Effects of leukocyte depletion and UVB irradiation on alloantigenicity of major histocompatibility complex antigens in platelet concentrates: a comparative study.

Recent technologic advancement enables us to prepare leukocyte-depleted or UVB-irradiated platelet concentrates for possible prevention of primary HLA alloimmunization. However, it is yet not known which of these two approaches is more efficacious. Because well-controlled studies cannot be easily conducted in human subjects to answer this question, a series of experiments were performed using a mouse transfusion model. The results showed that 100% of CBA mice with H2k haplotype developed antibody to donor H2d major histocompatibility complex (MHC) antigens after two weekly transfusions of platelet concentrates containing 2000 leukocytes/microL. In contrast, only 50% of the mice became alloimmunized after receiving platelets containing < or = 2 leukocytes/microL. More impressively, none developed anti-H2d antibodies after receiving two platelet concentrates irradiated with 1,200 mJ/cm2 UVB. UVB irradiation was found to be equally effective in reducing the alloantigenicity of platelet concentrates regardless of whether they contained more than a fully immunogenic dose of leukocytes. The antibody titers determined after five weekly transfusions also supported the observation that UVB irradiation was more efficacious than a 3-log leukocyte depletion in the prevention of primary alloimmunization to MHC antigens. In addition, the studies showed that only transfusions of UVB-irradiated platelet products could induce the suppression of immunologic responses to donor MHC antigens in recipients and the induced immunologic suppression could not be further enhanced by gamma irradiation or by leukocyte depletion.

Generation of tumor-specific transplantation antigens by UV radiation can occur independently of neoplastic transformation.

The purpose of this study was to determine whether the UV-associated antigens present on tumors induced in mice by chronic UV irradiation could be induced by in vitro irradiation of cells that were already tumorigenic, or whether their occurrence was associated with the primary neoplastic transformation event. Cells of a nonantigenic, spontaneous fibrosarcoma cell line were exposed to UV radiation in vitro, were cloned, and were tested for antigenic properties. A large number of the clones obtained after UV irradiation of the fibrosarcoma cells were highly antigenic (20 of 39), whereas clones derived from unirradiated cultures were not (0 of 10). The antigenic variants did not induce cross-protection among themselves, but induced only variant-specific immunity in vivo. Several antigenic variants were tested for susceptibility to the action of UV-induced suppressor cells, which seem to recognize a common determinant shared among UV-induced tumors. The variants tested were indeed subject to the activity of the UV-induced suppressor lymphocytes. These results demonstrate that the unique antigenic properties exhibited by UV-induced murine skin cancers are also exhibited by cells exposed to UV radiation in vitro. In addition, they imply that the UV-associated antigens arise as a consequence of exposing cells to UV radiation and that they can occur independently of an initial neoplastic transformation event.

Selection of cytotoxic T-cell precursors specific for minor histocompatibility determinants. I. Negative selection across H-2 barriers induced with disrupted cells but not with glutaraldehyde-treated cells: evidence for antigen processing.

Intravenous injection of CBA mice with H-2-compatible irradiated B10.BR spleen cells led to a sequence of negative and positive selection of the host T-cell response against the multiple foreign minor histocompatibility antigens (HA) on the injected cells. By 1 d posttransfer, thoracic duct lymphocytes (TDL) of the host had lost the capacity to differentiate in vitro into cytotoxic cells specific for the injected minor HA; spleen and lymph node cells, by contrast, gave normal or enriched responses at this time. By 5 d posttransfer, TDL were hyperresponsive to the injected antigens. Selection with disrupted (sonicated) cells gave similar findings. With injection of either irradiated of disrupted spleen cells, the H-2 haplotype of the minor HA-bearing cells had no apparent effect on the magnitude of selection. By contrast, treatment of spleen cells with glutaraldehyde before injection led to H-2 restriction of selection, i.e., negative selection of the CBA response to B10.BR was marked with injection of glutaraldehyde-treated H-2-compatible B10.BR cells but was minimal with H-2-different B10 or B10.D2 cells. These data are taken to imply that, at least in H-2-incompatible situations, the minor HA-bearing cells must be processed by host cells, i.e., to allow the antigens to become associated with self H-2 determinants. Circumstantial evidence from studies on the specificity of selection induced with glutaraldehyde-treated cells from mice of the B10 recombinant strains suggested that I region-restricted T cells may control the induction of H2K, D-restricted cytotoxic precursor cells.

Induction of allogeneic unresponsiveness in adult dogs. Role of non-DLA histocompatibility variables in conditioning the outcome of bone marrow, kidney, and skin transplantation in radiation chimeras.

Exposure to supralethal total body irradiation and transplantation of bone marrow from a DLA- and pedigree-identical donor have regularly produced successful engraftment and the establishment of stable long-term chimerism in beagles of the Cooperstown colony. Bone marrow allografts performed in pairs of dogs bearing identical DLA haplotypes derived from different pedigree origins (i.e., different classes of the same haplotype) yielded two different results. Depending upon the particular haplotype pedigree combination used, such transplants either led to long-term chimerism or to failures of engraftment, secondary disease, and death of the recipients (i.e., pedigree-incompatible combinations). Radiation chimeras given bone marrow from a DLA-and pedigree-identical donor were challenged within 8-12 h after marrow transplantation with a renal allograft obtained from another DLA- and pedigree-identical donor. The recipients have remained unresponsive to such renal allografts and have survived indefinitely with normal renal function. In contrast, renal allografts obtained from donors bearing the same DLA haplotypes derived from pedigree-incompatible sources were rejected within 25-50 days after transplantation. The long-term surviving recipients have also been unresponsive to skin allografts obtained from their donor of marrow and the kidney donor. Skin grafts obtained from other DLA- and pedigree-identical dogs were rejected within 13-41 days, and grafts from DLA-incompatible donors survived for 10-25 days. These results highlight the potential importance of genetically controlled histocompatibility determinants other than DLA in conditioning allograft reactivity. The determinants uncovered in the present study appear to be linked to the DLA complex, as demonstrated by the ability of the pedigree origins of DLA haplotypes present in individual dogs to serve as an effective marker system for such non-DLA antigen(s). The results also point to the potential usefulness of the early postirradiation period for the induction of allogeneic unresponsiveness in large adult mammals.

Vesicular stomatitis virus-infected L1210 murine leukemia cells: increased immunogenicity and altered surface antigens.

Homogenates of L1210 cells infected in vitro with vesicular stomatitis virus (VSV) were immunogenic agains a tumor graft of 100 times the LD50 dose of L1210 cells" whereas those of uninfected cells were not. The immunogenicity of intact X-irradiated L1210 cells was distinguishable from that of VSV-infected cell homogenates on the basis of the susceptibility of immunogenicity to experimental procedures used in preparation of the immunogenic homogenates: Homogenization of intact X-irradiated cells or their infection with VSV prior to irradiation led to loss of immunogenicity. In addition, uninfected cell homogenates were not made immunogenic nor was the immunogenicity of VSV-infected cell homogenates eliminated by X-irradiation. At the time of tumor challenge, sera from mice that were effectively immunized with VSV-infected cell homogenate showed a high VSV-neutralizing titer but no complement-dependent cytotoxicity for L1210 cells. Quantitative absorption studies demonstrated that VSV infection led to a marked reduction in L1210 surface antigens recognized by cytotoxic alloantibody; spatial association between these antigens and VSV antigens was not demonstrable on VSV-infected cells. Antigens recognized by heterologous antiserum to L1210 cells were also reduced following VSV infection.

Comparison of the allospecific and viral-specific immune responses to irradiated versus formaldehyde-fixed allogeneic Moloney lymphoma cells in CBA mice.

Two groups of adult CBA mice were immunized with 10-7 allogeneic Moloney lymphoma (YAC) cells. These YAC (H-2a) cells, which were either irradiated with 6000 R (Group i) or were formaldehyde fixed (Group II), were injected i.p. at weekly intervals for 3 weeks. Four days following the last injection, sera and lymphocytes were collected and tested in vitro for activity against either allospecific antigens (H-2d target cells) or viral-specific antigens, namely, Moloney leukemia virus (MLV). Both groups of animals developed measurable cellular and humoral immunity to the virally determined antigens. However, only the animals in Group i, immunized with irradiated cells, developed detectable immunity to H-2d. Immune and control lymphocytes were tested in microcytotoxicity tests and by 51Cr release. Antibody was assessed by complement-dependent cytotoxicity, indirect membrane immunofluorescence, virus neutralization, and antibody-dependent lymphocyte cytotoxicity. Group I serum, which had both anti-MLV and anti-H-2 antibodies, was absorbed with either living or formaldehyde-fixed YAC cells. The living cells were able to remove both H-2 and MLV antibodies. On the other hand, the formaldehyde-fixed cells removed no H-2 antibody but were able to remove MLV antibody, although less efficiently than living cells. These data indicate that formaldehyde fixation selectively impaired the H-2 antigens, leaving the viral antigenicity relatively intact. Differences between the immune responses to MLV-determined antigens and to H-2 antigens were demonstrated in many of the parallel in vitro tests.