Development of T-cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse.

Leukemia relapse remains the major cause of allogeneic hematopoietic stem cell transplantation (HCT) failure, and the prognosis for patients with post-HCT relapse is poor. There is compelling evidence that potent selective antileukemic effects can be delivered by donor T cells specific for particular minor histocompatibility (H) antigens. Thus, T-cell receptors (TCRs) isolated from minor H antigen-specific T cells represent an untapped resource for developing targeted T-cell immunotherapy to manage post-HCT leukemic relapse. Recognizing that several elements may be crucial to the efficacy and safety of engineered T-cell immunotherapy, we developed a therapeutic transgene with 4 components: (1) a TCR specific for the hematopoietic-restricted, leukemia-associated minor H antigen, HA-1; (2) a CD8 coreceptor to promote function of the class I-restricted TCR in CD4+ T cells; (3) an inducible caspase 9 safety switch to enable elimination of the HA-1 TCR T cells in case of toxicity; and (4) a CD34-CD20 epitope to facilitate selection of the engineered cell product and tracking of transferred HA-1 TCR T cells. The T-cell product includes HA-1 TCR CD4+ T cells to augment the persistence and function of the HA-1 TCR CD8+ T cells and includes only memory T cells; naive T cells are excluded to limit the potential for alloreactivity mediated by native TCR coexpressed by HA-1 TCR T cells. We describe the development of this unique immunotherapy and demonstrate functional responses to primary leukemia by CD4+ and CD8+ T cells transduced with a lentiviral vector incorporating the HA-1 TCR transgene construct.

Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish.

The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the human TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, IL-1R and IL-18R genes and one copy of the adaptor genes MyD88, MAL, TRIF and SARM. In contrast to data for the pufferfish Fugu rubripes, zebrafish has two genes that are highly similar to human TLR4. In addition, one fish-specific TLR group can be distinguished that is closely related to the Drosophila melanogaster Toll-9 gene. The sequence of cloned cDNAs for TLR4, TLR2 and MyD88 show the same intron-exon organisation as in the human counterparts. Expression analysis using reverse transcriptase-PCR (RT-PCR) shows that 17 of the predicted zebrafish TLR genes and all the genes encoding adaptor proteins are expressed in the adult stage. A subset of the TLR genes are expressed at higher levels in fish infected with the pathogen Mycobacterium marinum. The induced genes include the homologues of the human TLR1 and TLR2 genes, whose functions are associated with mycobacterial infections, underscoring the suitability of zebrafish as a model for analysis of the vertebrate innate immune system.