Key Points Removal of αβ+ T and CD19+ B cells is an effective strategy for successful HLA-haploidentical hematopoietic stem cell transplantation. The high probability of disease-free survival renders this transplant option attractive for any child with a nonmalignant disorder.

Key Points• Children with AL given haplo-HSCT after ab T-and B-cell depletion are exposed to a low risk of acute and chronic GVHD and NRM.• The leukemia-free, GVHDfree survival of patients given this type of allograft is comparable to that of HLAmatched donor HSCT recipients.Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children with acute leukemia (AL) either relapsed or at high-risk of treatment failure. We developed a novel method of graft manipulation based on negative depletion of ab T and B cells and conducted a prospective trial evaluating the outcome of children with AL transplanted with this approach. Eighty AL children, transplanted between September 2011 and September 2014, were enrolled in the trial. All children were given a fully myeloablative preparative regimen. Anti-T-lymphocyte globulin from day 25 to 23 was used for preventing graft rejection and graft-versus-host disease (GVHD); no patient received any posttransplantation GVHD prophylaxis. Two children experienced primary graft failure. The cumulative incidence of skin-only, grade 1-2 acute GVHD was 30%; no patient developed extensive chronic GVHD. Four patients died, the cumulative incidence of nonrelapse mortality being 5%, whereas 19 relapsed, resulting in a 24% cumulative incidence of relapse. With a median follow-up of 46 months for surviving patients, the 5-year probability of chronic GVHD-free, relapsefree survival (GRFS) is 71%. Total body irradiation-containing preparative regimen was the only variable favorably influencing relapse incidence and GRFS. The outcomes of these 80 patients are comparable to those of 41 and 51 children given transplantation from an HLA-identical sibling or a 10/10 allelic-matched unrelated donor in the same period. These data indicate that haplo-HSCT after ab T-and B-cell depletion represents a competitive alternative for children with AL in need of urgent allograft. This trial was registered at www.clinicaltrials.gov as #NCT01810120. (Blood. 2017;130(5):677-685)

SummaryActivation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function ofantibodies complexed with the relevant antigen . Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC . Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation . Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes {precipitated by a second antibody or solubilized by complement) . Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism .

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK subsets. CAR.CD19-NK cells display higher anti-leukemic activity towards CD19 + cell lines and primary blasts obtained from patients with B-cell precursor ALL compared to unmodified NK cells. In vivo animal model data showed that the anti-leukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically-modified peripheral blood NK cells for effective 'off-the-shelf' immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

CD8 and CD4 lymphocytes control cytomegalovirus (CMV) infection in immunocompetent individuals, while patients with defective cellular immunity are prone to endogenous reactivation of latent CMV or, like seronegative subjects, prone to primary infection. Administration of CMV-specific CD8 lymphocytes was beneficial for immunocompromised hemopoietic stem cell (HSC) graft recipients. Since CD4 cells contribute to expansion of cytotoxic T lymphocytes (CTL), we defined new T(h) peptides on the immunodominant protein pp65 recognized by CD4 cells from HLA-typed subjects, in the perspective of complementing CTL administration with CMV-specific T(h) cells. Screening by ELISPOT on CD4 and CD8 subsets using overlapping peptides identified 10 novel CD4 peptides. To simplify procedures to generate T cell lines, we used a CD4 peptide library for T cell stimulation instead of ill-defined viral lysates, without the requirement of dendritic cells. This library stimulated CMV-specific CD4 cells. In fact, peptide-induced CD4 cells responded to pp65 and to the viral lysate. These cells were also devoid of alloreactivity after one stimulation cycle. Since Good Manufacturing Procedure-grade peptides can be synthesized, culture conditions are simplified and alloreactivity is rapidly lost, these procedures based on peptide stimulation can facilitate implementation of adoptive reconstitution of CD4 responses in immunocompromised patients also in the case when the HSC allodonor is available for generation of the T cell line.

BackgroundHLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αβ/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition.MethodsNinety healthy donors were evaluated. Single-dose MZ was given to 30 ‘poor mobilizers’ (PM) failing to attain ≥40 CD34+ HSCs/μL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient’s body weight.ResultsMZ significantly increased CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs.ConclusionsMZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0240-z) contains supplementary material, which is available to authorized users.

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Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.