blood mononuclear cells (PBMC) of HCL patients for their ability to: (1) bind and kill K-562 NK-sensitive targets at the single cell binding level; (2) release the NK cytotoxic factor (NKCF) under different in vitro stimuli, including K-562 and phytohemoagglutinin; and (3) kill K-562 targets in a lectin-dependent cellular cytoxicity (LDCC) assay. This study demonstrates that untreated HCL patients' PBMC show a low ability to form conjugates with K-562 targets at the single cell binding level (5.7% +/- 1.0%) with respect to patients studied after treatment (9.3% +/- 1.3%) and controls (15.0% +/- 4.0%); P less than .05 and P less than .001, respectively. A decreased ability to kill the bound target was demonstrated in untreated cases (1.2% +/- 1.1%) versus patients studied after treatment and controls (12.3% +/- 1.6%, 17.0% +/- 3.1% respectively); P less than .001 in both conditions. After activation of effector cells with interleukin-2 (IL- 2) in vitro, an increase in the ability of PBMC to form conjugates with K-562 targets and kill the bound target was demonstrated in each group of patients. Moreover, IL-2 was able to increase the cytotoxicity against NK-sensitive targets in all patients tested. Evaluation of NKCF production showed that untreated patients release low levels of NKCF when PBMC were incubated in the presence of K-562 stimulators (1.8% +/- 0.7%) with respect to patients after interferon-alpha (IFN-alpha) therapy (7.6% +/- 2.1%) and controls (12.9% +/- 2.2%); P less than .02 and P less than .001, respectively. When the recognition mechanisms were bypassed by triggering the cells with lectins in an LDCC assay, we demonstrated an increase of the lytic activity in both groups of patients with respect to the baseline values. However, the cytotoxic capacity observed in untreated patients was significantly lower than that observed in subjects after IFN-alpha therapy and controls (P less than .001). These findings suggest that the impaired NK activity observed in patients with HCL is related to defects both at the target and posttarget cell binding levels.

The recently discovered cytokine, interleukin-15 (IL-15), has been demonstrated to share several biologic properties with IL-2 in different cell systems, including T-cell and natural killer (NK) cell compartments. As for B lymphocytes, IL-15 has been shown to provide stimulatory activities in normal preactivated B cells that are mainly transduced through IL- 2 receptor (IL-2R) complex components. Since leukemic B cells from patients with chronic lymphoproliferative disorders (CLD) bear IL-2R and grow in response to IL-2, we investigated whether IL-15 triggers the proliferation of malignant B cells obtained from 12 patients with B-cell chronic lymphocytic leukemia (B-CLL) and five patients with hairy cell leukemia (HCL). Enriched B cells recovered from five healthy subjects were also studied as controls. IL-15 stimulated the proliferation of freshly isolated leukemic B cells, but not resting normal B lymphocytes, the latter being able to grow in the presence of IL-15 only after in vitro preactivation with phorbol myristate acetate. The proliferation elicited by IL-2 on leukemic cells was comparable to that determined by IL-15. Following addition of graded concentrations of IL-15 to low/intermediate-dose IL-2, resting leukemic B cells showed a higher stimulatory rate than that observed using the two cytokines separately. In normal resting B lymphocytes, this cumulative effect was not observed. The role of different IL-2R subunits in IL-15-driven growth of malignant B cells was investigated both by their expression on leukemic cells and by the block of different IL-2R subunits (p55, p75, and p64) with specific monoclonal antibodies (MoAbs). Using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses we demonstrated that both B-CLL and HCL leukemic B cells express the beta and gamma chains of the IL-2R system. The stimulatory activity achieved by IL-15 decreased significantly, blocking the beta and gamma chains of the IL-2R. Taken together, these findings demonstrate that IL-15 triggers the growth of leukemic B cells through IL-2R system subunits, pointing to the role of this novel cytokine in regulating the neoplastic proliferation in CLD.

Human immunodeficiency virus-1 (HIV-1) infection of CD8+ lymphocytes has been described in several in vitro culture systems, but whether CD8+ cells are a target and also serve as a reservoir for infection in vivo as yet is unknown. We addressed this issue in patients with acquired immunodeficiency syndrome (AIDS)-related lower respiratory tract chronic inflammation, which is characterized by a massive influx of CD8+ HIV-1-specific cytotoxic T lymphocytes (CTL). Proviral load in lung T lymphocytes and their subpopulations was evaluated by using the DNA-polymerase chain reaction (PCR) technique on cells retrieved by bronchoalveolar lavage. To avoid the possibility that the presence of HIV-1 DNA could be caused by contaminating CD4+ cells, serial dilutions of highly purified CD8+ cells were also analyzed by PCR. Our findings showed that lung CD8+ cells harbor and express HIV-1. To explore the possible mechanisms leading to pulmonary CD8+ lymphocyte infection, we evaluated CD4 gene expression on highly purified CD8+ cells by means of reverse transcriptase PCR. Despite the lack of membrane CD4 reactivity, we could show that CD8+ cells may express CD4 RNA. Coinfection of lung CD8+ cells harboring proviral HIV-1 sequences by viral agents capable of inducing CD4 expression (ie, HHV-6) was not detected. Our data indicate that not only CD4+ T lymphocytes and macrophages, but also CD8+ cells, may represent a target and/or a reservoir for HIV-1 in vivo, and suggest that lung CD8+ lymphocytes could derive from precursors equipped with enough CD4 molecules to become HIV-1 permissive. Aside from the cell-to-cell contact between activated HIV-1 specific CTL and relevant targets, the infection of precursors could represent an additional mechanism accounting for the infection of pulmonary CD8+ cells and their functional impairment.

To elucidate the mechanisms of alpha-interferon's (alpha-INF) therapeutic effect on clinical and laboratory findings in hairy cell leukemia, we sequentially monitored different immunologic parameters in three patients treated with recombinant alpha-INF. The most evident effect of this treatment on the immune system was the recovery of natural killer (NK) cell in vitro activity of peripheral blood lymphocytes, which was severely impaired before therapy. In particular, NK function began to improve after 3 months, and a complete recovery was obtained after 6 months in all cases. This increase parallels the improvement in clinical and laboratory findings.

Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) express tumor necrosis factor (TNF) and interleukin-2 (IL-2) receptors, but only a low proliferative response can be elicited in vitro by TNF alpha and IL-2. To investigate the functional properties of IL-2 and TNF alpha on leukemic B cells, we evaluated (1) the regulation of expression of TNF receptors (TNF-R) and IL-2 receptors on leukemic B cells after culture with TNF alpha and IL-2; (2) the effect of the combination of TNF alpha and IL-2 in a proliferative in vitro assay; and (3) the expression and regulation by these cytokines of receptors for hematopoietic factors, including IL-3, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage colony- stimulating factor (GM-CSF). Flow cytometry analysis showed that freshly isolated leukemic cells from B-CLL patients bear the 75-kD TNF- R and the 55-kD IL-2R; TNF alpha was able to upregulate the 55-kD IL-2R but not the 75-kD TNF-R. On the other hand, IL-2 was not able to modify the expression of the above-mentioned receptors. Although each cytokine alone was unable to induce a relevant proliferation of leukemic cells, a synergistic proliferative effect was detected when these cytokines were used in combination. Leukemic B cells from B-CLL patients bear receptors for hematopoietic factors (IL-3, G-CSF, and GM-CSF) that were upregulated in vitro by IL-2 via the 55-kD IL-2R. On the contrary, TNF alpha was unable to affect the expression of the above-mentioned receptors. These results indicate (1) that IL-2 and TNF receptors are related to each other on leukemic cells in B-CLL and (2) that the IL-2R is involved in the regulation of other structures, ie, CSF receptors, thus pointing to another functional role of this receptor complex and the related cytokine in leukemic cells.

By using an enzyme-linked immunosorbent assay, the presence of the soluble form of the interleukin-2 receptor (sIL-2R) was evaluated in the peripheral blood of 54 patients with B cell chronic lymphocytic leukemia (B-CLL). Serum levels of sIL-2R were correlated with clinical features, relevant hematologic and immunological data, and in some cases, with in vitro functional studies. In 51 patients (94.4%), the levels of sIL-2R were increased as compared with normal age-matched controls (1,781 U/mL +/- 231 v 276 U/mL +/- 26, respectively; P less than .001). Although this increase was observed in all stages of the disease and independently of several hematologic and immunologic parameters, a trend toward lower levels of sIL-2R was documented in patients with a less-invasive disease. When the values were correlated with the functional status of the residual T cell population, it was found that patients with the lowest levels of sIL-2R showed the best mitogenic response and helper capacity. It is suggested that in B-CLL patients the high levels of serum sIL-2R, capable of binding to its ligand, may block the T cell-produced IL-2, thus contributing toward a defective physiological action by this lymphokine. In turn, this defective availability of IL-2 may play a part in the abnormal immunoregulation that is implicated in the hypogammaglobulinemia, susceptibility to infections, and incidence of second neoplasias often observed in this disease.

Tumour necrosis factor alpha (TNF-alpha) is a cytokine with a variety of immunological properties. The identification of two receptors for this molecule, i.e. the 75 kDa and the 55 kDa TNF receptors (TNF-R), recently clarified the mechanisms through which this cytokine provides its wide range of immunomodulatory activities. In this study we have investigated the expression and the functional properties of these receptors on tumour-infiltrating lymphocytes (TILs) recovered from 17 patients with solid cancers (melanoma, colorectal carcinoma and lung cancer). To this end, TIL lines and freshly isolated TILs were evaluated for (a) the expression and the functional role of TNF receptors following culture in the presence of interleukin 2 (IL-2) and (b) the production of TNF-alpha following culture with IL-2 and the role of this cytokine in IL-2-driven TIL proliferation. Flow cytometry analysis demonstrated that TILs bear the 75 kDa TNF-R. Moreover, TIL lines express detectable messages for TNF-alpha and release this cytokine. Functional in vitro studies have shown that anti-TNF-alpha, as well as anti-75 kDa TNF-R antibodies, are able to inhibit the IL-2-induced TIL proliferation. These data demonstrate that TILs are equipped with a fully functional TNF-R system and suggest a putative role for this receptor and its ligand in the activation and expression of TILs following immunotherapy with IL-2.

A long term follow-up study has been undertaken in 33 patients with acute non-lymphoblastic leukaemia (ANLL) in order to establish whether a correlation exists between the clinical course and the immunologic pattern of lymphoid subpopulations. Peripheral blood lymphoid cells have been investigated longitudinally (each 1 to 4 months) during complete remission (CR), by morphologic, phenotypic and functional analyses. Particular attention has been paid to the evaluation of the natural killer (NK) cell compartment, by the detection of cells expressing an NK-related phenotype and by NK in vitro assay. Among the patients so far evaluable, 20 relapsed (R) and 10 are long survivors in CR 'off therapy' (LS). The most relevant finding was represented by statistically higher values of NK activity observed in LS vs. R patients (P less than 0.01). The removal of adherent cells before the NK assay, performed to investigate the possible inhibitory effect on NK function played by the macrophage component, abolished this difference, due to a selective increase of NK function in the R group. The longitudinal study revealed that NK activity tended to decrease in individual patients who subsequently relapsed. These data suggest a possible role of NK cells in the relapse control of ANLL, although it cannot be excluded that the low level of NK activity observed in the R group is the result of impending relapse rather than its cause.

Background Mediators released by alveolar macrophages, as well as by T cells, play an important part in modulating local immune processes in sarcoidosis. Among alveolar macrophage secretory products, arachidonic acid metabolites are known to regulate inflammatory and immune reactions. It has been suggested that cyclo-oxygenase and lipoxygenase pathway metabolites of arachidonic acid modulate the evolution of the granulomatous inflammatory response in the lung differently.Methods Alveolar macrophages recovered from the bronchoalveolar lavage (BAL) fluid of 32 patients with sarcoidosis in different states of disease activity and 10 normal subjects were evaluated for their ability to release prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄). Alveolar macrophages were cultured in the presence or absence of opsonised zymosan (500 μg/ml), and PGE₂ and LTB₄ levels in the culture supernatants were determined by enzyme immunoassay (EIA).Results Stimulated alveolar macrophages from patients with active sarcoidosis released higher LTB₄ levels than those from normal subjects, but no differences in PGE₂ release were observed between the two groups. The time course of LTB₄ release by activated alveolar macrophages showed that normal cells produced similar levels of the hydroxyacid during the early and late times of culture while LTB₄ release by activated cells from patients with sarcoidosis increased markedly after 60 minutes of culture, remaining elevated until 24 hours. Indomethacin (3×10^-6 M) caused the expected inhibition of PGE₂ formation without affecting LTB₄ release.Conclusions These results suggest that alveolar macrophages from the BAL fluid of patients with active sarcoidosis are primed to release LTB₄, which may contribute to the locally heightened immune response.

To elucidate the mechanisms of a-interferon's (a-INF) therapeutic effect on clinical and laboratory findings in hairy cell leukemia, we sequentially monitored different immunologic parameters in three patients treated with recombinant a-INF. The most evident effect of this treatment on the immune system was the recovery of natural killer (NK) cell in vitro activity of peripheral blood lympho- cytes, which was severely impaired before therapy. In particular, NK function began to improve after 3 months, and a complete recovery was obtained after 6 months in all cases. This increase parallels the improvement in clinical and laboratory findings. ® 1986 by Grune & Stratton, Inc.

Using monoclonal antibodies (MoAbs) termed GL183 and EB6, directed to a novel family of natural killer (NK) specific triggering molecules, four functional subsets of NK cells have been recently defined (GL183+EB6-; GL183+EB6+; GL183-EB6+; GL183-EB6-). In healthy individuals, all these subsets are represented in variable portion. The expression of EB6 and GL183 surface antigens has been analyzed in a series of 14 patients with lymphoproliferative disease of granular lymphocytes (LDGL) characterized by a chronic CD3-CD16+ lymphocytosis. Our data showed that in 11 of 14 cases, the proliferation was specifically sustained by one of the four possible subsets of granular lymphocytes (GLs) (seven cases: EB6-GL183-; three cases: EB6+GL183-; one case: EB6-GL183+). In the remaining three cases, a pattern was demonstrated that is consistent with that of healthy individuals (ie, the presence of all four subsets). When expressed on GL surfaces, in the majority of cases tested both EB6 and GL183 MoAbs behave as functional surface molecules as assessed in the redirected killing of P815 target cells. We also provided evidence that EB6+GL183+ proliferating cells show a definite (type 1) in vitro NK specificity as do their normal counterparts. The unique expansion of a defined subset of NK cells in most patients with LDGL suggests that the pathologic noxa leading to GL proliferation selectively acts on a specific subset of NK lymphocytes.

Two receptors for tumor necrosis factor (TNF) with different molecular weight (75-Kd and 55-Kd) and binding affinity have been recently discovered. To investigate the distribution and the functional role of these receptors on leukemic B cells from hairy cell leukemia (HCL) and B-cell chronic lymphocytic leukemia (B-CLL) patients, we evaluated: (1) the cytofluorimetric pattern of uncultured and cultured leukemic B cells incubated with utr-1 and htr-9 monoclonal antibodies (MoAbs), which specifically recognize the 75-Kd and 55-Kd TNF receptors (TNFR), respectively; (2) the effect of TNF-alpha and TNF-beta on leukemic B cells in an in vitro proliferation assay; (3) the role of anti-TNFR MoAbs on TNF-alpha and TNF-beta-driven B-cell growth; and (4) the proliferative effect of utr-1 and htr-9 MoAbs on in vitro cultured leukemic cells. Our study shows that the high affinity (75-Kd) but not the low affinity (55-Kd) TNFR molecules are expressed on freshly isolated leukemic B cells recovered from HCL and B-CLL patients. The expression of these receptors was neither upregulated nor downregulated by different stimuli, including TNF-alpha, TNF-beta, B-cell growth factor, and interleukin-2. TNF-alpha efficiently triggers the proliferation of HC and, to a lesser extent, the growth of B-CLL cells. TNF-beta was also able to transduce the proliferative signal in HCL, but not in B-CLL patients. TNF-alpha- and TNF-beta-driven B-cell proliferation was inhibited by the preincubation of leukemic B cells with utr-1 but not htr-9 MoAb. Moreover, anti-75-Kd, but not anti-55-Kd TNFR MoAb, was able to trigger the proliferation of leukemic B cells, and in particular of HC. These results show that leukemic B cells from patients with HCL and B-CLL are equipped with a fully functional high affinity TNFR.

In this study we describe the clinical, morphologic, immunologic, and genetic features of a chronic peripheral blood lymphocytosis associated with posttraumatic splenectomy in patients with human immunodeficiency virus-1 (HIV-1) infection. Among a series of 2,365 consecutive HIV-1 seropositive cases investigated, eight patients were selected for the presence of more than 4,000 lymphocytes/mm3. All cases were characterized by a lymphocytosis with cytoplasmic azurophilic granules; in three patients the hematologic picture was superimposable with that of lymphoproliferative disease of granular lymphocytes. Phenotypic analysis of lymphocytes showed a prevalent CD3+CD8+ pattern. In vitro evaluations, including the response to mitogens and interleukin-2 and the cytotoxic assays, showed an unimpaired lymphocyte function in the majority of our patients, even in those with advanced stages of the syndrome. The analysis of the configuration of the T-cell receptor (TCR) beta and gamma genes showed a polyclonal pattern of rearrangement. At the mean follow-up time of 45 +/- 8 months, one patient died of overdose when the clinical conditions were stable; all the other patients are alive, although disease progression was documented in two. Our results indicate that a chronic polyclonal lymphocytosis may be associated with HIV-1 infection; this finding seems to be restricted to patients who have undergone splenectomy. The demonstration of a still uncompromised immune system together with a silent clinical course in the patients under study also suggest that splenectomy per se does not favor an aggressive clinical behavior of HIV-1 infection.

The role of tumor necrosis factor-alpha (TNF-alpha) in the development of in vitro proliferative and cytotoxic abilities of granular lymphocytes (GL) in patients with lymphoproliferative disease of GL (LDGL) has been investigated. To this aim, taking advantage of the recent generation of specific monoclonal antibodies (MoAbs) reacting with the p55 and p75 TNF receptors (TNF-R) (htr-9 and utr-1 MoAb, respectively), we evaluated the expression and the functional role of each TNF-R in freshly isolated highly purified GL from a series of 10 LDGL patients (six CD3+ T-lineage GL and four CD3- natural killer [NK]- lineage GL). The expression of TNF-alpha transcripts and the release of TNF-alpha in the culture medium at resting conditions and following cell activation were also studied. Our data indicate that at resting conditions both CD3+ and CD3- GL express only the p75 TNF-R. Accordingly, a specific inhibition of phycoerythrin (PE)-conjugated TNF- alpha binding was demonstrated by the anti-p75 TNF-R utr-1 MoAb, but not by the anti-p55 htr-9 MoAb. Following activation with interleukin-2 (IL-2), anti-CD3, or anti-CD16 MoAbs, an increased expression of the p75 TNF-R and a slight induction of the p55 TNF-R was observed. Weak expression of specific TNF-alpha transcripts was detected at resting conditions and on unstimulated cells, whereas both IL-2 or anti-CD3 MoAb induced TNF-alpha mRNA. Under these in vitro conditions, detectable amounts of this cytokine were demonstrated in the culture supernatant of GL. The cytotoxic and proliferating activities mediated by IL-2 or anti-CD3 MoAb were dampened by anti-TNF-alpha antibody, suggesting a role for endogenous TNF-alpha in these functions. Both utr- 1 and htr-9 MoAbs showed a moderate inhibition of proliferative activity, whereas cytotoxicity was not reduced. Taken together, our results suggest that TNF-alpha plays a role in the mechanisms leading to CD3+ and CD3- GL in vitro activation in patients with LDGL.

In this study we provide evidence that the sera of patients with hairy cell leukemia (HCL) contain a factor that can prevent the binding of a monoclonal antibody specific for interleukin-2 receptor (IL-2R) to its target. This factor corresponds to the soluble form of IL-2R (slL-2R), as assessed by a specific enzyme-linked immunosorbent assay test, and appears to be released by neoplastic hairy cells. The serum sIL-2R levels were very high at diagnosis and significantly reduced during recombinant α-interferon (rIFNα2) therapy. Values of slL-2R appeared to be inversely related to the natural killer in vitro function displayed by peripheral blood mononuclear cells from the same patients. The presence of slL-2R in the serum of patients with HCL might be involved in the impairment of cell-mediated immunity observed in these patients and could represent a valuable marker for monitoring different phases of the disease and for modulating IFN therapy. © 1987 by Grune & Stratton. Inc.

This study was undertaken to investigate the pathways involved in the interleukin 2 (IL-2)-driven growth of tumour-infiltrating lymphocytes (TILs). For this purpose, TIL lines and freshly isolated TILs obtained from 16 patients with solid cancer (three melanoma, seven primary colorectal carcinoma, four hepatic metastases from colorectal cancer and two lung cancer) were evaluated for (a) expression of IL-2 receptor (IL-2R) both at the RNA level and on the cell surface by flow cytometric analysis and (b) their proliferative activity in response to IL-2 and the role of IL-2R subunits in the IL-2-driven TIL growth. Northern blot analysis showed that TILs express a strong message for both the p55 and the p75 IL-2R. Accordingly, flow cytometric analysis demonstrated that TILs bear both IL-2R chains. TILs cultured in vitro in the presence of rIL-2 were able to proliferate in response to different concentrations of this cytokine. Monoclonal antibodies (MAbs) specifically recognising the p55 and p75 IL-2R chains (anti-Tac and TU27 respectively) exhibited a marked inhibitory effect on IL-2-driven growth when added individually or in appropriate combinations. Our results demonstrated that TILs are equipped with a fully functional IL-2 receptor system, thus suggesting the involvement of this structure in the activation and expansion of TILs following immunotherapy with IL-2.

In this study we investigated the serum levels of a released soluble form of the interleukin-2 receptor (sIL-2R) in 42 patients with rheumatoid arthritis and in 12 cases of systemic lupus erythematosus. Data were evaluated in relationship to the clinical phase and compared with those observed in normal controls (N=56) and in osteoarthritis (N = 7). Increased levels were observed in both rheumatoid arthritis (mean ± SE, 604±49 U/ml) and systemic lupus erythematosus (1438±481 U/ml). These values were significantly higher than in control (256±15 U/ml;P<0.001) and in osteoarthritis (298±33 U/ml;P<0.001) groups. In addition, the highest values were associated with the active phases of both rheumatoid arthritis (active vs inactive, 771±78 vs 451±39 U/ml;P<0.001) and systemic lupus erythematosus (active vs inactive, 2108±489 vs 499±75 U/ml;P<0.001). Our findings suggest that the detection of sIL-2R in rheumatoid arthritis and in systemic lupus erythematosus may represent a good marker of disease activity, which indirectly indicates the ongoing activation and/or proliferation of immunoreactive cells which are involved in the pathogenetic events of these autoimmune conditions.

Using a panel of monoclonal antibodies (MoAbs), the frequency of cells bearing Class I and Class II major histocompatibility complex (MHC) determinants, transferrin receptor (TR) sites, and interleukin-2 receptors (IL-2R) has been evaluated on pulmonary alveolar macrophages (PAM) recovered from the bronchoalveolar lavage (BAL) fluid of 21 patients with pulmonary sarcoidosis (including 11 cases with active sarcoidosis and 10 cases with inactive disease), 8 patients with hypersensitivity pneumonitis (HP), and 6 normal non-smoking volunteers. When the frequency of Class II DR-positive cells was considered, 64.3% of control PAM expressed HLA-DR products. No statistically significant differences were observed between controls and sarcoid patients, while HP patients showed an enhanced proportion of DR+ PAM with respect to normal PAM (P<0.05). On the contrary, the frequency of PAM expressing HLA-DQ molecules was higher in both active sarcoidosis and HP patients with respect to patients with inactive sarcoidosis and normal subjects (P<0.001). A statistically significant increase in Class I antigen-positive PAM has been demonstrated in HP patients as compared to controls (P<0.05). Active sarcoid patients showed a higher number of PAM-bearing TR sites than controls and other groups of patients considered (P<0.001). An increase in the percentage of IL-2R-positive PAM has been demonstrated in active sarcoidosis (P<0.001). Our data suggest that (1) PAM of patients with the above-considered interstitial lung diseases are in a state of activation and exhibit structures which play a crucial role in antigenic recognition by T lymphocytes, such as HLA-DQ molecules; (2) the presence of TR in PAM of patients with active sarcoidosis could be related to a more advanced differentiation stage of these cells and/or to particular functional properties; and (3) a direct role of the IL-2/IL-2R system in the interaction between T cells and monocytes in sarcoid lung is crucial. Besides representing an additional parameter which differentiates BAL features of sarcoidosis from those of HP patients, these results could represent a useful tool in the evaluation of the macrophagic component of alveolitis by the BAL.

Summary Experiments herein reported were designed to clarify the degree of sensitivity of hairy cells to lysis in vitro. The cytotoxic capacity of peripheral blood lymphocytes from hairy cell leukemia patients against autologous and/or allogenic hairy cells was tested both at resting conditions and after in vitro stimulation. While effectors activated by interferon a, lectins, or interleukin-2 were unable to induce a lysis of hairy cells, in some cases we were successful in eliciting a definite lysis of these cells by triggering the effector cells with anti-CD3 monoclonal antibodies. Our results favour the interpretation that hairy cells are relatively, but not completely resistant to in vitro lysis. Furthermore, the evidence that anti-CD3 antibodies increase the efficacy of the cytotoxic machinery might support the use of these molecules in designing new immunotherapeutic approaches against tumor targets.

Background: FDG-PET scan performed early during chemotherapy (CT) is a powerful prognostic tool in lymphoma management. This study aimed to compare the predictive value on treatment outcome of the International Prognostic Score (IPS) with that of FDG-PET scan performed after two courses of standard CT in untreated advanced stage (AS) HL patients. Patients: From December 2001, 202 new AS HL patients were consecutively admitted to 11 Italian and 3 Danish hematological centers, on behalf of Intergruppo Italiano Linfomi and Danish Lymphoma Cooperative Group. The mean age was 35.5 years (14–79), the male/female ratio 105/97; AS (IIB–IVB) was present in 153, and unfavorable stage IIA (> 3 nodal sites involved or sub-diaphragmatic presentation or bulky disease or ESR > 40) in 49. Bulky and extranodal disease was recorded in 71 and 58 patients, respectively. All patients had FDG-PET at baseline (PET-0) and after 2 courses of CT (PET-2). 192 patients were treated with ABVD, 8 with ABVD-like CT, 2 with BEACOPP. 102 patients received consolidation radiotherapy after CT. All patients were given the therapy programmed at baseline, except in case of overt progression. Results: The mean time from the diagnosis to latest follow-up was 796 days (range 91–1716). 164 patients attained CR while 38 were chemoresistant: 34 showed disease progression during CT and 4 showed early relapse (within 6 months) after CR entry: (+28 – +178 days). 4 out of the 164 pts attaining CR relapsed later than 6 months. In univariate analyses, both PET-2 (p<0.0001) and IPS (p<0.001) were significantly associated with a higher probability of treatment failure. However, in a multivariate analysis only PET-2 was independently predictive of relapse/progression probability (51.0; 95 % C.I. 20.9 – 124.2). The sensitivity of PET-2 for treatment failure was 85%, the specificity 96% and the overall accuracy 94%. The 2-y FFS probability for PET-2 negative and for PET-2 positive patients were 96% and 14%, respectively (log Rank test, p<0.0001; Fig. 1). Conclusions: so far, FDG-PET after two cycles of CT is the most powerful tool available for predicting treatment outcome in AS HL. Figure Figure

Background: FDG-PET scan performed early during chemotherapy (CT) is a powerful prognostic tool in lymphoma management. This study aimed to compare the predictive value on treatment outcome of the International Prognostic Score (IPS) with that of FDG-PET scan performed after two courses of standard CT in untreated advanced stage (AS) HL patients.