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The standard-of-care (SOC) first-line therapy for ovarian cancer (OC) patients is plagued with high relapse rates. Several studies indicated the immune system’s prominent role changing the disease course in OC patients. Chemo-immunotherapy regimens, currently being explored, include oregovomab, which is a monoclonal antibody specific for the OC associated antigen carbohydrate/cancer antigen 125 (CA125) that yielded promising results when administered together with SOC in a previous study. The QPT-ORE-002 multi-site phase II randomized study demonstrated that in patients with advanced OC, oregovomab combined with first-line SOC improved overall and progression-free survival, compared to SOC alone. The study included an Italian cohort in which we demonstrated that adding oregovomab to SOC resulted in increased patient numbers with amplified CA125-specific CD8+T lymphocytes/ml peripheral blood counts, which might explain the improved therapeutic effect of SOC + oregovomab over SOC alone. Predictive for oregovomab efficacy was a less suppressive immune environment at baseline as indicated by low numbers of circulating myeloid-derived suppressor cells, subset type 4, and a low neutrophil-and-monocyte to lymphocyte ratio.

BackgroundIdentifying the patients who may benefit the most from immune checkpoints inhibitors remains a great challenge for clinicians. Here we investigate on blood serum amyloid A (SAA) as biomarker of response to upfront pembrolizumab in patients with advanced non-small-cell lung cancer (NSCLC).MethodsPatients with PD-L1 ≥ 50% receiving upfront pembrolizumab (P cohort) and with PD-L1 0–49% treated with chemotherapy (CT cohort) were evaluated for blood SAA and radiological response at baseline and every 9 weeks. Endpoints were response rate (RR) according to RECIST1.1, progression-free (PFS) and overall survival (OS). The most accurate SAA cut-off to predict response was established with ROC analysis in the P cohort.ResultsIn the P Cohort (n = 42), the overall RR was 38%. After a median follow-up of 18.5 months (mo), baseline SAA ≤ the ROC-derived cut-off (29.9 mg/L;n = 28/42.67%) was significantly associated with higher RR (53.6 versus 7.1%; OR15, 95% CI 1.72–130.7,p = 0.009), longer PFS (17.4 versus 2.1 mo;p p 29.9 mg/L. In multivariate analysis, low SAA positively affects PFS (p = 0.001) and OS (p = 0.048) irrespective of ECOG PS, number of metastatic sites and pleural effusion. SAA monitoring (n = 40) was also significantly associated with survival endpoints: median PFS 17.4 versus 2.1 mo and median OS not reached versus 7.2 mo when SAA remained low (n = 14) and high (n = 12), respectively. In the CT Cohort (n = 30), RR was not affected by SAA level (p > 0.05) while low SAA at baseline (n = 17) was associated with better PFS (HR 0.38, 95% CI 0.16–0.90,p = 0.006) and OS (HR 0.25, 95% CI 0.09–0.67,p ConclusionLow SAA predicts good survival outcomes irrespective of treatment for advanced NSCLC patients and higher likelihood of response to upfront pembrolizumab only. The strong prognostic value might be exploited to easily identify patients most likely to benefit from immunotherapy. A further study (FoRECATT-2) is ongoing to confirm results in a larger sample size and to investigate the effect of SAA on immune response in vitro assays.

To determine whether abagovomab induces protective immune responses in ovarian cancer patients in first clinical remission. The present analysis is a substudy of monoclonal antibody immunotherapy for malignancies of the ovary by subcutaneous abagovomab trial (NCT00418574). The study included 129 patients, 91 in the abagovomab arm and 38 in the placebo arm. Circulating CA125-specific cytotoxic T lymphocytes (CTL) were measured by a flow cytometry-based interferon-γ producing assay. Human antimouse antibody and anti-anti-idiotypic (Ab3) were assessed by ELISA. Patients were evaluated before starting the treatment and at different time points during induction and maintenance phases. A similar percentage of patients in both the placebo and abagovomab arms had CA125-specific CTL (26.3 and 31.8 %, respectively; p = 0.673 by Fisher’s exact test). Patients with CA125-specific CTL in both arms tended to have an increased relapse-free survival (RFS, log-rank test p = 0.095) compared to patients without. Patients (n = 27) in the abagovomab arm without CA125-specific CTL but that developed Ab3 above the cutoff (defined as median Ab3 level at week 22) had a prolonged RFS compared to patients (n = 24) that did not develop Ab3 above the cutoff (log-rank test p = 0.019). Abagovomab does not induce CA125-specific CTL. However, patients with CA125-specific CTL perform better than patients without, irrespective of abagovomab treatment. Abagovomab-induced Ab3 associate with prolonged RFS in patients without CA125-specific CTL. Further studies are needed to confirm these data and to assess the potential utility of these immunological findings as a tool for patient selection in clinical trial.

Interleukin-2 (IL-2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL-2 facilitates regulatory T (Treg) cell development. IL-21 is a type I cytokine acting as a potent T-cell co-mitogen but less efficient than IL-2 in sustaining T-cell proliferation. Using various in vitro models for T-cell receptor (TCR)-dependent human T-cell proliferation, we found that IL-21 synergized with IL-2 to make CD4(+) and CD8(+) T cells attain a level of expansion that was impossible to obtain with IL-2 alone. Synergy was mostly evident in naive CD4(+) cells. IL-2 and tumour-released transforming growth factor-β (TGF-β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin-21 hampered Treg cell expansion induced by IL-2/TGF-β combination in naive CD4(+) cells by facilitating non-Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL-21 did not modulate the conversion of naive activated CD4(+) cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down-modulation of IL-2/TGF-β-induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL-21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4(+) and CD8(+) T cells. Present data provide proof-of-concept for evaluating a combinatorial approach that would reduce the IL-2 needed to sustain T-cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T-cell response.

Introduction Despite encouraging phase I and II study results, vaccination of ovarian cancer patients with abagovomab – an anti-idiotypic mAb that mimics the ovarian cancer CA125 protein – failed to demonstrate efficacy in the phase III trial named MIMOSA (NCT00418574). We postulated that in this trial patients with a more robust immune system did respond to abagovomab but went undetected among a larger number of non-responders. We also postulated that assessment of the immune system status ahead of abagovomab administration might predict patients’ propensity to respond to abagovomab. Materials and methods The immune system status was assessed as percentage and absolute count of CD8+ T cells producing IFN-γ after stimulation with Staphylococcal Enterotoxin B (SEB) in 80 patients on abagovomab and 31 patients on placebo from the MIMOSA trial ahead of treatment. Optimal cutoffs of the two variables were calculated by the web application “Cutoff Finder” as the points with most significant (log-rank test) splits based on relapse-free survival (RFS). The Kaplan–Meier curves and log-rank test served to estimate and compare RFS in patients with percentage and absolute count of IFN-γ producing CD8+ T cells around the cutoffs. Results Patients on abagovomab with IFN-γ producing CD8+T cell percentage above the cutoff had a better RFS (p = 0.042) than those with IFN-γ producing CD8+T cell percentage below the cutoff. Patients on abagovomab with IFN-γ producing CD8+T cell absolute count above the cutoff had a better RFS (p = 0.019) than those with IFN-γ producing CD8+T cell absolute counts below the cutoff. Consistently, the RFS of patients on abagovomab with IFN-γ producing CD8+T cell percentage and absolute counts values below the respective cutoffs was identical to that of patients on placebo. Neither the percentage nor the absolute count of IFN-γ producing CD8+T cells correlated with RFS in patients on placebo. Conclusions A robust immune system is essential to obtain a clinical response in OC patients undergoing abagovomab immunotherapy whereas a robust immune system does not confer per se a survival advantage. Further work will clarify whether the results shown here apply only in the present setting or extend to other types of cancer and/or immunotherapeutic agents.

Purpose To assess how neoadjuvant chemoradiation regimens modulate the immune system state in tumor-draining lymph nodes (TDLN), in the setting of advanced cervical cancer. Methods and Materials Tumor-draining lymph nodes of patients undergoing chemotherapy only (nonirradiated, NI-TDLN) and chemoradiation with lower-dose (39.6 Gy, LD-TDLN) and higher-dose radiation (50 Gy, HD-TDLN) were analyzed by multicolor flow cytometry. Results Enlarging our previous data, LD-TDLN showed features overall indicative of an enhanced antitumor response as compared with NI-TDLN, namely a significant Th1 and Tc1 polarization and a lower amount of the potent CD4 + Foxp3 + CD25 high regulatory T cell (Treg) subset identified by neuropilin-1 expression. Conversely, compared with NI-TDLN, HD-TDLN showed features overall indicative of an impaired antitumor response, namely a significantly inverted CD4/CD8 cell ratio, a higher Nrp1 + Treg frequency, and a higher frequency of CCR4 + Treg, a Treg subset facilitated in migrating out from TDLN to suppress the immune response against distant cancer cells. Moreover, the Th1 and Tc1 polarization induced by LD radiation was lost, and there was an unfavorable tolerogenic/immunogenic dendritic cell ratio compared with LD-TDLN. Conclusions Even minor differences in radiation dose in neoadjuvant regimens for locally advanced cervical cancer are crucial for determining the balance between a tolerogenic and an efficacious antitumor immune response in TDLN. Because most of the anticancer immune response takes place in TDLN, the present findings also emphasize the importance of chemoradiation protocols in the context of immunotherapeutic trials.

We previously showed that platelet reactivity at rest is increased in patients with cardiac syndrome X (CSX), but that exercise reduces platelet reactivity in these patients. Adenosine was suggested to be involved in this phenomenon. In this study we investigated the effect of adenosine on adenosine diphosphate (ADP) and thrombin-induced platelet reactivity in CSX patients.We studied 15 CSX patients and a control group of 15 healthy subjects. Formation of monocyte-platelet (MONO-PLT) aggregates in vitro was assessed by flow cytometry: 1) at baseline; 2) after ADP (10(-7) M) stimulation alone; 3) after ADP stimulation in presence of adenosine (10(-5) M); 4) after thrombin (10(-11) M) stimulation alone; 5) after thrombin stimulation in presence of adenosine.In non stimulated samples there were no relevant differences between the two groups in cytometry variables. Compared to controls, ADP induced a higher increase in MONO-PLT aggregates in CSX patients (P0.01), which was significantly inhibited by adenosine (P0.01). Thrombin also induced a greater increase in MONO-PLT aggregates in CSX patients (P0.001), which was also significantly blunted by adenosine. Similar trends were observed for platelet CD41 (glycoprotein IIb-IIIa) receptor and for monocyte receptors CD142 ad CD162 in MONO-PLT aggregates.In CSX patients platelet reactivity is increased at rest, compared to healthy controls. Pre-incubation with adenosine reduces the agonist-induced platelet hyper-reactivity in these patients, suggesting that adenosine may be involved in the reduction of platelet reactivity observed in CSX patients after exercise in our previous study.

We compared the immune system state in metastatic tumour draining lymph nodes (mTDLN) and metastasis free TDLN (mfTDLN) in 53 early stage cervical cancer patients to assess whether the presence of metastatic tumour cells worsen the balance between an efficacious anti-tumour and a tolerogenic microenvironment.The immune system state was measured by immunophenotypic and functional assessment of suppressor and effector immune cell subsets.Compared to mfTDLN, mTDLN were significantly enriched in CD4(+)Foxp3(+) regulatory T cells (Treg), which, in addition, exhibited an activated phenotype (HLA-DR(+) and CD69(+)). Treg in mTDLN were also significantly enriched in neuropilin-1 (Nrp1) expressing cells, a subset particularly potent in dampening T cell responses. mTDLN tended to be enriched in a population of CD8(+)Foxp3(+)T cells (operationally defined as CD8(+)Treg) that showed a suppressor potency similar to Treg under the same experimental conditions. Plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) generally show distinct roles in inducing T cell tolerance and activation, respectively. In line with the excess of suppressor T cells, the ratio pDC to mDC was significantly increased in mTDLN. Immunohistochemical testing showed that metastatic tumour cells produced the vascular endothelial growth factor, a natural ligand for Nrp1 expressed on the cell surface of Nrp1(+)Treg and pDC, and therefore a potential mediator by which tumour cells foster immune privilege in mTDLN. Consistent with the overall tolerogenic profile, mTDLN showed a significant Tc2 polarisation and tended to contain lower numbers of CD45RA(+)CD27(-) effector memory CD8(+)T cells.The increased recruitment of suppressor type cells concomitant with the scarcity of cytotoxic type cells suggests that in mTDLN the presence of tumour cells could tip the balance against anti-tumour immune response facilitating the survival of metastatic tumour cells and possibly contributing to systemic tolerance.

We examined the phenotype and function of CD4+ T cells expressing the semaphorin III receptor neuropilin-1 (Nrp1) in human lymph nodes and peripheral blood. In lymph nodes, Nrp1 identified a small regulatory CD4+ CD25(high) T-cell subpopulation (Nrp1+ Treg) that expressed higher levels of Forkhead box P3 (Foxp3) message and protein than Nrp1- Treg, and various molecular markers of activated Treg, i.e. CD45RO, human leucocyte antigen (HLA)-DR and glucocorticoid-induced tumour necrosis factor receptor (GITR). Similarly to conventional Treg, Nrp1+ Treg proliferated poorly in vitro, and exerted contact-dependent in vitro suppression of T-cell proliferation and cytokine secretion. However, Nrp1+ Treg were more efficient than Nrp1- Treg at inducing suppression. Nrp1 was also expressed on a small subpopulation of CD25(int) and CD25- CD4+ T cells that expressed more Foxp3, CD45RO, HLA-DR and GITR than their Nrp1- counterparts. In contrast, in peripheral blood Nrp1 identified a minor CD4+ T-cell subset that did not display the phenotypic features of Treg lacking Foxp3 expression and marginally expressing CD25. Hence, the function of Nrp1+ CD4+ T cells seemingly depends on their anatomical location. In a previous report, we proposed that Treg may curb the anti-tumour T-cell response in cervical cancer. We show here that Treg and Nrp1+ Treg levels dropped in the tumour-draining lymph nodes of patients with cervical cancer following preoperative chemoradiotherapy in a direct relationship with the reduction of tumour mass, suggesting that suppressor cell elimination facilitated the generation of T cells mediating the destruction of the neoplastic cells left behind after cytotoxic therapy.

We analyzed thymocyte and thymic regulatory T cell (CD4SPCD25+Foxp3+cells, Treg) development in thymoma with and without myasthenia gravis (MG, MG-thymoma, non-MG-thymoma) and in MG-associated non-neoplastic thymus (MG-NNT). An increased number of immature CD4+CD8(-)CD3(-) thymocytes through the CD4+CD8+ to CD4+CD8(-) transition and an abnormal T cell receptor Vbeta (TCRVbeta) development through the CD4+CD8+ to CD4(-)CD8+ transition were seen both in MG-and non-MG-thymomas. Terminal thymopoiesis, i.e., CD45RA+ cells within the CD4+CD8(-)CD3+ and CD8+CD4(-)CD3+ subsets, was skewed towards the CD4+ compartment in MG-thymoma and CD8+ compartment in non-MG-thymoma, but thymic export was increased only in the latter in keeping with the hypothesis that CD8+ lymphocytes may play a role in the initial stages of autosensitization and in disagreement with the relevance of an increased output of CD4+ T lymphocytes in paraneoplastic MG. Treg level in normal thymus and MG-NNT and both MG- and non-MG-thymoma was similar, and TCRVbeta development in Treg cells was slightly altered in thymoma but irrespective of MG presence. Thus, the relevance of a defective Treg development in MG context remains to be established. Most alterations in thymopoiesis were corrected by therapeutic corticosteroid administration, and the effects of steroid administration may be mediated by thymic microenvironment.

Here we provide a description of lymphocyte populations in human lymph nodes (LN) with a special emphasis on the CD4+ lymphocyte population constitutively expressing CD25 at a high level and endowed with immunoregulatory properties [T regulatory (Treg) cells]. Lymph nodes were analysed by multicolour flow cytometry in parallel with correspondent peripheral blood (PB). Immunomagnetically purified Treg cells were tested for anergy and suppressive activity in a CD3/T-cell receptor (TCR)-driven proliferation assay. Compared to PB, there was a reduced T/B lymphocyte ratio in LN. Both LN and PB contained a similar proportion of CD4+ lymphocytes but, conversely, CD8+ lymphocytes were less represented in PB, with a consequent increase in the ratio of CD4+/CD8+ natural killer cells were2% (PB range 6-22%). No significant differences existed in the frequency of the other lymphocyte subpopulations examined (naïve-type CD4+ and CD8+ lymphocytes, activated B and CD4+ lymphocytes, and effector-type CD8+ lymphocytes). LN and PB contained similar percentages of CD4+ lymphocytes constitutively expressing intermediate or high levels of CD25. CD4+ CD25++ cells constitutively coexpressed high levels of CD152 and were therefore identified as Treg cells. Treg cells in LN and PB differed in terms of CD45RB, HLA-DR, CD45RO, and CD62L expression. Also the TCRVbeta repertoire diverged between Treg cells from LN and PB. Similar to Treg cells from PB, Treg cells from LN were anergic and efficiently inhibited other CD4+ and CD8+ lymphocyte proliferation. This study extends the information on the diversities in lymphocyte composition between human LN and PB, and reports for the first time a description of the phenotypic and functional characteristics of Treg cells in human LN, highlighting the importance of the LN microenvironment in shaping the surface phenotype of Treg cells.

Objective To explore the effects of intraperitoneal (i.p.) infusion of catumaxomab, a bispecific monoclonal antibody (anti-EpCAM×anti-CD3), on T cells, NK cells and macrophages in ascites of cancer patients and to understand how ascitic immune cells can be activated despite the pervasive immunosuppressive ability of ascites microenvironment. Methods Six patients with malignant ascites received i.p. catumaxomab infusion. Ascitic immune cells were profiled by flow cytometry and gene expression at baseline and after i.p. catumaxomab infusion. In vitro experiments enabled investigations on the adverse effect of ascites microenvironment on catumaxomab-stimulated immune cells. Results I.p. catumaxomab infusion enhanced the expression of the CD69 and CD38 activation molecules in CD4 + and CD8 + T cells, NK cells and macrophages, and favoured CD8 + T cell accumulation into the peritoneal cavity. An analogous immune cell activation as well as IFN-γ and IL-2 production were induced by catumaxomab in vitro. In vitro experiments showed that the immunosuppressive milieu of ascites abrogated all the immunostimulatory activities of catumaxomab. Adding EpCAM + tumour cells to the culture permitted both catumaxomab Fab regions to engage cognate antigens and restored immunostimulatory catumaxomab activity. Conclusions This is the first demonstration in a clinical setting that i.p. catumaxomab infusion activates NK cells and macrophages in addition to T cells in ascites and favours CD8 + T cell accumulation into the peritoneal cavity. Moreover, our findings indicate that the concomitant binding of both catumaxomab Fab regions delivers an activation signal that is strong enough to activate immune cells despite the prevailing immunosuppressive environment of malignant ascites.

BACKGROUND: The peripheral blood progenitor cell (PBPC) mobilization capacity of EPO in association with either G–CSF or sequential GM–CSF/G–CSF was compared in a randomized fashion after epirubicin, paclitaxel, and cisplatin (ETP) chemotherapy. STUDY DESIGN AND METHODS: Forty patients with stage IIIB, IIIC, or IV ovarian carcinoma were enrolled in this randomized comparison of mobilizing capacity and myelopoietic effects of G–CSF + EPO and GM–/G–CSF + EPO following the first ETP chemotherapy treatment. After ETP chemotherapy (Day 1), 20 patients received G–CSF 5 μg per kg per day from Day 2 to Day 13 and 20 patients received GM–CSF 5 μg per kg per day from Day 2 to Day 6 followed by G–CSF 5 μg per kg per day from Day 7 to Day 13. EPO (150 IU per kg) was given every other day from Day 2 to Day 13 to all patients in both arms of the study. Apheresis (two blood volumes) was performed during hematologic recovery. RESULTS: The magnitude of CD34+ cell mobilization and the abrogation of patients' myelosuppression were comparable in both study arms; however, GM–/G–CSF + EPO patients had significantly higher CD34+ yields because of a higher CD34+ cell collection efficiency (57.5% for GM–/G–CSF + EPO and 46.3% for G–CSF + EPO patients; p = 0.0009). Identical doses of PBPCs mobilized by GM–/G–CSF + EPO and G–CSF + EPO drove comparable hematopoietic recovery after reinfusion in patients treated with identical high-dose chemotherapy. CONCLUSION: The sequential administration of GM–CSF and G–CSF in combination with EPO is feasible and improves the PBPC collection efficiency after platinum-based intensive polichemotherapy, associating high PBPC mobilization to high collection efficiency during apheresis.

Apoptosis has been indicated as a mechanism of T cell depletion in HIV-infected subjects and useful in monitoring disease progression. We investigated for the presence of apoptotic T lymphocytes in 130 HIV subjects in various stages of disease by the newly developed cell permeant DNA dye Apostain. Blood was collected in EDTA, lysed in buffered ammonium chloride, fixed in freshly prepared 1% paraformaldehyde and stored in aliquots at −80°C. Samples were thawed and double stained with FITC conjugated-CD3 monoclonal antibody and Apostain. Flow cytometry was then performed and T cells gated on a CD3 versus side scatter dot plot. Normal samples treated in the same manner served to establish the boundary separating non-apoptotic from apoptotic cells. There was no statistically significant association between the proportion of subjects with detectable apoptotic cells and CDC clinical categories A, B and C at the time of admission to the study, although a trend toward a lower apoptotic rate in category A (A= 29%, B=40% and C=41%) was noticed. Conversely, CDC T cell categories 2 and 3 contained significantly higher proportions of Apostain positive patients (1=6%, 2=32% and 3=49%, P=0.072, by χ2 test). Most importantly, Apostain test identified subjects at risk of disease progression during a 3.5–7 months follow-up in CDC category B and 2 (P=0.008 and P=0.0003, by Fisher's exact test, respectively). A similar, albeit not statistically significant trend was observed also in the other categories. Not requiring extensive manipulation of fresh samples nor cumbersome culture techniques, Apostain test appears suitable for identifying HIV subjects at higher risk of disease progression in clinical settings.Cytometry (Comm. Clin. Cytometry) 42:67–73, 2000 © 2000 Wiley-Liss, Inc.

Objective: We performed a side-by-side comparison between three stain-then-lyse commercially available methods (Ortho-mune Lysing solution, FACS lysing solution and ImmunoPrep reagent system) and a lyse-then-stain method using hypotonic NH4Cl. The major difference between these methods is that only in the latter the aliquots of sample to be distributed into diverse tubes for the various antibody combinations were obtained from a lysis step performed in the same tube. Design and Methods: Lymphocytes from 20 healthy and 20 HIV+ subjects were phenotyped by dual color flow cytometry using a standard procedure that included the establishing of a lymphocyte gate on light scatter bit map and the use of the minimal acceptable antibody combinations, i.e., CD45/CD14, CD3/CD4 and CD3/CD8, according to CDC recommendations. All samples were processed in triplicate to assess tube-to-tube variability. Results: In healthy subjects, erythrocytes pre-lysing provided the highest purity and recovery in the lymphocyte gate, and allowed the best identification of CD4+ lymphocytes. Most remarkably, erythrocytes pre-lysing significantly outdid all other methods in reducing tube-to-tube variability. This allowed the attainment of highest correlation between CD3+ cells identified by CD3/CD4 and CD3/CD8 antibody combinations and the minimum variability between the sum of the %CD3+CD4+ and %CD3+CD8+, and the total %CD3+. This higher reliability of the pre-lysis method was particularly evident with HIV+ patients in which the lymphocyte gate was often and unpredictably contaminated by debris and other cell types. Conclusions: The present study demonstrates that lysing erythrocytes in a single tube and distributing aliquots of lysed blood into different tubes for the various antibody combinations provides superior results for routine immunophenotyping.

Paclitaxel, docetaxel and a series of new analogs synthesized from 14beta-hydroxy-10-deacetylbaccatin III (14-OH-DAB), a natural diterpene closely related to the core synthon of the 2 above prototypes, were tested in vitro for their growth-inhibitory activity on different human cancer cell lines, including some expressing the classic multidrug-resistant (MDR) phenotype (MCF-7 ADRr and CEM VBLr). The 14-OH-DAB derivatives showed enhanced anti-proliferative activity as compared to the parent compounds on the MDR-positive cancer cell lines. Particularly, IDN 5109 showed a 25- to 30-fold higher activity than paclitaxel. The fold change in activity between paclitaxel and analogs (IC50 paclitaxel/IC50 analogs) on the MDR-positive cell lines was calculated and a significant correlation observed. As far as the MDR-negative MDA-MB 231 cells are concerned, docetaxel and IDN 5109 exhibited a more potent activity than paclitaxel. On the basis of the data obtained on cell growth inhibition, we selected the most active compounds to study their effect on the cell cycle. Cell cycle analysis showed that all of the compounds tested were able to induce cell cycle block at G2/M in a concentration-dependent manner. The amount of cell block, measured as a G1/G2 ratio, was correlated significantly (p < 0.001) with apoptosis, as evaluated in the sub-G1 region (% of DNA fragmentation), thereby suggesting that the G2/M-blocked cells underwent apoptosis. To confirm the occurrence of apoptosis in this system, DNA gel agarose electrophoresis was performed and showed the typical ladder pattern.

Irradiated human peripheral blood lymphoid cells undergo apoptosis and progressively exhibit typical changes in light scatter and plasma membrane integrity that can be easily tracked by flow cytometry. Using this model, we assessed the capacity of a newly developed fluorochrome, Apostain, in identifying early apoptosis in unfixed samples. This probe is a plasma membrane permeant DNA dye that can be conveniently excited at 488 nm and has an emission wavelength >650 nm. To identify apoptotic cells, Apostain relies on the transient changes of chromatin texture that allow to accommodate more of a DNA dye occurring in early apoptosis. As early as 4 h after irradiation, a proportion of cells showed an enhanced Apostain uptake. Consistent with their initial apoptotic nature, these cells had a still integer plasma membrane, as assessed by ethidium bromide, and unaltered light scatter. With time, cells showing the enhanced Apostain uptake started to bind dimly Annexin-V and, later, reduced their forward scatter. After 18 h from irradiation, cells exhibiting a reduced forward scatter exhibited a bright staining with Annexin-V with a concomitant reduction in Apostain uptake, reflecting the gross chromatin disruption characterising the endpoint of apoptosis.

Human T cell clones (TCC) are antigen-presenting cells (APC) able to present peptides and superantigens (SAg) and to process and present intact proteins. TCC express major histocompatibility complex (MHC) class II antigens and molecules involved in the accessory signal delivery, such as B7.1 and B7.2/B70. Notwithstanding these observations, the role of professional APC has been often denied to T cells because anergy of responder T cells rather than proliferation has been observed following the TCC presentation in the absence of added professional APC. Here, we show that upon stimulation with free SAg, TCC undergo proliferative responses followed, after a 1-week culture, by an SAg-dependent unresponsiveness to T cell receptor (TCR)-mediated stimuli, but not to interleukin-2. The anergy induced by the SAg can not be prevented by the addition of autologous Epstein-Barr virus (EBV)-transformed B cells, indicating that the induction of anergy occurs also in the presence of conventional APC. Conversely, if the TCC are stimulated by SAg-prepulsed irradiated APC, either EBV and TCC, the induction of anergy is not observed. After a 1-week culture, in fact, TCC stimulated with APC-bound SAg responded to TCR-mediated stimuli, irrespective of the APC (EBV or TCC) used for the SAg presentation. Stimulation of TCC with free SAg in a semisolid medium that prevents T-T cell contacts resulted in an activation followed by a state of anergy, suggesting that anergy is the consequence of SAg recognition at the single T cell level. These data indicate that the anergy observed in TCC upon a 1-week culture in the presence of soluble SAg is not the result of an inherent inability of TCC to act as professional APC. Rather the phenomenon depends on the presence of soluble SAg, leading to T cell autostimulation.

Polymorphonuclear leukocytes (PMN), atrial natriuretic peptide (ANP) and leukotriene B4 (LTB4) reportedly play a major role in ischemia/reperfusion states of coronary artery disease. We sought to determine whether ANP and LTB4 cooperate in inducing PMN activation with consequent modulation of membrane molecules required for adherence to endothelium and myocardial cells, namely CD11b and L-selectin and the release of toxic oxygen radicals. ANP (from 10(-16) to 10(-8) M), LTB4 (from 10(-10) to 10(-6) M) and combinations of the two were incubated with normal PMN at 37 degrees C for 15 minutes. Membrane molecules modulation was measured by flow cytometry using specific monoclonal antibodies. Hydrogen peroxide production, an indicator of the capacity of PMN to release toxic oxygen species was quantified by flow cytometry using the peroxide-sensitive fluorescent probe dichlorofluorescein diacetate. ANP, uneffective when used alone, dose-dependently potentiated the PMN response to LTB4 (10(-9) M) as evidenced by an up-regulation of CD11b expression and peroxide production, and a down-regulation of L-selectin expression. These effects were prevented dose-dependently by the protein kinase C (PKC) inhibitor staurosporine (from 10 to 160 microM). Two carnitine congeners, palmytoylcarnitine (tested from 125 pg to 2 micrograms/ml) that also possesses an established ability to antagonise PKC and L-carnitine (tested from 12 to 200 ng/ml) were also effective. These data indicate that ANP potentiates LTB4 in inducing PMN mobilization and activation with a possible consequent detrimental effect on cardiac tissue and evisages the usefulness of PMN metabolism modulators.

In gingival pockets of patients with periodontal disease, polymorphonuclear leukocytes (PMN) are in contact with a peculiar exudate, the gingival crevicular fluid (GCF). Because of the pivotal role played by PMN in periodontal disease, we evaluated the ability of GCF in modulating normal human PMN. GCF was obtained from two gingival sites with severe periodontitis (SP) and two gingival sites with only mild periodontitis (MP) in 12 patients. Purified PMN were exposed to GCF from SP and MP sites and, as a control, to sterile culture medium. GCF activity was evaluated by monitoring the modulation of membrane molecules relevant to cell function. Compared to control medium, GCF from SP and MP sites was able to induce an activation status in PMN evidenced by an increased CD11b (62 +/- 9% and 28 +/- 7%, respectively) and f-Met-Leu-Phe (56 +/- 5% and 31 +/- 7%, respectively) receptor expression, with a concomitant reduction of CD62L expression (56 +/- 8% and 23 +/- 7%, respectively). Thus, reflecting the clinical status, GCF from SP sites was significantly more efficient in affecting PMN than GCF from MP sites. Cell size modifications, evaluated as an additional indicator of PMN activation, were consistent with membrane molecule modulation. The difference in PMN-activating capacity between SP and MP was abrogated by the successful completion of an appropriate periodontal therapy that dramatically improved clinical status. This is the first direct demonstration that GCF from periodontitis has the capacity to activate normal resting PMN and that this capacity reflects the magnitude of the inflammatory process that takes place in the gingiva.

Meningococcal meningitis is still a serious infectious disease with a mortality rate that can be as high as 10% even in developed countries. Military recruits are generally a high-risk group for meningococcal disease, with a reported incidence of four to ten times greater than that of the general population. In Italy the results of the National Meningitis Surveillance Programme showed a high attack rate of the disease among recruits in 1985 as well as in 1986, with 92 and 95% of the cases, respectively, caused by serogroup C and thus preventable. These findings led to the authorities' decision to make vaccination against meningococcal disease mandatory for recruits starting from January 1987. After almost 5 years from the introduction of meningococcal vaccination, we here sum up the epidemiological and immunological effects of the vaccination. From the epidemiological point of view we have observed a dramatic reduction of the prevalence of the disease. In 1987, the year in which we had 150,000 unvaccinated and 150,000 vaccinated recruits, the protective efficacy was 91.2%. From the immunological point of view, vaccination is highly effective, as seroconversion against polysaccharide (PS) A and C is 84 and 91%, respectively. The spectrotypic analysis of the sera before and after vaccination shows that the type of response is mainly oligoclonal, like the majority of the responses to PSs, and the antibodies induced by a sole PS are not qualitatively different from the antibodies induced by natural immunization. In addition, the efficacy is not modified by environmental factors like hypoxia, as demonstrated during permanence at 16,174 feet for 20 days.(ABSTRACT TRUNCATED AT 250 WORDS)

The low-affinity Fc receptor for IgE, CD23/FceRII, has been expressed in T cell lines and pathologic T cells, but its presence on normal human T cells is still debated. We studied the expression of CD

The effect of carnitine, a drug that plays an essential role in mitochondria metabolism, on some of the most important human polymorphonuclear leucocytes (PMN) activation steps including modulation of adhesion molecule density, reactive oxygen species production, and tumour necrosis factor-α (TNFα) production was investigated. The capability of carnitine in protecting PMN from deter ioration on storage was also studied. Data shows that carnitine exerts considerable effects on all PMN functions investigated. Although the ultimate effect was often donor dependent, TNFα production was exceptional in that carnitine was able to consistently reduce TNFα production in Staphylococcus aureus stimulated PMN in a clear dose-dependent fashion. It is concluded that carnitine may represent a useful active agent in situations characterized by PMN mobilization/activation.

Resistance of tumor cells to the antigrowth activity of several cytotoxic compounds has been associated with the expression of the so-called multidrug resistance protein or P-glycoprotein. This article addresses the question whether the expression of such protein could also affect the sensitivity of HIV to AZT. Our data indicate that this possibility does exist. In fact, multidrug-resistant CEM VBL100 cells, which express high levels of P-glycoprotein, are less sensitive to both the antiproliferative activity and the antiviral action of AZT. Additionally, our data suggest that this phenomenon is specifically mediated by P-glycoprotein since trifluoroperazine, which is known to circumvent multidrug resistance due to the action on P-glycoprotein, increases the intracellular accumulation of AZT and affects the sensitivity of HIV to AZT. Although the biological and clinical significance of these observations has still to be established, this study suggests that cellular factors, other than virus itself, should be taken into account to address the phenomenon of drug resistance of HIV.

Superantigens (SAg) interact with T lymphocytes bearing particular V beta sequences as part of their T cell receptor (TcR). The interaction, however, requires the presence of major histocompatibility complex (MHC) class II molecules on antigen-presenting cell (APC). In peculiar circumstances, MHC class II+ T cell clones (TCC) have been shown to present peptides and selected antigens interacting with antigen-specific TCC in the absence of APC. In this report we studied the capacity of SAg to mediate a T-T cell interaction, investigating the TCC ability to present a panel of staphylococcal enteroxins (SE) independently of the presence of added APC. Upon exposure to a broad range of SE concentrations, MHC class II+ TCC showed an intense proliferative response even in the absence of professional APC. Diverse SE optimally stimulated responder TCC at different concentrations. The proliferation was inhibited by anti-DR monoclonal antibodies, both in the presence and in the absence of APC. The SE activation of TCC in the absence of APC induced the same series of phenotypic variations as that observed following the TCC stimulation with APC. Irradiated TCC efficiently presented membrane-bound SE to responder TCC as well as professional APC. These results show that a single cell of a given clone effectively presents the SE to other cells of the same clone, and provide evidence that SAg can efficiently mediate T-T cell interaction. In addition, the possibility also exists that one cell of the clone can actually undergo an auto-stimulation via SAg-mediated interactions between its own TcR and MHC class II molecule. It has recently been suggested that the V beta-selective depletion of T cells observed in acquired immunodeficiency syndrome (AIDS) patients might be a consequence of the interaction between a human immunodeficiency virus (HIV)-encoded SAg and T cells expressing a SAg complementary V beta. We suggest that the hypothesized HIV-encoded SAg might mediate T-T cell interactions that could play a relevant role in the V beta-selective depletion of T lymphocytes observed in HIV-infected patients.

Accumulating evidence indicates an immunosuppressive role of the thymus-derived CD4+ T-cell population constitutively expressing high level of CD25, T regulatory (Treg) cells, in autoimmune diseases. Here we show that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age-matched healthy subjects, whereas it is normal or elevated in patients on immunosuppressive therapy (prednisone frequently associated with azathioprine). Therapeutic thymectomy (Tx) for either the thymoma or non-neoplastic thymic alterations that are often associated with myasthenia gravis provided unique material for studying intrathymic Treg cells and correlating them with their peripheral counterparts. We observed that Tx prevents the increase of Treg cells in the circulation that follows immunosuppressive therapy (particularly evident if the thymus is not neoplastic), indicating that the thymus contributes to Treg-cell normalization. However, thymic Treg cells are not modulated by immunosuppressive therapy and even in thymectomized patients Treg-cell numbers in the blood eventually recover. The present findings suggest that a deficiency in Treg cells favours the development of myasthenia gravis and that their normalization is an important clinical benefit of immunosuppressive therapy. Treg normalization appears to be largely thymus independent and possibly reflects the reported capacity of corticosteroids to promote Treg-cell development.

BACKGROUND: Peripheral blood progenitor cell (PBPC) transplantation (PBPCT) combined with post-PBPCT administration of myelopoietic growth factors is a valid therapeutic intervention to rapidly restore hematopoiesis after the delivery of intensive, myeloablative cancer chemotherapy. On the other hand, the best growth factor regimen to potentiate PBPC-mediated immunohematopoietic recovery has yet to be determined. STUDY DESIGN AND METHODS: In a randomized evaluation, the effects produced by post-PBPCT G–CSF and GM–CSF on myeloid/lymphoid recovery and transplant outcome in women with chemosensitive cancer were compared. Thirty-seven ovarian cancer patients and 34 breast cancer patients ranging in age from 24 to 60 years were treated with carboplatin, etoposide, and melphalan (CEM) high-dose chemotherapy and then randomly assigned to receive G–CSF (5 μg/kg subcutaneously) or GM–CSF (5 μg/kg subcutaneously) until Day 13 after PBPCT. Patients were compared in regard to hematopoietic recovery, posttransplant clinical management, and immune recovery. Finally, clinical outcome was estimated as time to progression and overall survival. RESULTS: Hematopoietic recovery and posttransplant clinical management were comparable in both the G–CSF and GM–CSF series. Conversely, significantly higher T-cell counts were observed in G–CSF-treated patients during the early and late posttransplant follow-up. Patients who received G–CSF showed a significantly longer median time to progression. A parallel analysis revealed that patients in whom a higher CD3+ count was recovered had a significantly longer overall survival and time to progression. CONCLUSION: The enhancement of post-PBPCT T-cell recovery observed in G–CSF-treated patients encourages the use of G–CSF to ameliorate immune recovery, which seems to play a role in post-PBPCT control of disease in cancer patients. GM–CSF might be administered to prolong immunosuppression after autologous PBPCT for autoimmune diseases or allogeneic PBPCT.

Publisher Summary The fate of a single hemopoietic progenitor (HP) is determined by both an intrinsic hemopoietic potential typical of each HP and the activity of various cytokines that regulate HP survival, proliferation, and differentiation. Maturation of HP into functional hemopoietic cells in vitro is monitored with success at the population level by the use of flow cytometry. This approach exploits the availability of monoclonal antibodies (MAb) to specific surface markers defining the maturative stage and the specific lineage a given precursor is committed to and the capability of certain fluorescent probes to track cell division history. In these systems, cells are loaded with a probe that equally redistributes into daughter cells at each round of cell division, generating progenies with progressively halved fluorescence signal. This chapter focuses on the problems the flow cytometrist faces when attempting to analyze variations in human HP phenotype and correlates them with carboxyfluorescein diacetate–succinimidyl ester (CFDA–SE) halving during in vitro cytokine-driven differentiation toward the major hemopoietic lineages, namely, granulocytic/monocytic, erythroid, and megakaryocytic.

Publisher Summary Lymphocyte activation and proliferation represent an essential step in the immune response. Lymphocytes are activated and then proliferate, expanding specific clones of cells that are reactive against foreign antigens. A disregulation of the response, either diminished or enhanced, is responsible for a variety of pathological states. Lack of response leads to immunodeficency, and the series of severe clinical symptoms following human immunodeficiency virus (HIV) infection is a typical, although not exclusive, example of the consequences of an impaired immune response. The measurement of [3H]thymidine incorporation into DNA performed with bulk lymphocyte cultures has been used for decades, and it probably remains the most commonly used method to quantify lymphocyte proliferation, although some doubts about its validity have been raised in some circumstances.

Dendritic cells are professional antigen-presenting cells able to prime naive T lymphocytes and regulate steadily the delicate balance between tolerance and activation during the immune response. In past years several reports have shown that genetically engineered dendritic cells (DCs) can be a powerful tool for inducing an antigen-specific immune response. The use of such modified antigen-presenting cells is a real working hypothesis in preclinical studies and in clinical vaccination approaches for cancer treatment. The definition of optimal transfection conditions for preserving DC survival and functionality is necessary to design a correct immunotherapeutic protocol. Different lipid-based transfection compounds were studied for their effects on DC survival, phenotype and functional properties. All the transfection procedures were able to select DCs with a higher expression of activation and costimulatory molecules (ie MHCII-DR, CD83, CD86, CD25) than the untreated DCs. However, only two compounds (LipofectAMINE PLUS and FuGENE 6), preserved or even increased the immunopotency of DCs as antigen-presenting cells. These protocols were applied to modify DCs in order to express an epithelial tumor-associated antigen, MUC1, and such cells were able to induce in vitro a specific immune response in healthy donors.

The in vitro effect of single or combined doses of zidovudine (AZT) and dideoxycytidine (ddC) on PHA-activated human peripheral blood mononuclear cells (PBMC) proliferative response and lymphoblastoid T cell line CEM cell growth was evaluated. Clinically relevant amounts (0.1, 1 and 10 μM) of AZT, ddC and AZT/ddC combination (10 + 10 μ M) inhibited 3 H TdR uptake in both cell models in a dose-dependent manner. The inhibitory effect on cell growth was confirmed by counting the amount of viable CEM cells recovered after 24, 48 and 72 h exposure to the drugs. On equimolar basis, ddC was considerably more efficient than AZT although the latter potentiates the activity of the former. Flow cytometric analysis of PBMC and CEM cells exposed to the dideoxynucleosides revealed a decrease in the rate of DNA synthesis (rate of passage through the S phase of the cell cycle) and a reduced number of cell generations, the latter assessed by measuring the halving of the fluorescent probe 5–6 carboxyfluorescein diacetate succinimidyl ester by flow cytometry. The analysis of CEM cells recovered after exposure to ddC or AZT/ddC combination (10 + 10 μ M), showed that in addition to perturbing cell cycle progression, ddC, and most efficiently the AZT/ddC combination, induced cell death by apoptosis. The latter was manifested as enhanced side scatter and decreased, sub-G 1 , DNA content by flow cytometry, and as DNA breakdown in nucleosomal fragments by gel electrophoresis. Present findings indicate that clinically relevant concentrations of dideoxynucleosides reduce cell growth by hampering DNA replication and inducing apoptosis.

Oxidized low density lipoproteins (ox-LDL) are known to behave as physiological pro-oxidants leading to the formation of intracellular reactive oxygen species. The presence of these altered lipoproteins in the human plasma has been associated with a number of morbid states, including atherosclerosis and immuno-deficiency. Common features of such pathological conditions seem to be represented by several alterations occurring in the immune system. In this work we analyze the in vitro effects of ox-LDL on both proliferative response and cytokine production of normal human peripheral blood mononuclear cells (PBMC). Our results indicate that ox-LDL significantly inhibit proliferative response and modulate cytokine network interfering both at protein secretion and mRNA synthesis level.

Extensive data demonstrate that polymorphonuclear leukocytes (PMN) are the predominant cell type involved in periodontal disease and that gingival crevicular fluid constituents are influenced by the inflamed gingiva. The aim of the present study was to evaluate the ability of gingival crevicular washing (GCW) (a dilution of gingival crevicular fluid) from periodontal sites in different clinical conditions of modulating the PMN membrane receptors involved in motility, adhesion and phagocytosis before and after periodontal treatment. 10 patients affected by adult periodontitis (AP) were selected. From each patient, 2 test sites (TS) were chosen on the basis of a probing depth > 5 mm and attachment loss, and 2 control sites (CS) with probing depth < 3 mm without. Modifications of membrane receptor density of PMN from healthy donors incubated with GCW harvested from TS and CS was evaluated using fluorescent probes and flow cytometry. Compared to CS-GCW, TS-GCW before therapy increased the expression of the beta 2 integrin CD11b and the chemotactic receptor for the oligopeptide N-formyl methionyl leucyl phenylalanine (FMLP-R) while it reduced the expression of L-selectin. GCW collected from the same TS after the successful completion of periodontal treatment did not influence PMN receptors, indicating that the clinical improvement paralleled the disappearance of the PMN modulating capability contained in TS-GCW before therapy. In conclusion, the present data illustrate the relevant modifications occurring at PMN membrane in chronic adult periodontitis exerted by GCW obtained by a simple fluid collection technique. Thus, monitoring gingival crevicular fluid PMN activating capability may help disclose the presence of chronic periodontitis and may be useful in assessing successful treatment.

Transgenic mice were produced in which human HLA-Cw4 is stably integrated, behaves as a single Mendelian trait, and, being under the transcriptional control of human CD2, is selectively and efficiently expressed in T lymphocytes. These mice were used as a model system to determine whether HLA-type C molecules can be exposed on the surface of activated lymphocytes as free heavy chains, non-associated with beta2-microglobulin (beta2m). In our transgenic mice we could identify HLA-Cw4 molecules either as free chains or as beta2m-associated molecules by the use of monoclonal antibodies specific for either conformation of HLA class I and nonreactive to mouse H2 molecules. Resting mouse lymphocytes were shown by western transfer analysis to contain sizeable amounts of HLA-Cw4 free chains, but they exposed on their surface HLA-Cw4 only in association with beta2m, as indicated by flow cytometric measurements. Conversely, where the content of total HLA-Cw4 was increased, lectin-activated mouse lymphocytes exposed on their outer cell membrane HLA-Cw4 molecules in both conformations, namely, also as free heavy chains. Isoelectrofocusing analysis confirmed the presence of both HLA-Cw4 molecular conformations in activated T cells and indicated that HLA-Cw4 heavy chains can bind to mouse beta2m with the same low affinity displayed for human beta2m. The results of our experiments led us to conclude that (1) association with beta2m is not necessary for the exposure of HLA-C on the surface of activated T lymphocytes and (2) cell activation affects the balance between the two conformational forms of HLA-C.

The clinical and immunological responses to typhoid vaccination with parenteral and oral vaccines in two groups of 30 adult male subjects were studied. Specific anti-Salmonella typhi cell-mediated immunity and total or specific anti-lipopolysaccharide faecal immunoglobulin (Ig) A titres in vaccinated subjects were monitored. Cellular antibacterial activity was significantly increased only in orally vaccinated subjects. Serum arming activity and inhibition experiments suggested an IgA-dependent cellular cytotoxicity in those orally vaccinated. In these subjects, a total and anti-lipopolysaccharide faecal IgA increase was observed lasting up to 8 months after completion of the vaccination schedule. In parenteral vaccinated subjects, an early onset transitory increase of IgM rheumatoid factor was observed. Oral vaccine was well tolerated and free of side effects, whereas 65% of parenterally vaccinated subjects reported side effects such as fever, headache, malaise and local tenderness in the injection site.

In this report we have analysed the kinetics of modulation of human peripheral blood T lymphocyte membrane molecules upon activation with optimal amounts of phytohaemagglutinin (PHA) and concanavalin A (ConA). The following activation-related and differentiation/adhesion molecules were selectively and concomitantly investigated on CD4+ and CD8+ subsets by dual colour flow cytometry: CD69, CD25 and CD71; CD2, CD45RA and L-selectin. Cultures were assayed after 24, 48, 72, 120 and 168 h of incubation with PHA and ConA. This approach allowed a comprehensive evaluation of membrane phenomena occurring during activation of normal resting human T lymphocytes. Data show that the kinetics of expression of these molecules follows a precise and consistent time-course with no major differences between CD4 and CD8 subsets. CD69 expression peaked at 24 h, whereas CD25 and CD71 expression peaked at 48/72 h with some differences between PHA and ConA activation. L-selectin expression started an evident decrease in step with culture time whose magnitude was dependent on the lectin used, being higher with PHA than with ConA. Conversely, the expression of CD45RA remained stable for 72 h and then briskly decreased with no major differences between PHA and ConA activation. CD2 molecules increased with time in number and density, although the percentage of positive cells remained essentially constant (greater than 85%). After 48/72 h of stimulation about 10% of cells co-expressed CD4 and CD8 molecules. To ascertain whether the phenomenon was restricted to cells in a particular activation state, the phenotype of cells in the diverse phases of the cell cycle was established. Results obtained show that only actively proliferating cells, that is cells in S and G2-M phases, co-expressed the two molecules, suggesting that such a phenomenon reflects a momentary dysregulation of the normal sequence of gene expression. The present data are also discussed in the light of the dynamic role of T lymphocyte activation and adhesion molecules in regulating cell-cell interactions, tissue localization and eventual immunological function.

This study was undertaken to gain insight into the functional properties of the expanded suppressor/cytotoxic lymphocytes characteristically found in actinic reticuloid. Peripheral blood cells, either whole mononuclear cell populations or selectively enriched populations, were obtained from a patient with well-established actinic reticuloid. The patient had an expansion of circulating T lymphocytes expressing the suppressor/cytotoxic phenotype, i.e., CD3+, CD4-, CD8+. A proportion of these cells also expressed the Fc receptor for IgG. Functional studies, including pokeweed mitogen-driven immunoglobulin synthesis, mitogen and alloantigen response, natural killer and antibody-dependent cellular cytotoxicity were within normal ranges and suggested a polyclonal rather than monoclonal expansion. The present functional data extend previous phenotypic studies and support the hypothesis that a chronic reactive immunoregulatory disorder is involved in actinic reticuloid, as has been hypothesized for other T cell chronic proliferations.

Cyclooxygenase (COX)-1 and -2 catalyze the formation of prostaglandins (PG). Given the role of COX and PG during intrathymic T cell development in the mouse, we investigated the expression and localization of these isozymes in the human thymus. mRNA and proteins correspondent to COX-1 and -2 were observed from whole thymus extracts. By immunohistochemistry, COX-2 was selectively localized in the medulla and it was predominant in a subset of stromal cells. By contrast, COX-1 was diffusely and exclusively present in the cortex, both in thymocytes at early stages of differentiation and in cytokeratin-positive epithelial cells, as demonstrated by double immunostaining and flow cytometry analysis. COX-2-positive cells in the medulla expressed cytokeratin and HLA-DR molecules, but they were negative for dendritic or macrophagic antigens. In addition, COX-2-positive cells expressed both the epidermal growth factor receptor and its ligand, the transforming growth factor-alpha. The inducible isoform of the PGE(2) synthase was also present in the same cells, while was absent from COX-1-expressing cells of the cortex. Finally, electron microscopy confirmed that COX-2 was mainly localized in the cytoplasm of cytokeratin-positive cells, along the rough endoplasmic reticulum. In conclusion, COX-2 and the inducible isoform of PGE(2) synthase appear to be constitutively and selectively present in medullary epithelial cells of the human thymus, whereas COX-1 is predominantly present in the thymic cortex, both in the stroma and in developing thymocytes.

Monocytic features can be induced in the myeloid cell line HL60 in order to provide a suitable in vitro model for the investigation of in vitro activity in mononuclear phagocytes. 1,25-Dihydroxyvitamin D3 (calcitriol) induced the HL60 cell line to express the monocytic differentiation antigen Leu M3 in about 30-50% of the cells along with an increase (up to 20%) in the expression of HLA-DR but not HLA-DQ class II antigen. Functional investigation showed that calcitriol-treated cells formed rosettes with sheep erythrocytes coated with an anti-sheep erythrocyte-specific IgG2a mouse MoAb and readily ingested them. In addition, these same sensitized erythrocytes were lysed in an 18-hr antibody-dependent cellular cytotoxicity (ADCC) assay. All together these data indicate the presence of functionally active Fc-IgG receptors (FcR). Sorting experiments demonstrated that only Leu M3+ HLA-DR+ cells contained the effector cell population; such was also the case for blood monocytes. This phenotypic profile was, however, not predictive per se of FcR presence and function, as 12,O-tetradecanoylphorbol-13-acetate (TPA)-induced HL60 cells neither formed rosettes nor phagocytosed nor exhibited ADCC activity, although they express Leu M3 and HLA-DR (as well as HLA-DQ) antigens. These results suggest that calcitriol and TPA cause the differentiation of HL60 cells along distinct pathways. On the other hand, different subpopulations with given predetermined differentiation capabilities may coexist in HL60 cell line. This hypothesis gains support by the observation that when TPA and calcitriol were added together to the undifferentiated cells, the resulting phenotypic pattern was representative of the different activities of both of the inducers as they were used separately.

Summary Eighteen healthy adult male subjects, living in a work environment with hepatitis B surface antigen (HBsAg) carriers, were vaccinated against hepatitis B with French vaccine. Immunological monitoring, performed on days 0, 15, 30, 45, 60, 90 and 120, concerned (1) specific humoral and cell-mediated immunity to HBsAg (cell-mediated immunity was explored in only 13 subjects by the leukocyte migration inhibitory test, or LMIT), (2) autologous mixed lymphocyte reaction (AMLR), (3) OKT3-, T4- and T8-positive lymphocytes, (4) the study of phagocytic respiratory activity, and (5) autoantibodies and circulating immune complexes (CIC). Absolute clinical, hepatic and immunological safety, as documented by the absence of clinical side-effects, no change in serum transaminase levels and the lack of appearance of CIC and/or autoantibodies, was observed. Specific seroconversion, absent on the 15th day, reached 89% in subjects by the 90th day, whereas specific cell-mediated conversion seemed to occur earlier (many subjects already showed LMIT positivity by day 15). For monoclonal antibodies and AMLR, an increase in OKT8+ lymphocytes on day 60, seen only among normal responders, was observed with a subsequent reduction in the OKT4/OKT8 ratio and a reduction of the proliferative response in AMLR on the 45th day (except in the only non-responding subject tested). No significant variations in granulocyte respiratory activity were evident. These results are discussed, focusing on the role of cellular immunity during anti-HBsAg vaccination.