F1 Hybrid Cells Research Articles

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.

Characterization of sequence determinants of enhancer function using natural genetic variation.

Sequence variation in enhancers that control cell-type-specific gene transcription contributes significantly to phenotypic variation within human populations. However, it remains difficult to predict precisely the effect of any given sequence variant on enhancer function due to the complexity of DNA sequence motifs that determine transcription factor (TF) binding to enhancers in their native genomic context. Using F1-hybrid cells derived from crosses between distantly related inbred strains of mice, we identified thousands of enhancers with allele-specific TF binding and/or activity. We find that genetic variants located within the central region of enhancers are most likely to alter TF binding and enhancer activity. We observe that the AP-1 family of TFs (Fos/Jun) are frequently required for binding of TEAD TFs and for enhancer function. However, many sequence variants outside of core motifs for AP-1 and TEAD also impact enhancer function, including sequences flanking core TF motifs and AP-1 half sites. Taken together, these data represent one of the most comprehensive assessments of allele-specific TF binding and enhancer function to date and reveal how sequence changes at enhancers alter their function across evolutionary timescales.

Characterization of donor chimerism, alloreactive host T cells and memory cell development in thymi from mice resistant to neonatal transplantation tolerance.

Certain B10 background mouse strains are resistant to tolerance induction after neonatal inoculation of class I/II MHC-disparate F1 hybrid cells. Despite initial thymic deletion of alloreactive cells, the majority (95%) of these mice reacquire the capacity to reject donor allografts. These current studies examined thymi of adult B10.S (H-2S/H-2E-) mice that neonatally received (B10.S x B10.A)F1 cells, before and after rejection or acceptance of B10.A (H-2k/d/H-2E+) skin grafts. Alloreactive thymic V beta 11+ and V beta 5+ T cells were often reduced in the injected recipients preceding allograft challenge. In some mice a single B10.A skin graft generated an increase in these T cell populations concurrent with allograft rejection. Injected mice that accepted B10.A skin grafts (i.e., tolerant) or that rejected two sequential B10.A grafts often had a reduction of these T cells. Thymic memory cells (CD44high+) were present before transplant in injected mice and reduced in the tolerant mice. Donor chimeric cells were identified in the majority of injected mice before transplant and diminished after B10.A graft application. A greater proportion of chimeric cells coexpressed CD11b or B220 in the tolerant mice. Thus, neonatally injected B10.S mice resistant to tolerance induction reacquire immunocompetent thymic T cells bearing characteristics of memory T cells that could mediate graft rejection. Finally, the initial presentation of chimeric cells (e.g., macrophages) that may efficiently present class I Ags through H-2E likely increases the possibility of adult tolerance.

The major histocompatibility complex class II-linked cim locus controls the kinetics of intracellular transport of a classical class I molecule.

The dominant trans-acting major histocompatibility complex (MHC)-linked class I modifier (cim) locus, previously recognized through its ability to determine altered alloantigenicity of a rat class I molecule, RT1.A3, is shown here to influence class I intracellular transport. The MHC recombinant laboratory rat strains PVG.R1 and PVG.R8 display unusually long retention of RT1.Aa within the endoplasmic reticulum or cis-Golgi. In appropriate F1 hybrid cells heterozygous for RT1.Aa and another class I MHC allele, RT1.Ac, only the RT1.Aa protein is subject to slow transport. The cim gene product therefore shows class I allele specificity in its action, cim appears to be a polymorphic locus whose product is directly involved in the processes of class I MHC assembly and/or intracellular transport.

Specific inhibition of natural killer (NK) activity against different alloantigens

Allogeneic lymphocyte cytotoxicity (ALC), i.e., rapid rejection of i.v. injected allogeneic lymphocytes in unprimed hosts, is an example of NK activity. Apparently anomalous rejection patterns, such as acceptance of F1 hybrid cells by parental hosts and rejection of parental cells by F1 hybrid hosts in many strain combinations, would fit the hypothesis that the effector cells in ALC recognize the absence of certain self-molecules (passwords) rather than the presence of nonself determinants. However, cold target inhibition studies showed that ALC displays allospecificity: when a mixture of radiolabeled AO and DA cells were injected i.v. into euthymic or athymic PVG rats, adding a surplus of cold DA cells reduced killing only of labeled DA cells and vice versa. Furthermore, semiallogeneic cold target cells were ineffective in inhibiting elimination of fully allogeneic cells, which supports the argument against a modification of the hypothesis that self-determinants inhibit a postbinding stage of lysis. Finally, (DA x AO)F1 cells injected into (DA x PVG)F1 hosts were rapidly rejected, despite the fact that donor and host shared expressed DA determinants. In sum, our results show that a hypothesis based on inhibition of killing by self-determinants can only be sustained with extensive modifications, and favor the alternative mechanism that the effector cells positively recognize the presence of allospecific determinants on the target cell surface.

The effect of T lymphocyte depletion on neonatal tolerance induction, graft-vs.-host disease and cellular chimerism.

Treatment with a monoclonal anti-Thy-1 antibody and complement completely prevented C57BL spleen cells from causing graft-vs.-host disease following their inoculation into newborn CBA mice. The proportion of mice that became tolerant to C57BL antigens, as measured by skin grafting, was significantly less compared with mice given (CBA X C57BL)F1 hybrid cells. This was not due to the elimination of T cells, for antibody-treated F1 cells induced tolerance as readily as complement-treated control F1 cells. To investigate whether the apparent superiority of F1 cells over C57BL cells is attributable to differences in the mechanism inducing and maintaining unresponsiveness, two approaches were followed. First, the level of donor cell chimerism in the spleens of tolerant animals was studied. Though no difference between F1 and C57BL cells was uncovered, the presence of T cells in the donor inocula favored the establishment of chimerism. Second, the involvement of suppressor T cells was examined in adoptive transfer experiments. Splenic suppressor T cells were associated with tolerance regardless of how it was elicited. Preliminary results with F1 cells show that the tolerogenic property is not confined to any one cell type. It is proposed that the greater tolerogenicity of F1 cells is brought about by the presence of host-type (self) antigens, which enable the tolerogenic signals to operate without recourse to antigen processing by host cells.

Fine specificity of auto- and alloreactive cytotoxic T-lymphocytes: heteroclitic cross-reactions between mutant and original H-2 antigens.

A novel class of cytotoxic T-lymphocytes that specifically lyse or bind target cells bearing self major histocompatibility complex (MHC) antigens was recently reported from several laboratories (Peck et al. 1977; Altman et al. 1979; Altman and Katz 1980a, b; Ishikawa and Dutton 1979b, c. 1980; Nakamura et al. 1979a; Nakano et al. 1981a; Schawaller et al. 1980). These CTLs are referred to as “autoreactive”, because no target specificities other than class I self MHC products could be demonstrated. Peck et al. (1977) showed that T-lymphocytes cultured in fetal bovine serum (FBS) generated CTLs specific for self H-2 and not for FBS, irrespective of priming Altman et al. found that normal spleen cells incubated in vitro with allogeneic effect factor resulted in induction of CTLs specific for self MHC (Altman et al. 1979; Altman and Katz 1980a, b). Ishikawa and Dutton (1979b, c,1980) obtained autoreactive CTLs by simply culturing spleen cells of F1 hybrid mice of certain ages, again in FBS-containing medium. According to Schawaller et al. (1980), cells of an alloimmune anti-H-2d CTL clone were cytotoxic not only for H-2Kd but also for H-2Kk targets, despite the H-2 k origin of the CTLs. In our laboratory, primary CTLs generated by H-2-heterozygous F1 hybrid spleen cells cultured with irradiated parental stimulator cells (Shearer and Cudkowicz 1975; Shearer et al. 1976a) were found to be specific for H-2 antigens shared and expressed by the F1 responder and parental stimulator cells (Nakamura et al. 1979a, 1982; Cudkowicz et al. 1979;Nakano et al. 1981a). For example, H-2 b/H-2d F1 hybrid cells responded to either H-2Db or H-2Dd region antigens, depending on whether parental stimulator cells were H-2 b or H-2 d (Nakano et al. 1981b; Nakamura et al. 1982).KeywordsMajor Histocompatibility ComplexFine SpecificitySpecific LysisMajor Histocompatibility Complex AntigenTarget Cell LysisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Capacity of "transplanted" lymphocytes to traverse the intestinal epithelium of adult rats.

Suspensions of viable lymph node cells (LNCs), both unlabeled and 3H-uridine labeled, from adult Fischer (FI) rats were inoculated into the lumens of established, surgically isolated segments of ileum in adult (FI x DA)F1 hybrid hosts. Vascular, lymphatic, and nerve supplies of the isolated segment were preserved. It was reasoned that subsequent hypertrophy of the draining mesenteric LN complex, on an immunogenetically specific basis (attributable to graft-versus-host (GVH) reactivity) and histological identification of labeled lymphoid cells in the intestinal wall of the isolated segment, would be indicative of the passage of inoculated cells across the intestinal epithelium. It was found that host mesenteric LNs were significantly larger on the 8th postinoculation day in animals that received 100 x 10(6) or 300 x 10(6) FI LNCs than in animals that were given similar numbers of syngeneic F1 hybrid cells (controls). This lymph node hypertrophy was dosage dependent and favorably influenced by the presence of Peyer's patches. The results of experiments involving introduction of 3H-uridine-labeled cells into isolated ileal segments and subsequent radioautography corroborated the conclusion that lymphocytes deposited in the intestinal lumen can gain access to host tissues, i.e., they are "naturally transplanted."

Addition of serum to the medium used for preparation of cell suspensions as a possible source of artifacts in cell-mediated reactions studied by means of the popliteal lymph node test.

Conditions for preparations of cells used in immunogenetic studies may substantially affect their properties and the results obtained. It was found that short-term incubation or a mere preparation of parental spleen cells in a medium containing either xenogeneic serum or concanavalin A (Con A) increase their ability (after footpad injection) to induce enlargement of the popliteal lymph node in (B10.LP x A/Ph)F1 hybrid recipients. Under similar conditions, enlargement of PLN could be induced also by syngenetic F1 hybrid cells. The greater lymphadenomegaly following the injection of parental or syngeneic cells prepared in serum-containing medium may be explained by the host reaction against additional foreign antigens rather than increased GVH reactivity or autoimmunity.

Quantitative Studies on the Precursors of Cytotoxic Lymphocytes

Abstract The cytotoxic response to TNP-modified syngeneic cells is small compared to the cytotoxic response to alloantigens. Limiting dilution analyses indicate that the number of lymphocytes in C57BL/6J(B6) lymph nodes responding to DBA/2J(D2) and B6-TNP are 1199 and 40 per 106 cells, respectively. These cytotoxic T lymphocytes (CL) produced as a result of stimulation of B6 lymphocytes with B6-TNP also lyse D2-TNP targets. Clonal analyses show that this cross-reactivity is a result of strong recognition of D2-TNP targets by 40% of B6-TNP specific CL and not relatively weak recognition of D2-TNP by all B6-TNP specific CL. When (C57BL/6J × DBA/2J)F1 mice (BDF1) were stimulated with BDF1-TNP, about 67% of the cytotoxic clones that lysed B6-TNP also lysed D2-TNP target cells; clones specific for either B6-TNP or D2-TNP were also produced. Cold target competition studies suggest that the higher degree of cross-reactivity in the latter case cannot be explained on the basis of the existence of antigenic specificities unique only to the F1 hybrid cells. Nor is the anomalously high F1 cross-reactivity due to the absence of parental antigenic specificities on the F1 cells. One possible explanation for these data is that the repertoire of cytotoxic precursors of the F1 hybrid for TNP-modified self antigens may be different from the sum of the repertoires for the parents.

Anti-parental-strain lymphocyte responsiveness of F1 hybrid-strain lymphocytes.

The interactions between immunocompetent parental‐strain tells and the F1 hybrid rat into which these cells have been injected were studied. This involved examination of the behavior of both parental‐ and host‐strain cells. Host lymphocytes reactive against parental‐strain lymphocytes appeared in the thoracic duct lymph within 12 h of the intravenous injection of parental thoracic duct lymphocytes or thymus cells. The activity of these F1 hybrid‐strain lymphocytes was directed preferentially against parental‐strain lymphocytes with anti‐F1 hybrid potential. This subpopulation of parental cells was absent from the thoracic duct but was well represented in the spleen, this enrichment being masked by a reversible F1 hybrid anti‐parental‐strain lymphocyte response. If the anti‐parental‐strain lymphocyte activity of F1 hybrid cells was interrupted by the destruction of host‐strain cells with antiserum, parental‐strain lymphocytes in the tissues of F1 hybrid rats could be reactivated. By this procedure it was possible to passage graft‐versus‐host reactions initiated by parental‐strain lymphocytes through several F1 hybrid rats, thereby implying that anti‐parental lymphocyte activity is of importance in limiting the serial passage of these reactions. Some of the anti‐parental lymphocyte activity observed on the part of F1 hybrid‐strain cells was probably mediated by anti‐idiotypic antibodies, but other phenomena, especially selective migration of both donor and host lymphocyte subpopulations, are of major importance in the interaction between parental‐strain lymphocytes and an F1 hybrid host.

ADDENDUM

Data have just been published by S. Dorsch & B. Roser (Nature (London) 1975, 258, 233–235) showing that thoracic duct lymphocyte populations obtained from neonatally injected tolerant rats, and consisting of almost pure T cells, transferred specific tolerance to skin allografts when injected into sublethally irradiated syngeneic recipients. Their data, which they believe to exclude the participation of F1 hybrid cells stemming from the tolerance-inducing inoculum, are convincing—except that the criteria of tolerance applied (survival of skin allografts “for at last 50 days”) do not guarantee that the cell donors had been fully tolerant.

The Elaboration of Leukotactic Mediators during the Interaction between Parental-Type Lymphocytes and F1 Hybrid Cells

Mixed leukocyte cultures consisting of white blood cells from (Lewis times BN) F1 hybrids and Lewis parents produced monocyte chemotactic factor. Elaboration of this material preceded incorporation of 3H-thymidine. Local graft vs host (GVH) reactions were induced by subcapsular injection of parental thoracic duct cells into F1 hybrids. Homogenates from these kidneys, but not from kidneys injected with syngeneic thoracic duct cells, contained monocyte chemotactic factor. Little or no neutrophil chemotactic factor was present. Ultracentrifugal analysis of the monocyte chemotactic factor indicated a distribution similar to that found previously in culture fluids of lymphoid cells stimulated by soluble antigens. Differential counts of inflammatory cells extracted in suspension form from kidneys undergoing a GVH reaction indicated the majority of cells to be lymphocytic in type, but with a significant proportion of monocytes. Virtually no neutrophils were present. These findings indicate that a monocyte chemotactic factor is produced by cultures of parental leukocytes stimulated by semiallogeneic cells and that a similar factor appears in GVH reactions in the rat kidney. This chemotactic factor may be relevant to the character of the cellular exudates.

DO F1 CELLS DIVIDE AND RESPOND TO PARENTAL CELLS? STUDIES ON THE MIXED LYMPHOCYTE REACTION OF CBA MICE

SUMMARYThe CBA/H mouse lymphocytes which divide when cultured together with allogeneic blood in mixed lymphocyte reactions (MLR) have been shown to be mainly of thymic origin with the aid of thymus grafted radiation chimaeras. By adaptation of a system used previously to quantity mitogen responsive cells, the relative number of cells in CBA/H mouse blood capable of responding in the MLR, and the effects of preimmunizing CBA/H mice with allogeneic cells have been quantified. In mixed lymphocyte reactions between parental and allogeneic F1 hybrid cells, substantial numbers of the latter cell type have been found in division. Attempts to elucidate the nature of these reacting F1 cells indicate that they are probably mainly of bone marrow origin and that they are responding in a non‐specific manner.

On the role of the H-2 histocompatibility complex in determining the specificity of cytotoxic effector cells sensitized against syngeneic trinitrophenyl-modified targets.

Spleen cells cultured with syngeneic trinitrophenyl (TNP)-modified stimulator cells display a cytotoxic effect against syngeneic TNP-modified targets, but not against modified targets from unrelated H-2 haplotypes. Targets that share the K and I region of the H-2 complex with the stimulator (or effector) cell are lysed to the same extent as the specific targets, while targets that share the I region only are not. When only the D region is shared, a weak cytotoxic effect is observed. Therefore, the stimulator (or effector) and target cell must share the K or D but not the I region of the H-2 complex in order for optimal cytotoxicity to occur. Spleen cells sensitized to irradiated TNP-modified H-2-allogeneic cells are cytotoxic to these specific cells. Coculture of F1 hybrid cells with irradiated TNP-modified parental cells result in a cytotoxic effect against only those specific parental cells and not TNP-modified cells from the other parent. The cytotoxic effect of the F1 effector cells in the cell-mediated lympholysis test is blocked by the addition of unlabeled TNP-modified targets that are H-2 syngeneic with the sensitizing parental strain, but not H-2 syngeneic with the other parental strain. These data demonstrate that the specificity of the effector cell in this syngeneic cytotoxicity system is directed against altered self H-2-controlled-gene products, rather than a requirement for sharing of histocompatibility genes between effector and target cell in order for lysis to occur. The role of H-2 antigens in determining the sensitivity of a target cell to T-cell-mediated lysis is discussed.

Leukaemic transformation of f1-hybrid cells after inoculation of parental leukaemic cells.

An aneuploid chromosomal anomaly was observed in more than 90% of the mitoses of a spontaneous acute lymphatic leukaemia in C57BL/6J mice preserved by passage in the strain of origin since 1967. The leukaemia is strain-specific and causes the death of its recipients within 9 days. After repeated transfers within F1-hybrids tagged with the T6-marker chromosome, mitoses containing both the chromosomal anomaly of the leukaemic cells and the T6-marker of the semiallogeneic host were observed. This evidence of malignant transformation is compared to leukaemic transformation as observed after clinical bone marrow transplantation.

SPECIFIC UNRESPONSIVENESS TO SKIN ALLOGRAFTS IN MICE

The strain-specific unresponsiveness to H-2 incompatible skin allografts induced by treatment of adult mice with single inoculations of donor strain liver extract and Bordetella pertussis vaccine, as well as three doses of antilymphocyte serum, has been investigated by several in vivo and in vitro methods, with a view to elucidating it mechanism. Lymphoid cells from mice with long surviving skin grafts were found to be reactive in graft-versus-host assays (as measured by splenomegaly or popliteal lymph node enlargement), and mixed lymphocyte culture tests gave positive results. Attempts to cause lethal runting of F1 hybrid mice injected at birth with spleen cells from unresponsive mice gave variable results. However, the injection of F1 hybrid cells into the footpads of unresponsive animals failed to elicit a significant host-versus-graft response. Although lymphoid cells from unresponsive animals did not include detectable numbers of cytotoxic cells, such cells could be generated by previous in vitro mixed lymphocyte culture stimulation or, to some degree, by the injection of the animals with F1 hybrid cells. Attempts to prevent mixed lymphocyte culture stimulation or cytotoxicity with serum from unresponsive mice failed at the serum concentrations used. The data indicate that long-term unresponsiveness in this system is maintained by the production in the hosts of factors that interfere with the cell-mediated response.

Histoincompatibility Recognition Reaction by Irradiated or Mitomycin-Treated Parental Cells against Hybrid Spleen Cells Produces Hybrid Cell Proliferation and Illusion of Reaction of Hybrid against Parent

Abstract The addition of heavily irradiated or mitomycin-treated spleen cells to cultures containing syngeneic spleen cells and foreign erythrocytes may stimulate to some extent the hemolytic plaque-forming cell responses of these cultures, but the addition of semi-allogeneic irradiated or mitomycin-treated spleen cells may cause much greater stimulation. This semi-allogeneic stimulation is completely reciprocal; the plaque-forming cell responses of F1 hybrid spleen cell cultures often are stimulated as much after the addition of non-proliferating parental cells as are the responses of parental spleen cell cultures after the addition of non-proliferating F1 hybrid cells. The possibility that an in vitro manifestation of hybrid resistance might be involved was ruled out. It was found that irradiated or mitomycin-treated cells, although unable to proliferate, were still able to initiate a recognition reaction against histoincompatible cells, releasing in the process a soluble factor which stimulated proliferation of cells of the antibody-forming series. The significance of this finding for “one-way” mixed lymphocyte reactions is discussed.

Modulation of the H-2 antigenicity on the surface of murine peritoneal cells.

Incubation of peritoneal cells in H‐2 antisera abrogated specifically their sensitivity to the cytotoxic effect of these H‐2 antisera and complement. Incubation of the cells in antisera to either the O‐ or Ly alloantigens did not affect their sensitivity to H‐2 antisera. In F1 hybrid cells, H‐2 antigens determined by one of the parental strains could be modulated by H‐2 antibodies, without affecting the expression of the allelic H‐2 antigens determined by the other parent. Modulation of the H‐2 antigenicity of peritoneal cells resulted from an active metabolic process in the treated cells. The phenomenon was inhibited by exposure of the cells to sodium azide, 2,4 dinitrophenol, iodoacetate, corticosteroid hormones, or colchicine. In contrast, cytosine‐arabinoside, cycloheximide, puromycin, and chloramphenicol had no significant inhibitory effect. It is suggested that modulation of the H‐2 antigenicity of peritoneal cells results from redistribution of the H‐2 antigenicity as a result of pinocytosis.

Quantitative studies on antigenic recognition. IV. Specific inhibition of the recognition process by alloantisera.

AbstractIn mixed spleen cell cultures in which one partner was of parental strain origin and capable of recognizing transplantation antigens of the other partner, which was of a corresponding F1 hybrid origin, recognition of antigens was revealed by the elaboration of a product of antigenic recognition (PAR) in culture super‐natants. Recognition of antigens could be blocked by neutralizing them with alloantisera obtained from animals that had rejected allogeneic skin grafts. Inhibition was highly specific in that serum treatment of F1 hybrid cells blocked only one or the other of the two antigenic specificities present. In none of the situations tested did these alloantisera interfere with recognition when used to treat parental strain cells. It could also be shown that putative neutralization of antigens on parental strain aggressor cells did not affect their ability to recognize.As revealed by inhibitory titers, alloantisera were of different strengths. Activity could be demonstrated in sera of highly incompatible strain combinations of rats and mice, but also in those of Syrian hamsters. Furthermore, alloantibody activities were shown to be present in sera of mice following rejection of non‐H‐2 different skin grafts and even in an antiserum resulting from the weak H‐Y determined incompatibility.