ALBERT

Description: Donor natural killer (NK) cells after haploidentical hematopoietic stem-cell transplantation (HSCT) and infusion of haploidentical NK-cells have demonstrated a therapeutic effect. NK alloreactivity resulting from appropriate Killer cell Ig-like receptor (KIR)-ligand disparity in human-leukocyte-antigen (HLA)-haplotype mismatched HSCT has resulted in improved engraftment and decreased incidence of leukemia relapse. Yet, not all patient-donor pairs benefit for an allogeneic NK-cell effect. To identify NK-cell donors with a suitable KIR-ligand mismatch, we have developed a functional assay to measure NK-cell killing through KIR-ligand interactions. NK-cell lysis of target cells is blocked by inhibitory KIR that recognize classical HLA class I allotypes and HLA mismatches of an altered allelic repertoire, as in haploidentical HSCT, leading to KIR-ligand mismatch and alloreactive NK cell-mediated target killing (Figure 1A). A cytotoxicity assay was developed based on the NK-cell target HLAnull 721.221 cells, and a panel of targets with enforced expression of HLA genes recognized by KIR. After the killing assay was optimized for high throughput and sensitivity, we used the panel of targets to determine whether bulk populations of donor NK cells could be predicted to kill based on KIR and HLA typing. The results demonstrate patterns of target-cell lysis for the KIR repertoires corresponding, for some donors, with predicted donor-versus-recipient NK-cell alloreactivity (Figure 1B). A relative inhibition of HLA+ target-cell lysis of 〉30% was associated with binding of KIR to introduced HLA class I molecules. The benefit of this assay to transplant physicians is a tool to actually measure phenotype (lysis), rather than relying on predictive models based on genotype. This assay will be combined with typing data to help identify donors with NK-cell killing function for recipients of haploidentical HSCT and infusion of haploidentical NK cells. Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box). Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box).

Description: Human herpesvirus 8 (HHV-8; Kaposi sarcoma–associated herpesvirus)–specific cytotoxic T-lymphocyte (CTL) and interferon-γ (IFN-γ) responses to proteins produced during the lytic cycle of HHV-8 replication are mediated by HLA class I–restricted, CD8+ T cells. We have characterized the fine specificity of the CD8+ T-cell response to 25 peptides derived from 5 HHV-8 lytic cycle proteins based on a prediction model for HLA A*0201 binding motifs. One of the 25 HLA A*0201 peptides derived from the glycoprotein B (gB) homolog of Epstein-Barr virus (gB492-500; LMWYELSKI; single-letter amino acid codes) bound to HLA A*0201 and stimulated IFN-γ responses in CD8+ T cells from HHV-8+, HLA A*0201 persons, but not HHV-8–seronegative or non–HLA A*0201 persons. The peptide also induced IFN-γ and CTL reactivity to naturally processed gB protein. The peptide was a major immunogenic epitope of HHV-8 as indicated by induction of IFN-γ responses in peripheral blood mononuclear cells from 5 of 5 HHV-8 seropositive, HLA A*0201 persons when gB492-500 was presented by autologous dendritic cells. T-cell reactivity to gB492-500 was not related to detectable HHV-8 DNA in the blood. These data show that CD8+ T cells recognize an HLA A*0201–restricted epitope for HHV-8 lytic cycle protein gB, particularly when presented by dendritic cells. This epitope may be important in control of HHV-8 infection by CD8+ T cells.