Close Association of Virus-Specific Cell Surface (S) and H-2 Antigens in Ad12-Infected and -Transformed Mouse Cells

A virus-specific cell surface (S) antigen in adenovirus type 12 (Ad12)-transformed mouse cells has been assumed to be a direct target for cytotoxic thymus-derived lymphocytes (CTL). In this study, the spatial proximity between the S and H-2 antigens was determined by three different methods, the proximity and co-capping tests, and the test for blocking of CTL-mediated lysis by anti-H-2 serum. In the proximity test with Ad12-infected thymic and splenic lymphocytes, and an Ad12-transformed line of C3H/He (H-2^k) mouse cells, anti-H-2^k and anti-S sera reciprocally inhibited fluorescent-antibody staining of the opposite antigens. By contrast, anti-Thy-1, 2 serum as well as anti-Ia and anti-Ig sera failed to show any appreciable effect in this test, when paired with anti-S serum. In addition, the S and H-2 antigens co-capped in the infected thymic lymphocytes, and CTL-mediated lysis of the transformed cells was abrogated equally by treatment of cells with anti-S and anti-H-2 sera. These results clearly demonstrate that there is a close proximity between the S and H-2 antigens on the surface of Ad12-infected and -transformed mouse cells.     

H-2 antigens incorporated into phospholipid vesicles interact specifically with allogeneic cytotoxic T lymphocytes

Subcellular fractions containing different H-2 antigens were tested for their ability to inhibit specific T cell-target cell conjugate formation. H-2-containing membrane vesicles, lentil-lectin-purified H-2 antigens solubilized with detergent (referred to in the text as high-density fraction) or incorporated into lipid vesicles, inhibited T cell-target cell conjugate formation effectively and specifically. However, two- to threefold more protein was required to inhibit T cell-target cell conjugate formation when detergent-solubilized lentil-lectin-purified H-2 antigens were tested. This suggests that a lipid matrix is advantageous for interaction with anti-H-2 T-cell receptors. Experiments were also undertaken to demonstrate specific binding of liposomes containing ¹²⁵I-labeled H-2 antigen to anti-H-2-specific cytotoxic T lymphocytes (CTLs). The binding of the ¹²⁵I-labeled H-2-containing liposomes was saturable and was specifically inhibited by unlabeled H-2 antigens. Monospecific anti-H-2 sera specifically inhibited the binding of liposomes containing H-2 antigen to the CTLs. The results suggest that a specific interaction can occur between serologically defined H-2 antigens and the receptor of anti-H-2 CTLs.     

The spatial relationship of the viral and H-2 antigens recognized by anti-viral CTLs

With the use of monospecific rabbit anti-G protein and mouse monoclonal anti-H-2K^k, we have analyzed the spatial relationship of the serologically defined H-2K^k antigens and the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) to those antigens recognized by B10.A (k, d) anti-VSV cytotoxic T lymphocytes (CTLs). The ability of monoclonal anti-H-2K^k or rabbit anti-G protein to inhibit specifically the cytolytic activity of B10.A anti-VSV CTLs indicates that the G protein and the H-2K^k molecules are in close proximity to the viral and H-2K^k antigens recognized by the anti-VSV (CTLs. By the method of sequential immunoprecipitation, we also demonstrated that only 10–30 percent of the serologically defined G and H-2K^k molecules are in theG-H-2K ^k complexes.Abbreviations used in this paper Con A Concanavalin A - cpm counts per minure - CTLs cytotoxic T lymphocytes - E: T ratio effector: target ratio - G major surface glycoprotein of VSV - MHC major histocompatibility complex - MOI multiplicity of infection - NP40 Nonidet-P40 - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - UV ultraviolet light     

Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 antigens.     

Biochemical evidence for a separate,MHC-linked locus encoding H-2.28 antigens

In comparing the tryptic peptide maps of the H-2L and H-2D glycoprotein antigens isolated from NP-40 lysates of RADA1 (H-2 ^a ) leukemic cells, no more than 37% of the observed arginine-containing tryptic peptides are found to be homologous. Thus, the primary amino-acid sequences of these two antigens are probably less than 90% homologous. This constitutes the strongest evidence to date that the MHC-linkedH-2L region encodes H-2L antigens separately from theH-2D region, even though H-2L antigens bear D-end-associated antigenic determinants of the H-2.28 family. The anti-H-2.28 alloantiserum (k×r anti h2) used to precipitate H-2L antigens in this investigation was the NIH contract antiserum D28b. As the tryptic peptide maps also surprisingly revealed, D28b precipitates H-2D antigens as well and, thus, anti-H-2.4 immunoadsorbants were employed to isolate H-2L free of H-2D antigens. In light of the dual specificity of D28b, its reactivity with BALB/c-H-2 ^dm2 mutant cells was re-examined. Even though mutant lymphocytes, which lack H-2L but not H-2D antigens, are not cytotoxically lysed by D28b (as are parental H-2^d cells), D28b appears to precipitate H-2D antigens from NP-40 extracts of mutant splenocytes.     

Interspecies exchange of β 2-microglobulin and associated MHC and differentiation antigens

Radiolabeled human ₂-microglobulin (₂m) can bind to mouse histocompatibility (H-2) antigens on the cell surface or to partially purified H-2 antigens in solution. The complexes containing human ₂m and H-2 antigens from C3H (H-2^k) mice could be immunoprecipitated specifically with alloantisera, rabbit anti-H-2 xenoantisera, and with monoclonal H-2-specific antibodies. Specific association with H-2 antigens was also observed with other haplotypes. The only exception was B10.D2 (H-2 ^d ) from which complexes containing human ₂M could only be precipitated with anti-H-2 xenosera. Thus radiolabeled human ₂M can be used as a specific label for mouse H-2 antigens in precipitation and radioimmunoassays. The application of this finding extends to major histocompatibility complex antigens of other species, and to differentiation antigens with primary association with ₂m.Abbreviations used in this paper MHC major histocompatibility complex - ₂m ₂-microglobulin - LcH Lens culinaris hemagglutinin     

Polymorphism of minor histocompatibility genes in wild mice

H-2^b-restricted cytolytic T lymphocytes (CTL) were generated against H-1, H-3, and H-4 antigens and tested against target cells of F₁ hybrids between wild mice and inbred H-2 ^b mice. The congenic strain combinations for the CTL production were such that they tested one allele each at the H-1 and H-4 loci and four alleles at the H-3 locus. Most of the wild mice tested came from Southern Germany, but a few mice came from other European countries and Egypt and Israel. Virtually all wild mice typed as positive with CTL directed against H-3^b and H-4^b antigens; 32% of the F₁ hybrids tested reacted with anti-H-1^cCTL and 9% reacted with anti-H-3^d CTL. The positive results were not caused by cross-reaction with allogeneic H-2 antigens controlled by the major histocompatibility complex (Mhc) genes of the wild mice. At least some of the H-3 and H-4 antigens detected by the CTL in the F₁ hybrid were not identical with antigens of the immunizing strains. These results suggest a relatively low degree of polymorphism of the tested minor H loci in wild mice and further support the notion that minor H loci are unrelated to the Mhc.     

Association between H-2 and vaccinia virus-induced antigens on the surface of infected cells.

The relationship between H-2 molecules and vaccinia virus-induced antigens on the surface of H-2d infected cells was investigated by the differential redistribution method and by the blocking capacity of monospecific anti-H-2 sera on an anti-vaccinia cell-mediated cytotoxicity (CMC). Capping of either H-2K or H-2D molecules upon addition of monospecific and anti-H-2 sera was followed by the complete redistribution of viral antigens, suggesting the formation, on the cel membrane, of complexes of H-2K, H-2D molecules and vaccinia virus-induced antigens. However, not all H-2 molecules were involved in this association since i) free H-2K and H-2D molecules still moved independently on the cell surface, and ii) capping of vaccinia virus-induced antigens failed to induce the redistribution of all the H-2K and H-2D molecules. In addition, either monospecific anti-H-2K or anti-H-2D antiserum was found to exert potent blocking activity on anti-vaccinia CMC, indicating also a close topographical relationship between H-2K, H-2D molecules and vaccinia virus-induced antigens.     

H-2 histocompatibility antigens of subcellular membranes of mouse liver

Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE.     

Lateral diffusion and patch formation of H-2Kk antigens on mouse spleen lymphocytes

We have studied the diffusion and aggregation of H-2K^k antigens labeled with a fluorescent anti-H-2K^k monoclonal antibody (IgG) on mouse splenic lymphocytes, employing fluorescence photobleaching recovery and fluorescence microscopy. The H-2K^k antigens were initially distributed homogeneously on all lymphocytes. Upon antibody binding, sub-micron patches were formed on 50–60% of the cells. A lateral diffusion coefficient, D, of 7.1·10^?10 cm²/s and a mobile fraction of 0.73 were found for H-2K^k antigens on diffusely-labeled cells, while these antigens were immobile (D?5·10^?12 cm²/s) on patched cells. The patched and nonpatched sub-populations did not correspond to B- and T-lymphocytes. Subjection to low temperature or treatment with NaN₃ or cytoskeleton-disrupting drugs did not affect the diffusion or patching of H-2K^k, indicating no involvement of metabolic energy or drug-sensitive cytoskeletal components. These findings could be related to the interactions of H-2 antigens on the cell surface, and to the different susceptibilities of various cells to lysis by cytotoxic T-cells.     

Isolation and analysis of H-2 and Ia alloantigens from wild mouse strains.

Immunoprecipitation and SDS-PAGE analysis were used to examine H-2 and Ia antigens from mouse strains with wild-derived MHC haplotypes. Antisera raised against the wild-derived strains contained anti-H-2 and anti-Ia antibodies which precipitated antigen molecules readily distinguishable by a single assay procedure. The antibodies to wild strains were cross-reactive with standard laboratory haplotypes. Evidence supporting the similar genetic organization of wild and standard haplotypes was found, including isolation of separate H-2K and H-2D molecules from a wild-derived strain, and isolation of two separate Ia molecules from a wild-derived strain.     

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

H-2 antigens incorporated into phospholipid vesicles elicit specific allogeneic cytotoxic T lymphocytes

Different H-2 antigen-containing subcellular fractions were tested for their ability to elicit specific anti-H-2 cytotoxic T lymphocytes (CTLs). Intact cells and membrane vesicles were capable of eliciting strong anti-H-2 primary and secondary CTL responses. However, detergent-solubilized H-2 antigens partially purified with lentil lectin were greatly reduced in their capacity to elicit secondary anti-H-2 (CTLs) and at all amounts tested did not elicit a primary CTL response. Lentil lectin-purified H-2 antigens incorporated into egg lecithin plus cholesterol (30% w/w) vesicles elicited a strong secondary anti-H-2 CTL response and a low but significant primary anti-H-2 CTL response. The results also indicate that T-cell-defined specificities are closely associated with serologically defined private and public specificities.     

Further studies on non-H-2 histocompatibility alloantibodies in the mouse

Immunofluorescence tests indicate that alloantibodies specific for mouse histocompatibility antigens H-1^a, H-3^a, and H-13^a have been produced, using four different immunizations. Furthermore, an immunization employing donors and recipients which were H-2^k at the MHC produced stronger anti-H-3^a and anti-H-13^a than did immunizations where donors and recipients were H-2^b at the MHC.     

Abelson virus-transformed lymphocytes: null cells that modulate H-2.

A-MuLV-transformed lymphoid cells from Balb/c mice had the properties of null lymphocytes. They did not secrete Ig and all but one did not have detectable cell-associated Ig; one line synthesized, but did not secrete, the mu chain of IgM. The cells expressed H-2D and H-2K, but not H-21 histocompatibility antigens or theta-antigen; they had Fc receptors. Most cell lines grew to form donor cell tumors after inoculation into (Balb/c X C57B1/6)F1 mice. The tumor cells have more H-2Dd than cells passaged in vitro. Cell lines carried in vitro progressively lost H-2Dd. A line in which 5-30% of the cells were lysable by anti-H-2Dd was cloned; all eleven clones had H-2Dd (13-69% lysable) demonstrating that H-2 modulates in vitro. A clone with little H-2Dd (10-15% lysable) was tumorigenic even after treatment with anti-H-2Dd sera; at least 50% of the tumor cells were lysed by anti-H-2Dd. Thus A-MuLV-transformed lymphocytes modulate H-2 in vivo to higher levels and in vitro to lower levels.     

Cell-Mediated Immunity to Mouse Adenovirus Infection

Migration of peritoneal exudate cells (PEC), which were prepared from mice immunized against mouse adenovirus (M-Ad), was inhibited upon exposure to the antigenic extract of M-Ad-infected cells. This inhibition was shown to be blocked when infected cells or their extracts were pretreated with antiserum against M-Ad-induced cell surface(s) antigen(s) or with antisera against alloantigens of infected cells. Immune spleen cell-mediated cytolysis of M-Ad-infected cells was also blocked in the presence of anti-S, anti-alloantigen or anti-β₂m serum. Immunofluorescent antibody staining of S antigen(s) was blocked when infected cells were pretreated with anti-alloantigen or anti-β₂m serum, whereas it was not blocked when they were pretreated with anti-mouse immunoglobulin or anti-Thy-1.2 serum. Conversely, immunofluoresent antibody staining of alloantigens was blocked when infected cells were pretreated with anti-S serum. These findings indicate that S and alloantigens are associated with each other or at least located very close to each other.     

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.     

Dissociation ofH-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2 ^b /H-2 ^b ). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (allH-2 ^b /H-2 ^b ), FY7 failed to induce the primary in vitro generation of anti-H-2^b CTL by (B10.A x A)F₁ (H-2 ^a /H-2 ^a or (B10.D2 x BALB/c)F₁ (H-2 ^d /H-2 ^d ) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2^b CTL. Quantitative absorption tests with H-2K^b and H-2D^b antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2K^b product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2K^b CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2K^b-gene product expressed by FY7.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PAGE polyacrylamide gel electrophoresis     

Differential lack of class I H-2d antigen expression by sublines of the BALB/c S49 T cell lymphoma

Five different sublines of the BALB/c murine S49.1 T cell lymphoma were found to exhibit distinct patterns of absence of detectable H-2d class I major histocompatibility antigen expression. The results were demonstrated and verified by a) the generation of H-2Kd-, H-2Dd,Ld-, and H-2Ld-specific cytotoxic T lymphocytes that were assayed on S49.1 target cell lines, b) antibody-mediated cytotoxicity with the use of anti-H-2d monoclonal reagents, and c) flow microfluorometry. The five lines investigated were S49.1, T-25, T-25ADH, Thy-1-, and 100/0. None of these lines expressed detectable levels of Ld. S49.1 expressed both Kd and Dd, T-25 and T-25ADH expressed Dd but not Kd or Ld, Thy-1- expressed Kd but not Dd or Ld, and 100.0 did not express any detectable amounts of Kd, Dd, or Ld. These results indicate that K and D (and L) antigens can be expressed independently of each other and suggest that expression of class I antigens is controlled in a locus-specific manner.     

Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.Abbreviations used in this paper MMTV mammary tumor virus - MuLV murine leukemia virus - MHC major histocompatibility complex - IEM immunelectron microscopy