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Neuromyelitis optica is an immune‐mediated disease characterized by a relapsing course, resulting in progressive disability. In children, given the long life expectancy, a disease‐modifying treatment could be particularly desirable. Unfortunately, the currently available treatment strategies with this potential are scarce. Very limited data are available about the use of allogeneic hematopoietic stem cell transplantation (HSCT) for autoimmune neurological diseases. In this report, we present a pediatric case successfully treated with allogeneic HSCT from an HLA‐haploidentical donor, after ex vivo TCR/CD19‐depletion of the graft. To the best of our knowledge, this is the first case of a pediatric patient to benefit from such a treatment.

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.

We report on the successful infusion of human adenovirus (HAdV)-specific T cells in a child with congenital amegakaryocytic thrombocytopenia, given T-cell-depleted hematopoietic stem cell transplantation (HSCT) from the HLA-haploidentical mother during extracorporeal membrane oxygenation (ECMO) for severe HAdV-related respiratory failure. Donor-derived, interferon (IFN)-γ-secreting HAdV-specific T cells were enriched using the cytokine capture assay, after in vitro stimulation with overlapping peptides from the immunodominant HAdV5 hexon protein. Two weeks after T-cell transfer, viral load decreased and ECMO was discontinued. T-cell responses to HAdV antigens were documented after four weeks and were associated with viral clearance, immune reconstitution and clinical amelioration. Pediatr Blood Cancer 2014;61:376–379. © 2013 Wiley Periodicals, Inc.

T helper (Th) cells and cytolytic T lymphocytes (CTL) play defined roles in the cellular immune response. This distinction wavered when Th lymphocytes were shown to kill antigen-presenting cells displaying the relevant antigen. Here we demonstrate that also the opposite can be true: CTL can exert helper functions. We noticed that certain CMV-specific CTL lines grew after antigen activation also without exogenous IL-2. These lines produced their own IL-2, which supported the expansion of other CTL and Th cell lines. High levels of helper cytokines like IL-4, IL-5 and IL-6 were detected in the culture supernatants. Thus, we set up a helper assay to study the functional interactions between T cells (or their supernatants) and B cells. Conditioned media from helper CTL lines induced secretion of antigen-specific antibodies by B cells pulsed with antigen as first signal. We conclude that it is possible to isolate CTL lines that exhibit helper functions for T cells and B cells. If this possibility is proven also in vivo, we should revise some of our views on the pathogenesis of diseases in which CD8 cells are key players, such as in viral infections, graft rejection and GVHD.

Measuring antigen-specific immune responses (MASIR) is essential for basic immunological research and in the clinical setting. Numerous techniques have been used and the recent years have witnessed a flourishing of flow cytometry based methods for the identification of antigen specific T cells, in addition to other methodologies. The second MASIR conference held in Santorini, Greece, from 14 to 18 June 2006 has been a forum for the discussion of methodological issues and for research or clinical applications of these techniques, as reviewed here. In addition to flow cytometry based techniques, other emerging techniques with different degrees of complexity can be applied. These novel methods are highly promising in numerous conditions to look for correlates of protection, to test responses to natural infections or to vaccination trials, to evaluate the immune status of immunocompromised patients and to monitor persistence and function of specific T cells administered as adoptive therapy.

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-γ 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.

Department of Molecular Biology, University of Siena, Siena, ItalyAntagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to pre-vent CD4 T cell activation by the original peptide. Here we show that antagonistic activitycan be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptasep66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engi-neering, rather than by introducing substitutions by synthesis. The glutathione S-transferase(GST)-peptide system has been used to produce molecules that display the peptide at theappropriate end of the GST carrier. When the gp120 peptide 191–205 (pep24) wasexpressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells wasmaintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar resultswere obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonismwas (1) specific; proliferation of a CD4 T cell line from the same donor responding to theenvelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; prolifer-ative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed withthe antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APCpulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysiswhile the antagonistic activity was preserved. Thus a minimal sequence that confers antago-nistic activity can be engineered or synthesized with peptides to antagonize undesired CD4responses as an alternative to the use of APL.

+T cell line in the context of MHC class II molecules. No significant differences were observed when the attenuated strains were trapped in the phagolysosome or impaired in the capacity to spread intracellularly or from cell to cell. Similar results were obtained using carrier proteins that were either secreted, associated with the bacterial surface, or restricted to the bacterial cytoplasm. A dominant expression of the TCR V 22 gene subfamily was observed in specific T cell lines generated after stimulation with the recombinant strains or with soluble gp120. Our data show that in this in vitro system L. monocytogenes can efficiently deliver antigens to the MHC class II pathway, in addition to the well-established MHC class I pathway. The eukaryotic cell compartment in which the antigen is synthesized, and the mode of display seem to play a minor role in the overall efficiency of epitope processing and presentation.