The German Hodgkin Study Group risk model is useful for Hodgkin lymphoma patients receiving radiotherapy after autologous stem cell transplant.

PURPOSE: To apply the German Hodgkin Study Group (GHSG) risk model in patients with recurrent/refractory Hodgkin lymphoma receiving involved-field radiotherapy after autologous stem cell transplantation. MATERIAL AND METHODS: The study consisted in the retrospective analysis of 30 consecutive patients with recurrent/refractory Hodgkin lymphoma who received involved-field radiotherapy after autologous stem cell transplantation. Our policy was of adding involved-field radiotherapy for patients with positive PET scan before autologous stem cell transplantation (23 out of 30 patients, 77%), and/or irradiating sites of bulky disease at relapse (11 out of 30 patients, 37%). Patients were stratified into four risk groups according to the presence of the five clinical risk factors identified by the GHSG; (1) stage IV disease; (2) time to relapse≤3 months; (3) ECOG-PS≥1; (4) bulk≥5cm; and (5) inadequate response to salvage chemotherapy. RESULTS: The median interval from autologous stem cell transplantation to involved-field radiotherapy was 3 months (range, 1-7 months), and the median involved-field radiotherapy dose was 35Gy (range, 12-40Gy). At a median follow-up of 35 months (range, 1-132 months), the 2-year progression-free survival in the entire series was 60%. When examining the four different GHSG risk groups, the progression-free survival rate at 2 years was 86%, 83%, 50%, and 36% for patients with score=0, score=1, score=2, and score=3 to 5, respectively (P=0,01). Among the 12 patients havingat leastthree risk factors who underwent thoracic involved-field radiotherapy, three (25%) developed pneumonitis. CONCLUSION: The adoption of the GHSG risk model at the time of recurrence/progression is a useful prognostic tool to select patients with Hodgkin lymphoma for consolidative involved-field radiotherapy after autologous stem cell transplantation.

NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components.

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

Susceptibility to lysis of pulmonary alveolar macrophages by human lymphokine-activated killer cells.

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.

Expression and functional role of the p75 interleukin 2 receptor chain on leukemic hairy cells.

Hairy cell leukemia is a chronic lymphoproliferative disorder characterized by the expansion of neoplastic B-cells expressing the p55 chain of the interleukin 2 receptor (IL-2R) system that is recognized by anti-CD25 monoclonal antibodies (mAb) and binds interleukin 2 (IL-2) with low affinity. In the present study we investigated leukemic hairy cells (HC) for the presence of the p75 IL-2R chain which binds IL-2 with intermediate affinity and plays a crucial role in transducing the message to the cell. For this purpose, we tested highly enriched leukemic HC from six hairy cell leukemia patients for the presence of IL-2R transcripts and for the expression of the p55 and p75 IL-2R chains on their surface membrane by flow cytometry and immunoprecipitation analyses. The functional role of IL-2 in the regulation of HC proliferation was also investigated. Our results indicate that freshly isolated HC express detectable messages for both the p75 IL-2R and the p55 IL-2R. Flow cytometry analysis demonstrated detectable levels of p75 IL-2R on the HC from all patients tested. A mixture of two specific mAb was able to immunoprecipitate detectable amounts of p75 IL-2R from leukemic HC. When leukemic HC were cultured in the presence of several concentrations of IL-2 a low proliferative response was observed. Moreover, the IL-2-driven proliferation of HC was markedly inhibited by anti-p75 IL-2R mAb and to a lesser extent by anti-p55 IL-2R mAb. These findings provide direct evidence of the expression of different IL-2 receptors on leukemic HC and suggest that these molecules might play a role in leukemic cell growth.

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia.

CD49d, the alpha-chain of the integrin heterodimer α4ß1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

Different mechanisms of activation of proliferating CD3+ cells in patients with lymphoproliferative disease of granular lymphocytes.

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.

Origin of the soluble interleukin-2 receptor in the serum of patients with hairy cell leukemia.

High levels of soluble IL-2 receptor (sIL-2R) are detectable in the serum of HCL patients. To determine the cell source of this molecule, we evaluated the presence of sIL-2R in the supernatants obtained from in vitro cultures of leukemic (hairy cell, HC) and non-leukemic lymphocytes from six untreated HCL patients and from an additional four patients under therapy with rIFN-alpha 2. We demonstrated that cultured HCs at resting conditions were able to spontaneously release the sIL-2R, whereas control enriched B cells did not. This phenomenon was present only when culturing HCs recovered from patients observed at the time of diagnosis but was not observed during treatment with rIFN-alpha 2. Following activation in vitro with a series of different stimulatory agents including BCGF, phorbol myristate acetate, and anti-human IgM antibody, cultured HCs increased their capability to shed the IL-2R molecules. On the other hand, the release of sIL-2R from enriched T cell populations from HCL patients did not significantly differ from the value obtained in controls. Taken together, these findings provide evidence that leukemic B cells represent the main source of sIL-2R in HCL patients and further emphasize the importance of evaluating this parameter as a relevant marker for monitoring the effectiveness of rIFN-alpha 2 therapy.

CD8 alveolitis in sarcoidosis: incidence, phenotypic characteristics, and clinical features.

PURPOSE: Although the accumulation of CD4 cells in the lung and other involved tissues is regarded as the distinctive immunologic feature of sarcoidosis, a few sarcoid patients can present with CD8 alveolitis. This study evaluates the incidence as well as the clinical and immunologic features of sarcoidosis presenting with CD8 alveolitis. PATIENTS AND METHODS: A total of 2,214 consecutive bronchoalveolar lavage (BAL) specimens obtained from 481 patients with sarcoidosis between January 1985 and December 1991 were retrospectively analyzed. Subjects who entered the study had the following characteristics: (1) lymphocyte alveolitis and (2) lung CD4/CD8 ratio less than 1.0. Only data obtained from patients with a first episode of pulmonary involvement were included in the analysis (394 patients). RESULTS: Fifteen of the 394 patients studied at the time of diagnosis showed CD8 alveolitis as the presenting manifestation; the incidence of this phenomenon was 3.8%. A follow-up study of BAL T-cell subsets demonstrated that patients who showed high-intensity CD8 alveolitis at the onset of the disease maintained the CD8 pattern of alveolitis during relapses. Phenotypic analysis of lung T cells revealed that the accumulation of CD8 lymphocytes was due to the discrete local increase of CD45RO+ "memory" cells equipped with a number of accessory structures, including adhesion molecules and class II major histocompatibility complex-related HLA-DR antigen. CONCLUSIONS: The accumulation of CD8 cells in the sarcoid lung is likely to reflect a homing of memory cells due to the ongoing immunologic response against the unknown antigen causing the disease. Although CD8 alveolitis can be considered a relatively rare event in sarcoidosis, the possibility that an increase of CD8 cells in the BAL fluid might be sustained by an underlying sarcoid inflammatory process should never be dismissed on clinical grounds in patients with interstitial lung disease.

Time-course of interleukin-2 receptor expression in interferon beta-treated multiple sclerosis patients.

The time-course of CD25 (the 55-kD/alpha subunit of the interleukin-2 (IL-2) receptor) expression on CD4+ T lymphocytes, and serum levels of soluble IL-2 receptors (sIL-2R) and IL-2 were evaluated in relapsing-remitting multiple sclerosis (RRMS) patients treated with interferon beta-1b (IFNbeta1b). Peripheral blood samples were collected before therapy (T0), and 1 (T1), 2 (T2), 3 (T3), 6 (T4), and 12 (T5) months after therapy initiation. While at T1 and T2, half the patients showed an increased number of circulating CD4+ CD25+ lymphocytes and an up-regulation of CD25 expression, at T3 this T-cell subset was significantly reduced in all the patients. From T4 to T5, however, the progressive return to pretreatment values was observed. Serum sIL-2R levels were not significantly affected by IFNbeta1b at any time point. IL-2 was detected in only a few patients at T0, and never at T1 to T5. The transient up-regulation of CD25+ expression that occurred in about 50% of the patients may explain the unchanged relapse rate observed during the first 2 to 3 months after starting IFNbeta1b therapy. Our study demonstrates that IFNbeta1b down-regulates CD25 expression in vivo. This effect, however, was observed only after 3 months of therapy, and was followed by the return to pretreatment values after 6 to 12 months. Taken all together, our findings suggest that IFNbeta1b only transiently affects CD25 expression in vivo, and that this effect cannot account for the reported long-lasting beneficial action of IFNbeta1b on RRMS.

Sysmex SE-9000 hematology analyzer: performance evaluation on leukocyte differential counts using an NCCLS H20-A protocol. National Committee for Clinical Laboratory Standards.

We evaluated the performance (ie, imprecision, inaccuracy, and analytic sensitivity) of the Sysmex SE-9000 commercial hematology analyzer (TOA Medical Electronics, Kobe, Japan) on differential leukocyte counts according to the National Committee for Clinical Laboratory Standards H20-A protocol. The results obtained were compared with those from the Bayer H6000 and H3 (Bayer Diagnostic Division, Tarrytown, NY), the Coulter MAXM (Miami, Fla), and the microscopic method. Altogether, samples from 462 subjects were analyzed. The results show a substantial superimposition of reference intervals between the methods. The imprecision of the SE-9000 is low for all the leukocyte subpopulations, with the exception of basophils (coefficient of variation: neutrophils, 3.35%; lymphocytes, 4.25%; monocytes, 7.9%; eosinophils, 9.5%; and basophils, 44.2%) and is consistently lower than that of manual counts. The correlation with other methods is high, with the exception of basophils (r2: neutrophils, 0.94-0.95; lymphocytes, 0.93-0.97; monocytes, 0.76-0.85; eosinophils, 0.96-0.99; and basophils, 0.02-0.56). When compared with the microscopic method, an overestimation of neutrophils is seen mostly at low concentrations (mean difference, 2.63), and an underestimation of lymphocytes is seen at high concentrations (mean difference, -3.1). The clinical sensitivity was good, with an agreement of 75.7% on morphologic and 89.6% on distributional abnormalities. With a new analytical channel for immature cells (IMI), the analyzer shows high sensitivity in detecting immature cells of the granulocytic lineage (from 94.4% for immature granulocytes to 96% for myeloblasts).

Flow cytometric reticulocyte counting. Parallel evaluation of five fully automated analyzers: an NCCLS-ICSH approach.

We performed a parallel evaluation of 5 automated reticulocyte analyzers. The guidelines were those proposed by the National Committee for Clinical Laboratory Standards and the International Council for Standardisation in Haematology. Duplicate analyses were performed for 225 healthy subjects and 115 patients affected by various diseases. The reference intervals were different for each method (ADVIA 120, 27-125 x 10(3)/microL [27-125 x 10(9)/L]; CELL DYN 4000, 25-108 x 10(3)/microL [25-108 x 10(9)/L]; GEN-S, 20-85 x 10(3)/microL [20-85 x 10(9)/L]; SE 9500 RET, 23-95 x 10(3)/microL [23-95 x 10(9)/L]; and VEGA RETIC, 30-130 x 10(3)/microL [30-130 x 10(9)/L]). The comparisons of percentage counts with the microscopic reference method were satisfactory for all automated methods. However, a tendency to overestimate at low counts was noted. This progressively increased from the SE 9500 RET to the VEGA RETIC. The imprecision was excellent for all the methods at normal and high concentrations. This was higher at low concentrations. When compared with the microscopic reference, the analyzers showed satisfactory sensitivity at low counts and excellent sensitivity at high counts. The overall agreement varied from 74.8% for the GEN-S to 86.1% for the SE 9500 RET.

Interferon-beta (INF-beta) antibodies in interferon-beta1a- and interferon-beta1b-treated multiple sclerosis patients. Prevalence, kinetics, cross-reactivity, and factors enhancing interferon-beta immunogenicity in vivo.

We analysed the role of dosage, route and frequency of administration of clinical grade interferon-beta (IFN-beta) preparations in inducing anti-IFN-beta antibodies (IFN-beta-Abs) in 5 groups of relapsing-remitting multiple sclerosis (RRMS) patients who were respectively treated as follows: 1) weekly intramuscular (i.m.) injections of 30 mg of recombinant IFN-beta1a (Avonex), 2) subcutis (s.c.) injections of 250 mg IFN-beta1b (Betaferon) every other day, 3) weekly i.m. injections of 250 mg IFN-beta1b (Betaferon), 4) s.c. injections of 22 mg of IFN-beta1a (Rebif) three times a week, and 5) i.m. injections of 22 mg of IFN-beta1a (Rebif) twice a week. IFN-beta-Abs were determined by ELISA. IFN-beta1b was more immunogenic than IFN-beta1a not only when administered s.c. every other day, but also when administered i.m. at a lower weekly dose; i.m. injection, however, significantly delayed the appearance, and induced lower serum levels of IFN-beta-Abs. In patients treated with s.c. IFN-beta1b, Ab levels peaked 3 to 9 months after therapy initiation, and then slowly, but progressively, declined to pre-therapy levels that in some patients were reached after three years. Patients treated with i.m. or s.c. IFN-beta1a only rarely developed IFN-beta-Abs, and then at very low titers. Overall, the i.m. weekly administration of IFN-beta1a was the less immunogenic treatment. In IFN-beta1b-treated patients, a wash-out period of two/three months was sufficient to bring the IFN-beta-Ab levels below the cut-off. Our findings suggest that the immunogenicity of IFN-beta1a is low, regardless of the route of administration and the dosage, while that of IFN-beta1b is high, and is significantly, but not completely reduced by i.m. administration. As IFN-beta-Abs are cross-reactive, a wash-out period is suggested when the preparation is changed from IFN-beta1b to IFN-beta1a in order to maintain the clinical benefits of the therapy.

Laboratory evaluation of the Miles H.3 automated reticulocyte counter. A comparative study with manual reference method and Sysmex R-1000.

OBJECTIVE: To evaluate the performance of the new commercial Miles H.3 RTX analyzer in counting reticulocytes. METHODS AND PATIENTS: The results from the counter were compared to those obtained from microscopic methods, following the National Committee for Clinical Laboratory Standards H44-P guidelines, and to the results from the Sysmex R-1000 counter. In total, 279 samples were analyzed in duplicate with each of the three methods. One hundred thirty-three samples were from healthy subjects, while 146 were from patients with various pathologies, 10 of whom presented with posttherapeutic aplasia of the bone marrow and 9 with iron-deficiency anemia. RESULTS: The reference intervals for the normal controls are different for each of the three methods (manual: 0.35-2.35%, 16 to 116 x 10(9)/L; Miles H.3: 0.65-2.30%, 35.1 to 112.0 x 10(9)/L; Sysmex R-1000: 0.6-1.85%, 28.0 to 85.0 x 10(9)/L). The overall imprecision was lower for the instruments than for the microscopic method (Miles H.3: coefficient of variation, 11.6%; R-1000: coefficient of variation, 4.2%; microscopic method: coefficient of variation, 24.2%). The Miles H.3 shows a good correlation with the other methods, yet it overestimated the low values with respect to both the microscopic method (intercept, 0.55; slope, 0.70) and the R-1000 (intercept, 0.44; slope, 0.78). This became particularly pronounced in patients with marrow aplasia. CONCLUSIONS: Miles H.3 can produce results with an acceptable degree of accuracy. The agreement with the dedicated fluorescence-based flow cytometer R-1000 at normal and high concentrations is also good. The possibility of providing reticulocyte indices as well as erythrocyte indices (mean volume, mean hemoglobin content, mean hemoglobin concentration) and the relative dispersion indices could be useful in understanding red cell pathophysiology in normal and iron deficient patients.

Reticulocyte quantification by Coulter MAXM VCS (volume, conductivity, light scatter) technology.

In this study the ability of the Coulter MAXM analyzer to quantify reticulocytes was evaluated. The results were compared with those obtained by a microscopic method according to NCCLS H44-P recommendations and with the results from the automated analyzer Sysmex R-1000. Duplicate samples from 330 patients were analyzed. The reference intervals obtained with the three methods were: MAXM 0.37-1.80%, median 0.83%; manual 0.40-2.30%, median 1.00%; R-1000 0.60-1.95%, median 1.06%. The imprecision (CV) at all concentrations is lower than the microscopic method (low 16.1% vs 67%; normal 16.9% vs 28.9%; high 9.5% vs 13.0%). The MAXM shows a good overall correlation with the microscopic method (intercept 0.01, slope 0.89, r2 = 0.87) despite evidence of a significant overestimation at low concentrations [difference (D) 0.30] and a moderate underestimation at normal (D -0.15) and high (D -1.04) concentrations; the same behavior is shown in comparison with results from the R-1000, with an overall underestimation by MAXM (D -0.26). When compared with the microscopic method, MAXM shows a modest sensitivity at low reticulocyte counts (54.8%) and satisfactory sensitivity for high counts (87.3%), with an overall agreement of 81.3%.

Phenotypic diversity of natural killer (NK) populations in patients with NK-type lymphoproliferative disease of granular lymphocytes.

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.

Gamma delta T cell receptor subsets in the lung of patients with HIV-1 infection.

In this study the frequency of gamma delta+ cells and their subsets has been assessed in bronchoalveolar lavage (BAL) cell populations recovered from 51 patients at various clinical stages of HIV-1 infection. Thirteen out of the 51 HIV-1-infected patients showed an increase in the percentage of TCR delta 1+ BAL T cells (25.5%). BAL lymphocytes bearing pan-gamma delta antigens were also quantitatively increased in 10 patients (19.6%). A strict correlation was observed between the degree of CD8 alveolitis and the increase of gamma delta T cells. Phenotypic study of BAL gamma delta cells revealed that (a) V delta 2-related BB3+ cells accounted for the majority of lung gamma delta T cells; (b) these cells were CD45RO+ memory cells and expressed a series of adhesion molecules; and (c) 29% of BAL gamma delta T cells expressed CD8 surface molecules. We also compared the distribution of V delta 2 and V delta 1 subsets in paired samples of peripheral blood and BAL fluid. Patients who showed an increased number of BB3+ cells in the BAL fluid presented a reversal of the V delta 2 to V delta 1 cell ratio in the peripheral blood. By contrast, in the lung of normal subjects pulmonary BB3+ and A13+ cells were present in approximately the same proportions found in the peripheral blood. Taken together these data demonstrate that a redistribution of T cells expressing V delta 2 TCR takes place in the lung of a subset of patients with HIV-1 infection and CD8 alveolitis. In the pulmonary microenvironment these cells might play a role in the local immune response against HIV-1 and/or opportunistic infections.