H-2 antigen requirements in the in vitro induction of SV40-specific cytotoxic T lymphocytes

T cytotoxic cells generated to syngeneic SV40 virus transformants lyse only SV40 target cells that are syngeneic at the H-2 locus. In contrast, SV40-specific tumor transplantation immunity shows no requirements for syngeneic H-2. Inoculation of allogeneic or even xenogeneic transformants will confer immunity to a challenge of syngeneic SV40 tumor cells. The experiments described here represent an attempt to reconcile these apparently conflicting observations. In our hands, generation of SV40-specific T cytotoxic cells in vitro requires both in vivo priming and secondary in vitro sensitization. We have found that priming for a secondary syngeneic-restricted response requires only that the cell employed be SV40 transformed. That is, priming may be accomplished with syngeneic, allogeneic, or xenogeneic SV40 transformants. Thus, the apparent lack of H-2 restriction in vivo immunity does not eliminate a role for the H-2-restricted cytotoxic T cell in tumor transplantation immunity.     

Xenogeneic mixed leucocyte response

Xenogeneic mixed leucocyte cultures composed of human, chimpanzee, baboon, goat, sheep, pig, and dog cells were set up with a variety of plasma culture supplements. The characteristics of the human leucocyte response to xenogeneic cells was similar to its response to allogeneic cells. Peak response to xenogeneic stimulation occurred on the same culture day as the peak response to allogeneic stimulation. Similar numbers of xenogeneic and allogeneic cells produced peak stimulation of cells from any one individual. There was, however, a wide variation in the response of human lymphocytes to both allogeneic and xenogeneic cells. A factor in the plasma supplement specific for responding or stimulating cells inhibited the mixed leucocyte response in some combinations and could be removed by absorption techniques.     

Lectin-dependent cell-mediated cytotoxicity in hamsters bearing syngeneic tumors

Spleen cells from LSH hamsters inoculated with xenogeneic, allogeneic, or syngeneic (PARA-7) tumor cells were assayed for their ability to mediate direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) in a 4-hr chromium release assay. Spleen cells from animals immune to xenogeneic or allogeneic cells demonstrated specific DCMC against homologous target cells in the absence of Con A and nonspecific LDCC against both homologous and heterologous target cells in the presence of Con A. Spleen cells from animals bearing syngeneic PARA-7 tumors (TBA) failed to express DCMC against homologous or heterologous target cells; however, significant lysis of all target cells occurred in the presence of Con A. LDCC was not detectable when nonsensitized spleen cells from normal animals were employed. The LDCC reaction was dependent on the concentration of Con A and the number of effector cells present in the reaction. The development of LDCC effector cells in the TBA appeared to parallel the development of both DCMC and LDCC effector cells in immune animals.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

Response of human fetal lymphocytes in xenogeneic mixed leukocyte culture: Phylogenetic and ontogenetic aspects

Cells from liver, thymus, and spleen of human fetuses at different stages of development were capable of a proliferation response against xenogeneic and allogeneic lymphocytes. The kinetics of fetal responses against rat lymphocytes were identical to those of fetal and adult responses against allogeneic cells. With all of the cell types studied, including adult lymphocytes, allogeneic responses were stronger than xenogeneic. Xenogeneic responses against lymphocytes from rat, mouse, or sheep were stronger than those against lymphocytes from rabbit, chicken, snake, or frog. These results are interpreted to indicate that recognition of foreign lymphocytes by human lymphocytes depends on the phylogenetic position of the species used as a source of stimulating cells. The degree of recognition decreases as the phylogenetic distance increases. Specific elimination of responding cells and restimulation with another cell population was used to study the specificity of proliferation responses against mouse and rat lymphocytes. Responses by prethymic liver cells from human fetuses were not due to the existence of specifically recognizing subpopulations. Thymus and spleen at 16 weeks' gestation contained specific subpopulations capable of differentiating between xenogeneic and allogeneic cells, as well as between xenogeneic cells with different intraspecies histocompatibility patterns. Generation of receptor diversity on T lymphocytes is discussed briefly in the light of these findings.     

Specificity of allogeneic and xenogeneic cell recognition in the fetal lamb.

Cells prepared from liver, thymus, and spleen of fetal lambs at different stages if gestation were confronted with allogeneic and xenogeneic cells in MLC. Specific elimination of the responding cells with BUdR and UV light together with a subsequent restimulation was used to study the specificity of the reaction. The response of fetal liver cells was not based on the existence of specifically recognizing cellular subpopulations; the response was concluded to be due either to stimulatory products released by the stimulating cells or to the multipotentiality of the responding cells. Specifically recognizing cells first appeared in the thymus at 58 days postconception and in the spleen at 70 days. In the response of sheep lymphocytes against allogeneic and xenogeneic (mouse, human) cells, a cross-reactivity occurred. Fetal lamb lymphocytes were also capable of recognizing intraspecies differences on the xenogeneic cells. This capacity developed simultaneously with the specific recognition of allogeneic cells. No clear difference was observed in the reactivity of fetal thymus cells and spleen cells when compared to that of adult peripheral blood lymphocytes. These findings indicate that immunologically specific recognition of foreign cells is created in the sheep during the early intrauterine development.     

Innate and adaptive immune responses to nonvascular xenografts: evidence that macrophages are direct effectors of xenograft rejection

Nonvascularized xenograft rejection is T cell mediated, but is dependent on initial macrophage (Mphi) infiltration. We developed an i.p. transplant model to define the roles of Mphi and T cells in xenograft rejection. Nonobese diabetic or BALB/c mice were injected i.p. with xenogeneic, allogeneic, or syngeneic cells, and the responding cells in subsequent lavages were assessed by flow cytometry and adoptive transfer. Neutrophils and monocytes/elicited Mphi were rapidly recruited in response to xenogeneic pig (PK15 or spleen) cells and, to a significantly lesser extent, allogeneic cells. These innate responses preceded T cell infiltration and occurred in their absence in SCID mice. Syngeneic cells induced negligible neutrophil or Mphi responses. Neutrophils and Mphi induced by xenogeneic cells in SCID mice stimulated T cell recruitment after transfer to immunocompetent mice. T cells in turn were required for Mphi activation and xenogeneic cell rejection. Thus, Mphi harvested from immunocompetent but not SCID mice injected with xenogeneic cells expressed activation markers and rejected xenogeneic cells when transferred into SCID mice. These findings demonstrate the interdependent roles of Mphi and T cells in xenograft rejection. The requirement for Mphi reflects their ability to mount a rapid, local innate response that stimulates T cell recruitment and, having received T cell help, to act as direct effectors of rejection.     

Graft-versus-host reactivity of mouse thymocytes: effect of in vitro treatment with anti-TL serum.

The lymph ducts efferent from prefemoral nodes of sheep were cannulated and the lymph flow monitored during immune responses to injected allogeneic lymphocytes or xenogeneic murine P815 mastocytoma cells. Changes in the lymph began 5–6 days after injection of allogeneic cells but at 3–4 days after injection of xenogeneic cells, in both systems the number of large cells in the lymph increased to reach peak values of up to 40% of the total. The in vitro cytotoxic activity of lymph cells, cell supernatants, or cell free lymph was determined by measuring the release of ⁵¹Cr from prelabeled target lymphocytes or P815 cells. The cytotoxic mechanisms that were detected in the allogeneic and xenogeneic systems were similar; in both cases the lymph cells were cytotoxic only during the large cell response, and when the immunoblast numbers had returned to normal levels in the lymph no further cell-mediated cytotoxic effects were detected. During the blast response lymphocytes alone caused some target cell damage but their cytotoxic effector function was greatly increased in cultures containing complement or normal blood white cells. It was concluded that the lymph immunoblasts caused some target cell damage by direct action, but the majority of their cytotoxic activity was associated with synthesis and secretion of complement-dependent antibody (C.D.A.) and leukocyte-dependent antibody (L.D.A.).     

Intracellular localization of anti-tumor immune RNA.

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.     

Detection of HLA-DRw (Ia-like) antigens on human T lymphocytes grown in tissue culture.

Human T lymphocytes that proliferate in the presence of conditioned medium from PHA-stimulated allogeneic peripheral blood cells were shown to express IPA antigens after the 8th culture day. Ia antigens as detected by xenogeneic antisera were present on 80 to 90% of the cultured cells which were also strongly reactive with xenogeneic antisera defining a human T cell antigen and formed E rosettes. Control cultures with PHA or no conditioned medium expressed T cell but not Ia antigens. These cultured T cells also express the same HLA-DRw determinants as the B cells of the donor they were derived from. Absorption of xenogeneic Ia, and HLA-DRw alloantisera with cultured T cells completely removed the reactivity of these sera for enriched peripheral blood B lymphocytes from normal donors.     

Antitumor resistance induced by immunization with normal definitive tissues]

Immunization of C3HA mice with homogenates of various normal definitive tissues, obtained from syngeneic, allogeneic and xenogeneic donors, resulted in an increase of antitumor resistance, as evidenced by a considerable retardation of growth of the transplantable strain-specific hepatoma 22a. The effect may be due to sensibilization of animals to the antigens of normal definitive cells, and, in particular to the tissue-specific antigens characteristic of cells of the tumor in question.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. I. Activation by tuberculin and by Staphylococcus filtrate

Normal lymphocytes activated by mitogens such as phytohemagglutinin (PHA), by Staphylococcus filtrate (SF), or lymphocytes from sensitized individuals stimulated by antigen (PPD, etc.) are cytotoxic to tissue culture cells of different origins. In this and the following paper, the results of a detailed quantitative analysis of the specificity of this cytotoxic reaction are presented. Effector cells were human or mouse lymphocytes, activated by PHA, SF, PPD, or serum factors in the culture medium. Cells from established cell lines of human, mouse, hamster, or rabbit origin, or primary human or rat embryonic fibroblasts were used as target cells. Lysis was quantitated by release of ⁵¹Cr from labeled target cells.Purified human blood lymphocytes, activated by PPD, SF, or otherwise, preferentially damaged allogeneic target cells. Lysis of xenogeneic target cells was weak or did not occur. A close correlation was noted between target cell destruction and blastoid transformation of the lymphocytes, but the slope of the regression lines of xenogeneic cytotoxicity was much smaller than that of allogeneic cytotoxicity when plotted as a function of blastoid transformation.Lymph node or spleen cells from CBA mice were stimulated by PPD to transformation and DNA synthesis. CBA lymphocytes also showed an increased degree of blast transformation in medium containing fetal calf serum or certain batches of fresh human serum. Mouse lymphocytes activated in these ways damaged allogeneic L cells but had no effects on xenogeneic Chang cells.These results indicate that lymphocytes activated by various means preferentially damage target cells from their own species. The recognition mechanisms which determine the specificity of the reactions are not known.     

Ultrastructural study of biochemically modulated ADCC in HSV-1 infected and uninfected Chang liver cells

The effect of cytochalasin B, ouabain and 25-OH cholesterol on specific lysis due to antibody dependent cellular cytotoxicity (ADCC) in allogeneic and xenogeneic systems was studied using Herpes simplex I infected Chang liver cells. Cytochalasin B reduced both cytotoxicity and lymphocyte/target (LT) binding in the allogeneic system whereas cytotoxicity but not LT binding was reduced in the xenogeneic system. Ouabain inhibited ADCC in both systems as well as LT binding in the allogeneic system; however, binding in the xenogeneic system was not significantly reduced. The 25-OH cholesterol produced a marked decrease in ADCC in both systems but had no significant effect on LT binding in either system. The biochemical and ultrastructural data suggest that the modulators act at different stages in the ADCC response and that there may be more than one mechanism of ADCC to handle different types of target antigens.     

Cytotoxic and proliferative lymphocyte responses to allogeneic and xenogeneic antigens in vitro.

In vitro lymphoproliferative responses to foreign histocompatibility antigens are phylogenetically restricted. Responses occur most readily to allogeneic or closely related xenogeneic leucocytes, but not to unrelated xenogeneic cells. Specific cytotoxic T cell responses to foreign histocompatibility antigens show the same phylogenetic restriction. This lack of xenoreactivity is not due to a lack of precursor cells for the xenoantigens; guinea-pig lymphocytes, although normally unresponsive to mouse antigens, have a similar precursor frequency for these antigens as do lymphocytes of allogeneic mouse strains. Specific cytotoxic responses of guinea-pig lymphocytes to mouse antigens can be generated if a factor released from con A stimulated guinea-pig spleen cells is added to the culture medium. The factor produced by con A-activated spleen cells (CS) is also phylogenetically restricted in its action; CS must be obtained from animals homologous with the donor of the responding lymphocytes.     

Effects of in vivo priming on in vitro induction of cytotoxicity. I. Non-specific augmentation by in vivo presensitization with allogeneic or xenogeneic cells.

In vivo presensitization of donor mice of responding cells with third party cellular antigens augmented in vitro generation of cytotoxic T lymphocytes in allogeneic and xenogeneic combinations. In vitro induction of detectable cytotoxicity in presensitized responding cells required the incubation period needed for in vitro primary response. However, such cytotoxic T lymphocytes were generated after in vitro stimulation with monolayers of methylcholanthrene-induced tumor cells, UV-irradiated or heated spleen cells which had proved to be effective in secondary but not in primary response. Presensitized responding cells exposed to 600R-irradiation did not augment in vitro induction of cytotoxicity in normal responding cells. The augmenting effect of presensitized responding cells may be attributable to radiosensitive T cells which are in a transitional state in differentiation from typical unprimed cells to typical primed cells.     

Development of an allogeneic whole-cell tumor vaccine expressing xenogeneic gp100 and its implementation in a phase II clinical trial in canine patients with malignant melanoma

A xenogeneic melanoma-antigen-enhanced allogeneic tumor cell vaccine (ATCV) is an appealing strategy for anti-cancer immunotherapy due to its relative ease of production, and the theoretical possibility that presentation of a multiplex of antigens along with a xenogeneic antigen would result in cross-reaction between the xenogeneic homologs and self-molecules, breaking tolerance and ultimately resulting in a clinically relevant immune response. In this study, we evaluated the efficacy of such a strategy using a xenogeneic melanoma differentiation antigen, human glycoprotein 100 (hgp100) in the context of a phase II clinical trial utilizing spontaneously arising melanoma in pet dogs. Our results demonstrate that the approach was well tolerated and resulted in an overall response rate (complete and partial response) of 17% and a tumor control rate (complete and partial response and stable disease of >6 weeks duration) of 35%. Dogs that had evidence of tumor control had significantly longer survival times than dogs that did not experience control. Delayed type hypersensitivity (DTH) to 17CM98 canine melanoma cells used in the whole cell vaccine was enhanced by ATCV and correlated with clinical response. In vitro cytotoxicity was enhanced by ATCV, but did not correlate with clinical response. Additionally, anti-hgp100 antibodies were elicited in response to ATCV in the majority of patients tested; however, this also did not correlate with clinical response. This approach, along with further elucidation of the mechanisms of tumor protection after xenogeneic immunization, may allow the development of more rational vaccines. This trial also further demonstrates the utility of spontaneous tumors in companion animals as a valid translational model for the evaluation of novel vaccine therapies. E.G. MacEwen deceased     

Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor.

NK lymphocytes lyse certain xenogeneic cells without prior sensitization. The receptors by which NK cells recognize xenogeneic targets are largely uncharacterized but have been postulated to possess broad specificity against ubiquitous target ligands. However, previous studies suggest that mouse NK cells recognize xenogeneic targets in a strain-specific manner, implicating finely tuned, complex receptor systems in NK xenorecognition. We speculated that mouse Ly-49D, an activating NK receptor for the MHC I ligand, H2-Dd, might display public specificities for xenogeneic target structures. To test this hypothesis, we examined the lysis of xenogeneic targets by mouse Ly-49D transfectants of the rat NK cell line RNK-16 (RNK. Ly-49D). Of the xenogeneic tumor targets tested, RNK.Ly-49D, but not untransfected RNK-16, preferentially lysed tumor cells derived from Chinese hamsters and lymphoblast targets from rats. Ly-49D-dependent recognition of Chinese hamster cells was independent of target N-linked glycosylation. Mouse Ly-49D also specifically stimulated the natural killing of lymphoblast targets derived from wild-type and MHC-congenic rats of the RT1lv1 and RT1l haplotypes, but not of the RT1c, RT1u, RT1av1, or RT1n haplotypes. These studies demonstrate that Ly-49D can specifically mediate cytotoxicity against xenogeneic cells, and they suggest that Ly-49D may recognize xenogeneic MHC-encoded ligands.     

Xenogeneic rejection among three botryllids (compound Ascidians)

Xenogeneic rejection was observed among colonies of three botryllids, Botryllus scalaris, Botryllus primigenus, and Botrylloides simodensis. Allogeneic recognition occurs in each of these species, but the manner of allogeneic rejection differs among them. We studied xenogeneic rejection reactions among these species under the following conditions: colony contact at natural growing edges, colony contact at artificially cut surfaces, and injection of xenogeneic blood plasma into a vascular vessel. In the first two cases, xenogeneic rejection occurred only in Botryllus primigenus and Botrylloides simodensis. The features of that xenogeneic rejection were similar to those of allogeneic rejection in each of these two botryllids. Injection of xenogeneic blood plasma induced responses similar to those of allogeneic rejection in all three botryllids. It is interesting to note that colonies of Botryllus scalaris never showed any response against injected blood plasma from allogeneic incompatible colonies, unlike the responses seen in colonies of the other two botryllids under the same conditions. On the basis of these results, the relationship between allogeneic and xenogeneic rejection in botryllids is discussed.     

Immunologic and Osteogeneic Properties of Xenogeneic and Allogeneic Demineralized Bone Transplants

Xenografting is increasingly being developed as a response to the shortage of human tissues. However, antigenic components of bone material eliciting immune responses – particularly of cellular nature – are blamed for the reduction of the osteoinductive properties of bone and bone-derived implants. The aim of our study was to compare the immunologic response and osteogenesis induced by antigen-depleted allogeneic and xenogeneic bone-derived implants to that induced by partially antigen-depleted material heterotopically placed (muscular pouch) in rats. Wistar rats received bone-derived implants of different antigeneic condition, from both xenogeneic (rabbit) and allogeneic (rat) origin. After sacrifice, animals were evaluated for osteogenesis and immune response. New bone formation was observed around all bone-derived implants, whether fully or partially antigen-extracted, and from both xenogeneic and allogeneic origin. No significant humoral response resulted following bone implantation. Cellular response showed a similar pattern in partial and fully antigen-extracted bone of both allogeneic and xenogeneic origin. Xenogeneic antigen-extracted bone from safe donor sources could be a suitable solution to human tissue shortage in a near future.     

Autochthonous, allogeneic, and exenogeneic cells as targets for vaccinia immune lymphocyte cytotoxicity

The cytotoxicity of vaccinia-immune rabbit spleen cells against autochthonous, allogeneic, and xenogeneic vaccinia-infected target cells was studied by using the 51Cr release assay. The results showed that whereas effector cells from rabbit spleen could not lyse the xenogeneic target cells, there was no consistent difference in lysis of autochthonous and allogeneic targets. Antibody-mediated cytotoxicity showed the resistance to cell-mediated lysis in this system not to be the result to reduced virus antigen on the cell surface. The data also showed the established cell line RK13 eas markedly more sensitive to immune spleen cell lysis than short-term rabbit skin fibroblast cultures. The data suggested that it is possible to develop standardized tests to evaluate cell-mediated immunity to virus infections in a noinbred population.