Claudia Semino

HLA Class I molecule expression is up-regulated during maturation of dendritic cells, protecting them from natural killer cell-mediated lysis

It has been widely demonstrated that natural killer (NK) cells are able to discriminate between normal and abnormal cells on the basis of the interaction of their NKRs with the MHC molecules expressed on the target cells. Recent studies showed that also normal dendritic cells are susceptible to NK cell-mediated lysis. In this work, the potential relationships between the expression of different surface molecules on both immature and mature DC and susceptibility to lysis have been investigated. The reduced density of HLA class I on the surface of immature DC resulted in the only permissive signal to NK cell mediated killing. On the contrary, the remarkable increase in HLA class I molecules detected during DC maturation was strictly related to the resistance to NK cell. Contemporary, no clear evidences of a role for other surface molecules modulated during DC maturation (CD80, CD83, CD86 and CD40), were obtained.

Role of major histocompatibility complex class I expression and natural killer-like T cells in the genetic control of endometriosis

OBJECTIVE To evaluate whether the expression of human leukocyte antigen (HLA) class I on eutopic and ectopic endometrial cells modify the susceptibility to lysis mediated by lymphocytes. DESIGN Evaluation of T lymphocyte cytotoxic activity and HLA class I expression on endometrial cells. SETTING Subjects were recruited at laparoscopy. PATIENTS Patients with endometriosis (n = 7). Healthy women as controls (n = 10). MAIN OUTCOME MEASURES Human leukocyte antigen class I molecule analysis of endometrial cells was carried out by immunofluorescence and flow cytometry. Phenotyping of T lymphocytes was performed to analyze T-cell subsets. Cytotoxicity was performed to determine cytolytic activity against endometrial cells. RESULTS In vitro culture of endometrial cells down-regulates the expression of HLA class I molecules and enhances the susceptibility to lysis mediated by natural killer (NK)-like T lymphocytes. Cytolytic T-cell clones, expressing the CD94 antigen, are inhibited by the HLA-B7 allele on endometrial cells. Ectopic endometrial cells modulate the expression of HLA class I molecules. CONCLUSIONS The resistance to lysis of endometrial cells is related to expression of surface HLA class I molecules, which send a negative signal for lysis mediated by NK-like T lymphocytes. The HLA-B7 allele inhibits the cytotoxic activity, suggesting that the growth of ectopic endometrial cells might be under a genetic control.

Engagement of CD33 surface molecules prevents the generation of dendritic cells from both monocytes and CD34+ myeloid precursors.

Although CD33 represents an important marker of myeloid cell differentiation, its function remains poorly defined. In view of its homology with p75 / AIRM1, a recently identified surface molecule which exerts a potent inhibition on NK cell function, we re‐evaluated the effect of CD33 engagement in defined myeloid cell functions. Addition of anti‐CD33 mAb to cultures of CD14+ monocytes supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), IL‐4 and TNF‐α, prevented the generation of dendritic cells. In these cultured cells, engagement of CD33 resulted in an increased surface binding of annexin‐V, followed by cell death. Mature dendritic cells were resistant to the CD33‐mediated effect. Also in CD34+ precursors, cultured in the presence of flt3‐ligand, c‐Kit‐ligand, GM‐CSF, IL‐4 and TNF‐α, addition of anti‐CD33 mAb prevented the recovery of mature dendritic cells. These data suggest a regulatory role of CD33 in the myeloid cell maturation and may offer a tool to interfere with the monocyte / macrophage cell function as well as with the development of dendritic cells.

T lymphocytes express B7 family molecules following interaction with dendritic cells and acquire bystander costimulatory properties

Dendritic cells (DC) play a pivotal role in the initiation, maintenance and regulation of the immune response. Here we obtained the first evidence that DC, in the absence of any foreign antigens, induce the expression of B7 family costimulatory molecules, such as CD80, CD86, B7‐H1, PD‐L2, B7‐H3, and B7RP‐1, on autologous T lymphocytes. Cell‐to‐cell contact between DC and T cellswas needed in order to obtain this expression on T cells. De novo expressed B7 molecules on T cells were functional since B7+ T cells were able to costimulate the proliferation of highly purified T cells. While both autologous and allogeneic DC were able to induce similar levels of costimulatory molecule expression, the chemokine receptor repertoire on B7+ T cells after interaction with DC varied depending on the presence of alloantigens during the interaction (CCR7–, CCR5+) or the absence of antigens (CCR7+, CCR5–). In accordance with this different pattern of chemokine receptors in the two conditions, we propose that, after the encounter with DC in lymphoid organs, this peculiar T cell population should reside in the T cellareas of the lymph nodes or migrate to peripheral sites of inflammation, providing a second signal for activating or switching off, respectively, naive or peripheral effector T cells.

Recombinant Interleukin‐2 Corrects In Vitro the Immunological Defect of Endometriosis

PROBLEM: It has been shown recently that women with endometriosis have several immunological defects. In particular, these patients have a defect in peripheral blood natural killer cell activity and have lost their capacity of recognizing and lysing autologous endometrial cells.

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically

Natural killer (NK) cells have the capability of lysing targets that have down‐regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV‐infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti‐p70, anti‐p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV‐infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV‐infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer‐cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV‐infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA‐A and HLA‐B and did not modify the expression of HLA‐C, but also that NK cell clones expressing the “activating” form of the anti HLA‐C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV‐infected autologous target cells. J. Med. Virol. 62:354–363, 2000.

Quantitative flow cytometric analysis of the effects of cetirizine on the expression of ICAM-1/CD54 on primary cultured nasal cells

An in vitro flow cytometric model has been developed to evaluate the effects of antiallergic drugs such as cetirizine (CTZ) on the expression of surface molecules on primary cultured normal cells. Quantitative analysis demonstrated that HLA class I and ICAM‐1/CD54 molecules are present on both epithelial and stromal cells, and that their expression is strongly enhanced by treatment with interferon‐gamma (IFN‐γ). Nevertheless, the IFN‐γ‐mediated upregulation of ICAM‐1/CD54 was inhibited by treatment with CTZ, demonstrating a direct effect on both cell types. This finding is particularly interesting because ICAM‐1/CD54 is the main rhinovirus receptor, and rhinoviruses are the principal cause of asthma exacerbation in children. Thus, according to data derived from this in vitro model, CTZ should have an important role in the reduction of infectious exacerbation of asthma in atopic patients.

A phase I study of intravesical continuous perfusion of recombinant interleukin-2 in patients with superficial bladder cancer.

A Phase I study was started to evaluate the locoregional and/or systemic toxic effects of the continuous perfusion of re-combinant interleukin-2 (rIL-2) in superficial bladder cancer. Three different dose levels were used: 3 × 106 IU/day (3 patients), 9 × 106 ID/day (3 patients) and 27 × 106 IU/day (3 patients). Two patients (one treated with 3 × 106 and another with 27 × 106 IU/day of rIL-2) had hematuria after the end of the treatment, one patient had fever (grade I) and 7 of 9 patients experienced hypotension (grade I-II). All effects were not dose related. Routine laboratory tests indicated that no significant variations of biochemical parameters occurred. A phenotypic analysis of white blood cells detectable in the bladder, showed an evident locoregional activation of lymphoid cells. In particular, T lymphocytes expressed activation antigens (such as CD25 and HLA-DR) following treatment with rIL-2. A 6− to 12-month clinical follow-up, showed that all patients but one (which recurred after 5 months) are alive and disease-free. This therefore indicates that the locoregional perfusion of rIL-2 is safe and gives clinical results similar to those obtained using Calmette-Guerin bacillus locoregional instillation, in patients who underwent a transurethral resection of superficial bladder cancer.

Treatment of stage IIIB non-small-cell lung cancer with surgery followed by infusion of tumor infiltrating lymphocytes and recombinant interleukin-2 : A pilot study

Stage IIIb non-small-cell lung cancer (NSCLC) has a poor prognosis. The median survival is approximately 6 months, and only 30% of patients are alive 1 year after diagnosis. The need for effective treatment is evident. The aim of this study was to evaluate whether the infusion of tumor-infiltrating lymphocytes (TILs), isolated from resected tumor, expanded in vitro and injected together with recombinant Interleukin-2, is feasible and may at least partially modify the poor prognosis in these patients. The infusion of TILs, derived from surgically resected NSCLC and expanded in vitro, together with subcutaneous (s.c.) injections of recombinant interleukin-2 (rIL-2) was attempted in a group of 11 patients. Treated patients were infused i.v. with in vitro expanded TILs (from 4 to 70 x 10(9) cells), and rIL-2 was injected s.c. at doses varying from 61 to 378 x 10(6) IU. Toxic side effects (fever and, in some cases, hypotension) were observed and limited the dose of rIL-2 infused. Follow-up was continued for 40 months. The mean survival time was 13.8 months. Three of five TIL-treated patients with residual disease have no evident disease after 1 year, and two of them are still alive and have no evidence of disease after 40 months. This pilot study suggests that the infusion of in vitro expanded TILs, derived from surgical samples, is feasible and seems to prolong overall survival and to control the residual disease in patients with advanced NSCLC.

Detection of oligoclonal T lymphocytes in lymph nodes draining from advanced non-small-cell lung cancer

Despite the combined use of surgery and chemoradiotherapy, the poor prognosis of advanced non-smallcell lung cancer (NSCLC) requires the definition of new therapeutic approaches. The presence of T lymphocytes, with peculiar phenotypic, functional and molecular characteristics within the tumour, suggested the possible use of these cells, expanded in vitro, in protocols of adoptive immunotherapy. We have described how a population of oligoclonal T lymphocytes, derived from advanced NSCLC, can be expanded in vitro and has the capability of lysing autologous cancer cells. What is more important, we observed that patients with advanced NSCLC, treated with TIL expanded in vitro and recombinant interleukin-2, seemed to have a disease-free period longer than that of patients treated with conventional chemoradiotherapy. in an attempt to find new sources of specific lymphocytes for immunotherapy, we describe the analysis of the phenotypic, functional and molecular characteristics of T lymphocytes, derived from lymph nodes draining advanced NSCLC. In this paper we show that these cells, have restriction patterns of T cell receptor β chain similar to those detectable in the population of infiltrating T lymphocytes. This finding suggests that T cells derived from draining lymph nodes of advanced NSCLC have peculiar characteristics and can be a suitable source of effector cells for protocols of adoptive immunotherapy in lung cancer treatment.