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.

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.

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.

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.

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.

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 virustransformed 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 SVYNTV ATL, of an A‫-1020ء‬restricted 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.

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.

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.