Possible role of natural killer cells in negative selection of mutant lymphocytes that fail to express the human leukocyte antigen-A2 allele

Abstract

Increased frequencies of cells carrying mutations at several loci have been found in the blood cells of atomic-bomb (A-bomb) survivors upon testing four or five decades after the bombing. Interestingly, though, we have been unable to demonstrate any radiation-associated increases in the frequencies of mutant blood cells in which human leukocyte antigen (HLA)-A expression has been disrupted; this is true both of preliminary tests on the T cells of a small subset of A-bomb survivors and of the much more extensive study reported here in which we screened a much larger group of survivors for HLA-A2 loss mutations in B cells and granulocytes as well as in T cells. In attempting to explain our inability to detect any increases in HLA-A2-negative cell numbers in HLA-A2 heterozygous individuals exposed to A-bomb irradiation, we decided to test the hypothesis that HLA-A mutant lymphocytes might well have been induced by radiation exposure in much the same way as every other type of mutant we encountered, but may subsequently have been eliminated by the strong negative selection associated with their almost inevitable exposure to autologous natural killer (NK) cells in the bloodstream of each of the individuals concerned. We now report that mutant B lymphocyte cell lines that have lost the ability to express the HLA-A2 antigen do indeed appear to be much more readily eliminated than their parental heterozygous counterparts during co-culture in vitro with autologous NK cells. We make this claim first because we have observed that adding autologous NK cells to in vitro cultures of HLA-A2 heterozygous B or T cell lines appeared to cause a dose-dependent decrease in the numbers of HLA-A2-negative mutants that could be detected over a period of 3 days, and second because when we used peripheral blood HLA-A2 heterozygous lymphocyte cultures from which most of the autologous NK cells had been removed we found that we were able to detect newly-arising HLA-A2 mutant T cells in substantial numbers. Taken together, these results strongly support the hypothesis that autologous NK cells are responsible for eliminating mutant lymphocytes that have lost the ability to express self-HLA class I molecules in vivo, and may well therefore explain why we have been unable to detect increased frequencies of HLA-A2 mutants in samples from any of the 164 A-bomb survivors whose HLA-A2 heterozygote status made their lymphocytes suitable for our tests.

Introduction

We have previously reported finding increased frequencies of cells bearing genetic mutations at various loci in the blood cells of atomic-bomb (A-bomb) survivors in samples taken as recently as four or five decades after the bombing [1], [2], [3], [4], [5], [6], [7]. However, the radiation-associated mutational changes that we were able to detect in our earlier studies involving a small subset of A-bomb survivors did not appear to include any in which T lymphocytes had lost the ability to express the (HLA)-A human leukocyte antigen [8]. This negative finding could be attributed to the relatively small size of our initial study cohort, but it could equally well be a reflection of the sequence of changes that T cells must normally undergo in responding to the selection that follows from antigen stimulation in vivo. In order to distinguish between these two possibilities, we expanded our original study population considerably and have now screened B cells and granulocytes as well as T cells for HLA-A2 loss mutations on the assumption that the former two cell types are much less likely than the average T cell to be subject to selection in vivo.

Despite the evidence for increased frequencies of somatic mutations in A-bomb survivors cited above, plots of the relationship between estimated exposure dose and mutation frequency tend to be shallow [4], [5], [6] and the variations in mutation frequencies between individuals tend to be quite large [2], [3], [7]. These results could perhaps be indicating that selection against mutant cells is not an unusual occurrence in vivo. One fairly obvious form of negative selection would prevent cells with mutations in housekeeping genes from continuing to proliferate in the absence of an essential gene product. Another could result from host T cells recognizing and subsequently eradicating cells that express mutant gene products in conjunction with self-HLA/peptide complexes. It may therefore be all but impossible to detect radiation-induced mutations of either of the above types in individuals whose relevant exposure to radiation occurred several weeks ago, let alone several decades ago [1].

Loss or downregulation of major histocompatibility complex (MHC)/peptide complexes has been observed in many malignancies and is believed to provide mutant and malignant cells with a way of evading host immune defense mechanisms [9]. It has also been suggested that natural killer (NK) cells may be able to recognize and kill cells that fail to express self-MHC class I molecules [10]. This NK-mediated elimination mechanism is believed to apply to cells with mutations affecting the cell surface as well as to malignant cells, and it appears that identification of MHC class I antigen-specific inhibitory receptors is an essential feature of NK cell recognition of its target cells [11], [12], [13], [14], [15], [16]. Although it has been claimed that human NK cells are able to kill allogeneic cell lines that lack HLA class I molecules expressed by the NK cell population under test [17], [18], it has yet to be shown that NK cells can also eliminate cells that are deficient in self-HLA class I expression in autologous systems.

To study the possible role of NK cells in the elimination of HLA class I mutant cells in a simple autologous system, we carried out a series of experiments in which we studied the interactions between HLA-A2-negative mutant lymphocytes and NK cells in vitro. We began by comparing the susceptibility to NK-mediated lysis of an HLA-A2 heterozygous (Het) B cell line with the individual susceptibilities of each of a series of HLA-A2-negative mutant B cell lines, and went on to test the possibility that autologous NK cells might be able to eliminate HLA-A2-negative mutant cells during growth of the HLA-A2 (Het) B and T cell lines in vitro. We then attempted to determine whether the removal of NK cells from lymphocyte preparations obtained from HLA-A2 (Het) individuals prior to culturing them in vitro would permit us to detect significantly increased numbers of HLA-A2-negative mutant T cells emerging in these cultures in the virtual absence of selective pressure. Our results suggest that NK cells may well be responsible for the elimination of autologous cells that have lost the ability to express self-HLA class I molecules, and might therefore explain why we have been unable to detect increased numbers of HLA-A2-negative mutations in the blood cells of A-bomb survivors.