Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) represents a valuable alternative for children with nonmalignant disease and ex vivo negative selection of TCR-alpha beta(+) cells is an emerging graft manipulation option that carries several potential advantages in terms of reduced risk of graft-versus-host disease (GvHD) and improved immune reconstitution. We report all consecutive patients with a diagnosis of nonmalignant disease who received a TCR-alpha beta(+) and CD19(+)depleted haplo-HSCT at "IRCCS Istituto Giannina Gaslini" from 2013 to 2019; the conditioning regimen was myeloablative or non-myeloablative, depending on underlying disease; all patients received antithymocyte globulin and rituximab. No post-transplantation GvHD prophylaxis was given in presence of a TCR-alpha beta(+) cell dose in the graft lower than the threshold of 1 x 10(5)/kg of the recipient's weight. Among 20 HSCTs, engraftment occurred in 17 (85%) after a median of 14 and 12 days from graft infusion for neutrophils and platelets, respectively. Primary graft failure was diagnosed in 3 (15%) patients, and 2 (10%) experienced secondary rejection; all of these patients underwent a second HSCT. The cumulative incidence of a-GvHD and c-GvHD was 15% (2 = grade 1, 1 = grade 4) at 90 days and 5% (1 = grade 1) at 7 months, respectively. Cytomegalovirus reactivation requiring pre-emptive treatment was observed in 9 patients (45%). One patient developed a JC virus-related progressive multifocal leukoencephalopathy, successfully managed with donor-derived virus-specific T-cell infusions. A complete immunological recovery was reached in most patients within 6 months. After a median follow-up of 4 years, 18 patients are alive, with a cumulative survival probability of 90%. Haplo-HSCT after ex vivo TCR-alpha beta(+)/CD19(+) negative selection may be considered a good option for children with nonmalignant diseases because it ensures a high engraftment rate with an acceptable risk of graft failure, very low incidence of significant GvHD, and good immune reconstitution with low frequency of severe virus-related disease. However, the control of viral infection/reactivation should be kept high to promptly provide pre-emptive treatments and approaches of antiviral adoptive immunotherapy. (C) 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Introduction.Despite progress obtained with current treatments,the event-free survival of high-risk neuroblastoma(HR-NB)patients does not exceed 40-50% and the prognosis in refractory or relapsed patients is poor,still representing a challenge for pediatric oncologist.Therapeutic Iodine-131 meta-iodobenzylguanidine(Th-131I-MIBG) is a recognized safe and potentially effective treatment in NB.Materials.In this retrospective study,we report outcome of 28 MIBG-avid NB patients with advanced disease,because refractory or relapsed,underwent,from 1996 to 2014,to Th-131I-MIBG administered shortly before(median of 17 days) high-dose chemotherapy with Busulfan and Melphalan(HD-BuMel) and autologous stem cell transplantation(ASCT)at Gaslini Institute in Genoa,with the aim to analyze feasibility,safety and efficacy of this approach.Results.Engraftment occurred in all patients after a median of 14(11-29)and 30 days(13-80)from ASCT for neutrophil and platelet respectively.No treatment-related deaths were observed.The main high grade(3-4)toxicity observed was oral and gastrointestinal mucositis in 78.6% and 7.1% of patients respectively,while high grade hepatic toxicity was observed in 10.7%;two patients developed veno-occlusive-disease(7.1%),completely responsive to defibrotide.Hypothyroidism was the main late complication occurred in 9 patients(31.1%).After Th-131MIBG and HD-BuMel, 19 patients(67.8%) showed an improvement of disease status.Over a median follow-up of 15.9 years,the 3-year and 5-year overall survival(OS)probability were 53%(CI 0.33-0.69)and 41%(CI 0.22-0.59)and the 3-year and 5-year rates of cumulative risk of progression/relapse were 64%(CI 0.47-0.81)and 73%(CI 0.55-0.88),respectively.MYCN amplification emerged as the only risk factor significantly associated with OS(HR 3.58;p0.041).Conclusion.Th-131I-MIBG administered shortly before HD-BuMel turned out to be a safe and effective regimen,suggesting it should be included in a sequential approach in patients with advanced MIBG-avid NB.These patients could be benefit to be manage in centers with proven expertise in these treatments.

Background Anti-N-methyl-d-aspartate receptor encephalitis is a central nervous system inflammatory autoimmune disease affecting adults and children. The use of first- and second-line immunotherapies is supported. Recent reports suggest the efficacy of bortezomib in severe anti-N-methyl-d-aspartate encephalitis in adult patients not responsive to second-line treatment; there are no data about pediatric patients. patient description We describe an eight-year-old child with anti-N-methyl-d-aspartate encephalitis not responsive to first- and second-line treatments who experienced marked clinical improvement after bortezomib administration. Discussion Bortezomib is a selective and reversible inhibitor of the 26S proteasome, which is used to treat oncologic and rare autoimmune disorders in pediatric patients. As observed in adult patients, bortezomib administration induced anti-N-methyl-d-aspartate antibody titer decline and clinical improvement with an acceptable risk profile. Conclusion This is the first report of the use of bortezomib in children with anti-N-methyl-d-aspartate encephalitis; it could be a useful therapeutic option in children with refractory anti-N-methyl-d-aspartate encephalitis.

This study report clinical course, etiology, management, and long-term outcome of children who developed toxic epidermal necrolysis-like reaction (TEN-LR) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively collected children with TEN-LR occurring after allo-HSCT performed in 2 pediatric bone marrow units between 2005 and 2014. We identified 6 cases of TEN-LR of 322 patients (1.8%). Possible triggers of TEN included antibiotics, antiepileptics, antimycotics, and Mycoplasma infection. In 3 patients TEN-LR occurred concurrently with severe multiorgan acute graft versus host disease. The management of TEN included administration of high doses of intravenous immunoglobulins and steroids (n=6), anti-tumor necrosis factor (n=3), and plasmapheresis (n=3) and whenever possible, discontinuation of the potentially causative drugs. Four patients (66%) reached a complete clinical response of TEN-LR after a median of 11.2 days. Two children (34%) are presently alive, 1 with long-term ocular sequelae. TEN-LR is a potentially lethal complication that may occur after HSCT also in pediatric patients. In our experience, TEN-LR and acute graft versus host disease probably coexisted and an overlap between the 2 forms is suggested. The multidisciplinary approaches involving specialized nurses, hematologists, dermatologists, burn surgeons, and infectious disease specialists is crucial to treat these patients.

Background:It has been recently shown that during therapeutic apheresis procedure, a large amount of soluble HLA class I molecules settles onto plastic apheresis circuits, inducing sustained TGFβ1 pre/post‐transcriptional modulation in activated patients' leukocytes. Reportedly, donors' leukocytes may be exposed to similar immunosuppressing activities during donor apheresis procedures. On this basis, it could be hypothesized that such events can cause immune modulation. It is uncertain which blood cell population is most impacted by these events. This study is focused on the effects on the T lymphocytes.Study design and methods:To assess if such events occur, lymphocytes from 20 apheresis donors collected before and after three closely timed plasma and platelet donation procedures were analyzed for sHLA‐I mediated immunomodulation.Results:The results confirmed that sHLA‐I molecules bind to the apheresis circuit surfaces. Circuits can also transiently activate donors' CD8+ T lymphocytes, to which sHLA‐I molecules can bind, thus modulating short‐lasting immune effects, such as transcriptional and post‐transcriptional TGFβ1 modulation and soluble Fas ligand release. However, no significant change in relative proportions, absolute number and cell viability of lymphocyte subpopulations was found and no ex vivo immune effect was detectable longer than 14 days after procedure in any cell type in all donors.Conclusion:Short‐lived sHLA‐I mediated immunomodulation was demonstrable in lymphocytes from every donor as a consequence of apheresis procedures, but no enrolled subject experienced any adverse reaction or showed any sign of immunosuppression during 24 months of follow‐up after the donations. J. Clin. Apheresis 30:204–211, 2015. © 2014 Wiley Periodicals, Inc.

In a previous study, lack of IL-12 signaling in il12rb2 knock-out mice was found to predispose to lung adenocarcinoma (LAC). We asked whether specific polymorphisms of the human IL12RB2 gene may confer susceptibility to LAC. We studied IL12RB2 single nucleotide polymorphisms (SNPs) spanning from the promoter to the first untranslated exon of the gene. Genotypes of 49 individuals with LAC were compared with those of 93 healthy subjects. Two allele variants were found to be associated with increased susceptibility to LAC. One haplotype (hap), hap18, was more frequent in patients (18%) versus controls (6%) and significantly associated with increased probability of disease occurrence. Furthermore, IL-12 driven STAT4 phosphorylation in T cell blasts from healthy individuals was found to correlate with both single allele variants and haplotypes. In conclusion, genetically determined low signaling activity of IL-12R predisposes to the development of LAC.

in the late plasma separation by membrane filtration was devised. this large proteinaceous molecules, such as auto-antibodies and immune-complexes, or even single blood cell populations, supposed to be pathogenic, could be directly removed from whole blood by different apheretic techniques, with benefit for the treatment of patients suffering from immune-mediated diseases . Contemporaneously, a growing body of evidence indicated that therapeutic apheresis can induce multilevel immune-modulation in other ways than simply through the removal of immune-relevant "agents", leading to the concept of extracorporeal immunomodulation However, despite on clinical efficacy, a thorough understanding of the mechanisms of apheresis-induced modification of disease course has yet to be Results of sHLA-I staining in the apheresis circuits revealed a homogeneous layer of sHLA-I molecules binding to the plastic surfaces during the extracorporeal blood/circuit interaction, consistent with extremely high sHLA-I values in the plasma samples drawn directly from the circuits at the end of each apheretic procedure, with a mean count of 28.5  the circuit due to the extracorporeal blood/circuit interaction during

Background aims. Hematopoietic stem cell cryopreservation significantly contributed to autologous stem cell transplantation (ASCT). Cryopreserved stem cell units (SCU) are expected to be used soon after harvesting for most purposes, but, in a number of cases, they remain stored for some time, creating an increasing load for SCU depositories. Disposal policies vary widely in each center, and the existing guidelines are insufficient. Methods. We conducted a survey of seven Gruppo Italiano Trapianto di Midollo Osseo centers to investigate the outcome of SCU harvested from January 2005 to December 2009 for ASCT. The data from 1603 collections were gathered, for a total of 5822 SCU. Results. In our cohort, 79% of patients collected >5 x 10(6) CD34+ cells/kg, and 3.4% collected <2 x 10(6) CD34+ cells/kg. Up to 21% of all the patients and 42% of those with acute leukemia did not undergo reinfusion, and 37% of the cryopreserved SCU were excess, resulting from patients not reinfusing or partially reinfusing. Less than one-third of the excess SCU was disposed, and the major causes of disposal were death and, in a minority of cases, withdrawal of the indication for ASCT. In our analysis, very few first reinfusions occurred after 2 years, and those after 5 years were exceptional. Through the use of a multivariate analysis, we sought to identify the risk factors for collection non-use, independent of the centers' policies. Non-use of SCU was significantly associated with patients with acute leukemia, collections of <2 x 10(6) CD34/kg and lower age groups. Conclusions. These data serve as a valid basis to support rational recommendations for cost-effective storage and disposal of SCU.

BackgroundRecently biosimilars of granulocyte–colony‐stimulating factor (G‐CSF) became available for prophylaxis and treatment of postchemotherapy neutropenia and for mobilization of peripheral blood CD34+ cells for either autologous or allogeneic hematopoietic stem cell transplant. Most of the data on the mobilization efficacy and safety of biosimilar G‐CSF are from adult patients, whereas no data are available in pediatric patients.Study Design and MethodsThis was a retrospective study on cases treated at three Italian pediatric transplant centers, from January 2011 to October 2013. Data were collected on all children undergoing first peripheral blood stem cell (PBSC) mobilization after stimulation with biosimilar G‐CSF and chemotherapy. The results were compared with a historical control group.ResultsTwenty‐nine children underwent mobilization with biosimilar G‐CSF. Peak peripheral blood CD34+ cell count of 20 × 106/L was achieved in 90% of patients, with a median value of 71 × 106/L. Eighty‐three percent reached the desired target (CD34+/kg) dose. The median number of collected CD34+ cells was 10 × 106/kg (range, 4.8 × 106‐68.8 × 106/kg). No difference was observed in comparison with historical control group mobilized with originator filgrastim. Moreover, no major and/or unexpected side effects were reported.ConclusionBiosimilar G‐CSF resulted as effective and safe as originator filgrastim molecule in mobilizing PBSCs in children, with the advantage of a reduced cost.

BackgroundAutologous stem cell transplantation (ASCT) requires collection and cryopreservation of hematopoietic progenitor cells (HPCs), which in turn may be partially or never reinfused. Thus, HPC storage has become a logistic, ethical, and economic issue. SIDEM, GITMO, and CNT/ISS endorsed a project aimed to define national criteria for HPC disposal aimed to guarantee appropriateness and equity.Study Design and MethodsA multidisciplinary panel was convened including HPC harvest and manipulation experts from apheresis units, hematologists with clinical expertise in ASCT, a representative of the national health authority, and a bioethicist. An analytic hierarchy process (AHP) was carried out to select disposal criteria.ResultsThe AHP selected two criteria for prompt disposal of freshly collected HPCs: an abnormal freezing procedure causing highly reduced viability or major microbiology contamination. Moreover, AHP selected six major criteria, each one of them allowing for the disposal of stored HPC units: patient death, withdrawal of consent to ASCT, contraindications or loss of indications to ASCT, a damaged label that prevents correct identification of the unit, and time elapsed since harvest longer than 10 years. Three minor criteria were additionally identified that allowed to anticipate disposal only provided that viability levels are below the limit of acceptance: a documented cold chain interruption, loss of bag integrity, and total amount of stored CD34+ cells lower than 1 × 106/kg or lower than 2 × 106/kg in patients with a successfully completed stem cell transplantation program.ConclusionsA formal consensus process allowed SIDEM and GITMO to propose a policy for autologous HPC disposal that fulfills clinical, ethical, and economic criteria.

Dear Sir, Some concerns have been raised regarding possible immunosuppressive features in plasmapheresis donors, including alterations of neutrophil function1. On the whole, no report has given clear evidence of immunosuppression at a clinical level and in general no long-term harmful effects have been observed in donors who have undergone cytapheresis2. Our group has long been concerned with the immunomodulatory role of soluble human leucocyte antigen class I molecules (sHLA-I) in transfusion medicine3. We recently showed that CD8+ T lymphocytes and neutrophils rolling and floating in the circuit during therapeutic apheresis are susceptible to the bioactivity of sHLA-I molecules bound to the synthetic polymers of the circuit’s surface during procedure4. The circuits for donor apheresis procedures are made from exactly the same biocompatible plastic as that used in circuits for therapeutic apheresis and so it is conceivable that also during donation a boundary layer could form as a result of adsorption of plasma proteins to plastic membranes made of various synthetic polymers. Accordingly, also during plasma and platelet donation procedures, neutrophils could be activated by rolling in the circuits1 and bound to sHLA-I molecules adsorbed to the circuits’ polymers with Ig-like-transcripts, thereafter becoming sensitive to the biological effects of sHLA-I, such as transcriptional/post-transcriptional modulation of transforming growth factor-β1 (TGFβ1)5. Indeed, in contrast to the situation in patients suffering from immune-mediated diseases, in whom immune-competent cells circulate mostly in an enduringly activated status, the leucocytes of healthy donors are only transiently activated by contact with circuits during the donor procedure and should, therefore, show only an ephemeral sensitivity to sHLA-I-mediated immunomodulation. In order to evaluate a possible ephemeral sHLA-I-mediated immunomodulation in productive aphaeresis, neutrophils from 20 donors (16 male) were analysed before, immediately after, and 7 and 14 days following three closely timed plasma and platelet donation procedures. Apheresis donor procedures were performed in accordance with the Italian law, in healthy donors fulfilling the criteria laid down by the Italian blood donation guidelines and SIMTI recommendations for apheresis donation. Informed consent was obtained from all the donors. Samples to perform a complete pre-procedure laboratory assessment were obtained from each donor. In addition to biochemical and serological tests required by law, C-reactive protein, erythrocyte sedimentation rate, protein electrophoresis, fibrinogen and ferritin were also evaluated in order to further exclude any sub-clinical inflammatory disorder possibly causing leucocyte activation. Follow-up assessments were performed every 6 months up to 2 years, by physical examination, history taking and the same biochemical/serological tests performed during the enrolment. Monoclonal antibodies and other reagents for research use were purchased from several renowned companies. Immunofluorescence tests of the return line of the apheretic circuits were performed as described elsewhere4. Flow cytometry assays for CD66b and TGFβ were conducted using standard methods. The expression of CD11b membrane molecules was evaluated as an indicator of neutrophil activation. All data were subjected to appropriate statistical analyses. Results of sHLA-I staining in apheresis circuits revealed a homogeneous layer consisting of sHLA-I molecules binding the plastic vessel surfaces during the extracorporeal blood/circuit interaction. Donor neutrophils were found to be transiently activated by the interaction with plastic circuits during the procedure regardless of the type of donation procedure. More to the point, the comparison between the findings at different times showed a statistically significant increase in the expression of CD11b molecules immediately after the procedures (p<0.001). The above-mentioned activation markers significantly decreased after another 7 days (p<0.001), confirming the transient nature of the leucocyte activation (Figure 1). Once again, regardless of the donation procedure, a significant up-regulation of intracytoplasmic TGFβ1 molecules was consistently observed in neutrophils from healthy subjects. Specifically, flow-cytometry analysis revealed a sharp and significant (p<0.001) up-regulation of TGFβ1 molecules at the end of each apheresis procedure. An evident reversion to baseline levels was detectable 7 days after each procedure. Matching the above data, real-time polymerase chain reaction analyses in donor neutrophils showed a sharp increase of TGFβ1-mRNA immediately after all the three procedures, followed by a similarly evident and significant decrease 7 days after the apheresis (Figure 2). Figure 1 Neutrophil activation. Figure 2 Pre/post-transcriptional modulation of TGFB1 molecule. On the whole these findings indicate that sHLA-I molecules adsorb to plastic apheresis circuits during donation procedures and interact with Ig-like-transcripts surface receptors of donors’ neutrophils. Moreover, neutrophils activated by donation procedures1 become sensitive to the biological effects of sHLA-I, such as transcriptional/post-transcriptional modulation of TGFβ15. Nevertheless, unlike what was reported with regards to therapeutic apheresis, the transcriptional/post-transcriptional TGFβ1 modulation detectable in neutrophils following plasma and platelet donation procedures was very ephemeral. In our opinion, the difference in leucocyte activation status is a possible explanation of our findings: therapeutic apheresis is performed in patients suffering from immune-mediated diseases, in whom immune-competent cells circulate mostly in an enduringly activated status4 and are, therefore, much more prone to a possible protracted sHLA-I-mediated immunomodulation. Conversely, donor apheresis is performed in healthy subjects whose leucocytes mostly circulate in a not activated status, and only after interaction with the plastic circuits of the apheresis equipment during the procedure do they become transiently sensitive to sHLA-I. In accordance with such short-lived immunomodulation, no subject enrolled experienced any adverse reaction during the procedures or showed any possible immunosuppression or viral, bacterial or neoplastic disease during 24 months of monitoring following the donations. Finally, at every time-point of the study, the relative proportions and absolute numbers of the neutrophils were evaluated by flow cytometry. These parameters showed no modifications with respect to basal conditions in all the donors examined and in each type of apheresis procedure (data not shown). Overall, our findings might contribute to confirm the conceivable “immunological” harmlessness of donor apheresis, highlighting the importance of a correct assessment of the donors’ healthy status in order to avoid, among other complications, any kind of prolonged sHLA-I-mediated immunomodulation following platelet or plasma donor apheresis.

In spite of the large number of procedures performed every year both in the peripheral blood stem cell transplantation (PBSCT) setting and for the treatment of acute and/or chronic GvHD by means of extracorporeal photopheresis (ECP), there are still some unresolved issues about the optimal strategies (1) to perform PBSC mobilization/collection and (2) to treat GvHD patients unresponsive to immunosuppressive therapies by means of ECP. To address these questions, two Italian scientific societies (SIdEM and GITMO), representing respectively physician working within apheresis units and clinical hematologists, committed to working groups of experts the developments of consensus recommendations about the above quoted issues.

BACKGROUND: A large heterogeneity in current mobilization and collection practices is perceived. Moreover, recent evidence introduced novel issues into some specific topics. Optimization of the clinical practice, through the adoption of clinical practice guidelines, previously proved to reduce health care resource use.STUDY DESIGN AND METHODS: Two Italian scientific societies, Società Italiana Di Emaferesi e Manipolazione Cellulare (SIDEM) and Gruppo Italiano Trapianto Midollo Osseo (GITMO), perceived the need of hematologists and transfusionists to share a common paradigm in the setting of hematopoietic stem cell transplantation (SCT). The aim of the current position paper is to provide common definitions and criteria for mobilization and collection of peripheral blood stem cells both in autologous and in the allogeneic setting. Current international and national standards (i.e., International Society of Hematotherapy and Graft Engineering) and recommendations (i.e., European Group for Blood and Marrow Transplantation) were harmonized with the Panel recommendations.RESULTS: The Expert Panel consisted of nine members (five transfusionists and four hematologists with both clinical and scientific experience of SCT in both pediatric and adult setting) and one methodologist and first convened on April 19, 2010: they in turn agreed on the questions to be answered by the project. Available literature was reviewed by one expert and the methodologist and presented to the other members. Statements were then formulated. SIDEM and GITMO planned an informal meeting of the Panel every 2 years to discuss relevant updates and possible changes to the recommendations.CONCLUSION: The efforts of the expert panel members allowed to set up and share a common approach to the mobilization, enumeration, and collection issues in the field of both autologous and allogeneic peripheral blood SCT.

A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24 h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.

Background. Telomerase complex genes mutations (DKC1, TERC, TERT, and NOP10) lead to premature telomere shortening and are responsible for different forms of dyskeratosis congenital. TERC and TERT mutations were also found in patients with aplastic anemia. The aim of this work is to analyze the possible involvement of the telomerase complex gene NOLA1, in a population of Italian AA patients. Procedure. DNA of 108 AA patients and 170 normal controls was amplified by PCR and analyzed by DHPLC. For each abnormal elution profile PCR products was directly sequenced using ABI prism 3100 Genetic Analyzer. Results. We identified, in two patients and two control, the new c.390A > T variation, which is not reported in GenBank, and leads to p.H28L amino acidic change. Telomere analysis shows that the subjects carrying the change have a telomere length comparable to that of healthy controls thus suggesting that this variation has no effect on telomerase complex activity. Conclusions. We did not find any clear disruptive mutation in NOLA1 gene. The non-conservative variation identified in Our sample has no effect on telomeres length. This result Suggests that heterozygous point Mutations in NOLA1 gene are not responsible for AA in Our patients at least acting via telomere. However, in our experience, molecular analysis of other telomerase complex gene (TERC, TERT) is important for AA patients and family members in order to set up an adequate therapeutic or surveillance program and identify carriers or exclude them as potential bone marrow donors. Pediatr Blood Cancer 2009;52:376-378. (C) 2008 Wiley-Liss, Inc.

Bullous pemphigoid (BP) is an autoimmune skin disease that occurs mainly in elderly patients; onset of BP is rare in childhood. Inflammatory bowel diseases (IBD), by contrast, have a pediatric onset in 25% of presenting cases, requiring expert multidisciplinary management. Here we report a pediatric case of IBD (involving stomach, duodenum, ileum, and colon-rectum) associated with a disseminated form of drug-resistant BP successfully treated by plasma exchange (PEX), extracorporeal photochemotherapy (ECP), and corticosteroid therapy. The addition of PEX and ECP to standard treatment induced no severe side effects, prompted a rapidly achieved complete and long-term remission, and allowed dose tapering of the immunosuppressive drugs over an 18-month follow-up.

BACKGROUND: Recent clinical trials have demonstrated the efficacy of adoptive cellular therapy with virus-specific lymphocytes in patients with defective cellular immune responses. Immunoreconstitution has become a challenge for cellular immunology and for transfusion medicine. In fact, both expertises are required to provide effective and safe cellular products. Because of in vitro manipulation, T-lymphocyte cultures are at risk of contamination even under good manufacturing procedure (GMP) conditions.STUDY DESIGN AND METHODS: To further improve the quality of these GMP cellular products, a procedure was designed for purification, stimulation, and expansion of antigen-specific CD4 and CD8 T-lymphocytes in a sealed, unbreached system. Leukopacks from the blood bank that fulfill the requirements of a GMP product were the starting material. Gradient separation and washing were performed in bags with sterile connecting devices on the bench-top, as well as addition of ingredients (antigen, interleukin-2) or transfer to larger bags.RESULTS: The method is described in detail, and it is shown that increase in number of cytomegalovirus-specific CD4 or CD8 T-lymphocytes was similar to procedures based on open culture systems. Cell expansion after 4 weeks ranged from 800- to 2400-fold for CD4 lymphocytes and 300- to 900-fold for CD8 lymphocytes. Antigen specificity and loss of alloreactivity were demonstrated on the expanded cells with proliferation, intracytoplasmic interferon gamma-gamma staining, cytolytic activity, and pentamer binding.CONCLUSION: This procedure can be applied to improve sterility under GMP conditions when T-cell lines are generated for adoptive immunotherapy and may increase biosafety for the staff when cell lines are generated from subjects infected with dangerous pathogens.