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. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle-dependent cytotoxic therapy. Global transcriptional profiling identified LS cell-specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS 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. To clarify the presentation of these epitopes in HIV-1-infected T cells, CTL recognition for the clade E-specific and cross-clade epitopes was investigated using CD4CXCR4 cells infected with an HIV-1 clade E clone. RESULTS: Three epitopes, which are identical among clades A-E, were recognized as cross-clade CTL epitopes in both individuals. Clade B and E sequences corresponding to three epitopes were recognized as clade-specific epitopes in clade B-infected and clade E-infected individuals, respectively. In contrast, clade E-specific peptides corresponding to two other clade B epitopes failed to elicit clade E-specific CTL. CTL specific for the three cross-clade and three clade E-specific epitopes effectively lysed target cells infected with HIV-1 clade E virus. CONCLUSIONS: These six epitopes are found to be processed naturally in HIV-1 clade E-infected cells. We show here that a strategy utilizing HIV-1 clade B epitopes is very useful for identifying clade E CTL epitopes.

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. Ninety-nine 8-mer to 11-mer peptides corresponding to the HLA-A*3303-binding peptide motif were selected from the HIV-1 SF2 sequence. Fifty-two of these 99 peptides bound to HLA-A*3303. Six of these binding peptides induced peptide-specific CTLs in PBMCs from at least one of two HIV-1-seropositive individuals. CTL clones specific for three Pol peptides and one Gag peptide killed HLA-A*3303-restricted target cells infected with HIV-1 recombinant vaccinia, indicating that these peptides were naturally processed HLA-A*3303-restricted CTL epitopes. SF2-Pol 594-602 (FYVDGAANR) and SF2-Gag 144-152 (MVHQAISPR) induced specific CTLs in 5 and 4 of 10 chronically HIV-1-infected individuals, respectively, whereas SF2-Pol 60-70 (TLWQRPLVTIR) and SF2-Pol 934-943 (KIQNFRVYYR) induced specific CTLs in 2 and 1 of 10 chronically HIV-1-infected individuals, respectively. Thus, the former are immunodominant epitopes whereas the latter are not. These epitopes are useful for studies of AIDS immunopathogenesis and vaccine development.

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. In contrast, those specific for the other four epitopes failed to kill HIV-1-infected CD4+ T cells and partially or hardly suppressed HIV-1 replication. The difference of the ability between these two types of CTLs may result from the difference of the number of HLA class I epitope complex on the surface of NL-432-infected CD4+ T cells.

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. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.

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 Vbeta5.6 were responsible for the peptide-specific response in the context of both HLA-B*3501 and HLA-B*5101, confirming single TCR-mediated dual specificity. The second TCR-alpha chain was not somehow expressed on the cell surface. Remarkably, the Valpha12.1/Vbeta5.6 TCR also recognized the same peptide presented by allogeneic HLA class I molecules that share the similar peptide-binding motifs, such as HLA-B*5301 and HLA-B*0702. The sensitivity of peptide recognition by the Valpha12/Vbeta5.6 TCR appeared to be comparable when the peptide was presented by syngeneic and allogeneic HLA class I molecules, with changes in T cell responsiveness caused largely by peptide-binding capacity. Moreover, the CTL clone bearing Valpha12.1/Vbeta5.6 TCR showed substantial cytolytic activity against the peptide-loaded cells expressing HLA-B*3501, HLA-B*5101, HLA-B*5301, or HLA-B*0702, providing further evidence that a single TCR complex can recognize the same peptide presented by a broad range of HLA class I molecules. A TCR with fine specificity for an HIV Ag but broad specificity to multiple HLA molecules may provide an advantage to the generation of allorestricted, peptide-specific T cells, and thus could be a potent candidate for immunotherapy against HIV infection.

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. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.

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. Interestingly, CTL clones stimulated with NL-432-infected CD4(+) T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4(+) T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4(+) T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8(+) T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8(+) T cells are able to partially but not completely control HIV-1 replication in vivo.

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. An inverse correlation between perforin and CD27 expression was found in all four CD28CD45RA subsets. Therefore, to achieve a phenotypic classification of CD8(+) T cells that more precisely reflects their function, the CD27(+) subset was divided into CD27(low) and CD27(high) subsets based on the expression level of CD27. Functional and flow cytometric analyses of CD27CD28CD45RA subsets showed that this phenotypic classification reflects functional classification of CD8(+) T cells. HCMV-specific CD8(+) T cells from healthy HCMV-seropositive individuals were predominantly found in effector and memory/effector subsets, indicating that HCMV-specific effector CD8(+) T cells are actively induced by HCMV replication in healthy HCMV carriers. Phenotypic analyses of CD8(+) T cells using this classification will enable the characterization of antigen-specific CD8(+) T 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. However, the high affinity subset did not show cytolytic activity, cytokine production, or proliferation activity toward HIV-infected cells, whereas the moderate affinity one showed potent activities. Furthermore, using ectopic expression of each of the TCR genes into primary human CD8 T cells, the CD8 T cells transduced with the high affinity TCR showed greater binding activity toward the tetramer and impaired cytotoxic activity toward HIV-infected cells, corroborating the results obtained with parental CD8 T cells. Taken together, these data indicate that impaired responsiveness of T cells toward HIV-infected cells can occur at the level of TCR-ligand interactions, providing us further insight into the immune evasion mechanisms by HIV.

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. There were no signs of allogeneic responses in these cells, although hybrid TCR dimers consisting of transduced TCR and endogenous TCR were suspected to have been formed in these cells, as the effect of transgene expression on the surface expression of the desired TCR was limited. Moreover, the TCR-transduced cells showed potent inhibitory activity against HIV-1 replication in vitro, although the differential surface expression of the desired TCR resulted in differential functional avidity of individual TCR-transduced cells toward the peptide-pulsed target cells. These data suggest that the reconstitution of HIV-specific immunoreactive T cells engineered by genetic transfer of HIV-specific TCR is a potential alternative to immunotherapeutic applications against HIV infections.

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. The results of a chemotaxis assay showed that the migration of CXCR1(+)CD8(+) T cells was induced by IL-8. These results suggest that the IL-8-CXCR1 pathway plays an important role in the homing of effector CD8(+) T 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. 1JalphaCalpha transcript was found in the clone. The clone showed cytotoxic activity and IFN-gamma production towards cells expressing HLA-B*3501 pulsed with an HIV Pol-derived epitope peptide (IPLTEEAEL). By flow-cytometric analysis ex vivo using an HLA-B*3501 tetramer, a fraction of Vdelta1(+)CD8(+)tetramer(+) cells was found in peripheral lymphocytes of an HIV-infected patient, indicating the existence of HLA-restricted and HIV-specific Vdelta1(+) CD8 T cells in vivo. Moreover, retrovirus-mediated transfer of the TCR-encoding genes into TCR-negative hybridoma cells showed that the transduced cells were stained by the tetramer and were activated in response to the Pol peptide, further confirming the ligand specificity of the TCR. Together, these results clearly demonstrate that Vdelta1(+) alpha beta TCR are restricted to engaging peptide antigens in the context of classical MHC class I molecules, highlighting the difference in the ligand specificity between Vdelta1(+) alpha beta TCR and Vdelta1(+) gamma delta TCR.

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. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+-->CCR5+CCR7+-->CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.

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. Thus, their escape is likely due to the changes that occur during the processing and presentation of epitopes in the infected cells. Mutations responsible for this mode of escape were located within the epitope regions rather than the flanking regions, and such mutations did not influence the virus replication. The results suggest that the impaired antigen processing and presentation often occur in HIV-1 field isolates and thus are one of the major mechanisms that enable HIV-1 to escape from CTL recognition. We emphasize the importance of testing HIV-1 variants in an endogenous expression system.

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. HBV-specific CD8+ T cells were not detected in PBMCs from individuals with chronic HBV infection except for one patient during acute exacerbation. HBV-specific CD8+ T cells were induced by in vitro peptide stimulation in PBMCs from chronic HBV carriers with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. CD28CD45RA phenotype analysis showed that HBV-specific CD8+ T cells in acute phase PBMCs predominantly express a memory T cell phenotype. CONCLUSIONS: HBV-specific memory CD8+ T cells may play a crucial role in complete clearance of HBV from patients with acute HBV hepatitis.