Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2r gamma(null) mice carrying a complete null mutation of the cytokine gamma c upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo.

Patterns of cytokine production in human immunodeficiency virus type 1 (HIV-1)-specific human CD8+ T cells after stimulation with HIV-1-infected CD4+ T cells.

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production.

Cutting Edge: Epitope-dependent effect of Nef-mediated HLA class I down-regulation on ability of HIV-1-specific CTLs to suppress HIV-1 replication.

It is believed that Nef-mediated HLA class I down-regulation is one of the mechanisms that allow HIV-1-infected cells to escape from being killed by HIV-1-specific human CTLs. In this study, we show that the effect of Nef-mediated HLA class I down-regulation on the ability of HIV-1-specific CTLs to suppress HIV-1 replication is epitope dependent. The CTLs specific for two Pol epitopes presented by HLA-B*5101, one of the HLA alleles associated with slow progression to AIDS, effectively killed HIV-1-infected CD4+ T cells and suppressed HIV-1 replication.

Reconstitution of anti-HIV effector functions of primary human CD8 T lymphocytes by transfer of HIV-specific alphabeta TCR genes.

We redirected the antigen specificity of primary human CD8 T cells by retrovirus-mediated transduction of genes encoding alphabeta TCR specific to HIV-1 Pol protein. A large polyclonal population of TCR-transduced CD8 T cells showed substantial cytotoxic and cytokine production activities toward target cells either pulsed with the peptide or infected with HIV-1, and their functional activities were comparable to those of the parental CTL clone. Peptide fine-specificity and promiscuous recognition of HLA class I supertypes of the parental CTL clone were also preserved in the TCR-transduced cells.

Functionally impaired HIV-specific CD8 T cells show high affinity TCR-ligand interactions.

We eventually isolated two different clonotypic CD8 T cell subsets recognizing an HIV Pol-derived epitope peptide (IPLTEEAEL) in association with HLA-B35 from a chronic HIV-infected patient. By kinetic analysis experiments, the subsets showed a >3-fold difference in half-lives for the HLA tetramer in complex with the Pol peptide. In functional assays in vitro and ex vivo, both subsets showed substantial functional avidity toward peptide-loaded cells.

Cutting edge: expression of chemokine receptor CXCR1 on human effector CD8+ T cells.

IL-8 is a potent inflammatory cytokine that induces chemotaxis of neutrophils expressing CXCR1 and CXCR2, thus indicating its involvement in the migration of these cells to inflammatory sites where bacteria proliferate. Presently, we showed that CXCR1(+) cells were predominantly found among CD8(+) T cells having effector phenotype, and that the expression of CXCR1 was positively correlated with that of perforin, suggesting that CXCR1 is expressed on effector CD8(+) T cells. Indeed, human CMV-specific CD8(+) T cells from healthy individuals, which mostly express the effector phenotype and have cytolytic function, expressed CXCR1, whereas EBV-specific CD8(+) T cells, which mostly express the memory phenotype and have no cytolytic function, did not express this receptor.

Phenotypic classification of human CD8+ T cells reflecting their function: inverse correlation between quantitative expression of CD27 and cytotoxic effector function.

Phenotypic classification of human CD8(+) T cells using three cell surface markers, CD27, CD28 and CD45RA, was recently suggested to be useful for identification of naive, memory and effector CD8(+) T cells. However, it still remains unclear whether such classification precisely reflects functional classification of CD8(+) T cells. To clarify this, we characterized each CD27CD28CD45RA subset of total and human cytomegalovirus (HCMV)-specific CD8(+) T cells by analyzing the expression of perforin and two chemokine receptors, CCR5 and CCR7, as well as their function.

Impaired processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape mechanisms from CTL immune pressure in human immunodeficiency virus type 1 infection.

Investigating escape mechanisms of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTLs) is essential for understanding the pathogenesis of HIV-1 infection and developing effective vaccines. To study the processing and presentation of known CTL epitopes, we prepared Epstein-Barr virus-transformed B cells that endogenously express the gag gene of six field isolates by adopting an env/nef-deletion HIV-1 vector pseudotyped with vesicular stomatitis virus G protein and then tested them for the recognition by Gag epitope-specific CTL lines or clones. We observed that two field variants, SLFNTVAVL and SVYNTVATL, of an A*0201-restricted Gag CTL epitope SLYNTVATL, and three field variants, KYRLKHLVW, QYRLKHIVW, and RYRLKHLVW, of an A24-restricted Gag CTL epitope KYKLKHIVW escaped from being killed by the CTL lines, despite the fact that they were recognized when the synthetic peptides corresponding to these variant sequences were exogenously loaded onto the target cells.

HLA class I-restricted recognition of an HIV-derived epitope peptide by a human T cell receptor alpha chain having a Vdelta1 variable segment.

A subset of human peripheral alpha beta T cells have been shown to express TCR alpha chains containing Vdelta1 segments, although what antigens the Vdelta1(+) alpha beta T cells recognize via these TCR is not known yet. We eventually established a human CD8 T cell clone that expressed alpha beta TCR and Vdelta1 antigens. Corroboratively, a unique in-frame Vdelta1.

Identification and characterization of HLA-A*3303-restricted, HIV type 1 Pol- and Gag-derived cytotoxic T cell epitopes.

HLA-A*3303 is one of the common HLA alleles in East and Southeast Asia. Identification of HLA-A*3303-restricted HIV-1 cytotoxic T lymphocyte (CTL) epitopes is therefore required to investigate the immunopathogenesis of AIDS and vaccine development in these areas, where AIDS is rapidly expanding. We attempted to identify HLA-A*3303-restricted CTL epitopes derived from relatively conserved proteins Pol, Gag, and Nef of HIV-1 clade B, using reverse immunogenetics.

Single T cell receptor-mediated recognition of an identical HIV-derived peptide presented by multiple HLA class I molecules.

A dual specific human CTL clone harboring one beta and two inframe alpha transcripts of TCR was previously reported to recognize an HIV Pol-derived nonapeptide (IPLTEEAEL) endogenously presented by both syngeneic HLA-B*3501 and HLA-B*5101. In the current study, a retrovirus-mediated TCR transfer of individual alpha- and beta-chains to TCR-negative hybridoma showed that Valpha12.1 TCR in complex with Vbeta5.

Different effects of Nef-mediated HLA class I down-regulation on human immunodeficiency virus type 1-specific CD8(+) T-cell cytolytic activity and cytokine production.

A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4(+) T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4(+) T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4(+) T cells infected with NL-M20A.

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype.

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells.

Cytotoxic T-cell recognition of HIV-1 cross-clade and clade-specific epitopes in HIV-1-infected Thai and Japanese patients.

Objective: To identify and characterize cytotoxic T-cell (CTL) epitopes for HIV-1 clade E using eight known HLA-A*1101-restricted HIV-1 clade B epitopes.

Methods: Induction of clade E-specific CTL was examined by stimulating peripheral blood mononuclear cells (PBMC) from clade E-infected Thai individuals with the clade E-specific peptide corresponding to the clade B epitopes. Cross-clade and clade-specific CTL recognition for these epitopes was analysed using CTL clones and bulk CTL specific for these epitopes.

Functional expression of the chemokine receptor CCR5 on virus epitope-specific memory and effector CD8+ T cells.

Because the chemokine receptor CCR5 is expressed on Th1 CD4(+) cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8(+) T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells.

The role of hepatitis B virus-specific memory CD8 T cells in the control of viral replication.

Background/aims: The aim of this study is to clarify the differences of host immune responses between acute self-limited and chronic persistent hepatitis B virus (HBV) infections by quantitative and qualitative analysis of HLA-A*2402-restricted HBV-specific CD8+ T cells.

Methods: HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from patients infected with HBV were analyzed by flow cytometry using two HLA-A*2402-HBV peptide tetrameric complexes.

Results: High numbers of HBV-specific CD8+ T cells were detected in acute phase PBMCs from most individuals with acute HBV infection while the number of these cells was greatly reduced in recovery phase PBMCs.