Effect ofH-2 incompatibility between recipient and donor on the magnitude of response to Thy-1.1 antigen

Mice were immunized intravenously with 4 × 10⁷ thymocytes from Thy-1 disparate, eitherH-2-compatible orH- 2-incompatible donors. The magnitude of the anti-Thy-1.1 response was measured by determining the number of PFC in spleens of animals 6 days after immunization. Regardless of the origin of immunizing and target thymocytes, the assay employed detected exclusively PFC-producing antibodies to the Thy-1.1 antigen. In almost all instances,H-2-compatible thymocytes elicited a significantly higher response than didH-2-incompatible thymocytes, although the latter occasionally evoked a high response. TheH-2 incompatibility between donor and recipient appeared to be responsible for the differences in responsiveness of the standard inbred mice and theirH-2 mutants immunized with thymocytes compatible with standard inbred strains. The phenomenon observed appears to have several features in common with antigenic competition. We propose that the requirement forH-2 compatibility in the anti-Thy-1.1 response may be the expression of a general requirement of T cells to recognize an antigen in the context of the H-2 molecule.     

Mentor pollen: Possible role of wall-held pollen growth promoting substances in overcoming intra- and interspecific incompatibility

Recent studies have shown that certain normally incompatible, interspecific combinations in the genus Populus, and intraspecific combination in Cosmos bipinnatus can be made compatible by mixing irradiation- or chemically-killed compatible pollen (mentor) with normal incompatible pollen at the time of pollination. Knox and associates have proposed that in the sporophytic system of incompatibility the compatible mentor pollen provides the so-called specific recognition substances to the incompatible pollen thereby making the latter compatible. In the present experiments mentor pollen was used in attempts to overcome both intra- and interspecific incompatibility in the genus Nicotiana which has a gametophytic intraspecific incompatibility system. Although not denying that recognition substances specific to pollenpistil incompatibility reactions are present in pollen walls, the results suggest that a role of mentor pollen in overcoming incompatibility may be to provide extra free pollen growth promoting substances (PGS). These substances are apparently able to promote just sufficient further growth to allow some fertilizations by certain pollen tubes with inherently weak expression of incompatibility.The present proposed PGS-Hypothesis also elucidates the physiological basis of the widely-held relationship found between genetic systems of self-incompatibility and sites of pollen inhibition.     

Study ofH-2 mutations in mice

The serologically defined H-2.5 specificity was tested on spleen cells and red blood cells (RBC) of theH-2 ^b haplotype and a number of its mutants. Thebm8 (bh) mutant was barely distinguishable fromb in a variety of tests made. On spleen cells ofbm1 (ba) the H-2.5 specificity seemed to be unchanged, while it was virtually absent from RBC of this mutant. Mutantsbm4 (bf),bm5 (bg1), andbm6 (bg2) were similar tobm1, with slight differences between them. The mutantbm3 (bd) retained an unchanged quantity of H-2.5 on its spleen cells, while the specificity was substantially increased on its RBC. The H-2.5 ofbm3 is not identical to that ofH-2 ^a . Possible mechanisms causing differential serology of theH-2 ^b mutants are discussed.     

Immunogenetic analysis ofH-2 mutations

A.BY, B10.LPa, and B10.129(5M) mice were presensitized in vivo against B10.A(5R) cells and then restimulated in vitro by the same cells in the standard CML assay. The effector cells thus generated lysed not only B10.A(5R), but also C57BL/6 targets, indicating that, in addition to anti-H-2D_d response [measured on the B10.A(5R) targets], response to minor histocompatibility (H) antigens (measured on the C57BL/6 targets) also occurred. The latter response was directed against multiple minor H antigens in the case of the A.BY effectors, and against H-1 and H-3 antigens in the case of B10.129(5M) and B10.LPa effectors, respectively. The sensitization against minor H antigens occurred in the context of H-2K^b H-2D^d antigens, but by testing the response on C57BL/6 targets, only cells reacting with minor H antigens in the context of H-2K^b were assayed. The same effector cells were then tested against H-2^b mutant strains, in which theH-2K ^b allele was replaced by a mutant one. All three effector types [A.BY, B10.LPa, and B10.129(5M)] behaved in a similar way: they all reacted with theH-2 ^bg1 mutant to the same degree as withH-2 ^b, they did not react at all or reacted only weakly with theH-2 ^bd andH-2 ^bh mutants, and they reacted moderately or strongly with theH-2 ^ba mutant. The degree of crossreactivity with the mutants reflects, with one exception, the degree of relatedness of these mutants toH-2 ^b, as established by other methods. The one exception is theH-2 ^ba mutant, which is the most unrelated toH-2 ^b, and yet it crossreacted strongly. Further testing, however, suggested that in this instance the crossreactivity was probably directed against H-2 antigens: the anti-H-2D^d effectors apparently crossreacted with the H-2K^ba antigens. This finding is an example of cell-mediated crossreactivity between the products of two differentH-2 genes (H-2K andH-2D). It is also an example of anH-2 mutation generating an antigenic determinant known to be present in another strain.     

Immunogenetic analysis ofH-2 mutations

Spleen cells carrying theH-2K ^b allele and sensitized against TNP-modified stimulator cells in vitro displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^b allele (haplotypesH-2 ^ba ,H-2 ^bd , andH-2 ^bf ). Similar crossreactivity in TNP-CML was observed in the reciprocal direction. Spleen cells carrying theH-2K ^k allele and sensitized against TNP-modified stimulators displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^k allele (haplotypeH-2 ^ka ) and vice versa. The effector cells in these assays were sensitive to anti-T cell serum in the presence of complement, and supernatants from immune cultures did not induce nonimmune cells to display a cytotoxic effect. Titration of effector cells from mutant and wild-type strains of theH-2 ^b haplotype indicated no detectable quantitative differences in their activities. These data demonstrate that crossreactivity in TNP-CML occurs in closely related allogeneic strains that have recently undergone mutation in theH-2 complex.     

Studies ofH-2K b mutant mice

Three newH-2 ^b mutant strains, B6.C-H-2 ^bm9 , B6.C-H-2 ^bm10 and B6.C-H–2 ^bm11 , are described. The three mutant strains are of the gain and loss type as they reject skin grafts reciprocally with the parental C57BL/6Kh. The mutations, which arose independently, are all allelic at the same locus as 11 other mutant strains already described. By complementation and other studies the mutated gene has been shown to beH-2K ^b . The strains were typed directly and by absorption with antisera specific for H-2K^b and H-2D^b private and public specificities and for Ia^b specificities. Each strain typed differently with these sera. The strain B6.C-H-2 ^bm9 was found to be serologically identical with C57BL/6. The strains B6.C-H-2 ^bm10 and B6.C-H-2 ^bm11 were found to have alterations in the private H-2K^b specificity, H-2.33, and in the public specificity, H-2.5, but to a different extent. B6.C-H- 2^bm10 had a marked decrease in the amount of H-2.33 expressed on the splenic cell surface as compared to C57BL/6 and also has a marked decrease in the expression of H-2.5 on both spleen and red blood cells. In comparison, B6.C-H-2 ^bm11 has a decrease in the expression of H-2.33 but an increase in the expression of H-2.5 on both splenic and red blood cells. The other H-2^b specificities appeared to be unaltered as compared with C57BL/6.     

The relationship between theH-2 loss mutations ofH-2da andH-2db in the mouse

The mouse strain B10.D2-H-2^da carries the mutantH-2^da allele, derived after chemical induction, and this has been shown to be a gain and loss mutation involving theH-2D^d locus.BALB/c- H-2^db, derived spontaneously, is a loss mutation only, and appears not to involve theH-2D^d, but rather theH-2L^d locus. The two mutations effectboth graft rejection and serologically detected H-2 specificities (Type II mutation). In the experiments described in this study, theloss mutations in theH-2^da andH-2^db mutants have been compared by skin grafting, and by direct and absorption serological techniques: (1) By skin grafting, using the well established complementation method, it has been shown thatH-2^da andH-2^db do not complement each other, i.e., the mutation in both occurred at the same locus. However, by appropriate selection of donor and recipient, it has become clear thatH-2^da had a greater loss than didH-2^db, althoughH-2^da includes the loss found inH-2^db. (2) Serological studies have demonstrated that H-2D.4 was altered inH-2^da, but not inH-2^db; H-2.28 (detected by D-28b and D-29) was decreased or lost in both mutants;H-2^db anti-BALB/c failed to react withH-2^da; both mutants reacted similarly with D-28 sera. In addition, sera made usingH-2^da as donor did not contain an anti-H 2.28 antibody. The loss mutation involvingH-2^da therefore appears to have led also to the loss of H-2.28 as found inH-2^db. We conclude that theH-2^da strain arose after a complex mutation or recombination event which involvedboth theH-2D^d locus and the closely linkedH-2L^d locus, whereasH-2^db affects only theH-2L locus.     

Characterization ofH-2 congenic strains using DNA markers

Congenic mouse strains are widely used in mapping traits to specific loci or short chromosomal regions. The precision of the mapping depends on the information available about the length of the differential segment—the segment introduced from the donor into the background strain. Until recently, very few markers flanking the differential locus were known and consequently the length of the foreign segment could only be determined imprecisely. Now, in an attempt to construct a map of the mouse chromosome 17, we have produced a set of DNA markers distributed along the chromosome. These markers provide a new opportunity to measure the length of the differential segment of the congenic strains and thus increase their usefulness for gene mapping. Here we examined the DNA of 96H-2 congenic strains using 30 DNA markers; of these, the most proximal is located roughly 1.5 centiMorgans (cM) from the centromere and the most distal is about 20 cM telomeric from theH-2 complex (the complex itself being some 20 cM from the centromere). The mapping depends on polymorphism among the input strains and can therefore establish only the minimal length of the differential segment. This point is emphasized by the fact that the average observed length of the differential segment is only about one half of the expected values. Offprint requests to: V. Vincek.     

Serology and polymorphism ofH-2L- locus encoded antigens

Thirty B10.W congenic lines were analysed serologically, both by direct cytotoxicity and by absorption, for the presence of H-2L antigens. Three new H-2L antigens, 73, 74, and 75, were discovered. The B10.W lines and the inbred strains can be classified into at least six H-2L phenogroups on the basis of their reactivity withH- 2^dm2 anti-H- 2^d serum: BALB/c, B10.BUA1, B10.GAA37, B10.BUA16. B10.KPB128, and the negative group. Twenty-oneD-end recom-binants were analysed for the possible separation ofH-2D andH-2L loci. The failure to find such a separation indicates that theH-2D andH-2L loci are tightly linked. Serological analysis also indicated that theH- 2^dm1 has lost most of its H-2L antigens but retained at least one specificity which can be detected byH- 2^dm2 anti-H- 2^d serum.     

Recombinant plasmids formed in vivo carrying and expressing two incompatibility regions.

The formation in vivo of recombinants between a plasmid of incompatibility group N (R1010-10) and plasmids of groups P (R751) and W (R388) is described. From examination of the molecular weights of these recombinant plasmids, they appear to be cointegrates. These cointegrates have the incompatibility properties of both 'parent' plasmids.     

The role of pro regions in protein folding

In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases.     

Genetic control of contact sensitivity in mice: Effect ofH-2 and nonH-2 loci

The genetic control of delayed-type hypersensitivity in mice was investigated by contact sensitization with picryl chloride. Distribution patterns of contact sensitivity in 11 inbred strains of mice showed significant differences among strains. Comparison of levels of response between congenic-resistant lines and their inbred partners, at 9 to 11 weeks of age, revealed a clear association betweenH-2 haplotype and the magnitude of response. Testing ofH-2 recombinants further suggested the influence of two genes mapping at either end of theH-2 complex. While theH-2K ^d andH-2D ^k alleles were associated with a high response, theH-2K ^k ,H-2K ^b ,H-2D ^d , andH-2D ^b alleles were associated with a low response. Analysis of the ontogeny of response suggested that theH-2 haplotype manifests its effect through the maturation of contact sensitivity. On both the C57BL/6By and C57BL/10Sn backgrounds, theH-2 ^d haplotype was associated with early maturation of response, while theH-2 ^b haplotype was associated with late maturation. Analysis of the response of congenic lines with different genetic backgrounds and of CXB recombinant-inbred lines further revealed the marked effects of yet other genes on this trait.     

Genetic control ofH-2 alloantigens as inferred from analysis of mutations

A number of studies of phenotypic effects of anH-2D mutant, B10.D2(M504), have been reported by different laboratories. Their results show that lymphocyte-defined (LD) and serologically-defined (SD) functions of an antigenic molecule can both be controlled by a single gene,H-2D. Possible explanations of the observed phenotypic effects of 504 and other knownH-2 mutations are also discussed.     

Hierarchy ofH-2 haplotypes governs inheritance of immune responsiveness to TNP-MSA

Production of indirect TNP-specific plaque-forming cells (PFC) in response to immunization with 2, 4, 6-trinitrophenyl (TNP) conjugated to autogenous mouse serum albumin (MSA) in complete Freund's adjuvant (CFA) is underH-2 control. On the C57BL/10 (B10) background,H-2 ^b andH-2 ^d strains of mice are high responders, whereasH-2 ^a ,H-2 ^k orH-2 ^y2 strains yield low levels of indirect TNP-specific PFC. An unusual pattern of inheritance has been revealed in B10 congenic mice: high responsiveness controlled byH-2 ^b is inherited recessively, while high responsiveness controlled byH-2 ^d is inherited dominantly. On the C3H and A strain backgrounds, high responsiveness controlled byH-2 ^b is partially recessive;H-2 ^b /H-2 ^a F₁ mice respond with 20%-40% of the high responderH-2 ^b response. Yet, high responsiveness controlled by theH-2 ^d haplotype remains dominant on the C3H background. A hierarchy of haplotypes in order of decreasing immune responsiveness to TNP-MSA is evident as follows:H-2 ^d >H-2 ^b >H-2 ^k ,H-2 ^a orH-2 ^y2 . The unusual patterns of inheritance in the TNP-MSA system reveal graded regulation of responsiveness attributable to bothH-2 and non-H-2 genes.     

The [Het-s] prion of Podospora anserina and its role in heterokaryon incompatibility

[Het-s] is a prion from the filamentous fungus Podospora anserina and corresponds to a self-perpetuating amyloid aggregate of the HET-s protein. This prion protein is involved in a fungal self/non-self discrimination process termed heterokaryon incompatibility corresponding to a cell death reaction occurring upon fusion of genetically unlike strains. Two antagonistic allelic variants of this protein exist: HET-s, the prion form of which corresponds to [Het-s] and HET-S, incapable of prion formation. Fusion of a [Het-s] and HET-S strain triggers the incompatibility reaction, so that interaction of HET-S with the [Het-s] prion leads to cell death. HET-s and HET-S are highly homologous two domain proteins with a N-terminal globular domain termed HeLo and a C-terminal unstructured prion forming domain (PFD). The structure of the prion form of the HET-s PFD has been solved by solid state NMR and corresponds to a very well ordered β-solenoid fold with a triangular hydrophobic core. The ability to form this β-solenoid fold is retained in a distant homolog of HET-s from another fungal species. A model for the mechanism of [Het-s]/HET-S incompatibility has been proposed. It is believe that when interacting with the [Het-s] prion seed, the HET-S C-terminal region adopts the β-solenoid fold. This would act as a conformational switch to induce refolding and activation of the HeLo domain which then would exert its toxicity by a yet unknown mechanism.