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Background Virus-specific CD4 and CD8 T lymphocytes from HLA-matched donors are effective for treatment and prophylaxis of viral infections in immune-compromised recipients of hematopoietic stem cell transplant recipients. Adoptive immune reconstitution is based on selection of specific T cells or on generation of specific T-cell lines from the graft donor. Unfortunately, the graft donor is not always immune to the relevant pathogen or the graft donor may not be available (registry-derived or cord blood donors). Study Design and Methods Since the possibility of using T cells from a third-party subject is now established, we screened potential donors for T-cell responses against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus, the viruses most frequently targeted by adoptive immune reconstitution. Specific T-cell responses against viral antigens were analyzed in 111 donors using a miniaturized interferon-γ release assay. Results Responders to CMV were 64%, to EBV 40%, and to adenovirus 51%. Simultaneous responders to the three viruses were 49%. CMV-specific CD4 and CD8 T-cell lines could be generated from 11 of 12 donors defined as positive responders according to the T-cell assay. Conclusions These data demonstrate that a large fraction of volunteers can be recruited in a donor registry for selection or expansion of virus specific T cells and that our T-cell assay predicts the donors' ability to give rise to established T-cell lines endowed with proliferative potential and effector function for adoptive immune reconstitution.

BACKGROUND: Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space. STUDY DESIGN AND METHODS: The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated. RESULTS: The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ±12.19%), going from 236 mL (range, 100-443 mL; SD, ±80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ±47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ±8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ±13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing. DISCUSSION: Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.

Background Umbilical cord blood (UCB) is a valid alternative to be used in transplanted patients. Limitations of the use of stem cells depends on the small number of cells available; this is the reason why UCB can be used only in very low-weight patients. In this study we have evaluated the efficacy of cellular manipulation before transplant and in particular, before thawing the units through the Rubinstein method. Methods We have evaluated the results obtained after thawing 40 UCB to be used for as many patients affected by several pathologies (21 ALL, 6 AML, 3 MDS, 2 LNH, 2 histiocytosis, 2 β-thalassemia, 1 Chediak-Higashi syndrome, 1 Fanconi anemia, 1 Wiskott-Aldrich syndrome and 1 Omenn syndrome). Results After thawing, nucleated cells (NC) mean recovery was 76.81% (SD ± 15.41). The quantity of NC obtained was 124.29 × 107 (SD ± 43.18) and in only 5 cases the number of NC after the procedure was lower than the requested graft dose. Among the last ones, in two cases only we did not achieve the target after manipulation. The post-manipulation cellular viability was 83.48% (SD ± 10.6). For all the units shipment complied with all the necessary procedures; in fact the temperature never rose above −120 °C. Conclusion In our study we highlighted the efficacy of UCB thawing technique, with the same method defined in 1995 at the New York Blood Centre that guarantees an excellent NC recovery and maintains a high level of cell viability.

BACKGROUND: Immunomagnetic selection of peripheral blood progenitor cells (PBPCs) in patients with tumoral infiltration in marrow makes it possible to reduce contamination of cellular concentrates, but this procedure cannot always be used, mainly because of the low cellular count in apheresis concentrates. STUDY DESIGN AND METHODS: In this case two cellular concentrates taken separately at two different times were selected and cryopreserved; they were thawed with an automatic instrument. RESULTS: After manipulation, a selected concentrate containing 24.16 × 106 CD34+ cells with a purity of 90.15 percent was obtained; vitality after thawing and selection was 88 and 96 percent, respectively. The engraftment was achieved on Day +17 from the infusion of the previously selected PBPCs, as the literature also shows us. CONCLUSION: The time passed between the infusion and the engraftment gives us evidence of the efficacy of immunomagnetic selection carried out after thawing 2 cell units that were collected at different times from the same patient. In this way, it has been possible to perform an autologous transplant in a patient in which CD34+ cells transplant is recommended, but from whom the number of collected cells after a single mobilization cycle would not have been sufficient for the engraftment.

Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory.

Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space.The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated.The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing.Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.

T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of T(reg) cells designated naturally occurring and induced. Interest in T(reg) cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding T(reg) cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127(low) FOXP3+ regulatory T cells.

High-dose chemotherapy followed by reinfusion of autologous stem cells harvested from peripheral blood has been increasingly applied for a variety of disorders. The critical importance of cell dose in the clinical outcome, after transplant, has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility.The aim of this study is to evaluate the efficacy of the Sepax S-100 device to process thawed HPC-A products in comparison with manual procedure.We have analysed viability, total nucleated cells (TNC), haematopoietic progenitors and CD34+ cells recovery.The TNC and CD34+ cells recovery in the automatic procedure was of 91.9% (73-100; SD ± 12.60) and 86.7% (69-100; SD ± 10.21), respectively. Instead the recovery of TNC and CD34+ cells using the manual method was of 84.7% (47-100; SD ± 22.9) and 80.29% (23-100; SD ± 25.96). The results, obtained from the assessment of viability of CD34+ both 7-AAD)+ and AnnV+ showed a high percentage of necrosis and apoptosis in this cell subset by using the manual procedure in respect to the Sepax automated system.Overall, our data suggest that the automated washing procedure is safe and suitable for processing of thawed HPC-A products and can be daily used in clinical routine.

Background aims: Immunomagnetic CD34 cell selection (ICS) is utilized in autologous and allogeneic transplants. In the fi rst case it is used to reduce the neoplastic contamination of concentrates, while in the second case it is needed to carry out a T-depletion of cell concentrates in order to reduce the incidence of graft-versus-host disease (GvHD) in patients who have undergone haplo-identical transplants. Methods: The effi cacy of CliniMACS technology, after reduction of platelet contamination, incubation of monoclonal antibodies (MAb) and successive washings of concentrates, performed in 16 ICS using the standard method without reducing platelet content, was compared with the use of the automated system CytoMate, which was carried out in 46 ICS. Results: In the group of ICS carried out after automatic manipulation, a signifi cant statistical difference in purity was noted (91.39% versus 83.57, P 0.017) compared with the group of ICS carried out with the standard procedure. The same signifi cant difference was noted in relation to the remaining percentages of CD3 and CD19 cells (2.31% versus 5.68%, P 0.012, and 1.58% versus 2.71%, P 0.014, respectively). Recovery of CD34 cells overlapped in the two groups (70.49% versus 68.39%, P 0.774). Conclusions: Immunomagnetic selection carried out using the automated procedure was more effi cient, producing a purer sample, more effi cient T-depletion and optimal reduction of B cells, without infl uencing cell recovery. Furthermore, conforming to good manufacturing practice (GMP) guidelines, the entire procedure with CytoMate took place in a contamination-controlled environment.

Background We wondered whether short-term coseasonal sublingual immunotherapy (SLIT) can reduce the development of asthma in children with hay fever in an open randomized study. Objective We sought to determine whether SLIT is as effective as subcutaneous immunotherapy in reducing hay fever symptoms and the development of asthma in children with hay fever. Methods One hundred thirteen children aged 5 to 14 years (mean age, 7.7 years) with hay fever limited to grass pollen and no other clinically important allergies were randomized in an open study involving 6 Italian pediatric allergy centers to receive specific SLIT for 3 years or standard symptomatic therapy. All of the subjects had hay fever symptoms, but at the time of study entry, none reported seasonal asthma with more than 3 episodes per season. Symptomatic treatment was limited to cetirizine, loratadine, nasal budesonide, and salbutamol on demand. The hay fever and asthma symptoms were quantified clinically. Results The actively treated children used less medication in the second and third years of therapy, and their symptom scores tended to be lower. From the second year of immunotherapy, subjective evaluation of overall allergy symptoms was favorable in the actively treated children. Development of asthma after 3 years was 3.8 times more frequent (95% confidence limits, 1.5-10.0) in the control subjects. Conclusions Three years of coseasonal SLIT improves seasonal allergic rhinitis symptoms and reduces the development of seasonal asthma in children with hay fever.

Abstract 343 Introduction. Allogeneic hematopoietic stem cell transplantation (HSCT) from a HLA-haploidentical relative is a suitable option for patients (pts) lacking a compatible donor, either related or unrelated. The two main approaches for overcoming the obstacles of HLA barriers are based either on the infusion of large numbers of T-cell-depleted HSC or on intensive pharmacological prevention of graft-versus-host disease (GVHD). While for many years T-cell depletion (TCD) of the graft has been based on either immunomagnetic positive selection of CD34+ cells or on physical removal of all subsets of T cells by virtue of mAb, we and other groups have recently developed a novel method of ex vivo TCD based on the selective elimination of αβ+ T cells through labeling with a biotinylated anti-TCRαβ Ab, followed by incubation with an anti-biotin Ab conjugated to paramagnetic beads (Miltenyi Biotec, Germany). This approach also allows the removal of B cells to prevent post-transplant EBV-associated lymphoproliferative disease (PTLD). Here, we report the results of graft manipulation using this approach. Methods. Twenty-two children entered the study, 15 with hematological malignancies and 7 with non-malignant disorders. No post-transplant GvHD prophylaxis was employed. HLA-haploidentical family donors received G-CSF (12–16 μg/kg of body weight) to mobilize HSC prior to large-volume leukapheresis, which was commenced when circulating CD34+ HSC were >20 cells/μl. Cell therapy products containing up to 60×109 white blood cells (WBC) were processed according to the manufacturer's protocol. In some cases, leukapheresis bags were stored overnight at 4°C in appropriate media at a WBC concentration Results. Median recovery of CD34+ HSC and median number of infused CD34+ HSC were 99.3% (range 55.4–100) and 14.7×106/kg (range 7.9–37), respectively. The graft contained 3.9×106 CD3+ T cells/kg (range 0.9–30.9) and 0.08×106 B cells/kg (range 0.002–0.32). The log-depletion of αβ+ T cells was 4.5 (range 3.2–6.1), with a median number of transplanted αβ+ T cells equal to 36×103/kg (range 1–139.9). Patients received 36×106 CD56+ NK cells/kg (range 1.6–80.3) and 4.1×106 γδ+ T cells/kg (range 0.9–30.8). A median of 40.8% NK cells (range 30.3–60.2) co-expressed Tim-3, an activating co-receptor promoting IFN-γ production and being scarcely expressed on NK cells that reconstitute after allogeneic HSCT. The αβ/CD19-depleted grafts also contained DC1 and DC2 precursors, which were identified based on BDCA-1 (0.23% of all nucleated cells, range 0.02–2.4), BDCA-3 (0.93%, range 0.57–9.4) and BDCA-2 (0.66%, range 0.49–1.28) or BDCA-4 expression (0.69%, range 0.02–1.5), respectively. Invariant NKT cells were enumerated using mAb reacting against the TCR Vα24-Jα18 chain and were detected in minute percentages ( Conclusions. The immunomagnetic removal of αβ+ T cells and B cells was effective and gave reproducible results. In all cases, a remarkable depletion of unwanted T and B cells was attained. The depleted graft contained high numbers of CD34+ HSC, as well as of immune effector cells implicated in the control of leukemia growth and GVHD, such as γδ+ T cells, NK cells, DC1, DC2 and non-classical CD14+CD16+ monocytes. From a clinical standpoint, the infusion of αβ/CD19-depleted grafts from HLA-haploidentical family donors was safe, resulting into sustained donor engraftment without life-threatening complications. Disclosures: No relevant conflicts of interest to declare.