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In recent years, considerable progress has been made in frontline therapy for elderly/physically unfit patients with CLL. The combination of obinutuzumab and chlorambucil (O-Clb) has been shown to prolong progression free survival (PFS, median PFS-31.5 months) and overall survival (OS) compared to chlorambucil alone. More recently, obinutuzumab given in combination with either ibrutinib or venetoclax improved PFS but not OS when compared to O-Clb. In this retrospective multinational, multicenter co-operative study, we evaluated the efficacy and safety of frontline treatment with O±Clb in unfit patients with CLL, in a "real-world" setting. Patients with documented del(17p13.1)/TP53mutation were excluded. A total of 437 patients (median age, 75.9years; median CIRS score, 8; median creatinine clearance, 61.1 mL/min)were included. The clinical overall response rate was 80.3% (clinical complete and partial responses in 38.7% and 41.6% of patients, respectively). Median observation time was 14.1 months and estimated median PFS was 27.6 months (95%CI, 24.2-31.0). In a multivariate analysis, high-risk disease [del(11q22.3) and/or IGHV-unmutated], lymph nodes of diameter >5cm, obinutuzumab monotherapy and reduced cumulative dose of obinutuzumab, were all independently associated with shorter PFS. The median OS has not yet been reached and estimated 2-year OS is 88%. In conclusion, in a "real-world" setting, frontline treatment with O-Clb achieves PFS comparable to that reported in clinical trials. Inferior outcomes were noted in patients with del(11q22.3) and/or unmutated IGHV and those treated with obinutuzumab-monotherapy. Thus, O-Clb can be still considered as legitimate frontline therapy for unfit CLL patients with low-risk disease. This article is protected by copyright. All rights reserved.

Recent evidence suggests that complex karyotype (CK) defined by the presence of >= 3 chromosomal aberrations (structural and/or numerical) identified by using chromosome-banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges toward the routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with >= 5 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcomes, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and/or TP53 mutations [TP53abs]), and the expression of somatically hypermutated (M-CLL) or unmutated immunoglobulin heavy variable genes. Thus, they contrasted with CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and 112,119 displayed an exceptionally indolent profile. Building upon CK, TP53abs, and immunoglobulin heavy variable gene somatic hyper-mutation status, we propose a novel hierarchical model in which patients with high-CK exhibit the worst prognosis, whereas those with mutated CLL lacking CK or TP53abs, as well as CK with 112,119, show the longest overall survival. Thus, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with >= 5 chromosomal aberrations emerges as prognostically adverse, independent of other biomarkers. Prospective clinical validation is warranted before ultimately incorporating high-CK in risk stratification of CLL.

We performed an observational study on the efficacy of ben-damustine and rituximab (BR) as first salvage regimen in chronic lymphocytic leukemia (CLL). In an intention-to-treat analysis including 237 patients, the median progression-free survival (PFS) was 25 months. The presence of del(17p), unmutated IGHV and advanced stage were associated with a shorter PFS at multivariate analysis. The median time-to-next treatment was 31.3 months. Front-line treatment with a chemoimmunotherapy regimen was the only predictive factor for a shorter time to next treatment at multivariate analysis. The median overall survival (OS) was 74.5 months. Advanced disease stage (i.e. Rai stage III-IV or Binet stage C) and resistant disease were the only parameters significantly associated with a shorter OS. Grade 3-5 infections were recorded in 6.3% of patients. A matched-adjusted indirect comparison with ibrutinib given second-line within Named Patient Programs in the United Kingdom and in Italy was carried out with OS as objective end point. When restricting the analysis to patients with intact 17p who had received chemoimmunotherapy in first line, there was no difference in OS between patients treated with ibrutinib (63% alive at 36 months) and patients treated with BR (74.4% alive at 36 months). BR is an efficacious first salvage regimen in CLL in a real-life population, including the elderly and unfit patients. BR and ibrutinib may be equally effective in terms of OS when used as first salvage treatment in patients without 17p deletion. (Registered at clinicaltrials.gov identifier: 02491398)

The accumulation of T cells and monocytes at sites of ongoing inflammation represents the earliest step in the series of events that lead to granuloma formation in sarcoidosis. In this study, we evaluated the pulmonary production of IFN-inducible protein 10 (IP-10), a CXC chemokine that stimulates the directional migration of activated T cells. Striking levels of IP-10 were demonstrated in the bronchoalveolar lavage (BAL) fluid of 24 patients with pulmonary sarcoidosis and lymphocytic alveolitis, as compared with patients with inactive disease or control subjects. A positive correlation was demonstrated between IP-10 levels and the number of sarcoid CD45R0+/CD4+ cells in the BAL. Immunochemistry, performed with an anti-human IP-10 polyclonal Ab in lymph nodes displaying prominent sarcoid granulomas, showed that cells bearing IP-10 were mainly epithelioid cells and CD68+ macrophages located inside granulomatous areas. Macrophages recovered from the BAL of sarcoid patients stained positive for IP-10 protein. Furthermore, alveolar macrophages isolated from sarcoid patients with T cell alveolitis and cultured for 24 h in presence of IFN-γ secreted definite levels of IP-10 capable of inducing T cell chemiotaxis. Interestingly, alveolar lymphocytes recovered from patients with active sarcoidosis were CD4+ T cells expressing Th1 cytokines (IL-2 and IFN-γ) and high levels of CXCR3. Taken together, these data suggest the potential role of IP-10 in regulating the migration and activation of T cells toward sites of sarcoid inflammatory process and the consequent granuloma formation.

Immunological defence mechanisms of the lung are provided by several different types of immunocompetent cell. Among these, the most important are lymphocytes, alveolar macrophages and neutrophils. This review focuses mainly on the pattern of physiological activities of pulmonary lymphocytes in the local host defences. The article provides an overview of the events leading to the recruitment, homing and activity of lymphocytes in the lower respiratory tract. The pathways through which the pulmonary lymphoid system recognizes, destroys and contributes to removing potentially harmful inhaled antigenic materials are also briefly discussed. The final section of the article highlights the pathogenetic role envisaged for lymphoid cells in disease states, in the context of the known biology of these cells.

Hypersensitivity pneumonitis (HP) is a lung disorder characterized by an exaggerated accumulation of CD8+ T lymphocytes in the pulmonary parenchyma. To investigate the mechanisms accounting for the T cell alveolitis taking place in the lung of HP patients and their pattern of growth, cells recovered from the bronchoalveolar lavage (BAL) of seven patients were evaluated for: 1) the expression of activation markers, including IL-2R (p55 and p75 subunits), HLA-DR and VLA-1 Ag; 2) the ability of IL-2 and IL-4 to induce in vitro proliferation; 3) the capability to synthesize and release IL-2 by determining the levels of IL-2 in BAL cell-free supernatants and by evaluating the presence of mRNA transcripts for IL-2; and 4) the molecular configuration of the beta- and gamma-genes of the TCR. This study demonstrates that a high number of BAL lymphocytes recovered from the lungs of HP patients express activation markers including the p75 chain of IL-2R, VLA-1, and HLA-DR Ag. These cells express the CD3+,CD8+,CD16-,CD56+ phenotype and proliferate in vitro in the presence of IL-2 but do not release this cytokine. Furthermore, IL-2 transcripts could not be detected in BAL resting T lymphocytes. No proliferation was observed in the presence of IL-4. The analysis of the configuration of the TCR beta- and gamma-genes showed a polyclonal pattern, with the exception of one case in which extra bands were observed following digestion with BamHI and EcoRI restriction enzymes. Taken together, our data suggest that the IL-2 system may play a central role in the mechanisms accounting for lymphocytic alveolitis in HP patients. Although the pattern of growth is usually polyclonal, such polyclonal recruitment seems to be biased toward cells that have rearranged and possibly expressed particular V beta or V gamma genes, thus leading to the hypothesis that the events that take place in the lung of these patients may occasionally elicit an oligoclonal expansion of the cells proliferating in lung parenchyma.

The CD5 molecule is expressed on T cells and, at a lower density, on a minor B cell subset (CD5+ B cells). The pan-B Ag CD72 was recently identified as the CD5 counterstructure, and several data suggest the involvement of this ligand pair in T-B cell cognate interaction. However, the functional role of CD5 and CD72 molecules within the B cell compartment is still unknown. In this work we studied umbilical cord blood CD5+ B cells (B-1a), adult splenic CD5- B cells (B-2), and CD5+ B cells from patients with chronic lymphocytic leukemia. Flow cytometry analysis and proliferation assays were used to determine 1) the ability of B-1a and B-2 cells to coexpress functionally relevant counterligands other than CD5 and CD72, and 2) the signaling capacity of CD5 and CD72 in terms of B cell activation and proliferation. To this purpose, freshly isolated or preactivated normal and neoplastic B cells were cultured with agonistic anti-CD5 or anti-CD72 mAbs in the presence or the absence of cytokines equipped with B cell activity. Our data demonstrate that CD5 and CD72 molecules are coexpressed with other ligand pairs usually involved in T-B cell cognate interaction on B-1a cells, but not on B-2 cells. CD5 and/or CD72 engagement delivers critical costimulatory signals in B-1a, B-2, and B cells from patients with chronic lymphocytic leukemia, but with different requirements and patterns. Besides suggesting the potential involvement of B-1a lymphocytes in B-B cell interactions during T-independent B cell responses, our results indicate that CD5 and CD72 counterstructures play a functional role in the B cell compartment.

Cells recovered from bronchoalveolar lavage (BAL) and tissue sections from transbronchial lung biopsies were studied in 16 patients with symptomatic hypersensitivity pneumonitis (HP) and in six subjects with a similar history of exposure but without features of disease by using a series of monoclonal antibodies (MoAb) detecting different lymphocyte subpopulations, including T and T subsets, B lymphocytes, and natural killer (NK) cells. Their functional activities in cytotoxic and suppressor assays and the microenvironment in the lung by using immunohistological techniques were also evaluated. It has been demonstrated that the majority of cells recovered from BAL of HP patients are represented by T8 lymphocytes, with a relevant imbalance of the T4/T8 ratio (p less than 0.001). HNK-1+ cells were markedly increased (p less than 0.001), whereas the frequency of cells bearing other NK-related markers (NK-15, VEP 13, Ab8.28, T10, M1, and Fc gamma R) were not significantly increased with respect to controls. Immunohistological study confirmed that the majority of cells infiltrating lung parenchyma are T8+ lymphocytes. The number of HNK-1+ cells detected on lung biopsies was very low in all cases, even in patients with the highest values on BAL suspensions. The evidence of cells bearing the proliferation-associated markers (Tac and T9 antigens) seems to support the hypothesis of a local proliferation in the lung. In terms of phenotypic analysis, the results observed in the group of asymptomatic individuals are qualitatively superimposable on those observed in the HP group, but the magnitude of the phenomenon is less prominent and therefore the data are not as statistically significant as that produced by the comparison between HP patients and the same controls. Functional analysis of BAL T cells from both HP patients and asymptomatic individuals showed suppressor activity in vitro, as determined by the ability to influence a pokeweed mitogen (PWM)-driven B cell differentiation assay. BAL cells from HP patients were also able to display a definite cytotoxic function in vitro, whereas BAL lymphocytes from asymptomatic subjects did not. Taken together, these data demonstrated that cells responsible for the alveolitis in patients with HP are characterized by the expansion of T cells with the phenotype and functions of both suppressor and/or cytotoxic lymphocytes. This expansion is likely to be related to a local immunologic response to the antigenic stimulus and may provide new insights into the pathogenetic mechanisms of this disease, its pathological pattern, and its management.

To investigate the role of p55 and p75 chains of interleukin-2 receptor (IL-2R) on the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), the cells obtained from 11 LDGL patients (belonging to the CD3+ group) were studied for (a) the surface expression and (b) mRNA transcripts of the p55 and p75 IL-2R after activation with anti-CD3 monoclonal antibody (mAb) or interleukin-2 (IL-2). The effects of mAbs specifically blocking the p55 and p75 IL-2R on the generation of proliferative and cytotoxic functions were studied following anti-CD3 mAb stimulation. A significant difference was observed in the expression of p55 and p75 antigens on LDGL cells under resting conditions: a low number of p55 IL-2R+ (mean 1.2 +/- 0.4%) and high values of p75 IL-2R+ cells (54.9 +/- 7.4%). Accordingly, a barely detectable message for the p55 IL-2R and a strong signal for the p75 IL-2R mRNA were demonstrated. Following activation with anti-CD3 or IL-2, different patterns of IL-2R expression were observed. Anti-CD3 mAb induced an increase in the expression of the p55 IL-2R both at the mRNA and antigen level, whereas the p75 values remained consistently raised. In contrast, IL-2 induced the expression of p55 IL-2R mRNA associated with only a slight expression of this antigen. This finding was associated with a decrease in the cell expression of the p75 IL-2R, whereas the amount of p75 mRNA was unchanged. Both anti-CD3 mAb and IL-2 induced cell proliferation and cytotoxicity against the K-562 target cells. Anti-p55 IL-2R mAb did not affect the cytotoxic activity mediated by anti-CD3, but it markedly inhibited cell proliferation. Anti-p75 mAb did not inhibit either lytic function or cell proliferation mediated by anti-CD3 mAb, suggesting that only the high affinity IL-2R (p55 plus p75) is involved in anti-CD3 mediated cell activation in LDGL patients. This mechanism is different from that responsible for the IL-2 activation of CD3+ GL in LDGL patients, which is achieved through the p75 IL-2R alone. These results provide new insights into the pathophysiology of proliferating GL in LDGL patients and may also contribute to further characterization of the normal CD3+ GL population.

B-chronic lymphocytic leukemia (B-CLL) is a malignant disorder characterized by the accumulation of the leukemic cells in the G0-G1 phase of the cell cycle and expressing high levels of the anti-apoptotic protein Bcl-2. Since we observed that the treatment of autoimmune complications with Cyclosporine A (CsA) determined in some CLL patients an improvement not only of the autoimmune phenomena, but also of the leukemic process, we evaluated the in vitro cytotoxicity of CsA as compared to Dexamethasone (Dex) on leukemic cells. Leukemic cells obtained from 32 B-CLL patients showed a heterogeneous pattern of spontaneous apoptosis at 24 h interval and this pattern permitted to identify: Group 1 (14/32) with high (20%) apoptotic rate and Group 2 (18/32) with low cell death. CsA and Dex increased cell death in both groups with a different timing by an apoptotic mechanism that does not involve Bcl-2. Furthermore, in Group 2, CsA-induced apoptosis was significant higher than that observed with Dex both at 4 and 24 h. We suggest that, in B-CLL, CsA has a significant pro-apoptotic activity manifested also in patients with low spontaneous apoptosis. Our observations might be taken into account to consider new therapeutic strategies in B-CLL.

In this paper, the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lung of patients with HIV-1 infection was evaluated. This cytokine has well recognized effects on granulocyte and macrophage growth and differentiation and plays some role in the mechanisms leading to the accumulation of alveolar macrophages (AM) in patients with interstitial lung disease. Detectable levels of GM-CSF (up to 10 pg/ml) were demonstrated in unconcentrated bronchoalveolar lavage fluid retrieved from HIV-1-seropositive patients, thus suggesting that the GM-CSF is released in vivo in the lung during HIV-1 infection. A statistically significant correlation was demonstrated between the bronchoalveolar lavage concentrations of GM-CSF and the absolute numbers of AM and lung neutrophils. Cell-free supernatants obtained from unstimulated 24-h cultured AM isolated from HIV-1-infected patients contained discrete amounts of GM-CSF, as demonstrated by an immunoenzymatic assay. AM lost the capability of releasing GM-CSF after 72 h of culture, thus suggesting that the production of GM-CSF is not constitutive in AM. After exposition of AM with LPS, the release of GM-CSF and the expression of its mRNA significantly increased with respect to the baseline values; interestingly, the amount of GM-CSF released by LPS-stimulated AM was more than 10-fold higher in HIV-1-infected patients than in healthy subjects. As demonstrated by flow cytometry analysis, more than 70% of freshly isolated AM efficiently bound phycoerythrin-GM-CSF, thus indicating that they express the receptor for GM-CSF. Determination of AM in G1, S, and G2+M by flow cytometry showed that, after 48 h of culture with GM-CSF, 5.5 to 7% of AM entered the proliferative phase of the cell cycle. Taken together, these findings suggest that AM might represent an important source of GM-CSF production in HIV-1 infection. In particular, the hypothesis is formulated that pulmonary opportunists might trigger AM to synthesize GM-CSF in situ. The local overproduction of this cytokine is likely to play a role in the pathogenic events leading to the local proliferation of AM and recruitment of neutrophils in AIDS-associated interstitial lung disease.

Several costimulatory molecules play a key role in the differentiation of B lymphocytes and in T-B-cell interactions. In this study, we addressed the question of whether different receptors and counter-receptors may be expressed on malignant B lymphocytes from chronic B-cell malignancies. Using flow cytometry and reverse transcription PCR analyses, the expression of molecules belonging to the tumor necrosis factor receptor (TNFR) and tumor necrosis factor ligand (TNFL) families, as well as the expression of CD80 and CD86 molecules, was analyzed in normal B cells and in different chronic lymphoproliferative disorders of B-cell type, including B-cell chronic lymphocytic leukemia (CLL), mantle cell lymphoma, hairy cell leukemia (HCL), and HCL variant. Different patterns of expression of TNFR and TNFL superfamily molecules were demonstrated among B-cell malignancies. In particular, CD40 was commonly observed on all B cells (both tumor and normal), whereas its ligand (CD40L), which is usually undetectable on resting normal B lymphocytes, was expressed in CLL and HCL but not in other chronic lymphoproliferative disorders. CD27 was not shown in normal B cells, although it was present in all malignancies and with particularly high density in mantle cell lymphoma. CD70 was widely distributed on tumor B lymphocytes, but not on the CD5+ normal counterpart. CD30 was strongly expressed in HCL variant and weakly in B-cell CLL, whereas its ligand showed a wide pattern of expression, including all neoplastic and normal B cells. TNFR II (CD120b) and CD80 were distributed on neoplastic B cells from all groups, usually at an intermediate to high degree of intensity, whereas the CD86 molecule was present at lower intensity than CD80. Finally, reverse transcription PCR analysis confirmed the presence of CD40L, CD30, and CD30L mRNAs in those B cells expressing the corresponding membrane-bound proteins at low density. Our data indicate that TNFR and TNFL molecules are of use clinically both in differentiating B-cell malignancies from the normal counterpart (i.e., CD27, CD70, CD40L, CD30, and CD80) and in defining different chronic B-cell disorders (i.e., CD40L, CD27, and CD30). Interestingly, the observation that several receptors and their ligands (i.e., CD40/CD40L, CD30/CD30L, and CD27/CD70) can be expressed on the same cell suggests that these molecules play a role in initiating and maintaining the neoplastic process by mediating B-T and B-B interactions.

In this study we addressed the question of whether lymphokine-activated killer (LAK) cells, besides killing neoplastic cells, may exert a certain degree of lysis on the normal counterpart; in particular we took into consideration the toxicity against pulmonary alveolar macrophages (PAM). We demonstrated that human LAK cells generated in vitro following incubation of peripheral blood mononuclear cells with recombinant interleukin 2 for 4 days were able to lyse normal PAM in a 4-h 51Cr release assay. Similarly, PAM recovered from patients suffering from nonneoplastic interstitial lung disorders, i.e., sarcoidosis and hypersensitivity pneumonitis, were shown to be susceptible to the cytotoxic function provided by LAK cells. Both autologous and allogeneic PAM were lysed by LAK cells, thus suggesting that the phenomenon we observed does not require a major histocompatibility complex restriction. Preincubation of PAM under study with gamma-interferon did not affect their susceptibility to the lysis mediated by LAK cells. Furthermore, cold target inhibition assay demonstrated that normal PAM could efficiently compete with both NK-sensitive and NK-resistant target lines for the binding sites on LAK cells, thus indicating that the putative receptor(s), or at least the mechanism of target recognition, is shared by PAM and these different target cell lines. The evidence herein provided that LAK cells are cytotoxic to normal, nontransformed PAM points out that the pathogenetic mechanisms involving this self-addressed lytic activity could account for some adverse reactions related to LAK/interleukin 2 immunotherapy.

To clarify the mechanisms accounting for the hairy cell proliferation, in five patients with hairy cell leukemia (HCL), we evaluated the ability of neoplastic cells to proliferate in vitro in response to tumor necrosis factor-alpha (TNF-alpha), B-cell growth factor (BCGF), and interleukin 4 (IL-4). In addition, supernatants recovered from cultures of unstimulated hairy cells were tested for the capability to induce the proliferation of allogeneic hairy cells. We demonstrated that TNF-alpha and BCGF were able to induce the in vitro growth of hairy cells (stimulation index (SI) ranging, at different concentrations, from 1.6 to 10.7 and from 1.6 to 25.1 for TNF-alpha and BCGF, respectively). IL-4 did not show any proliferative effect on hairy cells. When unstimulated hairy cell supernatants were tested for the proliferative activity on allogeneic hairy cells, we demonstrated a low proliferative effect that was not inhibited by an anti-TNF-alpha antibody. Our findings demonstrate that unstimulated hairy cells proliferate in vitro in response to TNF-alpha and BCGF, thus suggesting that these cytokines could be involved in an autocrine model of cell proliferation.