Selective expansion of human natural killer cells leads to enhanced alloreactivity

In allogeneic stem cell transplantation （SCT）, natural killer （NK） cells lacking their cognate inhibitory ligand can induce graft-versus-leukemia responses, without the induction of severe graft-versus-host disease （GVHD）. This feature can be exploited for cellular immunotherapy. In this study, we examined selective expansion of NK cell subsets expressing distinct killer immunoglobulin-like receptors （KIRs） within the whole human peripheral blood NK cell population, in the presence of HLA-Cw3 （C1） or Cw4 （C2） transfected K562 stimulator cells. Coculture of KIR＋ NK cells with C1 or C2 positive K562 cells, in the presence of IL-2＋ IL-15, triggered the outgrowth of NK cells that missed their cognate ligand. This resulted in an increased frequency of alloreactive KIR＋ NK cells within the whole NK cell population. Also, after preculture with K562 cells lacking their cognate ligand, we observed that this alloreactive NK population revealed higher numbers of CD107~ cells when cocultured with the relevant K562 HLA-C transfected target cells, as compared to coculture with untransfected K562 cells. This enhanced reactivity was confirmed using primary leukemic cells as target. This study demonstrates that HLA class I expression can mediate the skewing of the NK cell repertoire and enrich the population for cells with enhanced alloreactivity towards leukemic target cells. This feature may support future clinical applications of NK cell-based immunotherapy.

Peptide-specific,allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from pep-tide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434 pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85)was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±0.01 %, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.