Biomedical subjects
Publications and source records attributed to F H Bach.
Organ culture of xenogeneic thyroids leads to a prolonged survival time. This effect can be reversed when donor-specific cells are injected at the time of transplantation. The results of this experiment disprove the assumption that target antigens are modulated or masked by organ culture.
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Two tests, the mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML) tests, have been used as in vitro models of the in vivo allograft reaction. These tests have been applied to histocompatibility testing for transplantation as well as to assay of immune function. They involve the use of peripheral blood lymphocytes as "responding" cells in mixed leukocyte culture or as "stimulating" cells in the generation of a proliferative or a cytotoxic response. The proliferative events in MLC are primarily in response to LD antigens of the major histocompatibility complex; the cytotoxic cells use as their targets the SD antigens of that complex. A new method for defining the LD antigens, the primed LD typing (PLT) test, is based on in vitro sensitization of lymphocytes to certain LD antigens of the major histocompatibility complex and their subsequent restimulation with test cells.
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Five recipients of successful living related donor kidney transplants stopped azathioprine and prednisone against medical advice. Two recipients who were nonidentical by human leukocyte (HL-A) serotyping and mixed leukocyte culture (MLC) became uremic again after cessation if immunosuppression for periods of 2 and 6 months. Both patients died while refusing to reinstitute iunosuppression and their kidneys showed severe rejection histologically. Three recipients of MCL and HL-A identical kidneys were off azathioprine and prednisone for periods of 7, 12, and 30 months, respectively. None developed changes in renal function. Two patients currently are back on immunosuppression. The third recipient remains stable although off immunosuppression for 36 months; her cessation of immunosuppression was elicited by a mailed questionnaire sent to all recipients following detection of the first four. In addition to these five patients, three recipients of cadaver kidneys lost graft function after lapses in immunosuppression. This represents a 4% incidence of major lapses in immunosuppression (excluding known cessation during sepsis). The clinical outcome depended on degree of histocompatibility as best measured by MLC.
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The specificity of antigen recognition by in vitro sensitized human cytotoxic T lymphocytes (CTLs) has been studied using a sensitive cell-mediated lympholysis (CML) assay. Frequently, high levels of cytotoxicity are observed on third-party targets unrelated to sensitizing or responding cells; however, no cytotoxicity differing significantly from zero has been observed on targets autologous to the responding CTLs. This "cross-killing" of third-party target cells has been observed when stimulating and third-party cells bear no cross-reacting serologically defined (SD) antigens, thought to be the target antigens recognized by CTLs. CML-blocking studies, using unlabeled normal human lymphocytes to inhibit 51Cr release from radiolabeled target cells, have shown that cross-killing, even in the absence of shared SD determinants, results from CTLs recognizing antigens shared by the third-party targets and the initial stimulating population. Furthermore, these antigens have been mapped to the major histocompatibility complex (MHC). The ability of human CTLs to specifically recognize MHC-controlled antigens not detected serologically suggests that SD antigens may be recognized differently by alloantisera and CTLs, or that MHC antigens other than SD may be the targets of CTLs in CML.
The regulation of B-cell and T-cell immune responses has been extensively examined and in the experimental animal appears to involve regulatory or "suppressor" T cells (1-4). The limitations of in vitro experimentation have made comparable study of nonpathological human suppression quite difficult (5). We report here an in vitro method that generates and quantitates suppressor activity in man after antigen-specific activation in mixed leukocyte culture (MLC). The one-way MLC induces both a proliferative response (6) and the generation of cytotoxic T lymphocytes (CTLs) (7). Both of these responses are mediated by antigen-specific T-cell subpopulations (8,9) and have been correlated with recognitive and destructive phases of allograft rejection. Recent reports have examined the antigen reactivity of mouse (10,11), rat (12), or human (13,14) lymphocytes obtained after proliferation in MLC. In all cases, after the primary MLC proliferative peak, the recovered lymphocytes rapidly differentiate upon re-exposure to the initial stimulating population, but do so only weakly when exposed to a presumably noncross-reactive third-party stimulating population. Velocity sedimentation separation studies have shown that the blast cells produced in a primary MLC revert to small lymphocytes that rapidly differentiate into proliferating and/or cytotoxic T lymphocytes upon restimulation with the initial antigen (15). These findings demonstrate that positive selection for the responding population in primary MLC does exist and may account for at least part of the specificity of the secondary response. However, this positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection in not yet significant, suggesting that suppression may be overriding importance in the specificity of MLC-activated secondary responses.
While spleen cells from neonatal B10 (H-2b) are reactive (proliferate) in one-way mixed leukocyte culture, cell-mediated lympholysis reactivity does not arise until 7 days of age. When B10 cells are sensitized to B10.D2 (H-2d), cross-killing of third-party B10.BR (H-2k) target is always lower than the specific killing of B10.D2 targets and is not demonstrable until 27 days after birth.
A 7-month-old boy with severe combined immunodeficiency had no relative who was a suitable bone-marrow donor as determined by mixed-leucocyte-culture (M.L.C.) testing. In the general population an M.L.C.-nonreactive, unrelated donor was found among individuals who were identical with our patient at the Four locus. Following a bone-marrow transplant this child showed signs of rapid immunological reconstitution and only a mild graft-versus-host reaction. Unfortunately, the child died 31 days post-transplantation of cytomegalovirus infection. The findings support the hypothesis that matching for lymphocyte-defined antigens among unrelated individuals will permit successful immunological reconstitution.
When human lymphocytes are cultured for 9 to 14 days with stimulating cells of a family member differing by a single HL-A haplotype they become "primed" to recognize specific HL-A LD (mixed lymphocyte culture) antigens. These primed lymphocytes respond specifically and rapidly when "restimulated" with cells of a person that contain the same LD antigens as those of the priming haplotype. Specific HL-A LD antigens can be detected within 24 hours by this primed LD typing.
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"Transfer factor" was prepared by Sephadex G-25 chromatography of lymph node cell lysates from guinea pigs immunized with ovalbumin or bovine gamma globulin. Treatment of nonimmune peritoneal exudate cells with the transfer factor and specific antigen leads to inhibition of migration of the cells, whereas cells treated with the transfer factor alone or specific antigen alone are not inhibited from migrating. An average of 24-28% inhibition is observed in the presence of transfer factor and specific antigen, but only 5-15% inhibition in the presence of transfer factor and nonspecific antigen. The guinea pig transfer factor we have tested in vitro has some physical characteristics in common with human transfer factor.
Spleen cells from 2- to 3-month-old normal mice of some strains having a low incidence of spontaneous leukemia were found to lyse cells of the spontaneous AKR leukemia K36 in the 51Cr release assay. Incubation of 51Cr-labeled ADR K36 cells with spleen cells from normal C57BL/6, C57L, C57BL/10, and RF mice resulted in the release of significantly more 51Cr than that released in the presence of medium alone. In contrast, 51Cr released from AKR K36 cells after incubation with spleen cells from mice of the high leukemic strains AKR and C58 was less than that released spontaneously. The results of competitive inhibition tests when C57BL/6 spleen cells were incubated simultaneously with 51Cr-labeled AKR K36 target cells and varying numbers of nonlabeled cells demonstrated that the cytotoxic activity of normal C57BL/6 spleen cells was directed against an antigen(s) associated with several leukemias, but that was undetectable on normal thymocytes. Pretreatment of C57BL/6 spleen cells with carbonyl iron and a magnet, which removed phagocytic macrophages, did not decrease the cytotoxic acitivity for AKR K36 cells.
We describe a miniaturized cell-mediated lympholysis assay for measuring the activity of cytotoxic lymphocytes generated in mouse mixed leukocyte cultures (MLC), using either serum-free or mouse serum-supplemented media. Mixed reactions of strain combinations possessing various genetic differences at H-2 produced cytotoxic lymphocytes with cytolytic activity against cells of the donor used in the sensitization phase. Results suggest that both serologically defined and lymphocyte-defined determinants can act as effective target antigens on target cells. In contrast, no cytotoxic lymphocytes were generated against products of non-H-2 loci--M-locus, theta, or other non-H-2 antigens--that induce MLC activation.
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