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.     

Hybrid Ia antigens: Genetic,serologic, and biochemical analyses

Ia specificities 22 and 23 were found to be determinants on hybrid Ia molecules, formed by the noncovalent binding of a 26,000–28,000 dalton beta polypeptide chain (A_e) coded by the I-A subregion and a 32,000–35,000 dalton alpha chain (E_α) coded by the I-E subregion. For expression of Ia. 23 the A_e chain, coded by the I-A subregion, must be derived from the H-2^d haplotype, while A^b, A^s, or A^k can provide the complementing beta chain for the expression of Ia. 22. For expression of Ia. 22 and Ia. 23, most Ia. 7 positive strains can provide the complementing alpha chain (E_α), with the one exception of B 10. PL (E^u), which is Ia. 7 positive but will not complement with A^d to express Ia. 23. Antisera were also produced against hybrid Ia antigens by immunizing with F₁ cells expressing Ia. 22 or Ia. 23 generated by transcomplementation. These antisera detect the same specificities as conventional anti-Ia. 22 and anti-Ia. 23 sera produced against cis-complementing Ia antigens. It is postulated that hybrid Ia determinants are involved in recognition and generation of immune response to antigens under dual Ir gene control. It is also suggested that there are 2 types of Ia specificities: (1) allotypic Ia specificities expressed on the alpha or beta chains (for example, Ia. 7 on the E_α chain) and (2) hybrid Ia specificities, which are unique interaction determinants formed by the association of alpha and beta chains (for example, Ia. 22 and Ia. 23). These interaction gene products may be involved in antigen recognition and presentation.     

Evidence for structural homology between murine and human Ia antigens

The serological cross-reactivity and the structural homology of murine and human Ia alloantigens were analyzed. Both normal human peripheral blood B lymphocytes and chronic lymphocytic leukemia (CLL) cells were shown to be lysed in the presence of complement by both murine anti-Ia and human anti-HLA-DR alloantisera. A mouse A.TH anti-A.TL (anti-I ^k ) alloantiserum reacted with determinants expressed on all of the 20 normal human B cell populations tested. Only 3 of these 20 B cell populations were lysed with an A.TL anti-A.TH anti-I ^s alloantiserum. The frequency of cytotoxic cross-reactivity concordant with anti-I ^k appears to be greater for anti-I-EC ^k than for anti-I-A ^k alloreactivity. An immunochemical analysis demonstrated that Iaα-chain andβ-chain polypeptides may be immunoprecipitated from CLL cell lysates by either a mouse anti-I ^k alloantiserum or various human anti-HLA-DR alloantisera. The Ia molecules detected with the mouse and human antisera are coprecipitable as revealed by one-dimensional gel electrophoresis. Two-dimensional gel electrophoresis studies indicated that the human CLL cell Ia antigens analyzed possess considerable molecular heterogeneity. They are structurally more similar, with respect to molecular size and charge, to mouse Ia antigens determined by the murineH-2-linkedI-EC subregion rather than theI-A subregion. The structural, genetic and functional implications of these findings are discussed.     

Topographical relationships among H-2 specificities controlled by theD region

In the present work, we used the differential redistribution method to study the molecular expression of several H-2 specificities controlled by theD region of theH-2 ^a haplotype. We observed that: capping of the private specificity H-2.4 induced capping of the public specificities H-2.3, H-2.35, and H-2.36, and vice versa; capping of any one of these specificities did not induce capping of the public specificity H-2.28, controlled by the same region. By contrast, capping of the H-2.28 specificity induced capping of these specificities; redistribution of H-2K and H-2D private specificities or redistribution of H-2D private specificity and Ia specificities did not induce capping of the H-2.28 specificity. These data indicate that a part of a molecule carrying the H-2.28 specificity is linked to a molecule carrying H-2.4, H-2.3, H-2.35, and H-2.36 specificities and that a part of a polypeptide chain bearing the H-2.28 specificity is independent from that bearing other specificities controlled either by theD region (i.e., H-2.4, H-2.3, H-2.35, and H-2.36) or by theK andI regions. These results further strengthened the hypothesis of the existence of at least two genes controlling theD-region H-2 antigenic specificities.     

Genetic control of T-cell proliferative responses to poly(glu40ala60) and poly(glu51lys34tyr15): Subregion-specific inhibition of the responses with monoclonal Ia antibodies

The relationship betweenIr genes and Ia antigens was studied in the T-cell proliferative responses to two synthetic polypeptides poly(glu⁴⁰ala⁶⁰) (GA) and poly(glu⁵¹lys³⁴tyr¹⁵) (GLT¹⁵). The response to GA was found to be controlled by anIr gene in theI-A subregion, whereas the anti-GLT¹⁵ response was shown to be under dual control, oneIr gene mapping probably in theI-A subregion, and the other in theI-E subregion. We obtained two different lines of evidence suggesting identity ofIr and Ia genes. First, the presence of certain serologically identified allelic forms of the I-A-encoded A molecule correlated with the responder status to GA both in inbred strains and in B10.W lines, the latter carrying wild-derivedH-2 haplotypes. Thus the Ir and Ia phenotypes were not separable in strains of independent origin. Second, the anti-GA response was completely inhibited by monoclonal antibodies against determinants on the A molecule (Ia.8, 15, and 19), but not by a monoclonal antibody against a determinant on the E molecule (Ia.7). In contrast, the anti-GLT¹⁵ response was only inhibited by a monoclonal antibody against the E molecule, but not by antibodies against the A molecule. Our data support the hypothesis that Ia antigens, as restriction elements for T-cell recognition, may in fact be the phenotypic manifestation ofIr genes.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

B lymphocyte alloantigens controlled by theI region of the major histocompatibility complex in mice

An antiserum was produced by reciprocal immunization of congenic resistent inbred strains of mice which differed only with respect to theI andS regions of theH- 2 complex. This antiserum permitted the serological detection of lymphocyte alloantigens, designated Ia (=I region associated antigens). Ia determinants are only present on mature B lymphocytes. They could not be found on thymocytes, splenic or lymph node T cells, or on the majority of bone marrow cells. Absorption studies demonstrated existence of several Ia specificities which are associated with differentI region types. Thus, theI region of theH- 2 complex appears to control not only T-cellexpressed antigen specific immune response genes, but also B-cell-expressed Ia determinants. The relevance of the Ia alloantigen system for cellular interaction in immune reactions is discussed.     

Demonstration of Ia antigens on mouse intestinal epithelial cells by immunoferritin labeling

Several kinds of epithelial cells that express H-2 antigens were studied by immunoferritin labeling with an antiserum reacting only with antigens of theI region of theH-2 complex. Spleen lymphocytes were used to test the labeling system and the effect of the epithelial cell dissociation procedure on Ia antigens. Immunoglobulin-positive B10.BR lymphocytes were labeled with an anti-la^k serum (A.TH anti-A.TL serum absorbed with BALB/c and B10.D2 cells), while congenic B10.D2 lymphocytes were unlabeled. The distribution of labeled Ia antigens on living B10.BR lymphocytes was patchy, while on cells fixed in periodate-lysine-paraformaldehyde before labeling, the distribution of label was continuous. Fixation evidently immobilized Ia antigens in the lymphocyte membrane. Trypsin and collagenase, as used in the epithelial cell dissociation procedure, had no discernible effect on the Ia antigens of lymphocytes. The epithelial cells studied included the columnar absorptive cells of the small intestine, uterine lining epithelium, tracheal brush cells, and pancreatic exocrine and duct cells. These cells were fixed before dissociation from their respective tissues. Ia antigens were detected only on the columnar absorptive cells of the small intestine. These cells labeled equally well with an antiserum reacting only with theK -end of theH-2 complex. In both cases, congenic control intestinal cells were unlabeled. Thus, intestinal epithelial cells appear to express theIa, K, and presumablyD regions of theH-2 complex, while the other epithelial cell types express only the K and D antigens. On fixed intestinal epithelial cells, Ia and H-2K antigens were continuously distributed on the lateral and basal cell membranes including the zonula adherens, but the antigens were absent from the apical microvillous membrane and the zonula occludens.     

Identity of molecules bearing murine ia alloantigenic specificities Ia.7 and Ia.22: Evidence for a single type of I-E molecule in homozygotes

In homozygous mice bearingI regions derived from haplotypek, only a single type of Ia molecule bearing the alloantigenic specificities Ia.7 and Ia.22 was found using techniques of sequential immune precipitation and tryptic peptide analysis. As suggested at the fourth Ir Gene Workshop (Sachs 1978), Ia.7 is considered here to be an antigenic determinant associated with I-E-subregionencoded molecules, i. e., it is excluded from theI–C subregion. TheI–C subregion is currently defined mainly by functional traits. It is now known that the I-E molecules are composed of an chain encoded in theI–E subregion, and a chain encoded in theI–A subregion. Since theI–C subregion is not involved with the determination of these Ia molecules, and since in homozygotes there is apparently only a single type of molecule bearing both specificities Ia.7 and Ia.22, the term I-E/C molecule should probably be dropped in favor of the simpler designation I-E.     

Genetics of graft-versus-host reactions (GVHR)

Injection of 20×10⁶ donor lymph node cells (LNCs) into newborn allogeneic recipients incompatible with donors at theIC subregion of the mouseH-2 complex evoked both GVH splenomegaly and GVH mortality. The strength or severity of the allogeneic reactions induced varied as a function of the interallelic strain combination and was influenced particularly by properties of the recipientIC determinants. Thus,IC ^s determinants on recipient cells led to strong GVHR, whileIC ^d determinants induced a moderate GVHR, even when donors carrying differentIC alleles were used. However, responder donor genes also affected the degree of GVHR in some combinations. The effect on donor GVH potential of pre-exposing B10 donors to antirecipient antiserum (B10 anti-B10.A) was also studied. Spleen cells from B10 donors pre-exposed to alloantiserum for two to seven days exhibited a markedly reduced ability to cause GVH splenomegaly and GVH disease in newborn B10. A or (B10. A × B10) F₁ recipients. Inhibition of donor lymphocyte GVH potential waned eight to 14 days after antiserum pretreatment. Inhibition was shown to be specific for variousH-2 determinants and to be caused by antirecipient alloantibodies. Pre-exposure of donors to alloantiserum reduced the GVH potential of spleen cells but did not affect LNC reactivity.Ia antibodies and, to a lesser extent, anti-H-2D serum were shown to be able to inhibit GVHR. The results suggest that the observed reduction in donor GVH reactivity is caused by antibody-mediated central feedback inhibition. Anti-H-2 alloantibodies evidently play an important role in the network regulating allogeneic responses.     

H-2 typing of mice genetically selected for high or low antibody production

H and L inbred mouse strains were derived from animals selected respectively for the production of high and low titers of agglutinins against xenogeneic erythrocytes. L was found to beH-2 ^s . H was found to beH-2K ^d ,D ^q , with anI region derived from another (probably unknown) haplotype.     

Variable effect of anH-21 barrier on response to skin allografts in the mouse

(AQR×B10)F₁ mice were grafted with skin from donors differing in theK, I, KI, andISD regions of theH-2 complex. A dichotomy was observed in the fate of theH-2I-disparate grafts: either they were rejected acutely within the second week or were accepted indefinitely. Acceptances were much more common among male than female hosts. Acceptor status was limited to the I group, was unpredictable in occurrence, was not well-correlated with positive serum anti-Ia titers, and did not confer protection of grafts that were alike atH-2I but different atH-2K orH-2D. Since theH-2I barrier studied here elicited such divergent responses in genetically identical hosts, it is unlikely that any histocompatibility typing test could predict graft fate.Abbreviations used in this paper are MST median survival time - MHC major histocompatibility complex - CTL cytotoxic T lymphocyte - B10 C57 BL/10 - 6R BIO.T(6R) - B10.A BIO. ASn - H-2-Ia serologically detected antigens coded in theI region ofH-2 This term is used in preference toIa, since it has recently been shown that Ia-like alloantigens may be coded outside the MHC (Dickleret al. 1975).     

The mouse primed lymphocyte typing (mPLT) test

A murine primed lymphocyte typing (mPLT) assay, based on the sequential selective isolation of specific immunocompetent, alloantigen-reactive T blast cells, has been utilized to define the H-2-associated lymphocyte-stimulating (LS) determinants. Data obtained using mPLT cells indicate that both the Ia molecules of the J region and the SD molecules of theK or D regions possess LS determinants. Isolated Ia molecules as well as isolated SD molecules induce mPLT cell proliferation irrespective of the genetic background, thus revealing that both classes of H-2 LS antigens function in an autonomous manner. Restimulation data of mPLT cells sensitized toI-region gene products indicate that the LS determinants of the Ia molecules are the Ia specificities. However, whereas subregionI-E (I-C) determines one stimulating moiety, ia.7, subregionI-A determines multiple stimulating Ia determinants associated with each allelic product. Genetic analysis, in combination with known serology, suggests that each allelic product of theK andD regions possesses a unique LS determinant. Based on specific cross-reactivities exhibited by mPLT cells sensitized against SD molecules, the recognition of the SD-associated LS determinant appears to be distinct from the recognition of SD specificities by antibody and recognition of the target moiety by cytotoxic T lymphocytes. Thus, this mPLT assay provides a positive approach to defining the H-2 LS determinants as well as a technique for isolating cells with functionally restricted, clonal responses. Furthermore, we propose here a nomenclature for the designation of mPLT-defined LS determinants.     

Serological characterization of previously unknown H-2 molecules identified in the products of theK d andD k region

The molecular relationship between H-2 private and some public specificities was studied in C3H.OH (H-2 ⁰² ) mice using surface-antigen re-distribution methods. Besides the K^d- and D^k-region antigens, which can be capped by antisera against the private and public specificities characteristic for a given allele, a previously unknown type of molecule was found in the products of both theK ^d andD ^k regions. These can be capped by the respective anti-private serum but not by antisera against some public specificities. The two K^d-region molecules are provisionally named H-2K1^d and H-2K2^d. We detected them onH-2 ⁰² (K ^d ,I ^d ,S ^d ,D ^k ) and also onH-2 ^dx (K ^d ,I ^f ,S ^f ,D ^dx ) T lymphocytes. Similarly, the two types of molecules detected on the products of theD ^k region are provisionally named H-2D1^k and H-2D2^k. The serological characteristics of these molecules are described. When compared with the products of theD ^d region, in which we previously described three different molecules (H-2D^d, H-2M^d, and H-2L^d), the mutual relationship between H-2K1^d and H-2K2^d as well as between H-2D1^k and H-2D2^k appears to be similar to that between H-2D^d and H-2M^d. In the absence of relevant recombinants or informative biochemical data, it is, however, difficult to establish homology between molecules produced by differentK- andD-region alleles.     

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

BALB/c-H-2 db : A newH-2 mutant in BALB/cKh that identifies a locus associated with theD region

A newH-2 mutant, BALB/c-H-2 ^db , is described. This mutant originated in BALB/c, is inbred, and is coisogenic with the parental BALB/cKh strain. The mutation is of the loss type since BALB/c-H- ^db rejects BALB/c, but not vice versa. Complementation studies have localized the mutation to theD region of theH-2 complex. A cross between BALB/c-H-2 ^db and B10.D2-H-2 ^da failed to complement for either BALB/c or B10.D2 skin grafts, indicating that these are two separate mutations at the same locus (Z2). Direct serological analysis and absorption studies revealed that, with one exception, theH-2 andIa specificities of BALB/c and BALB/c-H-2 ^db are identical. In particular,H-2.4, the H-2D^d private specificity, is quantitatively and qualitatively identical in the two strains. The exception is that of the specificities detected by antiserum D28b: (k×r)F₁ anti-h, which contains anti-H-2.27, 28, and 29. These specificities appear to be absent from theH-2 ^db mutant since they are not detected directly or by absorption. Other public specificities are present in normal amounts,e.g., the reaction with antisera to H-2.3, 8, 13, 35, and 36. The reaction with antiserum D28 (f×k)F₁ anti-s, which contains antibodies to H-2.28, 36, and 42, is the same in both strains. Antiserum made between the two strains (H-2 ^db anti-H-2 ^d ) reacts like an anti-H-2 serum, in that it reacts with both T and B cells by cytotoxicity, but is not a hemagglutinating antibody. The serum reacts as does the D28b serum in both strain distribution and in cross-absorption studies. We conclude that theH-2 ^db mutation occurred at a locus in theD region, resulting in the loss of the H-2.28 public serological specificity and of a histocompatibility antigen. Whether these are one and the same antigen is not yet known. The data, in view of other evidence, imply that the public and private specificities are coded for by separate genes.Abbreviations used in this paper are as follows CML cell-mediated lysis - MLR mixed lymphocyte reaction - GVHR graft-versus-host reaction - RFC rosette-forming cells - RAM-Ig rabbit anti-mouse IgG     

The histocompatibility-2 system in wild mice

The I-region gene products of 29 wild-derivedH-2 haplotypes on a B10 background (B10.W congenic lines) were typed with alloantisera which detect 17 inbred I-region antigens. Five new I-region antigens were defined by expanding the inbred line panel ofH-2 haplotypes to includeH-2 ^u , H-2^v, andH-2 ^j . Based on serological analyses of the inbred and B10.W lines, the polymorphism of theIA gene (or genes) is estimated to be at a minimum of 15 alleles and theIE gene (or genes) at a minimum of 4 alleles. These results indicate that theIA subregion is more polymorphic than theIE subregion. By combining the I-region typing data with theH-2K andH-2D region typing data reported previously, a total of 11 new natural recombinants of inbredH-2 alleles were detected among the B10.W lines.     

A new H-2.1-PositiveD region allele,D dx,controlling two molecules,H-2Ddx and H-2Ldx

The inbred strains GRS/A and LIS/A carry the haplotypeH-2 ^dx , which had earlier been shown to have theK ^d ,I ^f ,S ^f , andG ^f alleles and a previously unknownD region allele,D ^dx . We show here that theD ^dx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterD ^k ) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the D^dx product comprises two molecules: H-2D^dx bearing the private specificity H-2.63, and H-2L^dx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated D^dx products shows that the H-2L^dx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000).     

Evidence for a third,Ir-associated histocompatibility region in theH-2 complex of the mouse

Skin grafts transplanted from B10.HTT donors onto (A.TL × B10)F₁ recipients are rapidly rejected despite the fact that the B10.HTT and A.TL strains should be carrying the sameH-2 chromosomes and that both the donor and the recipient contain the B10 genome. The rejection is accompanied by a production of cytotoxic antibodies against antigens controlled by theIr region of theH-2 complex. These unexpected findings are interpreted as evidence for a third histocompatibility locus in theH-2 complex,H-2I, located in theIr region close toH-2K. The B10.HTT and A.TL strains are postulated to differ at this hypothetical locus, and the difference between the two strains is explained as resulting from a crossing over between theH-2 ^t1 andH-2 ^s chromosomes in the early history of the B10.HTT strain. TheH-2 genotypes of the B10.HTT and A.TL strains are assumed to beH-2K ^s Ir ^s / ^k Ss ^k H-2D ^d andH-2K ^s Ir ^k Ss ^k H-2D ^d , respectively. Thus, theH-2 chromosomes of the two strains differ only in a portion of theIr region, including theH-2I locus. The B10.HTT(H-2 ^tt) and B10.S(7R)(H-2 ^th) strains differ in a relatively minor histocompatibility locus, possibly residing in theTla region outside of theH-2 complex.