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H-2 antigens are expressed in substantial amounts of murine blood platelets (for H-2 antigenic content 1 lymphocyte approximately 50 platelets) whereas Ia antigens are probably not expressed at all (minimal Ia antigenic content more than 35 times lower than for H-2). This property of blood platelets makes them very useful for the selective absorption of anti-H-2 antibodies from complex sera and for the preparation of specific anti-Ia antibodies from such sera. In 20 sera produced against the complete H-2 complex, 12 sera contained anti-Ia antibodies beside the expected anti-H-2 antibodies. In two sera, separation of the anti-Ia antibodies was easily obtained by absorption of the anti-H--2 antibodies on platelets. The analysis of one serum (C3H.Q X B10.D2) anti-C3H [(q X d) anti-k] showed that, in addition to be expected anti-H--2.23 and anti-Ia.2 antibodies, it contained at least three other Ia antibodies, separable by absorption on lymphocytes, which recognized three antigens--Ia.17, determined by the haplotypes k, f, s, r, j; Ia.18, determined by the haplotypes k, f, s; and Ia.19 determined by the haplotypes k and r. The genes are located in the I--A and/or I--B subregions of the H--2 complex.

The five H-2t haplotypes are all intra-H-2 recombinants which have identical K and D regions, but differ in the central regions of the complex. Analyses of H-2, Ia and Ss-Slp types indicate that the crossover in H-2t1 took place between the K and I regions, in the H-2t2 between G and D, in H-2t3 and H-2t4 between the I-B and I-C subregions and in H-2t5 between I and S. The set of recombinants will be very valuable for studies of traits which map in the I and S regions.

A series of genetic crosses conducted to map the Ss serum variant with respect to the H—2 complex is described in detail. Three intra-H—2 recombinants were detected. These crossovers, designated H—2al, H—2ol and H—2tl, position the Ss gene in the middle of the H—2 complex between the regions H—2K and H—2D. The production of eight new congenic resistant inbred strains is also described.

Detailed serological analyses of four intra-H—2 recombinants for 20 H—2 alloantigenic specificities were conducted. The H—2 alloantigenic profiles of these recombinants and resulting assignments to the H—2 genetic fine structure map are presented.

A hallmark of major histocompatibility complex (MHC) genes is their extraordinarily high level of polymorphism. Polymorphic residues on MHC molecules determine which peptide ligands they bind and present to effector T lymphocytes. Although the genetic mechanisms responsible for MHC polymorphism have been delineated, the timetable and the pathway of their diversification remain unclear. To trace MHC evolution, we have characterized a highly polymorphic microsatellite containing tandem repeats (TRs) of two tetranucleotide units, TGGA and GGCA, located at the 3' end of the second intron in the class II Eb gene of mouse. On the basis of length as well as sequence variations, 11 TR alleles were defined in 55 inbred mouse strains, which included MHC recombinant haplotypes and haplotypes derived from different subspecies of mouse. In this extensive sampling, a striking concordance was observed between the serologically identified class II proteins and the associated TR alleles. Examination of several strains carrying the same MHC haplotypes as well as strains carrying recombinant MHC haplotypes indicates that TR alleles are extremely stable. These observations suggest that TR polymorphism predates the separation of various subspecies of mouse. On the basis of sequence divergence, a genealogical tree has been constructed to depict evolution of the different TR alleles. Finally, evidence is presented that suggests this microsatellite polymorphism is generated by slipped-strand mispairing during DNA replication.

We have demonstrated in an anti-Ia serum the presence of specific antibodies reacting with T cells, as well as with B cells, using a highly sensitive dye exclusion test. This antiserum reacts with both spleen and lymph node in a characteristic biphasic titration curve killing up to 70% of these cells. It also reacts with cortisone-resistant thymocytes. The A.TH-α-A.TL serum can be absorbed with spleen, lymph node, cortisone-resistant thymus, or normal thymus cells. Further in vivo absorptions in BALB/c nude cannot remove all of the cytotoxic activity for normal BALB lymph node lymphocytes, while completely removing the activity for nude cells. A Thy-1 positive cell line derived from a C57Br leukemia is reactive with this anti-Ia serum.

Female mice of 15 inbred strains in which males express the H-2-linked sex-limited protein (Slp) were tested for the production of this protein. Four inbred strains (FM, LG/J, NZB, PL/J) were found in which females produce Slp in the absence of hormonal manipulation. Crosses have been made between strains FM, NZB, or PL/J and several Slp-negative strains. Slp-typing of the F1, F2, and backcross progeny, as well as of a number of recombinant inbred strains, indicates that production of Slp by normal females of these strains depends upon the concurrent presence of an Slp-positive, H-2-linked allele and of permissive alleles of one or two non-H-2 autosomal genes. Complementation studies with two of the strains (FM and PL) indicate that an identical genetic mechanism mediates expression of Slp in females of these two strains. FM-derived animals carrying the testicular feminization mutation (tfm) also express Slp, as do castrated NZB mice, indicating that Slp expression in these instances is not dependent upon testosterone as it is in other inbred strains. It is concluded from these results that genes distinct from the putative structural gene for Slp influence the sex-limitation of its expression.

Specificity H-2.7 is expressed predominantly on erythrocytes and controlled by a gene that maps within the H-2 gene complex at a locus, designated as H-2G, which apparently lies between regions S and D. Three phenotypes have been observed with respect to this antigen: a) positive by direct test and absorption (haplotypes 11-2 f, H-2 j, H-2 p, H-2~); b) positive only by absorption (H-2k); and c) negative (H-2 b, H-2 d, 11-2~ New crossover positions have been established for several H-2 recombinants based on classifications for the H-2G locus.

We have examined the MLR reaction in two sets of recombinants that differ in the I-J subregion. In both cases, significant stimulation was mediated by antigens controlled by genes in the I-J subregion. This stimulation was inhibitable by the addition of the culture of antisera directed against the I-J gene products on the stimulator cell. The specificity of this inhibition was shown by specific blocking of the relevant gene product on F1 hybrid stimulator cells. MLR stimulation was also eliminated by pretreatment of the stimulator population with anti-I-J sera plus complement. Pretreatment of F1 hybrid stimulator T cells with anti-I-J sera directed against either parental I-J product in the presence of complement, completely eliminated stimulation, indicating that there is no allelic exclusion of the relevant I-J products. Pretreatment with an anti-I-E/I-C serum and complement also eliminated stimulation, suggesting that the stimulating T cells express both I-J and I-E/I-C subregion products. This assay offers a potentially more direct and practical method for serological detection of the I-J products.

Direct microcytotoxicity testing and absorption analyses were employed to determine whether H-2K, H-2D, and Ia antigens are present on murine islet of Langerhans cells. Products of the H-2K and H-2D loci were found on beta-cells from three different mouse strains, but I-region (Ia) antigens were not detected. The absence of Ia gene products from islet cells may be of importance in explaining the survival of islet allografts across major histocompatibility barriers.

A significant within-strain correlation has been demonstrated between the levels of Ss and hemolytic complement (C) activity in two Ss-high strains. Mouse serum specifically depleted of Ss by absorption with F(ab')2 fragments of anti-Ss had negligible C activity. In control experiments, Ss-specific antigen-antibody complexes formed with F(ab')2 fragments did not fix rabbit C, and bovine serum albumin-specific antigen-antibody complexes formed with F(ab')2 fragments did not fix mouse C. Therefore the removal of C activity by anti-Ss [F(ab')2] was apparently not due to C fixation. These results suggest that the Ss protein is a necessary component of the C system.

Genes mapping in the I region of the H-2 complex control a system of lymphocyte alloantigens (Ia) which are expressed on a subpopulation of T cells and on most B cells. Specific anti-Ia serum in the presence of rabbit complement removed the splenic T-cell subpopulation responsive to Con-A, but did not affect the response to phytohemagglutinin (PHA) or Leucoagglutinin. Antibodies specific for Ia, H-2K, or H-2D membrane antigens were used without complement to pretreat spleen cells. These antibody pretreated cells responded normally to Con-A and PHA.

Absorption analyses were conducted on antisera which had previously been shown to detect cross reactivity between the H-2K and H-2D gene products. These analyses revealed that the cross reactions were due to known public H-2 specificities. Identification of cross reactive anti-H-2.3 and anti H-2.5 antibodies confirmed earlier predictions that genes controlling these specificities should be localized at opposite ends of the H-2 complex. The finding of H-2.29 activity on both sides of the S region clarified ambiguities encountered in assigning this specificity to certain recombinant types. Results with H-2.36 support the suggestion that this determinant may also be specified by both H-2K and H-2D genes.

The protective activity of anti-Listeria-immune T cells assayed in an adoptive transfer system in H-2 restricted. As shown in the present studies, the demonstration of the restriction is directly dependent on the dose and the relative protective activity of spleen cells. In addition, some H-2-unrestricted protection is conferred predominantly by other than immunoglobulin-negative spleen cells. Thus, the activity of Listeria-immune T cells appears to be 'absolutely' restricted and is in this respect comparable to in vivo T-cell-mediated anti-viral protection. The predominant genetic region of H-2 coding for the structures which are mainly involved in this restriction in T-cell immunity to this prototype intracellular bacterium is the I region. The specificity of Listeria-immune T cells is determined by the H-2 haplotype of the donor. Thus, F1 hybrids seem to possess at least two separable sets of T cells, each specific for one parental haplotype. As is true in the virus model, the results cannot distinguish between an altered-self or a dual recognition model of T-cell recognition to explain H-2 restriction. They are, however, compatible with the idea and I-coded cell surface structures may serve as receptors for cell-specific differentiation signals, which trigger direct or lymphokin-mediated activation of macrophages to manifest increased bactericidal capacity. The interesting parallels in self-marker recognition of T cells in the virus and intracellular bacterium systems, respectively, appear to be reasonably explained by the different types of signals transmitted by T cells to various target cells via the distinctly different self-markers employed (i.e., K or D vs I).

Allogeneic Con A induced suppressor cells differing in the I and S region but not H -2 K or H -2 D regions were as efficient as syngeneic cells in suppressing the secondary IgM and IgG response to burro erythrocytes. Con A activated suppressor cells were not sensitive to anti-Ia serum and complement. However, if the spleen cell population was treated with anti-Ia serum and complement before stimulation with Con A, suppressor cells were not generated.

Ia antigens from the I-A8 and I-Ck subregions of the B10.HTT (H-2t3) strain of mice were isolated by indirect immunoprecipitation of arginine-labeled, nonionic detergent-solubilized materials. After biochemical purification the electrophoretically homogeneous 28,000 dalton glycoprotein β chains from the Ia precipitates were digested with trypsin and the resultant radiolabeled tryptic peptides were compared by analytical ion exchange chromatography. These comparisons reveal that the β chains of Ia antigens from the A (I-A8) and C (I-Ck) subregions of B10.HTT share only two out of 12 to 14 of their arginine tryptic peptides. Thus these noncross-reactive Ia antigens are structurally quite diverse, and would possess sufficient structural variability to account for their lack of antigenic cross-reactivity.

Serological cross-reactivity between the products of the H-2K and H-2D genes has been demonstrated by a design in which antibody was produced against determinants controlled by one locus (e .g . H-2K(k)), and then tested against the product of the opposite locus (e .g . H-2D(d)). A total of 13 out of 18 such test combinations exhibited H-2K-H-2D cross-reactivity. The presence or absence of cross-reactivity was reciprocal in most cases (i.e. antibody directed against the H-2K(k) gene product reacted with H-2(d) determinants, and antibody directed against the H-2D(d) gene product reacted with H-2K(k) determinants). An Ia-like reaction was detected with one antiserum which implied possible cross-reactivity between the products of two discrete la genes.

Radiolabeled murine C4 and C4 precursor (pro-C4) from cultured peritoneal macrophages were purified by immunoprecipitation and preparative gel electrophoresis. The partial NH2-terminal amino acid sequences of the isolated subunits demonstrated that murine C4 alpha, beta-, and gamma-chains all show sequence homology with the corresponding subunits of human C4; of the 10 residues identified in murine C4, all are identical to those in human C4. These data extend to the primary structural level the homology between the murine serum substance (Ss) protein and human C4. Comparisons of the amino acid sequences of murine pro-C4 and the constituent polypeptide chains of secreted C4 indicate that pro-C4 and the C4 beta-chain have an identical amino acid sequence. Both molecules have lysine at position 1, leucine at positions 4, 5, and 6, and phenylalanine at position 7. No sequence homology was found between pro-C4 and either the alpha- or gamma-subunits. These results define the beta-chain as the NH2-terminal subunit in the C4 precursor molecules.

We have shown that the Ia determinants expressed on nylon wool-purified T lymphocytes reactive to concanavalin A (Con A) in serum-free media are coded in a single I subregion of the H-2 gene complex. This region, I-J, is defined by two pairs of intra-H-2 recombinant haplotypes: H-2t3, H-2t4 and H-2i3, H-2i5, carried by B10.HTT, B10.S(9R), B10.A(3R), AND B10.A(5R), respectively. No activity against Con A-reactive T cells has been detected in any antiserum that was produced in strain combinations which shared a common I-J region. This suggests that Ia antigens expressed on Con A-reactive T cells are restricted to the I-J subregion.

induced. Alternatively, the suppression of T helper cells may be perpetuated by an idiotype-restricted suppressor cell bearing either the idiotype or anti-idiotype receptor. Whatever mechanism is considered likely, the question of why it develops in some individuals but not in most remains unanswered. Some studies have suggested that susceptibility to leprosy may be associated with a particular HLA haplotype 9, whereas others have postulated a variety of predisposing environmental factors. It is likely that environmental factors will operate within the genetic background and the outcome will be determined by the interaction of the two. M. J. COLSTON

Membrane associated molecules that are probably glycoproteins could be specifically precipitated from NP-40 detergent solubilized extracts of radiolabeled mouse spleen or lymph node cells by antisera produced in congenic strain combinations differing only in the Ir gene region which is linked to the H-2 genes. These Ir region products were designated Lna (lymph node antigen) to conform to previous serological work. Sodium dodecyl sulfate—polyacrylamide gel electrophoresis of unreduced specific immune precipitates revealed the presence of a possible dimer form, while reduced samples showed only a single peak equivalent to 30,000 daltons. Thus the Lna molecules are clearly distinct from the H-2D and H-2K molecules, which are about 45,000 daltons. Anti-Lna antibodies of different specificity can be present in a single serum; there were at least two separate antigen molecules present in one haplotype tested.

The development of congenic mouse strains identical at the H-2K and H-2D loci but differing by I-region associated (Ia) determinants has permitted an association to be established between Ia determinants and stimulation in mixed lymphocyte culture reactions (MLR). The present experiments were undertaken to establish whether the Ir-coded control of MLR operated at the level of recognition or of stimulation. Reciprocal MLR were established between A.TH and A.TL mouse spleen cells in the presence or absence of anti-Ia sera directed either at determinants of the stimulating or responding cells. The number of T cells responding was assessed by the virus plaque assay. Anti-Ia sera directed against the responding cells were no more inhibitory of the MLR than normal mouse serum. In contrast, anti-Ia sera directed against determinants of the mitomycin-treated stimulating cells markedly inhibited activation of T cells in the MLR.

Adherent peritoneal cells, presumably macrophages, from mice of a number of congenic strains when cultured in vitro in medium containing 14C-labeled amino acids were found to synthesize the Ss protein (mouse C4), as revealed by immunoprecipitation with anti-Ss serum from 14C-labeled, 24-hr culture supernatants. Strain B10.WR7, which expresses the Slp allotype of the Ss protein, was shown by the same method also to synthesize the Slp antigen. SDS-solubilization and 2-ME-reduction of the B10.WR7 immunoprecipitates followed by separation on SDS polyacrylamide slab gels and autoradiography revealed three major polypeptide bands in Slp-positive Ss molecules and three major bands in Slp-negative Ss molecules with m.w. comparable to those of the α-, β-, and γ-chains of human C4. The Slp-negative Ss polypeptides of strain B10.WR7 had apparent m.w. of 98,000, 75,000, and 33,500 whereas the Slp-positive Ss molecules of B10.WR7 yielded three polypeptides with apparent m.w. of 105,500, 75,000, and 32,000. Immunoprecipitation analyses of normal, unlabeled B10.WR7 serum or plasma in SDS slab gels followed by staining with Coomassie Blue revealed similar differences in the m.w. of the α- and γ-polypeptides of the Slp-negative versus the Slp-positive Ss proteins. Such analyses of sera and plasmas of mice from a backcross linkage test and from a series of intra-H-2 recombinants showed clearly that the m.w. differences in α- and γ-chains are controlled by the S region of the H-2 complex. In addition, the Slp-negative Ss protein in strains other than B10.WR7 had an Ssα-chain of 103,000 daltons, indicating the existence of still further Ss polymorphism also controlled by the S region. An apparent intracellular precursor polypeptide of the Ss molecule with a m.w. of approximately 185,000 (comparable to the guinea pig Pro-C4 described recently) has also been demonstrated in lysates of cultured peritoneal cells. These results further confirm the structural homologies of the mouse Ss-Slp and human and guinea pig C4 proteins. They also indicate that Ss and Slp-antisera define two similar, but structurally distinct subclasses of mouse C4 that are apparently controlled by two discrete structural genes in the S regions of Slppositive strains. It is postulated that each of these structural genes codes for a single polypeptide precursor for the native Slp-positive and Slp-negative Ss molecules, and that the observed differences in the m.w. of the α- and γ-chains of these two Ss subclasses reflect a mutational shift in the site of the proteolytic cleavage step that releases the chains.

SUMMARY Several genetic models were considered to explain the distribution of qualitatively positive and negative children in 204 Caucasian families. A model which best describes the inheritance of the Lp antigenic expression involves a major genetic locus which distinguishes two overlapping continuously distributed modes of quantitative activity.

Ia specificities 1-10 were detected on LPS-stimulated splenic lymphocytes and on Con A-stimulated spleen, lymph node, and thymus blasts by direct cytotoxic tests. Since Ia antigens are not readily detectable on resting thymocytes, our results suggest that T cells require some signal before they exhibit full expression of Ia specificities. Absorption-elution studies indicated that most of the Ia specificities detected on T and B cells may be identical. Ia antigens detected by homologous antisera gave much stronger reactions than those detected by cross-reacting antisera.

Properties of the Ss serum protein (putatively the murine homologue of the fourth component of complement) and its allotypic form, Slp, were studied in a wild-derived variant, Ssw7. The Ssw7 allele is unique in that it confers non-sexlimited expression of the Slp allotype. During examination of the H-2 haplotypes of two congenic lines which derived their Ssw7 allele from a common origin, it was found that intra-H-2 recombination had occurred during the establishment of one of these lines. The sex-limited expression of Slp as determined by the Ssd allele was constrasted in several ways to the constitutive expression of Slp as determined by the Ssw7 allele. Slp expression in mice carrying the Ssd allele was found to be subject to control by the testicular feminization mutant, Tfm, while in mice carrying Ssw7 the Slp expression was independent of Tfm control. This implies that Slp is subject to a rather fundamental hormonal control mechanism in mice carrying Ssd and presumably all other Slp-positive Ss alleles, with the exception of Ssw7. It is proposed that Ssw7 may represent an “operator-constitutive” mutation of the Jacob-Monod type. Including the Ssw7 allele, there are now at least seven distinguishable Ss variants. Each of these Ss alleles is characterized by a unique level of Ss and/or Slp antigenic expression. The sex-limitation difference and quantitative differences in Ss and Slp expression associated with the Ssw7 allele were found to be codominant or intermediate when Ssw7 was in heterozygous combinations with other Ss alleles. This indicates a lack of trans effects and argues against genetic differences in regulatory proteins as the basis for these polymorphisms.

B6.C-H-2 ba [H (z1)] is a mutant derived from C57BL/6. The two strains mutually reject their skingrafts and are incompatible in the mixed leucocyte reaction (MLR) and in cell-mediated lympholysis (CML) assays. They are serologically indistinguishable. This report shows that H(z1) carries a new, privateK end CML specificity clearly distinguishable from that of B6 by a third party strain, HTG. Antisera directed against the private H-2K specificity of B6 present on H(z1) cells) can block CML between the two strains in either direction. The new CML specificities of H(z1) cross-react with (public) CML specificities controlled by bothK andD regions of other unrelated haplotypes. The results suggest that H(z1) carries a mutation in theH-2K locus itself or in a closely linked gene, the product of which is also physically associated with the H-2K molecule corresponding to the cis-configuration of the alleles in both loci. These findings indicate that T- and B-cell dictionaries for histocompatibility determinants are different.

Recent analyses, at the protein and DNA levels of structure, of the murine complement component C4 and the closely related sex-limited protein, Slp have led to new insights into theH-2/Sregion-linkedC4andSlpgenes and their products. The primary products are 200000 Da precursors which are cleaved, intracellularly and extracellularly, into the mature α—β—ϒ-subunit molecules of plasma. Precursor order of subunits is β—α—ϒ; a complementary DNA clone spanning the α—ϒ junction has been extensively analysed. The C-terminal of the α-chain is of particular interest because of post-secretion processing which differentiates ‘secreted’ and ‘plasma’ forms of C4, both apparently functional, and because allelic variants of C4 and the Slp protein, which differ substantially in molecular masses, owe their differences principally to different levels of glycosylation of the α-chain. Allelic variations in rate of C4 synthesis (C4-high compared with C4-low) have been analysed in cultures of hepatocytes and macrophages. Three distinct modes of genetic regulation of the expression of the Slp protein have been identified.

The S region of the mouse H-2 complex is genetically defined by a series of alleles, or pseudoalleles, which control the level of a serum globulin, Ss, and of its allotypic vairatn, Slp. In contrast with the products of other genes in the complex, no homologue of the Ss protein has been found in other species, except the rat. In the present study, a component in human plasma was identified which cross-reacts with anti-mouse Ss, and which also displays electrophoretic and size similarities to the mouse Ss protein. This component was isolated and characterized immunochemically as the fourth component of human complement (C'4 or beta1E globulin). Several antisera monospecific for human C'4 cross-react with the mouse Ss protein and detect its genetically determined quantitative variations.

Subpopulations of B lymphocytes have been shown to vary in their expression of Ia alloantigens and polyclonal responsiveness to thymic independent antigens. We have demonstrated that the polyclonal B cell antibody response to dextran sulfate is less sensitive to removal of Ia-positive cells than is the response to LPS. This is a consistent finding whether alloantibody and complement (C) pretreatment is directed toward cells bearing Ia antigens coded for by the entire I region or by the I-A or I-E subregions. Heterogeneity appears to exist within the dextran sulfate-sensitive population in that using high antibody:cell ratios during antibody and C-mediated cell selection results in an inhibition of the proliferative but not the antibody response. This result may indicate a differential expression of Ia antigens on dextran sulfate-sensitive B cells that respond by proliferation versus those cells that produce antibody. Alternatively, proliferative responses to dextran sulfate may be more dependent upon Ia-positive accessory cells than is the polyclonal antibody response.

Primary hepatocyte cultures prepared from the livers of inbred strains of mice show different rates of synthesis for the fourth component of complement (C4). The relative amounts of C4 synthesized by hepatocyte cultures correspond to the relative plasma levels of C4 among strains, a trait determined by the S region of the H-2 major histocompatibility complex. There is no evidence for increased intracellular catabolism of C4 in hepatocyte cultures from a strain with a low level of plasma C4. Processing and secretion of C4 proceed very slowly in vitro relative to other hepatocyte proteins.

We have reviewed the relationship between C4 and its related blood group and discussed the mechanisms whereby a fragment of C4 could become attached to erythrocytes (E). We hypothesize that there is chronic fluid-phase activation of C4 by either C1 to form C4b or spontaneous cleavage of the thioester to form iC4. These activated molecules bind to E. Proteolytic degradation of the bound C4b or iC4 would leave a covalently attached fragment of C4 on E and thereby give rise to the Ch and Rg blood group antigens. This system is of further immunopathologic interest since this 'normal' activation or turnover of C4 is closely regulated. In patients deficient in regulatory proteins, this spontaneous or normal turnover of C4 and C3 may initiate a pathologic condition.

Murine responses to both the male specific histocompatibility antigen H-Y and the erythrocyte alloantigen Ea-l are regulated by genetic factors. In each case a single major gene that controls the immune response has been identified, but additional modifying factors can be demonstrated if appropriate strain combinations are studied. A single gene controlling the response to Ea-1 antigens, which segregates when strains YBR and B10.D2 are crossed, has been shown not to be an allele of the It-2 locus. A new phenomenon has also been observed in the control of anti-Ea-1 antibody production in that the mating of two responding strains, YBR and HTG, produces an F 1 generation of complete nonresponders. By linkage tests it was shown that the responding strain HTG possesses the nonresponder allele at the It-2 locus, so there appear to be recessive genes in the background which are able to overcome the suppressive influence of this allele.

The alpha-chain of murine fourth component of complement (C4) secreted by cells in vitro and in vivo has a Mr that is larger by approximately equal to 4,000 than that of the alpha-chain of the principal form of C4 in plasma. By using in vivo labeling of C4 with [35S]methionine, C4 was shown to be first synthesized with the higher Mr ("secreted") alpha-chain, which was then quickly processed (t1/2 approximately equal to 1 hr) extracellularly to the mature ("plasma") C4 possessing the lower Mr alpha-chain. Both forms of C4 were functional as assayed by the ability of their alpha-chains to be cleaved by the protease C1, to bind methylamine, and to undergo denaturation-dependent autolysis. When secreted C4 and plasma C4 were activated to C4b, the Mr difference of 4,000 was maintained in the alpha'-chains. The Mr difference was localized to the carboxyl-terminal autolytic fragment of the alpha-chain and was unaffected by the removal of carbohydrate. C4 from resident peritoneal macrophage cultures could be converted to the plasma form by incubation with heparin/plasma. This conversion could be blocked by EDTA or 1,10-phenanthroline. These data suggest that an enzyme, presumably a neutral proteinase present in mouse plasma, cleaves the carboxyl terminus of newly synthesized C4 alpha-chains, thereby creating the major form of C4 in plasma.

The molecular basis for variation in the Mr of the C4 alpha-chain of the fourth component of murine complement from several strains was investigated. All strains were capable of incorporating radiolabeled mannose into the alpha-chain of C4 from peritoneal macrophage cultures. In most cases, carbohydrate was found on both alpha-chain autolytic fragments produced by denaturation of the native C4. However, mice bearing the H-2 haplotypes w7, w16, and w19 produce C4 with no carbohydrate on the larger (COOH-terminal) autolytic fragment. This lack of carbohydrate is sufficient to cause the difference in Mr seen in the C4 alpha-chain from these mice. The chemical removal of all carbohydrate abrogated this difference. The levels of C4 antigen in the plasma of mice from these three strains are normal while the levels of hemolytic activity are reduced 60-80%. This suggests a strong role for carbohydrate in the functional properties of C4.

The distribution of Ia antigens on the surfaces of lymph node lymphocytes of several mouse strains was investigated using indirect immunoferritin labeling and electron microscopy. The immunoferritin labeling results agreed with results of cytotoxic tests in strain distribution of reactivity, proportion of cells showing label, and cell populations reacting. Capping was induced by increased incubation temperature but conditions for Ia antigen mobilization varied somewhat between the two anti-Ia antisera employed. Uncapped specimens generally showed a denser, more evenly distributed antigen coating than is the case for H-2 antigens labeled by the same indirect immunoferritin method.