Specificity of H-2 antigens expressed on mouse blastocysts

A highly sensitive cytotoxicity assay was used to detect H-2 antigens on mouse blastocyst stage embryos of the b, a, k, and d haplotypes. The assay was based on the principle that live embryos incorporate 3H-thymidine into DNA whereas embryos killed with antiserum and complement do not. The use of specific alloantisera showed that blastocysts of different haplotypes express different H-2 antigens. Thus, positive evidence was obtained for the expression of Kd and Dk molecules and negative evidence for the expression of Db, Kk, and Dd molecules. Evidence was also obtained that blastocysts express different H-2 antigens than those found on adult lymphocytes. Unexpected cross-reactions were found when some of the alloantisera were tested on blastocysts of different haplotypes. It is proposed that the aberrant expression of H-2 antigens on embryos might facilitate their escape from surveillance by the maternal immune system.     

DNA sequence analysis of the C3H H-2Kk and H-2Dk loci. Evolutionary relationships to H-2 genes from four other mouse strains

We generated nucleotide sequences for H-2Kk and H-2Dk from the C3H mouse, as well as for a genomic clone of H-2Db, in order to conduct an evolutionary analysis of the H-2 genes from three haplotypes, k, d, and b. H-2Kk from both the C3H and AKR strains, H-2Kd, H-2Kb, H-2Dk, H-2Ld, H-2Dd, H-2Db, and H-2Dp DNA sequences were aligned, and the alignments used to construct phylogenetic trees inferring the evolutionary relationships among the nine genes by two independent methods. Both approaches yielded trees with similar topologies. In addition, the sequence alignments revealed patterns of nucleotide substitutions which implicate both point mutation and recombination in the divergence of the H-2 genes. Future considerations for evolutionary analysis of class I genes are discussed.     

Introduction of the H-2Dk gene into a class I-negative tumor cell line confers interferon-gamma inducibility upon the silent endogenous H-2Kk gene.

Kgv cells do not constitutively express class I mRNA or protein. Interferon (IFN)-gamma, but not IFN-alpha/beta, induces H-2Dk expression. IFN does not induce H-2Kk expression. We examined constitutive and IFN-inducible class I expression on Kgv cells stably transfected with genomic clones of H-2Kk or H-2Dk and on somatic cell hybrid lines constructed between Kgv cells and constitutively class I-positive cells of a distinguishable H-2 haplotype. Our results suggest that both the lack of constitutive class I expression and the inability of IFN-alpha/beta to induce class I expression on Kgv cells are primarily due to cis-regulatory mechanisms. However, stable introduction of the H-2Dk gene into Kgv cells conferred IFN-gamma inducibility upon the silent endogenous H-2Kk gene. Therefore, the failure of IFN-gamma to induce H-2Kk expression on Kgv cells is due, at least in part, to a trans-regulatory mechanism.     

Cytotoxic T-lymphocyte tolerance to minor H-43a alloantigen is induced exclusively in the context of the self major histocompatibility complex class I H-2Kb molecules

We elucidated previously that cytotoxic T lymphocyte precursors (CTLp) against H-43a allo-antigen, which we had discovered as a new mouse minor H antigen, were primed in H-43b mice only in the context of self H-2Kb restriction element, and that anti-H-43a CTLp tolerance was induced in H-43b mice by injection with H-43a spleen cells (SC) from H-43 congenic mice, i.e., under the condition of disparity at only the H-43 locus. The present study attempted to determine whether the H-2Kb restriction element for anti-H-43a CTLp priming is also implicated in the induction of anti-H-43a CTLp tolerance. For this purpose, we used a newly established H-43b C3W (H-2k) strain which is H-43 congenic to H-43a C3H/HeN. When (C3W X B10.MBR)F1 (H-43b, H-2Kk/b, Ik/k, Dk/q) mice were injected with H-43a-bearing (C3H/HeN X B10.AKM)F1 (H-43a/b;H-2Kk/k,Ik/k,Dk/q)SC, their selfH-2Kb-restricted anti-H-43a CTLp were were primed (cross-priming). By contrast, injection of H-43a-bearing (C3H/HeN X B10.MBR)F1 (H-43a/b; H-2Kk/b,Ik/k, Dk/q)SC, which differ from (C3H/HeN x B10.AKM) F1 SC solely at H-2K and possess H-2Kb molecules, did not prime but specifically inactivated the anti-H-43a CTLp of (C3W x B10.MBR)F1 mice. These results indicate clearly that anti-H-43a CTLp tolerance is induced exclusively in the context of the H-2Kb element expressed on the antigenic H-43a SC.     

Inability of mice to generate cytotoxic T lymphocytes to vesicular stomatitis virus restricted to H-2Kk or H-2Dk

H-2k mice are unable to generate cytotoxic T lymphocytes (CTL) to vesicular stomatitis virus (VSV). This apparent unresponsiveness is found for both major serotypes of VSV, VSV-Indiana and VSV-New Jersey. CTL unresponsiveness occurs despite the ability of H-2k mice to generate a humoral immune response against VSV that is comparable to that found in responder (H-2b and H-2a) strains. All H-2k mice regardless of background genes, including various Ig allotypes, were found to be nonresponders. H-2k-linked unresponsiveness mapped to both H-2Kk and H-2Dk and occurred despite the presence of responder alleles in (responder x nonresponder)F1 mice. The unresponsiveness cannot be attributed to an inability of VSV-infected H-2k target cells to express viral surface antigens of H-2 molecules. Further, unresponsiveness cannot be overcome by using secondary stimulation in vivo or in vitro. H-2k-linked unresponsiveness does not appear to be due to suppression, and no complementation has been found in various (nonresponder x nonresponder)F1 mice. Thus unresponsiveness to VSV in association with H-2Kk or H-2Dk appears to represent an extensive defect of immune responsiveness that probably occurs because VSV is not a natural mouse pathogen.     

Differential induction of H-2K vs H-2D class I major histocompatibility complex antigen expression by murine recombinant interferon-gamma

The results presented here indicate that recombinant murine interferon-gamma can cause a dramatic differential induction of two distinct class I MHC molecules. Thus, IFN-gamma treatment of the murine leukemia virus (MuLV)-induced AKR SL3 tumor, a cell line that normally expresses moderate levels of class I MHC antigens, resulted in a large increase in H-2Dk expression, but no change or a slight decrease in H-2Kk expression as measured by cytofluorography. Explanations of the selective enhancement of Dk expression based on increased Fc receptor display or differential kinetics of induction were ruled out. The phenomenon was observed over a wide range of doses of IFN-gamma and with two different monoclonal antibodies to Kk, the latter finding making it unlikely that an altered form of the Kk molecule was induced. The same differential induction of the Dk antigen was observed for the LBRM.5A4 tumor cell line. Because LBRM.5A4 is also MuLV+ but of congenic B10.BR (H-2k) origin, these results were consistent with the possibility that such differential induction was associated with the H-2k haplotype and/or MuLV. The implications of these results, as a possible mechanism of tumor cell escape from an immune surveillance system monitored by class I MHC-restricted T cells and as a useful model system to dissect the mechanism of IFN-gamma induction of class I MHC antigens, are discussed.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

Analysis of a defect in the H-2 genes of SV40 transformed C3H fibroblasts that do not express H-2Kk

C3H fibroblasts transformed in vitro with SV40 were adapted to in vivo growth. Several clones were isolated from a single, highly oncogenic tumor and those that displayed oncogenic potential also no longer expressed the H-2Kk molecule. Using the technique of Southern blot hybridization, the H-2 genes and integrated SV40 sequences present in the genomic DNA of several of these clones have been examined and compared with both the parent line and normal liver genomic DNA from C3H mice. All H-2Kk negative clones had altered H-2 genes that appeared as a gain and, depending on the restriction endonuclease, loss of hybridizing fragments compared to normal C3H DNA. A 5.5-kb fragment missing from the Sstl digests of the H-2Kk negative variants was mapped to the H-2Kk region of the major histocompatability complex with the use of congenic mice. This provided direct evidence that a mutation had occurred in the H-2Kk region. The integrated SV40 sequences were similar to those already seen in other SV40 transformed cells and not closely linked to any of the H-2 genes. There was no indication that the H-2 mutation was caused by integration of SV40.     

Deficient major histocompatibility complex-linked innate murine cytomegalovirus immunity in MA/My.L-H2b mice and viral downregulation of H-2k class I proteins

NK cells are key effectors of innate immunity and host survival during cytomegalovirus (CMV) infection. Innate murine CMV (MCMV) resistance in MA/My mice requires Ly49H/m157-independent H-2k-linked NK cell control. Here we show that replacement of MA/My H-2k with C57L H-2b susceptibility genes led to a remarkable loss of innate virus immunity, though NK gamma interferon was induced in H-2b and H-2k strains shortly after infection. Thus, H-2b genes expressed in C57L or MA/My.L-H2b are sufficient in alerting NK cells to intrusion but fail to support NK restraint of viral infection. In addition, novel H-2 recombinant strains were produced and utilized in a further refinement of a critical genetic interval controlling innate H-2k-linked MCMV resistance. Importantly, this analysis excluded the gene interval from Kk class I through class II. The responsible gene(s) therefore resides in an interval spanning Dk class Ia and more-distal major histocompatibility complex (MHC) nonclassical class Ib genes. Recently, the NK activation receptor Ly49P and MHC class I Dk proteins were genetically implicated in MCMV resistance, in part because Ly49P-expressing reporter T cells could specifically bind Dk molecules on MCMV-infected mouse embryonic fibroblasts (MEFs). However, as we found that H-2k innate resistance differs in the C57L or MA/My backgrounds and because MCMV very efficiently downregulates H-2k class I proteins in L929 cells and primary MEFs shortly after infection, a Ly49P/Dk model should not fully explain H-2k-linked MCMV resistance.     

Intracellular transport of membrane glycoproteins: two closely related histocompatibility antigens differ in their rates of transit to the cell surface

The intracellular transport of two closely related membrane glycoproteins was studied in the murine B cell lymphoma line, AKTB-1b. Using pulse-chase radiolabeling, the kinetics of appearance of the class I histocompatibility antigens, H-2Kk and H-2Dk, at the cell surface were compared and found to be remarkably different. Newly synthesized H-2Kk is transported rapidly such that all radiolabeled molecules reach the surface within 1 h. In contrast, the H-2Dk antigen is transported slowly with a half-time of 4-5 h. The rates of surface appearance for the two antigens closely resemble the rates at which their Asn-linked oligosaccharides mature from endoglucosaminidase H (endo H)-sensitive to endo H-resistant forms, a process that occurs in the Golgi apparatus. This suggests that the rate-limiting step in the transport of H-2Dk to the cell surface occurs before the formation of endo H-resistant oligosaccharides in the Golgi apparatus. Subcellular fractionation experiments confirmed this conclusion by identifying the endoplasmic reticulum (ER) as the site where the H-2Dk antigen accumulates. The retention of this glycoprotein in the ER does not appear to be due to a lack of solubility or an inability of the H-2Dk heavy chain to associate with beta 2-microglobulin. Our data is inconsistent with a passive membrane flow mechanism for the intracellular transport of membrane glycoproteins. Rather, it suggests that one or more receptors localized to the ER membrane may mediate the selective transport of membrane glycoproteins out of the ER to the Golgi apparatus. The fact that H-2Kk and H-2Dk are highly homologous (greater than or equal to 80%) indicates that this process can be strongly influenced by limited alterations in protein structure.     

Preferential response patterns of cytotoxic T lymphocytes specific for fluorescein isothiocyanate-[FITC] modified autologous cells

Murine cytotoxic responses to TNP-modified syngeneic cells (TNP-self) have been shown to exhibit preferential recognition of K or D end self products encoded by the H-2 complex. In the present study, a number of B10 congenic and recombinant mouse strains were investigated to determine the H-2K and H-2D-restricted FTC-self CTL response patterns, and these were compared with the CTL response patterns obtained for TNP-self. The results indicate that for strains possessing the H-2k,d,h2,h4 haplotypes, respectively, preferential CTL responses were observed against FTC recognized in association with Kk over Dk, Dd over Kd, and Kk over Db. These patterns of preferential CTL responses were the same as those reported for TNP-self as well as several anti-viral CTL responses. In contrast to the results obtained in the B10.A strain, in which Kk preference was observed over Dd for TNP-self CTL, no preferential CTL response was observed when FTC was recognized in association with Kk and with Dd. In this context, it was observed that the CTL response to FTC recognized in association with Dd was particularly strong. This strong D end-associated response was shown to involve D locus products, and no evidence was obtained indicating that L locus self products were involved. These studies are discussed with respect to the possibility that different haptens can be recognized by CTL in association with different self determinants encoded by the same H-2 gene products.     

Interferon sensitive and insensitive MHC variants of a murine thymoma differentially resistant to methotrexate-containing antibody-directed liposomes and immunotoxin

We evaluated the ability of methotrexate-containing liposomes or a ricin alpha-chain immunotoxin, both associated with monoclonal antibodies specific for the major histocompatibility complex-encoded class I molecule H-2Kk, to kill cells of the murine k haplotype thymoma RDM4. Cells were incubated with liposomes or immunotoxin in the presence or absence of interferon-gamma, which is known to augment the expression of the target class I molecules. The great majority of cells were killed by either of these reagents. Two types of mutant cells were obtained: type 1 cells, selected by methotrexate-containing liposomes, failed to express sufficient target H-2k molecules to be killed by liposomes in the absence of interferon-gamma. In the presence of interferon-gamma, these cells increased expression of all H-2 class I molecules and could be killed by targeted liposomes. Type 2 cells were immunoselected from cloned type 1 cells by liposomes in the presence of interferon. These cells failed to respond to interferon with expression of the H-2Kk molecule, but continued to augment H-2Dk expression in response to interferon. A third variant (type 3) selected from the wild type population by an H-2Kk specific immunotoxin in the absence of interferon phenotypically resembled type 1 cells. Type 1 but not type 2 cells respond to interferon by augmented synthesis of H-2Kk specific mRNA. The results suggest that for interferon-sensitive cell surface molecules of tumor cells, use of interferon improves the efficacy of targeted chemotherapy, but does not prevent development of mutants lacking the target molecule.     

Anomalous reactions of mouse alloantisera with cultured tumor cells. I. Demonstration of widespread occurrence using reference typing sera.

M0use alloantisera produced against different specificities of the K, I, and D regions of the H-2 gene complex reacted as immunogenetically anticipated with normal lymphoid target cells of different haplotypes in cytotoxicity and indirect immunofluorescence tests. These same alloantisera, however, produced anomalous positive reactions when tested on cultured MCA-induced sarcoma cells from B10 background H-2 congenic mice. Absorption experiments demonstrated that the anomalous activity in these sera was directed against a tumor membrane antigen(s) which was distinct from H-2 region specificities against which the reference alloantisera were produced, and which was shared in common by multiple cultured sarcoma lines. Similar anti-tumor antibody activity could be demonstrated in the serum of older (greater than 12 weeks) but not younger normal unimmunized mice of the strains used as recipients for alloantiserum production. It is suggested that the observed anamalous anti-tumor activity in these alloantisera may be due to the presence of antibodies reactive with envelope antigens of murine leukemia virus which are expressed on sarcoma cells maintained in culture.     

Primary in vitro cytotoxic response of F1 T lymphocytes against parental antigens.

Spleen cells from adult female (AKR/J x BALB/(c)F1 mice can respond to mitomycin C-treated spleen from AKR/J mice and can generate effector CTL in a 5-day primary in vitro culture. The response is comparable in magnitude to the response to allogeneic H-2K or H-2D antigens. The response is T cell mediated and is directed to antigen(s) present only on the parental cells. The target cell must be homozygous at H-2Kk to be lysed and H-2Dk antigens do not serve as a target in this response. Spleen cells from (B10.BR x B10.D2) hybrids that have been stimulated with AKR/J lyse B10.Br as well as AKR/J target cells. Similar H-2k/d hybrid F1 anti-H-2k parent responses are seen in certain other strain combinations. A number of possible interpretations of these responses are discussed.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. II. Monoclonal H-2-specific antibodies obtained from an unimmunized C57BL/KaLwRij (H-2b) mouse

A monoclonal 'natural' anti-H-2 IgM antibody produced by a hybridoma cell line OL-3.17 (H-2 m. 209) is described. The OL-3.17 monoclonal antibody was obtained by hybridization of spleen B cells from an unimmunized C57BL/Ka (H-2b) mouse in the serum of which simultaneously an IgM kappa paraprotein of high concentration and a natural H-2-specific antibody of high titer was detected. The monoclonal antibody OL-3.17 reacted strongly with H-2d and H-2s and weakly with H-2k,q,r lymphocytes, thereby detecting a hitherto unknown H-2 public determinant. The target molecules for OL-3.17 cocapped with class-I H-2 antigens, but immunoprecipitation of H-2 antigens was not achieved. This is the first monoclonal H-2-specific antibody obtained from a mouse without intentional immunization and, with high probability, was derived from a B-cell clone which produced natural H-2-specific antibodies detectable in the serum of the original mouse.     

Specificities of killing by T lymphocytes generated against syngeneic SV40 transformants: studies employing recombinants within the H-2 complex.

T lymphocyte effectors to syngeneic SV40-transformed cells, generated by secondary in vitro sensitization of immune spleen cells, lyse SV40 transformed targets that are syngeneic at the H-2 locus. In this study we have employed recombinants within the H-2 region to examine in detail this H-2 specificity. H-2b effectors were found to lyse SV40-transformed targets from recombinants bearing either H-2Kb or H-2Db.H-2k effectors recognized only SV40-transformed H-2Kk, and not H-2Dk target cells. By using the same protocol for sensitization, no effector cells could be detected in H-2d mice. Effectors generated in H-2 recombinant mice showed that the response capacity resides with K and D. For example, HTG, which is H-2d except at the D locus (H-2Db), produced effector cells specific for SV40-transformed H-2Db targets. Thus, the secondary in vitro response to SV40 transformants was found to depend only on the K and D alleles and not to be modified by the I region to any measurable extent.     

Selection of phage-resistent clones of streptococcus group A and their properties]

In studying the effects induced by virulent phage CAI in the sensitive cultures of streptococcus, group A, a possibility was shown of selection of phage-resistant clones with the altered enzymatic activity. These clones lost their capacity to produce proteinase and retained residual lipoproteinase activity. This evidence correlates with literature data indicating that phage-resistant streptococci served as good producers of M-protein--the main virulence factor. Infection of the culture producing streptokinase with phage CAI with a definite infection multiplicity led to an increase of the enzyme activity in the culture fluid. This process was accompanied by selection of the resistant strains characterized by greater streptokinase production and greater enzyme stability. As suggested, the latter could result from the absence of proteolytic activity in the phage-resistant clone.     

"Physiological interaction" does not explain the H-2 requirement for recognition of virus-infected cells by cytotoxic T cells.

B10.A (H-2Kk, H-2Dd) ectromelia-immune T cells from secondary responses in vitro were protent killers of both infected L929 (H-2Kk H-2Dk) and infected P-815 (H-2Kd, H-2Db) target cells. Specific competition with unlabelled targets showed that two separate T cell subsets were responsible for lysis of infected L929 and infected P-815 cells. One hypothesis to account for this (a form of "physiological interaction") is that T cells which kill one target e.g. infected L929) display only one out of two possible self-complementary recognition structures, in this example the H-2Kk alloantigen, not H-2Dd, whereas T cells that lyse infected P-815 targets display only H-2Dd, not H-2Kk. This hypothesis was tested and seems untenable because of the following results: A.TH (H-2Ks, H-2Dd) ectromelia-immune, secondary cytotoxic T cells which killed infected SJL/J (H-2Ks, H-2Ds) targets were themselves inactivated by pre-incubation with SJL/J cytotoxic T cells generated in one-way mixed lymphocyte reaction (MLR) against BALB/c (H-2Kd, H-2Dd). A.TL (H-2Ks, H-2Dd) ectromelia-immune secondary cytotoxic T cells which killed infected BALB/c targets were themselves inactivated by BALB/c cytotoxic T cells generated in MLR against SJL/J. Thus, virus-immune T cells which lyse infected targets by virtue of shared H-2K are also displaying H-2D alloantigen, and vice versa.     

Biochemical characterization of murine Ia antigens with xenoantisera. I. Identification of a second la molecule (I-E?) in H-2s haplotype

Rabbit anti-Ia sera was produced by immunization with detergent-solubilized extracts from splenic, lymph-node and thymus cells. The antisera contained activity against H-2 as well as Ia molecules. By a sequential immunoprecipitation assay it was shown that the rabbit anti-mouse H-2s serum precipitated a second Ia molecule in the H-2s haplotype. Previous studies with alloantisera have shown only one Ia molecule associated with this haplotype. Sequential precipitations with alloantiserum against the whole I region were used to show that this second Ia molecule is coded by genes within the I region. Since only I-A- and I-E-region coded molecules are immunoprecipitable in most haplotypes, we presume that the rabbit antiserum could be identifying the I-E-subregion coded molecule in the H-2s haplotype. The rabbit antiserum reacts with an isotypic specificity on the molecule. The studies suggest that the I-E subregion does exist in the H-2s haplotype even though alloantiserum cannot be produced to identify allotypic variants associated with this subregion.