Littérature scientifique sur le sujet « Allospecificity »

Abstract Hemopoietic allografts of normal and neoplastic origin are subject to NK cell-mediated resistance in mice. Susceptibility to this resistance is controlled by MHC-linked genes in a recessive manner. Several distinct specificities could be postulated to explain the strain-dependent pattern of resistance. These presumptive specificities for recognition are H-2 haplotype dependent, but the correspondence is not one-to-one. For example, resistance of H-2d or H-2b/d host to H-2 b graft operationally defines specificity-1, establishing its link with haplotype H-2b. To examine the molecular basis of specificity-1, spontaneous Dd-loss mutant clones were isolated from H-2b/d and H-2d hemopoietic cell lines, i.e., 416B of (C57BL/6 x DBA/2)F1 (B6D2F1) origin and L1210 of DBA/2 origin, both of which lack specificity-1. The expression of specificity-1 in the mutant clones was examined in vivo and in vitro. The results indicate that Dd-loss clones of 416B and L1210 lines express specificity-1. These data suggest that murine NK cell allospecificity-1 is defined primarily by the absence of the Dd molecule or other class I molecules sharing the protective motifs; no H-2b-associated genes play a relevant role. This conclusion is consistent with the missing self hypothesis of NK cell reactivity, and is in agreement with the observation that lysis of B6 targets by B6D2F1 NK cells is mediated mostly by cells that express Ly-49A and/or Ly-49G2.

Abstract The class II molecules of DR4, DR7, and DRw9 haplotypes were analyzed by immunoprecipitation, followed by two-dimensional gel electrophoresis and N-terminal amino acid sequencing. By using HLA-DR chain-specific monoclonal antibodies, two distinct DR beta-chains were identified. One beta-chain, designated DR beta 2, had a characteristic acidic mobility. In all three DR types the DR beta 2-chains were indistinguishable by two-dimensional gel electrophoresis and partial N-terminal sequencing. A second DR beta-chain designated beta 1 had a more basic mobility on two-dimensional gel electrophoresis, and differed from the DR beta 2-chains by the consistent presence of phenylalanine at position 18. In contrast to the DR beta 2-chains, the DR beta 1-chains were clearly polymorphic, with specific amino acid sequence differences characteristic of each DR type. The monoclonal antibodies 109d6 and 17-3-3S, recognizing distinct polymorphic epitopes similarly correlated with the DRw53 allospecificity, were found to react with different DR beta-chains. The epitope recognized by monoclonal antibody 109d6 was identified on the DR beta 2-chain in the prototypic DR4, DR7, and DRw9 cell lines. However, the DR7, Dw11, DQw3 cell line BEI was unreactive with antibody 109d6 by either immunofluorescence or immunoprecipitation despite the presence of the DRw53 allodeterminant on this cell line. The other DRw53-like monoclonal antibody, 17-3-3S, reacted with the DR beta 1-rather than the DR beta 2-chain in all DR4 and DR7 cell lines tested, including the cell line BEI. However, antibody 17-3-3S did not react with the DRw53-positive DRw9 cell line ISK. These studies suggest that the DRw53 allospecificity is more complex than previously thought and may comprise a number of distinct epitopes encoded by two different DR beta loci.

Le Tr1 sono un subset di cellule regolatorie CD4+ indotte in periferia e caratterizzate dalla produzione di IL-10. Nell’ultimo decennio molte risorse sono state impiegate nella ricerca di metodi efficaci per la produzione di cellule Tr1 in vitro. Il nostro gruppo ha dimostrato che Tr1 antigene-specifiche possono essere ottenute in vitro mediante l’utilizzo di IL-10 o di cellule dendritiche tolerogeniche (DC-10). Trials clinici nell’ambito di trapianti allogenici di cellule staminali ematopoietiche (allo-HSCT) hanno dimostrato il basso rischio di terapie cellulari basate sull’uso di Tr1. Il limite dei metodi sopra citati è la presenza di una frazione di non Tr1 che potrebbe limitarne l'efficacia. Per cui abbiamo sviluppato un protocollo per generare Tr1 (CD4IL-10) utilizzando un vettore lentivirale (LV) che codifica per IL-10 e . Abbiamo dimostrato che forzando l’espressione di IL-10 in CD4+ umane si ottengono cellule con fenotipo e funzioni sovrapponibili a quelle delle Tr1, inclusa la capacità di eliminare cellule mieloidi. Inoltre, il trasferimento di cellule CD4IL-10 in topi immuno-deficienti sopprime la reazione di xeno-GvHD (Andolfi e Fousteri, Mol Ther 2012). Tuttavia, non è ancora chiaro se la terapia cellulare con CD4IL-10 può influenzare l’attività anti-leucemica (GvL). Gli obiettivi del progetto di dottorato sono: 1. Definire se la lisi delle CD4IL-10 è sovrapponibile a quello di Tr1 e convalidare l'uso di CD4IL-10 policlonali come terapia cellulare in modelli pre-clinici di GvL e GvHD; 2. Sviluppare un protocollo in vitro per generare cellule allo-specifiche Tr1-like lentivirale LV-IL-10. Per raggiungere il primo obiettivo, l'attività citotossica di cellule policlonali CD4IL-10 è stata testata contro un pannello di blasti primari. Studi in vitro dimostrano che l'attività litica di cellule CD4IL-10 è dipendente dall’attivazione mediata da molecole di HLA di classe I e dal rilascio di granzimaB; inoltre, è specifica nei confronti di cellule CD13+, e richiede l'adesione mediata dal CD54 e l’espressione di CD112 sui blasti. Inoltre, studi in vivo dimostrano che il trasferimento di cellule CD4IL-10 in modelli umanizzati impedisce la reazione di xeno-GvHD mediata da cellule T allogeniche, senza inibirne la loro attività antileucemica (GvL). Inoltre, le CD4IL-10 hanno un’attività anti-leucemica anche in vivo. Per raggiungere il secondo obiettivo, CD4+ sono state messe in coltura con mDC. Durante la seconda stimolazione, le cellule T sono state trasdotte con LV-IL-10. Le CD4IL-10 sono state isolate, espanse, e caratterizzate. Dopo stimolazione allo-specifica, le CD4IL-10 secernono livelli più elevati di IL-10 e comparabili di IFN-γ rispetto a cellule di controllo; inoltre, mostrano un fenotipo anergico e soppressivo. Nel complesso, i risultati di questi studi forniscono un razionale per l'uso di CD4IL-10 per inibire la reazione di GvHD, preservando la GvL a seguito di allo-HSCT per curare tumori mieloidi e rappresentano il primo passo per lo sviluppo di cellule Tr1 allo-specifiche e incrementerà l'utilizzo delle Tr1 come terapia per indurre tolleranza verso antigeni selezionati, riducendo la soppressione immunitaria generale.
T regulatory type 1 (Tr1) cells are a subset of CD4+ regulatory T (Treg) cells induced in the periphery and characterized by IL-10 production. During the last decade much effort has been dedicated to establish suitable methods for Tr1 cell generation in vitro for Treg-cell based therapy. We demonstrated that Tr1 cells can be generated in vitro in an antigen-specific manner with recombinant IL-10 or IL-10-producing tolerogenic DC-10. Proof-of-principle clinical trials in allo-HSCT demonstrated the safety of Treg-based cell therapy with these Tr1 cells. However, Tr1 cell cultures generated with the above mentioned methods include a fraction of non-Tr1 cells that may limit the efficacy of immunotherapy with Tr1 cells. To overcome this limitation we developed a protocol to generate Tr1 (CD4IL-10) cells using a Lentiviral Vector (LV) encoding for human IL-10 and , as marker gene. We showed that enforced IL-10 expression confers Tr1 phenotype and functions to human CD4+ T cells, including killing of myeloid cells. Moreover, adoptive transfer of CD4IL-10 cells into immune-deficient mice suppresses xeno-GvHD (Andolfi G. and Fousteri G., Mol Ther 2012). However, it is still unclear whether adoptive therapy with CD4IL-10 cells can affect Graft versus Leukemia (GvL) activity. The aims of my PhD project are: 1. to define whether killing mediated by CD4IL-10 cells is super-imposable to that of classical Tr1 cells and to validate the use of polyclonal CD4IL-10 cells as cell therapy in humanized pre-clinical models of GvL and GvHD; 2. to develop a new in vitro protocol to generate an homogeneous population of allo-antigen specific IL-10-producing Tr1 cells by LV-IL-10 gene transfer. To achieve the first aim the cytotoxic activity of polyclonal CD4IL-10 cells has been tested against a panel of primary blasts. In vitro studies show that the cytolysis of CD4IL-10 cells is HLA-class I- and granzyme B-dependent, is specific for CD13+ cells, and requires CD54-mediated adhesion and CD112 expression on target primary leukemic blasts. Moreover, in vivo studies show that adoptive transfer of CD4IL-10 cells in humanized models prevents xeno-GvHD mediated by human allogeneic T cells, while sparing their GvL capacity. In addition, we prove that CD4IL-10 T cells have potent anti-leukemia effects also in vivo. To achieve the second aim human naive CD4+ T cells were co-cultured with allogeneic in vitro differentiated mature DC. During second stimulation T cells are transduced with LV-IL-10, and CD4IL-10 cells are selected, expanded, and functionally characterized. Upon allo-antigen specific stimulation, CD4IL-10 cells secrete significantly higher levels of IL-10 and comparable amounts of IFN- compared to control cells, and display an anergic and suppressive phenotype. Overall, results from these studies provide a strong rationale for the use of CD4IL-10 cells to prevent GvHD while preserving GvL in allo-HSCT to cure myeloid malignancies and represent the first step for the development of allo-antigen specific Tr1 cells and will contribute to increase the use of Tr1-based immunotherapy, inducing tolerance to selected antigens, while minimizing general immune suppression.