Biomedical subjects

R Zambello

Publications and source records attributed to R Zambello.

At least 37 records · Page 2Linked to original sources

PubMed1997

Seroreactivity to an envelope protein of human T-cell leukemia/lymphoma virus in patients with CD3- (natural killer) lymphoproliferative disease of granular lymphocytes.

Natural killer (NK) cells are CD3- large granular lymphocytes (LGL) responsible for immunity against viral infections. A chronic lymphoproliferative disorder of NK cells has been described in which the expanded NK cells display a restricted phenotype and cytotoxic activity. These data raise the hypothesis that proliferating LGL in these patients result from discrete expansions of NK cells responding to an unknown, perhaps viral, antigen. Recently, it was found that mice transgenic for the tax gene of human T-cell leukemia/lymphoma virus (HTLV) develop NK leukemia. Therefore, we studied 15 patients with chronic NK lymphoproliferative disorder for evidence of HTLV infection. Sera were tested using an HTLV-I/II-enzyme linked immunosorbent assay and a modified Western blot assay containing recombinant env proteins. None of the sera met conventional criteria for HTLV seroreactivity. However, sera from 11 patients (73%) reacted with the recombinant HTLV env protein p21E. The anti-p21E reactivity of these sera was then mapped employing the recombinant proteins GD21 and BA21. No reactivity to the immunodominant HTLV epitope GD21 was observed, suggesting that prototypical HTLV infection is unlikely in these patients. This was confirmed by finding no evidence for HTLV nucleic acids by PCR analyses employing primers specific for conserved regions in the env, pol, and pX genes. In contrast, 10 of the 15 sera reacted with the epitope BA21, documenting for the first time an association between a unique seroreactivity and disease. The high incidence of BA21 seroreactivity in these patients suggests that exposure to a protein containing homology to BA21 may be important in the pathogenesis of this lymphoproliferative disorder.

Animals↗

PubMed1997

Interleukin-15 triggers activation and growth of the CD8 T-cell pool in extravascular tissues of patients with acquired immunodeficiency syndrome.

The impairment of interleukin-2 (IL-2) production occurs very early after human immunodeficiency virus (HIV) infection as a consequence of the quantitative depletion of Th1 cells. Despite the shift in cytokine production, most individuals develop an oligoclonal expansion of major histocompatibility complex restricted, HIV-specific CD8+ cytotoxic T lymphocytes (CTL) in different organs, suggesting that other cytokines replace IL-2 in initiating the tissue infiltration of CD8+ T cells. In this study we show that IL-15, a product of monocyte-macrophages and non-T cells and which has overlapping biological activities with IL-2, is involved in local cell networks accounting for the activation and expansion of CD8+ T-cell pools in a highly affected organ, ie, the lung. IL-15 induced proliferation of T cells obtained from the lower respiratory tract of HIV-infected patients with T-cell alveolitis and severe depletion of CD4+ T cells. Lung lymphocytes were CD45R0+/CD8+ T cells spontaneously expressing activation markers (CD69 and HLA-DR) and equipped with the receptorial subunits which bind IL-15, notably the beta and gamma chains of the IL-2 receptor (IL-2R) and the recently identified IL-15 binding-protein termed IL-15R alpha. Similar phenotypic findings were obtained after incubation of normal T cells with IL-15, which induced CD8+ T cells to express activation markers and to proliferate. The block of the IL-2R beta/IL-2R gamma complex with specific monoclonal antibodies abolished the T-cell stimulatory activity of IL-15 while the combination of IL-15 and tumor necrosis factor-alpha upregulated the proliferative response of lung T lymphocytes. The hypothesis that the tissue growth of lung CD8+ lymphocytes may involve cytokines produced from cells other than T lymphocytes was confirmed by the evidence that pulmonary macrophages expressed high levels of IL-15 and that anti-IL-15 antibodies inhibited the accessory function of alveolar macrophages on mitogen-induced CD8+ T-cell proliferation. Together, these results suggest that macrophage-derived cytokines produced at sites of T-cell infiltration play a role in the activation of HIV-specific CD8+ T-cell-mediated immune response.

AIDS-Related Opportunistic Infections↗

PubMed1997

Interleukin-15 triggers the proliferation and cytotoxicity of granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes.

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 (beta) and the p64 (gamma) 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 beta and gamma 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 gamma-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 alpha 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 beta and gamma chains of the IL-2R, providing further suggestions for the interpretation of the mechanisms that lead to cell expansion in patients with LDGL.

Adult↗

PubMed1997

The lymphoproliferative disease of granular lymphocytes: updated criteria for diagnosis.

The lymphoproliferative disease of granular lymphocytes (LDGL), also referred to as LGL leukemia, is a heterogeneous disorder, but is clinically, morphologically, and immunologically distinct. Although LDGL has recently been included in the revised classification of lymphomas as an independent clinical entity, no consensus exists on the criteria to establish the diagnosis. The aim of this report was to refine the parameters needed to make the diagnosis of LDGL. We studied 11 patients with chronic granular lymphocytosis selected from among 195 cases observed by our institutions from three different geographic areas (North America, Europe, and Asia). These cases did not meet the current criteria for inclusion in LDGL, since all patients had less than 2,000 GL/microL. However, in each of these patients, we found evidence for expansion of a discrete GL population. Clonal rearrangement of the T-cell receptor (TCR) beta gene was found in peripheral blood mononuclear cells (PBMC) of all nine patients with CD3+ LDGL. Using recently generated monoclonal antibodies (MoAbs) against the TCR V beta gene regions, we identified a unique TCR V beta on GL from each of three patients studied. In two patients with CD3- LDGL, we also identified a restricted pattern of reactivity, by staining with MoAbs against p58 antigen found on normal natural killer (NK) cells. The clinical features of these 11 patients with relatively low absolute number of GL were similar to those reported previously for patients with greater than 2,000 GL/microL. These data demonstrate that newer techniques such as MoAbs against V beta gene regions and p58 molecules and molecular analyses are useful to identify expansions of discrete GL proliferations. Demonstration of an expansion of a restricted GL subset is evidence for the diagnosis of LDGL, even in patients with a relatively low GL count. Our results also contribute to distinguish between the end of normality and the beginning of pathology in the broad spectrum of GL lymphocytoses.

Aged↗

PubMed1997

Spontaneous resolution of p58/EB6 antigen restricted NK-type lymphoproliferative disease of granular lymphocytes: role of Epstein Barr virus infection.

We describe a patient with a CD3- lymphoproliferative disease of granular lymphocytes (LDGL) characterized by proliferation of CD3-CD16+ GL, restricted to the expression of p58/EB6 antigen and lacking the p58/GL183 antigen. Using PCR analysis we demonstrated the presence of EBV DNA in the peripheral blood mononuclear cells and purified CD16+ GL from the patient; a monoclonal episomic configuration of the virus could not be demonstrated with Southern blot analysis. The presence of EBV DNA was also detected by PCR in the serum; this finding was associated with a serological pattern consistent with a previous, already seroconverted, EBV infection. During a 4-year follow-up the lymphocytosis spontaneously disappeared; interestingly, in terms of the p58 antigen expression, we provided evidence of the reconstitution of a normal pattern of circulating NK subsets (i.e. p58/EB6+ p58/GL183-, p58/EB6+ p58/GL183+, p58/EB6- p58/GL183-, p58/EB6-p58/GL183+). At the time of resolution of lymphocytosis, EBV-PCR analysis still demonstrated the persistence of EBV DNA in peripheral blood mononuclear cells, but not in the patient's serum. By indicating that inciting agents (in this case EBV) are involved in inducing the GL proliferation, our data contribute insights into the pathogenetic mechanisms accounting for in vivo GL accumulation in LDGL. It appears that a second, still unknown, event is required to determine the neoplastic transformation.

Aged↗

PubMed1997

Selection of T lymphocytes bearing limited TCR-Vbeta regions in the lung of hypersensitivity pneumonitis and sarcoidosis.

Hypersensitivity pneumonitis (HP) and sarcoidosis are interstitial lung disorders (ILD) characterized by a lymphocytic alveolitis that, in the active phase of the disease, is sustained by different T-cell subsets, i.e., CD8+ cells in HP and CD4+ lymphocytes in sarcoid patients. To address the question of whether a bias in T-cell selection occurs in the lung of patients with HP and sarcoidosis, we analyzed the T-cell receptor beta chain variable region (TCR-Vbeta) repertoire by flow cytometry and polymerase chain reaction (PCR) analyses in blood and lung lymphocytes of 14 HP and 25 sarcoid patients. To verify whether these cells can be activated in vitro through the TCR, blood and lung lymphocytes were also assessed for their responsiveness to different superantigenic stimuli represented by staphylococcal enterotoxins, including SEA, SEB, SEC1, SEC2, SED, and SEE. Flow cytometry and PCR analyses demonstrated an overexpression of cells bearing Vbeta2, Vbeta3, Vbeta5, Vbeta6, and Vbeta8 gene segments in the lung of HP patients as compared with the peripheral blood. In sarcoid patients cells bearing Vbeta2, Vbeta5, and Vbeta6 gene segments in the lung of HP patients as compared with the peripheral blood. In sarcoid patients cells bearing Vbeta2, Vbeta5, and Vbeta6 gene segments were overrepresented in the lung rather than in the blood. Both in HP and sarcoid patients almost all T cells bearing the dominant Vbeta segment belonged to the T-cell subset that sustains the alveolitis, i.e., CD8 in HP patients and CD4 in sarcoid subjects. Follow-up studies demonstrated that the recovery of the alveolitis was characterized by the disappearance of cells bearing a limited T-cell repertoire. Interestingly, T-lymphocyte response to different superantigens demonstrated that the proliferation elicited by different staphylococcal toxins was more pronounced in the lung than in the blood. Taken together, our findings indicate a compartmentalization of cells bearing discrete Vbeta gene products in the pulmonary microenvironment and suggest that the expansion of specific Vbeta region subsets occurring in the lung might result from triggering by a specific antigen. In fact, the removal from exposure in HP patients or specific treatment in sarcoidosis resulted in the decrease of the overrepresented cell population accounting for the lymphocytic alveolitis.

Adult↗

PubMed1997

Interleukin-15: a novel cytokine with regulatory properties on normal and neoplastic B lymphocytes.

IL-15 is a recently discovered cytokine that shares biological activities with IL-2. Although the biological functions displayed by these two molecules overlap to some extent, they are produced by different cell types and bind to distinct receptorial structures. Both cytokines transduce signals through the beta (p75) and gamma (p64) chains of the IL-2R system, but IL-15, like IL-2, binds to its own specific alpha chain, referred to as IL-15Ralpha. Similarly to IL-2, IL-15 is able to trigger both the proliferation and immunoglobulin production by normal B-lymphocytes. These biological functions may be acquired however only when B-cells have been preactivated in vitro with polyclonal mitogens, or alternatively, when they are cultured in association with other stimuli. By contrast, leukemic cells from patients with chronic B-cell malignancies, including B-cell chronic lymphocytic leukemia and hairy cell leukemia, proliferate to IL-15 regardless of in vitro preactivation. This peculiar IL-15 responsiveness distinguishes malignant B-cells from normal B-lymphocytes. Furthermore, the proliferation elicited by IL-15 in B-CLL and HCL is mainly related to the presence of the beta and gamma chains of the IL-2R system on malignant B-lymphocytes.

B-Lymphocytes↗

PubMed1996

The CD5/CD72 receptor system is coexpressed with several functionally relevant counterstructures on human B cells and delivers a critical signaling activity.

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.

Adult↗

PubMed1996

Role of IL-15, IL-2, and their receptors in the development of T cell alveolitis in pulmonary sarcoidosis.

Recent data suggest that the newly discovered cytokine IL-15 cooperates with IL-2 in driving T cell-mediated immune responses. The aim of this study was to determine the role of IL-15 in the regulatory networks leading to the development of T cell alveolitis in the lung of patients with sarcoidosis. We demonstrated that alveolar macrophages (AMs) isolated from the bronchoalveolar lavage of patients with active sarcoidosis expressed IL-15 mRNA and membrane and cytoplasmic IL-15, while AMs from healthy subjects and patients with inactive sarcoidosis did not. Pulmonary CD4+ T cells from sarcoid patients were equipped with the IL-2R subunits, which are able to bind IL-15, i.e., the IL-2R beta/IL-2R gamma complex, and proliferated in response to IL-15. Interestingly, the T cell proliferation elicited by IL-15 was comparable with that determined by IL-2. Following the addition of graded amounts of IL-15, IL-2-pulsed T cells showed a significant increase in their stimulation. TNF-alpha up-regulated the IL-15-mediated proliferative response of bronchoalveolar lavage T lymphocytes. Following the block of the IL-2R beta- and gamma-chains with specific mAbs, the stimulatory activity of IL-15 was abolished. The evaluation of the IL-2R on sarcoid AMs demonstrated the constitutive expression of alpha- and gamma-chain mRNA and proteins. Taken together, these findings demonstrate that IL-15 triggers the growth of sarcoid T cells through the IL-2R beta/IL-2R-gamma complex and raise the possibility that AMs may deliver proliferative signals for the development of the T cell alveolitis. Modulation of IL-2R on AMs could represent a critical variable in regulating local inflammatory responses.

Adult↗

PubMed1996

Interleukin-15 promotes the growth of leukemic cells of patients with B-cell chronic lymphoproliferative disorders.

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.

Adult↗

PubMed1996

The natural killer-related receptor for HLA-C expressed on T cells from CD3+ lymphoproliferative disease of granular lymphocytes displays either inhibitory or stimulatory function.

Four patients with lymphoproliferative disease of granular lymphocytes (LDGL) coexpressing CD3 and the natural killer (NK)-related "p58" receptor for HLA-C alleles were studied. These CD3+p58+ LDGLs have been detected among a series of 44 CD3+ LDGLs analyzed. Two patients with LDGL (GI and BA) expressed only the p58 molecule defined by the GL-183 and CH-L monoclonal antibodies (MoAbs), while the cases of patients PU and MA also coexpressed the molecular form identified by EB6 anti-p58 MoAb. Three LDGL cases (GI, MA, and PU) displayed the CD8+4-CD16+ T-cell receptor (TCR)alpha/beta+ phenotype, while one patient (BA) was CD8+4+CD16+ TCRalpha/beta+. Freshly isolated granular lymphocytes (GL) from these cases displayed cytolytic activity in an anti-CD3 MoAb-triggered redirected killing assay against the Fcgamma-receptor+ (Fcgamma-R+) P815 target cell line. Lysis of P815 target cells, triggered by an anti-CD3 or by anti-CD16 MoAb, could be inhibited by the addition of anti-p58 MoAb in three fresh or interleukin (IL)-2-cultured GL tested (GI, MA, and PU). Triggering of cytotoxicity against the HLA-DR+ Fcgamma-R+ Daudi cell line induced by appropriate superantigens could also be inhibited by anti-p58 MoAb in patients PU and GI with LDGL. These data indicate that activation through the CD16, CD3, and TCR molecules can be modulated by p58 receptors in these LDGLs. On the contrary, IL-2-expanded cells of patient BA were induced to lyse P815 target cells by anti-p58 MoAb. In addition, anti-p58 MoAB enhanced anti-CD16 MoAb triggered lysis and did not inhibit activation via CD3. These data indicate that, in this particular patient with LDGL, p58 displays a stimulatory effect on cell triggering, rather than the typical inhibitory effect previously observed in p58+ T-cell clones derived from healthy donors. The anti-p58 MoAb did not induce CA++ mobilization in p58+ LDGLs and in a p58+CD3+ normal T-cell clone equipped with inhibitory p58 molecules, while Ca++ mobilization could be observed in cultured GL from patient BA, which could be activated by anti-58 MoAb. These findings suggest that stimulatory and inhibitory p58 molecules are equipped with different signal transducing properties, thus contributing to a better knowledge of the normal counterpart.

Antibodies, Monoclonal↗

PubMed1996

Predictor of the rate of CD4 lymphocyte loss in HIV-1-seropositive asymptomatic hemophiliacs by in vitro immunoglobulin synthesis.

The objective of the present study was to evaluate the prognostic utility in determining the risk of AIDS progression in HIV-1-infected asymptomatic hemophiliacs by in vitro immunoglobulin (Ig) synthesis. With this aim, a cohort of 28 HIV-1-seropositive hemophiliacs were studied. All showed the number of CD4 lymphocytes higher than 400 positive cells/mm3. In all cases the spontaneous and pokeweed mitogen-induced in vitro production of Ig by peripheral blood lymphocytes was evaluated at the beginning of the study and the ratio stimulated/spontaneous (Stim/Spon) synthesis was calculated. At the same time, the absolute CD8+ cell count, IgA serum immunoglobulin, p24 HIV-1 antigenemia, and beta2 microglobulin were calculated. These data were monitored during the 4-year follow-up of patients and compared with the stimulated/spontaneous Ig synthesis ratio to evaluate the predictive significance on the progression of HIV infection. According to the stimulated/spontaneous Ig synthesis ratio, hemophilic patients were separated into two categories. Group I included 12 subjects with a Stim/Spon ratio higher than 2 (the lowest value of normal controls) and group II included 16 cases with a ratio lower than 2. As control, in 36 HIV-1-negative hemophiliac individuals the stimulated/spontaneous Ig ratio ranged between 2 and 42; mean +/- SEM, 12.9 +/- 1.8. At the end of the 4-year follow-up, group I patients showed a CD4 count and clinical staging consistent with those of the first evaluation; in contrast group II demonstrated a significant decrease in CD4 lymphocytes and deterioration of clinical conditions. Our results show that a low Stim/Spon Ig ratio when the CD4 lymphocyte count was still normal appears to predict the depletion of this lymphoid subset and progression to AIDS before T CD8, IgA immunoglobulin, p24 HIV-1 antigenemia, and beta2 microglobulin abnormalities. In this setting, the stimulated/spontaneous Ig ratio may represent a useful tool for clinical decisions in HIV-1-infected hemophiliacs.

CD4-Positive T-Lymphocytes↗

PubMed1996

HIV and pulmonary immune responses.

The lung is a central organ in human immunodeficiency virus (HIV)-induced pathophysiology, becoming infected during the asymptomatic period of the disease. The presence of HIV elicits a discrete virus-specific cytotoxic T lymphocyte (CTL)-mediated immune response in the pulmonary microenvironment. Here, Carlo Agostini and colleagues provide a framework for understanding the mechanisms underlying the spread of the retrovirus and the consequent progression of disease in patients with AIDS-associated lung involvement.

Acquired Immunodeficiency Syndrome↗

PubMed1996

IL-12 is involved in the activation of CD3+ granular lymphocytes in patients with lymphoproliferative disease of granular lymphocytes.

We investigated the effects of Il-12 on functional properties of CD3+ CD8+ granular lymphocytes (GL) of of patients with lymphoproliferative disease of granular lymphocytes (LDGL). To this aim, in 10 cases with clonal CD3+ GL proliferation (nine cases with an associated TCR alpha/beta receptor and one case with a TCR gamma/delta receptor) we studied the proliferative and cytotoxic activities of resting and alpha CD3 monoclonal antibody (mAb) activated cells in the presence of rIL-12 and anti-IL-12 blocking antibodies. Specific mRNA for IL-12 p40 subunit was also investigated. Our results showed that rIL-12 increased the proliferation of alpha CD3 pre-stimulated GL (2 to 6 times). Further, anti-IL-12 antibodies partially inhibited alpha CD3-induced cell growth, suggesting a role for this cytokine in the alpha CD3-mediated GL activation. The addition of antibodies blocking the p55 and p75 chains of IL-2 receptor (IL-2R) did not inhibit the rIL-12-mediated cell proliferation, indicating that the activity of rIL-12 is dependent of IL-2 in the in vivo expanded GL of patients under study. Concerning the cytotoxic activity, rIL-12 increased the alpha CD3-mediated NK activity against K-562 target cells and alpha CD3 redirected cytotoxicity against P815 target cells. Molecular analysis pointed out that, following alpha CD3 stimulation, patients' GL increased the expression of specific mRNA for the p40 subunit of IL-12 as compared to baseline conditions. Our data indicate that IL-12 is involved in the mechanisms of activation of clonal CD3+ GL in patients with LDGL; these features are consistent with the possibility that this discrete subset of GL might represent in vivo primed cytotoxic T lymphocytes.

Antibodies, Monoclonal↗

PubMed1996

Lysis of pulmonary fibroblasts by lymphokine (IL-2)-activated killer cells--a mechanism affecting the human lung microenvironment?

In this study we investigated whether IL-2-activated killer cells may bind and exert lytic activity against non-transformed lung fibroblasts. We demonstrated that human lymphokine-activated killer (LAK) cells generated in vitro following incubation with recombinant IL-2 of either peripheral blood mononuclear cells (PB-LAK) or lymphocytes obtained from bronchoalveolar lavage (BAL-LAK), but not resting cells, can lyse normal lung fibroblasts obtained from transbronchial lung biopsies in a 4-h 51Cr release assay. Both autologous and allogeneic fibroblasts were consistently lysed by LAK cells, thus suggesting that the phenomenon we observed is not MHC-restricted. Since fibroblasts can bind IL-2 through specific receptors, we evaluated whether long-term culture with rIL-2 could modulate the susceptibility to lysis of target cells. Our data showed that autologous fibroblasts were more resistant to lysis than allogeneic fibroblasts when they were cultured with rIL-2. Since LAK cells have been demonstrated to release a series of different immunomodulatory cytokines, we evaluated the effect of short-term incubation of fibroblasts with different factors, including IL-1, IL-2, IL-3, IL-4, IL-6, tumour necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma), on the binding and the lysis mediated by LAK cells. These cytokines were not directly cytotoxic on fibroblasts. Only IFN-gamma was found to have a significant protective effect against the lysis. Our data support the concept that a self-directed cytotoxicity against pulmonary fibroblasts is generated during lymphocyte activation with rIL-2.

Cells, Cultured↗

PubMed1996

Skewing of the T-cell receptor repertoire in the lung of patients with HIV-1 infection.

OBJECTIVES: A bias of the use of T-cell receptor (TCR) V beta regions has been reported both in peripheral blood and in several tissues in patients with AIDS, including lymph nodes, spleen and salivary glands. Although the disease is frequently characterized by an infiltration of T cells in the lung interstitium, no information is presently available on the configuration of the TCR repertoire in this microenvironment. This study was performed to address the question of whether a bias in T-cell selection occurs in the lung of patients with AIDS. METHODS: TCR beta-chain variable region (TCR-V beta) repertoire was analysed by flow cytometry and polymerase chain reaction (PCR) analyses in blood and lung lymphocytes of 13 patients with HIV-1 infection at different stages of the disease. Blood and lung lymphocytes were also assessed for their responsiveness to different superantigenic stimuli represented by staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED and SEE). RESULTS: Flow cytometry analysis in AIDS patients demonstrated an overexpression of cells bearing V beta 2 and V beta 3 gene segments in the lung compared with peripheral blood of the same subjects, as well as to lung and blood lymphocytes of normal controls. PCR analysis performed in AIDS patients extended these observations and demonstrated a significant bias also in the use of T cells bearing V beta 7 and V beta 9 gene regions in the lung compartment with respect to the blood. Virtually all T cells bearing the overrepresented V beta segment belong to the CD8 subset. Interestingly, T-lymphocyte response to different superantigens demonstrated a low proliferative rate in the lung with respect to the blood in HIV-1-infected patients. CONCLUSIONS: These findings indicate a compartmentalization of cells bearing discrete V beta gene products in the pulmonary microenvironment of patients with AIDS and suggest that the expansion of specific-V beta region subsets occurring in the lung might result from triggering by a superantigen.

Adult↗

PubMed1996

Expression of tumor necrosis factor-receptor superfamily members by lung T lymphocytes in interstitial lung disease.

Recently, a novel receptor superfamily has been identified whose members interact with a parallel family of ligands showing homology to tumor necrosis factor (TNF). To investigate the role of these receptor structures in the pulmonary environment, we evaluated the expression of some members of the TNF-receptor (CD27, CD30, CD40, CD95/Fas, CD120a, and CD120b) and TNF-ligand (CD40L, CD70/CD27L, CD30L, and mTNFalpha) superfamilies by bronchoalveolar lavage (BAL) T cells recovered from healthy subjects and patients with interstitial lung disease (ILD). Lung T lymphocytes recovered from control subjects showed a slight expression of CD27 but did not bear CD30, CD40, CD120a, or CD120b antigens. CD27 expression was restricted to normal CD4+ cells. Fas antigen (CD95), which is involved in activation-driven T-cell suicide, and the ligand for CD27 (CD70) were weakly expressed by normal BAL T-cell subpopulations. In patients with sarcoidosis, the majority of pulmonary T lymphocytes were CD4+ cells that expressed low levels of CD27 antigen and an upregulation of CD95 and CD70 molecules. When we characterized lymphocytes accumulating in the lung of patients with HIV infection and hypersensitivity pneumonitis, we demonstrated that T cells accounting for the CD8 alveolitis bore TNF-receptor type 2 (CD120b) at high density and were CD70+ while CD40L, CD30L, or mTNF-alpha expression were not found. The discrete surface expression of the TNF-receptors and TNF-ligands on alveolar T-cell subsets suggests that these molecules play a role in the immune regulatory mechanisms that ultimately lead to the alveolitis in the pulmonary microenvironment of interstitial lung disease.

Adult↗

PubMed1996

Detection of Epstein-Barr virus by PCR analyses in lymphoproliferative disease of granular lymphocytes.

We assayed peripheral blood mononuclear cells (PBMC) of fifty-eight patients with lymphoproliferative disease of granular lymphocytes (LDGL) for Epstein-Barr viral sequences. Phenotype analyses of the leukemic cell population(s) were also performed with a panel of monoclonal antibodies (MAb) to determine the lineage of the affected cells. Patients were shown to have proliferations of either CD3+ (T cell)LGL or CD3-(NK cell)LGL. Clonal studies showed that all CD3+ leukemic cells were clonally derived while none of the CD3-populations were. The CD3-leukemic cells were further studied through the use of two MAb, EB6 and GL183, that identify specific subsets of NK cells. Remarkably, 14 of 16 patients studied had skewed NK cell populations as compared to normal controls. Six of these patients had EBV detectable in their PBMC; moreover, EBV was found in all three LDGL patients with an EB6 + GL183-NK phenotype. Of the CD3+ patients, only six of thirty-nine contained EBV sequences in their PBMC. These results indicate that EBV is not the cause of the LDGL seen in these patients; however, there may be a specific subset of NK cells that respond directly to EBV infection.

CD3 Complex↗