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A method was developed to apply flow cytometry analysis to the characterization of individual phagosomes. Macrophages were incubated with latex beads and homogenized to release the phagosomes. Intact cells and nuclei were removed by low speed centrifugation, and a crude phagosome preparation was fixed with paraformaldehyde. Distinct optical properties of latex bead phagosomes allowed their analytic isolation from other organelles and cell fragments by flow analysis using a narrow gate based on scatter parameters. Furthermore, separate gates were established for phagosomes containing one, two and even three beads, which were sorted and examined by electron microscopy (EM). EM showed that the phagosomal membrane was closely apposed to the latex bead in most phagosomes, but some more spacious phagosomes were also observed. Phagosomes were immunolabeled and subjected to flow analysis for MHC-I and MHC-II molecules and lysosomal membrane markers (LAMPs). The proportion of LAMP-positive phagosomes increased with incubation time, reflecting maturation of phagolysosomes. Significant staining for MHC-I and MHC-II was demonstrated and remained relatively constant with time. Flow analysis of phagosomes allows the characterization and comparison of individual phagosomes, and the identification of subpopulations of phagosomes with differing membrane compositions. It also provides the advantage of analytically isolating phagosomes from other components of the cell without the need for extensive prior physical purification. Thus, it can be used to rapidly assess changes in phagosomal membrane composition as a function of phagosome maturation.

The aim of this study was to determine what factors induce major histocompatibility complex (MHC) molecules on the mouse small intestinal epithelium by using immunohistochemical methods. In germ-free mice, although MHC class I molecules such as H-2K and thymus leukemia antigen (TLa) were expressed on the small intestinal epithelium, class II molecules were absent. The introduction of microorganisms into germ-free mice induced characteristic MHC molecules on the small intestinal epithelial cells. The I-A molecule was induced on the villus tip and crypt epithelial cells 7 days after conventionalization, and the I-E molecule was induced on the mid villus and crypt epithelial cells 14 days after conventionalization. The staining intensity of the H-2K molecules was increased 4 days after conventionalization. In contrast, TLa did not change during conventionalization of germ-free mice. These results suggest that the expression of MHC molecules, except for the TLa, is greatly dependent on the presence of intestinal microorganisms.

Hybrid resistance (HR) is primarily controlled by the genes of the Hemopoietic histocompatibility-1 (Hh-1) locus within the H-2 complex. HR is a consequence of the Hh-1-controlled target determinants in homozygous parental strain mice and their absence in heterozygous F1 hybrid mice. To examine the mechanism that controls the Hh-1 phenotype, three independent clones of somatic cell hybrids between parental lines EL-4 (C57BL/6 origin, H-2b) and R1 (C58 origin, H-2k) were studied. The line EL-4 is Hh-1b-positive and is subject to HR by H-2b heterozygous F1 mice, but R1 lacks the Hh-1b allele and is not susceptible to HR. Of the three hybrid clones, F263.2 is Hh-1b-positive, whereas the other two, F262.2 and F264.2, are Hh-1b-negative, as judged by these cells' capacity to compete in vivo with the grafted parental C57BL/6 bone marrow cells in the resistant (C57BL/6 x C3H)F1 mice. All three clones express the H-2b and H-2k class I antigens equally well, are susceptible to activated NK cells to the same extent, and all carry four copies of chromosome 17. However, Southern analysis reveals that clone F263.2 contains three copies of H-2b chromosome and one H-2k, whereas the other two clones carry two copies each of the parental chromosome 17. The results suggest that the relative copy number of specific alleles is the crucial determinant of the Hh-1 phenotype, and render unlikely both the gene dosage hypothesis and the trans-acting dominant suppression hypothesis to account for the noncodominant expression of the Hh-1 phenotype.

Trophoblast cells of the placenta represent the frontier of the conceptus at the feto-maternal interface, exposed to the maternal immune system. The presence or the absence of major histocompatibility (MHC) antigens on trophoblast cells, thus, is a key determinant of immune interactions between the mother and the allogeneic fetus during pregnancy. This article critically reviews the studies of the alloantigenic status of murine and human trophoblast cells, including the published data in the mouse and unpublished data in the human in the authors' own laboratory. It is shown that, in both these species, a significant proportion of trophoblast cells express Class 1 but not Class 2 MHC antigens in a manner directly accessible to the maternal immune system. However, the lack of immunogenicity of semiallogeneic trophoblast cells can not solely be explained on the basis of the absence of Class 2 molecules, despite the presence of Class 1 molecules. It is proposed either that the Class 1 MHC antigens on the trophoblast cells are presented in a nonimmunogenic manner to the maternal T cells or else that certain cells at the feto-maternal interface (eg, fetally derived trophoblast cells, maternally derived lymphoid cells, or decidual cells) elaborate potent immunosuppressor molecules capable of abrogating T cell alloreactivity.

Differences in the expression of minor histocompatibility (Hm) alloantigens on two mouse embryonal carcinoma (EC) cell lines and the PYS-2 and T.D.M.-1 differentiated derivatives have been demonstrated by their ability to elicit a cytolytic T-lymphocyte (CTL) response. Experiments involving the use of various responder-target strain combinations on the one hand and Recombinant Inbred (RI) mice strains on the other have shown that: (i) there are major differences in Hm expression on the EC cells compared with the differentiated derivatives whose Hm expression appears more akin to that of adult splenocytes; (ii) although both EC cell lines show reduced Hm immunogenicity compared with adult splenocytes, major differences in the expression and possibly presentation between the F9 and PCC3 EC cell lines can be detected both by in vivo priming and by in vitro cold competition target experiments. These results are discussed in connection with the unexpected finding that some EC cell lines are capable of specific competition effects for appropriate CTL effectors despite their inability to stimulate such effectors in vitro and the absence of major histocompatibility complex (MHC) products.

Increased expression of major histocompatibility complex (MHC) antigens may occur following liver transplantation. The mechanism by which this occurs is unclear. Postoperative complications may result in cholestasis. We have investigated the possibility that cholestasis may cause increased expression of MHC antigens in the liver. Cholestasis was induced in rat livers by bile duct ligation and transection. Total serum bilirubin rose markedly postoperatively and remained elevated over a period of 21 days. Samples of bile taken from rats were shown to remain sterile during the study period and there was no evidence of viral infection. Liver tissue taken 1, 3, 7, and 21 days postobstruction showed a marked increase in the expression of rat class I MHC antigens but not class II antigens. We suggest that cholestasis itself can induce increased MHC class I antigens in the liver. Therefore, if cholestasis occurs postoperatively in the human liver transplant recipient this might lead to increased expression of human class I MHC antigens and add to the overall cellular immune activity in a rejection reaction, although cholestasis probably cannot initiate rejection.

The UV-induced, C3H fibrosarcoma, 1591, expresses at least three unique MHC class I antigens not found on normal C3H tissue. Here we report the complete DNA sequence of the three novel class I genes encoding these molecules, and describe in detail the recognition of the individual products by tumor-reactive and allospecific CTL. Remarkably, although C3H does not appear to express H-2L locus information, this C3H tumor expresses two distinct antigens, termed A149 and A166, which are extremely homologous to each other and to the H-2Ld antigen from BALB/c. The gene encoding the third novel class I antigen from 1591, A216, is quite homologous to H-2Kk) throughout its 3' end. Since all three of these genes account for polymorphic restriction fragments not found in C3H, it is likely that they were derived by recombination from the endogenous class I genes of C3H. The DNA sequence homology of A149, A166, and H-2Ld is especially significant given the functional conservation observed between the products of these genes. Limited sequence substitutions appear to correlate with some of the discrete serological differences observed between these molecules. In addition, both A149 and A166 crossreact, but to differing extents, with H-2Ld at the level of T cell recognition. Our results are consistent with the view that CTL recognize complex conformational determinants on class I molecules, but extend previous observations by comparing a set of antigens with discrete and overlapping structural and functional differences.

We have attempted to generate in vitro lymphocytes cytotoxic to a widely studied model of ovarian cancer in C3HeB/FeJ mice. These attempts were unsuccessful with either syngeneic or allogeneic spleen cells. The following experimental results demonstrated that this murine ovarian tumor lacks histocompatibility antigens. (a) Tumor cells were not lysed by allogeneic lymphocytes presensitized to H-2k spleen cells. (b) Tumor cells did not specifically inhibit the cell-mediated lysis of H-2k spleen cells by presensitized allogeneic lymphocytes. (c) Histoincompatible (H-2b or H-2d) and syngeneic (H-2k) mice all died with identical tumor growth patterns within 25, 30, or 35 days following the i.p. inoculation of 10(6), 10(5), or 10(4) tumor cells, respectively. (d) Tumor cells were not lysed by an anti-H-2k antiserum and complement. (e) Absorption of the anti-H-2k antiserum with tumor cells did not decrease the cytotoxicity of the antiserum. (f) Competitive inhibition of a radioimmunoassay and polyacrylamide gel electrophoresis of immunoprecipitate of radiolabeled tumor extracts failed to demonstrate an H-2 heavy chain, although a normal amount of beta-microglobulin was present. This lack of histocompatibility antigens may explain the failure to generate lymphocytes cytotoxic to this tumor. Thus, this murine ovarian tumor, which has a serologically detectable tumor-associated antigen and can be cured by nonspecific immunotherapy, may provide an excellent model for the study of successful immunotherapy in the absence of histocompatibility antigens and associated cell-mediated reactions.

Calcified cartilage transplants induce bone formation. This process may be inhibited if the recipient is immunized by the transplant. To study the relation between the degree of antigen incompatibility between the donor and recipient and bone formation in more detail, chondrocytes isolated from cartilaginous epiphyses of five-day-old mice were transplanted within a fully compatible syngeneic system and across weak (H-Y, non-H-2) and strong (H-2) histocompatibility barriers. Reconstruction of cartilage occurred in all cases. In these transplants, which did not evoke immunologic reaction (fully compatible system, transplants across H-Y barrier in nonrejector strain), reconstructed cartilage hypertrophied, calcified, yielded to resorption by mesenchyme, and finally, was replaced by bone. When (independently of the degree of antigenic difference) cartilage was surrounded by mononuclear infiltration, bone formation did not occur or was delayed. The presence of infiltrations around transplants led to the degeneration of chondrocytes as well as of matrix in peripheral regions of cartilage. Immunologic infiltration may prevent endochondral osteogenesis by inhibiting cartilage invasion by vascularized mesenchyme, and/or interfering with matrix mineralization. The function of the chondrocyte is not yet defined, but in endochondral ossification, it plays more than a passive role.

A feature of the expression of the tissue-specific class I antigen Qa-2 is the quantitative variation among mouse strains. Recently, the class I gene Q7 has been shown to encode a protein product that is biochemically indistinguishable from the lymphocyte-bound Qa-2 molecule. Utilizing gene transfection, we have identified a second Qa-2 subregion class I gene (Q9), in H-2b mice, which encodes a polypeptide biochemically similar to the Q7 and the Qa-2 polypeptides. Furthermore, we have observed that cell lines transfected with the allelic forms of the Q7 gene from C57BL/10 (Qa-2hi) or BALB/C (Qa-2low) display quantitative differences in cell-surface expression. Based on these studies, we suggest that gene dosage and allele-specific variation in cell-surface expression contribute to the strain-specific variation in the levels of Qa-2 antigen expression.

We have shown that cytotoxic lymphocytes generated in primary cultures of NZB spleen cells with H-2-identical BALB/c or B10.D2 stimulator cells exhibit specificity for Qa-1b-associated antigenic determinants. This unidirectional cytotoxicity constitutes the initial demonstration of a primary in vitro response to antigens of the Qa-Tla system. Such responses do not require H-2 homology between effector and target cells in the assay system. In fact, when H-2Dd homologous target cells were employed there was little, if any, evidence for development of primary H-2-restricted responses to minor locus histocompatibility antigens or viral antigens. In view of the recently defined role of Qa-1+, Ly-1,2,3+ cells as regulators of antibody responses, and of the deficiency of such cells in NZB mice, the observation of hyperreactivity for determinants of this system may be relevant to the development of autoimmunity in these animals.

We have investigated, using serological and biochemical assays, the specificity of an A.TH anti-A.TL-derived monoclonal antibody (mAb), designated 40.B, directed at a highly conserved antigenic determinant expressed on the majority of murine and human MHC class II antigens. In the mouse, mAb 40.B defines a new specificity expressed on the Ia products of the H-2 haplotypes K, d, b, v, r, p, u, j and w3. Analysis of its reactivity with H-2 recombinant strains and the results of the competitive binding inhibition of 125I-labeled mAb 40.B to B10.BR cells with I-Ak and I-Ek specific mAb suggested recognition of a shared Ia determinant expressed on both I-Ak and I-Ek molecules. This has been confirmed by sequential immunoprecipitation studies which demonstrated the specificity of mAb 40.B for the Ak beta Ak alpha, Ab beta Ab alpha, Ad beta Ad alpha, Ek beta Ek alpha, Es beta Ek beta and Ed beta Ed alpha dimers. In humans, this mAb bound to and immunoprecipitated HLA-D/DR molecules expressed on lymphoblastoid cell lines carrying the MT1 and MT2 supertypic specificities. The possible implications of these findings with regard to an evolutionary model of MHC class II antigens are discussed.

The ability to generate specific cytotoxic responses using purified major histocompatibility complex (MHC) antigen in liposomes has made it possible to directly assess the importance of class I carbohydrate moieties in T cell recognition of alloantigen. Deglycosylation of affinity-purified H-2Kk to yield a single glycan-free product did not alter the specificity, the magnitude, nor the dose range of the cytotoxic T lymphocyte (CTL) response to the class I antigen. It can be concluded that carbohydrate moieties are not required to maintain the necessary conformation of the MHC protein, nor to interact with either the antigen-specific receptor or accessory proteins on precursor CTL.

Thymuses of various types of bone-marrow-chimeric mice have been examined by tissue section immunologic staining for the presence and distribution of major histocompatibility complex (MHC) antigens. Cortical and medullary thymic epithelial cells continue to express thymus genotype I-A and H-2K/D antigens for at least 6 months posttransplantation. The appearance of bone-marrow-type MHC antigens is limited to low levels of H-2K/D on cortical and medullary lymphocytes, and to dendritic cells in the medulla; the medullary dendritic cells express high levels of donor-type I-A antigens as soon as 3 weeks posttransplantation. The observed patterns support the concept that I-A antigens are synthesized by thymic epithelial cells but are acquired by thymocytes. The findings are of relevance to the understanding of the role of the thymus in the generation of MHC restriction.

There is increasing support for the concept that in all of their varied immunologic functions, T lymphocytes act through a dual specificity. In the case of intracellular viral infections, the recognition required for cytolysis involves both a "self" component, coded for by the genes that specify the major transplantation antigens, and an "X" component, provided by viral antigens expressed on the target-cell surface.

A large number of fibrosarcoma cell lines was established in vitro from a tumor mass induced freshly by inoculating 3-methylcholanthrene (MCA) subcutaneously (sc) into C3H/HeN mice, and more than five clones were isolated from each cell line by the limiting dilution technique. The present study investigated a) qualitative and quantitative comparison of the immunogenicity [tumor-associated transplantation antigen (TATA) activity] of different tumor clones and b) the relationship between such immunogenicity and the expression of H-2 class I antigens. When TATA were compared between different clones from the same tumor, these TATA were revealed to be cross-reactive to each other. On the other hand, the comparison of TATA between clones from different tumors demonstrated the existence of individually unique TATA in these clones. In addition to qualitative heterogeneity of TATA from different tumors, the magnitude of immunogenicity was also heterogeneous in the individual clones established. Whether or not such quantitative heterogeneity of immunogenic strength was related to the expression of H-2 (class I) antigens was examined by flow microfluorometry studies using anti-H-2k antibodies. The results demonstrated that there was no correlation between TATA activity capable of inducing in vivo tumor resistance and the expression of H-2 antigens. This contrasted with parallelism between the expression of H-2 antigens and inducibility of cytotoxic T lymphocytes (CTL) or lysability of tumor cell clones by CTL. These results are discussed in the context of the cellular mechanism of tumor cell eradication in vivo and the regulation of cell surface H-2 expression in vitro and in vivo.